Chris Mcdermott-Roe
email: cjmcdermottroe@msn.com

Juliette Martin

Sophie Collot-Teixeira

John L Mcgregor

Chris Mcdermott

THE CD36 AMINO-CYTOPLASMIC DOMAIN IS NOT REQUIRED FOR THE INTERNALISATION OF OXIDISED LOW DENSITY LIPOPROTEN

Keywords: OxLDL, CD36, HEK293, mutagenesis, endocytosis, atherosclerosis OxLDL, Oxidised low density lipoprotein, TSP-1, Thrombospondin-1, FITC, fluorescein isothiocyanate, DiI, 1,1

The uptake of oxidised low density lipoproteins (OxLDL) by CD36-expressing macrophages in the arterial intima and the ensuing 'foam cell' formation represents a crucial step in the initiation and development of atherosclerotic plaques. This study has addressed the function of the CD36 amino-cytoplasmic domain in the binding and internalisation of OxLDL. A selection of CD36 amino-cytoplasmic domain mutants were generated and stably expressed in HEK293 cells. The capacity of three mutants (CD36_C3/7-A, CD36_D4/R5-A and CD36_nCPD-) to bind and endocytose OxLDL was then studied using immunofluorescence microscopy and quantitative fluoremetry. Each of the CD36 constructs was expressed at differing levels at the cell surface as measured by flow cytometry and western blotting. Following incubation with DiI-OxLDL, cells bearing the CD36_wild-type, CD36_C3/7-A, CD36_D4/R5-A and CD36_nCPD -construct all internalized DiI-OxLDL into endosomal structures whereas empty vector-transfected cells failed to do so indicating that unlike the carboxy-cytoplasmic domain, the amino-cytoplasmic domain is not essential for the endocytosis of OxLDL. To conclude, the uptake of OxLDL by CD36 is not reliant on the presence of the CD36 amino-cytoplasmic domain. However, the amino-cytoplasmic domain may conceivably be implicated in the maturation of CD36.

INTRODUCTION

The uptake of oxidatively-modified low density lipoproteins (OxLDL) by macrophages in the arterial intima and the ensuing 'foam cell' formation represents a crucial step in the initiation and development of atherosclerotic plaques. Uptake of OxLDL is mediated by certain members of the scavenger receptor family, a group of widely expressed, multi-functional membraneembedded glycoproteins. A major receptor for OxLDL is CD36 [START_REF] Endemann | CD36 is a receptor for oxidized low density lipoprotein[END_REF] and evidence implicates it as a key-player in foam cell formation and atherosclerotic lesion development. For example, macrophages obtained from CD36-deficient individuals bind and endocytose OxLDL far less effectively than those from normal subjects [START_REF] Nozaki | Reduced uptake of oxidised low density lipoproteins in monocyte-derived macrophages from CD36-deficient subjects[END_REF] and CD36-deficient macrophages are impaired in their capacity to secrete pro-inflammatory cytokines following OxLDL treatment [START_REF] Janabi | Oxidized LDL-induced NF-kappa B activation and subsequent expression of proinflammatory genes are defective in monocyte-derived macrophages from CD36-deficient patients[END_REF], a hallmark event of atherogenesis. Furthermore, CD36-ApoE double-null mice develop markedly smaller atherosclerotic plaques than ApoE -/-mice [START_REF] Febbraio | Targeted disruption of the class B scavenger receptor CD36 protects against atherosclerotic lesion development in mice[END_REF].

Based on findings by the likes of Gruarin et al. [START_REF] Gruarin | Formation of one or more intrachain disulphide bonds is required for the intracellular processing and transport of CD36[END_REF] and Tao et al. [START_REF] Tao | CD36 is palmitoylated on both N-and C-terminal cytoplasmic tails[END_REF], CD36 is predicted to adopt a ditopic configuration meaning it comprises an extracellular domain (residues 29-438) flanked by two transmembrane domains (residues 7-28 and 439-460) and two cytoplasmic domains (residues 1-6 and 461-472). A 28 residue segment in the extracellular domain (155-183) has been found to mediate binding with OxLDL [START_REF] Navazo | Identification on human CD36 of a domain (155-183) implicated in binding oxidized low-density lipoproteins (Ox-LDL)[END_REF]. Interestingly, despite the compartmental separation between the extracellular domain and the carboxy-cytoplasmic domain, the latter has been found to be essential for the binding and uptake of OxLDL as well as signal transduction processes. Our group previously demonstrated that HEK293 cells expressing a construct lacking the last 6 residues (467-472) of the carboxy-cytoplasmic domain displayed an approximate 80 % decrease in their capacity to bind and internalise OxLDL. The carboxy-cytoplasmic domain also possesses signal transduction properties; Lipsky et al. reported a significant decrease in OxLDLmediated NF-kB activation in CHO cells expressing a CD36 construct lacking the carboxycytoplasmic domain compared to cells bearing the wild-type protein [START_REF] Lipsky | The carboxyl-terminal cytoplasmic domain of CD36 is required for oxidized low-density lipoprotein modulation of NF-kappaB activity by tumor necrosis factor-alpha[END_REF]. Similarly, the antiangiogenic effect of TSP-1 observed in CD36-transfected endothelial cells is abrogated if certain residues in the carboxy-cytoplasmic domain (Cysteine 464, Arginine 467, and Lysine 469) are mutated [START_REF] Primo | Identification of CD36 molecular features required for its in vitro angiostatic activity[END_REF].

To our knowledge, the CD36 amino-cytoplasmic domain has not been investigated with regards to its involvement in ligand binding and uptake. The oppositely charged aspartatearginine sequence at positions 4 and 5, respectively may provide a means for CD36 to interact with co-receptors and / or facilitate capping and endocytosis of CD36-ligand complexes.

Similarly, the palmitoyl group shown to be appended to each of the cysteine residues at positions 3 and 7 [START_REF] Gruarin | Formation of one or more intrachain disulphide bonds is required for the intracellular processing and transport of CD36[END_REF] may mediate an interaction with membrane or cytoplasmic proteins that is necessary for CD36 binding and endocytosis. Hence, to investigate the role of the amino-cytoplasmic domain in the binding and internalisation of OxLDL, a selection of CD36 amino-cytoplasmic domain mutants were generated and stably expressed in HEK293 cells. The capacity of these mutants to bind and endocytose OxLDL was studied using immunofluorescence microscopy and quantitative fluoremetry.

MATERIALS AND METHODS

Cell culture

Human epithelial kidney 293 (HEK293) cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) fetal calf serum, 2 mM glutamine, 10 µg/ml streptomycin and 10 units/ml penicillin in a 5 % CO 2 , 95 % air atmosphere at 37°C.

Plasmid construction

The CD36 coding sequence was PCR-amplified from an M13mp19 cloning vector containing the CD36 coding sequence using the wild-type forward and reverse primers shown in Table 1 and sub-cloned into the pcDNA3.1(+) expression vector. Three amino-cytoplasmic domain mutant constructs (Figure 1) were generated by PCR-mutation of the wild-type CD36 sequence using the primer sequences shown in Table 1. As with the wild-type construct, all mutated constructs were sub-cloned into the pcDNA3.1(+) expression vector and validated by DNA sequencing.

Stable expression of CD36 constructs in HEK293 cells

Transfection of plasmid DNA into HEK293 cells was carried out using Polyfect reagent (Qiagen) according to the manufacturer's instructions. 48 hours post-transfection, growth medium was removed and replaced with complete medium containing geniticin (500 µg/ml) in order to select for stably-transfected cells.

SDS-PAGE, Western blotting and immunodetection

Protein lysates were prepared in accordance to a Triton X-100 membrane protein-enrichment method. Protein concentration was determined using the bicinchoninic acid assay. SDS-PAGE, Western blotting and immunodetection of CD36 were carried out essentially as described by Malaud et al. [START_REF] Malaud | The terminal six amino-acids of the carboxy cytoplasmic tail of CD36 contain a functional domain implicated in the binding and capture of oxidised low-density lipoprotein[END_REF].

Flow cytometry

Cells (1x10 6 ) were incubated in PBS containing the mouse anti-human CD36 monoclonal antibody 10.5 (5 µg/ml) (Biocytex) for 1 hour at 4°C with vertical rotation. After a single wash with PBS, cells were incubated in PBS containing a mouse anti-human FITC-conjugated antibody (80 µg/ml) for a further hour at 4°C with vertical rotation. Finally, cells were washed to remove residual antibody, re-suspended in 1 % paraformaldehyde and analysed on a Becton Coulter FC 500 flow cytometer with Cytomics RXP Analysis software.

Visualisation of DiI-OxLDL uptake HEK293 cells expressing the wild-type CD36 construct as well as the various mutated CD36 constructs were seeded in 6-well plates (400,000 cells/well) and grown on cover-slips for 24 hours before being incubated with 50 µg/ml DiI-OxLDL (in complete media) for 16 hours. Cells were rinsed with PBS, fixed with 4 % paraformaldehyde, mounted on to microscope slides using Vectashield (Vector Laboratories) and viewed at the specified magnification using a fluorescence microscope. Quantification of DiI-OxLDL uptake HEK293 cells stably-transfected with plasmids detailed above were seeded in 6-well plates (400,000 cells/well) and cultured for 24 hours before being incubated with 0, 25 and 50 µg/ml DiI-OxLDL (in complete media) for 16 hours at 37°C. Uptake of DiI-OxLDL was quantified according to the method of Luan and Griffiths [START_REF] Luan | Ceramides reduce CD36 cell surface expression and oxidised LDL uptake by monocytes and macrophages[END_REF]. Cells were harvested with trypsin/EDTA, washed twice with PBS and lysed into 5 % Triton X-100. DiI-OxLDL fluorescence was measured at 590 nm following excitation at 520 nm using a Molecular Devices SpectraMax Microplate Reader. Fluorescence values were converted to µg DiI-OxLDL via a standard curve produced by measuring the fluorescence at 590 nm of lysis buffer containing 0.1, 0.2, 0.5, 1.0 and 1.5 µg DiI-OxLDL. The protein concentration of each sample was determined via the BCA assay. Finally, the quantity of DiI-OxLDL taken up by cells was calculated as amount of OxLDL per mg of cellular protein.

