Stimulation of 131 Integrins on Fibroblasts Induces PDGF Independent Tyrosine Phosphorylation of PDGF [3-Receptors

We report that integrin-mediated signaling induces a rapid and transient tyrosine phosphorylation of platelet-derived growth factor (PDGF) 13-receptors in human diploid foreskin AG 1518 fibroblasts. A transient tyrosine phosphorylation of PDGF i3-receptors was evident one and two hours after cells had been plated on collagen type I and fibronectin, as well as on immobilized anti-integrin subunit IgG, but not on polyL-lysine. In contrast EGF or PDGF a-receptors were not phosphorylated on tyrosine residues under these conditions. Tyrosine phosphorylation of PDGF 13-receptors induced by plating on collagen type I was inhibited by cytochalasin D and herbimycin A, unaffected by cycloheximide and enhanced by orthovanadate. Furthermore, a transient phosphorylation of PDGF 13-receptors occurred when AG 1518 fibroblasts were cultured in three-dimensional collagen lattices or exposed to external strain exerted through centrifugation. The latter effect was evident already after two minutes. Clustering of cell surface 131 integrins led to PDGF ~-receptor phosphorylation both in suspended and firmly attached AG 1518 fibroblasts. Plating of cells on collagen type I, fibronectin, and anti-i3rintegrin IgG resulted in the formation of PDGF 13-receptor aggregates as detected by immunofluorescence. Suramin or anti-PDGF-BB IgG had no effect on the plating-induced tyrosine phosphorylation of PDGF 13-receptors. PDGF-B chain mRNA, or protein, were not detected in AG 1518 fibroblasts. Our data suggest that a ligand-independent PDGF 13-receptor activation during cell adhesion and early phases of cell spreading is involved in integrin-mediated signaling in fibroblasts, and constitutes parts of a mechanism for cells to respond during the dynamic phases of externally applied tension as well as fibroblast-mediated tension during cell adhesion and collagen gel contraction. C ELL adhesion to extracellular matrix (ECM) 1 regulates a plethora of cellular activities (6, 35, 68). Adhesion of cells to ECM components depends on a set of transmembrane receptors belonging to the integrin family. Integrins are heterodimers of non-covalently associated a and ~ subunits (2, 29, 32, 51), which link the ECM with the cytoskeleton, and act as signal transducing receptors (13, 16, 19, 35, 44, 55). In many types of cells, integrin stimulation by ligand occupation or clustering by antibodies elicit changes in the intracellular pH, cytoplasmic-free calcium concentration, phosphoinositide synthesis, protein tyrosine phosphorylation, and expression of certain genes. Proteins that are tyrosine phosphorylated in response to integrin-mediated adhesion include the focal adhesion kinase (p125 FAK) (9, 21, 25, 38, 53), paxillin (9), and the MAP kinases, ERK-1 and ERK-2 (72). Many of the responses elicited by integrin-mediated adhesion are also evoked by activation of the PDGF 13-receptors (7, 11, Address all correspondence to K. Rubin, Department of Medical and Physiological Chemistry, University of Uppsala, BMC Box 575, S-751 23 Uppsala, Sweden. Ph.: (46) 18 17 41 16. Fax: (46) 18 17 48 75. 1. Abbrevia t ions used in this paper. ECM, extracellular matrix; PAE, porcine aortic endothelium. 71). Thus, PDGF stimulation leads to tyrosine phosphorylation of p125 FAK and paxillin, as well as MAP kinases (11, 48) The role of PDGF B-receptors, if any, in integrininduced signaling, is not known. In addition to its well-known growth-promoting activities, PDGF stimulates several processes that depend on integrin activity. Examples are PDGF stimulated and 0t2131-mediated chemotaxis through collagen type I--coated membranes (61) and fibroblast-mediated contraction of threedimensional collagen lattices (14, 24, 37, 42, 54). Conversely, PDGF 13-receptors become functionally refractory in fibroblasts that have contracted a collagen gel (43, 64). PDGF specifically stimulates the synthesis of the collagenbinding integrin et2131 (1) and increases the apparent activity of 131 integrins in fibroblasts (24). Thus, several experimental observations strongly suggest that PDGF 13-receptors modulate, and are modulated by, cellular processes dependent on integrin activity. Integrins are concentrated and co-localize with several cytoskeletal components at focal adhesion sites (8, 57, 67). Within focal adhesions, cytoskeletal components, as well as signaling molecules concentrate, and are believed to serve as regulatory complexes for integrin signaling. Stimulation of fibroblasts with PDGF induces a rapid and tran© The Rockefeller University Press, 0021-9525196/02H41112 $2.00 The Journal of Cell Biology, Volume 132, Number 4, February 1996 741-752 741 on July 9, 2017 jcb.rress.org D ow nladed fom sient change of the cytoskeletal structure, involving the formation of membrane and circular ruffles (45). Stimulation with this growth factor also results in a redistribution, as well as a change in the phosphorylation pattern of the focal adhesion protein vinculin (30), a process dependent