Identification of Gli1-interacting proteins during simvastatin-stimulated osteogenic differentiation of bone marrow mesenchymal stem cells

Simvastatin has been shown to promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Our study aimed to illuminate the underlying mechanism, with a specific focus on the role of Hedgehog signaling in this process. BMSCs cultured with or without 10⁻⁷ mol/L simvastatin were subjected to evaluation of osteogenic differentiation capacity. Osteogenic markers such as type 1 collagen (COL1) and osteocalcin (OCN), as well as key molecules of Hedgehog signaling molecules, were examined by Western blot and real-time polymerase chain reaction (PCR). Co-immunoprecipitation and mass spectrometry assays were applied to screen for Gli1-interacting proteins. Cyclopamine (Cpn) was used as a Hedgehog signaling inhibitor. Our results indicated that simvastatin increased alkaline phosphatase (ALP) activity; mineralization of extracellular matrix; mRNA expression of ALP, COL1, and OCN; and expression and nuclear translocation of Gli1. Contrasting effects were observed in Cpn-exposed groups, but were partially rescued by the simvastatin treatment. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that Gli1-interacting proteins were primarily associated with mitogen-activated protein kinase (MAPK) (P = 7.04E⁻⁰⁴), hippo, insulin, and glucagon signaling. Further, hub genes identified by protein-protein interaction network analysis included Gli1-interacting proteins such as Ppp2r1a, Rac1, Etf1, and XPO1/CRM1. In summary, the current study showed that the mechanism by which simvastatin stimulates osteogenic differentiation of BMSCs involves activation of Hedgehog signaling, as indicated by interactions with Gli1 and, most notably, the MAPK signaling pathway.     

shRNA-mediated silencing of Gli2 gene inhibits proliferation and sensitizes human hepatocellular carcinoma cells towards TRAIL-induced apoptosis

Aberrant activation of the Hedgehog (Hh) signaling pathway has been reported in various cancer types including hepatocellular carcinoma (HCC). As a key effector of this signaling, Gli2 plays a crucial role in carcinogenesis, including the activation of genes encoding apoptosis inhibitors and cell-cycle regulators. In this study, we examined the role of Gli2 proliferation and survival of HCC cells. First, the expression levels of Hh pathway components were detected in a subset of HCC cell lines. To establish the role of Gli2 in maintaining the tumorigenic properties of HCC cells, we developed small hairpin RNA (shRNA) targeting Gli2 and transfected it into SMMC-7721 cell, which was selected with high level of Hh signaling expression. Next, effects of Gli2 gene silencing, on cell proliferation and on the expression of cell cycle-related proteins were evaluated, then, whether down-regulation of Gli2 renders HCC cell susceptible to TRAIL was examined in vitro. Knockdown of Gli2 inhibited cell proliferation and induced G1 phase arrest of cell cycle in SMMC-7721 cell through down-regulation of cyclin D1, cyclinE2, and up-regulation of p21-WAF1. Also, Gli2 gene siliencing sensitized SMMC-7721 cell to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by reducing the expression of the long and short isoform of c-FLIP and Bcl-2, and then augmented the activation of initiator caspases-8/-9 and effector caspases-3, which induces PARP cleavage. In conclusion, our data suggest that Gli2 plays a predominant role in the proliferation and apoptosis resistance of HCC cells, and that knockdown of Gli2 may be a novel anticancer strategy for the treatment of HCC.     

POMP与SuFu相互作用调控Hedgehog信号通路的活性

Hedgehog信号通路在胚胎发育、组织再生中发挥重要的作用,且与癌症发生发展密切相关. 其胞内调控组分Suppressor of Fused（SuFu）蛋白通过结合转录因子Gli(s),负调控该信号通路,但其作用的分子机制仍不甚清楚. 在本项研究中,以人SuFu作为诱饵蛋白,利用酵母双杂交技术成功地筛选到1个新的相互作用因子-蛋白酶体成熟蛋白（POMP）. 通过免疫共沉淀、体外GST pull-down和免疫细胞化学实验验证其相互作用. 为了探究POMP与SuFu的相互作用对Hedgehog信号通路的影响,构建了POMP的过表达质粒和干扰质粒（miR-RNAi）以及转录因子Gli活性检测系统,即荧光素酶报告基因法,结果显示,过表达SuFu蛋白时POMP正调控Hedgehog信号通路,而下调POMP的表达则抑制Gli的活性. 该研究结果揭示了POMP新的生物学功能,为阐明Hedgehog信号通路的具体分子机制提供了新的线索.     

Gli1 interacts with YAP1 to promote tumorigenesis in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is the predominant esophageal cancer type in China. The aberrant activation of glioma-associated oncogene homolog1 (Gli1), a key factor in Hedgehog (Hh) signaling pathway, has been found in esophageal carcinoma. Moreover, Yes-associated protein 1 (YAP1), the major mediator of Hippo signaling pathway, has been linked to esophageal carcinoma progression. However, the precise roles and the underlying mechanism of both Gli1 and YAP1 in ESCC are unclear. Here, we found that Gli1 and YAP1 are overexpressed in ESCC and are associated with poor prognosis. In addition, we confirmed that knockdown of Gli1 or YAP1 suppresses ESCC cell growth, migration, and invasion in ESCC TE1 and EC109 cells. Significantly, Gli1 interacts with YAP1 in ESCC cells. Both Gli1 and YAP1 proteins are closely correlated with each other in human ESCC samples. Mechanistically, Gli1 upregulates YAP1 in a LATS1-independent manner. Conversely, YAP1 induces Gli1 by regulating phosphoinositide 3-kinase (PI3K)/AKT signaling pathway. Most importantly, we demonstrated that the interaction between Gli1 and YAP1 promotes ESCC tumor growth in vitro and in vivo. Our findings established a novel signaling mechanism by which the interaction between Gli1 and YAP1 promotes ESCC cell growth. This signaling regulation of the tumorigenesis provides a new therapeutic strategy for highly lethal ESCC.