Cyclin-dependent kinase inhibitor, p21Waf1, regulates vascular smooth muscle cell hypertrophy.

In the process of vascular diseases, smooth muscle cells (SMC) undergo not only hyperplasia but also hypertrophy, resulting in vascular remodeling. A cyclin-dependent kinase inhibitor (CDKI), p21Waf1, has been shown to play an important role in SMC hyperplasia. Here we investigated a potential role of p21Waf1 in SMC hypertrophy. An exposure of cultured rat SMC to serum drove the cell cycle progression with up-regulation of various cell cycle markers and increased activities of cyclin-dependent kinases, but did not cause SMC hypertrophy. In contrast, incubation of SMC for 48 h with angiotensin II (AII, 100 nmol/l) resulted in a significant increase in the cell size measured by flowcytometric forward-angle light scatter assay, in association with an increase in the ratio of [3H]leucine/[3H]thymidine uptake, indicating SMC hypertrophy. At 48 h, p21Waf1 expression was up-regulated in SMC exposed to AII but not in those exposed to serum. These results suggest that p21Waf1 may be involved in hypertrophy. To further investigate this issue, two manipulations of the p21Waf1 gene were performed. Adenovirus-mediated over-expression of p21Waf1 not only reduced S-phasic cells but also caused hypertrophy, despite the exposure to serum. Antisense oligodeoxynucleotide for p21Waf1 inhibited the hypertrophy of SMC exposed to AII. Our data suggest that p21Waf1 may play a role in SMC hypertrophy as well.     

Down-regulation of p21WAF1/CIP1 or p27Kip1 abrogates antiestrogen-mediated cell cycle arrest in human breast cancer cells

Estrogens and antiestrogens influence the G(1) phase of the cell cycle. In MCF-7 breast cancer cells, estrogen stimulated cell cycle progression through loss of the kinase inhibitor proteins (KIPs) p27 and p21 and through G(1) cyclin-dependent kinase (cdk) activation. Treatment with antiestrogen drugs, Tamoxifen or ICI 182780, caused cell cycle arrest, with up-regulation of both p21 and p27 levels, an increase in their binding to cyclin E-cdk2, and kinase inhibition. The requirement for these KIPs in the arrests induced by estradiol depletion or by antiestrogens was investigated with antisense. Antisense inhibition of p21 or p27 expression in estradiol-depleted or antiestrogenarrested MCF-7 led to abrogation of cell cycle arrest, with loss of cyclin E-associated KIPs, activation of cyclin E-cdk2, and S phase entrance. These data demonstrate that depletion of either p21 or p27 can mimic estrogen-stimulated cell cycle activation and indicate that both of these KIPs are critical mediators of the therapeutic effects of antiestrogens in breast cancer.     

Histone deacetylase inhibitor suppression of autoantibody-mediated arthritis in mice via regulation of p16INK4a and p21(WAF1/Cip1) expression

OBJECTIVE: To examine whether depsipeptide (FK228), a histone deacetylase (HDA) inhibitor, has inhibitory effects on the proliferation of synovial fibroblasts from rheumatoid arthritis (RA) patients, and to examine the effects of systemic administration of FK228 in an animal model of arthritis. METHODS: Autoantibody-mediated arthritis (AMA) was induced in 19 male DBA/1 mice (6-7 weeks old); 10 of them were treated by intravenous administration of FK228 (2.5 mg/kg), and 9 were used as controls. The effects of FK228 were examined by radiographic, histologic, and immunohistochemical analyses and arthritis scores. RA synovial fibroblasts (RASFs) were obtained at the time of joint replacement surgery. In vitro effects of FK228 on cell proliferation were assessed by MTT assay. Cell morphology was examined by light and transmission electron microscopy. The effects on the expression of the cell cycle regulators p16INK4a and p21(WAF1/Cip1) were examined by real-time polymerase chain reaction and Western blot analysis. The acetylation status of the promoter regions of p16INK4a and p21(WAF1/Cip1) were determined by chromatin immunoprecipitation assay. RESULTS: A single intravenous injection of FK228 (2.5 mg/ml) successfully inhibited joint swelling, synovial inflammation, and subsequent bone and cartilage destruction in mice with AMA. FK228 treatment induced histone hyperacetylation in the synovial cells and decreased the levels of tumor necrosis factor alpha and interleukin-1beta in the synovial tissues of mice with AMA. FK228 inhibited the in vitro proliferation of RASFs in a dose-dependent manner. Treatment of cells with FK228 induced the expression of p16INK4a and up-regulated the expression of p21(WAF1/Cip1). These effects of FK228 on p16INK4a and p21(WAF1/Cip1) were related to the acetylation of the promoter region of the genes. CONCLUSION: Our findings strongly suggest that systemic administration of HDA inhibitors may represent a novel therapeutic target in RA by means of cell cycle arrest in RASFs via induction of p16INK4a expression and increase in p21(WAF1/Cip1) expression.     