RESULTS

Expression of CD36 constructs in HEK293 cells

HEK293 cells were stably-transfected with a plasmid encoding the CD36 wild-type sequence as well as plasmids encoding the desired amino-cytoplasmic domain mutant constructs. All constructs were expressed, although at differing levels, as found by Western blotting (Figure 2). Interestingly, the anti-CD36 antibody 10.5 recognised two distinct bands in proteins derived from cells expressing the CD36_nCPD -construct. These may represent differentially-glycosylated forms of CD36 and therefore would suggest an involvement of the amino cytoplasmic domain region in the maturation process. Each of the four CD36 constructs was expressed at differing levels at the cell surface as measured by flow cytometry (Figure 3a). The C3/7-A construct was marginally more abundant than the wild-type construct whilst the D4/R5-A and the nCPD - constructs were approximately twice and three times more abundant, respectively than the wildtype construct (Figure 3b).

Visualisation of DiI-OxLDL uptake by HEK293 cells bearing CD36 mutants

To determine whether the CD36 amino-cytoplasmic domain is required for binding and internalization of OxLDL, the passage of fluorescently-labelled (DiI)-OxLDL was tracked in HEK293 cells stably expressing each of the described CD36 constructs using immunofluorescence microscopy. Following incubation with 10 µg/ml DiI-OxLDL for 16 hours at 37°C, cells bearing the CD36_wild-type, CD36_C3/7-A, CD36_D4/R5-A and CD36_nCPD - construct all internalized DiI-OxLDL into endosomal structures whereas ev-transfected cells failed to do so (Figure 4) indicating that unlike the carboxy-cytoplasmic domain, the aminocytoplasmic domain is not essential for the endocytosis of OxLDL.

Quantification of DiI-OxLDL uptake by HEK293 cells bearing CD36 mutants

Despite the apparent expendability of the amino-cytoplasmic domain for OxLDL uptake, it was considered that different constructs may still display variation with respect to their binding and internalization efficacy. In order to compare the relative effectiveness of each construct to bind and internalize OxLDL, cells stably-expressing each construct were incubated with DiI-OxLDL as described in the Materials and Methods section. Each of the cell-types accumulated DiI-OxLDL in a dose-dependent manner, again indicating that the amino-cytoplasmic domain is not essential for OxLDL binding and uptake. HEK293-CD36_wt cells accumulated approximately twice as much DiI-OxLDL as ev-transfected control cells but an equal amount to HEK293-CD36_C3/7-A cells whilst HEK293-CD36_D4/R5-A and HEK293_nCPD -cells accumulated approximately three and four times more DiI-OxLDL, respectively than ev-transfected control cells (Figure 5a). This variation was thought to be resultant of the different levels of expression of each construct. Indeed, when data was normalized to compensate for the differential

DISCUSSION

Class B scavenger receptors such as CD36 are composed of an extracellular domain, two transmembrane domains and two cytoplasmic domains. The carboxy-cytoplasmic domain of CD36 has previously been shown to be crucial in the binding and internalization of OxLDL [START_REF] Malaud | The terminal six amino-acids of the carboxy cytoplasmic tail of CD36 contain a functional domain implicated in the binding and capture of oxidised low-density lipoprotein[END_REF] and the Gram positive bacterium Staphylococcos aureus [START_REF] Stuart | Response to Staphylococcus aureus requires CD36-mediated phagocytosis triggered by the COOHterminal cytoplasmic domain[END_REF], and is also required for TSP-1-induced anti-angiogenic signalling [START_REF] Primo | Identification of CD36 molecular features required for its in vitro angiostatic activity[END_REF] as well as NF-kB trans-activation [START_REF] Lipsky | The carboxyl-terminal cytoplasmic domain of CD36 is required for oxidized low-density lipoprotein modulation of NF-kappaB activity by tumor necrosis factor-alpha[END_REF]. To the best of our knowledge, the role of the CD36 amino-cytoplasmic domain in such events has yet to be addressed.

The amino-cytoplasmic domain of CD36 comprises the following residues: H 2 N-Met-Gly-Cys-Asp-Arg-Asn-Cys (where H 2 N refers to the amino-most residue). Two regions of this sequence were felt to be of interest. Firstly, the cysteine residues (at positions 3 and 7) are known to carry palmitate groups. Such moieties have previously been demonstrated to mediate proteinprotein and protein-lipid interactions. Secondly, the oppositely charged aspartate (-ve)-arginine (+ve) sequence (at positions 3 and 4, respectively) may also permit interactions with other receptor proteins and/or cytosolic proteins. To investigate the role played by the aminocytoplasmic domain (with specific emphasis on the abovementioned regions) in the expression of CD36 and the internalisation of OxLDL, three different mutants were generated by standard PCR-mutagenesis techniques. In the first mutant (C3/7-A), the two cysteine residues were converted to alanine; in the second mutant (D4/R5-A), the aspartate and arginine residues were converted to alanine; in the third mutant (nCPD -), the entire amino-cytoplasmic domain was removed. The three CD36 mutants as well as the wild-type construct were then stably-transfected in HEK293 cells. All constructs (including the wild-type) were synthesized and expressed at the cell surface as determined by flow cytometry, albeit at differing levels; the wild-type and the C3/7-A construct were expressed at the cell membrane at a similar level whereas the D4/R5-A construct and the nCPD -constructs were expressed approximately 2-and 3-fold higher, respectively than the wild-type construct. The fact that the amino-cytoplasmic domain constructs were expressed more highly than the wild-type protein at the cell surface would suggest that this region may suppress the migration of the CD36 protein from the cytoplasm to the plasma membrane, perhaps in order to lengthen its exposure to the various post-translational modification enzymes needed for tertiary structure acquisition and attachment of extra groups such as sugar moieties. Indeed, this may explain why the nCPD -construct seemed to comprise two species when analysed by western blotting; instead of a single band being present at approximately 85 kDa, two bands were present -one at approximately 85 kDa and one at approximately 80-82 kDa. The smaller band may represent an incompletely glycosylated species and would may indicate that part of or the entire amino-cytoplasmic domain is required for glycosylation of CD36. Assessment of the effect of removing glycosylation sites in CD36 on its membrane trafficking would be advantageous. It should be noted that in our hands, PNGase F-mediated deglycosylation of wild-type CD36 eliminated recognition by the anti-CD36 monoclonal antibody 10.5 (results not shown) suggesting that the epitope recognised by 10.5 comprises sugar groups as well as protein. Indeed, the region of CD36 thought to become bound by 10.5 holds one N-glycan group.

Immuofluorescence microscopy was next used to track the movement of fluorescentlylabelled (DiI)-OxLDL in the transfected HEK293 cells expressing the various CD36 mutants. DiI-OxLDL was consistently observed to accumulate in cells expressing the wild-type CD36 construct as well as the three mutated forms but not in ev-transfected (control) cells following an overnight incubation indicating that the amino-cytoplasmic domain is not essential for OxLDL uptake. A fluorescence-based assay adapted from Luan and Griffiths [START_REF] Luan | Ceramides reduce CD36 cell surface expression and oxidised LDL uptake by monocytes and macrophages[END_REF] was utilized to quantify the uptake of DiI-OxLDL by cells expressing each of the CD36 constructs. Consistent with the immunofluorescence work, cells bearing the different mutants were capable of binding and endocytosing OxLDL. In such analyses, there was a notable difference in the quantity of DiI-OxLDL taken up by each cell type. Cells expressing the wild-type and C3/7-A constructs both internalised approximately two-fold more DiI-OxLDL than ev-transfected cells, whilst cells expressing the D4/R5-A and nCPD -constructs internalised approximately four-fold and five-fold more DiI-OxLDL, respectively than the ev-transfected cells. These differences were felt to be due at least partially to the fact that the different constructs were expressed at different levels at the cell surface of transfectants. As a means of compensating for the disparity in construct expression, all DiI-OxLDL uptake values were normalised against the amount of CD36 expressed by each cell line. After doing this, there was a negligible difference in the efficacy of each celltype to take up DiI-OxLDL, again suggesting that the CD36 amino-cytoplasmic domain is not involved in the endocytotic mechanism. To conclude, the uptake of OxLDL by CD36 is not reliant on the presence of the CD36 amino cytoplasmic domain. However, this region may somehow be implicated in the maturation of CD36. Specifically, it could be involved in retarding the movement of the CD36 polypeptide -following translation -from the cytoplasm to the plasma membrane in order for it to attain its final, native state structure and receive additional moieties such as sugar chains and palmitoyl groups.

CD36 (1-472)

Figure 1: Sequence of generated CD36 amino-cytoplasmic domain mutants. All constructs were generated using primers specified in Table 1 by PCR-mutation and directionally-cloned into the pcDNA3.1(+) expression vector between the Kpn I and Xba I restriction sites.

Table 1: Primers used for generation of CD36 wild-type and amino-cytoplasmic domain mutant constructs.

The italic region in the forward and reverse sequences denote Kpn I and Xba I restriction sites, respectively.

Construct

Bioscience

  Reports Immediate Publication. Published on 18 Jun 2008 as manuscript BSR20080045 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080045 S t a g e 2 ( a ) P O S T -P R I N T

Bioscience

  Reports Immediate Publication. Published on 18 Jun 2008 as manuscript BSR20080045 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080045 S t a g e 2 ( a ) P O S T -P R I N T expression of each construct, the efficacy of each cell-type to accumulate DiI-OxLDL is approximately equivalent (Figure 5b).

