1 Renal Cell Biology Laboratory and Vascular Biology Institute, and 2 Department of Medicine, University of Miami School of Medicine, Miami, Florida, 3 GRECC and Research Service, VA Medical Center, Miami, Florida, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods. Cells were exposed to increasing doses of CsA (0, 0.5, 1 and 5 µg/ml). Proliferation was evaluated by bromodeoxyuridine (BrdU) incorporation, viability by Trypan Blue exclusion and apoptosis by ELISA. Type I collagen was measured by ELISA and reverse transcription-polymerase chain reaction (RT-PCR), matrix metalloproteinases (MMP) by zymography and RT-PCR, and tissue inhibitors of MMP (TIMP) by reverse zymography.
Results. CsA exposure for 48 h decreased osteoblastnumber in a dose-dependent manner in the absence of apoptosis or cytotoxicity. CsA at a dose of 5 µg/ml for 72 h caused decreased collagen type I mRNA expression and protein accumulation. While MMP-2 remained unaffected, MMP-9 activity increased. TIMP-1 activity was unaffected, while a dose-dependent increase of TIMP-2 was observed.
Conclusions. These data suggest that CsA alters ECM synthesis and degradation in MC3T3-E1 osteoblasts by decreasing type I collagen production and increasing MMP-9 activity. The combination of increased MMP-9 with unchanged TIMP-1 activity could reduce the osteoid matrix available for mineralization. In addition, decreased proliferation could further reduce the number of cells synthesizing new osteoid matrix and thus contribute to the process of bone loss.
Keywords: cyclosporine; extracellular matrix; osteoblasts
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The aim of this study was to investigate if CsA has a direct effect on osteoblast proliferation, viability and/or ability to synthesize or degrade ECM.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Growth curve studies and CsA treatment
Since experiments were performed with cells grown in media supplemented with 0.1% FBS, a concentration not previously reported to support MC3T3-E1 osteoblast growth, growth curves were compared between cells supplemented with either 0.1 or 10% FBS. Cells were plated into 24-well dishes at a density of 10 000 cells per well. After 24, 72 and 120 h, cell morphology was observed, cell counts were performed using a Coulter Counter (Coulter, Inc., Miami, FL) and cell viability was evaluated by Trypan Blue exclusion. Growth curves with different doses of CsA (0.5, 1, 5 and 10 µg/ml) in the culture media were performed and expressed as absolute cell number. This dose range was established according to a previous report [9]. CsA (IVAX Corporation, Miami, FL) was dissolved in dimethylsulphoxide (DMSO) at a final concentration of 0.1%. The same concentration of DMSO in medium was used as control. Cell counts and cell viability were evaluated as above for each of the doses tested after 24, 72 and 120 h. Concentration of CsA that did not affect cell viability (between 0.5 and 5 µg/ml) were chosen for all experiments.
Bromodeoxyuridine (BrdU) incorporation
Cells were plated and treated as described above for either 24 or 48 h. A colorimetric immunoassay for the quantification of cell proliferation based on the measurement of BrdU incorporation was used according to the manufacturer's instructions (Boehringer Mannheim, Indianapolis, IN). The absorbance values were in the linear range of a curve obtained when BrdU incorporation was performed on cells grown at densities ranging from 10 000 to 120 000/cm2.
Apoptosis
Cells were plated and treated as described above. Cell layers were collected after a total of 24, 48 and 72 h of treatment. Apoptosis was evaluated by ELISA for the determination of histone-associated DNA fragments released in the cytoplasm during the apoptotic process according to the manufacturer's instructions (Cell Death Detection ELISA PLUS, Boehringer Mannheim).
Alkaline phosphatase activity
Cells were plated in 96-well plates at 10 000 cells per well in -MEM supplemented with 10% FBS, allowed to attach for 46 h, then the media was changed to media supplemented with 0.1% FBS and containing DMSO (0.1%) or CsA (1 or 5 µg/ml). The cells were subsequently incubated for up to 72 h, and alkaline phosphatase activity was assayed directly using a plate reader as reported previously [10]. Data are presented as per cent of vehicle-treated (0.1% DMSO) cells, with n=6 wells per group in all cases.
Elisa for collagen type I
The number of cells plated was adjusted in order to reach a comparable density at the time of collection. After 12 h, CsA was added to duplicate wells at concentrations of 0.5, 1 and 5 µg/ml. Control cells were exposed to 0.1% DMSO. Sample collection and ELISA were performed as described previously [11], with some modifications described below. Briefly, the standards were plated in the linear portion of the curve that ranged between 3 and 0.094 ng/µl of collagen type I (Collaborative Biomedical Products). The plates were incubated at 37°C for 2 h, then 30 min at room temperature in blocking solution (PBS/0.05% Tween-20/0.25% BSA), followed by an overnight incubation at 4°C with a rabbit anti-mouse type I collagen IgG antibody diluted 1:2000 (Biodesign, Kennebunk, ME). A 1:2000 dilution of a biotinylated goat anti-rabbit IgG antibody was used as secondary antibody (Biosource International, Camarillo, CA) and incubated for 2 h at room temperature. Final values were expressed in ng/100 000 cells.
