EGFR signaling in retrovirus mediated gene transfer and cell-scattering
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Intracellular signaling network, a complex network of receptor-ligand mediated phosphorylation-dephosphorylation reactions that senses extracellular environment and relays the signal to cell nuclei resulting in appropriate response, is the key to understanding an underlying cellular behavior, e.g., guided migration of immune cells to a site of infection, or epidermal cells to a site of burn or injury, development of organs or blood vessels, expression of oncogenes or mutations and misregulation of growth factor receptors in development of cancers. A cell that senses an extracellular stimuli not only activates its own signaling network but also sends signals to other cells by secreting growth factors, cytokines, and small chemical molecules (ions and neurotransmitters) triggering a coordinated response to foreign injury. Because of their pluripotency, growth factors are key to understanding intracellular signaling network and due to high efficiency of gene transfer, recombinant retroviruses are key to genetic manipulation of cellular machinery-expression of a mutant gene, a therapeutic recombinant protein, or siRNAs to inhibit expression of an endogenous protein. Although a lot of progress has been made in molecular design of retrovirus vectors, the interactions of recombinant retrovirus with host cells remain largely elusive. The inability of recombinant retrovirus to transduce non-dividing cells prompted several studies to determine optimal cocktails of growth factors and/or extracellular matrix molecules to promote gene transfer to slowly diving cells and stem cells. In contrast to previous reports that growth factors increased gene transfer, we found that treatment of human epidermal keratinocytes and several cell lines with EGFR ligands EGF, TGF-α or HB-EGF decreased gene transfer. Conversely, treatment with an EGFR function blocking antibody or inhibition of EGFR tyrosine phosphorylation enhanced gene transfer in a dose dependent manner. In addition, blocking PKC-δ but not PKC-ζ, with chemical inhibitors or siRNA reversed the effects of EGF and restored gene transfer, indicating that the effect of EGFR activation is mediated through PKC-δ. Lastly cell cycle analysis showed that the effect of EGFR activation on retroviral gene transfer was independent of the cell cycle status of target cells. Our results implicate EGFR and PKC-δ in retroviral infection and may have wide implications for retrovirus gene transfer or design of antiretroviral therapies. Understanding of this tool helped us efficiently manipulate signaling pathways and understand physiological effect of growth factors on cellular function. Specifically, this study focuses on effect of epidermal growth factor, EGF, on adherens junction and cell-scattering. EGF is known to affect adherens junctions and disrupt cell-cell adhesion in a variety of carcinomas but the underlying mechanisms are not completely understood. Using human tumor epithelial cells overexpressing EGFR we demonstrated that EGF-induced cell scattering was mediated by protein kinase C-delta (PKC-δ). Reducing PKC-δ expression by stable delivery of siRNAs targeting three different regions of the PKC-δ mRNA inhibited EGF-induced internalization of E-cadherin into the cytoplasm and blocked cell scattering. EGF phosphorylated PKC-δ at Y311, which binds to E-cadherin. Y311F mutant of PKC-δ did not bind to E-cadherin and blocked EGF-induced cell-scattering. Moreover, blocking src or p38, the kinases known to phosphorylate PKC-δ at Y311, blocked its binding to E-cadherin and cell-scattering. Finally, EGF reduced expression of the tight junction protein, occludin, and this effect was also mediated by PKC-δ. Therefore, PKC-δ mediated the effects of EGF on adherens and tight junctions thereby playing an important role in cell-cell adhesion with possible wider implications in tumor metastasis or epithelial-to-mesenchymal transition.