Bioscience

  Reports Immediate Publication. Published on 18 Jun 2008 as manuscript BSR20080045 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080045 S t a g e 2 ( a ) P O S T -P R I N T

Forward ( 5 Figure 2 :

 52 Figure 2: Detection of CD36 constructs in stably-transfected HEK293 cells by Western blotting.Triton X-100-soluble proteins obtained from stable transfectants were separated by SDSPAGE and transferred to a nitrocellulose membrane. The membrane was then blocked and incubated simultaneously with an anti-CD36 monoclonal antibody (10.5) and an anti-α tubulin monoclonal antibody followed by an HRPconjugated goat anti-mouse secondary antibody. HEK293-CD36_wt (A), HEK293-ev (B), HE293-CD36_C3/7-A (C), HEK293-CD36_D4/R5-A (D), and HEK293-CD36_nCPD -(E).

Figure 3 :Figure 4 :

 34 Figure 3: Analysis of CD36 construct expression in stably-transfected HEK293 cells by flow cytometry. (a) Histograms displaying qualitative expression of each construct (grey area) relative to ev-transfected cells (white over-lay). (b) Bar chart showing fluorescence (taken as an indicator of abundance) of each CD36 construct. The contribution of ev-transfected cells has been subtracted from each construct. Presented data are means + S.D. of 3 different experiments.

Figure 5 :

 5 Figure 5: Accumulation of DiI-OxLDL in HEK293 cells bearing wild-type and mutant CD36 constructs. (a) HEK293 cells stably expressing the specified CD36 constructs were incubated with 0, 25 and 50 µg/ml DiI-OxLDL for 16 hours. Cells were then lysed into 5 % Triton X-100 and DiI-OxLDL fluorescence was measured at 590 nm following excitation at 520 nm using a Molecular Devices SpectraMax Microplate Reader. Presented data are means + S.D. of three experiments. (b) Data from (a) after correction for CD36 abundance. Note that error bars are omitted from (b) to aid visualisation of individual data points.

  concentration (µg/ml) oxLDL uptake (µg) / mg cell lysate protein HEK293-ev HEK293-CD36_nCPD-Bioscience Reports Immediate Publication. Published on 18 Jun 2008 as manuscript BSR20080045 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080045 S t a g e 2 ( a ) P O S T -P R I N T

-472) Met Gly Ala Asp Arg Asn Ala Met Gly Cys Ala Ala Asn Cys

	FIGURES
	mutants mutants	nCPD -C3/7-A D4/R5-A Wild-type nCPD TMD nCPD -C3/7-A D4/R5-A Wild-type nCPD TMD	Met Gly Ala Asp Arg Asn Ala Met Gly Cys Ala Ala Asn Cys -------Met Gly Cys Asp Arg Asn Cys TMD ECD ------Met Gly Cys Asp Arg Asn Cys CD36 (1-TMD ECD	cCPD cCPD

Bioscience Reports Immediate Publication. Published on 18 Jun 2008 as manuscript BSR20080045 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080045 S t a g e 2 ( a ) P O S T -P R I N T

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

ACKNOWLEDGEMENTS

CMcDR, JM and SCT were supported at the Thrombosis Research Institute by a Garfield Weston Foundation Grant.


Giridhar Mudduluru

Prof. Dr. med Heike Allgayer
email: heike.allgayer@chir.ma.uni-heidelberg.de

The human receptor tyrosine kinase Axl gene -promoter characterization and regulation of constitutive expression by Sp1, Sp3, and CpG methylation

Keywords: Axl receptor tyrosine kinase, Axl promoter, Methylation, 5-aza-dC, Sp1, Sp3

Axl-receptor-tyrosine-kinase promotes antiapoptosis, mitogenesis, invasion, angiogenesis, and metastasis, and is highly expressed in cancers. However, the transcriptional regulation of this important gene has never been characterized. The present study was initiated to characterize the promoter, first cis-acting elements, and promoter methylation driving expression of Axl. The 2.4kb sequence upstream of the translational start site, and sequential 5'-deletions were cloned and revealed a minimal GC-rich region (-556/+7) to be sufficient for basal Axl-promoter activity in Rko, HCT116, and HeLa cells. Within this minimal region, five Sp-binding sites were identified. Two sites (Sp-a, Spb) most proximal to the translation start site were indispensable for Axl-promoter activity, whereas mutation of three additional upstream (Sp-c, Sp-d, Sp-e) motifs was of additional relevance. Gelshift and chromatin-immunoprecipitation identified especially Sp1 and Sp3 bound to all 5 motifs, mutations of all motifs abolishing binding. Mithramycin, inhibiting binding of Sp-factors to GC-rich sites, dramatically reduced Axl-promoter activity, Axl-, Sp1-, and Sp3-expression. In Drosophila Schneider SL2-cells, exogenous expression of Sp1/Sp3 increased Axl-promoter activity. Sp1/Sp3-siRNAs significantly reduced Axl-promoter activity and protein in Rko and HeLa cells. Methylation-bisulfite sequencing detected methylated CpG-sites within three Sp-motifs (Sp-a, -b, -c) and GC-rich flanking sequences, demethylation by 5-aza-2'-deoxycytidine upregulating Axl-and Sp3-expression in low Axl-expressing Colo206f/WiDr, but not high Axl-expressing Rko-cells. These data suggest that Axl gene expression in cancer-cells 1) is constitutively driven by Sp1/Sp3 bound to 5 core-promoter motifs, 2) is restricted by methylation within/around Sp-binding sites. This might enhance understanding and treatment of essential mechanisms associated with cancer and other diseases.

Introduction

Axl (from the Greek word 'anexelekto,' or uncontrolled) belongs to the Tyro 3 family of receptor tyrosine kinases (RTKs) which includes Mer and Sky, and was originally identified as a transforming gene in human leukemias [START_REF] O'bryan | Axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase[END_REF]. These RTKs are characterized by the presence of two immunoglobulinlike domains and two fibronectin-type III domains in the extracellular region (a unique extra-cellular structure, resembling neural cell adhesion molecules), and a distinctive intracellular kinase domain [START_REF] O'bryan | Axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase[END_REF]. Human Axl is a 140 kDa protein with roughly equal distribution of amino acids on either side of the plasma membrane [START_REF] O'bryan | Axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase[END_REF][START_REF] Lai | Structure, expression, and activity of Tyro 3, a neural adhesion-related receptor tyrosine kinase[END_REF]. The main ligand for Axl RTK family members is Gas6, the product of growth-arrest-specific gene 6 [START_REF] Nagata | Identification of the product of growth arrest-specific gene 6 as a common ligand for Axl, Sky, and Mer receptor tyrosine kinases[END_REF], a soluble member of the vitamin K-dependent protein family [START_REF] Manfioletti | The protein encoded by a growth arrest-specific gene (gas6) is a new member of the vitamin K-dependent proteins related to protein S, a negative coregulator in the blood coagulation cascade[END_REF], which binds to Tyro3 family members with different affinities. Gas6 was first discovered as an upregulated gene in growth arrested cells [START_REF] Schneider | Genes specifically expressed at growth arrest of mammalian cells[END_REF], suggesting its role in preventing cellular stresses, such as apoptosis. Subsequent studies have suggested its role in cell survival [START_REF] Melaragno | Gas6 inhibits apoptosis in vascular smooth muscle: role of Axl kinase and Akt[END_REF][START_REF] Van Ginkel | Expression of the receptor tyrosine kinase Axl promotes ocular melanoma cell survival[END_REF], proliferation [START_REF] Stenhoff | Vitamin K-dependent Gas6 activates ERK kinase and stimulates growth of cardiac fibroblasts[END_REF][START_REF] Sainaghi | Gas6 induces proliferation in prostate carcinoma cell lines expressing the Axl receptor[END_REF], stimulation of cell migration [10], and cell-cell adhesion via Axl [START_REF] Mccloskey | GAS6 mediates adhesion of cells expressing the receptor tyrosine kinase Axl[END_REF]. Intracellular signalling of Axl is also activated by homophilic and heterophilic interactions [START_REF] Budagian | A promiscuous liaison between IL-15 receptor and Axl receptor tyrosine kinase in cell death control[END_REF], mediated mainly by a multi-substrate docking site [START_REF] Braunger | ntracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site[END_REF]. Over-expression of Axl can transform fibroblasts even in the absence of a ligand [START_REF] Burchert | Determinants for transformation induced by the Axl receptor tyrosine kinase[END_REF]. Different signalling molecules are activated by Axl in different cell types, among them being phosphatidylinositol 3-kinase (PI3K), Akt, Src, extracellular signal-regulated kinase (ERK), and nuclear factor kappaB (NF-kB) [START_REF] Goruppi | Requirement of phosphatidylinositol 3-kinase-dependent pathway and Src for Gas6-Axl mitogenic and survival activities in NIH 3T3 fibroblasts[END_REF][START_REF] Lee | Akt is required for Axl-Gas6 signaling to protect cells from E1A-mediated apoptosis[END_REF]. Gas6/Axl signaling is well studied in NIH3T3 mouse fibroblasts, where it is known to transduce antiapoptotic signals via NF-kB activation, and increased protein levels of Bcl-xl [START_REF] Demarchi | Gas6 antiapoptotic signaling requires NF-kappa B activation[END_REF]. An increased expression of Axl is associated with invasion, metastasis, and angiogenesis in various cancer cells, and is found in metastatic colon and prostate carcinoma, gastric, endometrial, certain types of breast cancers, and sarcomas [START_REF] Hafizi | Gas6 and protein S. Vitamin K-dependent ligands for the Axl receptor tyrosine kinase subfamily[END_REF].

