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Cardiovascular Research 2001 52(3):429-437; doi:10.1016/S0008-6363(01)00391-1
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Copyright © 2001, European Society of Cardiology

Serum depletion induces cell loss of rat cardiac fibroblasts and increased expression of extracellular matrix proteins in surviving cells

Monika Leicht*, Wilfried Briest, Alexander Hölzl and Heinz-Gerd Zimmer

Carl-Ludwig-Institute of Physiology, University of Leipzig, Liebigstrasse 27, D-04103 Leipzig, Germany

* Corresponding author. Tel.: +49-0341-971-5500; fax: +49-0341-971-5509 leicht{at}medizin.uni-leipzig.de

Received 4 January 2001; accepted 28 June 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 References
 
Objective: Since reduced nutrient supply is one component of ischemia, we have studied the effect of serum depletion on the survival of fibroblasts isolated from adult rat hearts and on the expression and degradation of extracellular matrix (ECM) proteins. Furthermore, we measured the role of the cAMP-dependent pathway in these processes. Methods: Isolated cardiac fibroblasts were grown to confluency in 10% serum containing medium. Serum was then removed and cell number was measured by use of a Coulter Counter. The activity of the cAMP response element binding protein (CREB) was investigated by Western blotting and subsequent use of the specific antibody which binds to the active form of the protein. The expression of colligin, collagen I and III, matrix metalloproteinases 2 (MMP-2), and tissue inhibitor of matrix metalloproteinase 2 (TIMP-2) was examined by ribonuclease protection assay (RPA) and Western blotting. Zymographic measurements were done to investigate gelatinase activity of MMP-2. Results: Serum withdrawal caused the death of 36% of the cells during the first 8 h. CREB was strongly phosphorylated 5 min after serum removal. Activation persisted up to 8 h and decreased thereafter. The mRNA abundance of colligin, collagen I and III, MMP-2, and TIMP-2 started to increase after 5 and 10 h, respectively, reaching a maximum after 20–30 h and decreasing thereafter. Protein levels of collagen I, collagen III, colligin and TIMP-2 were higher after 24 h until up to 96 h. MMP-2 zymographic activity was elevated 15-fold after 72 h. Application of the protein kinase A (PKA) blocker RpcAMPS suppressed the increase in phosphorylation of CREB. The increase in collagen III and MMP-2 mRNA abundance and elevation of collagen I and III, and TIMP-2 protein was diminished by RpcAMPS. The rise of colligin protein was completely suppressed. The increase in MMP-2 zymographic activity was also attenuated. RpcAMPS improved survival rate from 56 to 84%. Conclusions: Serum depletion led to cell death of isolated cardiac fibroblasts. Survival was associated with the increase in the expression of various ECM proteins. The transcription factor CREB was activated after serum removal. Inhibition of PKA improved the serum depletion induced decrease in the survival rate. The increase in collagen I, collagen III, MMP-2, TIMP-2, and colligin evoked by serum depletion was also diminished by PKA inhibition.

KEYWORDS Cell culture/isolation; Extracellular matrix; Gene expression; Ischemia; Signal transduction; Apoptosis; Protein kinases


   1. Introduction
Top
 Abstract
 1. Introduction
2. Material and methods
 3. Results
 4. Discussion
 References
 
Sustained myocardial ischemia leads to increased expression of collagen I and III in the infarcted region resulting in scar formation. As a consequence, the pumping capacity is reduced, and the risk of congestive heart failure and death rises [1]. Infarction also leads to cell loss which is predominantly due to the death of cardiomyocytes. In this process, apoptosis of myocytes may also be involved, as has been shown in heart failure [2], in hypoxia [3] and in myocardial infarction [4].

