Pedro Iñarrea

Hadi Moini

Derick Han

Daniel Rettori

Ignacio Aguiló

Maria Angeles Alava

María ITURRALDE ‡ Enrique Cadenas

Ph.D Pedro Inarrea
email: inarrea@unizar.es

Mitochondrial Respiratory Chain and Thioredoxin Reductase Regulate Intermembrane CU,ZN-Superoxide Dismutase Activity: Implications for Mitochondrial Energy Metabolism and Apoptosis

Keywords: Cu, Zn-superoxide dismutase, mitochondrial intermembrane space, reactive oxygen species, thioredoxin reductase, disulfide bond, aconitase

Intermembrane space Cu,Zn-superoxide dismutase (IMS Cu,Zn-SOD) is inactive in isolated intact rat liver mitochondria and is activated following oxidative modification of its critical thiol groups. The present study aimed to identify biochemical pathways implicated in the regulation of IMS Cu,Zn-SOD activity and to assess the impact of its functional state on key mitochondrial events. Exogenous H 2 O 2 (5 μM) activated Cu,Zn-SOD in intact mitochondria. However, neither H 2 O 2 alone nor H 2 O 2 in the presence of mitochondrial peroxiredoxin III activated Cu,Zn-SOD, which was purified from mitochondria and subsequently reduced by DTT to an inactive state. The reduced enzyme was activated following incubation with superoxide generating system, xanthine and xanthine oxidase. In intact mitochondria, the extent and duration of Cu,Zn-SOD activation was inversely correlated with mitochondrial superoxide production. Presence of thioredoxin reductase-1 (TxrR-1) was demonstrated in the mitochondrial IMS by western blotting. Inhibitors of TxrR-1, CDNB or auranofin, prolonged duration of H 2 O 2induced Cu,Zn-SOD activity in intact mitochondria. TxrR-1 inactivated Cu,Zn-SOD purified from mitochondria in an active oxidized state. Activation of IMS Cu,Zn-SOD by exogenous H 2 O 2 delayed CaCl 2 -induced loss of trans-membrane potential, decreased cytochrome c release, and markedly prevented superoxide-induced loss of aconitase activity in intact mitochondria respiring at state-3. These findings suggest that IMS Cu,Zn-SOD activity is reversibly regulated by H 2 O 2 , superoxide, and TxrR-1 and that the active enzyme is implicated in protecting vital mitochondrial functions.

INTRODUCTION

Recent studies on the subcellular distribution of superoxide dismutase (SOD) demonstrated that Cu,Zn-SOD is localized in not only cytosol [START_REF] Mccord | Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein)[END_REF] but also in the mitochondrial intermembrane space (IMS) of rat liver [START_REF] Okado-Matsumoto | Subcellular distribution of superoxide dismutases (SOD) in rat liver: Cu,Zn-SOD in mitochondria[END_REF][START_REF] Inarrea | Purification and determination of activity of mitochondrial cyanide-sensitive superoxide dismutase in rat tissue extract[END_REF] and yeast [START_REF] Sturtz | A fraction of yeast Cu,Zn-superoxide dismutase and its metallochaperone, CCS, localize to the intermembrane space of mitochondria. A physiological role for SOD1 in guarding against mitochondrial oxidative damage[END_REF]. Comparative kinetic studies, however, revealed that Cu,Zn-SOD is inactive in isolated intact rat liver mitochondria, unless the outer membrane is disrupted by digitonin [START_REF] Tyler | Polarographic assay and intracellular distribution of superoxide dismutase in rat liver[END_REF][START_REF] Peeters-Joris | Subcellular localization of superoxide dismutase in rat liver[END_REF][START_REF] Rotilio | Intracellular localization of superoxide dismutase and its relation to the distribution and mechanism of hydrogen peroxide-producting enzymes[END_REF][START_REF] Inarrea | Redox activation of mitochondrial intermembrane space Cu,Zn-superoxide dismutase[END_REF]. Presence of alkylating agents during disruption of the outer membrane prevents activation of the enzyme whereas exposure of intact mitochondria to low concentrations of H 2 O 2 (5 μM) activates Cu,Zn-SOD in the IMS [START_REF] Inarrea | Redox activation of mitochondrial intermembrane space Cu,Zn-superoxide dismutase[END_REF]. These findings suggest that intra-subunit disulfide bond necessary for the active state of Cu,Zn-SOD has not been formed in mitochondria [START_REF] Inarrea | Redox activation of mitochondrial intermembrane space Cu,Zn-superoxide dismutase[END_REF].

The biochemical pathways catalyzing reversible redox activation of Cu,Zn-SOD are not defined in mitochondria. Activation of Cu,Zn-SOD following exposure of mitochondria to low levels of H 2 O 2 suggest involvement of an enzyme-mediated thiol oxidation rather than direct oxidation of the enzyme by H 2 O 2 . Potential candidates to catalyse such a reaction are mitochondrial peroxiredoxins (III and/or V), a family of peroxidases that reduce H 2 O 2 to water through reducing equivalents provided by thiolcontaining proteins [START_REF] Seo | Identification of a new type of mammalian peroxiredoxin that forms an intramolecular disulfide as a reaction intermediate[END_REF][START_REF] Wood | Peroxiredoxin evolution and the regulation of hydrogen peroxide signaling[END_REF]. Thioredoxin reductase (TxrR) and glutahione reductase are major mitochondrial systems catalyzing reduction of protein disulfide bonds through reducing equivalents provided by thioredoxin (Txr) and glutathione (GSH) [START_REF] Rigobello | Distribution of protein disulphide isomerase in rat liver mitochondria[END_REF][START_REF] Hurd | Disulphide formation on mitochondrial protein thiols[END_REF]; hence, they may be implicated in reduction, and as a corollary inactivation of IMS Cu,Zn-SOD.

The role of IMS Cu,Zn-SOD in mitochondrial functions is not known. However, its localization in the IMS suggests that the enzyme has a functional relationship with the occurrence of superoxide in this compartment. Several mitochondrial sites are primary targets of regulation by fluctuating levels of superoxide and/or H 2 O 2 . Enzymes containing 4Fe-4S clusters, such as aconitase and fumarase, are target of inactivation by superoxide [START_REF] Gardner | Inactivation-reactivation of aconitase in Escherichia coli. A sensitive measure of superoxide radical[END_REF][START_REF] Gardner | The Oxygen Paradox[END_REF]. Superoxide and/or H 2 O 2 also mediate the opening of permeability transition pore (PTP) leading to the loss of mitochondrial transmembrane potential (Ψm), cytochrome c release, and apoptosis [START_REF] Kowaltowski | Opening of the mitochondrial permeability transition pore by uncoupling or inorganic phosphate in the presence of Ca2+ is dependent on mitochondrial-generated reactive oxygen species[END_REF][START_REF] Chernyak | Redox regulation of the mitochondrial permeability transition pore[END_REF]. Therefore, IMS Cu,Zn-SOD activity may potentially play an important role in key mitochondrial events (e.g. energy metabolism, transemembrane potential, and cytochrome c release) to maintain the integrity of mitochondria. Hence, the aims of this study were to elucidate the biochemical pathways regulating IMS Cu,Zn-SOD activity, and to establish a functional link between its activity and key mitochondrial events.

EXPERIMENTAL

Chemicals.

p-hydroxyphenylacetate, Bovine Cu,Zn-superoxide, fatty acid free bovine serum albumin (BSA), 3'-{1-[(phenylamino)-carbonyl]-3,4-tetrazolium}-bis (4-methoxy-6nitro) benzenesulfonic acid hydrate (XTT), digitonin, iodoacetamide (IAM), xanthine oxidase, catalase, reduced glutathion, Txr, TxrR-1, antimycin, horseradih peroxidase, NADP+, isocitrate dehydrogenase, rotenone, and rhodamine 123 were from Sigma Chemical Co. (St. Louis, MO, USA).

Isolation of liver mitochondria.

