Abstract
Cell-mediated contraction of collagenous matrices is modulated by various growth factors and cytokines, such as platelet-derived growth factor-BB (PDGF-BB). Here we used a genetic cell model to delineate defined signaling pathways that enhance collagen gel contraction downstream of ligand-stimulated platelet-derived growth factor receptor-β (PDGF-Rβ). Our data show that PDGF BB-enhanced activations of phosphatidylinositol 3′-kinase (PI3K) and phospholipase Cγ (PLCγ) were necessary for PDGF-enhanced collagen gel contraction. Importantly, other defined signaling pathways down-stream of PDGF-Rβ were, however, dispensable. The decisive roles for PI3K and PLCγ were corroborated by experiments using selective inhibitors. Furthermore, we show that de-phosphorylation and thereby activation of cofilin that is important for the turnover of actin filaments, is depended on PI3K and PLCγ down-stream of PDGF-Rβ. Moreover, inhibition of protein kinase C (PKC) by GÖ6976 and bisindolylmaleimide-II abolished cofilin de-phosphorylation, as well as PDGF-enhanced contraction. In contrast, activation of the PKC protein family by 4β-phorbol 12-myristate 13-acetate (PMA) did not accelerate collagen gel contraction although it induced long-term cofilin de-phosphorylation, showing the need of a dynamic control of cofilin de-phosphorylation for PDGF-enhanced collagen gel contraction. Taken together, our data point to the involvement of a PI3K/PLCγ-PKC-cofilin pathway in both PDGF-enhanced cofilin de-phosphorylation and PDGF-enhanced collagen gel contraction.
Highlights
Cell-mediated contraction of the interstitial extracellular matrix (ECM) controls the interstitial fluid content[1]
We show that the phosphatidylinositol 3′-kinase (PI3K)/phospholipase Cγ (PLCγ)-protein kinase C (PKC) pathway links the activated platelet-derived growth factor receptor-β (PDGF-Rβ) to cofilin de-phosphorylation and this chain of signaling events is involved in PDGF-enhanced collagen gel contraction
Addition of the PI3K inhibitor LY294002 at 50 μM completely inhibited PDGF-enhanced contraction of collagen gels mediated by BJ fibroblasts, and partially reduced contraction occurred in the absence of platelet-derived growth factor-BB (PDGF-BB) (Fig. 1B)
Summary
Cell-mediated contraction of the interstitial extracellular matrix (ECM) controls the interstitial fluid content[1]. Studies using mutational or inhibitory approaches have provided evidence for a crucial role of phosphatidylinositol 3′-kinase (PI3K) in PDGF-enhanced actin turnover and chemotaxis[17, 18], cell growth[19, 20], as well as for collagen gel contraction in vitro and normalization of anaphylaxis-induced lowered dermal interstitial fluid pressure in vivo[12, 21]. PLCγ induces hydrolysis of membrane-associated PIP2 resulting in the formation of diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3) These products activate protein kinase C (PKC) and increase intracellular Ca2+ 27, 28. A PDGF-Rβ mutation that leads to an overactive PLCγ resulted in a PI3K-independent hyperchemotaxis[33, 34] These observations suggest a dynamic interplay between PI3K and PLCγ signaling during cell-mediated matrix contraction. Membrane-bound cofilin (not phosphorylated) is inactive when bound to PIP2 and can be locally activated through PLCγ-induced hydrolysis of PIP2 leading to the localized release of active cofilin[44]
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