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β1 Integrin Ligation Stimulates Tyrosine Phosphorylation of Phospholipase Cγ1 and Elevates Intracellular Ca2+in Pancreatic Acinar Cells
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
226, 876–882 (1996)
1443
b1 Integrin Ligation Stimulates Tyrosine Phosphorylation of Phospholipase Cg1 and Elevates Intracellular Ca2/ in Pancreatic Acinar Cells Robert W. Wrenn,*,1 Tony L. Creazzo,* and Lee E. Herman† *Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, Georgia 30912; and †Department of Medicine, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, North Carolina 27103 Received August 20, 1996 We have recently reported increased tyrosine (TYR) phosphorylation of a number of pancreatic acinar cell proteins following antibody ligation of b1 integrins (Wrenn and Herman, Biochem. Biophys. Res. Commun. 208, 1995, 978–984). Concurrent with this TYR phosphorylation was a marked activation of protein kinase C (PKC). This led us to investigate phospholipase Cg1 (PLCg1), a key enzyme responsible for diacylglycerol generation, as a target for integrin-mediated TYR phosphorylation. Staining with antiphosphotyrosine antibodies revealed increased TYR phosphorylation of immunoprecipitated PLCg1 prepared from b1 integrin-ligated acinar cells. Subsequent stripping and reprobing of Western blots with polyclonal anti-PLCg1 was confirmatory. Over this same time period, intracellular [Ca2/] increased from õ100 nM to 600 nM, further suggesting a functional relevance of integrin-linked phosphorylation as a regulatory mechanism in exocrine pancreas. q 1996 Academic Press, Inc.
A universal characteristic of cells is the ability to recognize and respond to signaling molecules in their external milieu. Historically, research has focused on signal transduction across the plasma membrane initiated by hormones, neurotransmitters, and paracrine agents (growth factors, nitric oxide). Recently, it has become apparent that cell responsiveness can be modulated by additional elements of the surrounding environment. Attachment of cells to the extracellular matrix and to neighboring cells through membrane-localized adhesion receptors may well provide a ‘‘context’’ for cell responsiveness [2]. By this mechanism a given tissue may influence how its constituent cells respond to external stimuli. A major family of adhesion receptors, the integrins, mediates cell association to matrix proteins as well as contact with adjacent cells [3]. Integrins comprise a family of integral membrane ab heterodimers that bind multiple ECM components. Some 16 a and 8 b subunits have been identified and although all possible combinations of subunits do not appear to exist, the number of known integrin dimers is large and continually increasing. This heterogeneity is increased by alternative splicing found in the cytoplasmic domains of many integrins [3]. Integrins exhibit considerable overlap in ligand specificity, and many ECM components bind to several integrins. Until very recently, the biological effects of ECM-integrin interactions were assumed to hinge upon physical effects of matrix-receptor binding upon parameters such as cell shape and other direct interactions with the cytoskeleton [2]. The versatility of the integrin family became manifest with the realization that these adhesion receptors are conduits for signal transduction, both from the exterior of the cell to the cytoplasm as well as from the cell interior to the surrounding environment [2,4,5]. Integrins also appear able to transduce signals that regulate gene expression [6,7]. It was initially suggested in 1989 by Nakamura and Yamamura 1
Corresponding author. 876
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[8] and Ferrell and Martin [9] that integrin-mediated adhesion was associated with protein tyrosine (TYR) phosphorylation. This was followed by the finding of increased TYR phosphorylation of a 125 kDa protein in human epidermal carcinoma (KB) cells subsequent to antibody ligation of b1 integrins [10] and in 3T3 cells adhering to fibronectin or anti-b1 integrin IgG [11]. This protein, subsequently identified as focal adhesion kinase (FAK) [12] is itself a tyrosine kinase. It was soon demonstrated that TYR phosphorylation of FAK increased its activity towards its exogenous substrates [11], such as the cytoskeleton-associated protein paxillin [5], suggesting that one consequence of cell-ECM interaction is the organization of a supramolecular cytoskeleton-associated complex that is capable of bringing together components of a signaling cascade. The b1 integrin receptor thus appears intimately coupled functionally with a tyrosine phosphorylating activity, although the cytoplasmic domains of neither subunit have intrinsic enzymatic activity and appear to function by coupling with intracellular proteins to form large complexes containing both cytoskeletal and signaling elements [13]. Work in our laboratory has dealt with signaling pathways activated in the pancreatic acinar cell following ligation of integrins using antibodies to the b1 receptor subunit [1]. The initial steps of this pathway putatively involve a b1 integrin receptor-associated tyrosine kinase exerting its intracellular actions via phosphorylation of several specific proteins. Concurrent with these phosphorylation changes, we reported a marked cytosol-to-membrane translocation of PKCa [1]. In the current work, we present evidence that a primary substrate for b1-integrinmediated TYR phosphorylation in pancreatic acinar cells is phospholipase Cg1 (PLCg1), and further that b1 integrin ligation results in a