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

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, p21(Cip1/Waf1) and p27(Kip1), 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.     

Checkpoint kinase 1 prevents cell cycle exit linked to terminal cell differentiation

Trophoblast stem (TS) cells proliferate in the presence of fibroblast growth factor 4, but in its absence, they differentiate into polyploid trophoblast giant (TG) cells that remain viable but nonproliferative. Differentiation is coincident with expression of the cyclin-dependent kinase (CDK)-specific inhibitors p21 and p57, of which p57 is essential for switching from mitotic cell cycles to endocycles. Here, we show that, in the absence of induced DNA damage, checkpoint kinase-1 (CHK1), an enzyme essential for preventing mitosis in response to DNA damage, functions as a mitogen-dependent protein kinase that prevents premature differentiation of TS cells into TG cells by suppressing expression of p21 and p57, but not p27, the CDK inhibitor that regulates mitotic cell cycles. CHK1 phosphorylates p21 and p57 proteins at specific sites, thereby targeting them for degradation by the 26S proteasome. TG cells lack CHK1, and restoring CHK1 activity in TG cells suppresses expression of p57 and restores mitosis. Thus, CHK1 is part of a "G2 restriction point" that prevents premature cell cycle exit in cells programmed for terminal differentiation, a role that CHK2 cannot play.     

Temporal and spatial patterns of expression of p35, a regulatory subunit of cyclin-dependent kinase 5, in the nervous system of the mouse

The protein p35 is a regulatory subunit of cyclin-dependent kinase 5. It has no recognized homology to cyclins but binds to and activates cyclin-dependent kinase 5 directly in the absence of other protein molecules. Cyclin-dependent kinase 5 was initially isolated by homology to the key cell cycle regulator cdc2 kinase and later identified as a neuronal kinase that phosphorylates histone H1, tau or neurofilaments. This kinase is localized in axons of the developing and mature nervous system. To understand the role of p35 as a regulator of cyclin-dependent kinase 5 activity in the CNS, we examined the pattern of expression of p35 mRNA in the nervous system of embryonic, early postnatal and adult mice. In separate experiments, we also examined the spatial distribution of cyclin-dependent kinase 5 mRNA and the activity of cyclin-dependent kinase 5/p35 kinase complex. Postmitotic cells express p35 mRNA immediately after they leave the zones of cell proliferation. It is also expressed in developing axonal tracts in the brain. Cyclin-dependent kinase 5 mRNA is present in postmitotic and in proliferative cells throughout the embryonic central nervous system. During early postnatal period signal for p35 mRNA declines while that for cyclin-dependent kinase 5 mRNA increases throughout the brain. In the adult brain although both p35 and cyclin-dependent kinase 5 mRNAs are expressed at relatively high levels in certain structures associated with the limbic system, considerable differences exist in the patterns of their distribution in other parts of the brain. These data suggest that the p35/cyclin-dependent kinase 5 complex may be associated with early events of neuronal development such as neuronal migration and axonal growth while in the limbic system of the mature brain it may be associated with the maintenance of neuronal plasticity.     

The multiple roles of the cyclin-dependent kinase inhibitory protein p57(KIP2) in cerebral cortical neurogenesis

The members of the CIP/KIP family of cyclin-dependent kinase (CDK) inhibitory proteins (CKIs), including p57(KIP2), p27(KIP1), and p21(CIP1), block the progression of the cell cycle by binding and inhibiting cyclin/CDK complexes of the G1 phase. In addition to this well-characterized function, p57(KIP2) and p27(KIP1) have been shown to participate in an increasing number of other important cellular processes including cell fate and differentiation, cell motility and migration, and cell death/survival, both in peripheral and central nervous systems. Increasing evidence over the past few years has characterized the functions of the newest CIP/KIP member p57(KIP2) in orchestrating cell proliferation, differentiation, and migration during neurogenesis. Here, we focus our discussion on the multiple roles played by p57(KIP2) during cortical development, making comparisons to p27(KIP1) as well as the INK4 family of CKIs.     

