Transforming growth factor-beta-dependent growth inhibition in primary vascular smooth muscle cells is p38-dependent

Vascular smooth muscle cells (VSMCs) constitute the major cellular component of the vessel tunica media. VSMC proliferation is a key feature in developing vessels and pathological states such as atherosclerosis and restenosis. Transforming growth factor (TGF)-beta is a key regulator of VSMCs, but its effect on VSMC proliferation and apoptosis are controversial. Here, we characterized TGF-beta effects on basal-, serum-, and platelet-derived growth factor-BB-induced primary mouse VSMC proliferation. TGF-beta led to potent growth inhibition of VSMCs isolated from normal mouse aortae without inducing apoptosis. Growth inhibition by TGF-beta was due to G0/G1 arrest. Next, we explored distinct signaling pathways activated by TGF-beta and the effects of pharmacological inhibition of these. TGF-beta led to activation of Smad2/3, p38, p42/44, and c-Jun NH2-terminal kinase (JNK) pathways, assessed by phosphorylation, immunofluorescence, and reporter gene analysis. TGF-beta-dependent growth inhibition was specifically attenuated by pharmacological blockade of the TGF-beta type I receptor (TbetaRI) kinase or p38 mitogen-activated protein kinase pathways, whereas blockade of p42/44 or JNK kinases did not influence the effect of TGF-beta. TbetaRI kinase inhibition blocked all downstream pathways including Smad and p38 phosphorylation. In contrast, p38 inhibition did not alter Smad function, as assessed by translocation or reporter gene expression, but selectively inhibited p38 activity. These results demonstrate that TGF-beta acts as a potent antiproliferative mediator in VSMCs, irrespective of the proliferative stimulus, without inducing apoptotic effects. The anti-proliferative effect of TGF-beta is due to G0/G1 arrest and mediated primarily by the p38 pathway, suggesting that p38 kinase is central to TGF-beta-mediated growth inhibition in primary mouse VSMCs.     

Transforming growth factor beta 1 selectively regulates early murine hematopoietic progenitors and inhibits the growth of IL-3-dependent myeloid leukemia cell lines

Transforming growth factor beta 1 (TGF-beta 1) has been shown to be associated with active centers of hematopoiesis and lymphopoiesis in the developing fetus. Therefore, the effects of TGF-beta 1 on mouse hematopoiesis were studied. TGF-beta 1 is a potent inhibitor of IL-3-induced bone marrow proliferation, but it does not inhibit the proliferation induced by granulocyte/macrophage, colony-stimulating factor (CSF), granulocyte CSF, and erythropoietin (Epo). TGF-beta 1 also inhibits IL-3-induced multipotential colony formation of bone marrow cells in soft agar, which includes early erythroid differentiation, while Epo-induced terminal differentiation is unaffected. In addition, IL-3-induced granulocyte/macrophage colonies were inhibited; however, small clusters of differentiated myeloid cells were consistently seen in cultures containing IL-3 and TGF-beta 1. Thus, TGF-beta 1 selectively inhibits early hematopoietic progenitor growth and differentiation but not more mature progenitors. TGF-beta 1 is also a potent inhibitor of IL-3-dependent and -independent myelomonocytic leukemic cell growth, while the more mature erythroid and macrophage leukemias are insensitive. Therefore, TGF-beta 1 functions as a selective regulator of differentiating normal hematopoietic cells, and suppresses myeloid leukemic cell growth.     

Smad4 is critical for self-renewal of hematopoietic stem cells

Members of the transforming growth factor beta (TGF-beta) superfamily of growth factors have been shown to regulate the in vitro proliferation and maintenance of hematopoietic stem cells (HSCs). Working at a common level of convergence for all TGF-beta superfamily signals, Smad4 is key in orchestrating these effects. The role of Smad4 in HSC function has remained elusive because of the early embryonic lethality of the conventional knockout. We clarify its role by using an inducible model of Smad4 deletion coupled with transplantation experiments. Remarkably, systemic induction of Smad4 deletion through activation of MxCre was incompatible with survival 4 wk after induction because of anemia and histopathological changes in the colonic mucosa. Isolation of Smad4 deletion to the hematopoietic system via several transplantation approaches demonstrated a role for Smad4 in the maintenance of HSC self-renewal and reconstituting capacity, leaving homing potential, viability, and differentiation intact. Furthermore, the observed down-regulation of notch1 and c-myc in Smad4(-/-) primitive cells places Smad4 within a network of genes involved in the regulation HSC renewal.     

