Study Of Expression And Function Of Hrg-1 Gene

Hypertension is a multiple gene disease caused by interaction of environmental and genetic factors. Vasoactive peptides and their receptors, growth factors and cytokines as well as their receptors, cell signal transduction proteins and cyclins are all involved in the occurrence of hypertension. All these genes which affect blood pressure are called hypertension-related genes.Hypertension-related gene (hrg-1) is a novel gene correlated with vascular smooth muscle cell (VSMC) proliferation, which was isolated by screening VSMC cDNA library of Wistar-Kyoto rats (WKY) and spontaneous hypertension rats (SHR) using differential display method, it was named because its expression activity in VSMCs of SHR was weaker than that of WKY. Recent findings have shown that hrg-1 was expressed not only in VSMCs, but also widely distributed in various rat tisssues including heart, kidney, brain, lung, liver, leucocyte and so on. It has been discovered that transfecting hrg-1 expression plasmids into VSMCs could inhibit the activities of Raf-1 and mitogen activated protein kinase (MAPK) and decrease the expression of anti-apoptosis gene bcl-2 and proliferating cell nuclear antigen (PCNA). At the same time, it also inhibited cell proliferation. At present, the mechanisms whereby hrg-1 gene products regulate VSMC proliferation, migration and differentiation were poorly understood. It was little known about the subcellular location and function of hrg-1 in embryo development. In the present study, we investigated the role of hrg-1 in the process of VSMC redifferentiation, cell signal transduction, oocyte cleavage and early embryo development. Furthermore, we studied the effect of hrg-1 overexpression on VSMC proliferation, migration, and cell cycle processes at cellular, protein and molecular levels. 1 Study of expression and function of hrg-1 gene during VSMC redifferentiationIn order to study the relationship between hrg-1 expression and VSMC redifferentiation and explore the function of hrg-1 in regulation of cellular biological behavior, the redifferentiation of the dedifferentiated VSMCs was induced by serum deprivation and all-trans retinoic acid (atRA). We observed the relationship among hrg-1 gene expression, VSMC phenotypic marker gene (SM22α,SM α-actin) expression, cell morphology and cellular biological behavior by RT-PCR, Western blotting and cell migration assay. The results as following: 1.1 Differentiated (contractile phenotype) VSMC marker gene SM22α expression increased dramatically after dedifferentiated (synthetic phenotype) VSMCs being treated with atRA for 24 hours, and reached peak after VSMCs were induced for 48 hours by atRA, which was increased by 2.5 fold compared with that of control cells. And its expression activity maintained at high level until 72 hours. Expression of another differentiated VSMC marker gene SM α-actin also increased about 2 times as high as that of control one after VSMCs being treated with atRA for 24 hours, and continuously increased thereafter. In addition, the results of morphological observation showed that dedifferentiated VSMCs exhibited morphology like epithelioid, while the cells treated with atRA were characterized by morphological elongation. The expression of phenotypic marker genes and change of morphology in VSMCs treated with serum deprivation were similar to those treated with atRA. These results suggested that both serum deprivation and atRA treatment could induce VSMC transition from synthetic phenotype to contractile phenotype, i.e. redifferentiation.1.2 Compared with the control cells, hrg-1 mRNA expression was enhanced by 2.38-fold and 2.05-fold, respectively, after cultured VSMCs were treated with serum deprivation or atRA for 24 hours, and maintained at a relatively high level until 72 hours, which was consistent with the result of Western blotting analysis for HRG-1 protein. On the other hand, the result of immunocytochemistry staining showed that positively stained particles of HRG-1 dramatically increased in cytoplasm of VSMCs...