TY - JOUR
AU - Newby, Andrew, C
AB - Abstract Objective: We sought to understand why smooth muscle cell proliferation is effectively repressed in intact rat aortic tissue. Methods: Quiescent isolated rat aortic smooth muscle cells and segments of intact rat aorta were stimulated with 10% serum and the time course of expression and activity of proteins involved in cell cycle control were determined. Results: After serum stimulation, smooth muscle cells in intact aortic tissue exhibit no proliferation, whereas isolated cells entered S phase 14–16 h later. Activation of ERKs 1 and 2, and induction of cyclin D1 occurred both in isolated cells and aortic tissue. Regulation of Cdk4, cyclin E and Cdk2 protein levels was also not different. Levels of the cyclin-dependent kinase inhibitors (CKIs), p16 and p27, were initially high in quiescent isolated cells and tissue; levels were downregulated by serum in isolated cells but not in aortic tissue. Cyclin D1/Cdk4, and cyclin E/Cdk2 kinases were active before S phase entry in isolated cells, but remained inactive in aortic tissue. Conclusions: Cell cycle entry is prevented in aortic tissue, and this is associated with an inability to downregulate p16 and p27 CKIs, and therefore to activate cyclin D1 and cyclin E associated kinase activities. Cell culture/isolation, Extracellular matrix, Protein kinases, Signal transduction, Smooth muscle Time for primary review 27 days. 1 Introduction Vascular smooth muscle cell (VSMC) proliferation is an important factor in the pathophysiology of atherosclerosis, angioplasty restenosis and vein graft failure. In the healthy mature artery, smooth muscle cells (SMCs) are contractile in phenotype and exhibit extremely low rates of proliferation [1], even though endogenous growth factors may be present [2]. Furthermore, exposure of intact arterial segments to exogenous growth factors in vitro [3] or in vivo [4,5] does not lead to rapid cell proliferation. This suggests that constraints to proliferation usually exist in the normal vessel wall, which prevent the VSMCs from responding to growth factor stimulation. Growth factors and fetal calf serum (FCS) initiate VSMC proliferation by activating several signal transduction pathways, including importantly the extracellular regulated protein kinases, ERKs 1 and 2 [6]. Subsequently, cells enter the G1 phase of the cell cycle, which is positively regulated by a group of serine/threonine kinases controlled by association with a regulatory cyclin subunit [7,8]. The activity of cyclin dependent kinases (Cdks) is further regulated by phosphorylation/dephosphorylation [9] and association with inhibitory proteins, collectively known as the cyclin-dependent kinase inhibitors (CKIs [10]). There are two classes of CKIs, based on sequence homology and target specificity. The Ink4 family, consisting of p15INK4b, p16INK4a, p18INK4c and p19INK4d, all bind and inhibit Cdk4 and Cdk6 specifically, and therefore target cyclin D associated kinase activity. The Cip/Kip family, including p21waf1, p27kip1 and p57kip2, has a broader spectrum of activity, inhibiting both Cdk2- and Cdk4-containing complexes, and therefore inhibiting both cyclin D and cyclin E associated activity [10]. In outline, the sequence of events is as follows: In response to the early signal transduction pathways, cyclin D1 expression is induced, and active cyclin D1/Cdk4 complexes are assembled [11–13]. Activation of cyclin E/Cdk2 and a reduction in the total protein level of p16 and p27 subsequently occurs [7,10]. Loss of CKIs aids activation of cyclin/Cdk complexes by making them accessible to the cyclin-dependent kinase activating kinase, CAK [14]. The active cyclin/Cdk complexes then complete the transition to S-phase by phosphorylating pRb and releasing E2F to transactivate target genes [8]. It is possible that the constraints to VSMC proliferation impact on one or more stages of the SMC cell cycle. For example, heparin inhibits c-jun activation and hence activator protein-1 DNA binding [15], implying that it interferes with the early signal transduction pathways leading to exit from the G0 phase of the cell cycle. By contrast, polymerised collagen does not inhibit the early transduction pathways or G1 progression but inhibits downregulation of p27kip, thereby inhibiting S-phase entry [16]. It has also been suggested recently, that elevation of p27kip expression contributes to the re-establishment of VSMC quiescence following balloon injury [17]. However, the mechanisms underlying suppression of VSMC proliferation in the normal, intact vessel wall are presently unknown. We therefore compared ERK1/2 activity, cyclin and Cdk protein expression and activity, and regulation of CKIs in intact rat aortas and isolated rat aortic VSMCs in response to serum stimulation. 