BMP-2 inhibits proliferation of human aortic smooth muscle cells via p21Cip1/Waf1

Gail A. Wong1,4, Vincent Tang1,4, Faten El-Sabeawy1, and Robert H. Weiss2,3,4

1 Divisions of Endocrinology and 2 Nephrology, Department of Internal Medicine, and 3 Cell and Developmental Biology Graduate Group, University of California, Davis 95616; and 4 Department of Veterans Affairs, Northern California Health Care System, Mather, California 95655


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Bone-morphogenetic proteins (BMP)-2 and -7, multifunctional members of the transforming growth factor (TGF)-beta superfamily with powerful osteoinductive effects, cause cell cycle arrest in a variety of transformed cell lines by activating signaling cascades that involve several cyclin-dependent kinase inhibitors (CDKIs). CDKIs in the cip/kip family, p21Cip1/Waf1 and p27Kip1, have been shown to negatively regulate the G1 cyclins and their partner cyclin-dependent kinase proteins, resulting in BMP-mediated growth arrest. Bone morphogens have also been associated with antiproliferative effects in vascular tissue by unknown mechanisms. We now show that BMP-2-mediated inhibition of platelet-derived growth factor (PDGF)-stimulated human aortic smooth muscle cell (HASMC) proliferation is accompanied by increased levels of p21 protein. Antisense oligodeoxynucleotides specific for p21 attenuate BMP-2-induced inhibition of proliferation when transfected into HASMCs, demonstrating that BMP-2 inhibits PDGF-stimulated proliferation of HASMCs through induction of p21. Whether p21-mediated induction of cell cycle arrest by BMP-2 sets the stage for osteogenic differentiation of vascular smooth muscle cells, ultimately leading to vascular mineralization, remains to be investigated.

bone morphogenetic protein-2; vascular smooth muscle cell; antisense


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

ONE OF THE EARLY EVENTS in the development of atherosclerotic lesions is the proliferation of vascular smooth muscle cells (VSMCs) in response to a variety of luminal injuries (24). A number of growth-promoting proteins, including platelet-derived growth factor (PDGF), fibroblast growth factor, and insulin-like growth factor, have been identified in VSMCs as being important in the pathogenesis of these vascular lesions (2, 25). Elucidating the mechanism by which VSMCs commit to either cellular proliferation or cell cycle arrest is therefore critical to understanding the genesis of the cellular response to vascular injury and its subsequent contribution to atherosclerotic plaque formation, angioplasty restenosis, and vascular calcification.

The cyclin-dependent kinases (CDKs) are a family of serine-threonine kinases that act in conjunction with their partner cyclins in cascade fashion in response to mitogenic stimulation, ultimately leading to cell cycle progression through the G1/S phase transition (3). In many cell systems, cell cycle arrest is associated with p21Cip1/Waf1-mediated inhibition of CDK and cyclin activities and subsequent downstream dephosphorylation of retinoblastoma protein (8). More recently, we have shown that p21 is important in promoting assembly of cyclin D/CDK4 complexes in VSMCs (31).

In this context, p21 may be permissive for cell cycle progression through the G1/S phase and therefore significant in maintaining cellular integrity and survival (16, 31). Thus p21 may act differently in various cell types in response to specific cytokines to promote growth arrest or cell cycle progression. Because multiple cytokines are present simultaneously in the local environment, it is possible that different signaling pathways can be concurrently invoked and result in divergent effects in the same target cells.

Bone-morphogenetic proteins (BMPs) are powerful osteoinductive proteins thought to be integral to the process of differentiation, proliferation, and osteoblastic activation by which new bone is made (22, 23). BMP-2 is the most potent of the multiple cytokines and growth factors that interact sequentially to induce the differentiation of osteoprogenitor cells into osteoblasts (28, 30). Additionally, it is known that BMP-2 can elicit diverse responses in a number of cell systems, ranging from differentiation to apoptosis (12, 14, 20). Recent studies have implicated the CDK inhibitors (CDKIs) of the cip/kip family, p21 and p27 (6, 7, 12), in BMP-mediated actions. Subpopulations of aortic smooth muscle cells have been shown to have osteoblastic potential under specific conditions. These cells resemble pericytes morphologically and can be induced to differentiate into osteoblast-like cells, able to produce mineralized bone nodules in culture (29). In this study, we examined the potential of BMP-2 to inhibit PDGF-stimulated proliferation in a human aortic smooth muscle cell (HASMC) line and asked whether p21 plays a causative role in this process. In light of its powerful osteoinductive properties, the actions of BMP-2 on HASMCs in response to mitogenic triggers may thus have important implications with respect to the potential of a selected population of VSMCs to differentiate into osteogenic cells. p21 May therefore prove to be a useful target for future therapies aimed at preventing vascular calcification.