慢性低氧对肺动脉高压大鼠肺血管ERK 1/2、p38MAPK表达的影响

目的：通过建立慢性低氧性肺动脉高压大鼠模型,研究慢性低氧对大鼠肺血管细胞外信号调节蛋白激酶(ERK1/2)、p38MAPK蛋白表达的影响。方法建立慢性常压低氧肺动脉高压大鼠模型,将雄性SD大鼠随机分为正常对照组、低氧1d、3d、7d、14d和21d组,应用免疫组织化学技术检测肺动脉高压形成过程中大鼠肺血管 ERK1/2、p38MAPK 蛋白表达水平。结果①RVSP 和 RV/(LV+S)比值较正常对照组明显增加(P<0.05),低氧后3 d、7 d、14 d和21 d后大鼠肺血管明显增厚;②ERK1/2、p38MAPK蛋白广泛分布于肺血管内皮细胞、平滑肌细胞和成纤维细胞中,且随着低氧时间的延长,ERK1/2、p38MAPK蛋白表达量增加。结论 ERK1/2、p38MAPK 蛋白表达量的上调可能参与了慢性低氧诱导的大鼠肺动脉高压肺血管重塑的发生、发展过程。     

Cell cycle inhibition by the anti-angiogenic agent TNP-470 is mediated by p53 and p21WAF1/CIP1

Angiogenesis has been demonstrated to be essential for tumor growth and metastasis, and inhibition of angiogenesis is emerging as a promising strategy for treating cancer. Among the most potent inhibitors of angiogenesis is the fumagillin family of natural products. An analog of fumagillin, known as TNP-470 or AGM-1470, has been undergoing clinical trials for treating a variety of cancers. TNP-470 has been shown to block endothelial cell cycle progression in the late G(1) phase. Although the direct molecular target for TNP-470 has been identified as the type 2 methionine aminopeptidase (MetAP2), how inhibition of this enzyme leads to cell cycle arrest has remained unclear. We report that treatment of endothelial and other drug-sensitive cell types leads to the activation of the p53 pathway, causing an accumulation of the G(1) cyclin-dependent kinase inhibitor p21(WAF1/CIP1). The requirement of p53 and p21(WAF1/CIP1) for the cell cycle inhibition by TNP-470 is underscored by the observation that cells deficient in p53 and p21(WAF1/CIP1) are resistant to TNP-470. These results shed significant light on the mechanism of cell cycle inhibition by TNP-470 and suggest an alternative method of activating p53 in endothelial cells to halt angiogenesis and tumor progression.     

Histone deacetylase inhibitor suppression of autoantibody‐mediated arthritis in mice via regulation of p16INK4a and p21WAF1/Cip1 expression

Objective

To examine whether depsipeptide (FK228), a histone deacetylase (HDA) inhibitor, has inhibitory effects on the proliferation of synovial fibroblasts from rheumatoid arthritis (RA) patients, and to examine the effects of systemic administration of FK228 in an animal model of arthritis.

Methods

Autoantibody‐mediated arthritis (AMA) was induced in 19 male DBA/1 mice (6–7 weeks old); 10 of them were treated by intravenous administration of FK228 (2.5 mg/kg), and 9 were used as controls. The effects of FK228 were examined by radiographic, histologic, and immunohistochemical analyses and arthritis scores. RA synovial fibroblasts (RASFs) were obtained at the time of joint replacement surgery. In vitro effects of FK228 on cell proliferation were assessed by MTT assay. Cell morphology was examined by light and transmission electron microscopy. The effects on the expression of the cell cycle regulators p16^INK4a and p21^WAF1/Cip1 were examined by real‐time polymerase chain reaction and Western blot analysis. The acetylation status of the promoter regions of p16^INK4a and p21^WAF1/Cip1 were determined by chromatin immunoprecipitation assay.

