Fibronectin promotes cell cycle entry in smooth muscle cells in primary culture

Smooth muscle cell proliferation after arterial injury is regulated by growth factors and components of the extracellular matrix. We have previously demonstrated that fibronectin promotes a phenotypic modulation of freshly isolated rat smooth muscle cells from a contractile to a synthetic phenotype in primary culture and supports the ability of the cells to respond to growth factors. Here, we analyzed if fibronectin promotes cell cycle entry in freshly isolated rat aortic smooth muscle cells during primary culture. Cell cycle analysis showed that cells seeded on fibronectin remained in the G(0)/G(1) phase of the cell cycle during the first 6 days of culture. During this period, there was an increased expression of cyclin D1 and p27(KIP1) in the absence of exogenous growth factors. Addition of serum was followed by enhanced cyclin D1 expression, decreased p27(KIP1) levels, hyperphosphorylation of Rb protein, induction of cyclin A and cyclin D3 expression, and cell cycle progression into S phase. The results indicate that fibronectin initiates cell cycle entry in freshly isolated smooth muscle cells by promoting the induction of cyclin D1 and thereby facilitates further cell cycle progression together with growth factors.     

NFATc1 targets cyclin A in the regulation of vascular smooth muscle cell multiplication during restenosis

Platelet-derived growth factor BB (PDGF-BB) induced cyclin A expression and CDK2 activity in vascular smooth muscle cells (VSMC). Inhibition of nuclear factors of activated T cell (NFAT) activation by cyclosporin A (CsA) and VIVIT suppressed PDGF-BB-induced cyclin A expression and CDK2 activity, resulting in blockade of VSMC in the G(1) phase. In addition, CsA- and VIVIT-mediated inhibition of NFATs and small interfering RNA-targeted down-regulation of cyclin A levels suppressed PDGF-BB-induced VSMC DNA synthesis. PDGF-BB also induced cyclin A mRNA levels in VSMC in an NFAT-dependent manner. Cloning and bioinformatic analysis of rat cyclin A promoter revealed the presence of NFAT-binding elements, and PDGF-BB induced the binding of NFATs to these regulatory sequences in a CsA- and VIVIT-sensitive manner. Chromatin immunoprecipitation analysis showed that NFATc1 binds to the cyclin A promoter in response to PDGF-BB in a VIVIT-sensitive manner. Furthermore, PDGF-BB induced cyclin A promoter-luciferase reporter gene activity in VSMC, and it was inhibited by both CsA and VIVIT. Balloon injury induced cyclin A expression and CDK2 activity in rat carotid arteries, and these responses were also blocked by VIVIT. In addition, VIVIT attenuated balloon injury-induced SMC proliferation, resulting in reduced restenosis. Down-regulation of NFATc1 by its small interfering RNA inhibited PDGF-BB-induced cyclin A expression and DNA synthesis both in rat and human VSMC. Together, these findings demonstrate that the cyclin A-CDK2 complex may be a potential effector of NFATs, specifically NFATc1, in mediating SMC multiplication leading to neointima formation. Therefore, NFATs may be used as target molecules for the development of therapeutic agents against vascular diseases such as restenosis.     

Ceramide 1-phosphate induces neointimal formation via cell proliferation and cell cycle progression upstream of ERK1/2 in vascular smooth muscle cells

Ceramide 1-phosphate (C1P) is a novel bioactive sphingolipid formed by ceramide kinase (CERK)-catalyzed phosphorylation of ceramide. It has been implicated in the regulation of such vital pathophysiological functions as phagocytosis and inflammation, but there have been no reports ascribing a biological function to CERK in vascular disorders. Here the potential role of CERK/C1P in neointimal formation was investigated using rat aortic vascular smooth muscle cells (VSMCs) in primary culture and a rat carotid injury model. Exogenous C8-C1P stimulated cell proliferation, DNA synthesis, and cell cycle progression of rat aortic VSMCs in primary culture. In addition, wild-type CERK-transfected rat aortic VSMCs induced a marked increase in rat aortic VSMC proliferation and [³H]-thymidine incorporation when compared to empty vector transfectant. C8-C1P markedly activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) within 5 min, and the activation could be prevented by U0126, a MEK inhibitor. Also, K1, a CERK inhibitor, decreased the ERK1/2 phosphorylation and cell proliferation on platelet-derived growth factor (PDGF)-stimulated rat aortic VSMCs. CERK expression and C1P levels were found to be potently increased during neointimal formation using a rat carotid injury model. However, ceramide levels decreased during the neointimal formation process. These findings suggest that C1P can induce neointimal formation via cell proliferation through the regulation of the ERK1/2 protein in rat aortic VSMCs and that CERK/C1P may regulate VSMC proliferation as an important pathogenic marker in the development of cardiovascular disorders.     