Transforming growth factor beta is a potent inhibitor of interleukin 1 (IL-1) receptor expression: proposed mechanism of inhibition of IL-1 action

Transforming growth factor beta (TGF-beta) acts as a potent inhibitor of the growth and functions of lymphoid and hemopoietic progenitor cells. Cell proliferation depends not only on the presence of growth factors, but also on the development of specific receptor-signal transducing complexes. We therefore investigated whether the inhibitory actions of TGF-beta could be mediated by inhibition of growth factor receptors. TGF-beta inhibited the constitutive level of interleukin 1 receptor (IL-1R) expression on several murine lymphoid and myeloid progenitor cell lines, as well as IL-1R expression induced by interleukin 3 (IL-3) on normal murine and human bone marrow cells. Furthermore, treatment of bone marrow progenitor cells with TGF-beta concomitantly inhibited the ability of IL-1 to promote high proliferative potential (HPP) colony formation as well as blocked IL-1-induced IL-2 production by EL-4 6.1 cells. These findings provide the first evidence that the inhibitory action of TGF-beta on the growth and functional activities of hematopoietic and T cells is associated with a reduction in the cell surface receptor expression for IL-1.     

DNA replication of first-generation adenovirus vectors in tumor cells

A major role of the early gene 1A and 1B products (E1A and E1B) in adenovirus infection is to create a cellular environment appropriate for viral DNA replication. This is, in part, achieved by inactivation of tumor suppressor gene products such as pRb or p53. The functions of these same cellular proteins are also frequently lost in tumor cells. Therefore, we hypothesized that tumor cell lines with deregulated p53 and/or pRb pathways might support replication of E1A/E1B-deleted, first-generation adenovirus vectors (AdE1(-)). Here, we analyzed the impact of virus uptake, cell cycling, and the status of cell cycle regulators on AdE1(-) DNA synthesis. Cellular internalization of AdE1(-) vectors varied significantly among different tumor cell lines, whereas nuclear import of incoming viral DNA appeared to be less variable. Replication assays performed under equalized infection conditions demonstrated that all analyzed tumor cell lines supported AdE1(-) synthesis to varying degrees. There was no obvious correlation between the efficiency of viral DNA replication and the status of p53, pRb, and p16. However, the amount of virus attached and internalized changed with the cell cycle, affecting the intracellular concentration of viral DNA and thereby the replication efficacy. Furthermore, infection with AdE1 - vectors caused a partial G(2)/M arrest or delay in cell cycle progression, which became more pronounced in consecutive cell cycles. Correspondingly, vector DNA replication was found to be enhanced in cells artificially arrested in G(2)/M. Our findings suggest that cell cycling and thus passing through G(2)/M supports AdE1(-) DNA replication in the absence of E1A/E1B. This has potential implications for the use of first-generation adenovirus vectors in tumor gene therapy.     

Transforming growth factor beta mimetics: discovery of 7-[4-(4-cyanophenyl)phenoxy]-heptanohydroxamic acid,a biaryl hydroxamate inhibitor of histone deacetylase

Transforming growth factor beta (TGF-beta) is a multifunctional protein that has been shown to possess potent growth-inhibitory activity. To identify small molecular weight compounds with TGF-beta-like activities, high throughput screening was performed using mink lung epithelial cells stably transfected with a TGF-beta-responsive plasminogen activator inhibitor 1 promoter/luciferase construct. Biaryl hydroxamate compounds were identified that demonstrated TGF-beta-like activities. 7-[4-(4-cyanophenyl)phenoxy]-heptanohydroxamic acid (A-161906) demonstrated complete TGF-beta-like agonist activity in the plasminogen activator inhibitor 1/luciferase construct. A-161906 inhibited the proliferation of multiple cell lines in a concentration-dependent manner. Cells were growth arrested at the G1-S checkpoint similar to TGF-beta. Consistent with the G1-S arrest, A-161906 induced the expression of the cyclin-dependent kinase inhibitor p21waf1/cip1. A-161906 produced many cellular effects similar to that of TGF-beta but did not displace labeled TGF-beta from its receptors. Cells with mutations in either of the TGF-beta receptors I or II were growth-arrested by A-161906. Therefore, the site of action of A-161906 appears to be distal to the receptors and possibly involved with the signaling events controlled by TGF-beta. The TGF-beta mimetic effect of A-161906 can be partially, if not entirely, explained by its activity as a histone deacetylase (HDAC) inhibitor. A-161906 demonstrated potent HDAC-inhibitory activity (IC50 = 9 nM). A-161906 is a novel small molecular weight compound (< 400 MW) having TGF-beta mimetic activity as a result of its potent HDAC-inhibitory activity. These results and those of others demonstrate the importance of HDACs in regulation of the TGF-beta signaling pathway(s).     

Adenoviral delivery of E2F-1 directs cell cycle reentry and p53-independent apoptosis in postmitotic adult myocardium in vivo.