2 Methods 2.1 Antibodies Anti-cyclin D1 (Sc-6281 Santa Cruz), Cdk4 (Sc-601-G), p27 (Sc-528-G), cyclin E (Sc-481), p16 (Sc-1207), Cdk2 (Sc-6248) total ERK1/2 (PC54 Oncogene), active ERK1/2 (V-6671 Promega), BrdU (11200 ICN) were used. Horseradish peroxidase, biotin, and fluorescin conjugated secondary antibodies, were obtained from Dako. 2.2 Aortic tissues Wistar rats (male, between 350 and 400 g) were anaesthetized with sodium pentabarbitone, followed by retrograde perfusion of 100 ml of phosphate-buffered saline (PBS; 155 mM NaCl, 7.5 mM Na2HPO4·12H2O, 1.9 mM NaH2PO4·2H2O, pH 7.4) via the abdominal aorta, at a constant pressure of 120 mmHg (1 mmHg=133.322 Pa). Where specified, to cause injury, a 2F balloon embolectomy catheter was inflated with water and passed three times through the aorta with rotation. The thoracic aorta was then carefully excised, cut into 4 mm segments, and placed in DMEM (with sodium pyruvate and 1000 mg/L glucose) containing 0.25% α-lactalbumin hydrosylate for 24 h. The tissue was then stimulated with DMEM plus 10% FCS in the presence, where specified of either 1 μCi/ml [3H]thymidine or 10 μM BrdU for up to 4 days. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). 2.3 Cell culture and measurement of [3H]thymidine or BrdU incorporation into DNA SMCs were cultured from the medial layer of aortas by a modification of the explant method, as previously described [18], and cells were used between passages 6 and 9. 7·104 isolated SMCs were plated in six-well plates and left for 2 days, at which time they had reached approximately 70% coverage, and are referred to throughout as low-density cultures. Cells were then synchronized in G0 by serum deprivation, and stimulated with 10% FCS containing 1 μCi/ml [3H]thymidine. At specific time points following stimulation, cultures were washed with PBS, and 10% trichloroacetic acid (TCA) was added to precipitate nuclear DNA. Precipitates were treated for combined measurement of [3H]thymidine, DNA concentration, and ATP concentration as described previously [18]. Segments of rat aorta were similarly cultured in the presence of 10% FCS containing 1 μCi/ml [3H]thymidine. At the same time points following stimulation, each tissue segment was rinsed twice in PBS and cut into 1 mm2 pieces. These pieces were then pooled and treated for combined measurement of [3H]thymidine, DNA concentration, and ATP concentration, as was performed for the isolated cells. For studies looking at the response of high-density cultures to 10% FCS, 7·104 isolated SMCs were plated in six-well plates. After 2 days, 10% FCS was replaced and cells grown for a further 3 days, at which time complete coverage of the substratum was observed. These cells are referred to throughout as high-density cultures. Cells were then synchronized in G0 by serum deprivation, and stimulated with 10% FCS 48 h, with 1 μCi/ml [3H]thymidine present for the last 24 h. Cultures were washed with PBS, and DNA and thymidine assays were performed as described above. For comparison, this experiment was also performed on low-density cultures, again with [3H]thymidine present for the last 24 h. 2.4 Western analysis, immunoprecipitation and kinase activity 7·104 isolated SMCs were plated in six-well plates and left for 2 days. Cells were then synchronized in G0 by serum deprivation, and stimulated with 10% FCS. Cells were lysed at specific time points thereafter in ice-cold lysis solution (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2.5 mM EDTA, 2.5 mM EDTA, 10 mM β-phosphoglycerate, 1 mM NaF, 0.1 mM NaVO4, 1 μg/μl leupeptin, 18 μg/ml aprotinin), and homogenized by passing through a 20-gauge needle several times. Cellular debris was removed by centrifugation, and the supernatant was used for protein measurement (using a BCA assay kit from Pierce), or Western blotting. Protein samples were prepared in 3× loading buffer [150 mM Tris, pH 6.8, 6% sodium dodecyl sulfate (SDS), 0.3% bromophenol blue, 30% glycerol], containing 15% β-mercaptoethanol (βME). A 10-μg amount of total protein was separated per lane on SDS–polyacrylamide gels, and electrophoretically transferred to hydrophobic nitrocellulose membranes (Amersham). Membranes were blocked in TBS-T (0.020 M Tris, 0.137 M NaCl, 0.100% Tween, pH 7.6) containing 5% milk powder, and incubated with primary antibody diluted in the same buffer for 1 h. The following antibody dilutions were used: anti-cyclin D1, anti-p27, anti-cyclin E, anti-p16, anti-Cdk2, all used at 1:500, total ERK1/2, 1:1000, active ERK1/2, 1:2000. Membranes were washed several times in TBS-T/5% milk powder, and subsequently incubated with a HRP conjugated secondary antibody diluted 1:2000 in TBS-T/5% milk powder for 1 h. Peroxidase activity was