Assembly of fibronectin fibrils selectively attenuates platelet-derived growth factor–induced intracellular calcium release in fibroblasts

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 PDGFcalcium 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 ␤, AKT Ser/Thr kinase, phospholipase C␥1, 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.

A Matricryptic Conformation of the Integrin-Binding Domain of Fibronectin Regulates Platelet-Derived Growth Factor-Induced Intracellular Calcium Release

“…Fluorescence intensity was recorded as an indicator of intracellular calcium concentration. As demonstrated previously [10,48], PDGF evoked a single, transient increase in intracellular calcium that peaked ~2.5 min after PDGF addition (Figure 2A, +PBS; +PDGF). No significant change in fluorescence intensity was observed after the addition of vehicle only (Figure 2A, +PBS; +Vehicle).…”

“…No significant change in fluorescence intensity was observed after the addition of vehicle only (Figure 2A, +PBS; +Vehicle). As reported [10], cells pre-exposed to either fibronectin or FNIII1H,8-10 exhibited an attenuated response to PDGF compared to the respective controls (Figure 2A,B: +FN versus +PBS; +FNIII1H,8-10 versus +GST). In contrast, pre-exposing cells to FNIII8-10 did not alter PDGF-induced intracellular calcium release (Figure 2A,B).…”

“…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]. A similar inhibition of PDGF-induced calcium release was observed following treatment of FN-null MEFs with the recombinant fibronectin matrix mimetic, FNIII1H,8-10 [10]. In the present study, a series of fibronectin fragments was used in conjunction with FN-null MEFs to localize the sites in FNIII1H,8-10 responsible for this inhibitory activity.…”

“…However, a difficulty in targeting aberrant PDGF activity therapeutically arises from the complexity of the cellular response to PDGF, wherein broadly up- or down-regulating PDGF signaling often has undesirable consequences [1]. 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]. The PDGF–phospholipase C-gamma 1 (PLC-γ1)–PI3K–calcium signaling axis controls cell differentiation, proliferation, migration, and changes in gene transcription [1].…”

“…FNIII1H,8-10 is composed of a fragment of FNIII1 (FNIII1H), truncated at the N-terminus to expose the cryptic, heparin-binding core, that is coupled directly to the cell-binding, FNIII8-10 modules [18]. FNIII1H,8-10 binds to α5β1 integrins [35] and mimics the ability of ECM fibronectin to attenuate PDGF-induced intracellular calcium release [10]. In the present study, we used fibronectin-null mouse embryonic fibroblasts (FN-null MEFs), together with a series of recombinant FNIII1H,8-10 derivatives that selectively bind α5β1 or αvβ3 integrins, to determine the mechanism by which FNIII1H,8-10 inhibits the PDGF-PI3K-calcium signaling axis.…”