Irreversible exit from the cell cycle precludes the ability of cardiac muscle cells to increase cell number after infarction. Using adenoviral E1A, we previously demonstrated dual pocket protein- and p300-dependent pathways in neonatal rat cardiac myocytes, and have proven that E2F-1, which occupies the Rb pocket, suffices for these actions of E1A. By contrast, the susceptibility of adult ventricular cells to viral delivery of exogenous cell cycle regulators has not been tested, in vitro or in vivo. In cultured adult ventricular myocytes, adenoviral gene transfer of E2F-1 induced expression of proliferating cell nuclear antigen, cyclin-dependent protein kinase 4, cell division cycle 2 kinase, DNA synthesis, and apoptosis. In vivo, adenoviral delivery of E2F-1 by direct injection into myocardium induced DNA synthesis, shown by 5'-bromodeoxyuridine incorporation, and accumulation in G2/M, by image analysis of Feulgen-stained nuclei. In p53(-)/- mice, the prevalence of G1 exit was more than twofold greater; however, E2F-1 evoked apoptosis and rapid mortality comparably in both backgrounds. Thus, the differential effects of E2F-1 on G1 exit in wild-type versus p53-deficient mice illustrate the combinatorial power of viral gene delivery to genetically defined recipients: E2F-1 can override the G1/S checkpoint in postmitotic ventricular myocytes in vitro and in vivo, but leads to apoptosis even in p53(-)/- mice.     

A crucial role of caspase-3 in osteogenic differentiation of bone marrow stromal stem cells

Caspase-3 is a critical enzyme for apoptosis and cell survival. Here we report delayed ossification and decreased bone mineral density in caspase-3-deficient (Casp3(-/-) and Casp3(+/-)) mice due to an attenuated osteogenic differentiation of bone marrow stromal stem cells (BMSSCs). The mechanism involved in the impaired differentiation of BMSSCs is due, at least partially, to the overactivated TGF-beta/Smad2 signaling pathway and the upregulated expressions of p53 and p21 along with the downregulated expressions of Cdk2 and Cdc2, and ultimately increased replicative senescence. In addition, the overactivated TGF-beta/Smad2 signaling may result in the compromised Runx2/Cbfa1 expression in preosteoblasts. Furthermore, we demonstrate that caspase-3 inhibitor, a potential agent for clinical treatment of human diseases, caused accelerated bone loss in ovariectomized mice, which is also associated with the overactivated TGF-beta/Smad2 signaling in BMSSCs. This study demonstrates that caspase-3 is crucial for the differentiation of BMSSCs by influencing TGF-beta/Smad2 pathway and cell cycle progression.     

Decitabine,a DNA methyltransferases inhibitor,induces cell cycle arrest at G2/M phase through p53-independent pathway in human cancer cells

Decitabine (5-aza-2′-deoxycytidine), an inhibitor of DNA methyltransferases, has a wide range of anti-metabolic and anti-cancer activities. Decitabine also induces cell cycle arrest at G2/M phase and apoptosis in human cancer cells. However, the cellular and molecular mechanisms of this cell cycle arrest are poorly understood. In the present study, we investigated the roles of the tumor suppressor p53 and the cyclin-dependent kinase (Cdk) inhibitor p21 following decitabine-induced G2/M arrest in human cancer cells. DNA flow cytometric analyses indicated that decitabine induced a G2/M arrest in AGS gastric and A549 lung carcinoma cell lines, which have wild type p53. Western blot analyses using whole cell lysates from AGS cells demonstrated that decitabine treatment did not change the steady-state level of Cdks and Cdk inhibitor p27, but it partially inhibited expression of cyclin A, cyclin B1, and Cdc25C proteins. However, similar results were found using the A549 cell line, where decitabine induced a dramatic up-regulation of both p53 and p21 expression, and the increased levels of p21 were associated with increased binding of p21 with Cdks, cyclin A, and cyclin B1. Knockdown of p53 by small interfering RNA (siRNA) markedly abolished p53 induction by decitabine in AGS cells, yet p53 siRNA had no attenuating effect on p21 induction. In addition, depletion of p21 expression with siRNA, but not p53, significantly attenuated decitabine-induced G2/M arrest. We also observed that decitabine strongly induced G2/M arrest associated with p21 induction in both p53 allele-null (–/–) HCT116 and wild type p53 (+/+) HCT116 cell lines. Therefore, our data indicated that p21 plays a crucial role in decitabine-induced G2/M arrest and operates in a p53-independent manner.     

EBV立刻早期蛋白Zta基因对Daudi细胞周期的影响及其机制研究

目的 探讨EBV立刻早期蛋白Zta基因对Daudi细胞周期的影响及其可能机制.方法 构建Zta基因真核表达载体,通过电穿孔将该载体转染Daudi细胞,用流式细胞术检测细胞周期的变化,用蛋白印迹试验检测细胞周期蛋白p21、Rb、E2F-1的表达.结果 成功构建了 Zta基因表达载体,转染Zta基因可抑制Daudi细胞的增殖,并促进Daudi细胞由G0/G11期[(30.0±3.4)％)]向S期[(47.7±1.1)％]的转化.同时转染Zta基因可下调Rb的表达、上调E2F-1、p21的表达.结论 转染Zta基因使Daudi细胞周期由Go/G1期向S期转变,其机制可能与Rb的表达下降、E2F-1和p21表达上调有关.