rapid and sustained increase in intracellular free Ca2/ ([Ca2/] METHODS Materials. Monoclonal antibody to phosphotyrosine (PY20) was from Transduction Laboratories. Monoclonal antiphospholipase Cg1-agarose as well as polyclonal anti-phospholipase Cg1 were obtained from Upstate Biotechnology. Anti-b1 integrin (chick), antimouse IgG (unlabelled and horseradish peroxidase-labelled) and antirabbit IgG (peroxidase-labelled) were from Sigma. Rhodamine-coupled antimouse IgG was from Southern Biotechnology. Acinar cell preparation and b1 integrin receptor ligation. Pancreatic acinar cells were prepared from male Wistar rats by two-step collagenase digestion [14]. b1 integrin receptor ligation was carried out essentially as described by Kornberg et al. [10]. Briefly, dispersed acinar cells were incubated in HEPES-Ringer buffer, pH 7.4, in the presence of mAb to chick b1 integrin (Sigma; 2 ug/ml) for 30 minutes on ice followed by exposure to goat antimouse IgG at 377C for 30 minutes. Control cells received similar treatment, omitting the b1 integrin Ab. Cells were then analyzed as noted with subsequent figures. Western blotting and detection of tyrosine phosphorylated proteins and PLCg. Acinar cells (approx. 5 mg total protein) were sonicated in radioimmune precipitation buffer (RIP; 50 mM Tris, pH 7.4, 150 mM NaCl, 0.5 mM Na3VO4 , 5 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% deoxycholic acid, 20 mg/ml leupeptin, 10 mM PMSF, 100 units/ml aprotinin, 25 mU/ml a2-macroglobulin, 10 mg/ml p-nitrophenylphosphate). Following brief centrifugation (yielding no visible pellet), addition of SDS sample buffer and boiling, samples of the extract (40-50 mg protein/lane) were subjected to SDS-PAGE (10% gel) and blotted to PVDF membranes. Membranes were washed initially with Tris-buffered saline (TBS; 10 mM Tris-Cl, pH 7.2, 150 mM NaCl), blocked with TBS containing 3% calf serum, 1% nonfat dry milk and 0.5% Tween-20 and probed for phosphotyrosine-containing proteins or PLCg1 using the appropriate primary antibody (1:2500 dilution) and horseradish peroxidase-coupled IgG as secondary Ab (1:2000) in blocking buffer, rinsed sequentially (14) with TBS containing 0.5% Tween -20, 0.25% Tween-20 (13) and finally in TBS alone. Reactive proteins in all blots were visualized using chemiluminesce/autoradiography (Amersham ECL). Immunoprecipitation of phospholipase Cg1. Antibody ligation of b1 integrins was carried out as described. Control cells were exposed to only the second antibody. Cells were homogenized in immunoprecipitation buffer (10 mM TrisCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 0.2 mM Na3VO4 , 2 mM PMSF, 10 mg/ ml aprotinin, 0.5% NP-40). Immunoprecipitation of phospholipase Cg1 (PLCg1) was carried out using a mixed monoclonal preparation of anti-PLCg1 coupled to agarose (Upstate Biotechnology). The immunoprecipitate was subjected to SDS-PAGE, Western blotted to PVDF and the membrane probed initially with an antibody to phosphotyrosine (PY20) with visualization using chemiluminescence. The membrane was then stripped and reprobed for PLCg1 using a polyclonal antibody (UBI). Fura 2-AM loading. Acinar cells, prepared as described, were loaded in 1 ml of Ringer solution with 2 mM fura 2-acetoxymethyl ester for 20 min at 37 C in a rotating water bath. The primary antibody (anti-b1 integrin) was added 877 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Clustering of b1 integrins by antibody ligation in pancreatic acinar cells. Integrin ligation was carried out as described (Methods) using monoclonal anti-b1 integrin and rhodamine-conjugated anti-mouse IgG as second antibody. Fluorescently labelled integrins (left panel) were visualized and photographed using appropriate microscopy (Zeiss). Right panel: visible light micrograph of the same field. Results shown are typical of three separate experiments.
and deesterification of the fura 2 carried out for 15 min on ice and an additional 15 min at room temperature. The second antibody was added and single cell changes in fura 2 fluorescence measured. Fluorescence measurements. The epifluorescence setup consisted of a Photon Technology International (PTI) microspectrofluorometer, with dual monochrometers (10nm bandwidth) and projected onto single acinar cells via a 40X oil-immersion objective with a numerical aperture of 1.3 (X40 Fluor; Nikon, Tokyo, Japan) and using a 430 nm dichroic mirror. Fluorescent light was transmitted through a bandpass filter at 510 nm (bandwidth 10 nm) to a PTI photomultiplier attached to the video side port of the microscope. The photomultiplier output was digitized and stored in a microcomputer for later analysis. Fura 2 transients reported are the ratio of fluorescence transients measured at 340 and 380 nm. At the end of the experiment, acinar cells were exposed to 10 mM ionomycin (Ca2/ salt) for determination of Rmax and bmax , and then exposed to 25 mM ethylene glycol-bis (b-aminoethyl ether)-N,N,N*,N*tetraacetic acid (EGTA) for determination of Rmin and bmin . Fura 2 transients were then calibrated in terms of intracellular Ca2/ concentration ([Ca2/]i) by the ratiometric procedure of Grynkiewicz et al. [15] as modified by Brotto and Creazzo [16], using the equation:
[Ca2/]i Å Kd 1 b 1 (R0Rmin)/Rmax0R)
where Kd is dissociation constant for fura 2 (224 nM), b is the ratio of the fluorescence signal at 380 nm of a solution with high Ca2/ (bmax) and low Ca2/ (bmin), R is the observed ratio at 340/380 nm, Rmin is the ratio at 340/380 when the cell is in the presence of EGTA (low Ca2/), and Rmax is the ratio at 340/380 when the cell is in the presence of ionomycin (high Ca2/).
