NG2, a novel proapoptotic receptor, opposes integrin α4 to mediate anoikis through PKCα-dependent suppression of FAK phosphorylation

Abstract

Disruption of cell-matrix interactions can lead to anoikis -apoptosis due to loss of matrix contacts. Altered fibronectin (FN) induces anoikis of primary human fibroblasts by a novel signaling pathway characterized by reduced phosphorylation of focal adhesion kinase (FAK). However, the receptors involved are unknown. FAK phosphorylation is regulated by nerve/glial antigen 2 (NG2) receptor signaling through PKCa a point at which signals from integrins and proteoglycans may converge. We found that an altered FN matrix induced anoikis in fibroblasts by upregulating NG2 and downregulating integrin a4. Suppressing NG2 expression or overexpressing a4 rescued cells from anoikis. NG2 overexpression alone induced apoptosis and, by reducing FAK phosphorylation, increased anoikis induced by an altered matrix. NG2 overexpression or an altered matrix also suppressed PKCa expression, but overexpressing integrin a4 enhanced FAK phosphorylation independently of PKCa. Cotransfection with NG2 cDNA and integrin a4 siRNA did not lower PKCa and pFAK levels more than transfection with either alone. PKCa was upstream of FAK phosphorylation, as silencing PKCa decreased FAK phosphorylation. PKCa overexpression reversed this behavior and rescued cells from anoikis. Thus, NG2 is a novel proapoptotic receptor, and NG2 and integrin a4 oppositely regulate anoikis in fibroblasts. NG2 and integrin a4 regulate FAK phosphorylation by PKCa-dependent and -independent pathways, respectively. Cell Death and Differentiation (2008) The extracellular matrix (ECM) glycoprotein fibronectin (FN) affects adhesion, migration, survival, and other cellular functions. FN fragments found in vivo and associated with disease or comparable recombinant fragments trigger apoptosis/anoikis in primary human fibroblasts via a novel pathway regulated by decreases in focal adhesion kinase (FAK) phosphorylation and downregulation of p53.1-3 However, the receptors involved are not known. Two classes of cellsurface receptors, integrins and proteoglycans, interact with FN and could mediate the anoikis. This process is regulated by FAK, an integrin-dependent non-receptor tyrosine kinase, consistent with a role for integrins. Also proteoglycans are involved in this apoptotic mechanism. 1Integrins are a and b heterodimeric cell-surface receptors that mediate cell-ECM interactions and regulate cell adhesion, migration, proliferation, and survival. Interactions between FN and integrins promote the survival of many cell types. We showed that blocking antibodies against the a4 integrin subunit induced apoptosis-like cell rounding in human fibroblasts, similar to that induced by anoikis in response to an altered FN matrix fragment.2 Integrins mediate those responses by binding ECM ligands and activating signaling cascades that promote these actions. The a4 integrin subunit can bind to at least three interaction sites on FN. These include the arginine-glycine-aspartic acid site on the central cell-binding domain, the V region, and the heparin-binding domain. Once engaged, integrins ...

Activation of the Integrin Effector Kinase Focal Adhesion Kinase in Cancer Cells Is Regulated by Crosstalk between Protein Kinase Cα and the PDZ Adapter Protein mda-9/Syntenin

“…This may explain the relationship between activation of PKCα and formation of focal adhesions to FN (28)(29)(30)(31). Our findings also support the hypothesis that mda-9/syntenin functions as an adaptor protein for the FN-induced assembly of multimeric signaling complexes clustered at high concentrations on the plasma membrane.…”

“…PKCα is essential for integrin-mediated adhesion, migration, and signaling events through participation in integrin β 1 -associated protein complexes downstream of FN (28)(29)(30)(31)34). In particular, binding of integrin α 5 β 1 to FN activates PKCα, and inhibition of PKCα, but not PKCδ, suppresses focal adhesion formation and cell migration mediated by α 5 β 1 (31).…”

“…In particular, binding of integrin α 5 β 1 to FN activates PKCα, and inhibition of PKCα, but not PKCδ, suppresses focal adhesion formation and cell migration mediated by α 5 β 1 (31). FN binding to integrin α 5 β 1 requires a proteoglycan coreceptor, syndecan-4, for focal adhesion formation and cell migration (29)(30)(31). Syndecan-4-dependent activation of PKCα is essential for focal adhesion formation and cell migration in cells that adhere to FN through integrin α 5 β 1 (31).…”

“…PKCα activation is upstream FAK phosphorylation in response to FN. Inhibition of PKCα suppresses focal adhesion formation, cell migration, and FAK phosphorylation at Tyr 397 during adhesion to FN (26,(29)(30)(31). However, the mechanism by which links between PKCα activation and FAK phosphorylation remains poorly understood.…”

Three-Dimensional In Vitro Models in Glioma Research - Focus on Spheroids

“…It has been found in human acute myeloid leukemia and in gliomas, where it in the latter seems to increase tumor cell proliferation in vitro and promote angiogenesis in vivo. Brekke et al, 2006;Chekenya et al, 1999;Chekenya et al, 2002a;Chekenya et al, 2002b;Chekenya et al, 2008;Chekenya & Immervoll, 2007;Chekenya & Pilkington, 2002;Joo et al, 2008;Petrovici et al, 2010;Stallcup & Huang, 2008 www.intechopen.com Fig. 2.…”