J R Mackley

J Ando

P Herzyk

S J Winder
email: s.winder@sheffield.ac.uk

Prof Steve Winder

Phenotypic responses to mechanical stress in fibroblasts from tendon, cornea and skin

Keywords: Fibroblast, mechanical stimulation, phenotype, microarray, mechanotransduction Biochemical

Primary fibroblasts isolated from foetal mouse cornea, skin and tendon were subjected to linear shear stress and analysed for morphological parameters and by microarray as compared with unstimulated controls. Approximately 350 genes were either up or down regulated by a significant amount, with 51 of these being common to all 3 cell types. Approximately 50% of altered genes in tendon and cornea fibroblasts were changed in common with one of the other cell types, with the remaining approximately 50% being specific to tendon or cornea. In skin fibroblasts however, less than 25% of genes whose transcription was altered were specific only to skin. The functional spectrum of genes that were up or down regulated was diverse, with apparent house-keeping genes forming the major category of up-regulated genes, however a significant number of genes associated with cell adhesion, extracellular matrix and matrix remodeling, as well as cytokines and other signaling factors were also affected, somewhat surprisingly in these latter categories the trend was towards a reduction in mRNA levels. Verification of the mRNA quantity of a subset of these genes was performed by RT-PCR and was found to be in agreement with the microarray analysis.

These findings provide the first in-depth analysis of phenotypic differences between fibroblast cells from different tissue sources and reveal the responses of these cells to mechanical stress.

Introduction

Fibroblasts are stromal cells that constitute the predominant cell type in mesenchymal tissues and are considered to be the primary source of most extracellular matrix (ECM) components [START_REF] Gabbiani | The Fibroblast[END_REF]. The ECM is composed of a variety of proteins, including collagens, glycosaminoglycans, proteoglycans, and adhesive proteins such as fibronectin and laminin, all of which are secreted by fibroblasts and are eventually assembled into an organized meshwork based upon the functional requirement of the given tissue. Fibroblasts are also capable of producing and secreting various proteolytic enzymes, such as collagenases [START_REF] Birkedal-Hansen | Synthesis and release of procollagenase by cultured fibroblasts[END_REF][START_REF] Werb | A specific collagenase from rabbit fibroblasts in monolayer culture[END_REF] and serine-proteases [START_REF] Wilson | Molecular species of plasminogen activators secreted by normal and neoplastic human cells[END_REF], as well as some of their specific inhibitors [START_REF] Laiho | Enhanced production and extracellular deposition of the endothelial-type plasminogen activator inhibitor in cultured human lung fibroblasts by transforming growth factor-beta[END_REF], which allows fibroblasts to exert further control on the composition and turnover of the ECM. In addition to the synthesis and regulation of the ECM, fibroblasts play essential roles in wound healing and have been found to be involved in the pathogenesis of fibrotic processes [START_REF] Sappino | Differentiation repertoire of fibroblastic cells: expression of cytoskeletal proteins as marker of phenotypic modulations[END_REF].

Despite their broad functional roles and wide-spread use in culture, fibroblasts are very poorly defined. To date, there is no known marker for fibroblasts, mainly due to the fact that all potential markers have also been identified in other members of the connective tissue cell family, such as osteoblasts, smooth muscle cells and adipocytes [START_REF] Wolf | Intraclonal plasticity for bone, smooth muscle, and adipocyte lineages in bone marrow stroma fibroblastoid cells[END_REF]. To make matters more complicated, the term "fibroblast" sometimes refers to a phenotype or a fibroblast-like cell, rather than a specific cell type. This morphological definition is based upon the fact that fibroblasts are traditionally considered to have a relatively uniform morphology, appearing elongated and spindleshaped, with clear leading and trailing edges, a morphology sometimes shared in cells that aren't of fibroblast origin.

The observation that all fibroblasts are created morphologically equal has since been shown to be untrue, as demonstrated by the morphological differences observed between fibroblasts analyzed in vivo and those cultured in vitro [START_REF] Gabbiani | The Fibroblast[END_REF][START_REF] Pinto Da Silva | Gap junctions in normal and transformed fibroblasts in culture[END_REF][START_REF] Ross | Electron microscopy: attachment sites between connective tissue cells[END_REF]. Phenotypic plasticity in fibroblasts is further supported by findings that fibroblasts isolated from distinct tissues demonstrate unique behavior in culture, such as sensitivity to trypsin and EDTA, replication rate, saturation density, attachment efficiency, and proliferative capacity [START_REF] Banes | Cell populations of tendon: a simplified method for isolation of synovial cells and internal fibroblasts: confirmation of origin and biologic properties[END_REF][11][START_REF] Harper | Human skin fibroblasts derived from papillary and reticular dermis: differences in growth potential in vitro[END_REF][START_REF] Riederer-Henderson | Attachment and extracellular matrix differences between tendon and synovial fibroblastic cells[END_REF], discernible morphology [START_REF] Brown | Loading paradigms-intentional and unintentional--for cell culture mechanostimulus[END_REF][START_REF] Conrad | Differences in vitro between fibroblast-like cells from cornea, heart, and skin of embryonic chicks[END_REF][START_REF] Banes | Mechanical load stimulates expression of novel genes in vivo and in vitro in avian flexor tendon cells[END_REF], differential synthesis of extracellular matrix proteins [11,[START_REF] Riederer-Henderson | Attachment and extracellular matrix differences between tendon and synovial fibroblastic cells[END_REF][START_REF] Evans | An in vitro comparison of human flexor and extensor tendon cells[END_REF], and distinct cell-surface antigen presentation and surface receptors [START_REF] Garrett | Fibroblast-like cells from embryonic chick cornea, heart, and skin are antigenically distinct[END_REF]19].

In order to test the hypothesis that fibroblasts from different tissues are phenotypically distinct from one another, we have subjected tendon, skin and corneal

Experimental

Fibroblast Isolation and Culture

All cell culture reagents were obtained from Gibco (Paisley, UK). Embryos used for fibroblast isolation were obtained from a time mated CD1 mouse. At 19-days post conception, the pregnant mouse was euthanized by CO 2 asphyxiation and immediately swabbed with 70% ethanol in a sterile hood. Tendon, corneal and skin fibroblasts were isolated according to Spector et al [START_REF] Spector | Cells: a laboratory manual[END_REF]. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% FCS in a 5% CO 2 humidified atmosphere at 37 °C, and were subcultured when they reached approximately 80% confluence. Cells were cultured until five population doublings, at which point they were seeded onto a gelatin-coated (1% w/v) glass plate (7cm x 10cm) and allowed to adhere for approximately seven hours prior to stimulation.

Mechanical stimulation

Fluid flow was applied to cells using a parallel plate flow chamber described previously [25]. Wall shear stress (τ w ) was calculated according to the equation τ w = 6µQ/(bh 2 ), where µ is the fluid viscosity (0.001 Pa s), Q is the fluid flow rate (ml/s), and b and h are the width (5.5 cm) and height (0.04 cm) of the flow channel, respectively.

The assembled system was maintained at 37 °C in a 5% CO 2 humidified incubator. In this study, tendon, corneal and skin fibroblasts were subjected to a shear stress of 0.1 dynes/cm 2 for 14 hours, with a flow perfusate of DMEM supplemented with 2% FCS and 1% penicillin/streptomycin.

RNA isolation and purification

RNA extractions were carried out with the Absolutely RNA RT-PCR Miniprep kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions. Isolated total RNA was quantified and qualified by measuring its optical density at 260 nm and 280 nm. RNA samples were stored at -80°C until use.

Microarray analysis

RNA processing and microarray analysis was carried out at the Sir Henry Wellcome Functional Genomics Facility (SHWFGF, University of Glasgow, UK). RNA samples from 3 separate control and stimulated experiments for each of the three tissue

Statistical analysis

Gene expression in the stimulated and control groups for each of three tissues were compared using FunAlyse, a newly-established automated pipeline in SHWFGF (http://www.gla.ac.uk/functionalgenomics/rp/affy_analysis.html). As a first step of this analysis, all 18 samples were normalized using Robust Multichip Average (RMA) method [START_REF] Irizarry | Exploration, normalization, and summaries of high density oligonucleotide array probe level data[END_REF] implemented as module affy 1.2 in the Bioconductor library (http://www.bioconductor.org/). Subsequently, differentially expressed genes were identified using the Rank Products (RP) method [START_REF] Breitling | Rank products: a simple, yet powerful, new method to detect differentially regulated genes in replicated microarray experiments[END_REF] that is particularly powerful for experiments involving small number of biologically replicated samples. For every comparison, this method ranks the genes according to differential expression measured by the non-parametric RP-statistic and assesses the statistical significance by producing false discovery rates (FDR). The differentially expressed functional gene classes assigned using GeneOntology annotations (http://www.geneontology.org/) were identified using iterative Group Analysis (iGA) [28]. The A and B arrays of the Affymetrix GeneChip Mouse Expression Set 430 were analysed separately.

The RP-generated lists of differentially expressed genes cut using FDR 10% were further compared manually to identify the highest up-and lowest down-regulated genes in one or more cell lines. Altered genes were manually classified into one of nine, broad functional groups based upon their functional annotation in the SOURCE [29], GenBank [START_REF] Benson | GenBank: update[END_REF], and Mouse Genome Informatics [START_REF] Blake | MGD: the Mouse Genome Database[END_REF] databases.

Semi-quantitative RT-PCR

RNA isolated from mechanically stimulated and control fibroblasts was reverse transcribed and amplified using the Titan One Tube RT-PCR System (Roche, Lewes, East Sussex, UK) according to the manufacturer's instructions. 10 ng of total RNA was used as the template in all reactions. The reverse transcription reaction was carried out at 50 °C for 30 minutes, followed by denaturation for 2 min at 94 °C. The thermocycling conditions included 35 cycles of 10 seconds at 94 °C, 30 seconds at 45-65 °C (depending upon the melting temperature of the primer pairs used), [START_REF] Roelofsen | Mechanical stimulation by intermittent hydrostatic compression promotes bone-specific gene expression in vitro[END_REF] Copyright 2006 Biochemical Society

Results

Isolated cells are of mesenchymal origin and demonstrate fibroblastic morphology

Because there is no known universal marker for fibroblasts, we used the presence and/or absence of intermediate filament proteins to substantiate the mesenchymal origin of the isolated tendon, corneal and skin cell lines. Cell lysates of HeLa, Swiss 3T3 cells, C2C4 myoblasts and the isolated tendon, corneal and skin fibroblasts were resolved on 12% polyacrylamide gels and immunoblotted for desmin, keratin and vimentin (Fig. 1A). The isolated cell lines lack desmin and keratin intermediate filaments, markers for cells of muscle and epithelial origin, respectively, demonstrating a lack of any substantial contamination by these cell types. Tendon, corneal and skin fibroblasts did, however, contain a significant amount of vimentin, an intermediate filament prevalent in cells of mesenchymal origin. Cells appeared to have a typical fibroblast-like morphology in culture with a flattened irregular outline, on contact inhibition cells remained in the same overall pattern with no significant polarization or change in density as seen with many epithelial derived cells data not shown, see also Fig. 1B.