Insulin-like growth factor I triggers nuclear accumulation of cyclin D1 in MCF-7S breast cancer cells

Stimulation of the breast cancer-derived MCF-7S cell line with insulin-like growth factor I (IGF-I; 20 ng/ml) leads to enhanced expression of cyclin D1, hyperphosphorylation of pRb, DNA synthesis, and cell division. 17beta-Estradiol (E(2); 10(-9) m) is not able to stimulate proliferation of MCF-7S cells, although addition of E(2) to serum-starved cells does result in induction of cyclin D1. However, in combination with submitogenic amounts of IGF-I (2 ng/ml), E(2) induces cell proliferation. We have previously shown that the synergistic action of E(2) and IGF-I emanates from the ability of both hormones to induce cyclin D1 expression and that IGF-I action is required to induce activity of the cyclin D1-CDK4 complex, which triggers cell cycle progression. Here, we show that IGF-I (but not E(2)) is able to induce nuclear accumulation of cyclin D1 by a phosphatidylinositol 3-kinase-dependent mechanism. Nuclear accumulation of cyclin D1 and cell cycle progression were also observed when LiCl, a known inhibitor of GSK3beta, was added to E(2)-stimulated cells. Thus, inhibition of GSK3beta activity appears to trigger nuclear accumulation of cyclin D1 and cell cycle progression. This notion was confirmed by overexpression of constitutively active GSK3beta, which blocks IGF-I-induced nuclear accumulation of cyclin D1 as well as S phase transition.     

PDK1 regulates cell proliferation and cell cycle progression through control of cyclin D1 and p27Kip1 expression

PDK1 (3-phosphoinositide-dependent protein kinase 1) is a key mediator of signaling by phosphoinositide 3-kinase. To gain insight into the physiological importance of PDK1 in cell proliferation and cell cycle control, we established immortalized mouse embryonic fibroblasts (MEFs) from mice homozygous for a "floxed" allele of Pdk1 and from wild-type mice. Introduction of Cre recombinase by retrovirus-mediated gene transfer resulted in the depletion of PDK1 in Pdk1(lox/lox) MEFs but not in Pdk1(+/+) MEFs. The insulin-like growth factor-1-induced phosphorylation of various downstream effectors of PDK1, including Akt, glycogen synthase kinase 3, ribosomal protein S6, and p70 S6 kinase, was markedly inhibited in the PDK1-depleted (Pdk1-KO) MEFs. The rate of serum-induced cell proliferation was reduced; progression of the cell cycle from the G(0)-G(1) phase to the S phase was delayed, and cell cycle progression at G(2)-M phase was impaired in Pdk1-KO MEFs. These cells also manifested an increased level of p27(Kip1) expression and a reduced level of cyclin D1 expression during cell cycle progression. The defect in cell cycle progression from the G(0)-G(1) to the S phase in Pdk1-KO MEFs was rescued by forced expression of cyclin D1, whereas rescue of the defect in G(2)-M progression in these cells required both overexpression of cyclin D1 and depletion of p27(Kip1) by RNA interference. These data indicate that PDK1 plays an important role in cell proliferation and cell cycle progression by controlling the expression of both cyclin D1 and p27(Kip1).     

Inhibition of cell cycle progression and migration of vascular smooth muscle cells by prostaglandin D2 synthase: resistance in diabetic Goto-Kakizaki rats