Liver mitochondria were isolated from adult male Wistar rats by differential centrifugation as described previously [START_REF] Petit | Disruption of the outer mitochondrial membrane as a result of large amplitude swelling: the impact of irreversible permeability transition[END_REF]. Rat livers were excised, chopped into fine pieces, washed with 0.25 M sucrose and homogenized in isolation buffer consisted of 210 mM mannitol, 70 mM sucrose, 2 mM Hepes, pH 7.4, and 0.05% BSA. The homogenate was centrifuged at 800g for 8 min, the pellet was removed, and the centrifugation process was repeated. The supernatant was centrifuged at 8,000g for 10 min, the pellet was washed with the isolation buffer, and the centrifugation was repeated. The pellet containing mixture of organelles were further fractionated by centrifugation at 8,500 g for 10 min in Percoll gradient consisted of three layers of 18%, 30%, and 60% (w/v) Percoll in sucrose-Tris buffer (0.25 M sucrose, 1 mM EDTA, and 50 mM Tris), pH 7.4.

Mitochondria were collected from the interface of 30% and 60% Percoll and washed with the sucrose-Tris buffer. Mitochondrial proteins were determined by Bradford assay [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF].

Isolation of mitochondrial IMS contents.

Mitochondrial outer membrane was selectively disrupted by treating mitochondria (40 mg/ml) with 0.11 mg of digitonin per mg of protein [START_REF] Saidha | Localization of a sulphateactivating system within Euglena mitochondria[END_REF]. The IMS contents were then freed of mitoplasts by centrifugation at 10,000g for 15 min.

Measurement of SOD activity.

SOD activity was assayed by generating O 2

.

-with the xanthine/xanthine oxidase system in the presence of sulphonated tetrazolium salt, XTT [START_REF] Ukeda | Spectrophotometric assay for superoxide dismutase based on tetrazolium salt 3'--1--(phenylamino)carbonyl--3, 4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate reduction by xanthine-xanthine oxidase[END_REF]. Cyanide at 5.0 mM was used to selectively inhibit Cu,Zn-SOD activity.

Measurement of H 2 O 2 .

Fluorescence measurements were performed using Perkin-Elmer LS-5

spectrofluorometer. H 2 O 2 formation was measured by monitoring horseradish peroxidase-catalysed H 2 O 2 -dependent oxidation of p-hydroxyphenylacetate (λ ex = 320 nm; λ em = 400 nm) [START_REF] Hyslop | A quantitative fluorimetric assay for the determination of oxidant production by polymorphonuclear leukocytes: its use in the simultaneous fluorimetric assay of cellular activation processes[END_REF].

Temporal kinetics of IMS

Detection of TxrR-1 in mitochondrial IMS.

Mitochondrial IMS and cytosolic fractions were resolved on polyacrylamide gels and immunoblotted onto nitrocellulose membranes using anti-TxrR-1 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Cytosolic contamination was assessed by using anti-β-actin antibody (Sigma Chemical Co., St. Louis, MO, USA.).

Measurement of aconitase activity.

Mitochondria were incubated in the presence or absence of 5 µM H 2 O 2 for 5 minutes at 37 o C. The state 3 respiration was then initiated by addition of 2 mM ADP, 4 mM MgCl 2 , 5 mM KH 2 PO 4 , and 10 mM succinate. Aliquots were taken at indicated times to determine mitochondrial aconitase activity. Aconitase activity was assayed at room temperature by following formation of NADPH at 340 nm for 10 minutes in a reaction mixture containing 50 mM Tris HCl, pH 7.4, 30 mM sodium citrate, 0.6 mM MnCl 2 , 0.2 mM NADP + and 1 U/ml of isocitrate dehydrogenase. One nmole NADPH formed per min was corresponded to 1 mU of aconitase [START_REF] Drapier | Aconitases: a class of metalloproteins highly sensitive to nitric oxide synthesis[END_REF].

Measurement of mitochondrial membrane potential and cytochrome c release from mitochondria.

Mitochondria (1 mg/ml) were incubated in a buffer containing 0.250 mM sucrose, 10 mM Hepes/KOH, pH 7.4, 0.1% fatty-acid free bovine serum albumin, 6 mM succinate, 1 µg/ml rotenone, and 1 µM Rhodamine 123 for 5 min at 25ºC in order to equilibrate mitochondria with the cationic dye. Mitochondria were incubated with 5 µM

Data presentation

Data were reported as mean ± SD of at least three independent experiments performed in triplicate. Analysis of variance and Duncan's Multiple Range Test was used to analyse the difference among group means. A P value of < 0.05 was considered a statistically significant difference.

RESULTS

Superoxide and H 2 O 2 promote activation of IMS Cu,Zn-SOD.

In order to determine the biochemical processes involved in the activation of IMS Cu,Zn-SOD, a series of experiments were conducted with intact mitochondria and the purified inactive enzyme. Low micromolar concentrations (5 μM) of exogenous H 2 O 2 activated IMS Cu,Zn-SOD at a rate of 2 units/mg mitochondrial protein/min (Fig. 1A).

The enzyme activity reached to its maximum level, 10 units/mg mitochondrial protein, after 5 minutes of mitochondria exposure to H 2 O 2 , decreased sharply at 10 minutes, and ultimately returned to the basal levels at 30 minutes (Fig. 1A). The possibility of direct interaction between IMS Cu,Zn-SOD and H 2 O 2 was assessed by exploiting Cu,Zn-SOD, which was purified from mitochondria and subsequently reduced by DTT to inactive state. The specific activity of the purified IMS Cu,Zn-SOD was determined as 3200 ± 270 units/mg enzyme/min following rupture of the outer membrane with digitonin alone but prior to reduction with DTT. Neither H 2 O 2 alone nor H 2 O 2 in the presence of mitochondrial peroxiredoxin III activated the reduced Cu,Zn-SOD (Fig. 1B). However, the enzyme was markedly activated following incubation with superoxide generating system, xanthine and xanthine oxidase, and to a lesser extent with glucose and glucose oxidase, a system producing superoxide, in addition to H 2 O 2 , with a rate dependent on the pH and O 2 concentration [START_REF] Liochev | Superoxide from glucose oxidase or from nitroblue tetrazolium?[END_REF]. Lesser efficiency of glucose/glucose oxidase system is possibly because of a lower rate of superoxide production compared to xanthin/xanthine oxidase system. Presence of bovine erythrocyte SOD (300 U/ml) in the assay mixture abolished this activation whereas catalase was ineffective (Fig. 1B). Examination of kinetics of the enzyme activation revealed that steady state levels of superoxide (0.7-0.8 nmol/ml/min) generated by xanthine/xanthine oxidase activate the reduced IMS Cu,Zn-SOD at a rate of 66 units/mg enzyme/min (Fig. 1C). The enzyme specific activity was ultimately reached to a plateau of 3200 units/mg enzyme at 45 min. These findings suggest that both H 2 O 2 and superoxide may act as activators of IMS Cu,Zn-SOD. While superoxide can directly act upon the enzyme, H 2 O 2 requires additional mitochondrial component(s) to trigger activation of Cu,Zn SOD.

Superoxide generated by mitochondrial respiration activates Cu,Zn-SOD in intact mitochondria

In order to verify whether superoxide generated during mitochondrial respiration can activate Cu,Zn-SOD in the IMS, superoxide production was promoted by antimycin in mitochondria respiring on state 4 (Fig. 2A). Mitochondrial superoxide production was dose-dependently increased with antimycin concentrations between 0.01 to 0.1 μg/mg mitochondrial protein, generating hydrogen peroxide ranging from 0.30 ± 0.03 to 0.65 ± 0.04 nmoles/min/mg (equivalent to superoxide ranging from 0.60 ± 0.06 to 1.35 ± 0.08 nmoles/min/mg). An inverse relationship was, however, found between the levels of mitochondrial superoxide production and the extent and duration of Cu,Zn-SOD activation in the IMS. Cu,Zn-SOD was more potently activated for a significantly longer duration when mitochondria generated lower levels of superoxide 0.60 ± 0.06 nmoles/mg mitochondrial protein/min compared to a lesser and shorter duration of the enzyme activity when mitochondria generated high levels of superoxide 1.35 ± 0.08 nmoles/mg mitochondrial protein/min (Fig. 2B). These findings suggest that mitochondrial respiration regulates Cu,Zn-SOD activity in the IMS.