    MATERIAL AND METHODS
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

HASMCs and SmGM growth medium were purchased from Clonetics (San Diego, CA). These cells are isolated from the aortic arch, stain positively for alpha -smooth muscle actin and negatively for von Willebrand's factor. Dulbecco's modified Eagle's medium (DMEM), Opti-MEM medium, Fungizone, and lipofectin were purchased from Invitrogen (Carlsbad, CA). Recombinant human (rh) PDGF-BB and mouse monoclonal p21Waf1/Cip1 antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-caspase-3 rabbit polyclonal and horseradish peroxidase (HRP)-conjugated anti-mouse antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti-rabbit IgG antibody was purchased from Bio-Rad. rhBMP-2 was purchased from R&D Systems (Minneapolis, MN). [3H]thymidine and the enhanced chemiluminescence (ECL) detection kit were purchased from Amersham Biosciences (Piscataway, NJ). Fetal bovine serum, penicillin-streptomycin, trichloroacetic acid (TCA), and scintillation fluid were purchased from Fisher Scientific (Pittsburg, PA). All other reagents and kits, including mouse monoclonal smooth muscle alpha -actin antibody, were purchased from Sigma (St. Louis, MO).

Cell Culture and Incubations

HASMCs were grown in SmGM growth medium with 5% FBS and growth factors, as recommended by the vendor, until 70-80% confluent and were used between passages 5 and 7 for all experiments.

Cell Proliferation Assays

HASMCs were grown in supplemented SmGM medium on 24-well dishes until 70-80% confluent. They were synchronized in G0 by an overnight incubation in serum-free medium (DMEM) containing 0.1% BSA. Cells were then incubated for 24 h at 37°C with the indicated concentrations of rhBMP-2 in serum-free medium alone or in the presence of 40 ng/ml PDGF-BB. During the last 6 h of incubation, the cells were labeled with [3H]thymidine at a concentration of 1 µCi/ml. After 6 h, incorporated [3H]thymidine was precipitated with 15% TCA for 30 min. Cells were solubilized by 1 N NaOH, followed by neutralization with an equal volume of 1 N HCl. Incorporated [3H]thymidine was quantitated in a liquid scintillation counter, and proliferation response was expressed as cpm corrected for background.

Western Blotting

HASMCs were grown in supplemented SmGM medium in 60-mm dishes until 70-80% confluent and then incubated in serum-free medium with 0.1% BSA for 24 h as above. PDGF-BB (40 ng/ml) and/or rhBMP-2 (150 ng/ml) was added for 4, 8, and 24 h. The cells were then lysed with RIPA buffer (50 mM HEPES, 1% Triton X-100, 10 mM Na4P2O7, 100 mM NaF, 4 mM EDTA, 2 mM Na3VO4, 2 mM PMSF, 1 µg/ml aprotinin) for 10 min on ice. After scraping, the cells were homogenized by passage three times through a 25-gauge needle and centrifuged for 10 min at 4°C at 15,000 rpm. Equal quantities of protein, determined by Bio-Rad DC Protein Assay, were separated on a 12% SDS-polyacrylamide gel for 20 min at 60 V and then for 1 h and 45 min at 100 V. The proteins were transferred to nitrocellulose for 1 h at 100 V. The membrane was incubated for 1 h at room temperature in blocking solution [1× TBS, pH 7.6, and 0.05% Tween 20 containing 10% nonfat dry milk (TBS-T)]. After three 5-min washes in TBS-T, the membrane was sectioned and incubated overnight at 4°C in a 3% blocking solution with the primary antibodies for smooth muscle alpha -actin (1:1,000) and p21Cip1/Waf1 (1:200). After three additional washes in TBS-T, the membrane was incubated with HRP-conjugated secondary antibody (1:50,000) in TBS-T for 1 h at room temperature. The antigen-antibody complex was detected using the ECL kit per the manufacturer's instructions.

Measurement of Apoptosis

Hoechst staining. HASMCs were grown in growth medium supplemented with 10% FBS on 6-well plates until 70-80% confluent. The cells were synchronized in G0 in serum-free medium for 24 h and then incubated in 40 ng/ml PDGF-BB and/or 150 ng/ml BMP-2 for 24 h. The cells were fixed with methanol for 10 min. After the methanol was aspirated, 1 µg/ml Hoechst 33258 in H2O with 10 µg of nonfat dry milk was added to the wells. The cells' nuclear morphology was viewed under a Zeiss Axioskop fluorescence microscope.

