Abstract
Cellular responses to platelet-derived growth factor (PDGF) are altered in a variety of pathological conditions, including cancers, fibroses, and vascular diseases, making PDGF-induced signaling pathways important therapeutic targets. The limited success of therapies designed to impact PDGF pathways may be overcome with a clearer understanding of how cells integrate signals from PDGF and the extracellular matrix (ECM). Here, we assessed the effects of fibronectin matrix assembly on the responsiveness of mesenchymal cells to PDGF. Our results indicate that fibroblast-mediated assembly of fibronectin fibrils attenuates intracellular calcium release in response to PDGF. The dose-dependent inhibition of PDGF-induced intracellular calcium release was specific to the ECM form of fibronectin. Further, a recombinant protein engineered to mimic ECM fibronectin similarly attenuated intracellular calcium release in response to PDGF. Of note, fibronectin attenuated the PDGF-calcium signaling axis at the level of phosphoinositide 3-kinase (PI3K) activation. Interestingly, ECM fibronectin did not alter other intracellular signals activated by PDGF, including activation of PDGF receptor β, AKT Ser/Thr kinase, phospholipase Cγ1, and extracellular signal-regulated kinase 1/2 (ERK1/2). Rather, fibronectin inhibited activation of the p55 regulatory subunit of PI3K in response to a variety of stimuli, indicating that ECM fibronectin selectively attenuates the intracellular calcium release cascade while leaving intact other PDGF signaling pathways. Selective regulation of calcium signaling by ECM fibronectin via the p55 regulatory subunit of PI3K represents a mechanism by which cells tune their response to PDGF and may therefore serve as a target to selectively regulate one branch of PDGF signaling.
Highlights
Cellular responses to platelet-derived growth factor (PDGF) are altered in a variety of pathological conditions, including cancers, fibroses, and vascular diseases, making Platelet-derived growth factors (PDGFs)-induced signaling pathways important therapeutic targets
The difficulties in employing PDGF signaling as a therapeutic target arise from the complexity of the cellular responses to PDGF, 3 The abbreviations used are: PDGF, platelet-derived growth factor; PDGF-R, platelet-derived growth factor receptor; ER, endoplasmic reticulum; EGF, epidermal growth factor; ECM, extracellular matrix; FGF-2, fibroblast growth factor-2; FN, fibronectin; FNIII, fibronectin type III repeat; MEF, mouse embryonic fibroblast; GST, glutathione S-transferase; IP3, inositol trisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; normal human dermal fibroblasts (NHDFs), normal human dermal fibroblast; PI3K, phosphoinositide 3-kinase; PLC␥1, phospholipase C␥1; Src homology region domain– containing phosphatase-1 (SHP1), Src homology region 2 domain-containing phosphatase 1; RIPA, radioimmunoprecipitation assay; ANOVA, analysis of variance; pAb, polyclonal antibody
In the absence of fibronectin, the addition of PDGF to FNnull MEFs evoked a single transient increase in intracellular calcium that became apparent ϳ1 min post-PDGF treatment and peaked ϳ3.5 min post-PDGF treatment (Fig. 1A). This PDGF-induced intracellular calcium release was observed in the presence of the calcium chelator, EGTA, indicating release from intracellular stores [21]
Summary
Cellular responses to platelet-derived growth factor (PDGF) are altered in a variety of pathological conditions, including cancers, fibroses, and vascular diseases, making PDGF-induced signaling pathways important therapeutic targets. Fibronectin inhibited activation of the p55 regulatory subunit of PI3K in response to a variety of stimuli, indicating that ECM fibronectin selectively attenuates the intracellular calcium release cascade while leaving intact other PDGF signaling pathways. Selective regulation of calcium signaling by ECM fibronectin via the p55 regulatory subunit of PI3K represents a mechanism by which cells tune their response to PDGF and may serve as a target to selectively regulate one branch of PDGF signaling. Fibronectin attenuates intracellular calcium release wherein broadly up- or down-regulating PDGF signaling often produces off-target effects due to the number and impact of signaling pathways affected. A greater understanding of the mechanisms by which PDGF signaling is regulated and, in particular, how cells exert selective control over which PDGF-induced signaling pathways are activated will allow for the development of more targeted therapies
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