TxrR inactivates IMS Cu,Zn-SOD.

Presence of TxrR-1 in the IMS of rat liver mitochondria was detected by western blotting (Fig. 3). In order to determine potential biochemical pathway(s) implicated in the inactivation of IMS Cu,Zn-SOD, the enzyme was purified from mitochondria in an active, oxidized state by rupturing the outer membrane with digitonin alone. Incubation with TxrR-1 from rat liver significantly decreased activity of the oxidized Cu,Zn-SOD (Fig. 4A). The initial rate of inactivation of the oxidized enzyme was estimated 60 units/mg enzyme/min (Fig. 4B). Presence of Txr in the assay mixture did not potentiate inactivation of the oxidized enzyme by TxrR-1 (Fig. 4A). Furthermore, neither Txr nor GSH alone affected superoxide scavenging activity of the oxidized form of Cu,Zn-SOD.

These findings suggest that mitochondrial TxrR-1 is implicated in the inactivation of IMS IMS Cu,Zn-SOD protects against oxidative loss of aconitase activity in matrix.

Rat liver mitochondria are known to produce superoxide when respiring with complex II-linked substrates on state 3 [START_REF] Taylor | Reactive oxygen species produced by liver mitochondria of rats in sepsis[END_REF][START_REF] Venditti | Effect of thyroid state on H2O2 production by rat liver mitochondria[END_REF]. Direct aerobic inactivation of matrix aconitase was also recently reported in yeasts lacking Cu,Zn-SOD. Incubation of rat liver mitochondria respiring on state 3 at 37 o C resulted in loss of aconitase activity by 33 ± 5% and 42 ± 4% after 5 and 15 min, respectively, (Fig. 5). However, activation of IMS Cu,Zn-SOD following incubation of intact mitochondria with H 2 O 2 (5 μM) significantly prevented loss of aconitase activity in mitochondrial matrix (Fig. 5).

IMS Cu,Zn-SOD diminishes apoptotic capacity of mitochondria.

CaCl 2 progressively promoted dissipitation of Ψm in mitochondria respiring at state 4 (Fig. 6A). Loss of Ψm is known to precede the release of mitochondrial apoptotic factors, such as cytochrome c. Analysis of the mitochondria incubated with CaCl 2 for 30 min demonstrated significant loss of IMS cytochrome c accompanying with its release into extramitocondrial space (Fig. 6B The transient nature of the enzyme activation could be also attributed to the biochemical pathways inactivating IMS Cu,Zn-SOD. One such enzyme detected in the IMS was TxrR-1. The rate of Cu,Zn-SOD inactivation (0.1 μM/min) was, however, slower than its activation with either H 2 O 2 or superoxide. Whether additional enzymatic pathways are implicated in inactivation of IMS Cu,Zn-SOD remained to be determined.

Alternatively, faster inactivation of Cu,Zn-SOD in intact mitochondria could be potentially due to the substantially higher IMS TxrR-1 concentration compared to that under the assay conditions presented in Fig. 4 (20 U/ml). Moreover, CDNB and auranofin, selective TxrR-1 inhibitors, prolonged duration of H 2 O 2 -induced IMS Cu,Zn-SOD activation. These findings demonstrate that TxrR-1 is implicated in the inactivation of IMS Cu,Zn-SOD. In agreement with these findings, eukaryotic Cu,Zn-SOD expressed in prokaryotic organisms was found active only when the cytosolic expression of TxrR-1

was prevented [START_REF] Battistoni | In vivo formation of Cu,Zn superoxide dismutase disulfide bond in Escherichia coli[END_REF].

IMS Cu,Zn-SOD protects vital mitochondrial functions.

Yeast mutant lacking or deficient in Cu,Zn-SOD presents direct aerobic inactivation of enzymes, such as aconitase, homoaconitase and succinate dehydrogenase, containing superoxide sensitive matricial 4Fe-4S cluster. These findings suggest that superoxide released into the IMS may pass mitochondrial inner membrane and damage matricial 4Fe-4S cluster containing enzymes even in the presence of Mn-SOD [START_REF] Wallace | Superoxide inhibits 4Fe-4S cluster enzymes involved in amino acid biosynthesis. Cross-compartment protection by CuZn-superoxide dismutase[END_REF].

Superoxide (pK 4.8) is likely to be protonated at the external surface of inner membrane due to the protons extrusion, and diffuse into the matrix [START_REF] Guidot | Mitochondrial respiration scavenges extramitochondrial superoxide anion via a nonenzymatic mechanism[END_REF], or it may react with mitochondrial nitric oxide to produce peroxynitrite (pK 6.8), which can be then diffuse into the matrix [START_REF] Liochev | Cross-compartment protection by SOD1[END_REF]. Our findings indicate that active IMS Cu,Zn-SOD protects the matricial aconitase activity against superoxide-mediated inactivation.

It has long been known that Ca 2+ overload leads to the state of mitochondrial membrane permeability transition [START_REF] Kowaltowski | Opening of the mitochondrial permeability transition pore by uncoupling or inorganic phosphate in the presence of Ca2+ is dependent on mitochondrial-generated reactive oxygen species[END_REF][START_REF] Chernyak | Redox regulation of the mitochondrial permeability transition pore[END_REF] and the opening of a mitochondrial membrane pore (PTP) following increased mitochondrial production of reactive oxygen species [START_REF] Castilho | Permeabilization of the inner mitochondrial membrane by Ca2+ ions is stimulated by t-butyl hydroperoxide and mediated by reactive oxygen species generated by mitochondria[END_REF]. The opening of PTP leads to cytochrome c release into cytosol, which then triggers apoptotic process [START_REF] Pardo | Apoptotic pathways are selectively activated by[END_REF]. *Significantly different from the activity at 0 min, P < 0.05.

H 2 O

 2 2 for 3 minutes at 25 o C. CaCl 2 (12 or 25 µM) was then added to induce dissipitation of membrane potential. Membrane potential was assessed fluorimetrically by measuring the membrane potential dependent uptake of Rhodamine 123 (1 µM) with excitation at 505 nm and recording at 534 nm. [23]. Pellets and supernatants were treated with SDS sample buffer and boiled, and aliquots were resolved on polyacrylamide gesl and immunoblotted onto nitrocellulose membranes using anti-cytochrome c antibodies (BD Bioscience, San Jose, CA, USA.).

Fig. 1 -

 1 Fig. 1-H 2 O 2 and superoxide promote activation of IMS Cu,Zn-SOD. (A) Temporal

Fig. 2 -Fig. 3 .

 23 Fig. 2-Mitochondrial respiration regulates Cu,Zn-SOD activity in the IMS. (A)

Fig. 4 -

 4 Fig. 4-TxrR-1 inactivates the oxidized IMS Cu,Zn-SOD. Cu,Zn-SOD was purified

Fig. 5 .

 5 Fig. 5. IMS Cu,Zn-SOD prevents oxidative loss of aconitase activity in matrix.

Fig. 6 .

 6 Fig. 6. IMS Cu,Zn-SOD decreases apoptotic capacity of mitochondria. (A) Delay in the loss of Ψm induced by CaCl 2 . Mitochondria (1 mg/ml) were incubated at 37 º C for 3

Fig. 7 .

 7 Fig. 7. Putative biochemical pathways regulating IMS Cu,Zn-SOD activity and

Cu,Zn-SOD activation by H 2 O 2 .

	Mitochondria (2 mg/ml) were incubated with 5 µM H 2 O 2 for 5, 10, 20, 30 minutes
	at 37 o C, washed with sucrose-Tris buffer, and then incubated with digitonin in the
	presence of iodoacetamide (IAM) for 1 hour at room temperature. Cu,Zn-SOD activity
	was assayed in the mitochondrial IMS preparations.