Results

A single intravenous injection of FK228 (2.5 mg/ml) successfully inhibited joint swelling, synovial inflammation, and subsequent bone and cartilage destruction in mice with AMA. FK228 treatment induced histone hyperacetylation in the synovial cells and decreased the levels of tumor necrosis factor α and interleukin‐1β in the synovial tissues of mice with AMA. FK228 inhibited the in vitro proliferation of RASFs in a dose‐dependent manner. Treatment of cells with FK228 induced the expression of p16^INK4a and up‐regulated the expression of p21^WAF1/Cip1. These effects of FK228 on p16^INK4a and p21^WAF1/Cip1 were related to the acetylation of the promoter region of the genes.

Conclusion

Our findings strongly suggest that systemic administration of HDA inhibitors may represent a novel therapeutic target in RA by means of cell cycle arrest in RASFs via induction of p16^INK4a expression and increase in p21^WAF1/Cip1 expression.

     

Attenuation of matrix protein secretion by antisense oligodeoxynucleotides to the cyclin kinase inhibitor p21(Waf1/Cip1)

Progressive fibrosis in major organs, including the heart, the kidney and the vascular tree, plays an important role in mediating chronic disease and atherosclerosis. Production of extracellular matrix proteins, in many cases regulated by the growth factor TGF-beta is an essential component of this process. In a parallel manner to TGF-beta, the cyclin kinase inhibitors (CKIs; which are induced by TGF-beta) regulate transit through the cell cycle, and their effect on growth has been shown to be bimodal in the case of vascular smooth muscle (VSM) cells. Using an antisense oligodeoxynucleotide to the CKI p21(Waf1/Cip1), developed in our laboratory and shown to specifically inhibit p21(Waf1/Cip1) protein levels, we asked whether attenuation of the CKI p21(Waf1/Cip1) by transfection of this oligodeoxynucleotide results in the abolition of TGF-beta-mediated growth inhibition and/or diminished matrix protein production and secretion in the presence or absence of TGF-beta. Specific inhibition of p21(Waf1/Cip1) protein with the antisense oligodeoxynucleotide markedly reduces the production and secretion of the matrix proteins fibronectin and laminin, both in the presence and absence of TGF-beta stimulation, in VSM cells as observed by Western blotting of cell lysate and conditioned medium. In addition, TGF-beta-mediated cell growth inhibition, though attenuated by this oligo, is preserved. Due to the relative ease and safety of transfecting antisense oligodeoxynucleotides into VSM, we believe that this work unmasks a potentially powerful technique for inhibition of matrix protein synthesis in VSM and related cell lines, and may lead to new treatment strategies for atherosclerotic as well as other systemic diseases characterized by aberrant matrix protein secretion.     

Role of the cyclin-dependent kinase inhibitor p27(Kip1) in androgen-induced inhibition of CAMA-1 breast cancer cell proliferation

Androgens are known to inhibit the growth of breast cancer cells, but the molecular mechanism of androgen-induced growth inhibition remains unknown. To address this question, we examined functional and quantitative alterations in cell cycle regulators in the E-responsive CAMA-1 breast cancer cell line. We report here that the androgen 5 alpha-dihydrotestosterone inhibits the proliferation of CAMA-1 breast cancer cells. This inhibition of cell proliferation was dose dependent, and maximal inhibition of E2-stimulated proliferation was observed at the concentration of 1 nM 5 alpha-dihydrotestosterone. 5 alpha-Dihydrotestosterone-induced growth arrest was accompanied by an increase in the proportion of cells in the G(1) phase of the cell cycle. Compared with control cells, 5 alpha-dihydrotestosterone-treated cells showed an increase in the relative proportion of hypophosphorylated retinoblastoma protein consistent with G(1) arrest. In CAMA-1 cells, 5 alpha-dihydrotestosterone caused an accumulation of the cyclin-dependent kinase inhibitor p27(Kip1). Cyclin E-cyclin-dependent kinase-2-associated kinase activity was strongly inhibited in 5 alpha-dihydrotestosterone-treated cells, and immunoprecipitation-Western blot analysis showed an increase in the amount of p27(Kip1) associated with cyclin E-cyclin-dependent kinase-2 complexes. These results suggest that inhibition of breast cancer cell growth by androgens may be mediated at least in part by inactivation of the cyclin E-cyclin-dependent kinase-2 complexes by p27(Kip1).     