The Axl gene is located at human chromosome 19, encoded by 20 exons, and distributed over a region of 44 kb. Different isoforms of Axl mRNA are generated by alternative splicing of exon 10 [START_REF] Schulz | The genomic structure of the human UFO receptor[END_REF]. Until now, no functional differences were found for these isoforms. In one initial study, the Axl 5' upstream region has been shown to be GC-rich, lack TATA and CAAT boxes, and harbour multiple transcriptional start sites. These transcription start sites include the major start site at -169, and a second major start site at -460 bp upstream of the translation start site [START_REF] Schulz | The genomic structure of the human UFO receptor[END_REF]. However, although Axl has been shown to influence different major cell physiological properties, and to be associated with diverse cancers and cancer-specific properties, surprisingly little is known about its transcriptional regulation, and no further studies on the characterisation of its promoter have been published so far. Therefore, in this first and comprehensive study, we characterize the Axl promoter and its methylation status in different colorectal cell lines. We identify binding sites for various transcription factors within the -556 to -182 bp core region upstream to the Axl translational start codon, among them five binding sites for Sp1/Sp3, one for MZF1, and one for AP1. Furthermore, we show that the Sp binding sites, or adjacent CpG sites, are partially methylated in low Axl-expressing in contrast to high Axlexpressing cells. The results presented here are the first to show that Sp1 and Sp3 transcription factors have an essential role in the basal expression of the Axl gene, and that the methylation status of CpG sites in, or in close proximity to, Sp binding motifs modulates Axl gene expression in cultured colon cancer.

For luciferase assays, 0.13 × 10 6 Rko, 0.05 × 10 6 HeLa or 0.2 × 10 6 HCT116 cells were plated in 24 well plates. Luciferase reporter plasmids (1 μg of each) were transfected in optimum medium using Lipofectamine 2000 (Invitrogen). For all luciferase reporter assays, pRL-TK (50 ng, Renilla Luciferase; Promega) was also co-transfected and measured to normalize transfection efficiency. After 6 hours of transfection, optimum medium was replaced with regular medium, and the cells were incubated at 37 o C for 48 h. Cells were washed twice with PBS and lysed with 100 μl passive lysis buffer (Promega) for 20 min. Luciferase activity of 20 μl of cell lysate was measured via the Dual-Luciferase reporter assay system (Promega) according to the manufacturer's instructions. Assays for all samples were performed in triplicate, and the results were averaged.

Luciferase Assays in Drosophila melanogaster SL2 Insect Cells

For luciferase assays, at the time of transfection 0.5x10 6 cells were plated in a 24 well plate. 250 ng of AxlP3 were co-transfected with 150 ng of either Sp1 or Sp3 along with Renilla (Internal control for transfection efficiency) with effectin transfection reagent (Quiagen). After 48 h of transfection, cells were collected in Eppendorf tubes and lysed with 100 μl of passive lysis buffer (Promega). Luciferase activity of 50 μl of cell lysate was measured via the Dual-Luciferase reporter assay system (Promega) according to the manufacturer's instructions. Assays for all samples were performed in triplicate, and the results were averaged.

Chromatin Immunoprecipitation Assay

Chromatin immunoprecipitation (ChIP) assays were performed via a commercially purchased chromatin immunoprecipitation kit (Upstate Biotechnology, Lake Placid, NY), using either anti-Sp1 (sc-59 X), anti-Sp3 (sc-644 X) or anti-IgG (as control, sc-2338 X) antibodies. Rko cells were first cross-linked for 10 min by adding formaldehyde directly to tissue culture medium to a final concentration of 1% at room temperature, and then neutralized with 125mM Glycine for 10 min. Cells were then washed twice with cold PBS (with protease inhibitors), scraped, pelleted, resuspended in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1), and incubated for 10 min on ice. The lysates were then sonicated 10 times, each sonication for 15 sec at 30% amplitude, resting on ice for 1 min between sonication. After sonication, the samples were centrifuged, the supernatants diluted 10-fold in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl, and 16.7 mM Tris-HCl (pH 8.1)) with protease inhibitors, and precleared with 80 μl salmon sperm DNA/protein A Agarose-50% for 30 min at 4°C. Cross-linked chromatin was incubated overnight with 3 μg Sp1, 3 μg Sp3, or control IgG in a total volume of 1 ml at 4°C. Antibody-protein-DNA complexes were isolated by immunoprecipitation with 60 μl salmon sperm DNA/protein A. After extensive washing, pellets were eluted by freshly prepared elution buffer (1% SDS, 0.1 M NaHCO 3 ). Formaldehyde cross-linking was reversed by 6h incubation at 65°C. Samples were purified through PCR purification kit columns (Qiagen) and used as a template in PCR. ChIP primers: Region1: sense 5′-CCTGGCCCCTTTAAGAAAG-3′; antisense 5′-CTGCCTCCTTCCCTCACT-3′ and Region2: sense 5'-GTGTGTGTGTGTCCTTGTCC-3'; antisense 5' GGGCTCTGTGTCTGGTAAAC 3' were used to amplify a 159 bp and 168 bp fragment corresponding to the core Axl promoter, respectively. As negative control, a region in the urokinase receptor (uPAR) promoter region -937/-745 was amplified with the following set of primers, sense 5´ GCAGACATCAATAACATTCACC-3´; antisense 5´-GCCTGGCAACAGAACAAGAC-3´). Amplification of soluble chromatin prior to immunoprecipitation was used as an input control.

Knockdown of Sp1 or Sp3 via RNA Interference

Short interfering RNAs (siRNAs), designed to target human Sp1 and Sp3 mRNAs, were from Ambion. Briefly, 0.13X10 6 of Rko or 0.05X10 6 of HeLa cells were plated in either a 24 well plate for Luciferase assay, or 0.75X10 6 and 0.3X10 6 cells in 6 well plates for Western blot analysis the day before transfection. AxlP3 reporter plasmids were co-transfected with 25 or 50 nM of Sp1/Sp3 or 50 nM of scrambled RNA (scRNA), and luciferase assays were performed 48h after transfection. Rko or HeLa cells were transfected with either 50 nM, 100 nM of Sp1, Sp3, or combinations of Sp1 and Sp3 with different concentrations (50 nM or 75 nM Sp1 and Sp3) with Lipofectamin (Invitrogen). After 48 h, cells were washed with PBS and harvested for protein lysate. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

5-aza-2'-deoxycytidine (5-aza-dC) treatment

Cells were plated in a 6 well plate on Day 0 in respective medium supplemented with 10% FCS, and allowed to grow stably for 24 hours. Cells were treated either with a final concentration of 10, 20 or 40µM of 5-aza-dC for 5 days, changing the drug for every 24 hours, or with 10% FCS medium as a control. On the fifth day, cells were washed with PBS and grow without drug for 24 hours. Finally, cells were washed with ice cold PBS and total RNA isolated using the Quigen Mini R-Neasy Kit.

Sodium bisulfite conversion of DNA 1µg of WiDr or Rko genomic DNA was used for bisulfite conversion, using the EpiTect Bisulfite Kit (Qiagen) according to the manufacturer's instructions. Bisulfite treated DNA was amplified with two sets of methylated specific primers: Axl 5'-flanking region-specific primers Set one (amplified product -376bp to -98bp) 5'-GGGTATAGTTATTAGGGTTTTTTTT-3' (sense) and 5'-TTTAACACTACCTACCTAACACAAC-3' (antisense), and Set two (amplified product -669bp to -357bp) 5'-TGTTTTAGTTTGTGTGTGTTAGTGA-3' (sense) and 5'-AAACCCTAATAACTATACCCCCTATC -3' (antisense). The PCR reaction was carried out at 94°C for 2 minutes, 30 cycles at 94 °C for 30 seconds, annealing at 62 °C for 30 seconds, extension at 72 °C for 45 seconds, and final extension at 72 °C for 5 minutes. Purified PCR products were used for pGMT -easy cloning. 10 clones were sent for sequencing from each cell line.

Statistical analysis

Statistical analyses were performed using the SPSS statistical software (statistical package for social sciences, SPSS, Inc., Chicago, IL). Differences between groups were calculated with Student's t-test, and P-values of ≤0.05 considered statistically significant.

Results

Cloning and putative regulatory elements of the Axl 5'-flanking region

To define the Axl promoter and identify potential cis-elements involved in the transcriptional regulation of Axl, we isolated the 5' flanking region of 2376 bp upstream of the translation start site (TSS). The sequence does not contain typical TATA and CAAT boxes or an initiator sequence (Supplementary Fig. a). As published by others, the promoter has different transcriptional start sites including two major sites located at -169 bp and -460 bp upstream of the TSS, respectively [START_REF] Schulz | The genomic structure of the human UFO receptor[END_REF]. A TRANSFAC database (GenBank accession no: BC032229) search revealed several putative binding sites for transcription factors including MZF1, AP1, and multiple Sp binding sites. Based on potential cis-elements and major transcriptional start sites, we generated a series of 5' promoter deletion constructs as outlined in Fig 1a . Rko, HCT116 and HeLa cell lines, characterised by high basal mRNA amounts of Axl (Supplementary Fig. b), were used for this study. In the following, we will concentrate on characterizing the minimally required core promoter for constitutive expression of the Axl gene.

