Epidermal growth factor induces acute matrix contraction and subsequent calpain‐modulated relaxation

Allen, Fred; Asnes, Clara F.; Chang, Philip; Elson, Elliot L.; Lauffenburger, Douglas A.; Wells, Alan

doi:10.1046/j.1524-475x.2002.10701.x

Cited by 37 publications

(33 citation statements)

References 50 publications

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“…In addition to ET-1, other factors present in serum, namely, epidermal growth factor, platelet-derived growth factor (PDGF)-BB, transforming growth factor-␤ (TGF␤), and connective tissue growth factor (CTGF), act on fibroblasts to promote matrix contraction (Grinnell, 1994;Shi-Wen et al, 2000;Allen et al, 2002). Thus, in vivo, elevation of these factors in bronchial lavage fluid or serum of patients with fibrotic disease (Ludwicka et al, 1995;Sato et al, 2000;Leask et al, 2004) might collectively contribute to the enhanced contractile phenotype of fibroblasts in these individuals.…”

Section: Discussionmentioning

confidence: 99%

Endothelin-1 Promotes Myofibroblast Induction through the ETA Receptor via a rac/Phosphoinositide 3-Kinase/Akt-dependent Pathway and Is Essential for the Enhanced Contractile Phenotype of Fibrotic Fibroblasts

Chen

Denton

et al. 2004

MBoC

326

307

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The endothelins are a family of endothelium-derived peptides that possess a variety of functions, including vasoconstriction. Endothelin-1 (ET-1) is up-regulated during tissue repair and promotes myofibroblast contraction and migration, hence contributing to matrix remodeling during tissue repair. Here, we show that addition of ET-1 to normal lung fibroblasts induces expression of proteins that contribute to a contractile phenotype, including ␣-smooth muscle actin (␣-SMA), ezrin, moesin, and paxillin. We confirm that ET-1 enhances the ability of lung fibroblasts to contract extracellular matrix, a function essential for tissue repair, through induction of de novo protein synthesis. Blockade of the Akt/ phosphoinositide 3-kinase (PI3-kinase) pathway with LY294002 and wortmannin prevents the ability of ET-1 to induce ␣-SMA, ezrin, paxillin, and moesin and to promote matrix contraction. Dominant negative rac and Akt blocked the ability of ET-1 to promote formation of ␣-SMA stress fibers. Using specific ET-1 receptor inhibitors, we show that ET-1 induces collagen matrix contraction through the ETA, but not the ETB, receptor. Relative to normal pulmonary fibroblasts, fibroblasts cultured from scars of patients with the fibrotic disease systemic sclerosis (scleroderma) show enhanced ET-1 expression and binding. Systemic sclerosis lung fibroblasts show increased ability to contract a collagen matrix and elevated expression of the procontractile proteins ␣-SMA, ezrin, paxillin, and moesin, which are greatly reduced by antagonizing endogenous ET-1 signaling. Thus, blocking ET-1 or the PI3-kinase/Akt cascades might be beneficial in reducing scar formation in pulmonary fibrosis. INTRODUCTIONA complex histological and architectural structure is a prerequisite for effective lung function. In the lung, specialized structures, the alveoli, increase the surface area of the lung, allowing for efficient gas exchange. The maintenance of these specialized structures is in turn dependent on the underlying connective tissue, comprised principally of fibroblasts and extracellular matrix (ECM; for review, see Gadek et al., 1984), which is essential for the mechanical and structural integrity of the lung. As a response to environmental insults, or as a consequence of local inflammatory processes, structural damage to the lung can occur, resulting in a wound healing response. This response consists of an integrated series of biochemical, immunological, and structural changes that result in the de novo synthesis of a new epithelium, blood vessels, and connective tissue (Razzaque and Taguchi, 2003). The proper repair of connective tissue requires synthesis of new ECM components, such as collagen and fibronectin (Badylak, 2002). In addition, repair of connective tissue requires the proper reconstitution of its support function; that is, an appropriate tensile strength must be recreated. This tensile strength results from the remodeling of the newly formed ECM through a combination of cell locomotion and translocation of the flexible collage...

