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J Biol Chem, Vol. 274, Issue 48, 34361-34368, November 26, 1999

Soluble Collagen VI Drives Serum-starved Fibroblasts through S Phase and Prevents Apoptosis via Down-regulation of Bax^*

Martin Rühl, Ergün Sahin, Manfred Johannsen, Rajan Somasundaram, Dirk Manski, Ernst Otto Riecken, and Detlef Schuppan^§¶

From the Department of Medicine I, Klinikum B. Franklin, Free University of Berlin, Hindenburgdamm 30, 12200 Berlin and the ^§ Department of Medicine I, University of Erlangen-Nuernberg, Krankenhausstraße 12, 91054 Erlangen, Germany

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We previously showed that soluble, pepsin-solubilized collagen VI increases de novo DNA synthesis in serum-starved HT1080 and 3T3 fibroblasts up to 100-fold compared with soluble collagen I, reaching 80% of the stimulation caused by 10% fetal calf serum. Here we show that collagen VI also inhibits apoptotic cell death in serum-starved cells as evidenced by morphological criteria, DNA laddering, complementary apoptosis assays (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, enzyme-linked immunosorbent assay, and fluorescence-activated cell sorting), and quantification of apoptosis-regulating proteins. In the presence of starving medium alone or collagen I, the proapoptotic Bax was up-regulated 2-2.5-fold, compared with soluble collagen VI and fetal calf serum, whereas levels of the antiapoptotic Bcl-2 protein remained unaffected. In accordance with its potent stimulation of DNA synthesis, soluble collagen VI carries serum-starved HT1080 and Balb 3T3 fibroblasts through G₂ as shown by fluorescence-activated cell sorting analysis, whereas cells exposed to medium and collagen I where arrested at G₁-S. This was accompanied by a 2-3-fold increase in cyclin A, B, and D1 protein expression. Collagen VI-induced inhibition of apoptotic cell death may be operative during embryogenesis, wound healing, and fibrosis when elevated tissue and blood levels of collagen VI are observed, thus initiating a feedback loop of mesenchymal cell activation and proliferation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Apoptosis or programmed cell death is a distinct form of active self-destruction. It is an important mechanism by which cells can be eliminated from the organism, e.g. during tissue remodeling, embryogenesis, and immune elimination. Apoptosis is characterized by typical morphological changes and triggered by biochemically defined events (1). Morphologically, apoptotic cells are characterized by a reduction in cell size, chromatin condensation, and membrane blebbing. DNA fragmentation into multiples of 180 base pairs is regarded as the hallmark of apoptosis, but degradation into larger DNA fragments has also been described (2). Intracellular effectors of apoptosis are the caspases, enzymes that degrade key structural and functional molecules of the cell, such as nuclear lamins, cytoskeletal proteins, protein kinases, and DNA repair enzymes (3). The Bcl-2 family of proteins plays an important role in the regulation of apoptosis upstream of caspases. Their antiapoptotic members close and their proapoptotic members open mitochondrial membrane pores and thus regulate the release of cytochrome C into the cytosol. Cytochrome c can then activate procaspase 9 via formation of a complex with Apaf-1 (4). Bcl-2 family proteins function as homo- and heterodimers with the ratio of death promoters to death suppressors presumably determining if a cell enters the apoptotic pathway (5).

Apoptosis depends on signaling processes that derive from extracellular or intracellular events. The tumor suppressor gene p53, regarded as "guardian of the genome," is an example of intracellular regulation, because it is up-regulated in response to DNA damage and, in turn, can slow cell cycle progression and/or promote apoptosis by inducing the expression of the Bcl-2 family member Bax (6). Extracellularly, death factors such as tumor necrosis factor and Fas ligand, by binding to their receptors, trigger a caspase cascade via adaptor proteins (7). Other extracellular factors (e.g. interleukin-2 and -3 as well as nerve growth factor and insulin-like growth factor-1) provide survival rather than death signals for specialized cells (8).

Whereas much is known about regulation of apoptosis by growth factors, cytokines, and hormones, the role of the extracellular matrix (ECM)¹ in regulating apoptosis, although well recognized, is yet little understood (9, 10). The most common approach to study the effect of ECM on apoptosis is to plate cells on precoated ECM components (11). Using this approach, many investigators have demonstrated that integrin-mediated adhesion and spreading on ECM molecules is necessary for the survival of many cell lines. Adhesion-dependent cells that are deprived of anchorage die by an apoptotic mechanism called anoikis (12, 13). The basement membrane has been shown to prevent apoptosis through ₁-integrin signaling (14), and several distinct ECM molecules such as fibronectin or collagens have been found to facilitate survival through integrin-mediated signal transduction pathways (15).