Preparation of the oxidized and reduced IMS Cu,Zn-SOD.

	dialysis. The activity of the oxidized or reduced Cu,Zn SOD was measured following
	incubation with various agents for indicated times at 37 o C.
	Rat liver IMS Cu,Zn-SOD was purified in the oxidized state as described
	previously [2]. The reduced enzyme was prepared following incubation of the purified
	enzyme with 4 mM dithiotreitol (DTT) at 37ºC overnight. DTT was then removed by

Measurement of IMS Cu,Zn-SOD activation by mitochondrial superoxide.

	Mitochondria were incubated with various concentrations of antimycin ranging
	from 0.01 to 0.1 µg/mg of mitochondria respiring at state 4 in the presence of 1 mM p-
	hydroxyphenylacetate. H 2 O 2 formation was measured by monitoring horseradish
	peroxidase-catalyzed (5 U/ml) H 2 O 2 -dependent oxidation of p-hydroxyphenylacetate.
	Mitochondria were also incubated with 0.01-0.1 µg/mg of antimycin for indicated times
	at 37 o C, washed with sucrose-Tris buffer, and incubated with digitonin in the presence of

iodoacetamide (IAM) for 1 hour at room temperature. Cu,Zn-SOD activity was assayed in the mitochondrial IMS preparations.

  It is likely that mitochondrial factor(s) involved in H 2 O 2 -induced activation of IMS Cu,Zn-SOD are sensitive to alkylation by CDNB under this experimental condition.

	mitochondria with CDNB completely abolished activation of IMS Cu,Zn-SOD by H 2 O 2
	(Fig. 4C).
	and inhibit IMS Cu,Zn-SOD. Addition of CDNB to intact mitochondria at 4 th min of
	H 2 O 2 treatment substantially prolonged duration of H 2 O 2 -induced IMS Cu,Zn-SOD
	activity (Fig 4C). In a similar set of experiments, auranofin, a potent inhibitor of
	selenocystein-containing enzymes [25], comparably prolonged the enzyme activity
	induced by H 2 O 2 (data not shown). These findings suggest that TxrR-1 play a role in
	inactivation of Cu,Zn-SOD in intact mitochondria. However, pre-incubation of intact

Cu,Zn-SOD.

CDNB is an alkylating agent that selectively inhibits TxrR-1at low concentrations (250 μM). Pre-incubation of purified IMS Cu,Zn-SOD, which was reduced with DTT and subjected to dialysis to remove excess of DTT, with CDNB at 37ºC for 10 min did not affect activation of the reduced enzyme by xanthine/xanthine oxidase system (data not shown). This finding suggest that CDNB, at concentration used here, is unable to alkylate

  , upper panel). Prior activation of IMS Cu,Zn-SOD by exposing mitochondria to H 2 O 2 (5 μM) significantly delayed loss of Ψm and prevented cytochrome c release induced by CaCl 2 (Fig.6). These findings suggest that active state of IMS Cu,Zn-SOD may regulate events leading to the loss of mitochondrial integrity and cell death. following exposure to H 2 O 2 and superoxide. Superoxide generated by X/XO or mitochondrial respiration activated Cu,Zn-SOD at a lower rate (0.6 μM/min) than H 2 O 2 (5 μM/min). Higher rate of enzyme activation by H 2 O 2 may reflect enzyme-catalysed nature of this reaction. However, temporal kinetics of the enzyme activation by H 2 O 2 and superoxide were similar in the intact mitochondria. Both H 2 O 2 and superoxide elicited a transient activation of Cu,Zn-SOD, although the extend and duration of enzyme activation was longer with superoxide than with H 2 O 2 . It is noteworthy that the activity of Cu,Zn-SOD was inversely correlated with the level of mitochondrial superoxide production, a similar phenomenon observed following exposure of intact mitochondria to exogenous H 2 O 2 . These findings suggest that IMS Cu,Zn-SOD is active in a narrow window of physiologically relevant concentrations of superoxide or H 2 O 2 . The underlying reason for the lower level of the enzyme activity in the presence of higher concentrations of superoxide or H 2 O 2 remains to be elucitaed.

Journal Immediate Publication. Published on 30 Mar 2007 as manuscript BJ20061809

	Taken altogether, these findings suggest that IMS Cu,Zn-SOD activity is granzyme A and/or granzyme B in CTL-mediated target cell lysis. J. Cell Biol.
	167, 457-468.
	reversibly regulated by H 2 O 2 , superoxide, and TxrR-1 and that the enzyme is implicated
	in protecting vital mitochondrial functions.
	Transient activation of IMS Cu,Zn-SOD significantly decelerated
	dissipitation of mitochondrial m and decreased release of cytochrome c, suggesting that
	IMS Cu,Zn-SOD may function to prevent apoptosis during episodes of increased
	mitochondrial superoxide levels.

20

Biochemical

Biochemical Journal Immediate Publication. Published on 30 Mar 2007 as manuscript BJ20061809 © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 30 Mar 2007 as manuscript BJ20061809 © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 30 Mar 2007 as manuscript BJ20061809 © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 30 Mar 2007 as manuscript BJ20061809 © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 30 Mar 2007 as manuscript BJ20061809 © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 30 Mar 2007 as manuscript BJ20061809 © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 30 Mar 2007 as manuscript BJ20061809 © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 30 Mar 2007 as manuscript BJ20061809 © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 30 Mar 2007 as manuscript BJ20061809 © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 30 Mar 2007 as manuscript BJ20061809 © 2007 Biochemical Society

© 2007 Biochemical Society

DISCUSSION

The findings of this study suggest that IMS Cu,Zn-SOD activity is regulated by mitochondrial respiratory chain and TxrR-1. Figure 7 illustrates a network of biochemical pathways, which may regulate or be affected by IMS Cu,Zn-SOD activity. It appears that IMS Cu,Zn-SOD is implicated in protecting intermediate metabolism against oxidative damage and regulating apoptosis.

Regulation of Cu,Zn-SOD activity in the IMS.

In order to elucidate the biochemical pathways involved in reversible activation of IMS Cu,Zn-SOD, a series of experiments were conducted with intact mitochondria and the purified Cu,Zn-SOD reduced by DTT to an inactive state. While H 2 O 2 transiently activated Cu,Zn-SOD in intact mitochondria, it did not affect the activity of the reduced enzyme once it was purified from the IMS, suggesting that additional mitochondrial component(s) are necessary to catalyze the intra-subunit disulfide formation. In spite of localization of peroxiredoxin III in mitochondrial matrix and in the IMS, our findings suggest that peroxiredoxin III is not implicated in the activation of Cu,Zn-SOD. However, given that H 2 O 2 is readily diffusible across membranes, these findings suggest that IMS Cu,Zn-SOD is responsive to fluctuations of physiologically relevant concentrations of H 2 O 2 in a cell.

Mitochondria are the major source of superoxide. The mitochondrial inner membrane vectorially releases superoxide into the IMS [START_REF] Han | Mitochondrial respiratory chaindependent generation of superoxide anion and its release into the intermembrane space[END_REF] upon autoxidation of the ubisemiquinone at the outer coenzyme Q pool. Direct activation of IMS Cu,Zn-SOD by superoxide constitute a typical example of enzyme regulation in response to changes in the level of its substrate. The rate of Cu,Zn-SOD activation was, however, different


Dr Nadia Wahab
email: nadia.wahab@imperial.ac.uk

Dimity Cox

Abigail Witherden

Roger M Mason

Connective Tissue Growth Factor (CTGF) Promotes Activated Mesangial Cell Survival via Upregulation of MAP Kinase Phosphatase-1 (MKP-1)

Keywords: CTGF, CCN2, MKP-1, diabetic nephropathy, mesangial cell

Activated mesangial cells are thought to play a pivotal role in the development of kidney fibrosis in chronic pathological conditions, including diabetic nephropathy (DN). Their prolonged survival may enhance the development of the disease since they express increased amounts of growth factors and extracellular matrix proteins (ECM). CTGF is one of the growth factors produced by activated mesangial cells and is reported to play a key role in the pathogenesis of DN.