detected using the enhanced chemiluminescence detection kit (Amersham). For tissue samples, the procedure was identical except that samples were crushed to a fine powder under liquid nitrogen before addition of lysis solution and homogenisation. For cyclin E and p16, proteins were immunoprecipitated from cell and tissue lysates. A 100-μg amount of total cell or tissue protein was incubated with 15 μl of 50% slurry of protein A Sepharose in lysis solution for 30 min. The protein A Sepharose was removed by centrifugation, and the supernatant incubated with 10 μl of primary antibody overnight at 4°C. A 15-μl volume of a 50% slurry of protein A Sepharose in lysis solution was then added to each sample for 4 h with continuous agitation. Protein A bound proteins were harvested by centrifugation, and boiled in 1× reducing buffer (50 mM Tris, pH 6.8, 2% SDS, 0.1% bromophenol blue, 10% glycerol), containing 5% βME. Protein A was pelleted by centrifugation, and supernatants were loaded onto SDS–polyacrylamide gels. The procedure then continued as described above for Western analysis. For kinase assays, Cdk4/cyclin D1 and Cdk2/cyclin E holoenzymes were immunoprecipitated using the anti-cyclin D1 and anti-cyclin E antibodies. A 150-μg amount of total cell or tissue lysate was incubated with 10 μl of antibody overnight at 4°C. A 15-μl volume of a 50% slurry of protein A Sepharose in lysis solution was then added to each sample for 4 h with continuous agitation. Bound proteins were harvested by centrifugation, and pellets washed three times in lysis solution (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2.5 mM EDTA, 2.5 mM EDTA, 10 mM β-phosphoglycerate, 1 mM NaF, 0.1 mM NaVO4, 1 μg/μl leupeptin, 18 μg/ml aprotinin), and two times in kinase wash buffer (50 mM Tris–HCl, pH 7.4, 10 mM MgCl2, 10 mM β-phosphoglycerate, 2.5 mM EGTA, 1 mM NaF, 0.1 mM NaVO4, 0.1 M dithiothreitol). Pellets were then collected and resuspended in 20 μl kinase hot mix [50 mM Tris–HCl, pH 7.4, 2.5 mM EDTA, 10 mM β-phosphoglycerate, 10 mg/ml bovine serum albumin (BSA), 5 mM ATP, 0.1 M dithiothreitol, 3 μCi [32P]gamma ATP], containing 2 μg of histone (Sigma) for cyclin E/Cdk2 assays, and 0.8 μg of purified retinoblastoma protein (Santa-Cruz) for Cdk4/cyclin D1 assays, for 30 min at 30°C. The reaction was terminated by the addition of 10 μl of 3× reducing buffer. Samples were heated to 80°C for 10 min, and the pellets removed by centrifugation. Supernatants were separated on 8% polyacrylamide gels. The gels were then dried onto 3 mm Whatman paper using a Bio-Rad Model 583 gel dryer at 80°C for 1.5 h. The Whatman paper was then exposed to auto-radiography film for up to 48 h at −80°C. This procedure was based on that previously described [19]. For Western analysis of post-confluent cells, 7·104 isolated SMCs were plated in six-well plates. After 2 days, 10% FCS was replaced and cells grown for a further 3 days. Cells were then synchronized in G0 by serum deprivation, and stimulated with 10% FCS. After 24 h, cultures were washed with PBS, and protein extracted and treated as described above. 2.5 Immunocytochemical analysis Transverse sections (3 μm) were cut from formalin-fixed, paraffin-embedded tissues and were deparaffinised and rehydrated, as previously described by George et al. [20]. For antigenic unmasking slides were treated twice by covering in 10 mM sodium citrate buffer, pH 6.0, and heating in a microwave for 5 min. After rinsing in PBS, sections were blocked with 20% goat serum, 1% (w/v) BSA in PBS, for 1 h at room temperature. Slides were then incubated with anti-cyclin D1 antibody (1:50), or anti-active MAPK (1:250) diluted in 0.05% pontamine sky blue, 1% (w/v) BSA/PBS at 4°C overnight. After washing 3×1 min in PBS, and a biotin conjugated secondary antibody, diluted 1:200 in 1% (w/v) BSA/PBS, was applied for 30 min. Slides were washed for 3×1 min in PBS, and then incubated with a fluorecein isothiocyanate (FITC)-conjugated tertiary antibody, diluted 1:200 in PBS for 15 min a dark chamber. Following extensive washing in PBS, slides were mounted in Vectashield (Vector Laboratories, Burlingame, CA, USA. Slides were inspected using an Olympus BX40 fluorescence microscope, and photographed using an Olympus OM-4 Ti camera and slide film (Kodak). For BrdU staining, endogenous peroxidase in deparaffinized and rehydrated sections was quenched by placing slides in ice cold 1% (v/v) H2O2 in methanol for 15 min. Sections were washed 3×1 min in PBS, and antigenic unmasking performed by enzymatic digestion with 0.1% (w/v) trypsin from porcine pancreas, 0.1% (w/v) CaCl2/PBS, pH 7.8, for 10 min at 37°C. Sections were then washed 3×1 min in PBS, and placed in 2 M HCl for 10 min at 37°C to denature DNA. HCl was removed by washing for 3×1 min in PBS. Sections were then incubated in anti-BrdU primary antibody (ICN) diluted 1:10 in 1% horse serum, 1% (w/v) BSA/PBS, for 1 h at room temperature. After washing 3×1 min in PBS, sections were then incubated with a biotin conjugated horse anti-mouse IgG diluted 1:200 in 1% (w/v) BSA/PBS, for 30 min, washed 3×1 min in PBS and finally incubated with an extravidin horse radish peroxidase conjugated tertiary antibody (Sigma UK) diluted 1:200 in PBS for 30 min. Sections were washed 2×1 min in PBS, and incubated with 0.05% (w/v) DAB (3,3-diaminobenzidine), 0.3% (v/v) H2O2/PBS for up to 10 min to allow colour development. Nuclei were counterstained for 2 min with haematoxylin (BDH). 