The regulation of vascular smooth muscle cell (VSMC) proliferation, migration, and apoptosis plays a clear role in the atherosclerotic process. Recently, we reported on the inhibition of the exaggerated growth phenotype of VSMCs isolated from hypertensive rats by lipocalin-type prostaglandin D₂ synthase (L-PGDS). In the present study, we report the differential effects of L-PGDS on VSMC cell cycle progression, migration, and apoptosis in wild-type VSMCs vs. those from a type 2 diabetic model. In wild-type VSMCs, exogenously added L-PGDS delayed serum-induced cell cycle progression from the G₁ to S phase, as determined by gene array analysis and the decreased protein expressions of cyclin-dependent kinase-2, p21^Cip1, and cyclin D1. Cyclin D3 protein expression was unaffected by L-PGDS, although its gene expression was stimulated by L-PGDS in wild-type cells. In addition, platelet-derived growth factor-induced VSMC migration was inhibited by L-PGDS in wild-type cells. Type 2 diabetic VSMCs, however, were resistant to the L-PGDS effects on cell cycle progression and migration. L-PGDS did suppress the hyperproliferation of diabetic cells, albeit through a different mechanism, presumably involving the 2.5-fold increase in apoptosis and the concomitant 10-fold increase of L-PGDS uptake we observed in these cells. We propose that in wild-type VSMCs, L-PGDS retards cell cycle progression and migration, precluding hyperplasia of the tunica media, and that diabetic cells appear resistant to the inhibitory effects of L-PGDS, which consequently may help explain the increased atherosclerosis observed in diabetes. apoptosis; atherosclerosis; insulin resistance     

BCR-ABL and interleukin 3 promote haematopoietic cell proliferation and survival through modulation of cyclin D2 and p27Kip1 expression

Although it is evident that BCR-ABL can rescue cytokine-deprived hematopoietic progenitor cells from cell cycle arrest and apoptosis, the exact mechanism of action of BCR/ABL and interleukin (IL)-3 to promote proliferation and survival has not been established. Using the pro-B cell line BaF3 and a BaF3 cell line stably overexpressing BCR-ABL (BaF3-p210), we investigated the proliferative signals derived from BCR-ABL and IL-3. The results indicate that both IL-3 and BCR-ABL target the expression of cyclin Ds and down-regulation of p27(Kip1) to mediate pRB-related pocket protein phosphorylation, E2F activation, and thus S phase progression. These findings were further confirmed in a BaF3 cell line (TonB.210) where the BCR-ABL expression is inducible by doxycyclin and by using the drug STI571 to inactivate BCR-ABL activity in BaF3-p210. To establish the functional significance of cyclin D2 and p27(Kip1) expression in response to IL-3 and BCR-ABL expression, we studied the effects of ectopic expression of cyclin D2 and p27(Kip1) on cell proliferation and survival. Our results demonstrate that both cyclin D2 and p27(Kip1) have a role in BaF3 cell proliferation and survival, as ectopic expression of cyclin D2 is sufficient to abolish the cell cycle arrest and apoptosis induced by IL-3 withdrawal or by BCR-ABL inactivation, while overexpression of p27(Kip1) can cause cell cycle arrest and apoptosis in the BaF3 cells. Furthermore, our data also suggest that cyclin D2 functions upstream of p27(Kip1), cyclin E, and cyclin D3, and therefore, plays an essential part in integrating the signals from IL-3 and BCR-ABL with the pRB/E2F pathway.     

Calmodulin-Mediated Cell Cycle Regulation: New Mechanisms for Old Observations

The significance of divalent calcium ions (Ca2+) to cell cycle progression has been a subject of study for several decades, with a regulatory role for Ca2+ suggested in distinct cell types and multiple organisms. Our interest in proliferative vascular diseases led us to focus on mammalian vascular smooth muscle cells (VSMC) in particular, in which we and others had shown that a coordinate elevation in the intracellular free Ca2+ concentration is required for G1 to S phase cell cycle progression. However, the molecular basis for this Ca2+-sensitive cell cycle transition was not known.Our recent discovery of a functional protein-protein interaction between the late G1-active cyclin E1 and the major calcium signal-transducing factor Calmodulin (CaM) sheds new light on the mechanism(s) through which Ca2+ concentrations regulate cell cycle. Having identified a CaM-binding site on cyclin E1, our studies support a direct role for CaM in mediating Ca2+-sensitive cyclin E/cdk2 activity and G1 to S phase transitions in VSMC. The CaM binding site identified on cyclin E1 has a Kd for CaM consistent with that of known CaM-binding proteins, and is composed of a 22 amino acids N-terminal sequence that is highly conserved across several mammalian species. Deletion of this binding site abolished CaM binding and Ca2+-sensitive cyclin E/cdk2 activity.Here we provide our perspectives on the literature supporting a role for Ca2+ in cell cycle regulation, focusing on the evidence implicating CaM in this functionality, and discuss the potential for therapeutic modulation of CaM-dependent cell cycle machinery.     

