Bmpr1a is required for proper migration of the AVE through regulation of Dkk1 expression in the pre-streak mouse embryo

Here, we report a novel mechanism regulating migration of the anterior visceral endoderm (AVE) by BMP signaling through BMPRIA. In Bmpr1a-deficient (Bmpr-null) embryos, the AVE does not migrate at all. In embryos with an epiblast-specific deletion of Bmpr1a (Bmpr1a^null/flox; Sox2Cre embryos), the AVE cells migrate randomly from the distal end of embryos, resulting in an expansion of the AVE. Dkk1, which is normally expressed in the anterior proximal visceral endoderm (PxVE), is downregulated in Bmpr-null embryos, whereas it is circumferentially expressed in Bmpr1a^null/flox; Sox2Cre embryos at E5.75-6.5. These results demonstrate an association of the position of Dkk1 expressing cells with direction of the migration of AVE. In Bmpr1a^null/flox; Sox2Cre embryos, a drastic decrease of WNT signaling is observed at E6.0. Addition of WNT3A to the culture of Bmpr1a^null/flox; Sox2Cre embryos at E5.5 restores expression patterns of Dkk1 and Cer1. These data indicate that BMP signaling in the epiblast induces Wnt3 and Wnt3a expression to maintain WNT signaling in the VE, resulting in downregulation of Dkk1 to establish the anterior expression domain. Thus, our results suggest that BMP signaling regulates the expression patterns of Dkk1 for anterior migration of the AVE.     

Accumulation of Mutant p53 Modulates the Growth, Clonogenicity, and Radiochemosensitivity of Malignant Glioma Cells Independent of Endogenous p53 Status

Alterations of the p53 gene have been attributed a major role in the development and resistance to therapy of several human cancers. Accumulation of p53 in tumor cells may result from mutations associated with prolonged half-life or from stabilization of wild-type p53 by different mechanisms. To address the role of p53 accumulation in the response of malignant glioma cells to radiochemotherapy, we expressed the p53 mutant p53^V143A in five human malignant glioma cell lines with different genetic and functional p53 status. Accumulation of p53^V143A modulated proliferation in three and clonogenicity in four of five cell lines without a clear pattern with regard to their endogenous p53 status. p53^V143A inhibited the camptothecin-induced accumulation of p21^WAF1/CIP1 in cell lines with p53 functional wild-type activity, but not in cell lines lacking p53 activity, consistent with a transdominant-negative effect of p53^V143A. Irradiation induced a moderate G2/M arrest in all cell lines, irrespective of the p53 status, that was unaffected by p53^V143A. Radiosensitivity as well as sensitivity to BCNU, teniposide (VM26), topotecan, vincristine, Taxol, and cisplatin both in cytotoxic cell death and in clonogenic cell death was unchanged in p53^V143A-transfected cells with few exceptions. These data do not support the hypothesis that accumulation of mutant p53 is a major determinant of the response to adjuvant radiochemotherapy in human malignant glioma cells.     

PKCalpha mediates TGFbeta-induced growth inhibition of human keratinocytes via phosphorylation of S100C/A11

Growth regulation of epithelial cells is of major concern because most human cancers arise from them. We demonstrated previously a novel signal pathway involving S100C/A11 for high Ca2+-induced growth inhibition of normal human keratinocytes (Sakaguchi, M., M. Miyazaki, M. Takaishi, Y. Sakaguchi, E. Makino, N. Kataoka, H. Yamada, M. Namba, and N.H. Huh. 2003. J. Cell Biol. 163:825-835). This paper addresses a question whether transforming growth factor beta (TGFbeta) shares the pathway with high Ca2+. On exposure of the cells to TGFbeta1, S100C/A11 was phosphorylated, bound to nucleolin, and transferred to the nucleus, resulting in induction of p21WAF1/CIP1 and p15INK4B through activation of Sp1. Protein kinase C alpha (PKCalpha) was shown to phosphorylate 10Thr of S100C/A11, which is a critical event for the signal transduction. The TGFbeta1-induced growth inhibition was almost completely mitigated when PKCalpha activity was blocked or when S100C/A11 was functionally sequestered. These results indicate that, in addition to the well-characterized Smad-mediated pathway, the PKCalpha-S100C/A11-mediated pathway is involved in and essential for the growth inhibition of normal human keratinocytes cells by TGFbeta1.     

Mitochondrial ribosomal protein L41 mediates serum starvation-induced cell-cycle arrest through an increase of p21(WAF1/CIP1)

Ribosomal proteins not only act as components of the translation apparatus but also regulate cell proliferation and apoptosis. A previous study reported that MRPL41 plays an important role in p53-dependent apoptosis. It also showed that MRPL41 arrests the cell cycle by stabilizing p27(Kip1) in the absence of p53. This study found that MRPL41 mediates the p21(WAF1/CIP1)-mediated G1 arrest in response to serum starvation. The cells were released from serum starvation-induced G1 arrest via the siRNA-mediated blocking of MRPL41 expression. Overall, these results suggest that MRPL41 arrests the cell cycle by increasing the p21(WAF1/CIP1) and p27(Kip1) levels under the growth inhibitory conditions.     