2.6 Statistical methods Western blot films were analysed by densitometric scanning (using a Bio-Rad GS 690 imaging densitometer and computer program “molecular analyst”), enabling both identification and quantitative analysis to be performed. The data were further analysed using a non-parametric or unpaired t-test using the program Instat. 3 Results 3.1 Inhibition of SMC proliferation in intact aortic tissue Stimulation of low-density quiescent isolated SMCs with 10% FCS resulted in entry into S-phase as determined by [3H]thymidine (Fig. 1A). From the point of inflection on the time course in Fig. 1A, the length of the G1 was phase was estimated to be 14–16 h, in agreement with previous findings [21]. After 48 h serum stimulation, 44±3% (mean±S.E.M., n=3) of the isolated cells had entered S-phase as measured by BrdU incorporation. On the contrary, stimulation of 4 mm segments of uninjured aortic tissue with 10% FCS, for up to 48 h led to [3H]thymidine incorporation approximately 100-fold lower than in isolated cells (Fig. 1A), and no cells labelled with BrdU were observed in two sections each from three aortas. The viability of the tissue over this 48 h period was not compromised because the concentration of ATP in tissue which had been cultured for 48 h was not significantly lower than that of freshly isolated tissue. The ATP concentration was 0.35±0.10 nmoles/μg DNA in freshly isolated tissue and 0.33±0.02 nmoles/μg DNA after 48 h (n=6), consistent with published values for aortic tissue [18]. When the time course was expanded to 4 days, BrdU incorporation into uninjured aortas (n=4) was detectable (0.9±2.1%), however this was not different from aortas incubated in the absence of serum (1.3±0.4%). By contrast, segments from balloon-injured aortas showed approximately eightfold more BrdU incorporation (7.3±1.7%, p<0.05 vs. uninjured) when incubated with 10% serum, which was also significantly more than when injured segments were incubated without serum (1.0±0.7%). These data showed that smooth muscle cell proliferation in response to serum was suppressed in intact aorta compared to isolated cells but that it could be partially relieved by injury. We therefore investigated which components of the cell cycle machinery were affected differently in isolated rat aortic SMCs and cells within tissue. Fig. 1 Open in new tabDownload slide (A) Incorporation of [3H]thymidine into isolated smooth muscle cells and cells in aortic tissue. Data are means of three separate experiments, ±S.E.M. Fig. 1 Open in new tabDownload slide (A) Incorporation of [3H]thymidine into isolated smooth muscle cells and cells in aortic tissue. Data are means of three separate experiments, ±S.E.M. 3.2 Activation of ERK1/2 is not prevented in aortic tissue Prolonged activation of ERK1/2 has been associated with cell proliferation [12]. We therefore measured the activation status of ERK1/2 by Western blotting throughout the period corresponding to the G1 phase of the cell cycle, using an antibody that recognizes the dual phosphorylated active form. We found that activation of ERK1/2 occurred within 15 min of adding serum to aortic tissue (Fig. 2A) [ERK1 was activated from 0.95±0.4 to 2.13±0.1 arbitrary units (AU), and ERK2 was activated from 0.7±0.5 to 2.0±0.1 within 15 min of adding serum, p<0.5 and n=3 in both cases]. However, activation of ERK1/2 remained elevated over pre-stimulation values at all time points up to 24 h in three separate experiments (Fig. 2B), with ERK1 being 2.67±0.85, and ERK2 1.67±0.42 AU after 24 h. In isolated SMCs, activation of ERK1/2 also occurred within 15 min, after which highly active levels persisted up to 12 h (Fig. 2B). ERK1 was 3.6±1.1 and ERK2 1.1±0.8 AU in pre-stimulated cells, these were activated to 12.0±2.4 and 10.6±1.9 AU after 15 min, respectively. After 12 h, values for ERK1 were 6.7±1, and ERK2 2.3±0.6 AU. Total protein levels of ERK1/2 did not change in aortas or isolated cells during stimulation with serum (Fig. 2C). Overall, our data demonstrated that for isolated cells and intact tissue the time course of MAPK