Fibroblasts represent the majority of cardiac cells. They are known to produce and deposit the extracellular matrix of the heart, collagen I and III, fibronectin, collagenase, and trophic factors for myocytes, such as transforming growth factor (TGF) β [5–8]. Fibroblasts do also play an important role in the repair of the infarcted heart muscle. In ischemia-reperfusion, cardiac fibroblasts undergo hyperplasia and increased collagen production (reviewed in Ref. [9]). During ischemia, cells do not only experience oxygen deficiency, but also restricted supply of nutrients. Several studies have investigated the effect of serum removal on the disintegration of various cell types and the regulation by signaling proteins. In vascular smooth muscle cells, it was shown that serum deprivation enhanced the mRNA levels of type I and III collagens [10]. Matrix metalloproteinases (MMPs) are involved in the regulation of cardiac remodeling. Increased expression and activity of various MMPs has been detected after myocardial infarction [11–14]. During pathological fibrosis, not only the level of collagen I, but also that of its chaperone colligin is elevated [15].

In the present study, we have established a cell culture model in which isolated cardiac fibroblasts were deprived of serum. The aim of the study was to investigate the significance of the protein kinase A (PKA) signal transduction pathway in cell survival and the expression of ECM proteins. We tested the hypothesis that nutrient depletion induces changes in ECM composition in this cell culture model.


   2. Material and methods
Top
 Abstract
 1. Introduction
 2. Material and methods
3. Results
 4. Discussion
 References
 
Animals used in this study were maintained in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996). RpcAMPS (adenosine 3',5'-cyclic monophosphorothiolate-Rp) was obtained from Calbiochem (Bad Soden, Germany).

2.1 Cell culture
Cardiac fibroblasts were isolated from 200–250 g female Sprague–Dawley rats (Charles River, Sulzfeld) as previously described [16]. Cells were grown in Dulbecco’s minimal essential medium (DMEM)/Ham’s F-12 (Biochrom, Berlin, Germany)/10% fetal calf serum (FCS, Biochrom)/1% penicillin/streptomycin to confluency and were then passaged once. Immunofluorescence and Western blotting with anti {alpha}-smooth muscle actin (Progen, Heidelberg, Germany) and anti β-actin antibodies (Immunotech, Marseille, France) were used to test for contamination with smooth muscle cells. Tests for contamination with endothelial cells were done by staining with anti-human factor VIII (DAKO, Glostrup, Denmark), for pericytes with anti-desmin (Sigma, Munich, Germany) and for macrophages with anti-ED 1 (Pharmingen, Hamburg, Germany) antibodies. Cardiac myocytes can easily be characterized by their morphology. Positive immunostaining was obtained by staining with anti-vimentin antibodies (DAKO). The culture contained more than 95% fibroblasts.

For all experiments subsequently described, cells were seeded at 7–8x103 cells/cm2 and were again grown to confluency. Serum containing medium was then removed, and fresh DMEM/1% penicillin/streptomycin without FCS was added for the indicated periods of time.

2.2 Determination of cell number
Cells were grown on 12 well plates. At the time points given in the figures, cells were washed with PBS, detached from the dish by treatment with 0.5% trypsin-EDTA and suspended in 1 ml PBS. The number of viable cells was determined 48 h after the addition of the agents using a Coulter Counter Channelyzer (Coulter Electronics, Krefeld, Germany). Measurements were done in triplicate in volumes of 200 µl each. The orifice tube had an aperture size of 100 µm.

2.3 Gel electrophoresis and immunostaining
SDS–PAGE and Western blotting was performed as previously described [17]. Cells were lysed in 50 mM Tris–Cl, pH 6.7, 2% sodium dodecyl sulfate (SDS), 2% mercaptoethanol, and 1 mM sodium orthovanadate (Laemmli buffer) followed by digestion of nucleic acids with benzonase. The lysates were heated for 10 min at 95°C, and the protein content was quantified according to [18]. Gels of 0.75 mm thickness were loaded with 10 µg protein per lane for the detection of CREB and colligin. For the detection of collagen I, collagen III and TIMP-2, gels of 1.5 mm thickness were loaded with 50 µg protein per lane. To assess the release of collagen I and III into the media, 100 µg/ml β-aminoproprionitrile (Sigma) was applied to the media during serum starvation to prevent collagen crosslinking. Proteins were precipitated by addition of 30% ammoniumsulphate, and lysates were prepared as described above.