RESULTS
Figure 1 illustrates the clustering of acinar cell b1 integrins following sequential exposures of dispersed cells to anti-b1 integrin primary antibody (monoclonal) and a rhodamine-coupled second antibody (goat anti-mouse IgG). While the figure depicts an essentially maximal response, clustering was noted as soon as 3 minutes following exposure to the second antibody (data not shown). We have recently reported increased phosphorylation on tyrosine (TYR) of several acinar 878
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FIG. 2. SDS-PAGE/Western blot of acinar cell proteins stained for phosphotyrosine or phospholipase Cg1. Control (lane 1) or b1 integrin-ligated (lane 2) acinar cells were sonicated in RIP buffer, processed for SDS-PAGE/Western blotting and stained for phosphotyrosine as described (Methods). An adjacent lane (lane 3) was stained using antiPLCg1 as described (Methods). Result shown is representative of three separate experiments.
cell proteins [1] following antibody ligation of b1 integrins. Figure 2 demonstrates this (lanes 1 and 2). In addition, a companion lane (lane 3) was stained using polyclonal anti-PLCg1, which is shown to comigrate with the largest b1 integrin ligation-dependent TYR phosphorylated protein (approx. 123 kD). To more definitively demonstrate PLCg1 as a substrate for b1-integrin-dependent tyrosine kinase activity, immunoprecipitation of PLCg1 was carried out, using extracts from experimental (b1 integrin-ligated) and control acinar cells using agarose-conjugated monoclonal antiPLCg1 (Methods). The results depicted in Fig. 3 demonstrate increased TYR phosphorylation of immunoprecipitated PLCg1 following ligation of b1 integrin. Staining with a polyclonal antibody confirmed the immunoprecipitated protein as PLCg1 (Fig. 3; right lane). Recently, Somogyi demonstrated increased acinar cell levels of DG and IP3 , and elevated [Ca2/]i following exposure of cells to soluble type IV collagen [17]. These investigators postulated that the interaction of type IV collagen with b1 integrin might result in increased
FIG. 3. SDS-PAGE/Western blot of phospholipase Cg1 (PLCg1) immunoprecipitate from pancreatic acinar cells stained for phosphotyrosine and subsequently for PLCg1. Ligation of b1 integrins was carried out as described (Methods). Control cells were exposed to only the second antibody. Cells were homogenized as described (Methods) and immunoprecipitation carried out using a mixed monoclonal preparation of anti-PLCg1 coupled to agarose (Upstate Biotechnology; Methods). Following SDS-PAGE/Western blotting to PVDF, the membrane was probed with an antibody to phosphotyrosine as indicated. The membrane was then stripped and reprobed for PLCg1 (as indicated; rightmost lane) using a polyclonal antibody (UBI). Visualization was by chemiluminescence. Results shown are typical of three separate preparations. 879
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FIG. 4. Representative [Ca2/]i response in b1 integrin-ligated and control pancreatic acinar cells. Acinar cells were loaded with fura 2-AM, visualized and integrin ligation carried out (or not) as described (Methods). Single cell changes in fura 2 fluorescence were determined as detailed (Methods). Fluorescence changes in integrin-ligated cells are depicted by the dark solid line, and in control cells by the light broken line. Results shown are typical of four separate experiments.