Caspase-3 activation. HASMCs were grown in growth medium until 70-80% confluent. Cell lysates were prepared as discussed above after an overnight incubation in serum-free medium, followed by a 24-h incubation in 40 ng/ml PDGF-BB and/or rhBMP-2. Western analysis of procaspase-3 cleavage to caspase-3 was performed as described above, using 1:1,000 dilutions of anti-caspase-3 rabbit polyclonal antibody as the primary antibody (1:1,000 dilution) and goat anti-rabbit IgG as the secondary antibody (1:15,000). A Jurkat HL60 cell lysate was used as a positive control for caspase cleavage.

Cell Transfections with p21Waf1/Cip1 Antisense Oligonucleotide

Phosphorothioate antisense and random sequence oligodeoxynucleotides (ODN) were synthesized by Oligos, Etc. (Wilsonville, OR). The p21 antisense sequence 5'-ATCCCCAGCCGGTTCTGACAT-3' was designed around the start codon of human p21Cip1/Waf1. The randomly generated control sequence was 5'-TGGATCCGACATGTCAGA-3'. HASMCs were grown in supplemented SmGM growth medium on 60-mm dishes until 70-80% confluent and rinsed with sterile PBS. Random sequence or p21 antisense ODNs were mixed in 5.3 µl lipofectin/ml Opti-MEM medium to a final concentration of 200 nM. The cells were transfected with lipofectin/Opti-MEM alone, random sequence ODN, or p21 antisense ODN for 4 h at 37°C. Control cells were not transfected. After a rinse and an overnight incubation in serum-free medium, cells were incubated with or without PDGF-BB (40 ng/ml) for the indicated times in the presence and absence of BMP-2 (150 ng/ml) and used to prepare either cell lysates for immunoblotting of p21 protein or in cell proliferation experiments as described.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

BMP-2 is Associated with Decreased PDGF-Stimulated and Unstimulated HASMC Proliferation

To determine whether BMP-2 decreases cell cycle transit in HASMCs, we examined [3H]thymidine incorporation in an established line of HASMCs under PDGF-stimulated and unstimulated conditions in the presence or absence of simultaneously added BMP-2 at physiological concentrations. HASMCs were serum starved overnight and then stimulated to proliferate with PDGF (40 ng/ml) in the presence of increasing concentrations of rhBMP-2. DNA synthesis, as a measure of cell cycle transit, was assessed by [3H]thymidine incorporation into DNA. rhBMP-2 at 50, 150, and 300 ng/ml inhibited PDGF-induced HASMC proliferation by 35, 46, and 64%, respectively, compared with PDGF-stimulated control cells (Fig. 1A). A similar inhibitory effect was seen in serum-starved cells in the absence of PDGF stimulation (Fig. 1B). Thus BMP-2 decreases cell cycle progression in serum-starved HASMCs under both unstimulated and PDGF-stimulated conditions.


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Fig. 1.   Bone-morphogenetic protein (BMP)-2 inhibits human aortic smooth muscle cell (HASMC) proliferation both with and without platelet-derived growth factor (PDGF) stimulation. HASMCs were grown to 70-80% confluence, serum starved overnight, and incubated for 24 h with the indicated concentrations of recombinant human (rh)BMP-2 in the absence (A) and presence (B) of 40 ng/ml PDGF. DNA synthesis was asssessed by [3H]thymidine incorporation, as described in MATERIALS AND METHODS. Error bars represent SE, and results are representative of 3 separate experiments. *P < 0.05 compared with (-)BMP-2.

BMP-2-mediated cell cycle arrest is not associated with increased apoptosis. A possible explanation of the observed decrease in [H3]thymidine incorporation in response to BMP-2 is that BMP-2 is causing apoptosis of HASMCs. To address this possibility, we utilized two distinct methods to assess apoptosis. Hoechst staining and caspase-3 activation in HASMCs were performed in PDGF-treated and untreated cells after 24 h of incubation in the presence and absence of 150 ng/ml BMP-2, a concentration of BMP-2 that resulted in significant growth arrest (see Fig. 1, A and B). There were no changes in Hoechst staining under all conditions examined (Fig. 2). In addition, no activation of procaspase-3 to caspase-3 was apparent under all tested conditions (Fig. 3). Thus, in HASMCs, BMP-2 treatment results in decreased PDGF-mediated cell proliferation by directly inhibiting new DNA synthesis and subsequent G1/S phase progression, rather than by causing apoptosis.


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Fig. 2.   BMP-2 does not cause apoptosis at 24 h by Hoechst staining. HASMCs were grown to 70-80% confluence and then serum starved for 24 h. After a 24-h incubation with or without rhBMP-2 (150 ng/ml) in the presence and absence of PDGF (40 ng/ml), cells were fixed, stained with Hoechst 33258, and same representative fields were visualized under fluorescence (left) or phase-contrast (right) microscopy.