Collagen VI (CVI) is a large, multidomain ECM protein composed of a triple-helix of the chains ₁, ₂, and ₃, which, via tetramerization and end-to-end association, assemble into a microfibrillar network in vivo and in vitro (16). Its pattern of distribution and its unique structure and expression compared with other ECM molecules indicate that CVI might fulfill specialized tasks in tissue organization and cell functioning (17-19). In this line, CVI interacts with various other matrix components including hyaluronic acid (20), syndecan (21), decorin (22), von Willebrand factor (23), and collagens, types I, IV and XIV (19, 24). CVI has been shown to be highly expressed around mesenchymal cells during development of liver (17) and eyes (25), suggesting a role in cellular differentiation and survival (26). In this line, strong expression of CVI has been reported around malignant tumors like melanoma and glioma, which show more invasive behavior than those not expressing CVI (27, 28). Native, triple-helical CVI as well as its single chains promote cell attachment, spreading, and proliferation through at least partially integrin-independent mechanisms (29, 30).

Here we describe the unique effect of soluble CVI, in the absence of growth factors, to promote cell survival through down-regulation of the proapoptotic Bax protein. Furthermore, CVI sets the stage for cell cycle progression by up-regulating cyclins A, B, and D1.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

If not stated otherwise, all reagents were obtained from Sigma and were of the highest purity grade available.

Collagen Isolation-- Human collagen I (CI) and CVI were isolated from human placentas by pepsin digestion, fractional salt precipitation in acidic and neutral buffers, ion exchange, and molecular sieve chromatography as described previously (30). All preparations were tested for purity by SDS-PAGE and amino acid analysis after hydrolysis in 6 N HCl under nitrogen for 24 h at 110 °C. Purified collagens were lyophilized and redissolved in 0.15 or 0.5 M acetic acid before use. Growth factor contamination of CVI was excluded by pepsin digestion, molecular sieve chromatography with a cut-off of 100 kDa in a buffer containing 6 M guanidine, and the use of neutralizing antibodies against fibroblast growth factor, epidermal growth factor, PDGF AB, and transforming growth factor- as described previously (30).

Cell Culture-- Balb 3T3 cells (mouse fibroblasts, ATCC CCL-163) and HT1080 cells (human fibrosarcoma cells, ATCC CCL-121) were cultured in 80-cm² flasks using Dulbecco's modified Eagle's medium with high glucose (4.5 g/liter) and L-alanyl-L-glutamine (0.862 g/liter; Life Technologies, Inc., Eggenstein, Germany) supplemented with penicillin (107 units/liter), streptomycin (10 mg/liter), ascorbic acid (50 mg/liter), and 10% fetal calf serum (FCS) (Biochrom, Berlin, Germany) under standardized conditions (37 °C, 8% CO₂) in a humidified atmosphere. Cells were then serum-starved for 24 h, with no FCS (starving medium), followed by dissociation with 500 µl of 0.05% trypsin-EDTA (Biochrom) and neutralization with 30 ml of starving medium containing 1 g/liter soybean trypsin inhibitor and 1 g/liter ovalbumin. Alternatively, cells were scraped carefully with a cell scraper (Costar, Cambridge, MA) to exclude any potential effect of trypsin. After centrifugation at 800 × g for 10 min, cells were washed twice with 20 ml of PBS (without Ca²⁺ and Mg²⁺; Biochrom), resuspended in starving medium, and plated at a density of 1.5-2 × 10⁶ in 1.7 ml of starving medium, followed by treatment with starving medium alone, 100 µl of CI (20 mg/liter), 100 µl of CVI (20 mg/liter), or 10% FCS in six-well plates (NUNC, Wiesbaden, Germany).

For experiments on defined adhesive or antiadhesive surfaces, one million cells were seeded into six-well-plates (NUNC) coated either with 3.1 mg/cm² poly(2-hydroxyethylmethacrylate) (poly-HEMA) in 95% ethanol or with 1% BSA in sterile PBS as nonadhesive surfaces (30, 31) or with 60 µg/2 ml of CI or CVI overnight at 4 °C. These saturating concentrations of the coating substances were determined in pilot studies. Wells were washed three times with 2 ml of PBS/Tween 0.05% at room temperature, followed by a final wash with plain PBS. In integrin-blocking experiments freshly set cells were incubated with anti ₁-integrin antibody P4C10 (1:1000; Biomol, Hamburg, Germany), followed by immediate treatment with solubilized collagens. After 24 h, cells were subjected to FACS analysis.

Morphology-- 24 h after plating and treatment, cells were photographed using a Zeiss phase contrast microscope at a 1:40 magnification and Kodak Ektachrome 64 film.

Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling-- 24 h after plating and the addition of collagens or FCS, cells were harvested by trypsinization or scraping, suspended in starving medium containing trypsin inhibitor (100 mg/liter), centrifuged for 10 min at 800 × g, adjusted to the same cell number, washed twice with 10 ml of 1% BSA in PBS, and resuspended in 1 ml of PBS/BSA. Then cells were transferred to 1.5-ml Eppendorf tubes and fixed in 4% paraformaldehyde for 30 min at room temperature, centrifuged at 1,600 rpm for 10 min, washed twice with PBS, and kept in 70% ethanol for storage at 20 °C or used immediately. For further processing, ethanol was removed, and cells were washed with PBS. Permeabilization was done by adding 500 µl of PBS containing 0.1% Triton X-100 on ice for 2 min followed by two washes with ice-cold PBS. 50 µl of reaction buffer containing terminal deoxynucleotidyltransferase and fluoresceine-labeled dUTP were used for in situ labeling of fragmented DNA (In Situ Cell Death Detection; Roche Molecular Biochemicals). After incubation at 37 °C for 1 h, cells were washed twice with 500 µl of PBS, cytospun onto microscope slides, and visualized under a fluorescence microscope (Zeiss, Germany).

DNA Laddering-- After a 24-h treatment with medium, CI, CVI, or 10% FCS, cells were trypsinized and adjusted to equal cell number (1.5 × 10⁶). The Apoptotic DNA Ladder Kit (Roche Molecular Biochemicals) was used according to the manufacturer's instructions, leading to isolation of oligonucleosomes. The isolated DNA in each group was electrophoresed on a 1.5% agarose gel at 75 V for 1.5 h, stained with ethidium bromide, and visualized via a UV light source (302 nm). As a positive control, apoptotic DNA from HL60 cells (promyelocytic leukemia) was used.

Cell Death Detection ELISA-- 4 × 10⁵ cells were plated in six-well plates and treated with starving medium or medium plus CI, CVI, or FCS. After 6, 12, or 24 h, floating and adherent cells were pooled and lysed with 500 µl of lysis buffer (1% SDS, 10 mM Tris, pH 7.4) for 30 min at 4 °C. Lysed cells were transferred to 1.5-ml Eppendorf tubes and centrifuged at 13,000 rpm for 20 min to separate low molecular weight DNA (oligonucleosome-sized fragments derived from apoptotic cells) from high molecular weight DNA (from viable cells). 400 µl of the supernatant containing oligonucleosomes were carefully transferred to another Eppendorf tube and diluted 1:5 with lysis buffer. A 100-µl aliquot was used for the ELISA, which was performed according to the manufacturer's instructions (Cell Death Detection ELISA; Roche Molecular Biochemicals). In brief, antibodies against histones were coated onto 96-well, 100-µl lysate samples containing oligonucleotides, and histones were added and incubated for 1 h. After washing, the wells were incubated with anti-double strand DNA antibody coupled to peroxidase for 1 h. After reaction with peroxidase substrate, absorbance was measured at 405 nm using an ELISA reader. Background values (lysis buffer alone) were subtracted, and results are shown as enrichment factor (enrichment factor = OD (optical density) of the sample (apoptotic cells) divided by the OD of the corresponding control (viable cells, FCS group)). A defined positive control was prepared using a hypertonic buffer, containing 10 mM Tris, 400 mM NaCl, 5 mM CaCl₂, and 10 mM MgCl₂, instead of starving medium.

FACS Analysis-- 1.5 × 10⁶ freshly set cells were treated with medium, CI, CVI, or FCS for 24 or 48 h as described before. After trypsinization, cells were adjusted to same cell number, washed with 10 ml of PBS, fixed in 70% ethanol for 30 min at 20 °C, and suspended in 500 µl of PBS, containing RNase A (250 mg/liter) for 30 min at 37 °C. Fixed cells were stained in a final volume of 1 ml with propidium iodide (50 mg/liter) before being subjected to FACS analysis as described (31).

Western Blot Analysis-- Trypsinized and washed cells were centrifuged at 800 × g and lysed by the addition of 100 µl of lysis buffer (1% SDS, 10 mM Tris-HCl, pH 7.4). DNA was sheared with an insulin syringe (Braun-Melsungen, Germany) by up and down pipetting. Membrane fragments were removed by centrifugation at 13,000 rpm for 10 min, and protein concentration in each sample was determined using the BCA method (Pierce). Equivalent amounts of protein were removed, and volumes were adjusted to 20 µl. After the addition of 2× sample buffer, the aliquots were boiled for 5 min and subjected to 12% SDS-PAGE.