Previous studies have shown that addition of exogenous CTGF to human mesangial cells (HMC) rapidly activates ERK 1/2 MAPK and JNK MAPK, but not the p38 MAPK, despite the activation of the upstream kinases, MKK3/6. The aim of this study was to investigate whether the lack of phosphorylated p38 MAPK by CTGF has an anti-apoptotic effect on activated HMC.

We show that in HMC CTGF induces the rapid transcriptional activation and synthesis of MAPK phosphatase-1 (MPK-1), a dual specificity phosphatase which dephosphorylates p38 MAPK. This in turn prevents the anti-apoptotic protein, bcl-2, from being phosphorylated and losing its function, leading to the survival of the cells. Knockout of MKP-1 protein in mesangial cells treated with CTGF, using siRNA or antisense oligonucleotides, allows p38 MAPK activation and induces mesangial cell death.

Introduction

Glomerular mesangial cells are usually quiescent under normal physiological conditions. During acute kidney injury, these cells are thought to undergo a process termed "activation or dedifferentiation", the result of which is that they become myofibroblast -like cells [START_REF] Johnson | The activated mesangial cell: a glomerular "myofibroblast[END_REF]. These have contractile properties, produce 3 high levels of cytokines, chemokines and ECM proteins, and are involved in matrix remodeling. These differentiated cells may then undergo spontaneous apoptosis, in keeping with data indicating that apoptosis mediates clearing of myofibroblasts from wounded skin [START_REF] Desmouliere | Apoptosis mediates the decrease in cellularity during the transition between granulation tissue and scar[END_REF] and of activated hepatic stellate cells following transient liver tissue injury [START_REF] Enzan | Proliferation of Ito cells (fat-storing cells) in acute carbon tetrachloride liver injury. A light and electron microscopic autoradiographic study[END_REF]. However prolonged survival of these cells in chronic injury conditions may lead to the mesangial hypercellularity and glomerular scarring which occurs in DN, for example.

Numerous studies have shown that elevated levels of TGFβ support the initiation and progression of the mesangial cell's differentiation program. However, in the last 5-6 years it has become clear that CTGF, which is rapidly induced by TGFβ, as well as by other factors generated during chronic injury (e.g. ROS, hypoxia, etc.), plays a key role in this program [START_REF] Park | Hydrogen Peroxide Is a Novel Inducer of Connective Tissue Growth Factor[END_REF][START_REF] Higgins | Hypoxic induction of Ctgf is directly mediated by Hif-1[END_REF][START_REF] Mason | Extracellular Matrix Metabolism in Diabetic Nephropathy[END_REF][START_REF] Wahab | Connective tissue growth factor and renal diseases: some answers, more questions[END_REF].

Previously we have shown that CTGF mediates several TGFβ -dependent changes in mesangial cells which are also reported to occur in DN. These are: i) induction of the cellular hypertrophy due to induction of the cyclin-dependent kinase inhibitors (CDKIs) p15, p21 and p27, leading to cell cycle arrest in the G 1 phase [START_REF] Abdel-Wahab | Connective Tissue Growth Factor and Regulation of the Mesangial Cell Cycle: Role in Cellular Hypertrophy[END_REF], ii) increased expression of extracellular matrix proteins (ECM] [START_REF] Wahab | Role of connective tissue growth factor in the pathogenesis of diabetic nephropathy[END_REF], iii) increased expression of integrins on the cell surface to facilitate the deposition and assembly of ECM proteins [10].

In a previous study we showed that addition of exogenous CTGF to mesangial cells activates several intracellular signalling pathways including mitogenactivated protein kinases ERK 1/2 MAPK and JNK [START_REF] Wahab | Connective Tissue Growth Factor CCN2 Interacts with and Activates the Tyrosine Kinase Receptor TrkA[END_REF]. Interestingly, p38 MAPK was not activated. In the present study we show that p38 MAPK inactivation is critical for the survival of mesangial cells in the presence of CTGF. The underlying mechanism seems to involve the rapid induction of an immediate early response gene encoding the dual specificity protein phosphatase 1 (MKP-1). MKP-1 preferentially inactivates the p38 MAPK and thus prevents phosphorylation of the anti-apoptotic protein bcl-2 thereby promoting the survival of HMCs.

Materials and Methods

Cell Cultures, Antibodies and Reagents

Primary normal adult human mesangial cells (HMC) (CC-2259, lot OF1548) were purchased from Cambrex Bio Science (www.cambrex.com), maintained in culture as described previously [START_REF] Wahab | Role of connective tissue growth factor in the pathogenesis of diabetic nephropathy[END_REF], and used at passage 9-10. Recombinant CTGF was expressed as a fusion protein with V5 tag as described previously [START_REF] Wahab | Role of connective tissue growth factor in the pathogenesis of diabetic nephropathy[END_REF].

Phosph-ERK1/2 pathway sampler, phosphor-JNK pathway sampler and phosphor-p38 MAPK pathway sampler antibodies were from New England BioLabs (www.neb.uk.com). BCL-2 monoclonal antibody was from Santa Cruz (www.autogenbioclear.com). Anti-MKP-1 antibody was from Sigma (www.sigmaaldrich.com). The MKP-1 ANTISENSE-RNAi-Combination-Kit was from Biognostik GmbH (www.biognostik.com).

RNA Extraction and RT-PCR Analysis

Total RNA was extracted from mesangial cells using the RNAzol B method [AMS Biotechnology (UK) Ltd., Oxfordshire, UK]. Equal amounts of total RNA (3µg) from each sample were reverse transcribed into cDNA using Superscript II RNase H+ reverse transcriptase (Gibco BRL, Paisley, Scotland, UK) and random primers. Equal amounts of the reverse transcription reaction were subjected to PCR amplification as previously described [START_REF] Wahab | Role of connective tissue growth factor in the pathogenesis of diabetic nephropathy[END_REF]. Amplificatino was started with 5 min denaturation at 94°C followed by 30 PCR cycles. Each cycle consisted of 60 s at 94°C, 60 s at 55°C and 60 s at 72°C. The final extension was for 10 min at 72°C. A control omitting the RT step was also performed. The sequences of the primers used to amplify MKP-1 were 5'-CCGGAGCTGTGCAGCAAA-3' and 5'-CTCCACAGGGATGCTCTT-3', yielding a PCR product of 284bp.

Western Blotting

Cells grown on coverslips were lysed in reducing SDS-PAGE loading buffer and immediately scraped off the plate. Cell lysates were sonicated for 10 sec to shear DNA. Samples were boiled for 5 min and resolved on 4-12% gradient gels by SDS-PAGE. Proteins were transferred onto a polyvinylidene difluoride membrane filter (Immobilin-P,Millipore, Bedford, UK) using a BioRad transfer apparatus. Blots were incubated in blocking buffer containing 1x TBS, 0.1% Tween-20 with 5% (w/v) non-fat dry milk, for 1 h. Immunodetection was performed by incubating the blots in primary antibody at the appropriate dilution in antibody dilution buffer (1x TBS, 0.1% Tween-20 with 5% BSA), overnight at 4°C. Blots were then washed 3 times with washing buffer (1x TBS, 0.1% Tween-20) and incubated with secondary horseradish peroxidase (HRP)-conjugated antibodies for 1 h at room temperature. Bound antibodies were visualized using the enhanced chemiluminescence reagent Luminol (Autogen Bioclear UK Ltd,Wiltshire, UK). Prestained molecular weight standards (Amersham International PLC, Amersham, UK) were used to monitor protein migration.