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Fig. 3.   BMP-2 does not cause activation of procaspase-3 to caspase-3. Cell lysates from HASMCs incubated for 24 h with and without rhBMP-2 (150 ng/ml) in the presence and absence of PDGF (40 ng/ml) were subjected to Western analysis for levels of procaspase-3 and caspase-3 by use of 1:1,000 dilutions of anti-caspase-3 rabbit polyclonal antibody as the primary antibody and goat anti-rabbit IgG as the secondary antibody (1:15,000). A Jurkat HL60 cell lysate was used as a positive control for caspase-3 cleavage. No activation of procaspase-3 to caspase-3 was appreciated under any condition tested.

BMP-2 Increases p21 Protein Levels

The CDK inhibitor p21 is associated both with cell cycle arrest through its inhibitory effects on CDK2 (8) and with cell cycle progression by both promoting assembly of cyclin D1-CDK4 complexes and by its antiapoptotic actions (16, 31). To determine whether p21 is increased by BMP-2, HASMCs were treated with 150 ng/ml rhBMP-2 for 4, 8, and 24 h under PDGF-stimulated and unstimulated conditions. Exposure to PDGF caused an increase in p21 protein levels in the absence of BMP-2 after 4, 8, and 24 h of treatment, consistent with our previous observations (10). BMP-2 treatment of HASMCs resulted in increased p21 protein levels above those seen with PDGF at both 4 and 8 h (Fig. 4), with the effect disappearing in PDGF-stimulated cells after 24 h of incubation. p21 Levels increased in response to BMP-2 at 4, 8, and 24 h in unstimulated cells. Identification of the p21 protein band was confirmed by immunoblotting using HeLa cell lysate as a positive control for p21 (Fig. 4), and smooth muscle alpha -actin was used as a loading control.


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Fig. 4.   BMP-2 increased p21 protein levels at 4 and 8 h in PDGF-treated cells. HASMCs were grown to 70-80% confluence and treated concurrently with PDGF-BB (40 ng/ml) and rhBMP-2 (150 ng/ml) for the times indicated. Unstimulated cells were treated with rhBMP-2 (150 ng/ml) as noted. Immunoblotting of the cell lysates was performed using monoclonal antibodies to mouse p21Waf1/Cip1 and to smooth muscle alpha -actin antibody. HeLa cell lysate was electrophoresed as a control for p21 mobility. Total protein (25 µg) was added to each lane. Optical density was performed and expressed as the ratio of relative density units of the p21 protein band/actin band.

p21 Antisense ODN Attenuates BMP-2-Mediated Inhibition of Cell Proliferation

To determine whether inhibition of HASMC proliferation by BMP-2 involves p21, we generated a phosphorothioate-modified antisense ODN sequence to human p21. PDGF-stimulated and unstimulated HASMCs were untransfected (control) or transfected with lipofectin alone (no ODN), random sequence (scrambled) ODN control, or antisense ODN specific for human p21 (anti-p21 ODN). Cell lysates were prepared, and p21 protein levels were then analyzed by Western immunoblotting. Partial but significant inhibition of p21 protein was seen in cells transfected with p21 antisense ODN after 4 h of PDGF exposure, a time before the G1/S restriction point (Fig. 5).


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Fig. 5.   Antisense oligodeoxynucleotide (ODN) to p21 attenuates p21 protein levels. HASMCs were transfected with lipofectin alone (no ODN), random sequence (scrambled) control ODN, or p21 antisense ODN (anti-p21 ODN), as indicated. Control cells were not transfected. PDGF-BB (40 ng/ml) or saline was added 24 h after transfection, and cell lysates were prepared after incubation after 4 h. p21 Protein and actin levels were examined by immunoblotting as described. Optical density was performed and expressed as the ratio of relative density units of the p21 protein band to the actin band. Data are representative of 3 separate experiments.

We then asked whether BMP-2-mediated inhibition of HASMC proliferation was attenuated in cells transfected with p21 antisense ODN. Cell proliferation by [3H]thymidine incorporation was measured in PDGF-stimulated (Fig. 6A) and unstimulated (Fig. 6B) HASMCs in the presence and absence of BMP-2 (150 ng/ml). Cells were untransfected (control) or transfected with lipofectin alone (no ODN), random sequence (scrambled control) ODN, or p21-antisense ODN (anti-p21 ODN). BMP-2 inhibited [3H]thymidine incorporation in untransfected cells (control) and after transfection with lipofectin and random sequence ODN by 48, 35, and 46%, respectively, in PDGF-stimulated cells. However, after transfection with antisense to p21, the degree of BMP-2-mediated inhibition decreased to 7% in PDGF-stimulated HASMCs. In unstimulated HASMCs, BMP-2 similarly caused a decrease in cell proliferation in untransfected cells (control) and after transfection with lipofectin alone and scrambled control ODN. However, no significant attenuation of BMP-2-mediated growth inhibition was seen in unstimulated cells after transfection with p21 antisense ODN (Fig. 6B). Therefore, p21 is a significant mediator of antiproliferative effects of BMP-2 in PDGF-stimulated HASMCs but not in unstimulated, serum-starved cells. In addition, PDGF stimulation appears to be a requirement for p21-mediated BMP-2 antiproliferative actions in this system. In unstimulated cells, BMP-2 growth-inhibitory actions appear to be p21 independent.