The following antibodies were used: anti-phospho-CREB and anti-CREB (both 1 µg/ml, New England Biolabs, Schwalbach, Germany), anti-colligin (hsp47, 1 µg/ml, Stressgen Biotechnologies, Victoria, British Columbia, USA), anti-collagen I and anti-TIMP-2 (all 0.5 µg/ml, Santa Cruz Biotechnology, Heidelberg), anti-collagen III (1 µg/ml, Southern Biotechnology, Birmingham, AL, USA). Horseradish peroxidase labeled sheep-anti-mouse IgG, goat-anti-rabbit IgG antibodies, and rabbit-anti-goat were from Dianova (Hamburg, Germany).

2.4 RNase protection assay
Total RNA isolation was performed according to a modified phenol/guanidiniumthiocyanat (GTC) method of Chomczynski and Sacchi [19]. As previously described [20], 5 µg of total RNA was used in the RNase protection assay (RPA) with the probe template set labeled with RiboQuant®In Vitro Transcription Kit (Pharmingen; final probe concentration: 4x105 cpm/µl) and [{alpha}-32P] UTP (3000 Ci/mmol, Amersham, Freiburg, Germany), as described by the manufacturer. After hybridization (56°C; 12–16 h), the unhybridized riboprobe was digested with a mixture of RNases A and T1 (RiboQuant® RPA Kit, Pharmingen), according to the manufacturer’s instructions. Protected probes were displayed by electrophoresis on a denaturing gel containing 5% polyacrylamide/8 M urea followed by visualization with the Molecular Imager (BioRad, Munich, Germany). The densitometric quantification of the individual bands of the RPA assays was performed by the Multi-Analyst program version 1.1 (BioRad). The probe template set contained the following cDNA (probe length in bp/protected): rat collagen I (504/449), rat TIMP-2 (404/326), rat MMP-2 (360/269), rat collagen III (286/211), rat colligin (225/161), rat ARPP (143/113) and rat GAPDH (128/82).

2.5 Preparation of cell culture samples and zymography
For gelatinase measurements, aliquots of cell culture supernatants of fibroblasts cultured with FCS or after serum depletion were used. Myocardial matrix metalloproteinase activity in the gel was measured as described by Tyagi and coworkers [21]. Gelatin (0.1% (w/v), Merck, Darmstadt, Germany) was added to standard Laemmli acrylamide polymerization mixture. Culture supernatants were mixed 1:16 or 1:2 for supernatants after serum depletion with substrate gel sample buffer (10% (w/v) SDS, 4% (w/v) sucrose, 0.25 mM Tris–Cl pH 6.8 and 0.1% (w/v) bromphenol blue), 0.5 µg of protein of supernatant were loaded immediately without boiling. Gels were run at 20 mA at 4°C. Following electrophoresis, the gels were soaked in 2.5% (w/v) Triton X-100, incubated overnight at 37°C in substrate buffer (50 mM Tris–Cl pH 8.5 mM CaCl2 and 0.02%/w/v) NaN3), stained for 15–30 min in 0.05% Coomassie Blue R-250 in acetic acid:methanol:water (1:4.5:4.5 by volume), destained in 10% acetic acid/5% methanol and scanned for lysis band intensity. The lysis band intensity was used to estimate the collagenase activity. A gelatinase zymography standard (human MMP-2 and -9, Chemicon, Hofheim, Germany) was used to detect the correct band.

