Epidermal Growth Factor Induces Fibroblast Contractility and Motility via a Protein Kinase C δ-dependent Pathway

Abstract

Myosin-based cell contractile force is considered to be a critical process in cell motility. However, for epidermal growth factor (EGF)-induced fibroblast migration, molecular links between EGF receptor (EGFR) activation and force generation have not been clarified. Herein, we demonstrate that EGF stimulation increases myosin light chain (MLC) phosphorylation, a marker for contractile force, concomitant with protein kinase C (PKC) activity in mouse fibroblasts expressing human EGFR constructs. Interestingly, PKC␦ is the most strongly phosphorylated isoform, and the preferential PKC␦ inhibitor rottlerin largely prevented EGF-induced phosphorylation of PKC substrates and MARCKS. The pathway through which EGFR activates PKC␦ is suggested by the fact that the MEK-1 inhibitor U0126 and the phosphatidylinositol 3-kinase inhibitor LY294002 had no effect on PKC␦ activation, whereas lack of PLC␥ signaling resulted in delayed PKC␦ activation. EGF-enhanced MLC phosphorylation was prevented by a specific MLC kinase inhibitor ML-7 and the PKC inhibitors chelerythrine chloride and rottlerin. Further indicating that PKC␦ is required, a dominant-negative PKC␦ construct or RNAi-mediated PKC␦ depletion also prevented MLC phosphorylation. In the absence of PLC signaling, MLC phosphorylation and cell force generation were delayed similarly to PKC␦ activation. All of the interventions that blocked PKC␦ activation or MLC phosphorylation abrogated EGF-induced cell contractile force generation and motility. Our results suggest that PKC␦ activation is responsible for a major part of EGF-induced fibroblast contractile force generation. Hence, we identify here a new pathway helping to govern cell motility, with PLC signaling playing a role in activation of PKC␦ to promote the acute phase of EGF-induced MLC activation.Cell motility induced by activation of epidermal growth factor receptor (EGFR), 1 and related receptor tyrosine kinases, can be deconstructed into a series of orchestrated events: lamellipodial extension, formation of forward adhesions, exertion of contractile forces to pull the cell body forward, and detachment of the rear (1). While each process is required for net cell locomotion, it is not necessarily the case that signals downstream of receptor activation must concomitantly be involved in triggering all of the processes. Despite longstanding anecdotal indications, only recently have formal demonstrations emerged that signaling via EGFR actually elicits cell contractile force generation (2, 3) along with the other biophysical processes (4 -6). Because of the central importance of growth factor-induced cell motility in physiological and pathological applications, such as organogenesis, wound repair, and tumor invasion, determination of key pathways involved in connecting EGFR activity to contractile force generation, as well as the other processes underlying motility, is a crucial undertaking. Myosin motors operating on cytoskeletal actin filaments are presumed to be involved in growth factor-induced cell motility in manne...

Cooperation of Amphiregulin and Insulin-like Growth Factor-1 Inhibits Bax- and Bad-mediated Apoptosis via a Protein Kinase C-dependent Pathway in Non-small Cell Lung Cancer Cells

“…To date, the PKC pathway has not been described to directly propagate the EGF receptor signaling. However, the EGF receptor triggers PKC activity (46) at least in part downstream of phospholipase signaling (47), and EGF has recently been shown to induce fibroblast contractility and motility via a PKC␦-dependent pathway (48). In our model of NSCLC cells, the antiapoptotic activity of H358 CM, mediated by AR/IGF1 cooperation, is prevented by PKC inhibitors but not by PI3K or MAPK inhibitors, demonstrating the activation of an original PKC-dependent survival pathway (Fig.…”

PLCγ contributes to metastasis of in situ-occurring mammary and prostate tumors

“…Second, some low-level EGF-induced motility is noted in PLCg-1 devoid cells (Ji et al, 1998a). this is owing to other pathways that regulate motility-associated processes other than protrusion, such as ERK MAP kinase/ m-calpain (Chen et al, 1996;Glading et al, 2000) and PKCd/MLC (Iwabu et al, 2004), as well some upregulation of PLCg-2 expression. It should also be noted that while the targeted inhibition of PLCg is being discussed as a downstream effector required for EGFR-mediated motility, we cannot be certain that the extracellular trigger for breast and prostate cancer dissemination are the EGFR ligands.…”

Rho/ROCK and MEK/ERK activation by transforming growth factor-α induces articular cartilage degradation

“…In addition, we observed simultaneous MLC2 activation by TGFa, suggesting that contractility may be induced in articular chondrocytes by EGFR activation. 38 Although stress fiber formation and contractility is normal in some cell types such as fibroblasts, 39 these events represent unfavorable modification of the articular chondrocyte phenotype through activation of Rho/ROCK signaling. Furthermore, these data suggest that inhibition of Rho/ROCK signaling would favor articular cartilage maintenance and repair.…”