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Fig. 6.   p21 Antisense attenuates BMP-2 growth-inhibitory effects on PDGF-stimulated HASMCs. HASMCs (70-80% confluent) were serum starved overnight and then transfected with lipofectin alone (no ODN), random sequence (scrambled control) ODN, or p21-antisense ODN (anti-p21 ODN). Control cells were not transfected. After an overnight incubation in serum-free medium, cells were treated for 24 h with 40 ng/ml PDGF (A) or saline (B) in the presence or absence of rhBMP-2 (150 ng/ml). DNA synthesis was determined by [3H]thymidine incorporation added during the last 6 h of incubation, as described in MATERIALS AND METHODS. Data are representative of 3 experiments. *P < 0.05 compared with (-)BMP-2.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

BMP-2 has strong osteoinductive properties and, in addition, appears to mediate growth inhibition via the CDK inhibitors in a number of cell types. Whether BMP-2 is important in inducing osteogenesis in vascular cells in vivo, subsequently contributing to vascular calcification, however, is unknown. Intuitive to the progression of cells from one phenotype to another is the induction of growth inhibition of the precursor population. Vascular cells proliferate in response to injury and inflammation, but that proliferation must be arrested if they are to become osteoblast-like in nature (21).

In light of the antiproliferative effects of BMP-2 and other members of the TGF-beta family of cytokines in a number of cell systems (18, 19, 27), we asked whether BMP-2 also inhibits the proliferation of HASMCs. We observed a strong antiproliferative effect of BMP-2 on VSMC proliferation under both PDGF-stimulated and serum-free conditions. BMP-2 treatment of HASMCs resulted in decreased PDGF-stimulated DNA synthesis and cell proliferation in a dose-dependent manner over a wide physiological range of concentrations. BMP-2-mediated antiproliferative effects were also appreciated in unstimulated cells but were independent of p21. The decrease in proliferation mediated by BMP-2 was not due to increased apoptosis but appeared to result directly from the inhibition of new DNA synthesis. This is the first report of BMP-2-mediated inhibition of HASMC proliferation in vitro and thus may have broad implications for addressing the mechanism by which osteogenesis occurs in vascular tissue in response to pathogenic insult.

BMP-2, a member of the TGF-beta superfamily, is a powerful osteoinductive protein with diverse effects in a variety of cell lines. For example, BMP-2 and a member of the interleukin-6 (IL-6) family, leukemic inhibitory factor, act in synergy to induce neuroprogenitor cells to differentiate into astrocytes (20). In contrast, BMP-2 and IL-6 interact to cause apoptosis in mouse hybridoma cells (14). BMP-7, which has close sequence and structural homology to BMP-2, induces cell cycle arrest of anaplastic thyroid cancer cells (6). Studies in vascular tissue suggest an inhibitory role of the bone morphogens on vasculoproliferative responses. For example, in an in vivo model of arterial balloon-induced injury in the rat, virally transfected BMP-2 results in inhibition of intimal hyperplasia (19). In similar fashion, BMP-7 has been shown to inhibit smooth muscle cell proliferation in an established HASMC line (5).

In other cell types, BMP-associated growth-inhibitory responses are transmitted, at least in part, by the CDK inhibitor p21Cip1/Waf1. Apoptosis of myeloma cells and inhibition of breast cancer cell growth by BMP-2 and induction of cell cycle arrest in thyroid cancer cells are mediated by p21 (6, 7, 12). In response to DNA damage and subsequent activation of p53, increased p21 binding to cyclin E/CDK2 complexes occurs (8), resulting in inhibition of CDK activity, decreased phosphorylation of Rb protein, and subsequent cell cycle arrest in G1. In addition to its critical role in promoting cell cycle arrest in response to DNA damage, however, p21 has recently been invoked as an important mediator in proper assembly, stabilization, and nuclear transport of cyclin D/CDK4 complexes, thus potentially promoting cellular viability and integrity (15, 31). Furthermore, p21 can function as an antiapoptotic survival protein in tumor cells (16) as well as in VSMCs (Davis BB and Weiss RH, unpublished observations). Thus it appears that p21 can function differentially, depending on the cell's requirement for cycle arrest vs. cycle progression, in response to diverse conditions as well as time of activation relative to cell cycle events. In addition, the behavior of p21 may be different in cells transformed by mutagenic DNA damage than in its response to vascular insult.