TYR phosphorylation and activation of PLCg, resulting in altered DG, IP3 and [Ca2/]i . Since our current work directly demonstrates that b1 integrin ligation results in increased TYR phosphorylation (and presumed activation) of PLCg1, we undertook experiments to examine the effects upon acinar cell [Ca2/]i of integrin binding. As shown in Fig. 4, following addition of the second (ligating) antibody just prior to timeÅ0, [Ca2/]i rapidly increased from resting levels (approximately 70-150 nM) to over 200 nM within one minute and continued to rise, reaching a plateau of approximately 550 nM-600 nM within 10 min. DISCUSSION
The work described presents additional significant information concerning the nature of a signal transduction system in exocrine pancreas linked to the integrin family of cell surface receptors, the existence of which we recently reported [1]. b1 integrin receptors have been demonstrated in the human pancreas to be localized to the acini and exocrine ducts, but were not noted in islets [18]. Other integrin subtypes noted in the same study included a2, a3, a6, aV, b4 and b5 [18]. Differing subtype patterns were noted in pancreatic tumors [18]. In the current work we identify a major substrate protein of this b1 integrin-linked TYR phosphorylation system as phospholipase Cg1 (PLCg1), and further report that activation of this signaling pathway by antibody ligation of b1 integrin leads to a rapid and sustained increase in cytosolic [Ca2/]i , initiated presumably by increased PLCg1-generated inositol trisphosphate (IP3) In the inositol lipid/Ca2/ signaling process, there are two major receptor-mediated pathways for stimulating formation of 1,2-diglyceride (DG) and IP3 . In the first, G-protein-linked receptors activate members of the PLCb family, as is thought to occur in the case of the muscarinic cholinergic receptor. In the second, members of the PLCg family are stimulated by TYR kinase-linked receptors [19]. Increased PLCg1 activity has been shown subsequent to its TYR phosphorylation following activation of lymphocyte function-association antigens and some growth factor receptors [20]. Extracellular matrix binding has also been indicated to affect the 880
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PI cycle through PLC activation [21]. As shown in Fig. 3, the major anti-PLCg1 immunoprecipitable band was heavily phosphorylated on TYR following b1 integrin ligation, indicating PLCg1 as a substrate for the integrin-linked TYR kinase system. It may also be suggested that PLCg1 activity may be regulated by this pathway. Activated PLCg would then catalyze the hydrolysis of phosphatidylinositol-bisphosphate (PIP2) to DG and IP3 . The DG formed would activate specific protein kinase C isoforms, initiating associated SER/THR phosphorylation cascades, while IP3 acted to mobilize intracellular sequestered Ca2/, elevating cytosolic levels of the ion ([Ca2/]i). Our current findings, as well as our recent report linking activation of integrin-dependent TYR phosphorylation in acinar cells with translocation and activation of PKCa in these cells [1], are consistent with activated PLCg1. The observed changes in acinar cell [Ca2/]i following b1 integrin ligation (Fig. 4) are typical of those seen in non-excitable cells following challenge with a Ca2/-mobilizing agonist, consisting of a rapid [Ca2/]i increase as the ion is released from intracellular stores, followed by a sustained plateau due to an influx of extracellular Ca2/ [22]. In line with this reasoning, Somogyi et al. found a lack of effect of verapamil on their observed collagen-induced increase in acinar cell [Ca2/]i , and reported the sustained plateau in elevated [Ca2/]i was prevented by exposure to 2mM extracellular EGTA [17]. It is notable, moreover, that our observed increases in [Ca2/]i (Fig. 4) were 3-4 fold greater in absolute magnitude than those noted by Somogyi in acinar cells following exposure to collagen IV, possibly due to more effective or longerlasting activation of integrin-linked signaling pathways by antibody clustering. The b1 integrin-mediated pathway provides a basis for insight into how surrounding matrix constituents influence cellular activity directly and via interaction with other established signaling systems. We are currently directly assessing activity of PLCg1 in acinar cells in the presence and absence of activating conditions for b1 integrin. We have also begun comparison of the effects of ligation of various integrins present in acinar cells. In studies currently ongoing, we have noted divergent proteins phosphorylated on TYR following ligation of a3, b1 and b5 integrins in acinar cells (data not shown). The demonstration of b1 integrin-mediated tyrosine phosphorylation of PLCg1, taken in conjunction with the observed translocation/activation of PKC following b1 integrin ligation, offers new insights into the complex signaling network in the acinar cell. Further studies will investigate the potential role of integrin-mediated signal transduction in the maintenance and regulation of pancreatic exocrine secretory function. ACKNOWLEDGMENTS The authors gratefully acknowledge the excellent technical assistance of Jarrett Burch and Judy McCoy.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Wrenn, R. W., and Herman, L. E. (1995) Biochem. Biophys. Res. Commun. 208, 978–984. Nathan, C., and Sporn, M. (1991) J. Cell. Biol. 113, 981–986. Hynes, R. O. (1992) Cell 69, 11–25. Schwartz, M. A. (1994) in Integrins: Molecular and Biological Responses to the Extracellular Matrix (Cheresh, D. A., and Mecham, R. P., Eds.), pp. 33–47. Academic Press, San Diego. Clark, E. A., and Brugge, J. S. (1995) Science 268, 233–239. Damsky, C. H., and Werb, Z. (1992) Curr. Opin. Cell Biol. 4, 772–781. Rosales, C., O’Brien, V., Kornberg, L., and Juliano, R. (1995) Biochim. Biophys. Acta 1242, 77–98. Nakamura, S. I., and Yamamura, H. (1989) J. Biol. Chem. 264, 7089–7091. Ferrell, J. E., and Martin, G. S. (1989) Proc. Natl. Acad. Sci. USA 86, 2234–2238. Kornberg, L. J., Earp, H. S., Turner, C. E., Prockop, C., and Juliano, R. L. (1991) Proc. Natl. Acad. Sci. USA 88, 8392–8396. Guan, J.-L., Trevithick, J. E., and Hynes, R. O. (1991) Cell Regulation 2, 951. Kornberg, L. J., Earp, H. S., Parsons, J. T., Schaller, M., and Juliano, R. L. (1992) J. Biol. Chem. 267, 23439– 23442. 881
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13. 14. 15. 16. 17. 18. 19. 20.