2.6 Statistical analysis
All data were analysed and expressed as mean±S.E.M. The data were first compared by analysis of variance (ANOVA). Evaluation of statistical significance was performed by use of a post hoc test employing the Student–Newman–Keuls method (SigmaStat 2.0®, Jandel Corporation). A value of P<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
4. Discussion
 References
 
3.1 Measurement of cell number
Fig. 1 shows a reduction in cell number by 36% which occurred during the first 8 h following serum removal. It started with a delay of 5 h, occurred rapidly up to 7 h, and slowed down thereafter. The cell number remained constant at approximately 54% of control up to 38 h. After 56 h, cell number had decreased to 45% of control.


Figure 1
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  		Fig. 1 Number of cardiac fibroblasts after serum removal. Primary cultures were passaged after 6 days and seeded at 7–8x103/cm2 on 12 well plates. Cells were grown to confluency in DMEM supplemented with 10% calf serum. The medium was then replaced by DMEM without serum. After the indicated time intervals, cells were trypsinized, and the number of viable cells was determined by use of a Coulter Counter Channelyzer. The cell number which fibroblasts had reached at the start of the experiment was set to 100%. Each point represents the mean±S.E.M. of five separate experiments. Measurements were done in duplicate. * P<0.05 vs. FCS treated, time matched controls.

 
3.2 Expression of ECM proteins
Fig. 2A shows the result of ribonuclease protection assay (RPA) analysis of the expression of collagen I, collagen III, colligin, matrix metalloproteinase 2 (MMP-2), and tissue inhibitor of matrix metalloproteinases 2 (TIMP-2) mRNA in cardiac fibroblasts after the indicated time periods of serum deprivation. The mRNA abundance of collagen I and its chaperone colligin started to increase after 10 h, the levels of collagen III, MMP-2, and TIMP-2 mRNA already after 5 h (Fig. 2A). The mRNA levels reached a maximum after 24 h. Collagen I mRNA was increased 2.1-fold of control level, colligin mRNA 1.7-fold, collagen III 2.4-fold, and TIMP-2 1.9-fold. MMP-2 mRNA was elevated 2.8-fold of control after 30 h. After 72 h, mRNA levels had returned almost to the respective control levels. Collagen I, collagen III, colligin, and TIMP-2 protein levels were investigated by Western blotting (Fig. 2B). For collagen I, collagen III, and TIMP-2, there was hardly any or no immunoreactivity in control cells. These proteins were detected already after 1 day of serum starvation. The maximum was reached for all proteins after 48–72 h. Colligin protein, however, was detected in untreated cells. Its level increased 2.7-fold of control after 24 h. The maximum was 3.4-fold after 72 h. The release of collagen I and III to the media is also shown in Fig. 2B (bottom). Again, there was hardly any immunoreactivity in controls, while a release was detected for both collagen I and III after 72 h.


Figure 2
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  		Fig. 2 Temporal profile of mRNA and protein abundance of various ECM proteins and of MMP-2 activity. Cardiac fibroblasts were grown to confluency in 10 cm petri dishes, and serum-free DMEM was given for the indicated time intervals. (A) mRNA abundance. RNA was prepared as described in Materials and methods and the abundance of collagen I-, collagen III-, colligin-, TIMP-2, and MMP-2-mRNA was investigated by RNase protection assay. Data were normalized to GAPDH mRNA, and expressed as percentage increase with non-starved controls being set to 100%. (B) Protein levels. Cells were lysed as described in Materials and methods, and the lysates were subjected to Western blotting analysis using the specific antibodies. Top: cell-lysates; bottom: lysates from cell culture media. Detection was performed by chemoluminescence. The data are representative of five experiments. (C) MMP-2 activity. Cell culture supernatants were collected after the indicated time periods of serum depletion. Gelatinase activity was tested as described in Materials and methods. A representative gel is shown (top). Gels were quantified by laser scanning densitometry. The level of non-starved controls is set to 100%. All data are means of five experiments. Means±S.E.M. * P<0.05 vs. non-starved cells and supernatants of non-starved cells, respectively.