We employed an established cell line of HASMCs in our experiments as one model for VSMC phenotype. These cells are derived from the aortic arch and stain positively for alpha -actin and negatively for von Willebrand factor. HASMCs have been used by other investigators in a number of published reports examining regulatory events in cell cycling (5, 9, 26). Using these cells, we found p21 protein increased in PDGF-stimulated cells after 4, 8, and 24 h of treatment, consistent with our previous work in rat VSMCs and demonstrating a growth-permissive role of p21 in this context (31). BMP-2 treatment resulted in an additional increase in p21 above that seen with PDGF after 4 and 8 h. This increase was not observed after 24 h of treatment of BMP-2, suggesting that p21-mediated BMP-2 effects occur earlier in the cell cycle, likely at the G1/S boundary.

In the case of BMP-2, reduction of p21 levels by antisense techniques attenuated growth-inhibitory actions when observed 24 h after PDGF stimulation. In addition, treatment of PDGF-stimulated HASMCs with p21 antisense in the absence of BMP-2 resulted in a nonsignificant decrease in p21 levels and proliferative response, consistent with previous observations that p21 is important in PDGF-mediated cell proliferation. No attenuation of BMP-2-mediated growth arrest by p21 antisense occurred in unstimulated cells, however, suggesting that BMP-2 may act to modulate the PDGF-mediated upregulation of p21 that occurs early in the cell cycle. Our findings suggest that BMP-2 acts to promote cell cycle arrest in HASMCs stimulated by PDGF by further induction of the CDK inhibitor p21Cip1/Waf1 to promote growth arrest of these cells. It is conceivable that p21 may act differentially depending on its location. PDGF may induce cytoplasmic p21, promoting its function as a survival factor (1) and mediating PDGF-associated cell proliferation. However, further induction of p21 by BMP-2 and subsequent nuclear translocation of p21 from the cytoplasm may result in growth arrest in this system. This is consistent with the findings of two independent studies in a balloon injury model in the rat showing that adenovirally mediated transfer of either BMP-2 (19) or p21 (4) results in decreased intimal hyperplasia of carotid artery in response to luminal insult. Thus p21 is a critical downstream mediator of BMP-2-induced antiproliferative actions on PDGF-stimulated HASMCs. In contrast, BMP-2-mediated growth-inhibitory actions on cells in the basal state occur via a p21-independent mechanism and may be autocrine in nature.

Both endogenous and exogenous factors have been recently identified as causing growth arrest in VASMCs. p21 Has been invoked as an important mediator in suppression of VSMC proliferation by salicylates, nitric oxide, and cAMP (9, 11, 17). The mechanism is thought to be similar to that in nonvascular cells, i.e., a direct p21-mediated inhibition of cyclin E/CDK2 responses during G1/S progression leading to cell cycle arrest. Our findings and these results, taken together, suggest that p21 may act under specific conditions as a common mediator upon which disparate pathways converge to activate downstream signaling events that lead ultimately to growth arrest of VSMCs. This pathway may be induced by factors that differ from each other as widely as do salicylates, BMPs, and nitric oxide. Clearly, induction of such a pathway has important clinical implications in the regulation of vasculoproliferative response to arterial injury in vivo.

The mechanism by which TGF-beta , and BMPs in particular, cause cell cycle arrest has not been clearly elucidated. Induction of osteoblast differentiation and regulation of embryogenesis by BMP-2 occurs by signaling through the family of transcription factors known as the Smads (21). Recently, however, MAPK pathways have been demonstrated to be important in BMP-mediated actions. Activation of the TAK1-p38 pathway is required for BMP-2-mediated apoptosis of mouse hybridoma cells (14). Moreover, recent evidence suggests that cross talk between the Smad and MAPK pathways plays a significant role in downstream signaling events. In hybridoma cells, Smad6 has been shown to physically interact with TGF-beta -activated kinase-1 (TAK1), preventing activation of the TAK1-p38 pathway and abolishing apoptosis induced by BMP-2 (14). Notably, a recent report showing that p38 and Jnk1 stabilize p21 by phosphorylation (13) lends support to the potential role of upstream MAPK-signaling proteins in modulating BMP-2-mediated antiproliferative effects in VSMCs through p21. In this study, we show that BMP-2 causes cell cycle arrest in human VSMCs and that its growth-inhibitory action is mediated by p21, a CDK inhibitor involved in the mechanism utilized by other growth-inhibitory factors in VSMCs.