After blocking for 1 h at room temperature in a buffer containing 10 mmol of Tris-HCl, pH 7.5, 100 mmol of NaCl, 0.1% Tween 20, and 3% BSA, nitrocellulose membranes were incubated either with polyclonal rabbit anti-human Bax (1:1000, sc-493; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), monoclonal mouse anti-human Bcl-2 (1:250, B46620; Transduction Laboratories, Lexington, KY), polyclonal rabbit anti-human Bcl-2 (1:1000, sc783; Santa Cruz Biotechnology) or polyclonal rabbit anti-human -actin (1:4000, A2066; Sigma), polyclonal anti-cyclin A and anti-cylin B (1:250, sc-751 and sc-752, respectively; Santa Cruz Biotechnology), and monoclonal anti-cyclin D1 (1:250, 14561a; Pharmingen, Hamburg, Germany). The membranes were then washed three times for 10 min in a buffer containing 10 mmol of Tris-HCl, pH 7.5, 100 mmol of NaCl, and 0.1% Tween 20 and incubated with anti-mouse-IgG coupled to peroxidase (1:1000, A4416; Sigma) for 1 h at room temperature. Reactive bands were detected with the ECL chemiluminescence reagent (Amersham Pharmacia Biotech). After stripping in 0.1 M glycin-HCl, pH 2, for 20 min at room temperature, membranes were reprobed with appropriate primary and secondary antibodies. Band intensities were analyzed by densitometry. Results are shown as percentage of the control (10% FCS) after adjustment of band intensity to that of -actin, which served as internal control.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Soluble Type VI Collagen Inhibits Apoptosis in Serum-starved Fibroblasts-- Previous studies have shown that CVI strongly induces cellular attachment and spreading (24, 29, 32). To test the hypothesis that soluble CVI inhibits programmed cell death, we induced apoptosis in transformed and nontransformed fibroblasts by serum withdrawal (33). HT1080 and Balb 3T3 cells were synchronized over 24-48 h by culturing in Dulbecco's modified Eagle's medium containing no FCS, trypsinized thereafter, and seeded for the different assays as described under "Experimental Procedures." After plating, cells were treated with plain medium or 20 mg/liter pepsin-solubilized CI as negative controls, with 10% FCS as positive control or with 20 mg/liter CVI. Initial experiments included exposure of cells also to soluble collagens III, IV, V, or XIV; to fibronectin; or to laminin, which were ineffective in inhibiting apoptosis and of which CI was chosen as negative control for all subsequent experiments. After an additional 24-48 h, cells were harvested and subjected to several complementary assays to assess apoptosis.

The morphological features of apoptosis (i.e. cell shrinkage and nuclear condensation) were evident with medium alone or CI, whereas cells exposed to CVI or FCS remained well spread and viable after 24 h of treatment (Fig. 1). Initially, cells in each group adhered and spread to an equal extent, but, whereas cells exposed to medium or CI detached after 24 h, cells treated with 10% FCS or CVI remained well spread for up to 96 h (data not shown).

View larger version (191K): [in this window] [in a new window]   Fig. 1.   Serum-starved HT1080 fibroblasts remain viable upon exposure to soluble collagen VI. 2 × 106 HT1080 cells were plated on six-well plates in 1.7 ml of starving medium, which was changed immediately to a medium supplemented with 10% FCS, collagen I (20 mg/liter) or collagen VI (20 mg/liter) or changed to starving medium alone for 24 h. Sections were photographed with a Zeiss phase-contrast microscope at a magnification of 1:100. The morphological features for apoptotic cells include cell shrinkage and nuclear condensation, as is evident in the medium alone and the CI groups, whereas cells exposed to CVI and FCS remain well spread and viable after 24 h of treatment. Similar results were obtained for Balb 3T3 cells.

After 24 h the in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay that highlights apoptotic cells was clearly positive in 15-20% of cells treated with medium alone or CI, contrasting with less than 2% positive cells in the cultures treated with CVI or 10% FCS (Fig. 2).

View larger version (146K): [in this window] [in a new window]   Fig. 2.   Collagen VI prevents in situ DNA fragmentation in HT1080 cells. 2 × 106 HT1080 cells were plated on six-well plates in 1.7 ml of starving medium, which was changed after 2 h to a medium supplemented with 10% FCS, collagen I (20 mg/liter), collagen VI (20 mg/liter) or changed to starving medium alone for 24 h. Cells were harvested by trypsinization, adjusted to the same cell numbers, fixed in 4% paraformaldehyde and 70% ethanol, permeabilized with 0.1% Triton X-100, and 5'-end-labeled with fluoresceine-labeled dUTP. Cytospun cells were visualized under a fluorescence microscope, with apoptotic cells showing bright yellow, condensed nuclei in the groups treated with medium alone or CI.