Gelatin zymography and reverse zymography
Cells were plated and treated in 24-well plates as described above. Collection of supernatants was performed and cell number was determined after 24, 48 and 72 h. Conditioned media was centrifuged to remove cellular debris. The media was diluted appropriately with regular media (0.1% FBS) to normalize to an identical cell number (5000 cells). Zymography and reverse zymography were performed as described previously [12]. The gels were analysed on a Macintosh computer using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/ nih-image/). Data from each gel were normalized for average integrated density.
Reverse transcription-polymerase chain reaction (RT-PCR)
Cells were plated into six-well plates in 10% FBS. The media was changed to 0.1% FBS media 12 h after seeding and CsA at either 0.5 or 1 µg/ml was added. Total mRNA was collected after 72 h with Tri-Reagent (MRC, Inc, Cincinnati, OH). Reverse transcription and PCR were performed as described previously [12]. To exclude genomic DNA contamination, one RT reaction for each sample was performed without enzyme (AMV). In addition, all primer pairs used were located on different exons. The mRNA expression of 1 type I collagen (697 bp), MMP-2 (760 bp), MMP-9 (414 bp) and MMP-13 (942 bp) were analysed and normalized to GAPDH (562 bp), used as a housekeeping gene. The primer sequences used were published previously [11,12].
Data analysis
Each experiment was performed in duplicate or triplicate, and three to four independent experiments were repeated. Results are expressed as mean±SD. The results were analysed using analysis of variance (ANOVA). When ANOVA showed a statistically significant difference, a group-by-group comparison was performed using a t-test with a Tukey's correction for multiple comparison.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effect of CsA on cell growth
Growth curves were performed with increasing concentrations of CsA (0.5, 1, 5 and 10 µg/ml) in the culture media. DMSO 0.1% was used as control. After 120 h of CsA exposure, cell number decreased for all concentrations of CsA when compared with DMSO treatment. DMSO alone did not affect cell growth (Figure 1). Trypan Blue exclusion showed that CsA reduced cell viability at only the highest concentration tested (10 µg/ml) after 72, 96 and 120 h of treatment (24, 31 and 40% Trypan Blue uptake, respectively). Trypan Blue uptake was not significantly increased in cells treated with 5, 1 and 0.5 µg/ml at any of the time points tested. Therefore, the maximal concentration of CsA chosen for subsequent experiments was 5 µg/ml.
|
Effect of CsA treatment on cell proliferation
BrdU incorporation was performed after 24 and 48 h of treatment with different doses of CsA (0.5, 1 and 5 µg/ml) or DMSO 0.1% to investigate if the decrease in cell number observed after CsA treatment was due to reduced DNA synthesis.
After 24 h (Figure 2a), only the maximal dose of 5 µg/ml reduced DNA synthesis compared with DMSO treated cells (0.74±0.05 vs 1.23±0.11, respectively; P<0.05), while after 48 h of treatment (Figure 2b
) both 1 and 5 µg/ml CsA were associated with decreased DNA synthesis (1.00±0.06 and 0.75±0.05, respectively, vs 1.19±0.04 control; P<0.05 and P<0.001, respectively).
|
Effect of CsA on cell death
In order to investigate whether increased cell apoptosis contributed to the reduction in cell number secondary to CsA exposure, we examined apoptosis after adding CsA (1 µg/ml). Apoptosis was considered as the ratio between the test samples and a positive control provided by the manufacturer and was found to be 5±3% both for treated and untreated cells after 24, 48 and 72 h exposure to 1 µg/ml CsA.
Effect of CsA on alkaline phosphatase activity
Alkaline phosphatase activity is a measure of functional activity of osteoblasts. Our results show a time- and dose-dependent decrease in alkaline phosphatase activity as a function of CsA treatment. There was no effect after 24 h of treatment. After 48 h, CsA at a concentration of 5 µg/ml significantly inhibited alkaline phosphatase activity (90.7% of vehicle-treated control; P<0.05), and after 72 h CsA was inhibitory at concentrations of both 1 µg/ml (96.1% of control; P<0.05) and 5 µg/ml (89.0% of control; P<0.05).