Inhibition of Cdk6 expression through p38 MAP kinase is involved in differentiation of mouse prechondrocyte ATDC5

Because a temporal arrest in the G1-phase of the cell cycle is a prerequisite for cell differentiation, this study investigated the involvement of cell cycle factors in the differentiation of cultured mouse prechondrocyte cell line ATDC5. Among the G1 cell cycle factors examined, both protein and mRNA levels of cyclin-dependent kinase (Cdk6) were downregulated during the culture in a differentiation medium. The protein degradation of Cdk6 was not involved in this downregulation because proteasome inhibitors did not reverse the protein level. When inhibitors of p38 MAPK, ERK-1/2, and PI3K/Akt were added to the culture, only a p38 MAPK inhibitor SB203580 blocked the decrease in the Cdk6 protein level by the differentiation medium, indicating that the Cdk6 inhibition was mediated by p38 MAPK pathway. In fact, p38 MAPK was confirmed to be phosphorylated during differentiation of ATDC5 cells. Enforced expression of Cdk6 in ATDC5 cells blocked the chondrocyte differentiation and inhibited Sox5 and Sox6 expressions. However, the Cdk6 overexpression did not affect the proliferation or the cell cycle progression, suggesting that the inhibitory effect of Cdk6 on the differentiation was exerted by a mechanism largely independent of its cell cycle regulation. These results indicate that Cdk6 may be a regulator of chondrocyte differentiation and that its p38-mediated downregulation is involved in the efficient differentiation.     

The oncogenic forms of N-ras or H-ras prevent skeletal myoblast differentiation.

Differentiation of skeletal muscle involves withdrawal of myoblasts from the cell cycle, fusion to form myotubes, and the coordinate expression of a variety of muscle-specific gene products. Fibroblast growth factor and type beta transforming growth factor specifically inhibit myogenesis; however, the transmembrane signaling pathways responsible for suppression of differentiation by these growth factors remain elusive. Because ras proteins have been implicated in the transduction of growth factor signals across the plasma membrane, we used DNA-mediated gene transfer to investigate the potential involvement of this family of regulatory proteins in the control of myogenesis. Transfection of the mouse skeletal muscle cell line C2 with the oncogenic forms of H-ras or N-ras completely suppressed both myoblast fusion and induction of the muscle-specific gene products nicotinic acetylcholine receptor and creatine kinase. Inhibition of differentiation by activated ras genes occurred at the level of muscle-specific mRNA accumulation. In contrast, proto-oncogenic forms of N-ras or H-ras had no apparent effects on the ability of C2 cells to differentiate. Myoblasts transfected with activated ras genes exhibited normal growth properties and ceased proliferating in the absence of mitogens, indicating that ras inhibited differentiation through a mechanism independent of cell proliferation. These results demonstrate that activated ras gene products mimic the inhibitory effects of fibroblast growth factor and type beta transforming growth factor on myogenic differentiation and suggest that each of these regulators of myogenesis may operate through a common intracellular pathway.     

Induction of G1 arrest and apoptosis in human jurkat T cells by pentagalloylglucose through inhibiting proteasome activity and elevating p27Kip1, p21Cip1/WAF1, and Bax proteins

Pentagalloylglucose, which is found in many medicinal plants, can arrest the cell cycle at G(1) phase through down-regulation of cyclin-dependent kinases 2 and 4 and up-regulation of the cyclin-dependent kinase inhibitors p27(Kip1) and p21(Cip1/WAF1) in human breast cancer cells. Pentagalloylglucose also induces apoptosis in human leukemic cells. However, the mechanisms by which pentagalloylglucose induces these effects is unclear. We now show that pentagalloylglucose inhibits the activities of purified 20 and 26 S proteasomes in vitro, the 26 S proteasome in Jurkat T cell lysates, and chymotrypsin-like activity of the 26 S proteasome in intact Jurkat T cells. The turnover of p27(Kip1) and p21(Cip1/WAF1), which is necessary for cell cycle progression mediated by proteasome degradation, was disrupted by treatment of human Jurkat T cells with pentagalloylglucose. This was shown by cycloheximide treatment and in vivo pulse-chase labeling experiments, and this effect correlated with the arrest of proliferation of Jurkat T cells at G(1). Inhibition of the proteasome by pentagalloylglucose and by the proteasome inhibitor MG132 caused accumulation of ubiquitin-tagged proteins in Jurkat T cells. The addition of pentagalloylglucose to Jurkat T cells enhanced the stability of the proteasome substrate Bax and increased cytochrome c release and apoptosis. Our findings suggest a mechanism for the effect of pentagalloylglucose on the cell cycle in human leukemic cells: that pentagalloylglucose down-regulates proteasome-mediated pathways because it is a proteasome inhibitor.     