In order to investigate the possible differential responses of tendon, corneal and skin fibroblasts, all three cell lines were subjected to mechanical stimulation by shear stress. Cells were subcultured until five population doublings, seeded onto gelatincoated glass plates, and were either subjected to 0.1 dynes/cm 2 of laminar fluid flow for 14 hours (stimulated), or maintained under standard tissue culture conditions (control).

Both control and stimulated fibroblasts from each cell line were imaged after treatment (Fig. 1B). Upon initial inspection, it is evident that the magnitude of stimulation did not displace cells from their substrate, and tendon, corneal and skin fibroblasts maintain typical elongated, spindle-like morphology even after stimulation. Furthermore, it does not appear that the stimulated fibroblasts re-oriented in the direction of flow. Rather, cells seem to be randomly oriented on the gelatin-coated substrate and display no overt morphological changes as a consequence of the mechanical stimulation (Fig. 1B).

Tendon, corneal and skin fibroblasts demonstrate differential gene regulation when subjected to an identical mechanical stimulus

To gain a comprehensive sense of the genetic responses of tendon, corneal and skin fibroblasts to mechanical stimulation, gene regulation was examined in stimulated and control cells by microarray analysis. Following stimulation, cells were lysed Copyright 2006 Biochemical Society directly on the glass plate, and total RNA was isolated and amplified prior to analysis.

The probe array used was the GeneChip Mouse Expression Set 430 (Affymetrix, High Wycombe, UK), which enabled the simultaneous quantification of expression of 34,000 genes in parallel.

Microarray analysis revealed that 344 genes were significantly differentially regulated with stimulation (Fig. 2a). Of these altered genes, 15% were common to all three cell lines, 17% were identified in two of the three cell lines, and 69% were differentially regulated in one cell line only. Interestingly, 34% and 27% of the total genes identified were found to be unique to either tendon or corneal fibroblasts, respectively. In contrast, only 7% of the genes identified were unique to skin fibroblasts suggesting a more specific phenotypic expression in tendon and corneal cells than in skin. Upon closer inspection, it is apparent that tendon and corneal fibroblasts have approximately 6% of the identified genes in common, while tendon and skin fibroblasts share 8% of differentially regulated genes. Given that corneal and skin fibroblasts maintain only 3% of the altered genes in common, this hints to the possibility that skin and corneal fibroblasts demonstrate the most differential phenotypic response to stimulation.

In order to examine the transcriptional response of these cell lines to stimulation more carefully, altered genes were manually classified into one of nine, broad functional groups based upon their functional annotation in the SOURCE[29], GenBank [START_REF] Benson | GenBank: update[END_REF], and Mouse Genome Informatics [START_REF] Blake | MGD: the Mouse Genome Database[END_REF] databases. An automated Iterative Group Analysis classification system[28] was also used with qualitatively similar results, data not shown. Upon initial inspection, it is clear that the functional spectrum of altered genes was diverse; transcripts were found to be implicated in apoptosis, cell division, ECM and cytoskeletal remodeling, housekeeping, cell signaling, stress response, transcription or cellular transport (Fig. 2b). Tendon fibroblasts demonstrated approximately equal numbers of up-and down-regulated genes with stimulation, whereas the majority of genes were down-regulated in corneal fibroblasts. When compared with the other two cell lines, skin fibroblasts showed a significantly lower number of altered genes with stimulation, the majority of which were up-regulated. It is also clear that housekeeping genes comprise the major category of up-regulated genes, while a significant number of genes associated with cell division, extracellular matrix and cytoskeletal remodeling, as well as signaling factors, showed reductions in mRNA levels. The proportional contribution of the functional spectrum of differentially regulated genes was also calculated (Fig. 2C). Upon consideration of the total number of altered genes, the proportion of altered apoptosis-and transcription-related genes was within 3% in all three cell lines. Tendon and corneal fibroblasts show a significantly higher proportion of alterations in genes involved in cell division and ECM and cytoskeleton remodeling as compared with skin fibroblasts. Furthermore, corneal fibroblasts demonstrate a lower contribution of genes involved in signaling when compared with the other two cell lines. Of the genes differentially regulated with stimulation in tendon and corneal fibroblasts, approximately 4% were involved in transport. This is in contrast to an approximate 8% contribution demonstrated by skin fibroblasts. Signaling-and housekeeping-related genes were altered in similar proportions in tendon and skin fibroblasts, whereas the proportion of signaling-related genes changed with stimulation in corneal fibroblasts was markedly less. Of the transcripts implicated in stress responses, skin fibroblasts demonstrated the largest proportion, followed by tendon and then corneal cells.

If one considers differentially up-and down-regulated genes separately, it becomes apparent that the majority of housekeeping, stress response and transport related transcripts are up-regulated with stimulation, whereas apoptosis-, cell divisionand signaling-related genes are largely down-regulated. Tendon fibroblasts have the largest proportion of up-regulated genes involved in ECM/cytoskeletal remodeling, whereas skin fibroblasts have a markedly larger number of up-regulated genes involved in signaling and transport. As far as down-regulated genes are concerned, all three cell lines show a similar proportion of genes involved in apoptosis and transport-related

genes, yet maintain differences in the proportions of other functional classifications. Tendon fibroblasts, for example, have a significantly higher proportion of ECM/cytoskeletal remodeling-, housekeeping-and signaling-related genes downregulated with stimulation, whereas skin fibroblasts have a higher proportion of upregulated stress response-and transcription-related genes.

RT-PCR and quantification

In order to validate the changes in gene expression identified by microarray analysis, a subset of 14 genes was examined by semi-quantitative RT-PCR, representing eight down-regulated and six up-regulated genes. These particular genes were chosen based upon their differential regulation in one or more cell lines and/or possible role in for RT-PCR. The resultant products were electrophoresed and the volume integration of each product, visualized by ethidium bromide staining, was quantified with NIH Image (Fig 3). In all cases, the generated RT-PCR products migrated at their expected size, and semi-quantitative RT-PCR substantiated the up-or down-regulation of transcription initially revealed by the microarray data; the direction of regulation (i.e. up-or downregulation) is conserved in all cases between RT-PCR and the microarray data. On occasion, the relative magnitude of regulation determined by RT-PCR appears to be different between different genes, but not within genes with respect to the microarray data. This is partly due to the limitations of determining a useful dynamic range for the number of PCR cycles in the RT-PCR that could be applied across all samples. For example, the RNA levels determined by RT-PCR for Neogenin and Serpin1b were relatively low. Consequently, upward changes with stimulation are disproportionately large compared to genes with a more robust RT-PCR signal, such as Gsta4 and Hmox1.

Accordingly, quantification of the RT-PCR reactions are listed in a semi-quantitative manner (Fig. 3).

Protein levels do not directly correlate with mRNA levels, but do change, in stimulated fibroblasts

In an effort to gain a more global understanding of the response of fibroblasts to mechanical stimulation, levels of expression of four proteins were also investigated (Fig. 4, Table I). To achieve this, control and mechanically stimulated fibroblasts were lysed immediately after treatment, sonicated and the protein concentration of the resulting lysate determined. Equal concentrations of lysates were electrophoresed and subsequently visualized by western blotting, using antibodies directed to lumican, dyxin, CRP1, and neogenin, and quantified using a β-actin loading control.

The lumican antibody identified four distinct bands, indicating proteins with apparent molecular weights of 73, 62, 30 and 26 kDa, corresponding to the intact glycoprotein, an alternatively glycosylated form, and two isoforms of the core protein typically found in fibroblasts, respectively [START_REF] Funderburgh | Isoforms of corneal keratan sulfate proteoglycan[END_REF][START_REF] Funderburgh | Unique glycosylation of three keratan sulfate proteoglycan isoforms[END_REF]. After quantification, it appears that stimulation has no effect on the levels of the 73, 30 or 26 kDa species in all three cell lines. However, the 62 kDa species, corresponding to the alternatively deglycosylated form of lumican, shows an increase of approximately 6-, 3.5-, and 7-fold with Copyright 2006 Biochemical Society stimulation in tendon, corneal and skin fibroblasts, respectively. The apparent increase in the 62 kDa lumican species is at odds with the decreased levels of lumican mRNA with stimulation, as determined by both microarray and RT-PCR. However, the appearance of an alternative glycosylated form upon stimulation is in itself interesting.

This points to a post-translational pathway that has effects in a different direction to the transcriptional control, which may reveal an important functional insight into the role of lumican. This aspect of the study warrants further investigation.

The dyxin antibody revealed a band of approximately 40 kDa, which is in agreement with previous reports [START_REF] Yi | Specific and potent RNA interference in terminally differentiated myotubes[END_REF]. After quantification and normalization against βactin, all three cell lines appear to demonstrate an increase in protein expression with stimulation. Skin and tendon fibroblasts show the greatest increase in protein levels, with an approximate increase of 2-fold when compared to controls. This increase in dyxin protein levels is in contrast to reducing amounts of mRNA transcripts following stimulation.

Probing cell lysates with the CRP1 antisera revealed a band of approximately 20 kDa. Levels of transcripts encoding CRP1 were found to decrease in stimulated tendon fibroblasts, and this trend correlated with a concomitant 3.8-fold decrease in CRP1 protein levels. Furthermore, despite the fact that differential expression of CRP1 mRNA was not shown to be significantly altered in stimulated corneal or skin fibroblasts, levels of CRP1 protein in these cell lines was also shown to decrease by approximately 3-fold.

Neogenin immunoblots revealed a prominent band at approximately 193 kDa;

this most likely differs from the protein's theoretical molecular weight of 150 kDa because of glycosylation [START_REF] Vielmetter | Neogenin, an avian cell surface protein expressed during terminal neuronal differentiation, is closely related to the human tumor suppressor molecule deleted in colorectal cancer[END_REF]. Shear stress resulted in the differential regulation of neogenin by skin and corneal fibroblasts. Neogenin was found to increase by approximately 2-fold upon the application of mechanical stimulation in skin fibroblasts, which correlated well with a 1.67-fold increase in neogenin transcript levels, as determined by microarray analysis. Corneal fibroblasts, on the other hand, displayed an almost 3-fold decrease in neogenin following stimulation. In this case, the decrease in protein levels occurs without any apparent change in gene expression. Tendon fibroblasts did not show any change in neogenin protein levels with stimulation.

Discussion

Whilst the word fibroblast is a relatively generic term applied to connective tissue cells of diverse origins, these cells non-the-less possess features specific to their Copyright 2006 Biochemical Society tissue of origin. In this study we have attempted to define the genes in common and genes that define that specificity by examining the response of fibroblasts from different tissues to mechanical stress.

Of the 344 genes shown to be differentially regulated in the three cell lines, a surprisingly low 15% of these were common to all three and less than a third were shared between any two cell lines. A summary of some of these genes are shown in Table II. Furthermore, the spectrum of genes that are altered in response to mechanical stress are perhaps intuitively not what one might have expected.