Functional delineation of the basal Axl promoter region

To determine the minimal Axl promoter region and narrow down cis-elements regulating basal Axl gene expression, we transfected a series of deletion luciferase constructs (Fig. 1a) into Rko, HCT116, and HeLa cells. The AxlP3 construct (-1276/+7) exhibited a maximum percent luciferase activity as compared to the promoterless luciferase vector pGL3 Basic (Fig. 1b). Further deletion constructs, AxlP4 (-1010/+7), AxlP5 (-727/+7), AxlP6 (-614/+7), AxlP7 (-556 /+7), and AxlP8 (-478 /+7), showed a gradual decrease in luciferase activity in Rko, HCT116, and HeLa cells, the activity of the last two constructs being comparable (Fig 1b). AxlP1 (-2376 /+7) and AxlP2 (-1759/ +7) contain more of the 5' flanking promoter region than AxlP3, however, these constructs do not show more promoter activity than AxlP3, indicating that the maximum promoter activity lies within -1272 /+7 bp. AxlD1 (-2376/ -338), which lacks the major transcriptional start site, showed similar luciferase activity when compared with AxlP1, suggesting that both of the major transcription start sites are important. Correspondingly, AxlD2 (-556/-338), also including the second major transcription start site, showed similar luciferase activity as AxlP7 (-556 /+7), which include both major transcription start sites. AxlP9 (-181/+7) and AxlD3 (-478/-338) do not contain any GC-rich regions and show a complete loss of luciferase activity, comparable to the pGL3-Basic control vector activity (Fig. 1b). Taken together, whereas maximum promoter activity lies within -1272 /+7 bp, data suggests that the 5' flanking region of Axl located between -556/-182 bp includes the minimally required core promoter, since activity of AxlP7 (-556/+7) and AxlP8 (-478/+7) are similar, and luciferase activity is lost completely downstream of -478. Region -556/-182 of the promoter is GC-rich, and by TRANSFAC database search is predicted to bind zink-finger proteins such as Sp family transcription factors (Fig. 1c), but also AP-1 and MZF-1. Overall, our observations generated with all deletion constructs were similar in the Rko, HCT116, and HeLa cell line, excluding cell-line specific biases.

Five Sp family transcription factor binding sites regulate Axl core promoter activity Deletion constructs and in-silico analysis of the promoter revealed putative Sp binding GC-rich regions within a core promoter region of -556/-181. Sp-motifs are defined as Sp a (-225/-219), Sp b (-250/-242), Sp c (-512/-505), Sp d (-524/-516), and Sp e (-529/-523). To analyse their functional relevance, each binding motif was mutated at four bases by site-directed mutagenesis within the AxlP3 (-1276 to +7) reporter plasmid, which showed maximum promoter activity. Different mutational constructs were generated, with mutations either in single regions (Sp a, Sp b, Sp c, Sp d, Sp e), or combinations of two or more regions (Sp ab, Sp cde, and Sp abcde) (Fig. 2a, Table 1). Mutated and wild type constructs were transfected into Rko and HeLa cell lines and compared for reporter activity. We observed that all single mutations and their combinations significantly reduced promoter activity as compared to the wild type in both cell lines, with the exception of Sp c activity in Rko cells (Fig. 2b). The single mutational constructs Sp a, Sp b, Sp d and Sp e showed 40%, 60%, 30%, and 30% reduced activity as compared to the wild type in Rko cells, and 40%, 30%, 60% and 60% in HeLa cells, respectively. Out of the combined mutational constructs, Sp ab (mutated for Sp a, and Sp b) and Sp abcde (mutated for all 5 motifs) showed the most dramatic (70%) reduction in promoter activity in both of the cell lines. Even though all motifs were mutated in Sp abcde, it didn't reduce promoter activity further when compared with the Sp ab mutational construct. However, the Sp cde combinational construct alone reduced reporter activity by 50% in both of the cell lines, implicating a cooperative function of these Sp-motifs. In addition to Sp mutations, other mutational constructs were generated based on MZF1 and AP1 consensus binding domains at bp (-564/-556, -297/-292), yet neither of them could affect basal promoter activity (data not shown). Taken together, data suggests that all Sp binding sites are relevant for Axl core promoter activity, among them Sp a, and b being essential.

Nuclear proteins of the Sp family bind different binding motifs of the Axl core promoter

To demonstrate the binding of Sp family member transcription factors to their respective binding sites in the core promoter of Axl, gel shift analysis was performed using Rko cell nuclear extracts, and radio-labelled wild type and mutant probes (Table 1). Sp c, d and e are in close proximity, therefore, a competition experiment was conducted with increasing concentrations of 25x and 50x of either individual (Sp c, Sp d, Sp e) or combination mutant (Sp cde) oligos (using the oligos of site directed mutagenesis), the Sp d wild type oligo being used for labelling, and 50x unlabelled oligos for cold competition (Fig. 3a). As shown in Fig. 3a, competition with mutated Sp c and d completely or partially abolished binding of Sp transcription factor complexes, implicating that the binding of some of the factors stayed intact, whereas with mutant Sp e as a competitor, most of the Sp transcription factors complexes stayed intact. Competition with an oligo mutated for Sp cde did not change the binding complexes, even with increasing concentrations. These data show that transcription factors are binding to this region, however, they are binding predominately to the Sp e site. The same kind of competition experiment was performed for the Sp a, and b sites with their respective labelled wild type oligos. We observed intact binding complexes even after adding increased concentrations of mutated oligos Sp a and Sp b (Fig. 3a). The wild type competition specifically abolished binding to Sp a and Sp b, implicating sequence specific binding of nuclear proteins. To confirm that the specific complexes bound in the EMSA are indeed composed of Sp family members, we performed supershift assays for the Sp a , b, and cde motifs using antibodies against Sp1, Sp2, Sp3, Sp4, and IgG as a control (Fig. 3b). For all three motifs, the incubation with Sp 1, 2 and 3 antibodies led to shift of the major bands, implicating the presence of these Sp family members. Individual super shift analysis of Sp c and e also showed binding of Sp 1 and 3 (data not shown).

In order to analyze the binding of Sp family members to the endogenous Axl promoter, we used functionally well characterised antibodies against Sp1 or Sp3 in ChIP analysis in Rko cells. PCR promoter without Sp binding sites as a negative control, resulted in single bands of sizes consistent with the respective expected regions of the Axl promoter, no specific band being amplified for the nonspecific control (Fig. 3c). These results confirmed that Sp1 and Sp3 are indeed binding to the Sp a, b, and cde motifs of the endogenous Axl promoter.

Mithramycin represses Axl promoter activity, and decreases Axl mRNA and protein levels

To further assess the functional relevance of Sp transcription factors on Axl gene expression, cells were treated with mithramycin which binds to GC-rich DNA sequences and impedes the binding of GC-specific transcription factors by steric hindrance [START_REF] Kingsley | Cloning of GT box-binding proteins: a novel Sp1 multigene family regulating T-cell receptor gene expression[END_REF]. Rko cells were transfected with AxlP3. After 12 hours of transfection, cells were treated with either medium alone, or various concentrations of mithramycin (25, 50, 100 and 200 nM) for 24 hours. Increasing concentrations of mithramycin gradually and significantly decreased Axl promoter activity (Fig. 4a). For example, the 200 nM treated sample showed a 50% reduction of promoter activity when compared with the medium treated control (Fig. 4a). EMSA analysis was performed to assess the ability of mithramycin to prevent Sp-binding to the GC-rich region of the Axl promoter in a dose dependent manner (50 to 400 nM concentrations). Pre-incubation with mithramycin inhibited the binding of Sp family transcription factors to all identified Sp binding regions of the Axl promoter (Fig. 4b). These observations confirm that the GCrich and Sp-binding sequences identified contain Axl core promoter activity, this being effectively blocked by mithramycin.

To investigate the mithramycin effect on Axl gene expression, Rko cells were treated with 100 nM mithramycin for 4, 8, 16, 24 h, as compared to 24 h of medium control. We found that treatment of Rko cells with mithramycin led to an almost 90% decrease in expression of Axl mRNA and protein after 16 hours (Fig. 4c,d). Since Sp1 and Sp3 genes also contain GC rich promoters driven by Sp family members, we additionally screened Sp1 and Sp3 mRNA and protein levels following mithramycin treatment. Sp1 and Sp3 mRNA amounts were decreased 4 hours after treatment, and completely un-detectable after 16 hours, which was paralleled by a decrease of Sp1/Sp3 protein amounts (Fig. 4c,d). This data suggests that mithamycin not only inhibits the binding of Sp1 and Sp3 transcription factors to the Axl promoter, but also represses the expression of Sp1 and Sp3 by binding to their GC rich promoter regions. The results further confirm Sp1 and Sp3 as essential regulators of Axl gene expression.

Sp1 or Sp3 increase Axl promoter activity and gene expression

Next, the effect of Sp1 and Sp3 overexpression on the activity of the Axl promoter in Rko cells was investigated. Rko cells were co-transfected with the AxlP3 (-1276/+7) construct, or the mutational constructs for Sp a, Sp b, Sp cde and Sp abcde, respectively, and either Sp1 or Sp3 expression vectors in increasing concentrations. Overexpression of Sp1 and Sp3 led to a significant increase of Axl wild type promoter activity in a dose dependent manner (Fig. 5a). In contrast, the mutants Sp a, Sp b, Sp cde, and Sp abcde showed a significant reduction of responsiveness to Sp1 or Sp3. Specifically, mutant Sp a and Sp b did not show any response to Sp3 over-expression as compared to their vector control, and the Sp cde mutant showed a 50% reduction of response to Sp3 when compared with AxlP3 wild type. The Sp1 response was reduced by 80%, 100%, and 50% for mutant Sp a, Sp b, and Sp cde, respectively, when compared with the AxlP3 wild type (Fig. 5b). The Sp abcde mutant did not show a significant response to Sp1 or Sp3 overexpression (Fig. 5b, panel Sp abcde). The activity of the Sp abcde mutant following Sp1/3 overexpression was dramatically reduced as compared to the AxlP3 wild type (Fig 5b, panel AxlP3 WT). These results confirmed that especially Sp a and b are mediating Axl promoter activity following Sp1/Sp3 overexpression, with an additional role for the Sp cde site.