Inhibition of vascular smooth muscle cell proliferation by chronic hemin treatment

Hemin, an oxidized form of heme, is an essential regulator of gene expression and cell cycle progression. Our laboratory previously reported (34) that chronic hemin treatment of spontaneously hypertensive rats reversed the eutrophic inward remodeling of small peripheral arteries. Whether long-term treatment of cultured vascular smooth muscle cells (VSMCs) with hemin alters the proliferation status of these cells has been unknown. In the present study, hemin treatment at 5 muM for 4, 7, 14, and 21 days significantly inhibited the proliferation of cultured rat aortic VSMCs (A-10 cells) by arresting cells at G0/G1 phases so as to decelerate cell cycle progression. Heme oxygenase (HO) activity and inducible HO-1 protein expression were significantly increased by hemin treatment. HO inhibitor tin protoporphyrin IX (SnPP) abolished the effects of hemin on cell proliferation and HO activity. Interestingly, hemin-induced HO-1 expression was further increased in the presence of SnPP. Hemin treatment had no significant effect on the expression of constitutive HO-2. Expression of p21 protein and the level of reactive oxygen species (ROS) were decreased by hemin treatment, which was reversed by application of SnPP. After removal of hemin from culture medium, inhibited cell proliferation and increased HO-1 expression in VSMCs were returned to control level within 1 wk. Transfection with HO-1 small interfering RNA significantly knocked down HO-1 expression and decreased HO activity, but had no effect on HO-2 expression, in cells treated with or without hemin for 7 days. The inhibitory effect of hemin on cell proliferation was abolished in HO-1 silenced cells. It is concluded that induction of HO-1 and, consequently, increased HO activity are responsible for the chronic inhibitory effect of hemin on VSMC proliferation. Changes in the levels of p21 and ROS might also participate in the cellular effects of hemin.     

c-Ski inhibits the proliferation of vascular smooth muscle cells via suppressing Smad3 signaling but stimulating p38 pathway

Proliferation of vascular smooth muscle cells (VSMCs) plays key roles in the progression of intimal hyperplasia, but the molecular mechanisms that trigger VSMC proliferation after vascular injury remain unclear. c-Ski, a co-repressor of transforming growth factor β (TGF-β)/Smad signaling, was detected to express in VSMC of rat artery. During the course of arterial VSMC proliferation induced by balloon injury in rat, the endogenous protein expressions of c-Ski decreased markedly in a time-dependent manner. In vivo c-Ski gene delivery was found to significantly suppress balloon injury-induced VSMC proliferation and neointima formation. Further investigation in A10 rat aortic smooth muscle cells demonstrated that overexpression of c-Ski gene inhibited TGF-β1 (1 ng/ml)-induced A10 cell proliferation while knockdown of c-Ski by RNAi enhanced the stimulatory effect of TGF-β1 on A10 cell growth. Western blot for signaling detection showed that suppression of Smad3 phosphorylation while stimulating p38 signaling associated with upregulation of cyclin-dependent kinase inhibitors p21 and p27 was responsible for the inhibitory effect of c-Ski on TGF-β1-induced VSMC proliferation. These data suggest that the decrease of endogenous c-Ski expression is implicated in the progression of VSMC proliferation after arterial injury and c-Ski administration represents a promising role for treating intimal hyperplasia via inhibiting the proliferation of VSMC.     

PYK2 signaling is required for PDGF-dependent vascular smooth muscle cell proliferation