A number of genes were found that have a role in the protection of cells against oxidative stress, a phenomenon generated upon the production of reactive oxygen that can lead to subsequent changes to cellular macromolecules including nucleic acids, proteins and lipids [START_REF] Yang | Protection of HLE B-3 cells against hydrogen peroxide-and naphthaleneinduced lipid peroxidation and apoptosis by transfection with hGSTA1 and hGSTA2[END_REF]. The overwhelming majority of these genes, glutathione Stransferase (Gsta2), heme oxygenase 1 (Hmox1), glutamate-cysteine ligase, modifier subunit (Gclm), xanthine dehydrogenase (Xdh) aldo-keto reductase family 1, member B3 (Akr1b3), secretory leukocyte protease inhibitor (Slpi), were up-regulated in various cell lines after stimulation. Given the method by which these cells were stimulated, it is not surprising that all three cell lines responded by up-regulating oxidative response

genes. An increase in these transcripts most likely serves to protect the cells from reactive oxygen species produced as a consequence of mechanical stimulation. It is not so much the fact that the cells are all mounting a similar stress response that is of particular interest, but rather the differential regulation of unique oxidative-stress genes.

All three cell lines demonstrated the differential regulation of genes implicated in adipocyte differentiation, which is of particular interest given the capability of fibroblast precursors to differentiate along various connective tissue cell-type lineages, such as adipocytes [START_REF] Wolf | Intraclonal plasticity for bone, smooth muscle, and adipocyte lineages in bone marrow stroma fibroblastoid cells[END_REF]. In the case of carbonic anhydrase 3 (Car3) or lipocalin 2 (Lcn2), which have both been found to be expressed at high levels in mature adipocytes [START_REF] Kim | Nucleotide sequence and structure of the mouse carbonic anhydrase III gene[END_REF]40], it appears that stimulation is effectively reinforcing the fibroblast phenotype, since levels of these genes were down-regulated with fluid flow. In contrast, however, angiopoietin-like 4 (Angptl4) and ectonucleotide pyrophosphatase/phosphodiesterase 2 (Enpp2) have been shown to be up-regulated during adipocyte differentiation [START_REF] Mandard | The direct peroxisome proliferator-activated receptor target fasting-induced adipose factor (FIAF/PGAR/ANGPTL4) is present in blood plasma as a truncated protein that is increased by fenofibrate treatment[END_REF][START_REF] Yoon | Peroxisome proliferator-activated receptor gamma Biochemical Journal Immediate Publication. Published on 22 Feb 2006 as manuscript BJ20060057 Copyright 2006 Biochemical Society target gene encoding a novel angiopoietin-related protein associated with adipose differentiation[END_REF][START_REF] Ferry | Autotaxin is released from adipocytes, catalyzes lysophosphatidic acid synthesis, and activates preadipocyte proliferation. Up-regulated expression with adipocyte differentiation and obesity[END_REF].

Because Angptl4 and Enpp2 were both up-regulated in this study in response to stimulation, this hints to the possibility that shear stress is inducing an adipogenic-shift in tendon, corneal and skin fibroblasts. While this picture is currently conflicting and Copyright 2006 Biochemical Society difficult to interpret, the implication of these genes in mesenchymal differentiation is nonetheless interesting and should be investigated further.

Because the method of stimulation used in this study was mechanical, genes that were implicated in mechanotransduction, or the conversion of mechanical signals into biochemical response via direct or indirect connections between the internal actin cytoskeleton and the ECM, were investigated. Not surprisingly, numerous studies have shown that cells subjected to mechanical stimuli alter both the gene and protein expression of ECM and cytoskeletal components [START_REF] Jalali | Integrin-mediated mechanotransduction requires its dynamic interaction with specific extracellular matrix (ECM) ligands[END_REF][START_REF] Roelofsen | Mechanical stimulation by intermittent hydrostatic compression promotes bone-specific gene expression in vitro[END_REF][START_REF] Lambert | Pretranslational regulation of extracellular matrix macromolecules and collagenase expression in fibroblasts by mechanical forces[END_REF][START_REF] Lambert | Distinct pathways in the over-expression of matrix metalloproteinases in human fibroblasts by relaxation of mechanical tension[END_REF]. Given the importance of the cytoskeletal -ECM linkage in maintaining cellular integrity and physically resisting mechanical stress the number of genes altered in this category were surprisingly few, accounting for less than 5% of genes overall. ECM structure might be expected to be altered in response to shear stress. Two genes in this category were shown to be markedly altered with stimulation, however lumican (Lum) was downregulated, whereas chondroitin sulphate proteoglycan 4 (Cspg4) was upregualted.

Of the relatively few genes implicated in ECM/cytoskeleton remodeling, three were found to encode LIM domain-containing cytoskeletal adaptor proteins: cysteine and glycine-rich protein (Crp1), LIM and cysteine-rich domains 1 (Dyxin) and LIM domain containing preferred translocation partner in lipoma (Lpp). The LIM domain is implicated in protein-protein interactions, may target proteins to distinct subcellular locations, and is thought to mediate the assembly of multimeric protein complexes [START_REF] Wang | Human cysteine-rich protein. A member of the LIM/double-finger family displaying coordinate serum induction with c-myc[END_REF].

Levels of Crp1, which itself has been shown to interact with alpha-actinin [START_REF] Harper | Fine mapping of the alpha-actinin binding site within cysteine-rich protein[END_REF], as well as associate with and possibly stabilize adhesion plaques through its binding to zyxin [START_REF] Chang | Cysteine-rich LIMonly proteins CRP1 and CRP2 are potent smooth muscle differentiation cofactors[END_REF]51], were down-regulated in tendon fibroblasts with stimulation. Both Dyxin and Lpp were found to be down-regulated in corneal fibroblasts after stimulation.

Other genes encoding proteins that either directly or indirectly interacted with the actin cytoskeleton were also found such as myosin light chain kinase (Mlck), Mlck transcripts were reduced in tendon fibroblasts upon stimulation. We have identified Mlck previously as a shear stress-response gene in endothelial cells [START_REF] Ando | Differential display and cloning of shear stress-responsive messenger RNAs in human endothelial cells[END_REF], however in this case, Mlck was up-regulated. Furthermore, in Mlck-inhibited cells, myosin light chain phosphorylation was blocked at the cell periphery but not at the centre of the cell, and zyxin-containing adhesions were not assembled at the cell periphery but focal adhesions were maintained in the centre of the cell. These cells continued to generate membrane protrusions around the cell, but turned over more frequently and migrated less effectively [START_REF] Totsukawa | Distinct roles of MLCK and ROCK in the regulation of membrane protrusions and focal adhesion dynamics during cell migration of fibroblasts[END_REF]. It is possible, then, that tendon fibroblasts demonstrated a reduction in Copyright 2006 Biochemical Society Mlck levels in order to reduce migration under such mechanical stresses. Corneal fibroblasts also demonstrated a decrease in the level of transcripts for the FERM domain containing 3 (Frmd3) gene. Interestingly, only one gene implicated in ECM/cytoskeleton remodeling, Adamts5, was common to two or more cell lines after mechanical stimulation, with the rest being unique to one of the three lines tested. This trend is seen only in this functional classification and suggests that ECM/cytoskeletonrelated genes may contribute more specifically to the differential response of tendon, corneal and skin cell lines to stimulation.

The fact that protein levels showed such variable correlation to microarray data is not surprising and has been documented in previous reports [START_REF] Lichtinghagen | Different mRNA and protein Biochemical Journal Immediate Publication. Published on 22 Feb 2006 as manuscript BJ20060057 Copyright 2006 Biochemical Society expression of matrix metalloproteinases 2 and 9 and tissue inhibitor of metalloproteinases 1 in benign and malignant prostate tissue[END_REF]. Such poor correlations between protein and mRNA levels most likely results from (1) the possibility of regulating protein levels at either transcriptional or translational levels, and/or (2) the rate of protein turnover in vivo [START_REF] Greenbaum | Comparing protein abundance and mRNA expression levels on a genomic scale[END_REF]. This study provides the first in-depth analysis of the tissue-specific transcriptional response of a cell type to a mechanical stimulation and its comparison to the same cell type in other tissues. These findings will provide an invaluable resource for further study of the factors that control cell and tissue specific mechanical stress responses and may provide avenues for the manipulation and improvement of tissue engineered prostheses and implants for reconstructive surgery.

Figure 1 .

 1 Figure 1. Confirmation of mesenchymal origin of isolated fibroblasts and morphological examination after mechanical stimulation. A, Lysates of cultured mouse tendon, corneal and skin fibroblasts were resolved by SDS-PAGE and submitted to immunoblot analysis with the indicated intermediate filament anti-sera. Lysates of HeLa cells, NIH 3T3 cells, and C2C4 myoblasts serve as positive controls for keratin, vimentin and desmin, respectively. Fibroblast cells from tendon, cornes and skin expressed only vimentin demonstraing a lck of contamiantion by epithelial or muscle tissues. B, Tendon, corneal and skin fibroblasts were seeded onto gelatin-coated glass plates and placed either in a single well plate (control) or subjected to 0.1 dynes/cm 2 fluid flow for 14 h (stimulated). Cells were subsequently imaged with phase contrast microscopy in order to examine cell directionality and morphology. Scale bar = 100 µm. Arrow = direction of fluid flow.

Figure 2 .

 2 Figure 2. Total number and functional classification of genes up-and downregulated in tendon, corneal and skin fibroblasts following mechanical stimulation. mRNA isolated form mechanically stimulated tendon, corneal and skin fibroblasts was subjected to microarray analysis. A, Venn Diagram representing 344 up-and downregulated genes in tendon, corneal and skin fibroblasts. The number of genes identified in a given cell line are designated, and overlap regions denote genes common to the indicated cell lines. B, Number and functional classification of genes up-or downregulated in three fibroblast cell lines in response to stimulation. Genes were categorized into one of nine broad categories based upon their functional annotation (GenBank). C, Proportional contribution of the functional classifications of genes to the total numbers of genes up-or down-regulated in response to fluid flow in each individual cell type.

Figure 3 .

 3 Figure 3. Validation of microarray data by semi-quantitative RT-PCR. Mouse skin, tendon, and corneal fibroblasts were subjected to 14 hours of laminar flow at a rate of 0.1 dynes/cm 2 . Total RNA was isolated from three replicate experiments of control (C)

  gene expression profiles of tendon (T), corneal (C) and skin (S) fibroblasts were assessed as described in "Experimental Procedures." The tabulated genes were manually clustered into the indicated functional groups based upon their annotation. The numerical intensity values shown are the fold-changes calculated by the FunaAlyse software as antilog of mean between-chip log-ratios, positive values correspond to up-regulation whilst negative values correspond to down-regulation. Triplicate microarray experiments were performed with each fibroblast cell line.

Figure 1A Figure 1B Biochemical

 1A1B Figure 1A

Biochemical Journal Immediate Publication. Published on 22 Feb 2006 as manuscript BJ20060057 Copyright 2006 Biochemical Society types

  were analysed on the Affymetrix GeneChip Mouse Expression Set 430 using standard Affymetrix protocols.