DNA laddering (degradation into multiples of 180-base pair-long fragments) has been considered as one of the most characteristic signs of apoptotic cell death (1). Serum-starved cells were plated and treated as described before. When the isolated DNA was loaded onto a 2% agarose gel, the cells exposed to medium and CI exhibited typical laddering, whereas no DNA fragmentation was visible in the presence of CVI or 10% FCS (Fig. 3).

View larger version (90K): [in this window] [in a new window]   Fig. 3.   Prevention of DNA laddering in serum-starved HT1080 cells by collagen VI. 2 × 106 serum-starved cells were plated and treated as described before isolation of DNA. After a 24-h treatment as detailed in the legends to Figs. 1 and 2, cellular DNA was extracted and subjected to agarose gel electrophoresis. The cells exposed to plain medium and CI exhibited typical laddering, whereas no fragmentation was visible in the presence of CVI and 10% FCS. standard, DNA ladder standard (180-base pair spacing); medium, starved cells treated with medium alone; CI, collagen I-treated cells (20 mg/liter); CVI, collagen VI-treated cells (20 mg/liter); 10% FCS, cells maintained in 10% FCS; pos. control, apoptotic HL60 cells (positive control). Shown is a representative example of three independent experiments.

To quantify apoptotic cell death and to better delineate its time course, we used the cell death detection ELISA (Fig. 4) and FACS analysis (Fig. 5). Both methods confirmed the antiapoptotic effect of soluble CVI at 20 mg/liter (60 nmol/liter), which was equipotent to 10% FCS. FACS analysis showed that after 24 h of treatment with CVI or 10% FCS, less than 1% of the cells could be identified as apoptotic compared with 21 and 15% in the medium and CI groups, respectively. After 48 h, the percentage of apoptotic cells rose to 47% in the medium and CI group and increased moderately in the CVI (12.5%) and 10% FCS (5.5%) groups, respectively. Additionally, compared with plain medium or CI, a higher number of cells in the CVI and 10% FCS groups were driven into the G₂ and S phase of the cell cycle, as evidenced by doubled or increased DNA content (Fig. 5). After 48 h of treatment, 18 and 19%, respectively, of the cells treated with CVI or 10% FCS could be detected in G₂ compared with 3.8 and 5% in the medium and CI groups.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Time course of apoptotic cell death in serum-starved HT1080 cells (cell death detection ELISA). 4 × 105 cells were cultured in six-well plates and treated with plain medium, CI or CVI (final concentration 20 mg/liter), or 10% FCS. After 6, 12, or 24 h, cells were lysed, and low molecular weight oligonucleosomal DNA (from viable cells) was detected by a sandwich ELISA with antibodies against histones and DNA. Background OD values (lysis buffer alone) were subtracted, and results are shown as enrichment factor as proposed by the manufacturer (enrichment factor = OD (optical density) of the sample (apoptotic cells) divided by the OD of the corresponding control (viable cells, FCS group)). Cells were exposed to the following. M, starving medium; CI, collagen I (20 mg/liter); CVI, collagen VI (20 mg/liter); FCS, 10% FCS. Apoptosis started between 6 and 12 h after serum starvation, reaching an enrichment factor of 9 and 5 after 24 h in the medium alone and CI-treated group, respectively. Treatment with CVI as well as treatment with 10% FCS resulted in strong inhibition of apoptosis during all treatment periods (enrichment factor 1-2). Shown are means ± S.E. of three experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Cell cycle analysis of HT1080 cells exposed to starving medium, collagens, or FCS. Serum-starved HT1080 cells were treated with medium alone, CI, CVI (both at 20 mg/l), or 10% FCS for 24 or 48 h. After fixation in 70% ethanol and RNase digestion, cellular DNA was stained with propidium iodide and subjected to FACS analysis. The integrated area of cellular DNA content was calculated as percentage of cells in G0-G1, G2-M, S phase transition, or apoptosis (hp, hypoploid cells). Shown is a representative of three independent experiments.

