Participation of C/EBPB transcription factor in the activation and differentiation of fibroblasts into myofibroblasts in experimental model of bleomicin- induced fibrosis

• Grant number: 13/01496-0
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Effective date (Start): August 01, 2013
• Effective date (End): March 31, 2014
• Field of knowledge: Health Sciences - Medicine - Pathological Anatomy and Clinical Pathology
• Principal Investigator: Vera Luiza Capelozzi
• Grantee: Vanessa Martins da Silva
• Supervisor: Sem Hin Phan
• Host Institution: Faculdade de Medicina (FM). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Research place: University of Michigan, United States
• Associated to the scholarship: 11/09181-2 - PULMONARY FIBROBLASTS IN PARENCHYMAL REMODELING IN EXPERIMENTAL MODELS OF FIBROSIS INDUZED BY BLEOMYCIN AND 3-5-di-tert-butyl-4-hydroxytoluene (BHT), BP.DR

Abstract

In the proposed project the hypothesis is that myofibroblast differentiation is a key factor in driving fibrosis and its de novo emergence is regulated by a complex mechanism involving transcriptional and epigenetic regulation of gene expression by both local and bone marrow derived cells. Two specific directions are proposed, namely analysis of transcriptional and epigenetic regulation of myofibroblast differentiation and the role of bone marrow derived cells in this process and overall pulmonary fibrosis. In the first part the role of the transcription factor CCAAT enhancer binding protein ² (C/EBP²) will be analyzed by identification of its target genes and evaluation of their potential roles in fibroblast activation and myofibroblast differentiation. Mice deficient in this factor exhibit a significantly reduced pulmonary fibrosis in response to bleomycin-induced lung injury. A putative CCAAT binding element at -66 to -58 has been identified in the promoter of the ±-smooth muscle actin (±-SMA) gene, a key marker of the differentiated myofibroblast. To determine the in vivo role of C/EBP² the impact of its deficiency on lung myofibroblast differentiation and pulmonary fibrosis in response to bleomycin-induced injury will be analyzed. Specifically, the role of C/EBP² in bone marrow vs. intrapulmonary derived cells will be assessed by use of reciprocal bone marrow transplantation from wild type to knockout mice and vice versa. Additional relevant C/EBP target genes in vivo in the bleomycin-induced model of pulmonary fibrosis, and in isolated lung fibroblasts from fibrotic lung (including IPF patients) will be identified. Conditional selective fibroblast C/EBP² knockout mice has been successfully developed and an initial study studying the effects of this selective depletion in mesenchymal (type I collagen expressing) cells have confirmed the importance of C/EBP² in these cells in pulmonary fibrosis using the bleomycin model. Several C/EBP² target genes with potential relevance to fibrosis have been identified using these knockout mice, including matrix metalloproteinase 12 (MMP12), resistin-like molecule ± (RELM±) and Dkks. The cloned MMP12 and RELM± promoters and reporter gene constructs will be used to confirm this regulation by C/EBP². Further studies will include chromosome immunoprecipitation (ChIP) and gel shift assays to prove the functional relevance of any binding interaction revealed by the promoter studies in vitro. Epigenetic analysis will include examination of the interaction of C/EBP² with histones in regulation of target genes, with primary focus on the ±-SMA gene. The effects on DNA methylation of the ±-SMA gene will also be analyzed.Second, the role of bone marrow derived cell migration and differentiation will be examined using C/EBP² deficient bone marrow chimera mice. Donor bone marrow from type I collagen expressing cell specific conditional C/EBP² knockout mice will be transplanted to wild type recipients and after stable engraftment will be analyzed for responsiveness to bleomycin-induced pulmonary fibrosis. The recruitment of bone marrow derived cells will be analyzed and the phenotype of recruited cells will be determined, especially with respect to hematopoietic origin and expression of type I collagen. Co-culture experiments will be undertaken to elucidate the mechanism of how these cells can regulate myofibroblast differentiation in lung fibroblast populations. (AU)