Mechanisms of Human Dermal Fibroblast Migration

University Of Southern California, Los Angeles CA

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Abstract

DESCRIPTION (provided by applicant): During wound repair, fibroblasts in the dermis proliferate and migrate into the wound bed in response to newly formed provisional matrices and newly released growth factors, predominantly platelet-derived growth factor (PDGF). Extracellular matrix (ECM) proteins and growth factors bind distinct cell surface receptors and elicit parallel intracellular sigaling pathways. These "DUAL SIGNALING" processes by ECM and growth factors determine optimal cell migration. Our studies showed that PDGF-BB promotes migration of human dermal fibroblasts (HDFs) on a collagen matrix as effectively as whole serum. This suggests that the main migration-promoting activity in serum for HDFs comes from PDGF-BB. After HDFs migrate into the wound bed, they then synthesize, deposit, and remodel the extracellular matrix necessary for further ingrowths of themselves and other cell types. However, despite the recognition of being the only FDA-approved growth factor for the treatment of human skin wounds, the mechanisms of PDGF-BB's action together with ECM in the control of HDF migration are poorly understood. The objective of this proposal is to understand the function of a novel signaling pathway, PDGFR > Rac >Nck >Pak, in the control of HDF motility in response to the "dual signaling" of PDGF-BB and a collagen matrix. We will: 1) study the specific role of Rac in PDGF-BB and collagen dual signaling processes that lead to HDF migration; 2) investigate how the Nck-Pak complex mediates PDGF-BB- and collagen-induced HDF migration; 3) identify the downstream targets of the Rac > Nck > Pak pathway that regulate PDGF-BB-induced collagen gene expression and migration. Gain-of-function and loss-of-function mutants iof Rac, Nck and Pak, as well as Pak's potential downstream targets ERK, p38 and JNK, will be used in these studies. Since we use primary HDFs as the model system, we have studied various transfection and viral infection procedures to ensure efficient transgene delivery into these cells. We found that human lentiviral vector delivery systems offer more than 94 percent gene transduction efficiency and sustained expression of transgenes for HDFs in culture. Using this gene delivery technique, we have demonstrated the importance of a list of signaling molecules in HDF migration in response to PDGF-BB and a collagen matrix.