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Section: Discussionmentioning

confidence: 99%

Endothelin-1 Promotes Myofibroblast Induction through the ETA Receptor via a rac/Phosphoinositide 3-Kinase/Akt-dependent Pathway and Is Essential for the Enhanced Contractile Phenotype of Fibrotic Fibroblasts

Chen

Denton

et al. 2004

MBoC

326

307

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“…While the time-scale integration of motility would not allow us to discern this 30 -60 min delay (46), we could parse this by isometric gel contraction (2). We found cells expressing the cЈ973 EGFR that fails to activate PLC␥ did induce robust contraction but that this response was delayed compared with cells expressing WT EGFR (Fig.…”

Section: Pkc␦ Is Required For Egf-induced Cell Motility Andmentioning

confidence: 98%

“…While each process is required for net cell locomotion, it is not necessarily the case that signals downstream of receptor activation must concomitantly be involved in triggering all of the processes. Despite longstanding anecdotal indications, only recently have formal demonstrations emerged that signaling via EGFR actually elicits cell contractile force generation (2,3) along with the other biophysical processes (4 -6). Because of the central importance of growth factor-induced cell motility in physiological and pathological applications, such as organogenesis, wound repair, and tumor invasion, determination of key pathways involved in connecting EGFR activity to contractile force generation, as well as the other processes underlying motility, is a crucial undertaking.…”

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confidence: 99%

“…PKC consists of a family of at least 11 isoforms. Specific isoforms of PKC are activated by phospholipids, diacylglycerol (DAG) generated by phospholipase C (PLC) or phospholipase D (PLD) from phosphatidylinositol 4,5-bisphosphate (PIP 2 ), fatty acids generated by PLA 2 , and calcium, depending on isoforms. Based on their structural and biochemical properties these PKC isoforms can be divided into three major groups: (i) the classical PKC (cPKC; ␣, ␤I, ␤II, and ␥), which are activated by DAG and are Ca 2ϩ -dependent; (ii) the novel PKC (nPKC; ␦, ⑀, , , and ), which are activated by DAG but Ca 2ϩ -independent, and (iii) the atypical PKC (aPKC; and ), which do not respond to either DAG or calcium.…”

mentioning

confidence: 99%

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Epidermal Growth Factor Induces Fibroblast Contractility and Motility via a Protein Kinase C δ-dependent Pathway

Iwabu

Smith

Allen

et al. 2004

Journal of Biological Chemistry

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130

115

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Myosin-based cell contractile force is considered to be a critical process in cell motility. However, for epidermal growth factor (EGF)-induced fibroblast migration, molecular links between EGF receptor (EGFR) activation and force generation have not been clarified. Herein, we demonstrate that EGF stimulation increases myosin light chain (MLC) phosphorylation, a marker for contractile force, concomitant with protein kinase C (PKC) activity in mouse fibroblasts expressing human EGFR constructs. Interestingly, PKC␦ is the most strongly phosphorylated isoform, and the preferential PKC␦ inhibitor rottlerin largely prevented EGF-induced phosphorylation of PKC substrates and MARCKS. The pathway through which EGFR activates PKC␦ is suggested by the fact that the MEK-1 inhibitor U0126 and the phosphatidylinositol 3-kinase inhibitor LY294002 had no effect on PKC␦ activation, whereas lack of PLC␥ signaling resulted in delayed PKC␦ activation. EGF-enhanced MLC phosphorylation was prevented by a specific MLC kinase inhibitor ML-7 and the PKC inhibitors chelerythrine chloride and rottlerin. Further indicating that PKC␦ is required, a dominant-negative PKC␦ construct or RNAi-mediated PKC␦ depletion also prevented MLC phosphorylation. In the absence of PLC signaling, MLC phosphorylation and cell force generation were delayed similarly to PKC␦ activation. All of the interventions that blocked PKC␦ activation or MLC phosphorylation abrogated EGF-induced cell contractile force generation and motility. Our results suggest that PKC␦ activation is responsible for a major part of EGF-induced fibroblast contractile force generation. Hence, we identify here a new pathway helping to govern cell motility, with PLC signaling playing a role in activation of PKC␦ to promote the acute phase of EGF-induced MLC activation.Cell motility induced by activation of epidermal growth factor receptor (EGFR), 1 and related receptor tyrosine kinases, can be deconstructed into a series of orchestrated events: lamellipodial extension, formation of forward adhesions, exertion of contractile forces to pull the cell body forward, and detachment of the rear (1). While each process is required for net cell locomotion, it is not necessarily the case that signals downstream of receptor activation must concomitantly be involved in triggering all of the processes. Despite longstanding anecdotal indications, only recently have formal demonstrations emerged that signaling via EGFR actually elicits cell contractile force generation (2, 3) along with the other biophysical processes (4 -6). Because of the central importance of growth factor-induced cell motility in physiological and pathological applications, such as organogenesis, wound repair, and tumor invasion, determination of key pathways involved in connecting EGFR activity to contractile force generation, as well as the other processes underlying motility, is a crucial undertaking. Myosin motors operating on cytoskeletal actin filaments are presumed to be involved in growth factor-induced cell motility in manne...