Sastry, S. K., and Horwitz, A. F. (1993) Curr. Opin. Cell Biol. 5, 819–831. Gardner, J. D., and Jackson, M. J. (1977) J. Physiol. 270, 439–454. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440–3450. Brotto, M. A. D.-P., and Creazzo, T. L. (1996) Am. J. Physiol. 270, H518–H525. Somogyi, L., Lasic, Z., Vukicevic, S., and Banfic, H. (1994) Biochem. J. 299, 603–611. Hall, P. A., Coates, P., Lemoine, N. R., and Horton, M. A. (1991) J. Pathology 165, 33–41. Berridge, M. J. (1993) Nature (London) 361, 315–325. Kanner, S. B., Grosmaire, L. S., Ledbetter, J. A., and Damle, N. K. (1993) Proc. Natl. Acad. Sci. USA 90, 7099– 7103. 21. Cybulsky, A. V., Carbonetto, S., Cyr, M-D., McTavish, A. J., and Huang, Q. (1993) Am. J. Physiol. 264, C323– C332. 22. Berridge, M. J., and Irvine, R. F. (1989) Nature (London) 341, 204.
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226, 876–882 (1996)
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b1 Integrin Ligation Stimulates Tyrosine Phosphorylation of Phospholipase Cg1 and Elevates Intracellular Ca2/ in Pancreatic Acinar Cells Robert W. Wrenn,*,1 Tony L. Creazzo,* and Lee E. Herman† *Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, Georgia 30912; and †Department of Medicine, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, North Carolina 27103 Received August 20, 1996 We have recently reported increased tyrosine (TYR) phosphorylation of a number of pancreatic acinar cell proteins following antibody ligation of b1 integrins (Wrenn and Herman, Biochem. Biophys. Res. Commun. 208, 1995, 978–984). Concurrent with this TYR phosphorylation was a marked activation of protein kinase C (PKC). This led us to investigate phospholipase Cg1 (PLCg1), a key enzyme responsible for diacylglycerol generation, as a target for integrin-mediated TYR phosphorylation. Staining with antiphosphotyrosine antibodies revealed increased TYR phosphorylation of immunoprecipitated PLCg1 prepared from b1 integrin-ligated acinar cells. Subsequent stripping and reprobing of Western blots with polyclonal anti-PLCg1 was confirmatory. Over this same time period, intracellular [Ca2/] increased from õ100 nM to 600 nM, further suggesting a functional relevance of integrin-linked phosphorylation as a regulatory mechanism in exocrine pancreas. q 1996 Academic Press, Inc.
A universal characteristic of cells is the ability to recognize and respond to signaling molecules in their external milieu. Historically, research has focused on signal transduction across the plasma membrane initiated by hormones, neurotransmitters, and paracrine agents (growth factors, nitric oxide). Recently, it has become apparent that cell responsiveness can be modulated by additional elements of the surrounding environment. Attachment of cells to the extracellular matrix and to neighboring cells through membrane-localized adhesion receptors may well provide a ‘‘context’’ for cell responsiveness [2]. By this mechanism a given tissue may influence how its constituent cells respond to external stimuli. A major family of adhesion receptors, the integrins, mediates cell association to matrix proteins as well as contact with adjacent cells [3]. Integrins comprise a family of integral membrane ab heterodimers that bind multiple ECM components. Some 16 a and 8 b subunits have been identified and although all possible combinations of subunits do not appear to exist, the number of known integrin dimers is large and continually increasing. This heterogeneity is increased by alternative splicing found in the cytoplasmic domains of many integrins [3]. Integrins exhibit considerable overlap in ligand specificity, and many ECM components bind to several integrins. Until very recently, the biological effects of ECM-integrin interactions were assumed to hinge upon physical effects of matrix-receptor binding upon parameters such as cell shape and other direct interactions with the cytoskeleton [2]. The versatility of the integrin family became manifest with the realization that these adhesion receptors are conduits for signal transduction, both from the exterior of the cell to the cytoplasm as well as from the cell interior to the surrounding environment [2,4,5]. Integrins also appear able to transduce signals that regulate gene expression [6,7]. It was initially suggested in 1989 by Nakamura and Yamamura 1
Corresponding author. 876