Biochemical Journal Immediate Publication. Published on 22 Feb 2006 as manuscript BJ20060057 Copyright 2006 Biochemical Society

  seconds at 68 °C, which was increased by 5 seconds for each cycle during cycles 11-25, and a

	final, prolonged elongation cycle of 7 minutes at 68 °C. PCR was performed using
	primers	specific	for	each	target	gene	(Supplementary	Table	I
	http://www.BiochemJ.org/bj/vvv/bjvvvppppadd.htm) and conditions were chosen such
	that all of the RNAs analysed were in the exponential phase of amplification. Aliquots
	of the PCR reactions were electrophoresed on 1% agarose gels containing 80 ng/ml
	ethidium bromide and photographed. The integrated density of the resultant bands was
	quantified for each sample (NIH Image).
	Western Blot Analysis
	Western blotting was used to confirm the developmental origin of isolated
	fibroblasts as well as evaluate levels of lumican, dyxin, CRP1, and neogenin, since the
	genes encoding these proteins were shown to be differentially regulated with shear
	stress. Cells were lysed in ice-cold lysis buffer (50 mM Tris pH 6.8, 1% (w/v) SDS,
	10% (v/v) glycerol) containing protease inhibitors (1 µM pepstatin, 1 mM PMSF, 100
	µM TPCK, 260 µM TAME, 10 mM benzamidine) and sonicated on ice. The protein
	concentration of cell extracts was quantified using the Micro BCA Protein Assay
	(Perbio Ltd, Cramlington, UK), and 10 µg were separated by 12% sodium dodecyl
	sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) [32] and electrophoretically
	transferred to a polyvinylidene difluoride membrane. Western blotting was carried out
	as described previously[33] with one of eight primary antibodies: mouse monoclonal
	anti desmin (1:200; Sigma), guinea pig polyclonal anti keratin (1:200; Sigma), goat
	polyclonal anti vimentin (1:400), rabbit polyclonal anti lumican (1:500 dilution; a
	generous gift from A Oldberg, University of Lund, Sweden), rabbit polyclonal anti
	dyxin (1:50 dilution; kindly provided by R Crosbie, UCLA), mouse monoclonal anti
	CRP1 (1:100 dilution; BD Transduction Laboratories), rabbit polyclonal anti neogenin
	(1:50 dilution; Santa Cruz Biotechnology Inc.), goat polyclonal anti-actin was used as
	loading control (1:500; Santa Cruz Biotechnology Inc.). Blots were developed with an
	appropriate secondary antiserum and detected with chromogenic or ECL substrates as
	previously described [33].
	Microscopy
	Morphology of control and stimulated fibroblasts was assessed by phase contrast
	microscopy on a DM IRE2 with Plan 10x/0.3 objective (Leica) at room temperature.

Biochemical Journal Immediate Publication. Published on 22 Feb 2006 as manuscript BJ20060057 Copyright 2006 Biochemical Society

	Images were captured with a DC350F CCD camera (Leica) using Leica QFluoro
	software (version 1.2.0) and Adobe Photoshop 7.0.

Biochemical Journal Immediate Publication. Published on 22 Feb 2006 as manuscript BJ20060057

Biochemical Journal Immediate Publication. Published on 22 Feb 2006 as manuscript BJ20060057

Biochemical Journal Immediate Publication. Published on 22 Feb 2006 as manuscript BJ20060057

Biochemical Journal Immediate Publication. Published on 22 Feb 2006 as manuscript BJ20060057 Copyright 2006 Biochemical Society ECM

  biogenesis or cytoskeletal organization. RNA previously isolated from three replicate mechanical stimulation experiments for each cell line was used as a template

Biochemical Journal Immediate Publication. Published on 22 Feb 2006 as manuscript BJ20060057

Biochemical Journal Immediate Publication. Published on 22 Feb 2006 as manuscript BJ20060057

Biochemical Journal Immediate Publication. Published on 22 Feb 2006 as manuscript BJ20060057

Biochemical Journal Immediate Publication. Published on 22 Feb 2006 as manuscript BJ20060057

Biochemical Journal Immediate Publication. Published on 22 Feb 2006 as manuscript BJ20060057

Biochemical Journal Immediate Publication. Published on 22 Feb 2006 as manuscript BJ20060057 Copyright 2006 Biochemical Society

  . Total RNA was isolated from three replicate experiments of control (C)

	Protein	Species	Tendon	Cornea	Skin
	Lumican	62 kDa	6.07 ± 1.16	3.50 ± 0.49	7.21 ± 4.80
	Dyxin	40 kDa	1.98 ± 0.44	1.24 ± 0.19	2.08 ± 0.69
	Crp1	20 kDa	-3.79 ± 1.62	-3.08 ± 0.93	-2.78 ± 1.22
	Neogenin	193 kDa	-	-2.98 ± 0.97	2.16 ± 1.15

and stimulated (S) tendon, corneal and skin fibroblasts and subjected to reverse transcription for 14 genes of interest. Samples were taken during the linear phase of amplification, resolved by electrophoresis on 1% agarose gels and visualised by

Table I . Quantification of immuno-blotting of ECM/cytoskeleton-related proteins differentially regulated with stimulation.

 I Values are represented as the mean fold change between stimulated and control cells ± SEM.

	Biochemical

Journal Immediate Publication. Published on 22 Feb 2006 as manuscript BJ20060057

			Cell Line
	Gene description	Function	T	C	S	Assession #
	A. Highest up-regulated genes
	heme oxygenase (decycling) 1 (Hmox1)	Housekeeping	20.2 25.3 18.2	NM_010442
	glutathione S-transferase, alpha 2 (Gsta2)	Stress Response	18.7 27.9 13.3	NM_008182
	neoplastic progression 3 (Npn3)	Transcription	11.9 9.8		NM_029688
	angiopoietin-like 4 (Angptl4)	Housekeeping	11.4		8.6	NM_020581
	glutamate-cysteine ligase , modifier subunit (Gclm)	Housekeeping	11.1 9.3 8.5	NM_008129
	heat shock protein 1A (Hsp72)	Stress Response		9.0		NM_010479
	proliferin (Plf)	Signalling			14.0	NM_031191
	B. Lowest down-regulated genes
	CCAAT/enhancer binding protein delta (Cebpd)	Transcription	-4.3			NM_007679
	carbonic anhydrase 3 (Car3)	Housekeeping	-4.3			NM_007606
	chemokine (C-C motif) ligand 20 (Ccl20)	Signalling	-4.0		-2.5	NM_016960
	pentaxin related gene (Ptx3)	Stress Response	-4.0		-2.6	NM_008987
	early growth response 3 (Egr3)	Transcription	-3.5			NM_018781
	forkhead box P2 (Foxp2)	Transcription		-3.6		NM_053242
	follistatin (Fst)	Signalling		-3.4		NM_008046
	cyclin E2 (Ccne2)	Cell Division		-3.3		NM_009830
	GULP, engulfment adaptor PTB domain containing 1 (Gulp1)	Apoptosis		-3.3		BB138485
	solute carrier family 4 (anion exchanger), member 4 (Slc4a4)	Transport		-3.1		NM_018760
	chemokine (C-X-C motif) ligand 5 (Cxcl5)	Signalling			-4.9	NM_009141
	thymic stromal lymphopoietin (Tslp)	Signalling			-2.8	NM_021367
	lipocalin 2 (Lcn2)	Housekeeping			-2.6	NM_008491
	C. Highest up-regulated genes found in only one cell line
	dual specificity phosphatase 4 (Dusp4)	Signalling	4.8			NM_176933
	xanthine dehydrogenase (Xdh)	Housekeeping	4.5			NM_011723
	mitogen activated protein kinase kinase kinase 4 (Map3k4)	Signalling	3.9			NM_011948
	dystonin (Dst)	ECM/Cytoskeleton 3.7			NM_133833
	growth arrest specific 5 (Gas5)	Cell Division	3.5			NM_013525
	gamma-aminobutyric acid receptor-associated protein-like 1 (Gabarapl1)	ECM/Cytoskeleton		4.4		NM_020590
	aldo-keto reductase family 1, member B3 (Akr1b3)	Housekeeping		3.7		BB469763
	sphingosine kinase 1 (Sphk1)	ECM/Cytoskeleton		3.3		NM_011451
	cytochrome b-5 (Cyb5)	Housekeeping		2.9		NM_025797
	ectonucleotide pyrophosphatase/phosphodiesterase 2 (Enpp2)	Housekeeping		2.8		NM_015744
	BTB (POZ) domain containing 11 (Btbd11)	Transcription			3.5	BC072592
	neogenin (Neo)	ECM/Cytoskeleton			2.7	NM_008684
	secretory leukocyte protease inhibitor (Slpi)	Stress Response			2.5	NM_011414
	endothelin receptor type B (Ednrb)	Signalling			2.5	NM_007904
	centaurin, gamma 2 (Centg2)	Transport			2.5	NM_178119
	D. Lowest down-regulated genes found in only one cell line
	carbonic anhydrase 3 (Car3)	Housekeeping	-4.3			NM_007606
	ELAV-like 2 (Hu antigen B) (Elavl2)	Cell Division	-3.0			NM_207685
	chemokine (C-X-C motif) ligand 14 (Cxcl14)	Signalling	-3.0			NM_019568
	early growth response 2 (Egr2)	Transcription	-3.0			NM_010118
	receptor activator of NF-κB Ligand (RANKL)	Signalling	-2.9			NM_011613
	forkhead box P2 (Foxp2)	Transcription		-3.6		NM_053242
	solute carrier family 4, member 4 (Slc4a4)	Transport		-3.1		NM_018760

Biochemical Journal Immediate Publication. Published on 22 Feb 2006 as manuscript BJ20060057 Copyright 2006 Biochemical Society

	RAD51 homolog (S. cerevisiae) (Rad51)	Stress Response	-2.7	NM_011234
	Nik-related kinase (Nrk)	Signalling	-2.7	NM_013724
	anillin, actin binding protein (Anln)	ECM/Cytoskeleton	-2.6	NM_028390
	lipocalin 2 (Lcn2)	Housekeeping	-2.6	NM_008491
	odd-skipped related 1 (Drosophila) (Osr1)	Transcription	-2.3	NM_011859
	endothelin 1 (Edn1)	Signalling	-2.3	NM_010104
	E. Genes of interest identified in one cell line
	lumican (Lum)	ECM/Cytoskeleton -2.5	NM_008524
	LIM and cysteine-rich domains 1 (Dyxin)	ECM/Cytoskeleton	-2.5	NM_144799
	cysteine and glycine-rich protein 1 (Crp1)	ECM/Cytoskeleton -2.4	NM_007791
	myosin, light polypeptide kinase (Mlck)	ECM/Cytoskeleton -2.0	NM_139300
	LIM domain containing preferred translocation partner in lipoma (Lpp)	ECM/Cytoskeleton	-2.1	NM_178665
	FERM domain containing 3 (Frmd3)	ECM/Cytoskeleton	-2.0	NM_172869
	A disintegrin and metalloprotease domain 33 (ADAM33)	ECM/Cytoskeleton 2.1	NM_033615
	chondroitin sulfate proteoglycan 4 (Cspg4)	ECM/Cytoskeleton 3.4	NM_139001
	F. Genes of interest identified in all cell lines
	a disintegrin-like & metalloprotease w/thrombospondin type 1 motif, 5
	(Adamts5)	ECM/Cytoskeleton -1.8 -2.2 -1.9	NM_011782
	serine (or cysteine) proteinase inhibitor, clade B, member 1b (Serpinb1b)	Housekeeping	5.6 4.5 6.7	NM_173052
	glutathione S-transferase, alpha 4 (Gsta4)	Stress Response	6.6 4.3 5.9	NM_010357

Table II . Genes differentially regulated in tendon, corneal and skin fibroblasts with mechanical stimulation. The

 II

Acknowledgements

We are grateful to A Oldberg, University of Lund and R Crosbie, UCLA for gifts of antisera. The research was supported by an MRC Career Establishment Grant to SJW, and JRM was in receipt of a PhD studentship from the University of Stirling.

ethidium bromide staining. The intensity of the resultant bands was quantified (NIH Image). Genes are represented as being up-or down-regulated in skin (S), tendon (T), or corneal (C) fibroblasts by less than 2-fold (-/+), greater than or equal to 2-fold, but less than 10-fold (--/ + +), or greater than or equal to 10-fold (---/ + + +). As means of comparison, the fold-change in gene expression as determined by microarray analysis (where statistically significant) is listed alongside the semi-quantitative integrated density results from RT-PCR.