Visualizing the dynamics of p21(Waf1/Cip1) cyclin-dependent kinase inhibitor expression in living animals

Although the role of p21(Waf1/Cip1) gene expression is well documented in various cell culture studies, its in vivo roles are poorly understood. To gain further insight into the role of p21(Waf1/Cip1) gene expression in vivo, we attempted to visualize the dynamics of p21(Waf1/Cip1) gene expression in living animals. In this study, we established a transgenic mice line (p21-p-luc) expressing the firefly luciferase under the control of the p21(Waf1/Cip1) gene promoter. In conjunction with a noninvasive bioluminescent imaging technique, p21-p-luc mice enabled us to monitor the endogenous p21(Waf1/Cip1) gene expression in vivo. By monitoring and quantifying the p21(Waf1/Cip1) gene expression repeatedly in the same mouse throughout its entire lifespan, we were able to unveil the dynamics of p21(Waf1/Cip1) gene expression in the aging process. We also applied this system to chemically induced skin carcinogenesis and found that the levels of p21(Waf1/Cip1) gene expression rise dramatically in benign skin papillomas, suggesting that p21(Waf1/Cip1) plays a preventative role(s) in skin tumor formation. Surprisingly, moreover, we found that the level of p21(Waf1/Cip1) expression strikingly increased in the hair bulb and oscillated with a 3-week period correlating with hair follicle cycle progression. Notably, this was accompanied by the expression of p63 but not p53. This approach, together with the analysis of p21(Waf1/Cip1) knockout mice, has uncovered a novel role for the p21(Waf1/Cip1) gene in hair development. These data illustrate the unique utility of bioluminescence imaging in advancing our understanding of the timing and, hence, likely roles of specific gene expression in higher eukaryotes.     

Laminar shear stress inhibits vascular endothelial cell proliferation by inducing cyclin-dependent kinase inhibitor p21(Sdi1/Cip1/Waf1)

Alterations in the functions of vascular endothelial cells (ECs) induced by fluid shear stress may play a pivotal role in both the development and prevention of vascular diseases. We found that DNA synthesis of bovine aortic and human umbilical vein ECs, determined by [(3)H]thymidine incorporation, was inhibited by steady laminar shear stress (5 and 30 dyne/cm(2)). This growth inhibition due to shear stress was associated with suppression of cell transition from the G(1) to S phase of the cell cycle. Therefore, we studied G(1)-phase events to find the molecules responsible for this cell cycle arrest. Shear stress inhibited the phosphorylation of a retinoblastoma protein (pRb) and the activity of cyclin-dependent kinase (cdk) 2 and cdk4, which phosphorylate pRb. The level of cdk inhibitor p21(Sdi1/Cip1/Waf1) protein, but not that of p27(Kip1), increased as a result of shear stress, and the amount of p21 protein associated with cdk2 also increased, although the protein level of cdk2 was unchanged. Shear stress markedly elevated the mRNA level of p21, and this elevation in mRNA faded after the release of cells from shear stress, concomitant with a recovery of DNA synthesis. These results suggest that steady laminar shear stress induces cell cycle arrest by upregulating p21. Derangement of the steady laminar flow may release cells from this inhibition and induce cell proliferation, which, in turn, may cause atherosclerosis through the induction of EC stability disruption.     