Immunofluorescence Staining

Cells were fixed with 3.7% paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS for 10 min at room temperature. Coverslips were then incubated overnight at 4°C with serum (5% in PBS) from the same species as that in which the secondary antibody was raised. After this they were incubated with primary antibodies (at optimum dilution in PBS containing 3% BSA) for 1 h at 37°C. Coverslips were then washed and incubated in the dark for 1 h with fluorescein-conjugated secondary antibody (Sigma Aldrich, Dorset, UK). After staining, the coverslips were mounted on glass slides with anti-fade mounting media (Vector Labs,Peterborough, UK) and examined using a fluorescence microscope.

Use of Antisense Oligonucleotides

Phosphothioate MKP-1 antisense oligonucleotide (2 µM, as recommended by the manufacturer; sequence ID: 2.05983, Biognostik), or a CG-matched randomised sequence oligonucleotide (2 µM, as a negative control; sequence ID: C5278), was added directly to cultures of equal numbers of cells, 24 hours prior to any other treatments.

siRNA Transfection

HMC were plated at equal number (2.4 x10 4 / well) in 24-well plates, 24 h before transfection. Cells were then transfected with MKP-1 siRNA, or with a luciferase siRNA as a nonspecific control, using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions with 0.7 µl Lipofectamine per well. Three different MKP-1 specific siRNAs were tested at different concentrations (10-50 nM) for their ability to reduce MKP-1 expression in HMC stimulated with 20 ng/ml CTGF for 1hr (quiescent cells express only very low levels of MKP-1). The optimum concentration of siRNAs in the cell culture medium was determined to be 30 nM. 6 hours post-transfection, growth medium was replaced with serum free medium. Cells were then incubated at 37 °C for 24 h before being assessed for MKP-1 gene knockdown. All three siRNAs caused a reduction in MKP-1 gene expression but to different levels. Silencer 20639 was the most effective and was used in the reported experiments (sense 5'-CCACCACCGUGUUCAACUUtt-3', antisense 5-'AAGUUGAACACGGUGGUGGtt-3').

Cell Viability Assay

Mesangial cell death was determined using the cell proliferation kit II (XTT) (Roche, www.roche-applied-science.com). The method is based on the cellular reduction of XTT by mitochondrial dehydrogenases to an orange formazan product that can be measured spectrophotometrically. Briefly, XTT labelling mixture (1ml XTT labelling reagent and 20 µl electron coupling reagent) was prepared immediately before use. Aliquots of 150 µl of XTT labelling mixture were added to cells grown in a 24-well plate in a final volume of 300 µl culture medium per well. Cells were then incubated for different periods of time at 37°C after which absorbance at 450 nm was measured with a multiwell spectrophotometer.

Statistical Analysis

Results were compared using Student's unpaired t-test. A p value of 0.05 or less was regarded as denoting a significant difference.

Results

CTGF induces p38 MAPK dephosphorylation

Stimulation of HMCs with 20 ng/ml recombinant CTGF rapidly activates the classical MAPK (ERK1/2) and JNK but not the p38 MAPK, as previously reported [START_REF] Wahab | Connective Tissue Growth Factor CCN2 Interacts with and Activates the Tyrosine Kinase Receptor TrkA[END_REF]. Figure 1a

CTGF induces the rapid expression of MKP-1 in HMC

It is known that MAPKs, once activated, can be inactivated by dual specificity protein phosphatases. P38 and JNK MAPKs are preferentially inactivated by MKP-1 [13]. However, in vitro studies have shown that MKP-1 is most selective for p38 MAPK at physiologic levels of expression where it directly binds to the p38 kinases [13,[START_REF] Hutter | Catalytic activation of mitogen-activated protein (MAP) kinase phosphatase-1 by binding to p38 MAP kinase: critical role of the p38 C-terminal domain in its negative regulation[END_REF]. At high levels of expression MKP-1 can also inactivate JNK and even ERK1/2 but at low levels [13,[START_REF] Hutter | Catalytic activation of mitogen-activated protein (MAP) kinase phosphatase-1 by binding to p38 MAP kinase: critical role of the p38 C-terminal domain in its negative regulation[END_REF]. MKP-1 is an immediate-early gene product that is potently induced in response to both growth factors and stress stimuli. Thus we investigated whether CTGF is able to induce the expression level of MKP-1 in HMC. MKP-1 is constitutively expressed at a very low level in serum starved HMC (fig 2 A andB). Upon CTGF stimulation MPK-1 mRNA levels were rapidly induced within 15 min and reach a maximum at about 60 min, followed by a gradual decline. Figure 2C shows that MKP-1 protein levels were also rapidly induced in CTGF stimulated HMC, mirroring the increase in the mRNA. However, MKP-1 protein was barely detectable in the untreated cells.

CTGF induction of MKP-1 was sustained, with the protein detectable up to 24 h after CTGF addition.

CTGF enhances the levels of Bcl-2 protein in HMC

The correlation between MKP-1 induction and the inactivation of p38 MAPK suggests that MKP-1 may play a significant role in mediating the dephosphorylation of this kinase in CTGF-stimulated HMC. p38 MAPK has been reported as one of the kinases responsible for the phosphorylation of the antiapoptotic protein Bcl-2, which leads to loss of its anti-apoptotic potential [START_REF] Haldar | Taxol induces bcl-2 phosphorylation and death of prostate cancer cells[END_REF] and an increase in its proteolytic degradation via a proteosomal pathway [16]. Thus we investigated the relative abundance of the Bcl-2 protein in HMC stimulated with CTGF. Figure 3 shows that CTGF stimulation rapidly enhances the level of Bcl-2 protein in HMC and that this increased level was maintained over the time course of the experiment. No clear shift in the mobility of bcl-2 protein was observed. Attempts to detect Bcl-X L protein expression level in HMC were not successful (data not shown).

To confirm the prominent role of MKP-1 in the dephosphorylation/inactivation of p38 MAPK and thus the protection of bcl-2 we decided to knock-down MKP-1 protein in cells stimulated with CTGF. For this we used an ANTISENSE-RNAi-Combination-Kit, which includes siRNAs and antisense oligonuleotides specifically designed for the human MKP-1 gene. This allowed us to crossvalidate our results, as both techniques work through independent mechanisms of action. Twenty-four hours after MKP-1 siRNA, or MKP-1 antisense oligonucleotide transfection, cells were incubated with or without 20 ng/ml CTGF for 1hr. To control for nonspecific effects of siRNA delivery, controls were included in which cells were either mock transfected (transfection reagent only), or transfected with Luciferase siRNA, while a CG-matched randomised sequence oligonucleotide was used as a control for the specificity of the antisense delivery.

MKP-1 expression was induced in response to CTGF stimulation in cells which were either mock transfected, or transfected with a nonspecific siRNA (figure 4a,b). In contrast, transfection with a specific siRNA led to a significant reduction of MKP-1 expression in HMC treated with CTGF for 1hr at both the mRNA and protein levels. MKP-1 RNA inhibition, correlates well with the high reduction in the protein level indicating that the response is a classical RNAi effect and not an miRNA effect.

Figure 4 (C) shows that MKP-1 antisense oligonucleotide, but not the control oligonucleotide, also induces a marked reduction in MKP-1 protein expression level in HMC treated with CTGF for 1hr.

After assessing the efficiency of both the siRNA and antisense approaches to knock-down MKP-1 protein in HMC, similar experiments were performed in which the cells were lysed after different periods of time and equal amounts of total protein analyzed for the expression level of phospho-p38MAPK and bcl-2. Interestingly, a second band of a higher molecular weight (~ 29kDa) that cross reacted with the bcl-2 antibody can also be seen in the lysates from cells which were treated with siRNA, but not in those which were mock treated. This may represent the p38 MAPK-dependent phosphorylated form of bcl-2. Similar results were obtained using the MKP-1 antisense approach. The data in figure 5 (b) confirm that inhibition of MKP-1 expression in HMC enhances CTGF-mediated p38 MAPK activation which in turn plays a key role in reducing the level of the cell survival factor bcl-2.

Modulation of MKP-1 protein levels is involved in CTGF-mediated survivalactivity in HMC.