Inhibition of PDGF-stimulated and matrix-mediated proliferation of human vascular smooth muscle cells by SPARC is independent of changes in cell shape or cyclin-dependent kinase inhibitors

Interactions among growth factors, cells, and extracellular matrix regulate proliferation during normal development and in pathologies such as atherosclerosis. SPARC (secreted protein, acidic, and rich in cysteine) is a matrix-associated glycoprotein that modulates the adhesion and proliferation of vascular cells. In this study, we demonstrate that SPARC inhibits human arterial smooth muscle cell proliferation stimulated by platelet-derived growth factor or by adhesion to monomeric type I collagen. Binding studies with SPARC and SPARC peptides indicate specific and saturable interaction with smooth muscle cells that involves the C-terminal Ca2+-binding region of the protein. We also report that SPARC arrests monomeric collagen-supported smooth muscle cell proliferation in the late G1-phase of the cell cycle in the absence of an effect on cell shape or on levels of cyclin-dependent kinase inhibitors. Cyclin-dependent kinase-2 activity, p107 and cyclin A levels, and retinoblastoma protein phosphorylation are markedly reduced in response to the addition of exogenous SPARC and/or peptides derived from specific domains of SPARC. Thus, SPARC, previously characterized as an inhibitor of platelet-derived growth factor binding to its receptor, also antagonizes smooth muscle cell proliferation mediated by monomeric collagen at the level of cyclin-dependent kinase-2 activity.     

Trans-differentiation of alveolar epithelial type II cells to type I cells involves autocrine signaling by transforming growth factor beta 1 through the Smad pathway

Type II alveolar epithelial cells (AEC II) proliferate and transdifferentiate into type I alveolar epithelial cells (AEC I) when the normal AEC I population is damaged in the lung alveoli. We hypothesized that signaling by transforming growth factor beta1 (TGF beta1), through its downstream Smad proteins, is involved in keeping AEC II quiescent in normal cells and its altered signaling may be involved in the trans-differentiation of AEC II to AEC I. In the normal lung, TGF beta1 and Smad4 were highly expressed in AEC II. Using an in vitro cell culture model, we demonstrated that the trans-differentiation of AEC II into AEC I-like cells began with a proliferative phase, followed by a differentiation phase. The expression of TGF beta1, Smad2, and Samd3 and their phosphorylated protein forms, and cell cycle inhibitors, p15(Ink4b) and p21(Cip1), was lower during the proliferative phase but higher during the differentiation phase. Furthermore, cyclin-dependent kinases 2, 4, and 6 showed an opposite trend of expression. TGF beta1 secretion into the media increased during the differentiation phase, indicating an autocrine regulation. The addition of TGF beta1 neutralizing antibody after the proliferative phase and silencing of Smad4 by RNA interference inhibited the trans-differentiation process. In summary, our results suggest that the trans-differentiation of AEC II to AEC I is modulated by signaling through the Smad-dependent TGF beta1 pathway by altering the expression of proteins that control the G1 to S phase entry in the cell cycle.     

Overexpression of integrin beta1 inhibits proliferation of hepatocellular carcinoma cell SMMC-7721 through preventing Skp2-dependent degradation of p27 via PI3K pathway

Integrins may play important roles in many cellular events, such as cell proliferation, differentiation, and apoptosis. We showed previously that overexpression of integrin beta1 inhibits cell proliferation in SMMC-7721 cells. Here we reported that one of the cyclin-dependent kinase (CDK) inhibitors, p27(Kip1) was involved in proliferation-inhibition induced by overexpression of integrin beta1. Overexpression of integrin beta1 upregulated p27(Kip1) at the protein level, but not mRNA level. The knock-down of p27(Kip1) expression restored cell growth in integrin beta1-overexpressing cells. Cycloheximide (Chx) treatment and pulse-chase experiments revealed that overexpression of integrin beta1 prolonged the half-life of p27(Kip1) by inhibiting its degradation. Proteasome inhibitor (MG132) treatment of the cells indicated that proteasome mediated degradation of p27, and Skp2-dependent degradation might be prevented. Overexpression of integrin beta1 decreased Skp2 at mRNA level, which was regulated by cell adhesion and the subsequent adhesion-dependent signaling. Overexpression of integrin beta1 reduced cell adhesion, accordingly, inactivated the phosphoinositide 3-kinase (PI3K) signaling. PI3K inhibitor LY294002 upregulated p27(Kip1) at post-translational level and downregulate Skp2 at mRNA level, which could mimic the effects of integrin beta1 overexpression on p27(Kip1) and Skp2. Together, these results suggested that overexpression of integrin beta1 inhibited cell proliferation by preventing the Skp2-dependent degradation of p27(Kip1) via PI3K pathway.     