 
3.3 MMP-2 activity
Fig. 2C shows the result of measurements of MMP-2 zymographic activity in the cell culture supernatant during serum starvation. Representative lysis bands which appear due to the gelatinase activity of MMP-2 are also provided. Gels were scanned, and the results of five experiments are summarized. There was a steep, almost linear elevation of MMP-2 activity up to 24 h and a less pronounced elevation thereafter with a maximum after 72 h (Fig. 2C).

3.4 Activation of CREB
Further experiments were carried out to elucidate a possible signaling pathway involved in the regulation of the processes described above. Serum starvation caused a strong activation of the cAMP response element binding protein (CREB) as assessed by Western blotting and use of a phospho-specific antibody (Fig. 3A). The strongest phosphorylation was measured after 5–60 min. After 5 h, the amount of phosphorylated CREB reached control-level again. The amount of CREB remained constant throughout the experimental period.


Figure 3
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  		Fig. 3 Time course of CREB phosphorylation. (A) CREB phosphorylation during serum removal. Confluent cells were deprived of serum. After the time periods indicated, cells were lysed, and the phosphorylation of CREB was investigated by Western blotting using the phospho-specific antibody (upper panel). The antibodies were then removed from the blot membrane, and the amount of CREB protein was detected by use of a specific CREB antibody. The result is shown in the bottom panel. Phospho-CREB and CREB were detected by chemoluminescence. (B) Effect of RpcAMPS on CREB phosphorylation during serum-starvation: 0.1 µM RpcAMPS was added 30 min before (0) and for the time periods of exposure to serum-free medium (5–60 min). Western blots were performed as described for (A). All Western blots are representative of five separate experiments.

 
3.5 Effect of PKA blockade on CREB phosphorylation, ECM expression and activity, and cell survival
In many cell types, CREB is phosphorylated by protein kinase A (PKA). To examine whether PKA may also activate CREB in cardiac fibroblasts, the specific PKA inhibitor RpcAMPS was used. Fig. 3B shows that 30 min preincubation with RpcAMPS (0.1 µM) prior to serum removal suppressed the activation of CREB. To investigate whether PKA and CREB regulate cell death and the expression of the ECM proteins, RpcAMPS was applied during serum removal. The concentrations used were those with the strongest effect and had no influence on non-starved cells. At first, we investigated whether PKA blockade reduced the increase in collagen I, collagen III, colligin, MMP-2, and TIMP-2 mRNA expression which appeared 24 h after serum depletion. A significant inhibition was found for collagen III and MMP-2 mRNA. After 24 h, 1 µM RpcAMPS had reduced the increase by 31 and 35% for collagen III and MMP-2 mRNA, respectively, over non-starved cells (Fig. 4A). However, no significant effect was found for collagen I, colligin and TIMP-2 mRNA. Fig. 4B depicts changes in the immunoreactivity of collagen I, collagen III, colligin, and TIMP-2. Compared to the protein levels obtained after 72 h, PKA-blockade by 1 µM RpcAMPS caused a reduction in collagen I and also of collagen III protein by 26%, and of TIMP-2 by 25%. The amount of colligin protein was determined after 66 h. The increase in colligin expression induced by serum depletion was completely suppressed (Fig. 4B). At shorter periods of PKA blockade, no difference to serum-deprived cells was found. The elevation of MMP-2 zymographic activity was suppressed by 0.1 µM RpcAMPS from 10.3- to 5.6-fold after 24 h (Fig. 4C).


