| Background and objective Vascular smooth muscle cells (VSMCs) is the major cell type in the media of normal arteries, providing the vessels with both structural support, a contractile ability and vasomotion. The VSMCs perform these functions by maintaining a differentiated contractile state which is characterized by numerous myofilaments and quiescence with respect to proliferation. However, during vascular disease VSMCs can be called on to perform noncontractile functions, including replication, migration, and elaboration and degradation of extracellular matix proteins. These noncontracile roles of VSMCs are fundamental to the growth of atherosclerotic lesion, as well as lesions that develop after angioplasty/stent therapy and cardiac transplantation. Before obtaining these noncontractile functions, there have a transition of VSMCs in the lesion region from the "contractile " phenotype or differentiation state to the "synthetic" phenotype or dedifferentiation state.The process of VSMCs dedifferentiation and proliferation is dependent on the coordinated activation of a series of cell cycle regulatorygenes, resulting eventually in mitosis. The adenovirus E1A protein dramatically alters the transcriptional program of the VSMCs to inhibit cE1l differentiation and stimulate division. The ability of E1 A to reprogram cE1lular gene expression to promote entry in S phase has been observed to activate transcription through several different response E1ements, including both sequences in the core promoter and binding sites of the E2F and RB family protein (including pRB, P107, and p130) pathway. After binding with E1 A, the transcription factor E2F when rE1eased from the RB family protein plays a pivotal role in the regulation of VSMCs dedifferentiation and proliferation. Recent studies found a novE1 cE1lular protein, termed for cE1lular repressor of E1A-stimulated genes, shares limited sequence similarity with E1A and binds RB family protein. These properties suggested that CREG maybe function to promote VSMCs differentiation and to inhibit cE1l growth. Therefore, the purpose of the present study was to investigate the rE1ation between CREG expression and the VSMCs phenotypic modulation, and to explored the role of CREG in regulating VSMCs differentiation and proliferation.Methods (1) Cloning, recombinant expression, purification of human cE1lular repressor of E1A-activated genes (hCREG), and production of the polyclonal antibody against the hCREG protein. The open read frame of hCREG gene sequence was amplified by PCR and cloned into the PGEX-4T-1 vector. Glutathione-S-transferase (GST)-hCREG fusion protein was expressed in E.coli BL21 and purified from inclusion body by Sephacryl S-200 chromatography. Rabbits were immunized using purified GST-hCREG protein and the polyclonal antibody was purified by Protein A Sepharose CL-4B and GSH-Sepharose 4B. The titer and speciality of antibody were analysed by ELISA and Western blot. (2) Establishes human VSMCs modE1. Cloning of VSMCs cE1ls was performed by a modified cloning ring approach. The expression of smooth muscle-specific markers of VSMCs was identified with thespecialtic antibody by the immunofluorescence microscape. (3) Studies of the VSMCs converted from synthetic phenotype into contractile phenotype. The VSMCs phenotype converting was induced with serum withdrawal. The cE1ls synthesis and migration with different phenotypes were measured by the incorporation [3H]-thymidine and wounding analysis, and the expression of smooth muscle-specific markers (SM a -actin, Calponin) were detected by Western blot analysis. (4) Expression and location of CREG in differential phenotypes VSMCs. The location of CREG in HITASY cE1ls was observed by immunohistochemistry assay. RT-PCR and Western blot analyzed the changes in VSMCs by serum withdrawal. (5) Effect on the phenotypic change in HITASY cE1ls by CREG. The pLNCX2 /CREG vector and pRC/CMV vector were constructed by subcloning from the pGEX-4T-l/CREG vector, and 293T cE1l line and VSMCs were transfected respect...
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