activation was similar. It was unlikely therefore to account for failure of aortic SMC proliferation. However, it was possible that ERK activation occurred only in a small population of cells that did not contribute significantly to overall thymidine incorporation. Immunocytochemistry (ICC) was used therefore to investigate the distribution of ERK1/2 activation using immunofluorescence (Fig. 3A), which was the only feasible method of ICC with the available antibodies. In Fig. 3A the background autofluorescence has been red shifted with Pontamine sky blue so that the only green fluorescence was due to specific staining for activated ERK1/2. Immunofluorescence confirmed the Western blotting results that activation occurred at times corresponding to early (1 h) and late (12 h) G1 in intact aortas. They also demonstrated that active ERK1/2 was distributed evenly throughout the medial layer of aortic sections, not in a small subpopulation of cells. Fig. 3 Open in new tabDownload slide Distribution of Active MAPK and cyclin D1 is even in aortic tissue sections. Rat aortic sections were investigated for expression of active MAPK (panel A) and cyclin D1 (panel B) as described in Methods. Tissue was prepared at the indicated time points (h) following serum stimulation. −ve panel was incubated with non-immune rabbit IgG for active MAPK and non-immune mouse IgG for cyclin D1. Results are an example of three similar experiments. White bar represents 250 μM, white arrows indicate the internal elastic lamina, black arrows the external elastic lamina. Fig. 3 Open in new tabDownload slide Distribution of Active MAPK and cyclin D1 is even in aortic tissue sections. Rat aortic sections were investigated for expression of active MAPK (panel A) and cyclin D1 (panel B) as described in Methods. Tissue was prepared at the indicated time points (h) following serum stimulation. −ve panel was incubated with non-immune rabbit IgG for active MAPK and non-immune mouse IgG for cyclin D1. Results are an example of three similar experiments. White bar represents 250 μM, white arrows indicate the internal elastic lamina, black arrows the external elastic lamina. Fig. 2 Open in new tabDownload slide (A) and (B) MAPK activation in aortic tissue (Ao) and isolated smooth muscle cells (SMCs). Western analysis of active MAPK in low-density isolated SMCs and aortic tissue. Cell and tissue lysates were prepared at the indicated time points (h=hours, or min=minutes) following serum stimulation as described in Methods. A 10-μg amount of total cell or tissue protein was separated on 8% SDS–polyacrylamide gels. Results are an example of three similar experiments. (C) Total MAPK in low-density isolated SMCs and aortic tissue. Cell and tissue lysates were prepared at the indicated time points (h=hours) following serum stimulation as described in Methods. A 10-μg amount of total cell or tissue protein was separated on 8% SDS–polyacrylamide gels. Results are an example of three similar experiments. Fig. 2 Open in new tabDownload slide (A) and (B) MAPK activation in aortic tissue (Ao) and isolated smooth muscle cells (SMCs). Western analysis of active MAPK in low-density isolated SMCs and aortic tissue. Cell and tissue lysates were prepared at the indicated time points (h=hours, or min=minutes) following serum stimulation as described in Methods. A 10-μg amount of total cell or tissue protein was separated on 8% SDS–polyacrylamide gels. Results are an example of three similar experiments. (C) Total MAPK in low-density isolated SMCs and aortic tissue. Cell and tissue lysates were prepared at the indicated time points (h=hours) following serum stimulation as described in Methods. A 10-μg amount of total cell or tissue protein was separated on 8% SDS–polyacrylamide gels. Results are an example of three similar experiments. 3.3 Induction of cyclin D1 and Cdk4 is not inhibited in aortic tissue The expression of cyclin D1 in isolated SMCs measured by Western blotting closely followed the pattern for active ERK1/2 in aortas and isolated cells. In aortic tissue and isolated cells, the mean peak of induction occurred within 1 h of serum stimulation. In both tissue and isolated cells, cyclin D1 levels then remained elevated above pre-stimulation levels throughout G1 (Fig. 4A), in each of three similar experiments. In pre-stimulated cells and aortic tissue, cyclin D1 levels were 2.0±1.5 and 1.47±0.9 AU, respectively. Levels were induced after 1 h to 8.7±3.7 AU in cells and 4.8±1.8 AU in aortic tissue. After 24 h cyclin D1 remained elevated above pre-stimulated levels, being 12.0±4.1 in cells and 6.1±1.4 AU in aortic tissue. These data show that the fold induction in cells and tissue is of a similar magnitude. As shown by immunofluorescence in Fig. 3B, increased