Soluble CVI Is More Effective than Immobilized CVI in Preventing Apoptosis-- In order to compare the antiapoptotic effect of added soluble CVI with that of immobilized CVI, we seeded HT1080 cells on the nonadhesive substrates poly-HEMA (Fig. 6B) or BSA (Fig. 6C) or on plastic, CI, or CVI, followed by the addition of soluble CI, CVI, plain medium, or FCS (Fig. 6A). After 24 h, propidium iodide-stained cells were subjected to FACS analysis. Here, soluble CVI was twice as efficient in preventing apoptosis as coated CVI and 4 times more efficient than coated or soluble CI (Fig. 6A). These experiments again showed that soluble CVI is almost as potent as 10% FCS in preventing apoptosis with 8 and 6% apoptotic cells, respectively.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Cell survival on antiadhesive or adhesive surfaces. 106 serum-starved HT1080 cells were seeded into uncoated () six-well plates or plates coated with collagens (CI and CVI) (A), poly-HEMA (pHema) (B), and 1% BSA in PBS (BSA) (C). D, cells were added to uncoated wells 2 h after the addition of 1-integrin receptor blocking antibody P4C10. Wells were then immediately treated with medium alone (), soluble CI (sCI) or CVI (sCVI), or 10% FCS (FCS). After 24 h, cells were subjected to FACS analysis as shown in Fig. 5. The area under the shoulder left of the peak that represents the G0-G1 fraction of the FACS histogram in D (hypopl, hypoploid cells) was taken as the percentage of apoptotic cells. The results shown are means ± S.E. or means of three or four independent experiments.

CVI Prevents Apoptosis via a Partially Spreading and ₁-Integrin-independent Mechanism-- Attachment and spreading were decreased by coating the wells with the nonadhesive substrata poly-HEMA (Fig. 6B) or BSA (Fig. 6C). Since anchorage-dependent cells undergo apoptosis under conditions that prevent attachment and spreading (14), we next investigated whether the anti-apoptotic effect of type VI collagen might be due to promoting attachment and spreading. Cells plated on poly-HEMA showed no spreading in any treatment group, and 40% of cells were apoptotic when treated with plain medium or CI after 24 h compared with 20 and 10% in the CVI-treated and 10% FCS groups, respectively. The survival-promoting effect of soluble CVI on cells plated onto BSA was even more pronounced: less than 1% of the cells died compared with 20-25% apoptotic cells treated with CI or plain medium. These results suggest that spreading-independent mechanisms contribute to the antiapoptotic effect of soluble CVI.

When ₁-integrin function was blocked with monoclonal antibody P4C10 shortly before the addition of CI or CVI, cell spreading was inhibited. Under these conditions, the ratios of apoptotic cells versus cells in G₁ were 3.0, 1.2, and 0.7 for plain medium, collagen I, and collagen VI, respectively, compared with complete suppression of apoptosis by FCS (Fig. 6D). This indicates an important but not exclusive role of ₁-integrins in antiapoptotic signals transduced by CVI.

Proapoptotic Bax Is Down-regulated by Soluble CVI-- To study whether soluble CVI may be regulating key proapoptotic or antiapoptotic proteins, such as Bax and Bcl-2 (4), we determined the levels of these two proteins by quantitative Western blot analysis. The ratio of Bax/Bcl-2 is thought to be crucial for cell survival. Fig. 7 illustrates that in HT1080 cells, relative to the internal standard -actin, Bcl-2 levels remained unaltered upon treatment with CVI or 10% FCS compared with medium or CI, whereas in Balb 3T3 cells, 10% FCS but not CVI caused a 3-fold up-regulation of Bcl-2 (data not shown). In contrast, CVI clearly down-regulated Bax 2-3-fold in both cell lines. This effect was comparable with the 3-fold down-regulation of Bax observed with 10% FCS compared with medium alone or CI. Thus, in HT1080 cells, the Bax/Bcl-2 ratio was decreased 3-fold upon CVI treatment, thus favoring cell survival.

View larger version (28K): [in this window] [in a new window]   Fig. 7.   Expression of Bax and Bcl-2 protein in collagen VI-treated HT1080 cells. HT1080 cells were treated with medium alone, CI, CVI, or 10% FCS for 24 h. Cell lysates were adjusted to equal protein contents, run on a 12% SDS-PAGE, and blotted onto nitrocellulose, followed by sequential incubation with polyclonal rabbit anti-human Bax, monoclonal mouse anti-human Bcl-2, or polyclonal rabbit anti-human -actin. Membranes were developed with anti-mouse-IgG coupled to peroxidase and detected with the ECL chemiluminescence reagent. After stripping in acidic glycine buffer, membranes were reprobed with appropriate primary and secondary antibodies. Bands were analyzed by densitometry, and results are shown as percentage of the 10% FCS-treated control, normalized to cellular -actin content. M, starving medium; CI, collagen I (20 mg/liter); CVI, collagen VI (20 mg/liter); FCS, 10% FCS. B, ratio of Bax/Bcl-2 for HT1080 cells. Shown is a representative example of four independent experiments.