BMP-2 has been shown to induce osteoblastic phenotype in a number of pluripotent cell lines and indeed is one of the most osteoinductive cytokines known. We now show that BMP-2 acts to promote growth inhibition in PDGF-stimulated HASMCs through a p21-mediated mechanism. Whether these observations can be extended to explain osteogenic differentiation of VSMCs in general, or whether induction by BMP-2 of p21-mediated growth arrest is a requirement for subsequent differentiation of mitogen-stimulated human VSMCs into those with osteoblastic potential, is currently under investigation in our laboratory.


    ACKNOWLEDGEMENTS

We thank Yao Dong and Benjamin B. Davis for technical assistance.


    FOOTNOTES

This work was supported by the Research Service of the Department for Veterans Affairs and a UC Davis Health System Award.

Address for reprint requests and other correspondence: G. A. Wong, Division of Endocrinology, Clinical Nutrition & Vascular Medicine, 4150 V St., PSSB G400, UC Davis Medical Center, Sacramento, CA 95817 (E-mail: gawong{at}ucdavis.edu).

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.

First published January 14, 2003;10.1152/ajpendo.00385.2002

Received 29 August 2002; accepted in final form 24 December 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Asada, M, Yamada T, Ichijo H, Delia D, Miyazono K, Fukumuro K, and Mizutani S. Apoptosis inhibitory activity of cytoplasmic p21(Cip1/WAF1) in monocytic differentiation. EMBO J 18: 1223-1234, 1999[Abstract/Free Full Text].

2.   Banskota, NK, Taub R, Zellner K, and King GL. Insulin, insulin-like growth factor I and platelet-derived growth factor interact additively in the induction of the protooncogene c-myc and cellular proliferation in cultured bovine aortic smooth muscle cells. Mol Endocrinol 3: 1183-1190, 1989[Abstract].

3.   Bartek, J, and Lukas J. Pathways governing G1/S transition and their response to DNA damage. FEBS Lett 490: 117-122, 2001[ISI][Medline].

4.   Chang, MW, Barr E, Lu MM, Barton K, and Leiden JM. Adenovirus-mediated over-expression of the cyclin/cyclin-dependent kinase inhibitor, p21, inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty. J Clin Invest 96: 2260-2268, 1995[ISI][Medline].

5.   Dorai, H, Vukicevic S, and Sampath TK. Bone morphogenetic protein-7 (osteogenic protein-1) inhibits smooth muscle cell proliferation and stimulates the expression of markers that are characteristic of SMC phenotype in vitro. J Cell Physiol 184: 37-45, 2000[ISI][Medline].

6.   Franzen, A, and Heldin NE. BMP-7-induced cell cycle arrest of anaplastic thyroid carcinoma cells via p21(CIP1) and p27(KIP1). Biochem Biophys Res Commun 285: 773-781, 2001[ISI][Medline].

7.   Ghosh-Choudhury, N, Ghosh-Choudhury G, Celeste A, Ghosh PM, Moyer M, Abboud SL, and Kreisberg J. Bone morphogenetic protein-2 induces cyclin kinase inhibitor p21 and hypophosphorylation of retinoblastoma protein in estradiol-treated MCF-7 human breast cancer cells. Biochim Biophys Acta 1497: 186-196, 2000[ISI][Medline].

8.   Harbour, JW, and Dean DC. The Rb/E2F pathway: expanding roles and emerging paradigms. Genes Dev 14: 2393-2409, 2000[Free Full Text].

9.   Hayashi, S, Morishita R, Matsushita H, Nakagami H, Taniyama Y, Nakamura T, Aoki M, Yamamoto K, Higaki J, and Ogihara T. Cyclic AMP inhibited proliferation of human aortic vascular smooth muscle cells, accompanied by induction of p53 and p21. Hypertension 35: 237-243, 2000[Abstract/Free Full Text].

10.   Hupfeld, CJ, and Weiss RH. TZDs inhibit vascular smooth muscle cell growth independently of the cyclin kinase inhibitors p21 and p27. Am J Physiol Endocrinol Metab 281: E207-E216, 2001[Abstract/Free Full Text].

11.   Ishida, A, Sasaguri T, Kosaka C, Nojima H, and Ogata J. Induction of the cyclin-dependent kinase inhibitor p21(Sdi1/Cip1/Waf1) by nitric oxide-generating vasodilator in vascular smooth muscle cells. J Biol Chem 272: 10050-10057, 1997[Abstract/Free Full Text].

12.   Kawamura, C, Kizaki M, Yamato K, Uchida H, Fukuchi Y, Hattori Y, Koseki T, Nishihara T, and Ikeda Y. Bone morphogenetic protein-2 induces apoptosis in human myeloma cells with modulation of STAT3. Blood 96: 2005-2011, 2000[Abstract/Free Full Text].

13.  Kim GY, Mercer SE, Ewton DZ, Yan Z, Jin K, and Friedman E. The stress-activated kinases p38alpha and Jnk1 stabilize p21cip1 by phosphorylation. J Biol Chem: 29792-29802, 2002.