CDK11p110 plays a critical role in the tumorigenicity of esophageal squamous cell carcinoma cells and is a potential drug target

Esophageal squamous cell carcinoma (ESCC) is a serious malignancy with limited options for targeted therapy. The exploration of novel targeted therapies for combating ESCC is urgently needed. Cyclin-dependent kinases (CDKs) play important roles in the progression of cancers; however, the function of CDK11^p110 (cyclin-dependent kinase 11^p110) in ESCC is still unknown. Here, we investigated the effects and molecular mechanisms of CDK11^p110 in the proliferation and growth of ESCC by examining the expression of CDK11^p110 in ESCC tissues and by detecting phenotypic changes in ESCC cells after CDK11^p110 knockdown or overexpression in vitro and in vivo. According to the tissue microarray analysis, compared with its expression level in normal tissues, the expression level of CDK11^p110 was significantly elevated in ESCC tissues; this result was in concordance with the data in TCGA (The Cancer Genome Atlas) datasets. In addition, RNAi-mediated CDK11^p110 silencing exerted a substantial inhibitory effect on the proliferation, clonogenicity and migration ability of ESCC cells. Further study indicated that CDK11^p110 knockdown arrested ESCC cells in the G2/M phase of the cell cycle and induced cell apoptosis. Moreover, stable shRNA-mediated CDK11^p110 knockdown inhibited tumor growth in an ESCC xenograft model, and overexpression of CDK11^p110 enhanced tumor growth. In addition, the Ki67 proliferation index was closely associated with the elevation or depletion of CDK11^p110 in vivo. In summary, this study provides evidence that CDK11^p110 play a critical role in the tumorigenicity of ESCC cells, which suggests that CDK11^p110 may be a promising therapeutic target in ESCC.

Abbreviations: CDKs: cyclin-dependent kinases; CDK11: Cyclin-dependent kinase 11; CDK11^p110: Cyclin-dependent kinase 11^p110, the larger isomer of cyclin-dependent kinase 11; ESCC: esophageal squamous cell carcinoma; FACS: fluorescence-activated cell sorting; FDA: the Food and Drug Administration; TCGA: The Cancer Genome Atlas; TMA: tissue microarray.     

Identification of multiple cell cycle regulatory functions of p57Kip2 in human T lymphocytes

The specific functions of p57(Kip2) in lymphocytes have not yet been fully elucidated. In this study, it is shown that p57(Kip2), which is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors, is present in the nuclei of normal resting (G(0)) T cells from peripheral blood and in the nuclei of the T cell-derived Jurkat cell line. Activation through the TCR results in rapid transport of cytoplasmic cyclin-dependent kinase 6 (cdk6) to nuclei, where it associates with cyclin D and p57(Kip2) in active enzyme complexes. Using purified recombinant proteins, it was shown in vitro that addition of p57(Kip2) protein to a mixture of cyclin D2 and cdk6 enhanced the association of the latter two proteins and resulted in phosphorylation of p57(Kip2). To probe further the function of p57(Kip2), Jurkat cells stably transfected with a plasmid encoding p57(Kip2) under control of an inducible (tetracycline) promoter were made. Induction of p57(Kip2) resulted in increased association of cdk6 with cyclin D3, without receptor-mediated T cell stimulation. The overall amounts of cdk6 and cyclin D3, and also of cdk4 and cyclin E, remained unchanged. Most notably, increased p57(Kip2) levels resulted in marked inhibition of both cyclin E- and cyclin A-associated cdk2 kinase activities and a decrease in cyclin A amounts. Therefore, although facilitating activation of cdk6, the ultimate outcome of p57(Kip2) induction was a decrease in DNA synthesis and cell proliferation. The results indicate that p57(Kip2) is involved in the regulation of several aspects of the T cell cycle.     

