| Fibrosis is the end result of chronic tissue injury of organs induced by a variety of harmful stimuli, which is defined by the overgrowth, hardening, and/or scarring of tissues and attributed to excess deposition of extracellular matrix components. Progression of fibrosis involves interstitial hypercellularity, excessive matrix accumulation, and atrophy of epithelial structures leading to deformation and dysfunction ultimately.We previously compared differentially expressed genes between normal skin and hypertrophic scar with microarray and founded a targeted gene, P311, was highly expressed in the early hypertrophic scar[1-2]. P311 does not belong to any known family of proteins, It has been demonstrated that P311 could induce a TGF-β1–independent, nonfibrogenic myofibroblast phenotype, and that adenovirus-mediated P311 expression could accelerate nerve regeneration of the axotomized facial nerve[3-4]. To investigate the potential molecular mechanisms of P311 in the formation of hypertrophic scar, we screened the possible interactive protein with P311 from the adult liver cDNA library with a yeast two-hybrid system and acquired a qualified one--ITGB4BP [5].ITGB4BP (alias eIF6, p27BBP) was identified by its ability to bindβ4 integrin[6]. ITGB4BP was present in the nucleolus of all kinds of cells. Data show that ITGB4BP was a necessary transacting factor for the biogenesis of eukaryotic 60S ribosomal subunits[7-9]. Meanwhile ITGB4BP was recruited by the RISC complex, enabling microRNA complexes to exert their repressive effects on protein translation[10]. In cancer tissues, ITGB4BP mRNA and protein was upregulated predicting malignant transformation[11,12]. Activation of most cells was accompanied by a several-fold increase expression of ITGB4BP [13].Transforming growth factorβ1 (TGF-β1) plays an important role in both physiologic and pathologic processes, such as cell growth, differentiation, migration, adhesion, angiogenesis, immunity[14-17]. TGF-β1 is one of the most important cytokines leading to organ fibrosis and extracellular matrix (ECM) metabolism. Numerous studies have demonstrated that TGF-β1 play a significant role in the occurrence, development and progression of organ fibrosis[18-23].Wnt signal pathway is a complex process and is critical for fibrosis development. A variety of cellular processes are mediated by Wnt signaling, which includes cellular proliferation, differentiation, survival, apoptosis, and cell moratlity[24,25]. Loss of regulation of these pathways can lead to a large number of diseases, such as cancer and fibrosis[26,27]. Several therapeutic strategies have been applied successfully to inhibit profibrotic TGF-β1 activity in experimental models of fibrosis. These approaches include inhibition of TGF-β1 by neutralizing antibodies, antisense oligodeoxynucleotides, soluble TGF-β1 receptors, and blockade of TGF-β1 activation by decorin[28-30]. Despite the impressive results obtained in experimental tissue fibrosis, TGF-beta1 blockade has not yet been translated into an effective and safe therapeutic methods in human patients[31]. Therefore, it remains important to explore the role of additional factors which are involved in regeneration and fibrosis.To better understand the mechanism of ITGB4BP on fibrosis, we carried out this study and the major results are summarized as below:1. ITGB4BP inhibited the the expression of TGF-β1 both at mRNA level and protein level in hypertrophic scar-derived fibroblast and human HEK293 cells. The recombinant adenoviral-ITGB4BP vector and its empty control adenovirus were transfected into the hypertrophic scar-derived fibroblasts or human HEK293 cells and then TGF-β1 expression was examined by means of Real-Time PCR and Westernblot. We firstly observed that ITGB4BP significantly reduced the gene and protein expressions of TGF-β1 in vivo.2. ITGB4BP interacted with TGF-β1 promoter and inhibited its transcriptional activity. To identify the transcriptional regulation of ITGB4BP on TGF-β1 gene, a series of luciferase reporter constructs containing various 5'flanking region deletions of TGF-β1 gene were generated by PCR amplification and were transiently co-transfected with ITGB4BP into HEK293 cells. Luciferase reporter assays showed that ITGB4BP strongly inhibited transcriptional activity of TGF-β1 promoter from nucleotides +11 to +271. Electrophoretic mobility shift assay (EMSA) and Mass spectrum (MS) revealed that ITGB4BP could interact with the +204 to +271 promoter region of TGF-β1 gene in vitro. 3. The influence of ITGB4BP on Wnt signal pathway. The recombinant adenoviral-ITGB4BP expression vector and its empty control vector were transfected into human HEK293 cells, we demonstrated that overexpression of ITGB4BP in HEK293 cells caused a decrease in activity of theβ-catenin/TCF responsive reporter, TOPFlash. Western blotting revealed that ITGB4BP could decrease the expression ofβ-catenin protein compared with empty control vector. Indirect Immunofluorescence assay were clearly revealed thatβ-catenin was positive both in cytoplasm and nuclear, which was consistent with the TOPflash reporter results.In summary, we for the first time demonstrated that ITGB4BP was involved in the TGF-β1 transcriptional regulation and Wnt/β-catenin pathway. It provides us an alternative new strategy for the treatment of organ fibrosis.
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