Aberrant vascular smooth muscle cell (VSMC) growth is associated with many vascular diseases including atherosclerosis, hypertension, and restenosis. Platelet-derived growth factor-BB (PDGF) induces VSMC proliferation through control of cell cycle progression and protein and DNA synthesis. Multiple signaling cascades control VSMC growth, including members of the mitogen-activated protein kinase (MAPK) family as well as phosphatidylinositol 3-kinase (PI3K) and its downstream effector AKT/protein kinase B (PKB). Little is known about how these signals are integrated by mitogens and whether there are common receptor-proximal signaling control points that synchronize the execution of physiological growth functions. The nonreceptor proline-rich tyrosine kinase 2 (PYK2) is activated by a variety of growth factors and G protein receptor agonists in VSMC and lies upstream of both PI3K and MAPK cascades. The present study investigated the role of PYK2 in PDGF signaling in cultured rat aortic VSMC. PYK2 downregulation attenuated PDGF-dependent protein and DNA synthesis, which correlated with inhibition of AKT and extracellular signal-regulated kinases 1 and 2 (ERK1/2) but not p38 MAPK activation. Inhibition of PDGF-dependent protein kinase B (AKT) and ERK1/2 signaling by inhibitors of upstream kinases PI3K and MEK, respectively, as well as downregulation of PYK2 resulted in modulation of the G(1)/S phase of the cell cycle through inhibition of retinoblastoma protein (Rb) phosphorylation and cyclin D(1) expression, as well as p27(Kip) upregulation. Cell division kinase 2 (cdc2) phosphorylation at G(2)/M was also contingent on PDGF-dependent PI3K-AKT and ERK1/2 signaling. These data suggest that PYK2 is an important upstream mediator in PDGF-dependent signaling cascades that regulate VSMC proliferation.     

Overexpression of cyclin E/CDK2 complexes overcomes FGF-induced cell cycle arrest in the presence of hypophosphorylated Rb proteins

FGF signaling inhibits chondrocyte proliferation and requires the function of the p107 and p130 members of the Rb protein family to execute growth arrest. p107 dephosphorylation plays a critical role in the chondrocyte response to FGF, as overexpression of cyclin D1/CDK4 complexes (the major p107 kinase) in rat chondrosarcoma (RCS) cells overcomes FGF-induced p107 dephosphorylation and growth arrest. In cells overexpressing cyclin D1/CDK4, FGF-induced downregulation of cyclin E/CDK2 activity was absent. To examine the role of cyclin E/CDK2 complexes in mediating FGF-induced growth arrest, this kinase was overexpressed in RCS cells. FGF-induced dephosphorylation of either p107 or p130 was not prevented by overexpressing cyclin E/CDK2 complexes. Unexpectedly, however, FGF-treated cells exhibited sustained proliferation even in the presence of hypophosphorylated p107 and p130. Both pocket proteins were able to form repressive complexes with E2F4 and E2F5 but these repressors were not translocated into the nucleus and therefore were unable to occupy their respective target DNA sites. Overexpressed cyclin E/CDK2 molecules were stably associated with p107 and p130 in FGF-treated cells in the context of E2F repressive complexes. Taken together, our data suggest a novel mechanism by which cyclin E/CDK2 complexes can promote cell cycle progression in the presence of dephosphorylated Rb proteins and provide a novel insight into the key Retinoblastoma/E2F/cyclin E pathway. Our data also highlight the importance of E2F4/p130 complexes for FGF-mediated growth arrest in chondrocytes.     

Overexpression of cyclin E/CDK2 complexes overcomes FGF-induced cell cycle arrest in the presence of hypophosphorylated Rb proteins

FGF signaling inhibits chondrocyte proliferation and requires the function of the p107 and p130 members of the Rb protein family to execute growth arrest. p107 dephosphorylation plays a critical role in the chondrocyte response to FGF, as overexpression of cyclin D1/CDK4 complexes (the major p107 kinase) in rat chondrosarcoma (RCS) cells overcomes FGF-induced p107 dephosphorylation and growth arrest. In cells overexpressing cyclin D1/CDK4, FGF-induced downregulation of cyclin E/CDK2 activity was absent. To examine the role of cyclin E/CDK2 complexes in mediating FGF-induced growth arrest, this kinase was overexpressed in RCS cells. FGF-induced dephosphorylation of either p107 or p130 was not prevented by overexpressing cyclin E/CDK2 complexes. Unexpectedly, however, FGF-treated cells exhibited sustained proliferation even in the presence of hypophosphorylated p107 and p130. Both pocket proteins were able to form repressive complexes with E2F4 and E2F5 but these repressors were not translocated into the nucleus and therefore were unable to occupy their respective target DNA sites. Overexpressed cyclin E/CDK2 molecules were stably associated with p107 and p130 in FGF-treated cells in the context of E2F repressive complexes. Taken together, our data suggest a novel mechanism by which cyclin E/CDK2 complexes can promote cell cycle progression in the presence of dephosphorylated Rb proteins and provide a novel insight into the key Retinoblastoma/E2F/cyclin E pathway. Our data also highlight the importance of E2F4/p130 complexes for FGF-mediated growth arrest in chondrocytes.