Figure 4
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  		Fig. 4 Effect of RpcAMPS on the increase in collagen I, collagen III, colligin, MMP-2, TIMP-2 mRNA and protein and MMP-2 activity induced by serum removal. To study this, 1 µM RpcAMPS was added 30 min before and at the time serum-free medium was given (starved+RpcAMPS). After 24 h, the cells were harvested. (A) RNA was subjected to RNase protection assay. The level of mRNA/GAPDH abundance of non-starved controls is set to 100%. (B) For the detection of protein levels, cell lysates were prepared after 72 h. Bands from Western blots on X-ray films were quantified by laser scanning densitometry. The levels of collagen I and III, and of TIMP-2 (left) are set to 100% (starved). For colligin (right), the level of non-starved cells is set to 100% (non-starved). (C) MMP-2 gelatinase activity was measured in cell culture supernatants collected 24 h after serum depletion (starved) and the addition of 0.1 µM RpcAMPS which was applied 30 min before and at the time serum-free medium was given (starved+RpcAMPS), respectively. Gels were quantified by laser scanning densitometry. MMP-2 activity in non-starved controls is set to 100%. All data are means of four experiments. Means±S.E.M. * P<0.05 vs. non-starved cells, + P<0.05 vs. starved cells.

 
3.6 Effect of PKA blockade on cell survival
The induction of cell death by serum removal was attenuated by PKA inhibition (Fig. 5). While only 64% of the cells survived after 24 h of serum depletion, preincubation and simultaneous addition of RpcAMPS (0.1 nM) increased the survival rate to 84%.


Figure 5
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  		Fig. 5 Effect of RpcAMPS on the decrease in cell number induced by serum removal. Confluent cells were deprived of serum (starved). To study this, 0.1 nM RpcAMPS was added 30 min before and at the time serum-free medium was given (starved+RpcAMPS). Cell number was determined after 24 h using a Coulter Counter Channelyzer. Each column represents the mean±S.E.M. of three separate experiments in duplicate determinations using cells from each isolation. The cell number which non-starved fibroblasts had reached at the end of the experiment (after 24 h) was set to 100%. * P<0.05 vs. non-starved cells, + P<0.05 vs. starved cells.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
References
 
4.1 Cell loss after serum removal
Our present investigation shows that nutrient deprivation induces a reduction in cell number. This result adds to many other studies which describe cell death after serum depletion for other cell types. The rate and kinetics of cell death are similar to the ones described here [22,23].

4.2 Increased expression of extracellular matrix proteins
We have provided evidence that the expression of the investigated ECM proteins, collagen I, its chaperone colligin, collagen III, matrix metalloproteinase 2 (MMP-2), and tissue inhibitor of matrix metalloproteinases 2 (TIMP-2) was greater in the surviving cells. Thus, serum depletion leads to the loss of about 50% in cardiac fibroblasts, while ECM production is strongly increased in the remaining cells. This process might provide a mechanism by which the space between surviving fibroblasts due to the occurring cell loss is refilled by ECM in order to reestablish cell–cell contact. Our findings are in line with data from Kindy et al. [10], who reported earlier that mRNA levels for collagen I and collagen III are increased in vascular smooth muscle cells (VSMC) after serum deprivation. The authors hypothesized that cell–cell contacts and cell growth state influence the expression of collagen genes. Further evidence of an inverse regulation of proliferation and the expression of extracelluar matrix is given by others [24,25]. Collagen synthesis in post-confluent and proliferating cells was investigated more precisely by Majors and Ehrhart [26]: The authors measured a decrease in collagen synthesis in post-confluent versus proliferating cells. However, the percentage of collagen production of total protein synthesis was higher in post-confluent cells. The impact of fibroblast cell death on the deposition of ECM has still to be investigated thoroughly during myocardial infarction where collagen I and collagen III mRNA are also increased [20,27]. The expression of colligin and collagen I is tightly coregulated [28]. Besides its function as a chaperone in the biosynthesis and secretion of procollagen, colligin, which has also proved to be a heat shock protein, carrying the name hsp47, might as well be involved in cell survival [15,29,30]. The increase in MMP-2 mRNA abundance and its elevated gelatinase activity which we measured zymographically in the cell culture supernatant might be necessary to reorganize the extracellular matrix via digestion of its components. Elevated expression and activity of MMPs, including MMP-2, was already detected after myocardial infarction [11–13,20]. There is a huge number of studies investigating the role of metalloproteinases in cardiac matrix remodeling (reviewed in Refs. [31,20]). TIMPs bind to the active site of MMPs thereby blocking the access to their substrate (reviewed in Ref. [32]). In the present study, we measured a parallel increase of TIMP-2 and MMP-2 expression and also of MMP-2 zymographic activity. A similar coincidence was observed after experimental infarction in rat hearts [20]. We have demonstrated here that in cardiac fibroblasts MMP-2 mRNA increased by 2.8-fold, while TIMP-2 mRNA was upregulated 1.9-fold (Fig. 2A). We also provided evidence for elevations on the protein level (Fig. 2B and C). If we assume that the increase in MMP-2 mRNA is translated into an increased protein level as was actually shown for TIMP-2 (Fig. 2B), this may represent one mechanism for an increase in MMP-2 activity that we measured zymographically [32]. Zymography, however, does not reveal the possible influence of regulating proteins, e.g. TIMPs, on MMP activity. We may therefore suggest that the parallel increase in TIMP-2 protein might suppress MMP-2 activity in the cell culture supernatant so that a net accumulation of collagens can still occur.