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[8] and Ferrell and Martin [9] that integrin-mediated adhesion was associated with protein tyrosine (TYR) phosphorylation. This was followed by the finding of increased TYR phosphorylation of a 125 kDa protein in human epidermal carcinoma (KB) cells subsequent to antibody ligation of b1 integrins [10] and in 3T3 cells adhering to fibronectin or anti-b1 integrin IgG [11]. This protein, subsequently identified as focal adhesion kinase (FAK) [12] is itself a tyrosine kinase. It was soon demonstrated that TYR phosphorylation of FAK increased its activity towards its exogenous substrates [11], such as the cytoskeleton-associated protein paxillin [5], suggesting that one consequence of cell-ECM interaction is the organization of a supramolecular cytoskeleton-associated complex that is capable of bringing together components of a signaling cascade. The b1 integrin receptor thus appears intimately coupled functionally with a tyrosine phosphorylating activity, although the cytoplasmic domains of neither subunit have intrinsic enzymatic activity and appear to function by coupling with intracellular proteins to form large complexes containing both cytoskeletal and signaling elements [13]. Work in our laboratory has dealt with signaling pathways activated in the pancreatic acinar cell following ligation of integrins using antibodies to the b1 receptor subunit [1]. The initial steps of this pathway putatively involve a b1 integrin receptor-associated tyrosine kinase exerting its intracellular actions via phosphorylation of several specific proteins. Concurrent with these phosphorylation changes, we reported a marked cytosol-to-membrane translocation of PKCa [1]. In the current work, we present evidence that a primary substrate for b1-integrinmediated TYR phosphorylation in pancreatic acinar cells is phospholipase Cg1 (PLCg1), and further that b1 integrin ligation results in a rapid and sustained increase in intracellular free Ca2/ ([Ca2/] METHODS Materials. Monoclonal antibody to phosphotyrosine (PY20) was from Transduction Laboratories. Monoclonal antiphospholipase Cg1-agarose as well as polyclonal anti-phospholipase Cg1 were obtained from Upstate Biotechnology. Anti-b1 integrin (chick), antimouse IgG (unlabelled and horseradish peroxidase-labelled) and antirabbit IgG (peroxidase-labelled) were from Sigma. Rhodamine-coupled antimouse IgG was from Southern Biotechnology. Acinar cell preparation and b1 integrin receptor ligation. Pancreatic acinar cells were prepared from male Wistar rats by two-step collagenase digestion [14]. b1 integrin receptor ligation was carried out essentially as described by Kornberg et al. [10]. Briefly, dispersed acinar cells were incubated in HEPES-Ringer buffer, pH 7.4, in the presence of mAb to chick b1 integrin (Sigma; 2 ug/ml) for 30 minutes on ice followed by exposure to goat antimouse IgG at 377C for 30 minutes. Control cells received similar treatment, omitting the b1 integrin Ab. Cells were then analyzed as noted with subsequent figures. Western blotting and detection of tyrosine phosphorylated proteins and PLCg. Acinar cells (approx. 5 mg total protein) were sonicated in radioimmune precipitation buffer (RIP; 50 mM Tris, pH 7.4, 150 mM NaCl, 0.5 mM Na3VO4 , 5 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% deoxycholic acid, 20 mg/ml leupeptin, 10 mM PMSF, 100 units/ml aprotinin, 25 mU/ml a2-macroglobulin, 10 mg/ml p-nitrophenylphosphate). Following brief centrifugation (yielding no visible pellet), addition of SDS sample buffer and boiling, samples of the extract (40-50 mg protein/lane) were subjected to SDS-PAGE (10% gel) and blotted to PVDF membranes. Membranes were washed initially with Tris-buffered saline (TBS; 10 mM Tris-Cl, pH 7.2, 150 mM NaCl), blocked with TBS containing 3% calf serum, 1% nonfat dry milk and 0.5% Tween-20 and probed for phosphotyrosine-containing proteins or PLCg1 using the appropriate primary antibody (1:2500 dilution) and horseradish peroxidase-coupled IgG as secondary Ab (1:2000) in blocking buffer, rinsed sequentially (14) with TBS containing 0.5% Tween -20, 0.25% Tween-20 (13) and finally in TBS alone. Reactive proteins in all blots were visualized using chemiluminesce/autoradiography (Amersham ECL). Immunoprecipitation of phospholipase Cg1. Antibody ligation of b1 integrins was carried out as described. Control cells were exposed to only the second antibody. Cells were homogenized in immunoprecipitation buffer (10 mM TrisCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 0.2 mM Na3VO4 , 2 mM PMSF, 10 mg/ ml aprotinin, 0.5% NP-40). Immunoprecipitation of phospholipase Cg1 (PLCg1) was carried out using a mixed monoclonal preparation of anti-PLCg1 coupled to agarose (Upstate Biotechnology). The immunoprecipitate was subjected to SDS-PAGE, Western blotted to PVDF and the membrane probed initially with an antibody to phosphotyrosine (PY20) with visualization using chemiluminescence. The membrane was then stripped and reprobed for PLCg1 using a polyclonal antibody (UBI). Fura 2-AM loading. Acinar cells, prepared as described, were loaded in 1 ml of Ringer solution with 2 mM fura 2-acetoxymethyl ester for 20 min at 37 C in a rotating water bath. The primary antibody (anti-b1 integrin) was added 877 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Clustering of b1 integrins by antibody ligation in pancreatic acinar cells. Integrin ligation was carried out as described (Methods) using monoclonal anti-b1 integrin and rhodamine-conjugated anti-mouse IgG as second antibody. Fluorescently labelled integrins (left panel) were visualized and photographed using appropriate microscopy (Zeiss). Right panel: visible light micrograph of the same field. Results shown are typical of three separate experiments.