Effect of CsA on type I collagen
Type I collagen is the main collagenous component of the osteoid matrix. Although preliminary experiments suggested that CsA exposure for 24 or 48 h was not associated with a reduction in type I collagen protein accumulation, there was a dose-dependent reduction in collagen type I protein and mRNA levels after 72 h. There was a decrease in the level of type I collagen protein in the supernatants and cell layers after exposure to 5 µg/ml CsA as assessed by ELISA (305.6±29.9 vs 510.8±21.2 ng/100 000 cells; P<0.05) (Figure 3). The mRNA expression for
1 type I collagen also decreased in a dose-dependent manner (Figure 4
).
|
|
Effect of CsA on collagen degradation
The effect of CsA exposure on collagen degradation was evaluated by examining MMP-2 and MMP-9 activity and mRNA expression, MMP-13 mRNA expression, and TIMP-1 and TIMP-2 activity. We initially examined MMP-2, MMP-9, TIMP-1 and TIMP-2 activity after 24, 48 and 72 h of CsA exposure and found that the earliest changes occurred after 72 h. MMP-2 and MMP-9 activity was studied on separate gels to avoid false negative results due to saturation of the gelatin digestion. MMP-2 activity (data not shown) and mRNA expression (Figure 4) remained unchanged after CsA exposure. MMP-9 activity was almost undetectable in untreated cells and increased after 72 h of CsA exposure (Figure 5
). Quantification of MMP-9 activity was not possible due to the weak signal by zymography. MMP-9 mRNA expression was unaltered by CsA (Figure 4
), as was the expression of MMP-13 mRNA (Figure 4
). MC3T3-E1 cells produced both TIMP-1 and TIMP-2 under control conditions. TIMP-1 activity was unaltered by CsA exposure, while there was a significant dose-dependent increase in TIMP-2 activity when cells were treated with 5 µg/ml CsA (1.67±0.2 integrated density vs 0.52±0.1 control; P<0.01) (Figure 6
).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Herein, we show that MC3T3-E1 osteoblasts proliferate in 0.1% FBS, a condition required for some of the experiments of the study. These cells are a well established non-transformed mouse osteoblastic cell line which retain many of the characteristics of primary cultures of osteoblasts in terms of cell maturation and differentiation [13]. CsA treatment resulted in a dose-dependent decrease in cell number, which was due to an antiproliferative activity rather than the induction of apoptosis. Only the highest concentration of CsA tested (10 µg/ml) affected cell viability. Our data are consistent with previous observations on rat osteosarcoma cells, where CsA was shown to have antiproliferative activity, without changes in cell viability [9]. The antiproliferative effect of CsA is in contrast to in vivo data, where an increase in the number of osteoblasts per trabecular surface area was found after CsA treatment [4]. However, a CsA-mediated decrease in trabecular bone [14] could have accounted for an increase in the relative number of osteoblasts.
Our data on alkaline phosphatase activity as an index of osteoblast function are also consistent with previous data on rat osteosarcoma cells, where an inhibitory effect of CsA was observed [9].
Our work is the first to investigate the effects of CsA on bone extracellular matrix synthesis and degradation in vitro. The earliest changes in the amount of type I collagen protein and MMPs activity were observed after 72 h, suggesting that long term, rather than acute, effects of CsA are responsible for altered collagen synthesis and degradation by osteoblasts. CsA treatment resulted in decreased collagen type I mRNA expression and net protein accumulation. The decreased accumulation of type I collagen could contribute to the decreased formation of non-mineralized bone.
Matrix degradation was less affected by CsA treatment. MMP-13 is the main degradative enzyme for type I collagen, while the potential ability of MMP-2 and MMP-9 to digest collagen type I is described in one single report for MMP-9 [15] and for MMP-2 [16]. We found that CsA did not alter mRNA expression of MMP-2, MMP-9 or MMP-13 by mouse osteoblasts. Similarly, the activity of MMP-2 was not altered by CsA treatment, while MMP-9 activity, almost undetectable at baseline conditions, increased. The discrepancy between MMP-9 activity and mRNA expression could result from post-transcriptional regulation of MMP-9, reflecting MMP-13-mediated activation of MMP-9 [17]. The effect of CsA on the activity of MMP-13 will be the subject of future studies. The increase in MMP-9 activity without modification of TIMP-1 could account for net increased bone degradation. However, an increase in TIMP-2 was also found. While TIMP-2 has been shown to protect stimulated bone resorption [18], the increased TIMP-2 may not have been sufficient to prevent the decrease in type I collagen. Further studies will be needed to address the contribution of MMP activity to the type I collagen accumulation observed in our study. Some of the CsA concentrations used in these experiments, i.e. 1 and 5 µg/ml, are above the plasma concentrations seen in patients of 100450 ng/ml. However peak levels in patients are likely to be in the range of 1 µg/ml, and the tissue concentration of CsA is not known.
In summary, we show that CsA exposure is associated with changes in type I collagen mRNA expression and net protein accumulation by non-transformed MC3T3-E1 osteoblastic cells. Decreased proliferation due to CsA could reduce the number of cells able to synthesize new osteoid matrix, which could further contribute to the process of bone loss. These changes could contribute to the osteopenia observed in CsA-treated patients.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|