Soluble CVI Induces Cell Cycle Progression through Up-regulation of Cyclins A, B, and D1-- Cyclins A, D, and E have been reported to be regulated by a coordinated stimulation of growth factors and ECM molecules in adhesion-dependent cells (34). Induction of cell cycle progression through up-regulation of specific cyclins by ECM molecules alone has not been reported. Therefore, we analyzed the expression of cyclins A, B, and D1 after the addition of various stimuli including CVI. Expression of the anchorage-dependent cyclin A is up-regulated during S phase, and cyclin B controls cell cycle progression into G₂ (35). Similarly, cyclin D1 controls entry into the G₁-S phase in many cell lines, including fibroblasts, but fails to do so in the absence of ECM signaling (36). Fig. 8 shows that CVI alone up-regulated cyclins A, B, and D1 2-3-fold compared with plain medium or CI, comparable with cells treated with 10% FCS. These results are in full agreement with our cell cycle analysis by FACS scan, which showed that only in the presence of CVI or FCS had a significant percentage of cells progressed to G₂ (Fig. 5).

View larger version (38K): [in this window] [in a new window]   Fig. 8.   Expression of cyclins A, B, and D1 in collagen VI-treated HT1080 cells. HT1080 cells were treated with medium alone, CI, CVI, or 10% FCS for 24 h. Equal amounts of cell lysate were run on 12% SDS-PAGE and blotted onto nitrocellulose, which was incubated with antibodies to (a) cyclin D1, (b) cyclin A, and (c) cyclin B followed by stripping and reprobing with polyclonal rabbit anti-human -actin. Membranes were developed with anti-mouse-IgG coupled to peroxidase and detected with the ECL chemiluminescence reagent. After stripping in acidic glycine buffer, membranes were reprobed with the appropriate primary and secondary antibodies. Bands were analyzed by densitometry, and results are shown as percentage of the 10% FCS-treated control, normalized to cellular -actin content. M, starving medium; CI, collagen I (20 mg/liter); CVI, collagen VI (20 mg/liter); FCS, 10% FCS. *, control cell lysate to test the specificity of antibodies. Shown is a representative example of three experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Adhesion of cells to several ECM proteins can prevent apoptosis under conditions of nutrient and growth factor deficiency. A recent report showed that the soluble, ECM-derived ₁-integrin ligand collagen I can rescue detached and serum-starved neuronal cells from apoptosis (37). Here, by using complementary methods, we could demonstrate that soluble, contrary to immobilized, CVI prevents apoptosis in serum-starved HT1080 and Balb 3T3 cells. The antiapoptotic effect of CVI is comparable with that provided by 10% fetal calf serum and far more potent than that contributed by single, classical growth factors for mesenchymal cells. Furthermore, soluble CVI induced a 2-3-fold down-regulation of the proapoptotic Bax, again comparable with 10% FCS, whereas expression of Bcl-2 was unaffected. In line with our previous observations of the strong activation of DNA-synthesis by soluble CVI (30), its antiapoptotic effect was accompanied by a 2-3-fold up-regulation, compared with medium control, of cyclins A, B, and D1, which are required for progression of the cell cycle from G₁ to G₂. This up-regulation reached 70-80% of cells cultured in 10% FCS.

When offered CVI as adhesive substrate, the viability of corneal cells is enhanced 2-fold compared with a matrix of CI (26). While our data support these findings for immobilized CVI, the soluble form of CVI, compared with soluble collagen I, is even more potent in preventing apoptosis in HT1080 and 3T3 cells.

One explanation for the more pronounced antiapoptotic effect of soluble CVI may be the creation of a pericellular microfilamentous CVI network due to in vitro polymerization (38) that resembles the pericellular CVI matrix in vivo and increases interactions with the cellular CVI receptors. Such interactions can subject cells to increased tension and are accompanied by enhanced spreading (39). The degree of spreading has been shown to correlate with cell survival and proliferation (40). Besides determining proliferation and apoptosis via cell shape alterations, generation of tensile stress may alter responsiveness to growth factors (41). Thus, fibroblasts that are cultured in collagen matrices respond to PDGF signaling with DNA synthesis, while the same cells fail to respond to the growth factor when stress is relaxed in a collagen gel (41). However, when cells were plated on nonadhesive surfaces, we observed no spreading after the addition of soluble CVI, despite a potent antiapoptotic effect, suggesting the existence of spreading-independent pathways of CVI-induced signal transduction.

Whereas the integrins ₂₁ and ₁₁ are receptors for native, triple helical CVI, and ₅₁, _v3, and _IIb3 recognize denatured CVI, they all promote cell migration and spreading after ligation of CVI (29). A nonintegrin CVI receptor, the chondroitin sulfate proteoglycan NG2 (42-45), has been shown to colocalize with the PDGF  receptor. Interestingly, PDGF  receptor signaling is disrupted by NG2 down-regulation, suggesting a cross-talk between CVI and PDGF receptors (46, 47). In accordance with these findings and previous reports (26, 30), the effects of CVI on proliferation and apoptosis were not solely dependent on ₁-integrins, since integrin-blocking antibodies only partially influenced CVI-induced proliferation or apoptosis as shown in Fig. 6D, suggesting that NG2 could be an important proliferation-inducing and antiapoptotic CVI receptor (42-45).