Claudia Piccoli

Rosella Scrima

Domenico Boffoli

Nazzareno Capitanio

The control of cytochrome C oxidase on the cellular oxidative phosphorylation system depends on the mitochondrial energy state

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

exported to the cytoplasm by the adenine nucleotides translocator, in exchange with ADP, to fulfil the cytoplasmic energy demand. If the availability of ADP in the mitochondrial matrix becomes limiting, the transmembrane electro-chemical H + gradient accumulates and in turn inhibits further unnecessary electron transfer along the respiratory complexes. This negative loop allows modulating the rate of respiration to the cellular energy demand and constitutes the basis of the mitochondrial respiratory control [START_REF] Chance | Respiratory enzymes in oxidative phosphorylation. III. The steady state[END_REF].

The multi-components structuring of the OXPHOS network [START_REF] Saraste | Oxidative phosphorylation at the fin de siecle[END_REF] poses the question to define which step(s) is(are) critical in controlling the efficiency of the overall process. The understanding of this specific point, besides its interest in basic research, is also important for elucidating the etiopathogenesis of a number of human diseases characterised by impairment in the OXPHOS efficiency [START_REF] Kang | Alterations of mitochondrial DNA in common diseases and disease states: aging, neurodegeneration, heart failure, diabetes, and cancer[END_REF]. The metabolic flux control analysis (MFCA) constitutes a solid theoretical and experimental tool that has contributed in our understanding of the molecular mechanism that determines how metabolic pathway fluxes are controlled [START_REF] Kacser | Rate Control of Biological Processes[END_REF][START_REF] Heinrich | A linear steady-state treatment of enzymatic chains. Critique of the crossover theorem and a general procedure to identify interaction sites with an effector[END_REF]. It has been extensively applied to the mitochondrial OXPHOS system to estimate the control strength exerted by specific enzymatic steps to the overall process (i.e. respiratory rate and/or ATP synthesis) [START_REF] Tager | Control of mitochondrial respiration[END_REF][START_REF] Mazat | Metabolic control analysis and threshold effect in oxidative phosphorylation: implications for mitochondrial pathologies[END_REF][START_REF] Rossignol | Tissue variation in the control of oxidative phosphorylation: implication for mitochondrial diseases[END_REF]. The survey of the results produced by these studies would indicate that the control of the OXPHOS system is shared among different steps with none of them showing a large control coefficient. As most of these studies have been carried out on isolated mitochondria and more recently on permeabilised cells [START_REF] Saks | Permeabilized cell and skinned fiber techniques in studies of mitochondrial function in vivo[END_REF][START_REF] Kunz | Flux control of cytochrome c oxidase in human skeletal muscle[END_REF], concern about the compliance of the conclusions to the "in vivo" situation has been raised [START_REF] Villani | In vivo control of respiration by cytochrome c oxidase in wild-type and mitochondrial DNA mutation-carrying human cells[END_REF][START_REF] Villani | Low reserve of cytochrome c oxidase capacity in vivo in the respiratory chain of a variety of human cell types[END_REF][START_REF] Villani | In vivo control of respiration by cytochrome c oxidase in human cells[END_REF][START_REF] Villani | In vivo measurements of respiration control by cytochrome c oxidase and in situ analysis of oxidative phosphorylation[END_REF].

Extension of the MFCA to intact cells has revealed, indeed, that cytochrome c oxidase (COX) exhibits a much lower reserve capacity than in isolated mitochondria thus promoting COX to a pivotal role in controlling respiration and OXPHOS.

It must be pointed out, however, that either "ex vivo" or "in vivo" MFCA of mitochondrial respiration, has been, almost exclusively, performed under uncoupled conditions (achieved in the presence of an excess of ADP or upon treatment of the sample with uncouplers of respiration) that limits the survey to a situation that does not encompass the physiological impact of the membrane potential on the mitochondrial OXPHOS performance. Therefore, the aim of this work was to explore if the depressing effect of the membrane potential on the overall mitochondrial respiration modifies the control strength exerted by a given isolated step (specifically COX) or simply superimposes a thermodynamic control without altering its kinetic reserve capacity.

MATERIALS AND METHODS

Cell culturing.

The human hepatoma cell line HepG2 was maintained in culture with DMEM with 10% fetal bovine serum. Cells were allowed to grow to 70-80% confluence before harvesting. Cells were detached from 150 mm Petri dishes with 2 ml of trypsin-EDTA and washed in 20 ml phosphate buffer pH 7.4 (PBS) with 5% calf serum, centrifuged at 500xg, resuspended in 200 µl PBS, counted and immediately used. Cell viability as determined by trypan blue exclusion was typically never below 98%.

Mitochondria isolation, cytochrome c oxidase purification and liposome reconstitution.

Mitochondria from bovine heart ("heavy" fraction) and rat liver were isolated by differential centrifugation as described in Ref.s 16 and 17respectively. Cytochrome c oxidase was purified from bovine heart mitochondria, as described in [START_REF] Errede | Preparation and properties of complex IV (ferrocytochrome c: oxygen oxidoreductase EC 1.9.3.1)[END_REF], tested for structural and functional parameters as in [START_REF] Capitanio | Redox-linked protolytic reactions in soluble cytochrome-c oxidase from beef-heart mitochondria: redox Bohr effects[END_REF] and reconstituted in small unilamellar vesicles by the "cholate-dialysis" method [START_REF] Papa | Characteristics of the redox-linked proton ejection in beef-heart cytochrome c oxidase reconstituted in liposomes[END_REF]. The respiratory control ratio of cytochrome c oxidase vesicles was tested as in [START_REF] Papa | Characteristics of the redox-linked proton ejection in beef-heart cytochrome c oxidase reconstituted in liposomes[END_REF] and resulted routinely higher than 10.

Polarographic measurements.

The rate of oxygen consumption was measured polarographically with a Clark-type oxygen electrode in a thermostated chamber equipped with a magnetic stirring device (the instrumental setting was computer-controlled) and a gas-tight plugger endowed with a narrow port for additions by an Hamilton microsyringe. The medium/buffer used for measurement was 50 mM KH 2 PO 4 , 10 mM Hepes, 1 mM EDTA, pH 7.4, air-equilibrated at 37 °C (volume 0.5 ml) and the concentration of viable HepG2 cells was typically 10⋅10 6 cells/ml. Inhibition titration of the cellular respiratory activity was carried out by adding sequentially 0.5-1 µl of freshly prepared KCN solutions. The respiratory activities were corrected for the instrumental and/or medium-linked oxygen consumption drifts.

Laser scanning confocal microscopy analysis.

Cells were seeded at low density onto fibronectin coated 35 mm glass bottom dishes. After adhesion, living cells were directly incubated for 20 min at 37 °C with MitoCapture (1/1000 dilution, following the manufacturer recommendation). Then after cells were washed twice with PBS and examined by a Nikon TE 2000 microscope (images collected using a 60X objective (1.

Metabolic control analysis.

For the estimation of the control coefficient c 0 exerted by cytochrome c oxidase over the cell respiration two procedures were adopted. In one the ratios of the initial slopes from the cyanide titration curves of the integrated flow and isolated step were calculated by least square linear regression analysis. In this case the flux control coefficient is operatively defined as:

c 0 = (∆J IF /∆[I]) [I]=0 /(∆J IS /∆[I]) [I]=0
where ∆J IF and ∆J IS are the changes of the respiratory rates of the integrated flow and isolated step respectively taken at the same interval of the inhibitor concentration, tending to 0.

The alternative procedure to calculate c 0 was that developed in [START_REF] Gellerich | Estimation of flux control coefficients from inhibitor titrations by non-linear regression[END_REF] for non-competitive inhibition. In this case the final equation given in [START_REF] Gellerich | Estimation of flux control coefficients from inhibitor titrations by non-linear regression[END_REF] was simplified as follow:

% J/J 0 = 100⋅E / (c 0 (E 0 -E) + E) with E = -0.5(α-√(α 2 + 4E 0 K D )) and α = [I] + K D -E 0
where: E and E 0 are the concentrations of the active enzyme at a given concentration of the inhibitor

[I] and at [I]=0 respectively; K D is the dissociation constant of the EI complex; J and J 0 are the respiratory rates measured at a given concentration of the inhibitor [I] and at [I]=0 respectively.

With respect to the equation given in [START_REF] Gellerich | Estimation of flux control coefficients from inhibitor titrations by non-linear regression[END_REF], an empirical exponent n was assumed to be 1 and as at high concentration of the inhibitor (i.e. E=0) the residual measurable respiratory rate was negligible, J i was assumed to be 0 (cf. with eq. 5 in ref. [START_REF] Gellerich | Estimation of flux control coefficients from inhibitor titrations by non-linear regression[END_REF]. Imposing E 0 =100, the entire data-set of the inhibitor titration curve for the integrated flow was fitted with the % of the un-inhibited activity given as a function of the concentration of the inhibitor. The parameters c 0 and K D were estimated by the program GraFit 4.0.13 (Erithacus Software Limited). The accuracy of the fitting procedure was further tested by analysing the data of the inhibition titration curve for the isolated step. In this case c 0 was imposed to be 1 and the estimated K D was verified to be comparable with that obtained from the fit of the integrated flow.

Kinetic simulation.

Modelling of a metabolic flow mimicking a multi-step electron transfer system was carried out with the freeware software package Chemical Kinetics Simulator (available at : http://www.almaden.ibm.com/st/ computational_science/ck/msim/). This program does not integrate sets of coupled differential equations to predict the time course of a chemical system.

Instead, it uses a general, rigorously accurate stochastic algorithm to propagate a reaction. The stochastic method is comparable in efficiency to integration for simple kinetic schemes, and significantly faster for stiff systems [START_REF] Gillespie | A general method for numerically the stochastic time evoluyion of coupled chemical reactions[END_REF].

Materials.

HepG2 cell line was from ATCC; DMEM, PBS, trypsin (0.05%)-EDTA(0.02%), penicillin (10000 U/ml)-streptomycin (10000µg/ml), fetal bovine serum from EuroClone; oligomycin, antimycin A, actractiloside, valinomycin, CCCP, cytochrome c from Sigma; ascorbate from Boehringer; TMPD from Fluka; Mitocapture from Biovision. All other chemicals were of the highest purity available.

