INTRODUCTION Adhesion to extracellular matrix (ECM) proteins initiates a wide range of signaling events (Clark and Brugge, 1995; Edwards and Streuli, 1999; Giancotti and Ruoslahti, 1999; Schwartz et al., 1995), many of which are also triggered or affected by growth factor (GF) stimulation (Schwartz and Baron, 1999; van der Geer and Hunter, 1994). Moreover, this interconnectivity between ECM- and GF-mediated signals extends to adhesion-mediated structural responses such as the formation of cortical actin structures (Aplin and Juliano, 1999) or focal adhesion complexes, in which not only clustered integrin adhesion receptors, but also growth factor receptors, are found (Miyamoto et al., 1996; Plopper et al., 1995). This high degree of signaling crosstalk gives rise to joint regulation of numerous cell responses by the ECM and soluble factors (Giancotti and Ruoslahti, 1999; Sastry and Horwitz, 1996). A major example of such coregulation occurs in cell cycle progression, in which most normal cells require adhesion to proliferate even in the presence of soluble mitogens. Deregulation of adhesion dependence results in uncontrolled cell proliferation and pathologies such as tumor formation. Thus, better understanding of how signaling events initiated by both ECM and GFs regulate cell cycle progression may offer potential strategies for the development of therapeutics. One signal implicated in GF-mediated regulation of proliferation involves the extracellular signal-regulated kinases 1/2 (ERK1/2) belonging to the mitogen-activated protein (MAP) kinase family (Lewis et al., 1998; Pages et al., 1993). Since these kinases are also activated in response to adhesion to ECM proteins (Chen et al., 1994; Morino et al., 1995), signaling crosstalk at ERK1/2 has been suspected to play an important role in the joint regulation of cell cycle progression by cell adhesion and GFs. In fact, cell adhesion to fibronectin (Fn) has been shown to enhance GF-mediated activation of ERK1/2 (Lin et al., 1997; Miyamoto et al., 1996; Moro et al., 1998; Renshaw et al., 1997), providing a possible mechanism by which adhesion stimulates GF-mediated proliferation. However, this hypothesis remains to be tested quantitatively by examining the relationship between the degree of ERK1/2 signal enhancement and the level of adhesion-dependent cell cycle progression. Does the magnitude of ERK1/2 activation 4499 Journal of Cell Science 113, 4499-4510 (2000) Printed in Great Britain © The Company of Biologists Limited JCS1997 Both the extracellular matrix and growth factors jointly regulate cell cycle progression via a complex network of signaling pathways. Applying quantitative assays and analysis, we demonstrate here that concurrent stimulation of Chinese hamster ovary (CHO) cells with fibronectin (Fn) and insulin elicits a DNA synthesis response that reveals a synergy far more complex than a simple additive enhancement of response magnitude. CHO cell adhesion to higher Fn density shifts the sensitivity of the DNA synthesis response to insulin concentration from smoothly graded to sharply ‘switch-like’ and dramatically decreases the insulin concentration required for half-maximal response by about 1000-fold. Conversely, treatment with insulin has a milder and less complex effect on the response to varying Fn concentrations. Governing this DNA synthesis response is a common requirement for a transient, cell area- independent extracellular signal-regulated kinase 2 (ERK2) signal. Moreover, we show that the time-integrated value of this ‘pulse’ signal provides an appropriate metric for quantifying the dependence of DNA synthesis on the degree of ERK2 activation. Indeed, in the absence of insulin, the adhesion-mediated response is linearly proportional to ERK2 activation over a broad range of stimulatory Fn and MEK inhibitor amounts. However, in the presence of both Fn and insulin, total integrated ERK2 activity (the sum of Fn- and insulin-mediated signals) no longer serves as a predictor of DNA synthesis, demonstrating that the signaling crosstalk underlying response synergism does not converge at ERK2 activation. Instead, adhesion to higher Fn density enhances insulin stimulation of DNA synthesis, not by increasing insulin-mediated ERK2 activation, but via parallel elevation of at least one other insulin-mediated signal such as IRS-1 phosphorylation. Key words: Adhesion, Cell cycle, DNA synthesis, Fibronectin, Insulin, ERK2 SUMMARY The role of transient ERK2 signals in fibronectin- and insulin-mediated DNA synthesis Anand R. Asthagiri 1, *, Cynthia A. Reinhart 2 , Alan F. Horwitz 4 and Douglas A. Lauffenburger 1,3 1 Department of Chemical Engineering, 2 Department of Biology and 3 Division of Bioengineering and Environmental Health, Cancer Research Center, Massachusetts Institute of Technology, Cambridge, MA, USA 4 Department of Cell Biology, University of Virginia, Charlottesville, VA, USA *Author for correspondence at present address: Department of Cell Biology, Harvard Medical School, Boston, MA, USA (e-mail: anand_asthagiri@hms.harvard.edu) Accepted 6 October; published on WWW 16 November 2000