4.3 Cellular signaling
The research of cell–cell contact regulation has gained great interest. The transduction of signals, e.g. activation of protein kinases, from the extracellular matrix to the cell is being investigated in several cell types [33,34]. We have found increased activity of the transcription factor cAMP response element binding protein (CREB) after serum withdrawal. This result adds to other publications which report a rise in the amount of cAMP during serum deprivation [35]. This signal started to occur 5 min after serum removal. By blocking protein kinase A (PKA), we found that there is a relation between the activation of PKA and CREB and the increased expression and activity of ECM proteins which was detected hours later. Our results show that in cardiac fibroblasts, PKA is involved in the regulation of cell death, since inhibition of its activity led to increased survival. This is in agreement with results obtained by others who found that cAMP dependent pathways are involved in the induction and enhancement of apoptosis in various cell types such as Jurkat T cells, Molt 4 T cells, cerebral cortical cells, leukemia and neuroblastoma cells [36–40]. The role of cAMP-dependent processes in the regulation of survival and apoptotic cell death is, however, controversial: In several other cell types, such as neuronal cells, VSMC, and PC12 cells, activation of cAMP-dependent signaling provides protection from apoptosis [41–44]. The function of cAMP may vary depending on whether the rise occurs after differentiation or during proliferation [45].

We have found that PKA inhibition also suppressed the serum depletion-induced elevation in colligin, collagen III, MMP2, and TIMP-2 expression, and MMP-2 activation. Earlier, CREB was identified as a regulating molecule for the expression and activity of MMP-2 in human melanoma cells and also of MMP-1 in synovial cell hyperfunction in patients with rheumatoid arthritis [46,47]. Activation of CREB-binding protein CBP was recently shown to be involved in the increased expression of collagen I in human skin fibroblasts [48]. While improved survival was already obtained with 0.1 nM RpcAMPS, the serum deprivation-induced increase in the expression of ECM proteins was reduced by 1 µM. Thus, different processes, cell loss and increased expression of ECM proteins, seem to exhibit different sensitivities to PKA activity.

4.4 Complex control by PKA
PKA and CREB activation are involved in the regulation of serum deprivation-induced cell death of cardiac fibroblasts, since suppression of its activity led to the elevation of the survival rate. The increased expression of ECM proteins in the surviving cardiac fibroblasts also seems, at least in part, to be due to PKA and CREB activation. Thus, the same signal transduction pathway seems to be responsible for two divergent responses to serum depletion.

Time for primary review 18 days.


   Acknowledgements
 
This study was supported by the Deutsche Forschungsgemeinschaft (Zi 199/10-1, Zi 199/10-3). The authors thank Mrs. Grit Marx for excellent technical assistance.


   References
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 References
 

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