Statistical analysis.

Two tailed Student's t-test was applied to evaluate the significance of differences measured throughout the data-sets reported.

RESULTS

Measurement of Endogenous Respiration and Cytochrome c Oxidase Activity in Intact Cells.

HepG2 is a human hepatoma cell line whose metabolism in the late exponential growth phase is mainly sustained by mitochondrial oxidative phosphorylation [START_REF] Pinti | Hepatoma HepG2 cells as a model for in vitro studies on mitochondrial toxicity of antiviral drugs: which correlation with the patient?[END_REF] and was therefore chosen for this study.

Fig. 1A shows a typical output of respirometric measurements for oxygen consumption, carried out on intact HepG2 cells. The respiratory rate was sustained by endogenous substrates and relied almost completely on the mitochondrial contribution as it was fully inhibited by KCN. The amount of the endogenous oxidiseble substrates was apparently never limiting since oxygen consumption was linear up to the instrumental detection limit (< 5% oxygen saturation). Addition of the ATP-synthase inhibitor oligomycin resulted in a marked depression of the oxygen consumption rate documenting the feature of an active phosphorylating state III for the endogenous respiration.

The respiratory control ratio (state III endogeneous /state IV oligomycin ) was 3.3 ±0.4 (n=10) (Fig. 1B).

Atractiloside, an inhibitor of the mitochondrial adenine nucleotide translocator, resulted in depression of the endogenous respiration to an extent comparable with that attained by oligomycin These results confirmed qualitatively, at the cellular level, the bioenergetic paradigms well established in classical experiments carried out on isolated mitochondria [START_REF] Chance | Respiratory enzymes in oxidative phosphorylation. III. The steady state[END_REF] and showed that transmembrane potential exerts "in vivo" a tight control on respiration.

Measurement of Mitochondrial Membrane Potential in Intact Cells.

The involvement of the membrane potential in the control of mitochondrial respiration in intact cells was directly assessed by laser scanning confocal microscopy using the probe MitoCapture (Fig. 2). Depending on the extent of the trans-membrane electrical potential the dye accumulates as monomer (green emitter) or dimer (red emitter, in iper-polirized mitochondria)

resembling other dual emitters ∆Ψ probe [START_REF] Di Lisa | Mitochondrial membrane potential in single living adult rat cardiac myocytes exposed to anoxia or metabolic inhibition[END_REF]. Fig. 2A shows that the fluorescence signal, due to accumulation of the probe in response to membrane potential was scantly detectable under condition of endogenous respiration but strongly increased (10 fold) when the cell samples were incubated with oligomycin. Coherently with the polarographic measurements Fig. 2 shows also that the membrane potential generated in the presence of oligomycin by COX was lower than that observed under endogenous respiration but however larger than that seen with the fully active ATPsynthase. This is not surprising as the proton-motive force generated by the cytochrome c oxidase is only a fraction of that produced by the entire respiratory chain which operates with the other two inseries proton pumping respiratory complexes (NADH dehydrogenase and cytochrome c reductase) [START_REF] Saraste | Oxidative phosphorylation at the fin de siecle[END_REF]. Moreover it has been shown that the coupling efficiency of the COX redox-linked proton pump is negatively controlled by the extent of the ∆p [START_REF] Capitanio | Factors affecting the H+/e-stoichiometry in mitochondrial cytochrome c oxidase: influence of the rate of electron flow and transmembrane delta pH[END_REF].

MCA of cytochrome c oxidase in intact cells.

The control exerted by COX over cell respiration and the effect on it of the mitochondrial membrane energization state was tested by applying the MFCA to intact cells. When the same analysis was carried out with oligomycin pre-treated cells, the KCN-titration curve of the integrated flow revealed a much higher resistance to inhibition when compared with that of the isolated step and a more sigmoidal shape (Fig. 3B). The calculated c 0 was around 0.25.

The insert in Fig. 3B shows that the KCN-inhibition titrations carried out on purified cytochrome oxidase reconstituted in liposomes under coupled (RCR ≥10) and uncoupled conditions resulted in super-imposable curves with practically identical Ki 50 for KCN. This control clearly ruled out the possibility of an effect of ∆Ψ on the accessibility/affinity of CN-to the COX active site [START_REF] Panda | Kinetics and mechanism for the binding of HCN to cytochrome c oxidase[END_REF].

Fig. 3C shows that addition of KCN (up to 300 µM) to oligomycin-treated endogenously respiring HepG2 cells did not cause any appreciable change in the mt∆Ψ-dependent accumulation of MitoCapture. Some decrease of the fluorescent signal was recorded at 1 mM of the inhibitor.

Thus, in spite of the inhibition of respiration the mt∆Ψ was substantially maintained throughout the cyanide titration assay (see Discussion).

When the experimental data were graphed as a threshold plot, i.e. percentage of the endogenous respiratory activity as a function of the percentage of cytochrome c oxidase inhibition at the same KCN concentrations (Fig. 3D), a difference emerged. Whereas in the absence of membrane potential, COX exerted a strong control over the respiratory metabolic flux, such that any degree of inhibition of the isolated step resulted in an almost identical level of inhibition of the integrated respiratory flux, in the presence of oligomycin (i.e. with an established ∆µH + ) the control strength of COX over the respiratory flux was reduced such that a 50-60 % inhibition of the isolated step resulted in only a 10 % decrease of the integrated flux. The threshold plots are usually characterized by a more evident biphasic behaviour with a net break point that allows for estimation of the threshold value [START_REF] Rossignol | Threshold effect and tissue specificity. Implication for mitochondrial cytopathies[END_REF]. Instead, the data presented here fit with a continuous curve which is however predicted by the metabolic control theory where it reveals the occurrence of compensatory changes of intermediates in the sequential array of enzymatic steps constituting the metabolic network [START_REF] Mazat | What do mitochondrial diseases teach us about normal mitochondrial functions that we already knew: threshold expression of mitochondrial defects[END_REF] (see Discussion). In the presence of oligomycin a tentative threshold value might be set at around 75% inhibition of the isolated step. It must be mentioned, however, that a continuous curve in the threshold plot is not excluded. Further information deriving from the analysis of the threshold plot is the maximal reserve capacity of the isolated step that can be estimated by Copyright 2006 Biochemical Society extrapolation of the linear part of the threshold plot at the highest degrees of inhibition of the isolated step [START_REF] Villani | Low reserve of cytochrome c oxidase capacity in vivo in the respiratory chain of a variety of human cell types[END_REF]. From the data shown in Fig. 3D it was estimated that the maximal reserve capacity of cytochrome c oxidase exceeded the theoretical capacity to sustain the full activity of the respiratory flux by 73% and 286% in the absence and in the presence of oligomycin respectively.

To exclude what observed was not due to undesired side-effect of oligomycin on cytochrome oxidase MFCA was carried out on HepG2 preincubated with oligomycin but in the presence of valinomycin plus CCCP. It can be seen that the fully uncoupling effect of the two ionophors resulted in a threshold curve that was undistinguishable from that obtained under endogenous respiration (Fig. 4 and cf. with Fig. 3D). Interestingly when the MCA was performed in the presence of oligomycin supplemented with sole valinomycin the threshold plot did not change with respect to that observed in the presence of oligomycin alone. As an enhancement of the DpH component of the ∆µH + is expected to compensate for the K + -valinomycin-mediated dissipation of the mt∆Ψ [START_REF] Cossarizza | A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the Jaggregate forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'tetraethylbenzimidazolcarbocyanine iodide (JC-1)[END_REF], the result presented in Fig. 4 would suggest that the relative decrease of the COXcontrol strength on the cell respiration in the presence of a mitochondrial transmembrane protonmotive force is independent on which of the two thermodynamical components is operative with ∆Ψ and ∆pH being equally effective.

MCA of Cytochrome c Oxidase in Isolated Mitochondria.

The evidence here shown on one hand confirms what has been reported for other cell types in culture [START_REF] Villani | In vivo control of respiration by cytochrome c oxidase in wild-type and mitochondrial DNA mutation-carrying human cells[END_REF][START_REF] Villani | Low reserve of cytochrome c oxidase capacity in vivo in the respiratory chain of a variety of human cell types[END_REF], and on the other hand contrasts with results of MFCA carried out on isolated mitochondria [START_REF] Tager | Control of mitochondrial respiration[END_REF][START_REF] Mazat | Metabolic control analysis and threshold effect in oxidative phosphorylation: implications for mitochondrial pathologies[END_REF][START_REF] Rossignol | Tissue variation in the control of oxidative phosphorylation: implication for mitochondrial diseases[END_REF] which depending on the mammalian organ source resulted in a flux control coefficient for COX of 0.15-0.25 on the respiratory flux fuelled with either NADH-linked substrates or succinate [START_REF] Rossignol | Tissue variation in the control of oxidative phosphorylation: implication for mitochondrial diseases[END_REF]. Therefore we extended (as intra-lab control) the MFCA on mitochondria isolated from beef-heart and rat-liver. To rule out differences in the coupling efficiency that may arise from diverse protocols in the separation procedures we performed the MFCA on fully uncoupled frozenthawed mitochondria. Fig. 5 illustrates the results obtained directly presented as threshold plot. It is shown that for the succinate-sustained respiratory flux COX displayed a very low reserve capacity (about 30%) and a c 0 of 0.7-0.8. As the freezing-thawing treatment of isolated mitochondria enables the substrate-binding site of complex I to be freely accessible it was also possible to apply the MFCA to respiratory flow directly fuelled by NADH. Also in this case, the threshold plot documented that for heart mitochondria a tight control of cytochrome oxidase with a maximal reserve capacity and control coefficient undistinguishable, on a statistical basis, from those measured for the succinate-sustained respiration. Addition of cytochrome c, to compensate for its possible loss from the mitochondrial intermembrane space, did not result in any significant Copyright 2006 Biochemical Society difference (data not shown). Surprisingly, when the same analysis was carried out on mitochondria isolated from rat liver the threshold curve for COX on the succinate-driven respiration was markedly different with a much lower control strength (both in terms of larger excess capacity and lower control coefficient). Table 1 summarizes the metabolic flux control parameters calculated for COX in all the conditions reported throughout this study.

DISCUSSION

The results here reported on the MCA of COX over the cell respiration resumed and extended previous studies carried out on intact cells. Under uncoupled conditions, it was confirmed that COX exhibits a low reserve capacity and a large control coefficient over the endogenous cell respiration but mimicking the state III of respiration by blocking the H + ATP-synthase clearly showed that COX decreased its apparent control efficiency and that this effect can be attributable to the establishment of a steady-state ∆µH + . To our knowledge, this is the first case of a study carried out in intact cells, under close physiological conditions, where a comparison was made under two different bioenergetic states.

To avoid misinterpretations of the results we have carefully considered a number of points.

First, a criticism that could be raised to our experimental approach is that the membrane potential changed during the KCN inhibitory titration. We checked this specific point, and found that the addition of up to 300 µM cyanide (which caused 40% inhibition of the endogenous respiration in the presence of oligomycin) showed no effect on the "in situ" extent of mitochondrial ∆Ψ. Some decrease of ∆Ψ was detected at 1 mM cyanide with a 70% inhibition of respiration.