Autocrine and exogenous transforming growth factor beta control cell cycle inhibition through pathways with different sensitivity

Human colon carcinoma cells HCT116 that lack transforming growth factor beta (TGF-beta) type II receptor (RII) demonstrated restoration of autocrine TGF-beta activity upon reexpression of RII without restoring inhibitory responses to exogenous TGF-beta treatment. RII transfectants (designated RII Cl 37) had a longer lag phase relative to NEO-transfected control cells (designated NEO pool) before entering exponential growth in tissue culture. The prolonged growth arrest of RII Cl 37 cells was associated with markedly reduced cyclin-dependent kinase (CDK)2 activity. Our results demonstrate that p21 induction by autocrine TGF-beta is responsible for reduced CDK2 activity, which at least partially contributes to prolonged growth arrest and reduced cell proliferation in RII Cl 37 cells. In contrast to RII transfectants, HCT116 cells transfected with chromosome 3 (designated HCT116Ch3), which bears the RII gene, restored the response to exogenous TGF-beta as well as autocrine TGF-beta activity. Autocrine TGF-beta activity in HCT116Ch3 cells induced p21 expression as seen in RII Cl 37 cells; however, in addition to autocrine activity, HCT116Ch3 cells responded to exogenous TGF-beta as decreased CDK4 expression and reduced pRb phosphorylation mediated a TGF-beta inhibitory response in these cells. These results indicate that autocrine TGF-beta regulates the cell cycle through a pathway different from exogenous TGF-beta in the sense that p21 is a more sensitive effector of the TGF-beta signaling pathway, which can be induced and saturated by autocrine TGF-beta, whereas CDK4 inhibition is a less sensitive effector, which can only be activated by high levels of exogenous TGF-beta     

Regulation of myogenic differentiation by type beta transforming growth factor

Type beta transforming growth factor (TGF beta) has been shown to be both a positive and negative regulator of cellular proliferation and differentiation. The effects of TGF beta also are cell-type specific and appear to be modulated by other growth factors. In the present study, we examined the potential of TGF beta for control of myogenic differentiation. In mouse C-2 myoblasts, TGF beta inhibited fusion and prevented expression of the muscle-specific gene products, creatine kinase and acetylcholine receptor. Differentiation of the nonfusing muscle cell line, BC2Hl, was also inhibited by TGF beta in a dose-dependent manner (ID50 approximately 0.5 ng/ml). TGF beta was not mitogenic for either muscle cell line, indicating that its inhibitory effects do not require cell proliferation. Inhibition of differentiation required the continual presence of TGF beta in the culture media. Removal of TGF beta led to rapid appearance of muscle proteins, which indicates that intracellular signals generated by TGF beta are highly transient and require continuous occupancy of the TGF beta receptor. Northern blot hybridization analysis using a muscle creatine kinase cDNA probe indicated that TGF beta inhibited differentiation at the level of muscle-specific mRNA accumulation. These results provide the first demonstration that TGF beta is a potent regulator of myogenic differentiation and suggest that TGF beta may play an important role in the control of tissue-specific gene expression during development.     

Regulation of cell proliferation by autocrine motility factor/phosphoglucose isomerase signaling

Autocrine motility factor (AMF)/phosphoglucose isomerase (PGI; EC 5.3.1.9) is a housekeeping cytosolic enzyme that plays a key role in both glycolysis and gluconeogenesis pathways. AMF/PGI is also a multifunctional protein that displays cytokine properties, eliciting mitogenic, motogenic, and differentiation activities, and has been implicated in tumor progression and metastasis. Because little is known about AMF/PGI-dependent signaling in general and during tumorigenesis in particular, we sought to study its effect on the cell cycle. To elucidate the functional role of PGI, we stably transfected its cDNA into NIH/3T3 and BALB/c 3T3-A31 fibroblasts. Ectopic overexpression of PGI results in the acquisition of a transformed phenotype associated with an acceleration of G1 to S cell cycle transition. These were manifested by up-regulation of cyclin D1 expression and cyclin-dependent kinase activity and down-regulation of the cyclin-dependent kinase inhibitor p27Kip1. The reduced p27Kip1 protein expression level in PGI-overexpressing cells could be restored to control levels by treatment with proteasome inhibitor. PGI-overexpressing cells also exhibited elevated expression of Skp2 involved in p27Kip1 ubiquitination and elevation in the levels of retinoblastoma protein hyperphosphorylation. Thus, we may conclude that the overexpression of AMF/PGI enhances cell proliferation together with up-regulation of cyclin/cyclin-dependent kinase activities and down-regulation of p27Kip1, whereas the induction of 3T3 fibroblast transformation by PGI is regulated by the retinoblastoma protein pathway.