Requirement for diacylglycerol and protein kinase C in HeLa cell- substratum adhesion and their feedback amplification of arachidonic acid production for optimum cell spreading

JS Chun and BS Jacobson

Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst 01003.

Release of arachidonic acid (AA) and subsequent formation of a lipoxygenase (LOX) metabolite(s) is an obligatory signal to induce spreading of HeLa cells on a gelatin substratum (Chun and Jacobson, 1992). This study characterizes signaling pathways that follow the LOX metabolite(s) formation. Levels of diacylglycerol (DG) increase upon attachment and before cell spreading on a gelatin substratum. DG production and cell spreading are insignificant when phospholipase A2 (PLA2) or LOX is blocked. In contrast, when cells in suspension where PLA2 activity is not stimulated are treated with exogenous AA, DG production is turned on, and inhibition of LOX turns it off. This indicates that the formation of a LOX metabolite(s) from AA released during cell attachment induces the production of DG. Consistent with the DG production is the activation of protein kinase C (PKC) which, as with AA and DG, occurs upon attachment and before cell spreading. Inhibition of AA release and subsequent DG production blocks both PKC activation and cell spreading. Cell spreading is also blocked by the inhibition of PKC with calphostin C or sphingosine. The inhibition of cell spreading induced by blocking AA release is reversed by the direct activation of PKC with phorbol ester. However, the inhibition of cell spreading induced by PKC inhibition is not reversed by exogenously applied AA. In addition, inhibition of PKC does not block AA release and DG production. The data indicate that there is a sequence of events triggered by HeLa cell attachment to a gelatin substratum that leads to the initiation of cell spreading: AA release, a LOX metabolite(s) formation, DG production, and PKC activation. The data also provide evidence indicating that HeLa cell spreading is a cyclic feedback amplification process centered on the production of AA, which is the first messenger produced in the sequence of messengers initiating cell spreading. Both DG and PKC activity that are increased during HeLa cell attachment to a gelatin substratum appear to be involved. DG not only activates PKC, which is essential for cell spreading, but is also hydrolyzed to AA. PKC, which is initially activated as consequence of AA production, also increases more AA production by activating PLA2.

Volume 4, Issue 3, pp. 271-281, 03/01/1993
Copyright © 1993 by The American Society for Cell Biology

This article has been cited by other articles:

				J. A. Green, R. A. Stockton, C. Johnson, and B. S. Jacobson 5-Lipoxygenase and cyclooxygenase regulate wound closure in NIH/3T3 fibroblast monolayers Am J Physiol Cell Physiol, August 1, 2004; 287(2): C373 - C383. [Abstract] [Full Text] [PDF]

				Y. Zhang, K. Chen, Y. Tu, A. Velyvis, Y. Yang, J. Qin, and C. Wu Assembly of the PINCH-ILK-CH-ILKBP complex precedes and is essential for localization of each component to cell-matrix adhesion sites J. Cell Sci., March 14, 2003; 115(24): 4777 - 4786. [Abstract] [Full Text] [PDF]

				P. A. Robinson, S. Brown, M. J. McGrath, I. D. Coghill, R. Gurung, and C. A. Mitchell Skeletal muscle LIM protein 1 regulates integrin-mediated myoblast adhesion, spreading, and migration Am J Physiol Cell Physiol, March 1, 2003; 284(3): C681 - C695. [Abstract] [Full Text] [PDF]

				W. H. Ziegler, U. Tigges, A. Zieseniss, and B. M. Jockusch A Lipid-regulated Docking Site on Vinculin for Protein Kinase C J. Biol. Chem., February 22, 2002; 277(9): 7396 - 7404. [Abstract] [Full Text] [PDF]

				R. A. Stockton and B. S. Jacobson Modulation of Cell-Substrate Adhesion by Arachidonic Acid: Lipoxygenase Regulates Cell Spreading and ERK1/2-inducible Cyclooxygenase Regulates Cell Migration in NIH-3T3 Fibroblasts Mol. Biol. Cell, July 1, 2001; 12(7): 1937 - 1956. [Abstract] [Full Text] [PDF]

				A. Alt, M. Ohba, L. Li, M. Gartsbein, A. Belanger, Mitchell. F. Denning, T. Kuroki, S. H. Yuspa, and T. Tennenbaum Protein Kinase C{{delta}}-mediated Phosphorylation of {{alpha}}6{beta}4 Is Associated with Reduced Integrin Localization to the Hemidesmosome and Decreased Keratinocyte Attachment Cancer Res., June 1, 2001; 61(11): 4591 - 4598. [Abstract] [Full Text] [PDF]

				R. Polanowska-Grabowska and A. R. L. Gear Activation of Protein Kinase C Is Required for the Stable Attachment of Adherent Platelets to Collagen but Is Not Needed for the Initial Rapid Adhesion Under Flow Conditions Arterioscler. Thromb. Vasc. Biol., December 1, 1999; 19(12): 3044 - 3054. [Abstract] [Full Text] [PDF]

