Increased cytoplasmic levels of CIS, SOCS1, SOCS2, or SOCS3 are required for nuclear translocation

We investigated the cellular localization of ectopically-expressed CIS, SOCS1, SOCS2 and SOCS3 proteins. We found that SOCS proteins localize to the nucleus where they reduce Stat3 proteins and that the presence of proteasome inhibitors increased SOCS nuclear localization. Our results indicate that increased nuclear localization resulted from increased levels of SOCS proteins in the cytoplasm. Finally, we demonstrate that the same effect occurs with endogenously-expressed SOCS proteins. These observations suggest that increased cytoplasmic levels of proteins in the SOCS family are regulated through nuclear translocation.     

Genetic regulation of MUC1 expression by Helicobacter pylori in gastric cancer cells

Helicobacter pylori infection of the stomach is associated with the development of gastritis, peptic ulcers, and gastric adenocarcinomas, but the mechanisms are unknown. MUC1 is aberrantly overexpressed by more than 50% of stomach cancers, but its role in carcinogenesis remains to be defined. The current studies were undertaken to identify the genetic mechanisms regulating H. pylori-dependent MUC1 expression by gastric epithelial cells. Treatment of AGS cells with H. pylori increased MUC1 mRNA and protein levels, and augmented MUC1 gene promoter activity, compared with untreated cells. H. pylori increased binding of STAT3 and MUC1 itself to the MUC1 gene promoter within a region containing a STAT3 binding site, and decreased CpG methylation of the MUC1 promoter proximal to the STAT3 binding site, compared with untreated cells. These results suggest that H. pylori upregulates MUC1 expression in gastric cancer cells through STAT3 and CpG hypomethylation.     

STAT3小干扰RNA的构建及对缺氧复氧人肾小管上皮细胞凋亡的影响

目的:构建信号转导与转录因子3(STAT3)小干扰RNA(siRNA)表达载体,并观察其对缺氧复氧后人肾小管上皮细胞(HKC)凋亡的影响。方法:设计3对人STAT3 siRNA靶序列,用DNA重组技术克隆至质粒pRNAT-U6.1/neo中,构建重组质粒pRNAT-U6.1-STAT3 siRNA,检测并筛选出最佳抑制效率的siRNA质粒载体。重组质粒转染至缺氧复氧后HKC细胞,Western blotting和Real Time-PCR测定STAT3蛋白和mRNA表达量,流式细胞仪测定细胞凋亡,间接荧光法测定Bcl-2和Bax表达的变化。结果:靶向STAT3基因表达的质粒载体构建成功,并筛选出抑制效率最佳的重组质粒。缺氧复氧后HKC细胞STAT3表达、凋亡率和Bax/Bcl-2比值增加;缺氧复氧后HKC细胞转染重组质粒后STAT3表达、凋亡率和Bax/Bcl-2比值明显降低。结论:成功构建并筛选最佳抑制效率的靶向STAT3的重组质粒载体。该载体可有效抑制缺氧复氧后HKC细胞中STAT3信号转导通路的活化,并进一步通过上调Bcl-2、下调Bax蛋白的表达,从而抑制细胞凋亡。     

HYS-32, a novel analogue of combretastatin A-4, enhances connexin43 expression and gap junction intercellular communication in rat astrocytes

HYS-32 [4-(3,4-dimethoxyphenyl)-3-(naphthalen-2-yl)-2(5H)-furanone] is a new analogue of the anti-tumor compound combretastatin A-4 containing a cis-stilbene moiety. In this study, we investigated its effects on Cx43 gap junction intercellular communication (GJIC) and the signaling pathway involved in rat primary astrocytes. Western blot analyses showed that HYS-32 dose- and time-dependently upregulated Cx43 expression. A confocal microscopic study and scrape-loading/dye transfer analyses demonstrated that HYS-32 (5 μM) induced microtubule coiling, accumulation of Cx43 in gap junction plaques, and increased GJIC in astrocytes. The HYS-32-induced microtubule coiling and Cx43 accumulation in gap junction plaques was reversed when HYS-32 was removed. Treatment of astrocytes with cycloheximide resulted in time-dependent degradation of by co-treatment with HYS-32 by increasing the half-life of Cx43. Co-treatment with HYS-32 also prevented the LPS-induced downregulation of Cx43 and inhibition of GJIC in astrocytes. HYS-32 induced activation of PKC, ERK, and JNK, and co-treatment with the PKC inhibitor Go6976 or the ERK inhibitor PD98059, but not the JNK inhibitor SP600125, prevented the HYS-32-induced increase in Cx43 expression and GJIC. Go6976 suppressed the HYS-32-induced PKC phosphorylation and increase in phospho-ERK levels, while PD98059 did not prevent the HYS-32-induced increase in phospho-PKC levels, suggesting that PKC is an upstream effector of ERK. In conclusion, our results show that HYS-32 increases the half-life of Cx43 and enhances Cx43 expression and GJIC in astrocytes via a PKC–ERK signaling cascade. These novel biological effects of HYS-32 on astrocyte gap junctions support its potential for therapeutic use as a protective agent for the central nervous system.     

Increased SOCS6 stability with PMA requires its N-terminal region and the Erk pathway via Pkcdelta activation

We investigated stability of the ectopically expressed the SOCS6 protein in HEK293T cells with PMA, which activates protein kinase C (PKC). The treatment of PMA could largely increase SOCS6 stability in HEK293T cells. But, we did not observe increased protein levels of SOCS3 or Erk1 with PMA. This result suggests that the increased stability of SOCS6 with PMA did not generally occur in other proteins. The stability of SOCS6 depended on the N-terminal region containing an unidentified domain. We then studied the role of signal pathways in SOCS6 stability with PMA. We found that both Erk and Pkcdelta activation were required for the increased SOCS6 stability by PMA. The Erk activation by PMA appeared to be downstream from the Pkcdelta activation. The increased SOCS6 stability and Erk activation by PMA were both conserved in another cell line, MCF7. In addition, we demonstrated that PMA, insulin, and PDGF increased both the stability of endogenous-expressed SOCS6 and Erk activation in MDA-MB231 cells. These observations suggest that Erk activation may be correlated in the cells with high expression of SOCS6.     

The nuclear localization of SOCS6 requires the N-terminal region and negatively regulates Stat3 protein levels

We determined that endogenous- and overexpressed- SOCS6 was localized in both the nucleus and cytoplasm. The localization of SOCS6 depended on amino acids 1-210 in the N-terminal region of the protein, which contains an unidentified domain. GFP-tagged SOCS6 or the N-terminal region, was exclusively localized and widely distributed throughout the entire nucleus, whereas the C-terminal region displayed a nuclear omission pattern. We also demonstrated that the SOCS6 protein could decrease the levels of the Stat3 protein in the nucleus, and that its negative regulation of the Stat3 protein level was dependent on its C-terminal region. These observations suggest that SOCS6 is composed of at least two functional domains required for its biological role in localizing and degrading Stat3 in the nucleus.