and deesterification of the fura 2 carried out for 15 min on ice and an additional 15 min at room temperature. The second antibody was added and single cell changes in fura 2 fluorescence measured. Fluorescence measurements. The epifluorescence setup consisted of a Photon Technology International (PTI) microspectrofluorometer, with dual monochrometers (10nm bandwidth) and projected onto single acinar cells via a 40X oil-immersion objective with a numerical aperture of 1.3 (X40 Fluor; Nikon, Tokyo, Japan) and using a 430 nm dichroic mirror. Fluorescent light was transmitted through a bandpass filter at 510 nm (bandwidth 10 nm) to a PTI photomultiplier attached to the video side port of the microscope. The photomultiplier output was digitized and stored in a microcomputer for later analysis. Fura 2 transients reported are the ratio of fluorescence transients measured at 340 and 380 nm. At the end of the experiment, acinar cells were exposed to 10 mM ionomycin (Ca2/ salt) for determination of Rmax and bmax , and then exposed to 25 mM ethylene glycol-bis (b-aminoethyl ether)-N,N,N*,N*tetraacetic acid (EGTA) for determination of Rmin and bmin . Fura 2 transients were then calibrated in terms of intracellular Ca2/ concentration ([Ca2/]i) by the ratiometric procedure of Grynkiewicz et al. [15] as modified by Brotto and Creazzo [16], using the equation:
[Ca2/]i Å Kd 1 b 1 (R0Rmin)/Rmax0R)
where Kd is dissociation constant for fura 2 (224 nM), b is the ratio of the fluorescence signal at 380 nm of a solution with high Ca2/ (bmax) and low Ca2/ (bmin), R is the observed ratio at 340/380 nm, Rmin is the ratio at 340/380 when the cell is in the presence of EGTA (low Ca2/), and Rmax is the ratio at 340/380 when the cell is in the presence of ionomycin (high Ca2/).
RESULTS
Figure 1 illustrates the clustering of acinar cell b1 integrins following sequential exposures of dispersed cells to anti-b1 integrin primary antibody (monoclonal) and a rhodamine-coupled second antibody (goat anti-mouse IgG). While the figure depicts an essentially maximal response, clustering was noted as soon as 3 minutes following exposure to the second antibody (data not shown). We have recently reported increased phosphorylation on tyrosine (TYR) of several acinar 878
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FIG. 2. SDS-PAGE/Western blot of acinar cell proteins stained for phosphotyrosine or phospholipase Cg1. Control (lane 1) or b1 integrin-ligated (lane 2) acinar cells were sonicated in RIP buffer, processed for SDS-PAGE/Western blotting and stained for phosphotyrosine as described (Methods). An adjacent lane (lane 3) was stained using antiPLCg1 as described (Methods). Result shown is representative of three separate experiments.
cell proteins [1] following antibody ligation of b1 integrins. Figure 2 demonstrates this (lanes 1 and 2). In addition, a companion lane (lane 3) was stained using polyclonal anti-PLCg1, which is shown to comigrate with the largest b1 integrin ligation-dependent TYR phosphorylated protein (approx. 123 kD). To more definitively demonstrate PLCg1 as a substrate for b1-integrin-dependent tyrosine kinase activity, immunoprecipitation of PLCg1 was carried out, using extracts from experimental (b1 integrin-ligated) and control acinar cells using agarose-conjugated monoclonal antiPLCg1 (Methods). The results depicted in Fig. 3 demonstrate increased TYR phosphorylation of immunoprecipitated PLCg1 following ligation of b1 integrin. Staining with a polyclonal antibody confirmed the immunoprecipitated protein as PLCg1 (Fig. 3; right lane). Recently, Somogyi demonstrated increased acinar cell levels of DG and IP3 , and elevated [Ca2/]i following exposure of cells to soluble type IV collagen [17]. These investigators postulated that the interaction of type IV collagen with b1 integrin might result in increased
FIG. 3. SDS-PAGE/Western blot of phospholipase Cg1 (PLCg1) immunoprecipitate from pancreatic acinar cells stained for phosphotyrosine and subsequently for PLCg1. Ligation of b1 integrins was carried out as described (Methods). Control cells were exposed to only the second antibody. Cells were homogenized as described (Methods) and immunoprecipitation carried out using a mixed monoclonal preparation of anti-PLCg1 coupled to agarose (Upstate Biotechnology; Methods). Following SDS-PAGE/Western blotting to PVDF, the membrane was probed with an antibody to phosphotyrosine as indicated. The membrane was then stripped and reprobed for PLCg1 (as indicated; rightmost lane) using a polyclonal antibody (UBI). Visualization was by chemiluminescence. Results shown are typical of three separate preparations. 879
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FIG. 4. Representative [Ca2/]i response in b1 integrin-ligated and control pancreatic acinar cells. Acinar cells were loaded with fura 2-AM, visualized and integrin ligation carried out (or not) as described (Methods). Single cell changes in fura 2 fluorescence were determined as detailed (Methods). Fluorescence changes in integrin-ligated cells are depicted by the dark solid line, and in control cells by the light broken line. Results shown are typical of four separate experiments.