				J. C. Adams, J. D. Clelland, G. D.M. Collett, F. Matsumura, S. Yamashiro, and L. Zhang Cell-Matrix Adhesions Differentially Regulate Fascin Phosphorylation Mol. Biol. Cell, December 1, 1999; 10(12): 4177 - 4190. [Abstract] [Full Text]

				C. K. Miranti, S. Ohno, and J. S. Brugge Protein Kinase C Regulates Integrin-induced Activation of the Extracellular Regulated Kinase Pathway Upstream of Shc J. Biol. Chem., April 9, 1999; 274(15): 10571 - 10581. [Abstract] [Full Text] [PDF]

				F. Uberall, K. Hellbert, S. Kampfer, K. Maly, A. Villunger, M. Spitaler, J. Mwanjewe, G. Baier-Bitterlich, G. Baier, and H. H. Grunicke Evidence That Atypical Protein Kinase C-lambda and Atypical Protein Kinase C-zeta Participate in Ras-mediated Reorganization of the F-actin Cytoskeleton J. Cell Biol., February 8, 1999; 144(3): 413 - 425. [Abstract] [Full Text] [PDF]

				J. R. Crawford and B. S. Jacobson Extracellular Calcium Regulates HeLa Cell Morphology during Adhesion to Gelatin: Role of Translocation and Phosphorylation of Cytosolic Phospholipase A2 Mol. Biol. Cell, December 1, 1998; 9(12): 3429 - 3443. [Abstract] [Full Text]

				D. Ilic, E. A.C. Almeida, D. D. Schlaepfer, P. Dazin, S. Aizawa, and C. H. Damsky Extracellular Matrix Survival Signals Transduced by Focal Adhesion Kinase Suppress p53-mediated Apoptosis J. Cell Biol., October 19, 1998; 143(2): 547 - 560. [Abstract] [Full Text] [PDF]

				L. S. Price, J. Leng, M. A. Schwartz, and G. M. Bokoch Activation of Rac and Cdc42 by Integrins Mediates Cell Spreading Mol. Biol. Cell, July 1, 1998; 9(7): 1863 - 1871. [Abstract] [Full Text]

				E.-S. Oh, A. Woods, S.-T. Lim, A. W. Theibert, and J. R. Couchman Syndecan-4 Proteoglycan Cytoplasmic Domain and Phosphatidylinositol 4,5-Bisphosphate Coordinately Regulate Protein Kinase C Activity J. Biol. Chem., April 24, 1998; 273(17): 10624 - 10629. [Abstract] [Full Text] [PDF]

				D. A. Nix and M. C. Beckerle Nuclear-Cytoplasmic Shuttling of the Focal Contact Protein, Zyxin: A Potential Mechanism for Communication between Sites of Cell Adhesion and the Nucleus J. Cell Biol., September 8, 1997; 138(5): 1139 - 1147. [Abstract] [Full Text] [PDF]

				E.-S. Oh, A. Woods, and J. R. Couchman Multimerization of the Cytoplasmic Domain of Syndecan-4 Is Required for Its Ability to Activate Protein Kinase C J. Biol. Chem., May 2, 1997; 272(18): 11805 - 11811. [Abstract] [Full Text] [PDF]

				J. M. Lewis, R. Baskaran, S. Taagepera, M. A. Schwartz, and J. Y. J. Wang Integrin regulation of c-Abl tyrosine kinase activity and cytoplasmic-nuclear transport PNAS, December 24, 1996; 93(26): 15174 - 15179. [Abstract] [Full Text] [PDF]

				J.-S. Chun, M.-J. Ha, and B. S. Jacobson Differential Translocation of Protein Kinase C epsilon during HeLa Cell Adhesion to a Gelatin Substratum J. Biol. Chem., May 31, 1996; 271(22): 13008 - 13012. [Abstract] [Full Text] [PDF]

				T. Udagawa and B. W. McIntyre ADP-ribosylation of the G Protein Rho Inhibits Integrin Regulation of Tumor Cell Growth J. Biol. Chem., May 24, 1996; 271(21): 12542 - 12548. [Abstract] [Full Text] [PDF]

				T. S. Panetti, S. A. Wilcox, C. Horzempa, and P. J. McKeown-Longo alpha(v)beta(5)Integrin Receptor-mediated Endocytosis of Vitronectin Is Protein Kinase C-dependent J. Biol. Chem., August 4, 1995; 270(31): 18593 - 18597. [Abstract] [Full Text] [PDF]

				V. Berthet, V. Rigot, S. Champion, J. Secchi, F. Fouchier, J. Marvaldi, and J. Luis Role of Endoproteolytic Processing in the Adhesive and Signaling Functions of alpha vbeta 5 Integrin J. Biol. Chem., October 20, 2000; 275(43): 33308 - 33313. [Abstract] [Full Text] [PDF]