These results can be rationalised in terms of a non-ohmic flow-force relationship explainable in terms of a progressive change of either the passive proton conductance of the membrane [START_REF] Brand | The causes and functions of mitochondrial proton leak[END_REF] and/or the intrinsic the proton pumping efficiency of the respiratory chain complexes [START_REF] Zoratti | Intrinsic uncoupling of mitochondrial proton pumps. 1. Non-ohmic conductance cannot account for the nonlinear dependence of static head respiration on delta microH[END_REF]. As the metabolic control coefficient c 0 was estimated from the ratio of the initial slopes of the integrated flow and the isolated step, it was, therefore, not affected by a decrease of the membrane potential that took place at much higher inhibition of the respiratory rates.

It might be argued that according to the summation principle a variation in the number of steps contributing to the metabolic flux would be expected to result in a re-distribution of the control coefficients whose sum must remain 1.0 [START_REF] Brand | The sum of flux control coefficients in the electron-transport chain of mitochondria[END_REF]. Application of a static-head ∆µH + on isolated mitochondria introduces, indeed, the H + back-leak as a new factor controlling the rate of respiration.

Nevertheless the experiment on valinomycin-treated cells presented in Fig. 4 shows that substitution Copyright 2006 Biochemical Society of the trans-membrane electrical potential with a pH gradient resulted in no change of the COX control. Given the large difference in membrane conductivity of protons with respect to other cations or anions (the membrane conductance for H + is at least 10 6 times higher than for K + [START_REF] Wrigglesworth | The proteoliposomal steady state. Effect of size, capacitance and membrane permeability on cytochrome-oxidase-induced ion gradients[END_REF]) one would expect a tighter control of ∆pH in the metabolic network and as a consequence a compensatory decrease of the control coefficients of the other steps. But this was not the case for COX (cf. Fig. 3D and4).

Another possibility that one could envision is that the membrane potential might change the catalytic features of cytochrome c oxidase (i.e. Km, Kcat) [START_REF] Petersen | Kinetics of the cytochrome c oxidase and reductase reactions in energized and de-energized mitochondria[END_REF]. Given that the other complexes of the mitochondrial respiratory chain (which contain electrogenic steps in their catalytic mechanisms)

would not escape the same possibility, differentiated variations in the relative catalytic features of the enzymatic steps in the network might cause a re-distribution of the control coefficients.

Although this sounds plausible, it is not immediately clear how the conversion of an electrical potential in a pH gradient might mechanicistically produce the same effect. This would be the case if a protogenic step in COX (as well as in the other respiratory complexes) coincided with an electrogenic step that was rate-limiting, leaving this possibility very unlikely [START_REF] Gregory | Independent control of respiration in cytochrome c oxidase vesicles by pH and electrical gradients[END_REF][START_REF] Capitanio | Protonmotive activity of cytochrome c oxidase: control of oxidoreduction of the heme centers by the protonmotive force in the reconstituted beef heart enzyme[END_REF].

The structural organization in the membrane of the many components of the mitochondrial OXPHOS system has been object in the past of a dispute with a model representing the enzymatic complexes acting as isolated entities in opposition to a model where the occurrence of a clustered structuring was considered [START_REF] Hackenbrock | The random collision model and a critical assessment of diffusion and collision in mitochondrial electron transport[END_REF][START_REF] Schagger | Respiratory chain supercomplexes of mitochondria and bacteria[END_REF]. The latter hypothesis has been recently re-appraised on more solid experimental basis indicating the presence, in mitochondria of different species, of assembled super-complexes with defined stoicheiometry [START_REF] Schagger | Respiratory chain supercomplexes of mitochondria and bacteria[END_REF][START_REF] Bianchi | The mitochondrial respiratory chain is partially organized in a supercomplex assembly: kinetic evidence using flux control analysis[END_REF].

The so called "respirasomes" would gain a higher catalytic efficiency because of the channelling of mobile intermediates (like ubiquinol and cytochrome c) and of the contiguity between the ∆µH + producers and utilizers. The alternative random collision controlled model, however, is not to be completely abandoned. Indeed one can envision a situation where the two organizational modes, respirasome vs isolated complexes, coexist in a dynamic equilibrium with the relative occupancy of the two structural subsets depending on the actual bioenergetic needs of the cell. The higher is the membrane potential the lower is, in principle, the energy demand of the cell because a slower rate of ATP utilization does not make available ADP for the ATP-synthase that in turn cannot dissipate the redox linked proton-motive force generated by the respiratory chain. Under this metabolic condition, the respirasome would dissociate in its constituting complexes and this would slow down the rate of oxygen consumption because diffusional steps between redox intermediates and enzymatic complexes are introduced. All the way around, when the cell hydrolyzes more ATP, because of a larger bioenergetic request, ADP is no longer a limitation for Copyright 2006 Biochemical Society the activity of the ATP-synthase, the transmembrane potential is promptly collapsed and the required higher rate of respiration satisfied by the assembly of the respiratory enzymes in supercomplexes. Under this condition, the channelling of the redox intermediates will speed up the rate of electron transfer.

To test the hypothesis that a change in the control strength of an individual step might be featured in terms of equilibrium between different organizational structures of the enzymatic steps constituting the integrated flow, a mathematical simulation was performed. A simple electron transfer process was designed with three putative redox catalysts (X, Y, Z) transferring in sequence an electron from an initial donor (De -) to a final acceptor (A). The three catalytic steps formed an assembled complex (XYZ) or alternatively were isolated from each other and the transfer of the electron was controlled by a channelled or collisional diffusion mechanism respectively (see schemes in Fig. 6). The results of a modelled inhibitory titration of the catalyst "Z" are shown in Fig. 6 as a threshold-like plot. One can see that when "Z" is part of the XYZ complex the overall rate of the flux correlates linearly with the amount of the controlling step, whereas when "Z"

operates as an isolated step, a decrease in its amount, results in a clear deviation from a linear impact on the flux. It must be pointed out that in the two modelled conditions the rate constants for each simulated catalytic step were identical thus the change in the control exerted by the catalyst "Z" depends simply on the fact that when the catalyst is not complexed, its partial inhibition results in an increase of the steady-state concentration of Ye -which reacting, accordingly to a second order reaction, with the residual fraction of the uninhibited "Z", compensates with a fractional increase of its actual transfer rate, the expected inhibition of the overall electron flux . This occurrence does not apply when "Z" is part of a complex because the intermediate XYe -Z inhibited cannot transfer e-to the un-inhibited XYZ complexes. Consequently, that step will loose in controlling strength. In the framework of the metabolic control theory an identical result is predicted in the so called "network attenuation" [START_REF] Heijnen | Metabolic flux control analysis of branch points: an improved approach to obtain flux control coefficients from large perturbation data[END_REF] which however applies when each individual step of the network is working at a velocity substantially lower than their maximal velocity.

The above-depicted scenario implies that a change in the ∆µH + itself might be at the same time the physiological signal and the trigger causing the structural re-organization of the enzymatic complexes of the mitochondrial OXPHOS system (see the model hypothesis in Fig. 7). Although the driving forces, leading to the assembly/disassembly of the super-complexes, have been not yet defined, it is not unconceivable that given the membrane-integrated nature of the single complexes, electrostatic as well as hydrophobic interactions must enter into play. Recent published observations have indicated for cardiolipin an essential role in promoting formation of mitochondrial respiratory supercomplexes [START_REF] Pfeiffer | Cardiolipin stabilizes respiratory chain supercomplexes[END_REF]. Knocking-out the gene coding for the CL-synthase in yeast resulted, indeed, Copyright 2006 Biochemical Society in disappearance of the mitochondrial respirasomes, without affecting the catalytic activities of the isolated complexes [START_REF] Zhang | Cardiolipin is essential for organization of complexes III and IV into a supercomplex in intact yeast mitochondria[END_REF]. Cardiolipin is a four acyl-tails anionic phospholipid with a large dipole potential, able to sense electrical gradient as well as pH (the pK a2 of the phosphate headgroup is ≥ 8, [START_REF] Haines | Cardiolipin: a proton trap for oxidative phosphorylation[END_REF]) and to alter the physical properties of the membrane [START_REF] Nichols-Smith | Electrostatic interactions between model mitochondrial membranes[END_REF]. Moreover binding sites on the known crystallographic 3D structures of cytochrome c reductase and oxidase have been clearly localised [START_REF] Lange | Specific roles of proteinphospholipid interactions in the yeast cytochrome bc1 complex structure[END_REF]46]. Therefore cardiolipin (almost exclusively localised in the inner mitochondrial membranes) might well be the molecular sensor of the energy state of the mitochondrial membrane enabling the complexes of the OXPHOS system to change their assembly in response to the ∆µH + .

Consistent with our proposal are the results obtained with bovine heart isolated mitochondria confirming the tight control exerted by COX on the uncoupled NADH-or FADH 2linked respiration. Cardiolipin, as well as the OXPHOS enzymatic-content, is known to vary in mitochondria from different organs with the heart being the richest one [START_REF] Agnati | How proteins come together in the plasma membrane and function in macromolecular assemblies: focus on receptor mosaics[END_REF]. Mitochondria isolated from tissue relatively poorer in cardiolipin (like liver) or that are more vulnerable to modification during the isolation procedure (i.e. peroxidation) would tend to maintain the complexes isolated even in the absence of a membrane potential thus resulting in an apparent low flux control by COX.

The cardiolipin-mediated(controlled) change in the electrostatic/hydrophobic interaction of the mitochondrial OXPHOS complexes might provide therefore a "bioenergetic signal transduction" system that resembles the described function of plasma membrane rafts in stabilising a local platform for integrating the many components of the signal transduction network [START_REF] Fleischer | Lipid composition of mitochondria from bovine heart, liver, and kidney[END_REF] though based on different membrane components.

