用酵母双杂交系统研究SMAD3和SMAD4的相互作用

利用酵母双杂交试验,鉴定了细胞内信号传导蛋白SMAD3和SMAD4的相互作用。通过SMAD3和SMAD4各突变体的同源和异源相互作用的双杂交反应,确定SMAD4介导信号传递的功能区在中间连接区,SMAD3的功能区在C末端。     

STAT and SMAD signaling in cancer

STAT and SMAD often exert opposite biological effects on diverse cellular functions. Recent studies have shown that STAT can interface with SMAD at molecular level and that some novel molecules, such as SOCS (also called CIS) and APRO6 (also called TOB), modulate this signaling. A cofactor p300/CBP might act as a bridging molecule to mediate the interface. Thus, STAT and SMAD signaling pathways may crosstalk each other with interweaved regulatory mechanisms. Interestingly, the importance of all the proteins' function has been shown by the increasing evidence of their involvement in cancer. These recent progresses have been made in attributing novel exciting functions. Accordingly, we would like to review the latest advances of those pathways on a cross-section in cancer signaling.     

SMAD3 regulates gonadal tumorigenesis

Inhibin is a secreted tumor suppressor and an activin antagonist. Inhibin alpha null mice develop gonadal sex cord-stromal tumors with 100% penetrance and die of a cachexia-like syndrome due to increased activin signaling. Because Sma and Mad-related protein (SMAD)2 and SMAD3 transduce activin signals in vitro, we attempted to define the role of SMAD3 in gonadal tumorigenesis and the wasting syndrome by generating inhibin alpha and Smad3 double mutant mice. Inhibin alpha and Smad3 double homozygous males were protected from early tumorigenesis and the usual weight loss and death. Approximately 90% of these males survived to 26 wk in contrast to 95% of inhibin-deficient males, which develop bilateral testicular tumors and die of the wasting syndrome by 12 wk. Testicular tumors were either absent or unilaterally slow growing and less hemorrhagic in the majority of double-knockout males. In contrast, development of the ovarian tumors and wasting syndrome was delayed, but still occurred, in the majority of the double-knockout females by 26 wk. In double mutant females, tumor development was accompanied by typical activin-induced pathological changes. In summary, we identify an important function of SMAD3 in gonadal tumorigenesis in both sexes. However, this effect is significantly more pronounced in the male, indicating that SMAD3 is the primary transducer of male gonadal tumorigenesis, whereas SMAD3 potentially overlaps with SMAD2 function in the ovary. Moreover, the activin-induced cachexia syndrome is potentially mediated through both SMAD2 and SMAD3 or only through SMAD2 in the liver and stomach. These studies identify sexually dimorphic functions of SMAD3 in gonadal tumorigenesis.     

Angiogenesis defects and mesenchymal apoptosis in mice lacking SMAD5

The transforming growth factor-beta (TGF-beta) signals are mediated by a family of at least nine SMAD proteins, of which SMAD5 is thought to relay signals of the bone morphogenetic protein (BMP) pathway. To investigate the role of SMAD5 during vertebrate development and tumorigenesis, we disrupted the Smad5 gene by homologous recombination. We showed that Smad5 was expressed predominantly in mesenchyme and somites during embryogenesis, and in many tissues of the adult. Mice homozygous for the mutation died between days 10.5 and 11.5 of gestation due to defects in angiogenesis. The mutant yolk sacs lacked normal vasculature and had irregularly distributed blood cells, although they contained hematopoietic precursors capable of erythroid differentiation. Smad5 mutant embryos had enlarged blood vessels surrounded by decreased numbers of vascular smooth muscle cells, suffered massive apoptosis of mesenchymal cells, and were unable to direct angiogenesis in vitro. These data suggest that SMAD5 may regulate endothelium-mesenchyme interactions during angiogenesis and that it is essential for mesenchymal survival.     

Using RNA interference to identify the different roles of SMAD2 and SMAD3 in NIH/3T3 fibroblast cells

Smad proteins are principal intracellular signaling mediators of transforming growth factor beta (TGF-beta) that regulate a wide range of biological processes. However, the identities of Smad partners mediating TGF-beta signaling are not fully understood. We firstly examined the expression of Smad2 and Smad3 induced by TGF-beta 1 in normal NIH/3T3 cells. The expression of Smad2 and Smad3 was assessed by RT-PCR and Western blotting. The results showed that the expression of Smad2 was increased after treatment with TGF-betaI, but Smad3 was more sensitive to TGF-betaI than Smad2. RNA interference (RNAi) provides a new approach for elucidation of gene function. Use of hairpin siRNA expression vectors for RNAi has provided a rapid and versatile method for assessing gene function in mammalian cells. Here, we have constructed Smad2 and Smad3 hairpin siRNA expression plasmids, and then transfected them into mouse NIH/3T3 cells. Endogenous Smad2 and Smad3 proteins decreased significantly at 48 h after transfection. We found the expression of Smad3 in Smad2-depleted cells was increased, however, the expression of Smad2 in Smad3-depleted cells was not changed. Consistently, the expression of Smad4 mRNA was also attenuated in Smad3-depleted cells. From these data, we suggest that Smad3, but not Smad2, may play a key role in TGF-beta signaling.     

SMAD4 contributes to chondrocyte and osteocyte development

Different cellular and molecular mechanisms contribute to chondrocyte and osteocyte development. Although vital roles of the mothers against decapentaplegic homolog 4 (also called ‘SMAD4’) have been discussed in different cancers and stem cell‐related studies, there are a few reviews summarizing the roles of this protein in the skeletal development and bone homeostasis. In order to fill this gap, we discuss the critical roles of SMAD4 in the skeletal development. To this end, we review the different signalling pathways and also how SMAD4 defines stem cell features. We also elaborate how the epigenetic factors—ie DNA methylation, histone modifications and noncoding RNAs—make a contribution to the chondrocyte and osteocyte development. To better grasp the important roles of SMAD4 in the cartilage and bone development, we also review the genotype‐phenotype correlation in animal models. This review helps us to understand the importance of the SMAD4 in the chondrocyte and bone development and the potential applications for therapeutic goals.     

Aberrations in SMAD family of genes among HNSCC patients

Head and neck cancer is a debilitating disease with several etiological factors. One of the main etiologies to be noticed is the alteration, which is either caused by genetic or environmental factors. Therefore, it is of interest to assess the effect of genetic alterations, especially the non-synonymous mutations of the SMAD gene family and its possible association with HNSCC. Data shows a significant novel mutation in the SMAD gene family in association with head and neck squamous cell carcinoma (HNSCC), which would aid in better diagnosis and treatment planning for cancer.