Next we investigated whether CTGF exerts a survival-promoting activity in HMC.

Twenty-four hours after MKP-1 siRNA, or MKP-1 antisense oligonucleotide transfection, equal numbers of cells were incubated with, or without, 20 ng/ml CTGF for 2, 4 and 6 hr. At the end of the incubation period, the proportions of surviving cells were quantified by XTT uptake and reduction, as described in the materials and methods. Figure 6 a) shows that cells treated with CTGF for 6hrs were 100% viable while cells in which MKP-1 phosphatase was knocked down by siRNA silencing show a drop in viability by 25-70% within 2-6 hrs of incubation with the same concentration of CTGF. The control luc siRNA had no effect on HMC viability. Loss of viable cells due to serum-deprivation, thereby decreasing the supply of survival factors of HMC that were not treated with CTGF was about 10-20% within 2-6 hr.

The role of MKP-1 in mediating CTGF survival-promoting activity was also confirmed using the MKP-1 antisense approach where cell viability dropped by ~15-67% within 2-6hrs of CTGF stimulation (fig 6b). Differentiated HMCs adopting a myofibroblast-like phenotype have been reported to express de novo α-SMA and SMemb (the embryonic form of myosin heavy chain) genes [START_REF] Makino | Phenotypic modulation of the mesangium reflected by contractile proteins in diabetes[END_REF]18]. To assess whether CTGF potentially protects differentiated/activated mesangial cells from apoptosis whilst promoting change to a myofibroblast phenotype, we investigated the expression of SMemb in HMC treated with 20 ng/ml CTGF for 6hr. RT-PCR results (fig 7) show that SMemb expression is clearly increased with this treatment.

Discussion

It is now clear that CTGF is implicated in the pathogenesis of DN and it has been shown to mediate a wide range of disordered functions in HMC [START_REF] Abdel-Wahab | Connective Tissue Growth Factor and Regulation of the Mesangial Cell Cycle: Role in Cellular Hypertrophy[END_REF][START_REF] Wahab | Role of connective tissue growth factor in the pathogenesis of diabetic nephropathy[END_REF]10,[START_REF] Wahab | Glomerular expression of thrombospondin-1, transforming growth factor beta and connective tissue growth factor at different stages of diabetic nephropathy and their interdependent roles in mesangial response to diabetic stimuli[END_REF].

However, the molecular mechanisms by which the growth factor functions are still poorly understood. In a previous study we observed that, in HMC, CTGF rapidly activates the intra-signalling pathways ERK 1/2 MAPK and JNK MAPK, but not the p38 MAPK, despite rapidly activating MKK3/6, the upstream activators of p38 MAPK. In the present paper, we confirmed this observation. As p38 MAPK has been reported to function as a pro-death signalling effector in some cell types [START_REF] Kaiser | Targeted Inhibition of p38 Mitogen-activated Protein Kinase Antagonizes Cardiac Injury and Cell Death Following Ischemia-Reperfusion in Vivo[END_REF] we addressed the hypothesis that CTGF-dependent p38 inactivation may protect mesangial cells from death. MKP-1 phosphatase is a member of the family of inducible dual specificity phosphatases, dsPTPs [START_REF] Keyse | An emerging family of dual specificity MAP kinase phosphatases[END_REF] and is a potential deactivator for p38MAPK [13].

While serum starved HMC express only very low levels of MKP-1, stimulation with CTGF resulted in a rapid and sustained induction of MKP-1 expression. This rapid induction is consistent with data reported in other experimental systems [START_REF] Duff | Mitogen-activated Protein (MAP) Kinase Is Regulated by the MAP Kinase Phosphatase (MKP-1) in Vascular Smooth Muscle Cells[END_REF][START_REF] Xu | Retinoic acid regulation of mesangial cell apoptosis[END_REF][24]. MKP-1 has also been reported to be involved in the anti-apoptotic effect of retinoids in mesangial cells incubated with H 2 O 2 [START_REF] Guo | Resistance to TNF-alpha cytotoxicity can be achieved through different signalling pathways in rat mesangial cells[END_REF] as well as in the restriction of TNF-induced apoptosis in rat mesangial cells [START_REF] Peinado-Ramon | MAP kinase phosphatase-1 mRNA is expressed in embryonic sympathetic neurons and is upregulated after NGF stimulation[END_REF].

Interestingly, it has been reported that activation of the Trk-A receptor may be crucial for MKP-1 gene expression, independently of the cellular origin or type on which the receptor is active [27]. HMC express the Trk-A receptor and CTGF has been shown to activate it [START_REF] Wahab | Connective Tissue Growth Factor CCN2 Interacts with and Activates the Tyrosine Kinase Receptor TrkA[END_REF]. Activation of the ERK1/2 MAPK pathway and protein kinase C were reported to be involved in the induction of MKP-1 gene expression [START_REF] Guo | Resistance to TNF-alpha cytotoxicity can be achieved through different signalling pathways in rat mesangial cells[END_REF]27].

P38 MAPK is one of several kinases including Cdc2, cdK6, PKC α and JNK which can phosphorylate the anti-apoptotic protein bcl-2 [START_REF] Pathan | Microtubule-Targeting Drugs Induce Bcl-2 Phosphorylation and Association with Pin1[END_REF][START_REF] Fan | Vinblastine-induced Phosphorylation of Bcl-2 and Bcl-XL Is Mediated by JNK and Occurs in Parallel with Inactivation of the Raf-1/MEK/ERK Cascade[END_REF][START_REF] Ojala | The apoptotic v-cyclin-CDK6 complex phosphorylates inactivates Bcl-2[END_REF][START_REF] Ruvolo | A Functional Role for Mitochondrial Protein Kinase Calpha in Bcl2 Phosphorylation and Suppression of Apoptosis[END_REF] at serine and threonine residues located in the loop region between the α1 and α2 helices of the molecule [START_REF] Scacheri | Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells[END_REF][START_REF] Chang | Identification of a novel regulatory domain in Bcl-X(L) and Bcl-2[END_REF]. This phosphorylation impairs the anti-apoptotic function of the protein by reducing its ability for heterodimerization with Bax, the proapoptotic protein [START_REF] Haldar | Taxol induces bcl-2 phosphorylation and death of prostate cancer cells[END_REF]. Homodimers of Bax then form pores in the mitochondrial membrane that release cytochrome c and other pro-apoptotic factors into the cytoplasm. Accordingly, Bcl-2 phosphorylation by p38MAPK can lead to cytochrome c release from mitochondria, caspase activation and apoptotic cell death [START_REF] Torcia | Nerve Growth Factor Inhibits Apoptosis in Memory B Lymphocytes via Inactivation of p38 MAPK, Prevention of Bcl-2 Phosphorylation, and Cytochrome c Release[END_REF]. On the other hand, continuous deactivation of p38 MAPK maintains the functional and structural properties of bcl-2 protein and was found to be important for the survival-promoting activity of the growth factor NGF in the lymphoblastoid CESS B cell line [START_REF] Torcia | Nerve Growth Factor Inhibits Apoptosis in Memory B Lymphocytes via Inactivation of p38 MAPK, Prevention of Bcl-2 Phosphorylation, and Cytochrome c Release[END_REF]. Phosphorylation of bcl-2 can also render the protein susceptible to proteolytic degradation [16]. In the present study we show that CTGF-mediated rapid and continuous deactivation of p38 MAPK is accompanied by enhanced levels of bcl-2. The direct role of MKP-1 in mediating this p38 MAPK inactivation was assessed by MKP-1 protein knock-down experiments. Due to the recent growing concerns about siRNAs causing offtarget effects by acting in a microRNA-like manner or by inducing an interferon response [START_REF] Scacheri | Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells[END_REF][START_REF] Doench | siRNAs can function as miRNAs[END_REF][START_REF] Snove | Many commonly used siRNAs risk off-target activity[END_REF][START_REF] Bridge | Induction of an interferon response by RNAi vectors in mammalian cells[END_REF], and since such effects are not caused by the antisense oligonucleotides, we used the antisense-RNAi-combination techniques to cross-validate our results. Here we show that selective blockade of MKP-1 expression in HMC by both techniques resulted in the same observed phenotype in response to CTGF. This phenotype is characterized by sustained activation of p38 MAPK and a decreased expression level of bcl-2, accompanied by a reduction in cell viability.