protein expression of cyclin D1 occurred throughout the medial SMC, not in a small population. These data are consistent with several recent reports suggesting that cyclin D1 is a major downstream target of the Ras>Raf>MEK>ERK1/2 cascade [11,12]. They provide further evidence that the early signal transduction pathways were activated in both aortic tissue and isolated cells. Fig. 4 Open in new tabDownload slide Cyclin D1 and Cdk4 expression in isolated SMCs and aortic tissue (Ao). Western analysis of cyclin D1 (A), and Cdk4 (B), in low-density isolated SMCs and aortic tissue. Cell and tissue lysates (10 μg of total cell or tissue protein) were prepared at the indicated time points (h) following serum stimulation as described in Methods. Results are an example of three similar experiments. Fig. 4 Open in new tabDownload slide Cyclin D1 and Cdk4 expression in isolated SMCs and aortic tissue (Ao). Western analysis of cyclin D1 (A), and Cdk4 (B), in low-density isolated SMCs and aortic tissue. Cell and tissue lysates (10 μg of total cell or tissue protein) were prepared at the indicated time points (h) following serum stimulation as described in Methods. Results are an example of three similar experiments. Levels of the catalytic partner of cyclin D1, Cdk4, increased steadily through the first 12 h after serum stimulation in both aortic tissue and isolated SMCs (Fig. 4B). Since the patterns of cyclin D1 and Cdk4 expression were similar in aortic tissue and isolated SMCs, it is unlikely that their regulation explain the dramatic difference in cell proliferation. 3.4 Induction of cyclin E and Cdk2 is not inhibited in aortic tissue We went on to investigate cell cycle components reported to be essential for mid to late G1 phase progression. Cyclin E was present in isolated quiescent cells and aortic tissue at t=0. Cyclin E was further upregulated in both isolated cells (1.4±0.1-fold induction, n=3) and aortic tissue (1.6±0.2-fold induction, n=3) within 30 min, and the levels then remained invariant throughout the time course (see Fig. 5A). We also investigated Cdk2, an essential G1 kinase that requires cyclin E binding for activity in mid to late G1 phase. In both isolated SMCs and aortic tissue, Cdk2 was induced in response to serum stimulation (2.2±0.4- and 1.7±0.1-fold induction after 24 h, respectively, n=3), with a time dependent accumulation observed (see Fig. 5B). Fig. 5 Open in new tabDownload slide Cyclin E and Cdk2 levels in isolated SMCs and aortic tissue (Ao). Western analysis of cyclin E in low-density isolated SMCs and aortic tissue. For cyclin E (panel A), 100 μg of total cell or tissue protein was immunoprecipitated with anti-cyclin E antibody at the indicated time points (h). For Cdk2 (panel B), 10 μg of total cell or tissue protein was separated. Data are representatives of three experiments. Fig. 5 Open in new tabDownload slide Cyclin E and Cdk2 levels in isolated SMCs and aortic tissue (Ao). Western analysis of cyclin E in low-density isolated SMCs and aortic tissue. For cyclin E (panel A), 100 μg of total cell or tissue protein was immunoprecipitated with anti-cyclin E antibody at the indicated time points (h). For Cdk2 (panel B), 10 μg of total cell or tissue protein was separated. Data are representatives of three experiments. 3.5 p16 and p27 CKIs are downregulated in response to serum stimulation in isolated cells but not aortic tissue The results described above demonstrate that inhibition of SMC proliferation in aortic tissue is not mediated by inhibition of ERK1/2 activation, or by cyclin D1, Cdk4, cyclin E or Cdk2 protein expression. However, the absence of proliferation in intact tissue suggested that active holoenzymes were not assembled. We therefore investigated the regulation of p16, which selectively inhibits Cdk4, and p27, which inhibits both Cdk4 and Cdk2. Levels of p16 (Fig. 6A) and p27 (Fig. 6B) were detected by Western analysis in aortas and quiescent isolated SMCs. In aortas, p16 and p27 were clearly detected at t=0 and no downregulation occurred in response to serum stimulation; levels remained elevated for the duration of the time course (Fig. 6A, B). After 24 h, p27 levels were 125±17% of pre-stimulation levels, and p16 levels were 90.2±11.8% of pre-stimulation levels, n=3, p>0.05). By contrast, both the CKIs were downregulated as the isolated SMCs progressed through the cell cycle (Fig. 6B). 