The Axl promoter is regulated by especially Sp1 or Sp3, both of which often being ubiquitously expressed in mammalian cells [START_REF] Kingsley | Cloning of GT box-binding proteins: a novel Sp1 multigene family regulating T-cell receptor gene expression[END_REF]. To gain further evidence for Sp1 or Sp3 being essential for promoter activity, the Drosophila SL2 cell line which is devoid of endogenous human Sp family members was cotransfected with AxlP3, and either PacSp1, or PacSp3 (insect expression vectors encoding human Sp1 or Sp3). Both Sp1 and Sp3 strongly induced (150%) AxlP3 promoter activity, suggesting that either of these transcription factors alone can positively drive Axl gene expression (Fig. 5d).

To gain evidence that Sp1 and Sp3-driven Axl gene expression is relevant at the protein level, Rko cells were transfected with 4 µg of Sp1, Sp3, or a mock control (Vector alone). Both Sp1 and Sp3 induced Axl protein amounts after 48 hours of overexpression (Fig. 5c) when compared with the control. Taken together, it is clear that Sp1 or Sp3 positively regulate Axl gene expression.

siRNA strategies against Sp1 and Sp3 downregulate Axl promoter activity and Axl gene expression

To provide evidence that inhibition of endogenous Sp1 or Sp3 transcription factors can inhibit Axl promoter activity and expression, siRNA knock-down experiments were performed. Either Rko or HeLa cells were transfected with either Sp1 or Sp3-specific small interfering oligonucleotides (siRNA) in a dose dependent manner. The knock down of Sp1 and Sp3 was confirmed by Western blotting (Fig. 6 c,d). Expression of a siRNA against Sp1 or Sp3 decreased Axl promoter activity to almost 80% and 70% in Rko, and to 50% and 40% in HeLa cells, respectively (Fig. 6 a,b). This was paralleled by a significant reduction of Axl protein amounts with increasing concentrations of siRNA for either Sp1 or Sp3, the protein amounts of Axl being even further reduced with a combination of si-Sp1 and si-Sp3 (Fig. 6). This confirms that the knockdown of endogenous Sp1 and Sp3 represses transactivation of the Axl promoter, and basal Axl gene expression in Rko and HeLa cells.

Axl gene expression is also dependent on DNA methylation in cultured colorectal cancer cells

In assessing human cancer cell lines for the concordance of Axl mRNA/protein and Sp1/3 expression (Fig. 7a,b), Rko, HCT116, SW480 (colorectal cancer) and HeLa (derived from cervical cancer) cells showed a high endogenous expression of Axl which was paralleled by high levels of Sp1 and Sp3 mRNA and protein. However, another group of colorectal cancer cell lines (HCT15, HT29, Geo, WiDr, (colo206f) showed an almost undetectable basal Axl gene expression despite of moderate expression levels of Sp1 and Sp3 mRNA and protein. This observation promoted us to speculate that, apart from Sp1 and Sp3, other, potentially epigenetic mechanisms, could be influencing basal Axl gene expression.

In order to investigate one potential epigenetic mechanism in terms of methylation of CpG sites, the colorectal cell lines Colo206f and WiDr characterized by a low constitutive Axl expression were treated with a selective inhibitor of DNA methyltransferases (5-aza-dC) with increasing concentrations. In 5-aza-dC treated cells, Axl and Sp3 expression was significantly induced in a dose dependent manner, whereas no significant differences were observed in Sp1 mRNA expression (Fig. 8a). To define methylated regions of the Axl promoter and to compare a potential methylation between high-and low Axl expressing colorectal cancer cells, 1 µg of genomic DNA from WiDr (low Axl) and Rko (high Axl) cells was subjected to bisulfite conversion. Sequencing revealed that the Sp sites a, b, and c were partially methylated in low Axl-expressing WiDr cells (Fig. ,8b,c areas 4, 5 and 8), but not in high Axl-expressing Rko cells. Furthermore, in WiDr cells, CG-rich flanking regions of GC-rich areas around the Sp-sites identified were also partially methylated (Reg. F, areas 6, 7, 9) (Fig. 8b,c). These data demonstrate that methylated CpG sites, in part within identified Sp-binding motifs, in part in GC-rich flanking regions, influence constitutive Axl gene expression apart from the binding of Sp1and Sp3 transcription factors.

Discussion

Axl as a member of the mammalian Tyro 3 receptor tyrosine kinase family, which is widely overexpressed in different cancers [START_REF] Hafizi | Gas6 and protein S. Vitamin K-dependent ligands for the Axl receptor tyrosine kinase subfamily[END_REF], and an increasing number of reports has shown an essential functional relevance of Axl in tumor progression, invasion, metastasis, antiapoptosis, angiogenesis, and immunological escape [START_REF] Budagian | A promiscuous liaison between IL-15 receptor and Axl receptor tyrosine kinase in cell death control[END_REF][START_REF] Shieh | Expression of axl in lung adenocarcinoma and correlation with tumor progression[END_REF][START_REF] Vajkoczy | Dominantnegative inhibition of the Axl receptor tyrosine kinase suppresses brain tumor cell growth and invasion and prolongs survival[END_REF][START_REF] Lemke | Macrophage regulation by Tyro 3 family receptors[END_REF][START_REF] Green | Overexpression of the Axl tyrosine kinase receptor in cutaneous SCC-derived cell lines and tumours[END_REF]. In our present work, we have systematically characterized the promoter of this highly important gene in diverse colorectal and also the HeLa cell line for the first time. In the present paper, we have focused on delineating the minimally required basal Axl promoter driving endogenous gene expression. In this context, we have clearly identified five different Spbinding regions within the minimally required basal promoter region -556/-183, bound especially with Sp1 and Sp3 transcription factors. The dominant role of Sp1 and Sp3 in controlling Axl gene expression was solidified by either overexpression or siRNA knockdown of Sp1 and Sp3, which led to decreased or increased promoter activity and Axl protein levels, respectively. This is supported by mithramycion experiments, showing a decrease of promoter activity, mRNA, and protein, and also Sp1 and Sp3 expression, brought about by this GC-rich region binding molecule. In addition to Sp1 and Sp3 as essential regulators of basal Axl gene expression, we show first evidence that the methylation of CpG sites within specific Sp1 motifs, and also in promoter regions flanking these motifs, is modulating Axl gene expression.

Sp1, and Sp3 are zinc finger proteins that belong to the Specificity Factor (Sp) family of transcriptional factors, regulating the transcription of many house-keeping genes, but also tissuespecific, viral, and inducible genes by binding and acting through the GC box motif "GGGCGG" [START_REF] Li | Gene regulation by Sp1 and Sp3[END_REF][START_REF] Suske | The Sp-family of transcription factors[END_REF]. Sp family members are characterized by a motif of three conserved C 2 H 2 zinc fingers, which form the DNA-binding domain [START_REF] Suske | The Sp-family of transcription factors[END_REF]. Among Sp family transcription factors, very little functional activities are known for Sp2, and Sp4. Sp2 is characterized by the glutamine-rich activation domain, indicating that it can act as a potential activator [START_REF] Kingsley | Cloning of GT box-binding proteins: a novel Sp1 multigene family regulating T-cell receptor gene expression[END_REF], whereas Sp4 is a tissue specific Sp family member predominately found in the brain. Since, in our present study, we found binding of Sp2 in addition to Sp1 and Sp3 to some of the five Sp-binding motifs identified within the Axl promoter, it is certainly possible that, besides Sp1 and Sp3, also Sp2 will transactivate the Axl promoter via these sites. Due also to a limited availability of suitable constructs and antibodies, however, our study, focused on Sp1 and Sp3, Sp1 being characterized by glutamine rich regions which act as strong activation domains [START_REF] Courey | Analysis of Sp1 in vivo reveals multiple transcriptional domains, including a novel glutamine-rich activation motif[END_REF], and a short C-terminal domain D, which is required for synergistic activation [START_REF] Kingsley | Cloning of GT box-binding proteins: a novel Sp1 multigene family regulating T-cell receptor gene expression[END_REF]. Sp3 has been shown to be able to act as either transcriptional activator [START_REF] Udvadia | Functional interactions between the retinoblastoma (Rb) protein and Sp-family members: superactivation by Rb requires amino acids necessary for growth suppression[END_REF][START_REF] Zhao | The human POLD1 gene. Identification of an upstream activator sequence, activation by Sp1 and Sp3, and cell cycle regulation[END_REF] or transcriptional repressor [START_REF] Allgayer | Transactivation of the urokinase-type plasminogen activator receptor gene through a novel promoter motif bound with an activator protein-2alpha-related factor[END_REF] by binding to GC rich regions. It has been suggested that the functional activation of Sp3 also depends on the presence of binding sites within target promoters, since it has been observed that promoters with a single binding site were strongly activated, whereas promoters with multiple binding sites were either inactive or weakly activated by Sp3 [START_REF] Birnbaum | Sp1 trans-activation of cell cycle regulated promoters is selectively repressed by Sp3[END_REF]. Sp1 and Sp3 share more than 90% sequence homology in the DNA-binding domain and bind to the same GC-rich elements. Therefore, it has been hypothesized that the ratio between Sp1 and Sp3, or the cellular background may lead to either activation or repression of gene expression [START_REF] Majello | Sp3 is a bifunctional transcription regulator with modular independent activation and repression domains[END_REF][START_REF] Yu | Stability of the Sp3-DNA complex is promoter-specific: Sp3 efficiently competes with Sp1 for binding to promoters containing multiple Sp-sites[END_REF]. For the cell lines investigated in our present paper, we did not find a significant correlation between the ratio of expression of Sp1 and Sp3 with the amounts of endogenous Axl mRNA or protein (data not shown), therefore, it is unlikely that a similar situation is relevant for the expression of Axl.