Finally the observations reported here provide a rationale to understand some aspects of the stress-related symptoms of patients suffering of neuromuscular diseases caused by defects of the mitochondrial cytochrome c oxidase [START_REF] Pecina | Genetic defects of cytochrome c oxidase assembly[END_REF] or, more generally, by impairments of the OXPHOS system [START_REF] Kang | Alterations of mitochondrial DNA in common diseases and disease states: aging, neurodegeneration, heart failure, diabetes, and cancer[END_REF][START_REF] Wallace | A Mitochondrial Paradigm of Metabolic and Degenerative Diseases, Aging, and Cancer: A Dawn for Evolutionary Medicine[END_REF]. Although the mitochondrial diseases encompass rare human inherited pathologies (1 case on 5000 newborns) the understanding of their ethiopatological mechanism may help in managing a larger number of more common pathologies where the involvement of defects in the mitochondrial respiratory chain has been defined [START_REF] Wallace | A Mitochondrial Paradigm of Metabolic and Degenerative Diseases, Aging, and Cancer: A Dawn for Evolutionary Medicine[END_REF]. The manifestation and severity of diseases linked to mutations in mtDNA is often correlated to its level of heteroplasmy [START_REF] Lightowlers | Mammalian mitochondrial genetics: heredity, heteroplasmy and disease[END_REF]. In the same cell of a given tissue, mitochondria harbouring mutated and/or wild-type DNA can coexist. When the amount of mutated mtDNA is such that the gene product provided by the wild-type mtDNA does not compensate the defect of the mutated mtDNA, the bioenergetic competence of the cell is compromised. Thus a genetic threshold for the mutated mtDNA must be overcome to cause the disease to manifest and a wide array of clinical symptoms with different degree of severity may arise from the same mtDNA mutation depending on its level of heteroplasmy [START_REF] Mazat | What do mitochondrial diseases teach us about normal mitochondrial functions that we already knew: threshold expression of mitochondrial defects[END_REF][START_REF] Lightowlers | Mammalian mitochondrial genetics: heredity, heteroplasmy and disease[END_REF]. A clinical condition a defective COX activity will cause, according to our data, an almost proportional decrease of the overall aerobic flux which if not able to satisfy the enhanced bioenergetic demand will result in an unbalanced anaerobic cell metabolism. In addition to this, it is conceivable that an accumulation of redox intermediates up-hill of COX will favour formation of oxygen free radicals with further aggravation of the already debilitated patient conditions [54]. At rest, the same level of COX deficit might not be relevant because the re-distribution of the flux control coefficients will attenuate its impact on the overall respiratory rate. ----------------------------

4 NA

 4 )) coupled to a Radiance 2100 dual laser (four-lines Argon-Krypton, single-line Helium-Neon) scanning confocal microscopy system (Biorad). The fluorescent signal of the double-emitter probe was examined sequentially, exciting first with the Ar-Kr laser beam (λ ex =488 nm) and then with the Biochemical Journal Immediate Publication. Published on 13 Mar 2006 as manuscript BJ20060077 Copyright 2006 Biochemical Society He-Ne laser beam (λ ex =543 nm). Confocal planes of 0.2 µm in thickness were examined along the z-axes, going from the top to the bottom of the cells. Acquisition, storage and analysis of data were made by using LaserSharp and LaserPix software from Biorad. Quantification of the emitted fluorescent signal was achieved by producing a xz-intensity profile of the average value of the pixels within marked edges, including a single cell, as a function of each focal plane. Correction was made for minimal background by repeating the procedure in a cell-free field. The integrated value of the xz profile was taken as a measure of the fluorescence intensity of that individual cell.

Fig.1A

  Fig.1A shows, also, that antimycin A, a specific inhibitor of cytochrome c reductase, completely inhibited endogenous respiration and that successive addition of ascorbate plus the membrane-permeant redox-recycling TMPD by-passed the block delivering electrons directly to cytochrome c oxidase via cytochrome c reduction. Unlike what was observed with the endogenous respiration, the ascorbate/TMPD-fuelled cytochrome c oxidase activity was only slightly blunted by oligomycin resulting in a respiratory control ratio of 1.3 ±0.1 (n=6).

  Fig. 3A shows the results of KCN titrations on the oxygen consumption rates sustained either by endogenous Biochemical Journal Immediate Publication. Published on 13 Mar 2006 as manuscript BJ20060077 Copyright 2006 Biochemical Society substrates or by ascorbate plus TMPD. One can see that inhibition of the cell respiratory flux, resulted in a normalized titration curve almost super-imposable with that of the functionally isolated step. The flux control coefficient c 0 measured either by the ratio of the slopes attained in the initial low inhibitor concentration ranges and by non-linear fitting of the entire experimental data set of the integrated flow, was 0.6-0.7.

Figure 1 .

 1 Figure 1. Measurement of O 2 consumption rates in HepG2. A: polarographic traces showing the endogenous respiration in intact cells (on the left) and the in situ activity of cytochrome c oxidase (on the right). In the latter case the endogenous respiration was inhibited by 1 µg/ml antimycin A and the cytochrome oxidase activity was rescued by addition 10 mM ascorbate plus 200 µM of the membrane permeant TMPD. Where indicated 2 µg/ml oligomycin and 2 mM KCN were added. The changes in the rates of O 2 consumption, throughout the assays, are given as first derivative in the panels below the corresponding traces. B: statistical evaluation of the effect of oligomycin on the activities of the respiratory endogenous flow (±S.E.M., n=10) and cytochrome c oxidase isolated step (±S.E.M., n=6). The respiratory control ratios of the rates of O 2 consumption in the absence and in the presence of oligomycin is also shown. Paired Student's t test analysis: *, P<0.05; **, P<0.001.

Figure 2 .

 2 Figure 2. Confocal microscopy analysis of respiration-driven mitochondrial membrane potential in HepG2. A: imaging of the fluorescence signal of the probe MitoCapture in live cells. The upper two panels show the analysis carried out under respiratory conditions sustained by endogenous substrates (on the left) and by activation of cytochrome c oxidase as isolated step (on the right). The lower two panels show the effect of pre-incubation of the cells with 2 µg/ml oligomycin on the mitochondrial membrane potential generation under endogenous (on the left) and isolated step (on the right) respiratory activities. The images shown are superimposition of confocal planes and are representative of 4 different experiments. B: single cells analysis of the green fluorescence intensity of MitoCapture under the four conditions described in A. The fluorescence intensities were quantified as described in Materials and Methods and normalised to the surface of the selected area; 20-30 cells were analysed for each condition and the results shown are averages (±S.E.M.) of the experiment shown in A. Results of paired Student's t test analysis for the fluorescence intensity of oligomycin-untreated vs oligomycin-treated cells: *, P<0.01; **, P<0.001.

Figure 3 .

 3 Figure 3. Metabolic control analysis of cytochrome c oxidase over the endogenous respiration in intact HepG2 cell line: effect of ∆µH + . Titration curves for KCN inhibition of the endogenous respiration (black circles) and of ascorbate/TMPD-dependent respiration (white circles) in the absence (A) and in the presence (B) of oligomycin. The residual activity is given as percentage of the un-inhibited respiratory rate. See legend to Fig. 1A and Materials and Methods for experimental details. The data points shown are the averages of at least 5 different titrations (± S.E.M.) for each condition. P represents the statistical significance provided by the Student's t test analysis when

Figure 4 .

 4 Figure 4. Effect of ionophores on the threshold plots for COX activity over the endogenous respiration in oligomycin-treated HepG2 cells. The experimental conditions for the inhibitor titrations are similar to those shown in Fig.1-panel B but cells were pre-incubated with 4 µg/ml

Figure 5 .

 5 Figure 5. Metabolic control analysis of cytochrome c oxidase in isolated mitochondria. 1 mg prot/ml of frozen-thawed mitochondria isolated from beef heart (BHM, closed symbols) or rat liver (RLM, open symbols) were fuelled with 200 µM NADH (squares) or 5 mM succinate (circles) (in the presence of 1µg/ml rotenone) and the respiratory rates subjected to KCN inhibition titration. Identical KCN range was used to titrate the activity of cytochrome c oxidase as isolated step sustained by 10 mM ascorbate plus 0.2 mM TMPD. The results of the inhibition titrations are directly shown as threshold plots of the COX activity on the NADH-and succinate-sustained respiration.

Figure 6 .

 6 Figure 6. Kinetic simulation of the metabolic flow throughout aggregated or isolated steps:effect on the control of a single step. A metabolic flow mimicking electron transfer throughout three enzymatic steps from an initial donor De-to a final acceptor A was simulated. Two models were tested: in one the three steps were assumed to be physically assembled in a single complex with an internal e-transfer (model A), in the other the three steps were assumed to be isolated and the e-transfer occurred by stochastic events controlled by random collision. In addition one of the three putative enzyme (Z) was possibly blocked by an inhibitor I. The rates of the overall simulated e-transfer to the acceptor was estimated in the presence of increasing concentrations of the inhibitor inducing a progressive 10% stepwise inhibition of the isolated step irrespective of its assembly state. The graph reports as threshold plots the results of the simulations for both models. The numerical values for the kinetic constants where: 1.0 for steps 1-4, 8 and 100 for steps 5-7, in simulation (A) and 1.0 for steps 1-4 and 100 for step 5, in simulation (B). See under Materials and Methods for details on the software used.

Figure 7 .

 7 Figure 7. Schematic working model illustrating the effect of the mitochondrial membrane energy state on the super-molecular structure of the enzymatic components of the OXPHOS system. The respiratory complexes are illustrated as boxes embedded in the inner mitochondrial

  Fig. 7

Biochemical Journal Immediate Publication. Published on 13 Mar 2006 as manuscript BJ20060077

Biochemical Journal Immediate Publication. Published on 13 Mar 2006 as manuscript BJ20060077 Copyright 2006 Biochemical Society

	(data not shown). Uncouplers of the oxidative phosphorylation (DNP or CCCP) stimulated the
	oligomycin (or actractiloside)-settled respiratory rate but never exceeded that elicited in the state
	III

endogeneous (data not shown).

Biochemical Journal Immediate Publication. Published on 13 Mar 2006 as manuscript BJ20060077

Biochemical Journal Immediate Publication. Published on 13 Mar 2006 as manuscript BJ20060077

Biochemical Journal Immediate Publication. Published on 13 Mar 2006 as manuscript BJ20060077

Biochemical Journal Immediate Publication. Published on 13 Mar 2006 as manuscript BJ20060077

Biochemical Journal Immediate Publication. Published on 13 Mar 2006 as manuscript BJ20060077

Biochemical Journal Immediate Publication. Published on 13 Mar 2006 as manuscript BJ20060077 Copyright 2006 Biochemical Society feature

  shared among patients suffering directly or indirectly of impairments of the COX complex is their intolerance to muscular work overload[52, 53]. An active state at the tissue-level (in terms of bioenergetic balance) means a condition of increased ATP consumption to perform work, which at the mitochondrial level produces a compensatory enhancement of the ATP-synthase activity given the larger availability of ADP and thus a decrease of the steady-state ∆µΗ + . Under this "stress"

Biochemical Journal Immediate Publication. Published on 13 Mar 2006 as manuscript BJ20060077

ACKNOWLEDGEMENTS

The authors are grateful to Dr. Giuseppe Capitanio for having provided samples of beef heart and rat liver mitochondria and cytochrome oxidase vesicles. N.C. thanks Prof. Sergio Papa for continuous encouragement and critical reading of the manuscript and Dr. Gaetano Villani for stimulating discussion on the issues dealt within this paper. This work was supported by the University of Foggia Funds for Research "Quota progetti 2003-2004". The authors have no conflicting financial interests.

MFCA of cytochrome c oxidase on the respiratory activity of intact cells and isolated mitochondria.

Summary of the results presented throughout the paper. c 0 : control coefficient calculated from the CN titration curves either by dividing the slopes attained in the initial low inhibitor concentration ranges and by non-linear fitting of the entire experimental data set of the integrated flow (see under Materials and Methods); the values reported are the averages of the results obtained by the two procedures differing by less than 10%. MRC: maximal reserve capacity, calculated from the threshold plots by extrapolating to 0% COX activity the experimental points attained at the highest inhibitor concentration. Thresholds values refer to the intercepts of the straight lines of the experimental points attained at the lowest and highest inhibitor concentrations and interpolated on the % COX inhibition axe. For experimental details see legends to Figures 345.

Cytochrome c Oxidase