14.   Kimura, N, Matsuo R, Shibuya H, Nakashima K, and Taga T. BMP2-induced apoptosis is mediated by activation of the TAK1-p38 kinase pathway that is negatively regulated by Smad6. J Biol Chem 275: 17647-17652, 2000[Abstract/Free Full Text].

15.   LaBaer, J, Garrett MD, Stevenson LF, Slingerland JM, Sandhu C, Chou HS, Fattaey A, and Harlow E. New functional activities for the p21 family of CDK inhibitors. Genes Dev 11: 847-862, 1997[Abstract].

16.   Li, Y, Dowbenko D, and Lasky LA. AKT/PKB phosphorylation of p21Cip/WAF1 enhances protein stability of p21Cip/WAF1 and promotes cell survival. J Biol Chem 277: 11352-11361, 2002[Abstract/Free Full Text].

17.   Marra, DE, Simoncini T, and Liao JK. Inhibition of vascular smooth muscle cell proliferation by sodium salicylate mediated by upregulation of p21(Waf1) and p27(Kip1). Circulation 102: 2124-2130, 2000[Abstract/Free Full Text].

18.   Moses, HL, Yang EY, and Pietenpol JA. TGF-beta stimulation and inhibition of cell proliferation: new mechanistic insights. Cell 63: 245-247, 1990[ISI][Medline].

19.   Nakaoka, T, Gonda K, Ogita T, Otawara-Hamamoto Y, Okabe F, Kira Y, Harii K, Miyazono K, Takuwa Y, and Fujita T. Inhibition of rat vascular smooth muscle proliferation in vitro and in vivo by bone morphogenetic protein-2. J Clin Invest 100: 2824-2832, 1997[Abstract/Free Full Text].

20.   Nakashima, K, Yanagisawa M, Arakawa H, and Taga T. Astrocyte differentiation mediated by LIF in cooperation with BMP2. FEBS Lett 457: 43-46, 1999[ISI][Medline].

21.   Ogasawara, TKM, Miura T, Susami T, Takato T, Nakamura K, and Okayama H. Osteoblast diffentiation by BMP2 is mediated by downregulation of CDK6 (Abstract). J Bone Miner Res 16: S203, 2001.

22.   Reddi, AH. Role of morphogenetic proteins in skeletal tissue engineering and regeneration. Nat Biotechnol 16: 247-252, 1998[ISI][Medline].

23.  Riley EH, Lane JM, Urist MR, Lyons KM, and Lieberman JR. Bone morphogenetic protein-2: biology and applications. Clin Orthop: 39-46, 1996.

24.   Ross, R. Atherosclerosis is an inflammatory disease. Am Heart J 138: S419-S420, 1999[ISI][Medline].

25.   Ross, R, Masuda J, Raines EW, Gown AM, Katsuda S, Sasahara M, Malden LT, Masuko H, and Sato H. Localization of PDGF-B protein in macrophages in all phases of atherogenesis. Science 248: 1009-1012, 1990[ISI][Medline].

26.   Sandirasegarane, L, and Kester M. Enhanced stimulation of Akt-3/protein kinase B-gamma in human aortic smooth muscle cells. Biochem Biophys Res Commun 283: 158-163, 2001[ISI][Medline].

27.   Sporn, MB, and Roberts AB. Transforming growth factor-beta: recent progress and new challenges. J Cell Biol 119: 1017-1021, 1992[ISI][Medline].

28.   Takuwa, Y, Ohse C, Wang EA, Wozney JM, and Yamashita K. Bone morphogenetic protein-2 stimulates alkaline phosphatase activity and collagen synthesis in cultured osteoblastic cells, MC3T3-E1. Biochem Biophys Res Commun 174: 96-101, 1991[ISI][Medline].

29.   Tintut, Y, Parhami F, Bostrom K, Jackson SM, and Demer LL. cAMP stimulates osteoblast-like differentiation of calcifying vascular cells. Potential signaling pathway for vascular calcification. J Biol Chem 273: 7547-7553, 1998[Abstract/Free Full Text].

30.   Wang, EA, Rosen V, D'Alessandro JS, Bauduy M, Cordes P, Harada T, Israel DI, Hewick RM, Kerns KM, LaPan P, Luxenberg DP, McQuaid D, Moutsatsos IK, Nove J, and Wozney JM. Recombinant human bone morphogenetic protein induces bone formation. Proc Natl Acad Sci USA 87: 2220-2224, 1990[Abstract].

31.   Weiss, RH, Joo A, and Randour C. p21(Waf1/Cip1) is an assembly factor required for platelet-derived growth factor-induced vascular smooth muscle cell proliferation. J Biol Chem 275: 10285-10290, 2000[Abstract/Free Full Text].


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