Within the -556/-181 base pair region minimally required for Axl promoter activity, we identified five Sp consensus binding sites within a generally GC rich region. With EMSA and chromatin IP studies, we clearly showed that especially Sp1 and Sp3 are strongly bound to oligonucleotides corresponding to all of the Sp binding motifs, and also to the endogenous GC-rich region. Functional mutagenesis studies for these Sp binding sites revealed that especially the Sp a and Sp b motif are indispensable for basal Axl promoter activity, whereas the Sp cde sites show an additional and modulating relevance for basal promoter activity. Still, since a construct mutated for all five Sp binding sites still exerted a minimal reporter activity as compared to an empty vector control, we cannot rule out the possibility that additional parameters besides Sp factors bound to the five Sp sites identified remotely co-regulate the Axl promoter. For example, Sp family members might bind to GC rich regions other than the Sp consensus motifs identified within the Axl core promoter. Moreover, other zinc finger proteins with a high affinity towards GC rich regions, such as BTEB1 (basis transcription element binding protein 1), TIEG1 or TIEG2 ( TGFβ-inducible early protein 1 and 2) [START_REF] Subramaniam | Identification of a novel TGF-beta-regulated gene encoding a putative zinc finger protein in human osteoblasts[END_REF][START_REF] Fautsch | TGFbeta-inducible early gene (TIEG) also codes for early growth response alpha (EGRalpha): evidence of multiple transcripts from alternate promoters[END_REF][START_REF] Cook | Molecular cloning and characterization of TIEG2 reveals a new subfamily of transforming growth factor-beta-inducible Sp1like zinc finger-encoding genes involved in the regulation of cell growth[END_REF] might bind to the GC rich Axl core promoter. In addition, an AP-1 and an MZF-1 motif also identified within the core promoter might contribute to a minimal activation, however, this is unlikely since mutations of either of the motifs did not change reporter activity in our luciferase experiments (data not shown). Still, our cotransfection experiments of Sp1 or Sp3 with the Axl promoter, showing a significant increase in promoter activity in the SL2 cell line (null for endogenous expression of human Sp1 and Sp3) [START_REF] De Wolf | The constitutive expression of anticoagulant protein S is regulated through multiple binding sites for Sp1 and Sp3 transcription factors in the protein S gene promoter[END_REF][START_REF] Liu | Regulation of histone deacetylase 4 expression by the SP family of transcription factors[END_REF], and our observation that Sp1 or Sp3 significantly increase Axl gene expression, whereas an siRNA knock down for either Sp1 or Sp3 dramatically reduced Axl promoter activity and expression, clearly imply the notion that both transcription factors are essential for Axl promoter transactivation, and endogenous gene expression [START_REF] Liu | Regulation of histone deacetylase 4 expression by the SP family of transcription factors[END_REF]. In this context, it is interesting to note that a second ligand of the Axl receptor, protein S, has also been shown to be constitutively regulated by Sp1 and Sp3 transcription factors [START_REF] De Wolf | The constitutive expression of anticoagulant protein S is regulated through multiple binding sites for Sp1 and Sp3 transcription factors in the protein S gene promoter[END_REF]. Although these experiments had been conducted mainly in the hepatocytic cell line HepG2, it is interesting to speculate that in gastrointestinal tumors, Sp1 and Sp3 may regulate the constitutive expression of a tyrosine kinase receptor highly relevant for tumor progression, invasion, and angiogenesis, in addition to upregulating one of its specific ligands, to enhance tumor progression.

Mithramycin, having been shown to reduce the activity of the Sp1 promoter as well as to inhibit Sp1 binding [START_REF] Lou | Identification of the promoter of human transcription factor Sp3 and evidence of the role of factors Sp1 and Sp3 in the expression of Sp3 protein[END_REF], in our present study reduced Axl promoter activity in a dose dependent manner, and almost abolished the expression of mRNA and protein of Axl, Sp1, and Sp3 within 24 hours [START_REF] Liu | Regulation of histone deacetylase 4 expression by the SP family of transcription factors[END_REF]. Mithramycin is a cell-permeable reagent frequently used to explore the sequence dependence of DNAbinding factors known to bind to GC-rich regions by steric hindrance. In our present study, overexpression or knock down of Sp1 or Sp3 had a significant, however not 100% impact on Axl promoter activity, whereas mithramycin treatment completely abolished the expression of Axl mRNA or protein. One explanation for this small discrepancy might be that all GC rich regions of the Axl promoter were completely blocked by mithramycin. Moreover, as already stated above, other transcription factors known to bind to GC rich regions and/or exhibit similar binding specificity as Sp1, such BTEB1, TIEG1 or TIEG2, might add to Axl promoter transactivation to a small extent, and might also have been blocked by mithramycin. At the present time, we cannot rule out the possibility of a remote additional impact of suchlike transcription factors apart from Sp1 or Sp3 in in vivo conditions.

In the present paper, we also provide the first systematic screening of a number of human colorectal cancer cell lines in addition to HeLa (cervical cancer cell line) for transcript and protein amounts of human Axl, Sp1, and Sp3. In Rko, HCT116, SW480 and HeLa cells, a high endogenous Axl expression was correlated with high amounts of endogenous Sp1 and Sp3. On the other hand, HCT15, HT29, Geo, WiDr and Colo206f cells did not show significant endogenous Axl gene expression, although they have detectable transcript and protein levels of Sp1 or Sp3. This certainly can be explained by the fact that Sp1 and Sp3 regulate many more genes that might also be of relevance for the latter cell lines, besides Axl. Moreover, the activity of Sp family transcription factors towards specific promoter motifs is certainly not only defined by their expression, but also by posttranslational modifications and protein-protein interactions. It has been well described that posttranslational modifications such as phosphorylation, glycosylation, or sumoylation modify the transactivating activity of, for example, Sp1 [START_REF] Chu | Sp1: regulation of gene expression by phosphorylation[END_REF]43]. Also, Sp1 can exert indirect effects on transcription via its interaction with, for example, chromatin remodelling factors (p300 or PCAF), and other DNA-binding proteins [START_REF] Li | Gene regulation by Sp1 and Sp3[END_REF][START_REF] Chu | Sp1: regulation of gene expression by phosphorylation[END_REF][START_REF] Xiao | p300 collaborates with Sp1 and Sp3 in p21(waf1/cip1) promoter activation induced by histone deacetylase inhibitor[END_REF][START_REF] Huang | Trichostatin A induces transforming growth factor beta type II receptor promoter activity and acetylation of Sp1 by recruitment of PCAF/p300 to a Sp1.NF-Y complex[END_REF]. Furthermore, as stated before, the ratio of Sp1 to Sp3 in certain cellular contexts may determine the degree of activation [START_REF] Hata | Transcription factors Sp1 and Sp3 alter vascular endothelial growth factor receptor expression through a novel recognition sequence[END_REF][START_REF] Wong | Decreased methylation and transcription repressor Sp3 up-regulated human monoamine oxidase (MAO) B expression during Caco-2 differentiation[END_REF]. Suchlike mechanisms certainly can explain the discrepancy between Axl, Sp1, and Sp3 expression levels found in some of the cell lines investigated.

Our finding that the methylation of some of the Sp binding sites, and their flanking regions, within the basal Axl promoter may modulate Axl gene expression, certainly merits discussion. CpG methylation is well known to be important for gene imprinting [START_REF] Bartolomei | Epigenetic mechanisms underlying the imprinting of the mouse H19 gene[END_REF], cell cycle control, tumor suppression, embryonic development, differentiation, and other phenomena [START_REF] Carlone | CpG binding protein is crucial for early embryonic development[END_REF]. Specifically, there have been many interesting reports showing that the methylation of CG sites within Sp transcription factor binding motifs, but also of their flanking regions, might affect binding and the transactivating potential of Sp1 and Sp3 [START_REF] Zhu | Methylation of adjacent CpG sites affects Sp1/Sp3 binding and activity in the p21(Cip1) promoter[END_REF][START_REF] Kudo | Tissue-specific transcriptional regulation of human leukosialin (CD43) gene is achieved by DNA methylation[END_REF][START_REF] Kitzazwa | Transcriptional regulation of rat cyclin D1 gene by CpG methylation status in promoter region[END_REF]. In the present study, we found that the basal Axl promoter is extremely GC rich, with 19 CpG sites within the region -669/-97, and harbours many putative CpG methylation sites. CpG methylation can inhibit Sp1 binding by two mechanisms, either by directly inhibiting the binding of Sp1, or by contributing to a competition with other factors like MeCP2 for binding sites [53,54]. In our present study, we found that some of the CpG sites within Sp family binding motifs, or within adjacent CG rich regions, were partially methylated in WiDr cells, which are characterized by an almost undetectable endogenous Axl gene expression. In contrast, in high Axl expressing Rko cells, no methylation of such sites was found. This observation suggests that CpG methylation of the basal Axl promoter is related to endogenous Axl gene expression. To substantiate this hypothesis, we found that a demethylating agent (5-aza-dC) was able to activate Axl gene expression in the low Axl expressing WiDr cell line. Moreover, not only Axl, but also Sp3 expression was induced by 5-aza-dC in Colo206f and WiDr cells in a dose dependent manner. This supports the hypothesis that in low Axl expressing colon cancer cells, transcription of the Axl gene is kept silent by partial methylation of CpG islands, within the basal promoter. The additional interesting observation that 5-aza-dC directly affected the expression of Axl and Sp3, but not Sp1, prompts us to speculate that the demethylating agent interferes with the ability of Sp1 and Sp3 to transactivate Axl gene expression in two different ways: in case of Sp3, it may directly interfere with Sp3 gene expression by demethylating sequences within its gene promoter. In contrast, it may directly modulate Sp1 binding to the Axl gene promoter by demethylating target sites within the Axl promoter sequence. Thus, the effect of 5-aza-dC on Sp1/Sp3 regulated Axl promoter activity might be both direct and indirect.

Taken together, this is the first study to characterize the Axl gene promoter, and to establish a major role for Sp1, Sp3, and also methylation of the core promoter, as key regulators of constitutive Axl gene expression in diverse colorectal and HeLa cancer cells. Further ongoing studies of our group will elucidate the function of further upstream transcriptional elements in the constitutive and inducible expression of this tyrosine kinase receptor, which is essential for many functions associated with cancer.