Differential expression of cyclin-dependent kinase inhibitors, p27Kip1 and p57Kip2, by corticotropin in rat adrenal cortex

An important role for the cyclin-dependent kinase inhibitors (CDKIs), p27Kip1 and p57Kip2, in the proliferation and differentiation of adrenal cells has been suggested by their knockout mice, which display adrenal hyperplasia. Adrenal development and function are primarily regulated by ACTH. In the present study, we investigated the effects of ACTH on the expression of p27Kip1, p57Kip2 and proliferating cell nuclear antigen (PCNA) in rat adrenals. Male Wistar rats were treated with dexamethasone (Dex) to inhibit endogenous ACTH secretion. ACTH was then administered to the rats, and the adrenals were examined by Western blot and immunohistochemical analyses. Dex treatment induced shrinkage of adrenals where no PCNA-expressing cells were detected, but most of the cells expressed p27Kip1. Subsequent ACTH treatment resulted in the marked suppression of p27Kip1 expression, specifically in adrenocortical cells at 12 h after the stimulus. At 48 h, the p27Kip1 suppression still continued in the cortex, while the PCNA-expressing cells appeared mainly around the zona glomerulosa and increased at 72 h. At this time, the p27Kip1-expressing cells also appeared in the same zone. In contrast to p27Kip1, the expression of p57Kip2 was not detected in the Dex-treated adrenal. However, its expression was markedly induced by ACTH in the zona glomerulosa at 48 and 72 h. The results demonstrate that the primary site for mitogenic action of ACTH in rat adrenocortex is the zona glomerulosa, and that ACTH modulates proliferation of adrenocortical cells by regulating p27Kip1 and p57Kip2 expression in a time- and site-specific manner.     

Interferon-gamma impairs physiologic downregulation of cyclin-dependent kinase inhibitor, p27Kip1, during G1 phase progression in macrophages

Cell cycle progression of mouse macrophage cells was impaired by interferon-gamma (IFN-gamma). The blockage of G1/S transition was associated with diminution of cyclin-dependent kinase-2 (CDK2)-associated kinase activities. The expression of p21Cip1 was not upregulated by IFN-gamma. Instead, the physiologic downregulation of p27Kip1 necessary for normal cell cycle progression did not take place sufficiently in the presence of IFN-gamma. During normal cell cycle progression, the levels of p27Kip1 were maximal at early G1 and then decreased gradually. In the presence of IFN-gamma, however, the levels of p27Kip1 discontinued to decrease at a late mid G1 point and were consistently as high as, or higher than, levels observed there. The steady, relatively high-level attachment of p27Kip1 to CDK2 contributed to the insufficient formation of active cyclin/CDK2, possibly deferring cells from entering S phase.     

Transforming growth factor beta 1 mediates cell-cycle arrest of primitive hematopoietic cells independent of p21(Cip1/Waf1) or p27(Kip1).

The regulation of stem cell proliferation is a poorly understood process balancing rapid, massive blood cell production in times of stress with maintenance of a multipotent stem cell pool over decades of life. Transforming growth factor beta 1 (TGF-beta 1) has pleiotropic effects on hematopoietic cells, including the inhibition of primitive cell proliferation. It was recently demonstrated that the cyclin-dependent kinase inhibitors, p21(Cip1/Waf1) (p21) and p27(Kip1) (p27), can inhibit the proliferation of hematopoietic stem cells and progenitor cells, respectively. The relation of TGF-beta 1 stimulation to p21 and p27 was examined using a fine-mapping approach to gene expression in individual cells. Abundant TGF-beta 1 expression and p21 expression were documented in quiescent, cytokine-resistant hematopoietic stem cells and in terminally differentiated mature blood cells, but not in proliferating progenitor cell populations. TGF-beta 1 receptor (T beta R II) was expressed ubiquitously without apparent modulation. Cell- cycle-synchronized 32D cells exposed to TGF-beta 1 demonstrated a marked antiproliferative effect of TGF-beta 1, yet neither the level of p21 mRNA nor the protein level of either p21 or p27 was altered. To corroborate these observations in primary cells, bone marrow mononuclear cells derived from mice engineered to be deficient in p21 or p27 were assessed. Progenitor and primitive cell function was inhibited by TGF-beta 1 equivalently in -/- and +/+ littermate controls. These data indicate that TGF-beta 1 exerts its inhibition on cell cycling independent of p21 and p27 in hematopoietic cells. TGF-beta 1 and p21 or p27 participate in independent pathways of stem cell regulation, suggesting that targeting each may provide complementary strategies for enhancing stem or progenitor cell expansion and gene transduction.