10.87±4% of p27 remaining after 24 h, and 28.1±10.1% of p16, n=3, p<0.05. Persistent expression of p16 and p27 in aortic tissue could suppress the activity of their target Cdks and might therefore explain the absence of cell proliferation. To test this hypothesis we conducted assays of cyclin D/Cdk4 and cyclin E/Cdk2 activity. Fig. 6 Open in new tabDownload slide p16 and p27 levels in isolated SMCs and aortic tissue (Ao). Western analysis of p16 (panel A), and p27 (panel B) in low-density isolated SMCs and aortic tissue. For isolated cells, 100 μg of total protein was immunoprecipitated with anti-p16 antibody at the indicated time points (h) following serum stimulation as described in methods. For studies looking at p16 in tissue, 20 μg of total protein was separated. For p27, 10 μg of total cell or tissue protein was used. Results are an example of three experiments. Panels C and D show p16 and p27 levels, respectively, following serum stimulation of high-density cultures. Lane 1 in each represents pre-stimulated quiescent high-density cultures, and lane 2 cultures which have been stimulated for 24 h. Fig. 6 Open in new tabDownload slide p16 and p27 levels in isolated SMCs and aortic tissue (Ao). Western analysis of p16 (panel A), and p27 (panel B) in low-density isolated SMCs and aortic tissue. For isolated cells, 100 μg of total protein was immunoprecipitated with anti-p16 antibody at the indicated time points (h) following serum stimulation as described in methods. For studies looking at p16 in tissue, 20 μg of total protein was separated. For p27, 10 μg of total cell or tissue protein was used. Results are an example of three experiments. Panels C and D show p16 and p27 levels, respectively, following serum stimulation of high-density cultures. Lane 1 in each represents pre-stimulated quiescent high-density cultures, and lane 2 cultures which have been stimulated for 24 h. 3.6 Cyclin D1 and cyclin E associated kinase activities are persistently inhibited in aortic tissue Induction of cyclin D1 (Fig. 7A) or cyclin E (Fig. 7B) associated kinase activity remained undetectable in aortic tissue even after 24 h of serum stimulation. By contrast, in isolated cells, both cyclin D1 and cyclin E associated kinase activity was increased in response to serum stimulation as expected (Fig. 7A, B). Levels were undetectable before stimulation and rose to 12.25±2.1 and 19.25±2.2 AU for cyclin D1/Cdk4 and cycle E/Cdk 2, respectively. Hence active kinase complexes were absent from quiescent isolated cells but increased as SMCs approached the G1/S phase transition. Fig. 7 Open in new tabDownload slide Cyclin D1 and cyclin E associated kinase activity in low-density isolated SMCs and aortic tissue (Ao). Radioactive kinase assay showing cyclin D1 associated kinase activity, (panel A), or cyclin E associated kinase activity, (panel B), in isolated SMCs and aortic tissue at the indicated time points (h) following serum stimulation. Lysates from isolated cells were used as positive controls for the tissue samples (24 +ve). Data are representative of three individual experiments. Fig. 7 Open in new tabDownload slide Cyclin D1 and cyclin E associated kinase activity in low-density isolated SMCs and aortic tissue (Ao). Radioactive kinase assay showing cyclin D1 associated kinase activity, (panel A), or cyclin E associated kinase activity, (panel B), in isolated SMCs and aortic tissue at the indicated time points (h) following serum stimulation. Lysates from isolated cells were used as positive controls for the tissue samples (24 +ve). Data are representative of three individual experiments. 3.7 Regulation of proliferation and CKIs in isolated smooth muscle cells grown to high density Although smooth muscle cells in culture do not exhibit growth arrest at confluence, their growth is downregulated at high cell density. Since smooth muscle cells in aortic tissue are arguably at higher density than isolated cultures, we examined the effect of high-density culture on proliferation and patterns of CKI protein expression. When high-density cultures of SMCs were stimulated with 10% FCS, the incorporation of [3H]thymidine was reduced 4.5-fold compared to cells which were stimulated at low density (5700±1800 vs. 25 600±7400 dpm/μg DNA, p<0.05, n=6). Interestingly, the levels of the CKIs were maintained in cells that had been stimulated at high density (Fig. 6C and D). Hence cells growing at high density recapitulated the key differences in proliferation rate and CKI downregulation between aortic tissues and low-density cultures. 