on changes in phosphoinositide turnover (18). It has been suggested that integrins can function as mechanochemical transducers conveying strain from the extracellular matrix to the cytoskeleton leading to effects on the cell signaling machinery (reviewed in 33, 56). In a recent communication, Wang et al. (69) demonstrated that integrin-mediated adhesions will restrain external force applied to the structural components linked to the cell surface via integrins. Given the background that integrins transduce chemical signals that converge with responses elicited by the PDGF g-receptor, and that the latter in turn influence integrin activity, it is reasonable to propose an involvement of PDGF 13-receptors in the mechanochemical transducing properties of integrins. Here we report that integrins and integrin-mediated adhesion processes induce an early and transient phosphorylation and internalization of PDGF 13-receptors in fibroblasts. These processes were independent of PDGF. The PDGF 13-receptor autophosphorylation was stimulated by increases in mechanical tension or stress exerted on the cells. Thus our findings suggest that autophosphorylation of PDGF 13-receptors constitutes parts of a mechanism for cells to respond during the dynamic phases of externally applied tension, as well as fibroblast-mediated tension during cell adhesion and collagen gel contraction. Furthermore, the data are compatible with that PDGF 13-receptor activation during cell adhesion and early phases of cell spreading may be a component in integrin-induced signaling in fibroblasts. Materials and Methods Antibodies and Other Reagents The characteristics of the PDGF 13-receptor specific mouse mAb, PDGFR-B2, have been described elsewhere (28, 52). PDGFR-B2 was used at a concentration of I ~g/ml (49, 65). Polyclonal rabbit anti-PDGFBB IgG (34) was kindly donated by Dr. C.-H. Heldin (Ludwig Institute for Cancer Research, Uppsala, Sweden). The anti-phosphotyrosine mouse mAb (4G10) (15, 36), and polyclonal rabbit anti-human PDGF type B-receptor serum raised against a synthetic peptide corresponding to amino acids 1013-1025 (12) was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). The anti-phosphotyrosine mouse mAb PY20 (20) was purchased from Transduction Laboratories (Lexington, KY). The anti-PDGF a-receptor mAb (anti-PDGF-Ra) (27) was purchased from Genzyme (Cambridge, MA). The anti-human EGF receptor mAb, clone 108, (3) was kindly donated by Dr. Andreas Batzer (New York University Medical Center, New York, NY). The mAb PGF 007, raised toward a synthetic peptide corresponding to amino acid residues 73-97 of the PDGF B-chain was purchased from Mochida Co. (Tokyo, Japan). The PGF 007 antibody specifically recognizes PDGF-AB and PDGF-BB (60). The anti-human fibroblast surface protein mAb (1B10) was purchased from Sigma Chemical Co. (St. Louis, MO). The anti-integrin al-subunit mAb TS2/7 (29) was kindly donated by Dr. Timothy Springer (Boston Blood Center, Boston, MA). The anti-integrin ot2-subunit mAb P1H5; anti-integrin as-subunit mAb P1B3; anti-integrin as-subunit mAb PID6; and the anti-integrin 13rsubunit mAb P4C10 (10, 63, 70) were all kindly donated by Dr. William Carter (Fred Hutchinson Cancer Research Center, Seattle, WA). Polyclonal rabbit anti-integrin 13t-subunit IgG was raised essentially as described (23, 50) with the modifications that purified rat hepatocytes were used as starting material and that collagen-binding proteins were eluted from collagen type I~Sepharose affinity columns by 10 mM EDTA. Cytochalsin D, cyclohexamide, herbimycin A, and sodium orthovanadate were purchased from Sigma Chem. Co. Rabbit anti-mouse IgG antibodies and goat anti-rabbit IgG antibodies were purchased from Sigma Chem. Co. and used at a concentration of 10 Ixg/ml. Affinity purified F(ab)2 fragments of goat anti-mouse immunoglobulins (IgG, IgA, and IgM) was purchased from Cooper Biomedical (Malvern, PA). Biotinylated horse anti-mouse IgG, biotinylated goat anti-rabbit IgG, and Texas red avidin D were from Vector Laboratories (Burlingame, CA). Fluorescein-conjugated goat anti-mouse IgG, and goat anti-rabbit IgG were from Becton and Dickinson (Mountainview, CA) and Sigma Chem. Co., respectively. All antibodies were diluted in PBS (130 mM NaCl, 10 mM Na-phosphate, pH 7.4), and used in optimal concentrations determined after serial dilutions. Recombinant human PDGF-AA and PDGF-BB (47a) was kindly donated by Dr. C.-H. Heldin. EGF was purchased from Sigma Chem. Co. Suramin, an agent which inhibits the binding of several growth factors to their receptors and is able to dissociate receptor-bound growth factors including PDGF (5, 31, 39), was kindly provided by Bayer (Leverkusen, Germany). Human plasma fibronectin was purified according to the method described by Miekka et al. (46). Calf skin collagen type I (Vitrogen 100) was obtained from Celltrix (Palo Alto, CA). DME, MCDB 104 medium used for serum-free cell culture and trypsin-EDTA were obtained from the National Veterinary Institute (Uppsala, Sweden). FBS was obtained from Sera-Lab limited (Sussex, UK).