In the present study we were not able to detect any mitogenic activity for CTGF in HMC (data not shown), consistent with our previous finding that the growth factor is a hypertrophic factor that promotes mesangial cell cycle arrest in the G 1 phase [START_REF] Abdel-Wahab | Connective Tissue Growth Factor and Regulation of the Mesangial Cell Cycle: Role in Cellular Hypertrophy[END_REF]. While CTGF has also been reported to have a non-mitogenic antiapoptotic function in chicken embryo fibroblasts (CEF) [38], it does not promote the survival of NIH/3T3 cells, [38]. Moreover it induces apoptosis in human breast cancer cells [39]. This may indicate that the survival-promoting function of CTGF may be cell-type specific, or that cells are CTGF-responsive only at a certain stage of their differentiation.

MC are usually quiescent but in response to injury, the cells undergo a process of differentiation into myofibroblast-like cells as an important step in the tissue repair program. These cells undergo spontaneous apoptosis during transient injury. In contrast, their prolonged survival during chronic injury may lead to hypercellularity and amplification of fibrosis. In the present study, we presented in vitro evidence that addition of recombinant CTGF at a concentration of 20 ng/ml (500 pM) promotes the survival of activated HMC. Since CTGF expression level is highly upregulated in the glomeruli of DN patients [START_REF] Wahab | Glomerular expression of thrombospondin-1, transforming growth factor beta and connective tissue growth factor at different stages of diabetic nephropathy and their interdependent roles in mesangial response to diabetic stimuli[END_REF], this may account for the mesangial glomerular hypercellularity seen in DN. In conclusion, we propose that CTGF orchestrates several cellular mechanisms in order to activate, and then protect the activated HMC from cell death. Figure 8 represents a model to illustrate this. Elevated expression of CTGF under diabetic conditions induces the re-expression of several embryonic genes including the SMemb myosin heavy chain isoform, and the fibronectin EDA + splice variant [10], leading to dedifferentiation of MC into myofibroblasts. At the same time, CTGF activates MKK3/6, the upstream kinases that activate p38 MAPK. Smad7, which is known to have an inhibitory effect on TGFβ signalling, also has an important role as a pro-apoptotic molecule and its overexpression in rat MC induces apoptosis [START_REF] Okado | Smad7 mediates transforming growth factor-[beta]-induced apoptosis in mesangial cells[END_REF]. Smad7 acts as a scaffolding protein, interacting with TAK1, MKK3 and p38 MAPK to facilitate p38 activation and apoptosis [START_REF] Edlund | Transforming Growth Factor[END_REF].

Figure 7 CTGF promotes differentiation of HMC

Equal numbers of HMC were incubated with or without 20 ng/ml r-CTGF for 6 hr.

At the end of the incubation period, total RNA was extracted, equal amounts were reverse-transcribed and used for semi-quantitative PCR analysis for the myofibroblast marker, SMemb, as well as for the house keeping gene GAPDH.

Results are representative of three separate experiments.

  confirms this finding. However, western blot analysis indicates that the addition of CTGF to serum-starved HMC cultures rapidly activates MKK3/6, the upstream kinases which are known to activate the P38 MAPK [12] (fig 1b). Western blot analysis also indicates that CTGF in fact induces a rapid dephosphorylation of the basal active p38 MAPK. This was maintained up to 24 hours after CTGF stimulation. Total p38 MAPK levels did not change over the time course of the experiment.

Figure 5 (

 5 Figure 5 (a) shows that in HMC treated with CTGF, inhibition of MKP-1 expression by RNAi is accompanied by enhanced phosphorylation of p38 MAPK and a clear reduction in Bcl-2 level. This suggests that p38 MAPK signalling may be coordinating bcl-2 gene expression, mRNA stability, translation efficiency or protein degradation. However, this was not investigated in the present study.

Figure legends Figure 1

 legends1 Figure legendsFigure 1 Activation of MAPK signaling pathways by CTGF in HMC A) Cells were grown on coverslips and serum-starved for 48 h prior to incubation in the absence or presence of 20 ng/ml r-CTGF for 5 or 30 min. Cells were fixed, permeabilized, probed with antiphospho -ERK1/2 , -JNK and -p38 MAKP primary antibodies, and then with fluorescein-conjugated secondary antibody, as described in the Methods. Results are representative of three separate experiments. B) Serum-starved HMC were incubated in the presence of r-CTGF for the periods of time indicated, after which the cells were lysed. Equal amounts of lysate protein were subjected to SDS-PAGE and analysed by Western blotting using phospho-specific antibodies against the p38 MAPK and the phosphokinases upstream of it, MKK3/MKK6, as well as antibody against the total p38 MAPK. Results are representative of three separate experiments

Figure 3 CTGF

 3 Figure 3 CTGF enhances the levels of Bcl-2 protein in HMC Serum-starved HMC were incubated in the presence or absence of 20 ng/ml r-CTGF for the periods of time indicated, after which the cells were lysed. A) Equal amounts of lysate protein were subjected to SDS-PAGE and analysed by Western blotting using anti Bcl-2 antibody. Membranes were stripped and reprobed with an anti β-actin antibody to control for sample loading. B) Densitometric analysis of bcl-2 expression from three separate experiments.Results are presented with mean ± SEM.

Figure 4

 4 Figure 4Efficiency of MKP-1 protein knock-down in HMC using siRNA and antisense oligonucleotide approaches Twenty-four hours after transfection with MKP-1 siRNA, or MKP-1 antisense oligonucleotides, cells were incubated with or without 20 ng/ml r-CTGF for 1hr.To control for nonspecific effects of siRNA delivery, controls were included in which cells were either mock transfected (transfection reagent only), or transfected with Luciferase siRNA, while a CG-matched randomised sequence oligonucleotide was used as a control for the specificity of the antisense delivery.At the end of the incubation time, total RNA was extracted and used for semiquantitative RT-PCR (a). In parallel experiments, cells were lysed and protein was subjected to SDS-PAGE and analysed by Western blotting using anti MKP-1 antibody (b, c). Membranes were stripped and re-probed with an anti β-actin antibody to control for sample loading. Results are representative of three separate experiments.

Figure 5

 5 Figure 5 Targeted inhibition of MKP-1 expression enhances p38MAPK activation and reduces Bcl-2 expression in HMC treated with r-CTGF Twenty-four hours after mock transfecting, or transfecting HMC with MKP-1 siRNA (a) or treating them with MKP-1 antisense and control antisense oligonucleotides (b), cells were incubated with or without 20 ng/ml r-CTGF for

Figure 6

 6 Figure 6 Modulation of MKP-1 protein levels is involved in CTGFmediated survival activity in HMC Twenty-four hours after (a) transfecting HMC with MKP-1 siRNA or Luc siRNA or (b) treating them with MKP-1 antisense or control antisense oligonucleotides, equal numbers of cells were incubated with or without 20 ng/ml r-CTGF for 2, 4 and 6 hr. At the end of incubation period, the proportion of surviving cells was quantified by XTT uptake and reduction, as described in the materials and methods. Results are means ± SEM of three experiments, each performed in triplicate. The cell number of cultures treated with CTGF did not change during the course of the experiment and is expressed as 100% cell viability at each time point.*P<0.0001 compared with cells treated with CTGF only.

Figure 8

 8 Figure 8Model showing the cellular mechanisms orchestrated by CTGF to protect activated HMC from apoptosis

Biochemical Journal Immediate Publication. Published on 10 May 2007 as manuscript BJ20061817 © 2007 Biochemical Society

Acknowledgements

We thank the Medical Research Council (UK) for the financial support.