HeLa细胞中Skp2的表达调控p21WAFCIP1稳定性

泛素 蛋白酶体抑制剂MG 132处理的HeLa和SaoS 2细胞中 ,低表达的p2 1WAF CIP1受泛素 蛋白酶体通路调控 .为探讨肿瘤细胞中高表达的E3连接酶底物结合亚基 Skp2和低表达的p2 1WAF CIP1之间的关系 ,在HeLa细胞中转染Skp2的反义寡核苷酸后 ,p2 1表达水平明显升高 .同时 ,相对于转染空载体和Skp2ΔF(Skp2的F box缺失突变体 )的真核表达载体 ,转染全长Skp2的HeLa细胞中 ,p2 1表达水平显著下降 ,说明肿瘤细胞中Skp2调节p2 1的稳定性 ,并且这种调控作用依赖于Skp2蛋白中的F box结构 .免疫荧光结果表明 ,Skp2和p2 1共定位于细胞核 ,这为两者间的相互作用提供了前提 ;体内相互免疫共沉淀结果表明 ,p2 1和其它SCFSkp2 的底物一样与Skp2直接结合 ,并且它们之间的结合区不在F box结构域 ,这一结果在体外的GST pulldown实验中得到验证     

SEK1-dependent JNK1 activation prolongs cell survival during G-Rh2-induced apoptosis

We provide here evidence that c-Jun N-terminal protein kinase 1 (JNK1) activity is differentially up-regulated during apoptosis of SK-HEP-1 cells after treatment with ginsenoside Rh2 (G-Rh2). The G-Rh2-mediated JNK1 activation that occurred for the first 10-30min was associated with SEK1 activity, but thereafter, the sustained activation was associated not with SEK1 activity, but with proteolytic cleavage of JNK1-associated p21(WAF1/CIP1). Supporting this is that the expression of the dominant negative SEK1 mutant effectively blocked the early JNK1 activation phase but did not alter the sustained activation phase or apoptosis. Furthermore, expression of p21D112N, an uncleavable mutant of p21(WAF1/CIP1), suppressed the later JNK1 activation. Moreover, the stable overexpression of ectopic JNK1 suppressed apoptosis while expression of the dominant negative JNK1 mutant promoted it. We propose that the early SEK1-associated JNK1 activation phase acts to prolong cell survival in response to apoptosis-inducing agents, thereby serving as an intervening checkpoint prior to the commitment to apoptosis.     

PKCdelta modulates p21WAF1/CIP1 ability to bind to Cdk2 during TNFalpha-induced apoptosis

Cyclin-dependent kinase 2 (Cdk2) activity is thought to be involved in cell death-associated chromatin condensation and other manifestations of apoptotic death. Here we show that during TNFalpha-induced apoptosis, PKCdelta is activated in a caspase-3-dependent manner and phosphorylates p21(WAF1/CIP1), a specific cyclin-dependent kinase inhibitor, on (146)Ser. This residue is located near a cyclin-binding motif (Cy2) that plays an important role in the interaction between p21(WAF1/CIP1) and Cdk2, and its phosphorylation modulates the ability of p21(WAF1/CIP1) to associate with Cdk2. The phosphorylation of p21(WAF1/CIP1) is temporally related to the activation kinetics of Cdk2 activity during the apoptosis. We propose that during TNFalpha-induced apoptosis, PKCdelta-mediated phosphorylation of p21(WAF1/CIP1) at (146)Ser attenuates the Cdk2 binding of p21(WAF1/CIP1) and thereby upregulates Cdk2 activity.     

Cyclical expression of the Notch/Wnt regulator Nrarp requires modulation by Dll3 in somitogenesis

Delta-like 3 (Dll3) is a divergent ligand and modulator of the Notch signaling pathway only identified so far in mammals. Null mutations of Dll3 disrupt cycling expression of Notch targets Hes1, Hes5, and Lfng, but not of Hes7. Compared with Dll1 or Notch1, the effects of Dll3 mutations are less severe for gene expression in the presomitic mesoderm, yet severe segmentation phenotypes and vertebral defects result in both human and mouse. Reasoning that Dll3 specifically disrupts key regulators of somite cycling, we carried out functional analysis to identify targets accounting for the segmental phenotype. Using microdissected embryonic tissue from somitic and presomitic mesodermal tissue, we identified new genes enriched in these tissues, including Limch1, Rhpn2, and A130022J15Rik. Surprisingly, we only identified a small number of genes disrupted by the Dll3 mutation. These include Uncx, a somite gene required for rib and vertebral patterning, and Nrarp, a regulator of Notch/Wnt signaling in zebrafish and a cycling gene in mouse. To determine the effects of Dll3 mutation on Nrarp, we characterized the cycling expression of this gene from early (8.5 dpc) to late (10.5 dpc) somitogenesis. Nrarp displays a distinct pattern of cycling phases when compared to Lfng and Axin2 (a Wnt pathway gene) at 9.5 dpc but appears to be in phase with Lfng by 10.5 dpc. Nrarp cycling appears to require Dll3 but not Lfng modulation. In Dll3 null embryos, Nrarp displayed static patterns. However, in Lfng null embryos, Nrarp appeared static at 8.5 dpc but resumed cycling expression by 9.5 and dynamic expression at 10.5 dpc stages. By contrast, in Wnt3a null embryos, Nrarp expression was completely absent in the presomitic mesoderm. Towards identifying the role of Dll3 in regulating somitogenesis, Nrarp emerges as a potentially important regulator that requires Dll3 but not Lfng for normal function.