Abstract
Platelet-derived growth factor (PDGF) signaling is dysregulated in a wide variety of diseases, making PDGF an attractive therapeutic target. However, PDGF also affects numerous signaling cascades essential for tissue homeostasis, limiting the development of PDGF-based therapies that lack adverse side-effects. Recent studies showed that fibroblast-mediated assembly of extracellular matrix (ECM) fibronectin fibrils attenuates PDGF-induced intracellular calcium release by selectively inhibiting phosphoinositol 3-kinase (PI3K) activation while leaving other PDGF-mediated signaling cascades intact. In the present study, a series of recombinant fibronectin-derived fusion proteins were used to localize the sequences in fibronectin that are responsible for this inhibition. Results demonstrate that attenuation of PDGF-induced intracellular calcium release by the fibronectin matrix mimetic, FNIII1H,8-10 requires α5β1 integrin ligation, but is not dependent upon the matricryptic, heparin-binding site of FNIII1. Intact cell-binding fibronectin fragments were also unable to attenuate PDGF-induced intracellular calcium release. In contrast, a novel integrin-binding fragment that adopts an extended and aligned conformational state, inhibited both PI3K activation and intracellular calcium release in response to PDGF. Taken together, these studies provide evidence that attenuation of PDGF-induced intracellular calcium release by fibronectin is mediated by a novel conformation of the α5β1 integrin-binding, FNIII9-10 modules, that is expressed by fibrillar fibronectin.
Highlights
Platelet-derived growth factor (PDGF) evokes a diverse array of intracellular signaling cascades that promote a variety of cell and tissue behaviors, including cell growth [1], survival [2], chemotaxis [3], extracellular matrix (ECM) deposition [4], collagen matrix contraction [4,5] and wound healing [6].Currently, recombinant PDGF is used for the clinical treatment of diabetic ulcers [7] and broad spectrum inhibitors of PDGF signaling are under development to treat pulmonary arterial hypertension [8]and pulmonary fibrosis [9]
Fibronectin matrix assembly by either FN-null MEFs or human dermal fibroblasts reduces the magnitude of intracellular calcium release in response to PDGF exposure [10]
To determine if integrin ligation alone attenuates PDGF-induced intracellular calcium release, collagen-adherent FN-null MEFs were pretreated with various fibronectin fragments spanning the integrin-binding domain, and stimulated with PDGF
Summary
Platelet-derived growth factor (PDGF) evokes a diverse array of intracellular signaling cascades that promote a variety of cell and tissue behaviors, including cell growth [1], survival [2], chemotaxis [3], extracellular matrix (ECM) deposition [4], collagen matrix contraction [4,5] and wound healing [6].Currently, recombinant PDGF is used for the clinical treatment of diabetic ulcers [7] and broad spectrum inhibitors of PDGF signaling are under development to treat pulmonary arterial hypertension [8]and pulmonary fibrosis [9]. Recombinant PDGF is used for the clinical treatment of diabetic ulcers [7] and broad spectrum inhibitors of PDGF signaling are under development to treat pulmonary arterial hypertension [8]. A difficulty in targeting aberrant PDGF activity therapeutically arises from the complexity of the cellular response to PDGF, wherein broadly up- or down-regulating. We recently demonstrated that the ECM form of fibronectin attenuates PDGF-induced intracellular calcium release by selectively inhibiting phosphoinositol 3-kinase (PI3K) activation while leaving other PDGF-mediated signaling cascades intact [10]. Understanding the mechanism by which ECM fibronectin selectively regulates this arm of PDGF signaling has the potential to inform the design of advanced targeted therapeutics
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