53 Lewis, J.D., Meehan, R.R., Henzel, W.J., Maurer-Fogy, I., Jeppesen, P., Klein, F., and Bird, A. (1992) Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA. Cell 69, 905-914 54 Ammanamanchi, S., and Brattain, M.G. (2001) 5-azaC treatment enhances expression of transforming growth factor-beta receptors through down-regulation of Sp3. J Biol Chem 276, 32854-32859

Footnotes

The abbreviations used are: ChIP, chromatin immunoprecipitation; RT-PCR, Reverse transctiption PCR; siRNA, small interfering RNA; scRNA, scrambled RNA; Tables Table 1. Oligos used for EMSA and Mutagenesis

S. No Name

Sequence of normal(N) and mutated(M) oligos (5' 3') Position (5'to3') Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Bioscience

  Reports Immediate Publication. Published on 03 Jun 2008 as manuscript BSR20080046 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080046 S t a g e 2 ( a ) P O S T -P R I N T

  Bioscience Reports Immediate Publication. Published on 03 Jun 2008 as manuscript BSR20080046 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080046 S t a g e 2 ( a ) P O S T -P R I N T amplification of the Sp 1/3 immunoprecipitated DNA using primers for Region I (covering Sp a and Sp b, 159bp), Region II (covering Sp cde, 168 bp) (Fig 3) and a non-specific region of the u-PAR

Fig 1 Fig 2

 12 Fig 1 Identification of the core promoter region of Axl. a) The schematic representation shows the design of pGL3 reporters to test the relative contribution of different portions of the Axl promoter. b) Luciferase reporter assay showing Axl promoter activity in Rko, HCT116 and HeLa. 1 µg of Axl constructs were co-transfected with a Renila luciferase plasmid, serving as internal control for transfection efficiency, into Rko, HCT116 and HeLa, and luciferase activity was measured 48 h post transfection. Activity of AxlP1 (-2376/+7), the largest deletion construct, was set to 100% (first bar), and activity of the other constructs was calculated and plotted as a percentage of this value. All samples were performed in triplicate, the bars representing the mean. Error bars, (± S.D). c) Schematic representation of maximum promoter activity showing promoter region AxlP3 (-1276/+7) with different transcription start sites, and putative Sp family binding sites.

Fig 4

 4 Fig 4 Axl promoter activity and expression is repressed by mithramycin.a) pGL3-basic and AxlP3 were transfected into Rko cells. 12 hours after transfection, cells were mocktreated or treated with 25,50,100, or 200 nM mithramycin. Another 24 h after treatment, all plates were harvested and the resulting luciferase activity was measured. Equal amounts of DNA (1µg) were transfected for each sample. Luciferase activity of cells transfected with AxlP3 and unexposed to mithramycin was set to 100% (first bar), and the activities of all other samples were calculated and plotted as a percentage of this value. Relative luciferase activity of each sample was normalized to the activity of a co-transfected Renilla luciferase plasmid. All assays were performed in triplicate, and the results were averaged. Error bars, (± S.D). The statistical difference of the promoter activity is shown against AxlP3 untreated transfected cells. **, p < 0.05. b) Mithramycin binding to GC-rich regions inhibits the binding efficiency of Sp family members. EMSA analysis was performed for the regions containing Sp a (-239/-202 bp), Sp b (-263/-228 bp) and Sp cde (538/-500 bp) with mithramycin incubation. γ-p33 labelled oligos were pre-incubated for 1 hour with mithramycin with increasing concentrations ( 50, 100, 200 and 400 nM), and 50x excess wild type oligos were pre-incubated for 20 min before adding labelled oligos. Sp transcription factor binding efficiency was reduced for all

Fig 5 Fig 6

 56 Fig 5 Overexpression of Sp1 or Sp3 induces Axl promoter activity and protein expression. a) Overexpression of Sp1 or Sp3 in Rko cells increases activity of the Axl promoter.Rko cells were cotransfected with 1 µg of AxlP3 (-1276/+7) and 0.5 µg and 1 µg of the Sp1 or the Sp3 expression vector, along with 50 ng of renilla luciferase plasmid. Sp1 and Sp3 increased Axl promoter activity in a dose dependent manner. Mock vector co-transfected AxlP3 luciferase activity was set to 100%, and activity of the other constructs was calculated and plotted as a percentage of this value. b) Rko cells were cotransfected with 1 µg of AxlP3 wild type or mutated luciferase reporter vectors and 1 µg of either Sp1 or Sp3 expressing vectors. AxlP3, mock treated activity was set to 100% (first bar), and activity of the mutated constructs activity were calculated and plotted as a percentage of this value. All samples were performed in triplicate and the results were averaged and plotted as shown. Error bars, (± S.D). c) Over-expression of Sp1 or Sp3 increases Axl protein amounts. Rko cells were transfected in a 6 well plate with 4 µg of pCMV-Sp1, pCMV-Sp3, and vector alone as a mock control. After 48 hours of transfection, cells were harvested and cell lysates immunoblotted for Sp1, Sp3, Axl, and actin (Loading control). Either Sp1 or Sp3 overexpression led to increased amounts of Axl protein. d) Either Sp1 or Sp3 can drive Axl promoter activity in SL2 cells. SL2 insect cells were tested for reporter activity under different conditions. Cells were plated in a 24 well plate and transfected with the AxlP3 luciferase reporter either with pPac, pPacSp1, or pPacSp3, along with Ranilla (which drives expression of Sp1 and Sp3 via an insect-specific promoter). As shown, Sp1 and Sp3 induces Axl promoter activity in the SL2 cell line. Renilla served as internal control for transfection efficiency. All samples were performed in triplicate and the results averaged and plotted as shown, with all results expressed as a percentage of the activity resulting from the AxlP3 promoter and pPac (which was set at 100%). Error bars, (± S.D). The statistical difference of the promoter activity in all panels is shown against AxlP3 mock transfected cells. **, p < 0.05.

Fig 7

 7 Fig 7 Screening for Axl, Sp1 and Sp3 expression in nine different cell lines a,b) All the cells were grown and treated under identical conditions: Cells were harvested in their exponential phase for either for RNA or protein lysate. Expression transcript levels of Axl, Sp1, Sp3 and GAPDH (Control) were screened through RT-PCR, and the lysates separated via SDS-PAGE, transferred to PVDF, and immunoblotted for Axl, Sp1, Sp3, and actin (loading control).

Fig 8

 8 Fig 8 Effects of 5-Aza-dC treatment on the expression of Axl, Sp1 and Sp3 mRNA in colorectal cancer cell lines, and Bisulfite sequencing of CpG sites within the Axl promoter. a) RT-PCR analysis for Axl, Sp1 and Sp3 mRNA expression in Colo 206 f and WiDr cells treated with 10, 20, 40 μM 5-Aza-dC for 6 d, showing that the demethylation reagent induces Axl and Sp3 gene expression but not Sp1. b) Schematic representation of the CpG sites of Axl. This sequence spans 572 bp between positions -669 to -97 relative to the translation start site, including 19 CpGs. There are three CpG-containing Sp binding sites within this sequence that are indicated with shaded circles, corresponding to Sp a,b, and Sp c, respectively (position 4, 5 and 8). c) DNA from Widr cells was treated with bisulfite, and the Axl promoter was PCR amplified. The PCR product was ligated into the pGMT easy vector by using the TA cloning system. Ten subclones were picked and sequenced. As a result, some of the CpG sites, some within the Sp a,b and c motif, others within GC-rich flanking regions, are partially methylated in WiDr cells.

Figures

  Figures

Table 2 . Primers used for delineation constructs

 2

	S.No Name	Sequence (5'-----------3')	5' position
	1	AxlF1	GAAGGTACCATGACAACCCAGGCAAAGTG	-2376
	2	AxlF2	GAAGGTACCACAAAGCAAGACTGGGTCTC	-1759
	3	AxlF3	GAAGGTACCCAGTCCCACCAGAAGGAGAG	-1276
	4	AxlF4	GAAGGTACCTGCCCGGCCAACAACTATTC	-1010
	5	AxlF5	GAAGGTACCTGTCTGTGGGCCAGTAGC	-727
	6	AxlF6	GAAGGTACCGTGCGTGTGTGTGTGTGTG	-614
	7	AxlF7	GAAGGTACCAATGAAGGGCCAAGGAGGC	-556
	8	AxlF8	GAAGGTACCAGGGGAGTGGAGTTCTGG	-478
	9	AxlF9	GAAGGTACCGGCAGGGGTGCTGAGAAG	-181
	10	AxlR1	GAACTCGAGACGCCATGGGTGCCAAAC	+7
	11	AxlR2	GAACTCGAGGAGCTGAGGAGGGGGAAG	-338
	AxlF: Axl Forward; AxlR: Axl Reverse

© 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080046 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Bioscience Reports Immediate Publication. Published on 03 Jun 2008 as manuscript BSR20080046 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080046 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements

HA was supported by Alfried Krupp von Bohlen und Halbach Foundation (Award for Young Full Professors), Essen, Wilhelm Sander Stiftung, Munich, Auguste-Schaedel-Dantscher-Stiftung, Garmisch, Hella-Bühler-Foundation, Heidelberg, Dr. Ingrid zu Solms Foundation, Frankfurt, B. Braun Foundation, Melsungen, and the Hector Foundation, Weinheim, Germany. This publication contains parts of the dissertation of Giridhar Mudduluru performed in partial fulfillment of the requirements for the PhD at the Medical Faculty Mannheim, University Heidelberg, and the DKFZ (German Cancer Research Center) Heidelberg. We thank Erika Hillerich for excellent help with the manuscript. We thank Dr. Guntram Suske, Philipps-Universität Marburg, Germany, for the generous gift of Sp1 and Sp3 human and insect expressing vectors.