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“…Indeed, the ELR-negative CXC chemokines IP-9 (CXCL11 or I-TAC) and IP-10 (CXCL10) inhibit the growth factor-induced motility of fibroblasts and endothelial cells and also induce the apoptosis of these supernumerary cells (27). These chemokines thereby induce the contraction of the matrix (28) and restore the physiologic paucicellularity, leading to the formation of a strong and organized dermis.…”

mentioning

confidence: 99%

m-calpain Activation Is Regulated by Its Membrane Localization and by Its Binding to Phosphatidylinositol 4,5-Bisphosphate*

Leloup

Shao

Bae³

et al. 2010

Journal of Biological Chemistry

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m-calpain plays a critical role in cell migration enabling rear de-adhesion of adherent cells by cleaving structural components of the adhesion plaques. Growth factors and chemokines regulate keratinocyte, fibroblast, and endothelial cell migration by modulating m-calpain activity. Growth factor receptors activate m-calpain secondary to phosphorylation on serine 50 by ERK. Concurrently, activated m-calpain is localized to its inner membrane milieu by binding to phosphatidylinositol 4,5-bisphosphate (PIP 2 ). Opposing this, CXCR3 ligands inhibit cell migration by blocking m-calpain activity secondary to a PKAmediated phosphorylation in the C2-like domain. The failure of m-calpain activation in the absence of PIP 2 points to a key regulatory role, although whether this PIP 2 -mediated membrane localization is regulatory for m-calpain activity or merely serves as a docking site for ERK phosphorylation is uncertain. Herein, we report the effects of two CXCR3 ligands, CXCL11/IP-9/I-TAC and CXCL10/IP-10, on the EGF-and VEGF-induced redistribution of m-calpain in human fibroblasts and endothelial cells. The two chemokines block the tail retraction and, thus, the migration within minutes, preventing and reverting growth factor-induced relocalization of m-calpain to the plasma membrane of the cells. PKA phosphorylation of m-calpain blocks the binding of the protease to PIP 2 . Unexpectedly, we found that this was due to membrane anchorage itself and not merely serine 50 phosphorylation, as the farnesylation-induced anchorage of m-calpain triggers a strong activation of this protease, leading notably to an increased cell death. Moreover, the ERK and PKA phosphorylations have no effect on this membrane-anchored m-calpain. However, the presence of PIP 2 is still required for the activation of the anchored m-calpain. In conclusion, we describe a novel mechanism of m-calpain activation by interaction with the plasma membrane and PIP 2 specifically, this phosphoinositide acting as a cofactor for the enzyme. The phosphorylation of m-calpain by ERK and PKA by growth factors and chemokines, respectively, act in cells to regulate the enzyme only indirectly by controlling its redistribution.Calpains are intracellular cysteine proteases involved in numerous physiological and pathological phenomena, such as embryo development and tumor invasion (1). Among the 15 members of the calpain family, the two ubiquitous calpains, calpain 1 and calpain 2, are the best described. They form with the calpain small subunit 1 (calpain S1) two heterodimers, -calpain for calpain 1 and m-calpain for calpain 2, that are strongly involved in the regulation of cell motility. Cell migration was previously described as a four-stage process: cell membrane protrusion, adhesion to the substrate, contraction of the cell body, and finally, release of the adhesion contacts at the rear of the cell (2-4). Cell migration is, thus, governed by a succession of adhesion and de-adhesion steps, and a balance between these two processes is required for an optimal cell mo...

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Epidermal growth factor induces acute matrix contraction and subsequent calpain‐modulated relaxation

Cited by 37 publications

References 50 publications

Endothelin-1 Promotes Myofibroblast Induction through the ETA Receptor via a rac/Phosphoinositide 3-Kinase/Akt-dependent Pathway and Is Essential for the Enhanced Contractile Phenotype of Fibrotic Fibroblasts

Endothelin-1 Promotes Myofibroblast Induction through the ETA Receptor via a rac/Phosphoinositide 3-Kinase/Akt-dependent Pathway and Is Essential for the Enhanced Contractile Phenotype of Fibrotic Fibroblasts

Epidermal Growth Factor Induces Fibroblast Contractility and Motility via a Protein Kinase C δ-dependent Pathway

m-calpain Activation Is Regulated by Its Membrane Localization and by Its Binding to Phosphatidylinositol 4,5-Bisphosphate*

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