4 Discussion We sought to understand the mechanisms underlying the unresponsiveness of intact as opposed to injured rat aorta to exogenous growth factors (see introduction). One hypothesis was that growth factor receptors are not present or are sequestered so that the immediate signal transduction from growth factor receptors is impaired. A second hypothesis suggests altered regulation of the cell cycle that results in failure to progress into S phase. To distinguish these possibilities, we compared the pattern of activation of the signal transducing ERKs and the changes in protein expression and activity of several key cell cycle proteins in isolated SMCs and SMCs in aortic tissue. Our objective was to define differences that would explain the contrasting SMC proliferative response to serum stimulation. Our key findings are that activation ERKs 1 and 2, and induction of cyclin D1 and cyclin E, early events in G1 progression, occur with a similar time course uninjured aortic tissue and isolated cells. By contrast, downregulation of p16 and p27 CKIs is prevented in aortic tissue in response to serum stimulation, and this is associated with a lack of cyclin D1/Cdk4, and cyclin E/Cdk2 kinase activity. Failure to downregulate CKIs and impairment of proliferation also occurred in isolated cells grown at high density. The potential importance of p27 in the maintenance of SMC quiescence, has been suggested previously by studies showing that arterial injury can promote differential inhibitor expression. For example, injury of porcine femoral arteries with a balloon catheter enhanced SMC proliferation, co-incident with a decline in p27 levels [17]. The reappearance of p27 at later time points in these arteries (3 weeks), correlated with a reduced proliferative capacity and re-establishment of quiescence [17]. This suggests that p27 is an important component in the reversing the increased proliferation that follows balloon injury. Our studies also demonstrated that in confluent cultures of SMCs, which are re-establishing quiescence, the levels of p27 are increased. Studies using gene transfer have demonstrated that significant inhibition of neointimal formation in animal models can be achieved by over expressing p27 [22] and p21 [23]. In particular, overexpression of p27, but not p16, has been shown to be sufficient to inhibit VSMC proliferation after balloon injury in pigs [22]. However, p16 overexpression did partially inhibit proliferation of isolated pig smooth muscle cells [22] and it was not tested whether overexpression of p16 might further enhance the inhibitory effect of p27. In the studies of Tanner et al., p16 was not detectable in normal or balloon injured porcine arteries [22]. However, we found p16 as well as p27 to be present in the rat aorta, and this may reflect species differences, or differences in protein extraction method. The presence of more than one inhibitory signal is consistent with findings for p27−/− mice. Cells grown from p27−/− mice are not impaired in their ability to enter G0 in response to antiproliferative signals such as serum starvation, TGF-β and rapamycin [24]. Moreover their vascular development is grossly normal. Our results show directly that the constitutive levels of p16 and p27 in the normal uninjured vessel wall are sufficient to suppress Cdk activity. They strongly imply that the maintenance of CKI levels contributes to the very low levels of proliferation seen in normal blood vessels. The potential mechanisms underlying the arrest of smooth muscle cell proliferation in arteries have been discussed in detail elsewhere [1]. Briefly, one explanation for the maintenance of p16 and p27 in rat aortic tissue and confluent SMCs in the presence of serum, is that cell–matrix or cell–cell contacts relay inhibitory signals to the cell cycle machinery. In the rat aorta, where medial SMCs are surrounded and attached to a cage of basement membrane consisting of laminin, collagen IV and heparan sulphate proteoglycans, the cells are prevented from responding to endogenous growth factors mainly because of cell–matrix interactions. For example, it has previously been demonstrated that cells grown on polymer collagen and laminin, which probably mimic the basement membrane, do not divide when exposed to mitogenic stimuli [16,25]. The reason for this is thought to be integrin initiated signalling pathways, the nature of which are not fully defined. Alternatively, the presence of basement membranes may prevent engagement of specific matrix molecules with cognate integrin receptors. Our data do not distinguish between these alternative mechanisms. Interestingly, our results showed that the proliferation response to serum of SMCs in the rat aorta is significantly increased after injury. In contrast, the proliferation of smooth muscle cells was not increased when the injured aorta was cultured in serum-free medium. 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TI - Mechanisms underlying maintenance of smooth muscle cell quiescence in rat aorta: role of the cyclin dependent kinases and their inhibitors
JF - Cardiovascular Research
DO - 10.1016/S0008-6363(01)00444-8
DA - 2002-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/mechanisms-underlying-maintenance-of-smooth-muscle-cell-quiescence-in-WORFifnoyW
SP - 242
EP - 252
VL - 53
IS - 1
DP - DeepDyve
ER -