Searching for proteins that are involved in the CVI-mediated prevention of apoptosis, we studied the expression of antiapoptotic Bcl-2 and proapoptotic Bax, the ratio of which determines the susceptibility to programmed cell death in various cell types (48, 49). CVI suppressed Bax up-regulation upon serum withdrawal to a similar degree as treatment with 10% FCS. In addition to growth factors, ECM-receptor interactions have been shown to alter the ratio of Bcl-2/Bax. Accordingly, one previous study showed that engagement of the integrin ₅₁ can promote survival of cells on fibronectin by up-regulation of Bcl-2 expression (50). Since soluble CVI leads to activation of the focal adhesion kinase (51), which has been shown to play an important role in preventing apoptosis (52), focal adhesion kinase could be a central mediator of the antiapoptotic CVI signaling.

Mammalian cells express various cyclin-dependent kinases that function at different stages of the cell cycle. Their function is modulated by the cell cycle-dependent expression of four distinct types of cyclins; in concert with cyclin-dependent kinases 4 and 6, the D-type cyclins drive cell cycle progression through the G₁ phase, which is also promoted by the cyclin E-cyclin-dependent kinase 2 complex, whereas the cyclin A-cyclin-dependent kinase 2 complex carries the cells through S-phase, and cyclin B-cyclin-dependent kinase 2 is responsible for progression through G₂/M. Growth factors and cell anchorage jointly regulate transit through G₁ in almost all cell types, but the molecular basis for this combined requirement remains largely unknown (35). Here, we show that soluble CVI alone is sufficient to drive cell cycle progression through up-regulation of cyclins A, B, and D1, which explains the previously observed stimulation of DNA synthesis by CVI (30). Since the mitogen-activated protein kinase, extracellular signal-regulated kinase-2, a major second messenger in mitogenesis, is activated 20-fold after exposure of HT1080 cells to CVI (51), the observed up-regulation of cyclin D1 might be linked to mitogen-activated protein kinase activation (53). We therefore hypothesize that CVI has intrinsic growth factor-like properties.

A prominent matrix of CVI is found around fibroblasts (16), neural crest (32), and hematopoietic stem cells (20), suggesting a role of this collagen for mesenchymal cell differentiation during development. Increased expression of CVI is found in the ECM of melanomas or gliomas (27, 28) or in chronic fibrotic conditions (54, 55). Thus, CVI-content is increased 10-fold in fibrotic livers, and CVI serum levels are highly elevated in adults and children with advanced renal and hepatic fibrosis (55-57). Therefore, enhanced proteolysis and release of CVI during continuous inflammation and matrix turnover could trigger mesenchymal cell proliferation in an autocrine and paracrine manner.

Considering the potential of CVI to serve as a survival factor for fibrogenic cells, novel strategies for antifibrotic treatment can be envisaged. Thus, CVI receptor-recognizing peptides may specifically target and antagonize activated myofibroblasts, which are the main producers of excess ECM in fibrosis of the liver, kidneys, lungs, and arteries. Recent in vivo experiments show that after intravenous injection more than 50% of a cyclic peptide with specificity for a CVI receptor (58) reaches activated hepatic stellate cells, the major fibrogenic cells in liver (59).

	ACKNOWLEDGEMENTS

We thank Renate Ackermann and Monika Schmid for expert technical assistance and Dr. S. Rosewicz, K. Detjens, and M. Welser for support in FACS analysis.

	FOOTNOTES

^* This project was funded by Sonderforschungsbereich 366 C5 and Deutsche Forschungsgemeinschaft Grant Schu 646/10-2. D.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

^¶ Recipient of a Hermann-und-Lilly-Schilling-professorship. To whom correspondence should be addressed: Med. Klinik I, Universität Erlangen-Nürnberg, Krankenhausstr. 12, 91054 Erlangen, Germany. Tel.: 49-91318533386; Fax: 49-91318536003; E-mail: detlef.schuppan@med1.med.uni-erlangen.de.

	ABBREVIATIONS

The abbreviations used are: ECM, extracellular matrix; BSA, bovine serum albumin; CI, collagen I; CVI, collagen type VI; FACS, fluorescence-activated cell sorting; FCS, fetal calf serum; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; poly-HEMA, poly(2-hydroxymethylmethacrylate); PDGF, platelet-derived growth factor; ELISA, enzyme-linked immunosorbent assay.

	REFERENCES

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