TYR phosphorylation and activation of PLCg, resulting in altered DG, IP3 and [Ca2/]i . Since our current work directly demonstrates that b1 integrin ligation results in increased TYR phosphorylation (and presumed activation) of PLCg1, we undertook experiments to examine the effects upon acinar cell [Ca2/]i of integrin binding. As shown in Fig. 4, following addition of the second (ligating) antibody just prior to timeÅ0, [Ca2/]i rapidly increased from resting levels (approximately 70-150 nM) to over 200 nM within one minute and continued to rise, reaching a plateau of approximately 550 nM-600 nM within 10 min. DISCUSSION
The work described presents additional significant information concerning the nature of a signal transduction system in exocrine pancreas linked to the integrin family of cell surface receptors, the existence of which we recently reported [1]. b1 integrin receptors have been demonstrated in the human pancreas to be localized to the acini and exocrine ducts, but were not noted in islets [18]. Other integrin subtypes noted in the same study included a2, a3, a6, aV, b4 and b5 [18]. Differing subtype patterns were noted in pancreatic tumors [18]. In the current work we identify a major substrate protein of this b1 integrin-linked TYR phosphorylation system as phospholipase Cg1 (PLCg1), and further report that activation of this signaling pathway by antibody ligation of b1 integrin leads to a rapid and sustained increase in cytosolic [Ca2/]i , initiated presumably by increased PLCg1-generated inositol trisphosphate (IP3) In the inositol lipid/Ca2/ signaling process, there are two major receptor-mediated pathways for stimulating formation of 1,2-diglyceride (DG) and IP3 . In the first, G-protein-linked receptors activate members of the PLCb family, as is thought to occur in the case of the muscarinic cholinergic receptor. In the second, members of the PLCg family are stimulated by TYR kinase-linked receptors [19]. Increased PLCg1 activity has been shown subsequent to its TYR phosphorylation following activation of lymphocyte function-association antigens and some growth factor receptors [20]. Extracellular matrix binding has also been indicated to affect the 880
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PI cycle through PLC activation [21]. As shown in Fig. 3, the major anti-PLCg1 immunoprecipitable band was heavily phosphorylated on TYR following b1 integrin ligation, indicating PLCg1 as a substrate for the integrin-linked TYR kinase system. It may also be suggested that PLCg1 activity may be regulated by this pathway. Activated PLCg would then catalyze the hydrolysis of phosphatidylinositol-bisphosphate (PIP2) to DG and IP3 . The DG formed would activate specific protein kinase C isoforms, initiating associated SER/THR phosphorylation cascades, while IP3 acted to mobilize intracellular sequestered Ca2/, elevating cytosolic levels of the ion ([Ca2/]i). Our current findings, as well as our recent report linking activation of integrin-dependent TYR phosphorylation in acinar cells with translocation and activation of PKCa in these cells [1], are consistent with activated PLCg1. The observed changes in acinar cell [Ca2/]i following b1 integrin ligation (Fig. 4) are typical of those seen in non-excitable cells following challenge with a Ca2/-mobilizing agonist, consisting of a rapid [Ca2/]i increase as the ion is released from intracellular stores, followed by a sustained plateau due to an influx of extracellular Ca2/ [22]. In line with this reasoning, Somogyi et al. found a lack of effect of verapamil on their observed collagen-induced increase in acinar cell [Ca2/]i , and reported the sustained plateau in elevated [Ca2/]i was prevented by exposure to 2mM extracellular EGTA [17]. It is notable, moreover, that our observed increases in [Ca2/]i (Fig. 4) were 3-4 fold greater in absolute magnitude than those noted by Somogyi in acinar cells following exposure to collagen IV, possibly due to more effective or longerlasting activation of integrin-linked signaling pathways by antibody clustering. The b1 integrin-mediated pathway provides a basis for insight into how surrounding matrix constituents influence cellular activity directly and via interaction with other established signaling systems. We are currently directly assessing activity of PLCg1 in acinar cells in the presence and absence of activating conditions for b1 integrin. We have also begun comparison of the effects of ligation of various integrins present in acinar cells. In studies currently ongoing, we have noted divergent proteins phosphorylated on TYR following ligation of a3, b1 and b5 integrins in acinar cells (data not shown). The demonstration of b1 integrin-mediated tyrosine phosphorylation of PLCg1, taken in conjunction with the observed translocation/activation of PKC following b1 integrin ligation, offers new insights into the complex signaling network in the acinar cell. Further studies will investigate the potential role of integrin-mediated signal transduction in the maintenance and regulation of pancreatic exocrine secretory function. ACKNOWLEDGMENTS The authors gratefully acknowledge the excellent technical assistance of Jarrett Burch and Judy McCoy.
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