sign in sign up
<<
Home , Cell adhesion, Integrin

COMMENTARY and Cellular Organization 2553 and proliferation: regulation of cyclin- dependent via cytoplasmic signaling pathways

Martin Alexander Schwartz1,* and Richard K. Assoian2 1Department of Vascular , The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA 2Department of Pharmacology, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, Philadelphia, PA 19104, USA *Author for correspondence (e-mail: [email protected])

Journal of Cell Science 114, 2553-2560 (2001) © The Company of Biologists Ltd

Summary Cell cycle progression in mammalian cells is strictly -dependent signals that are channeled into the regulated by both integrin-mediated adhesion to the regulation of the G1 phase CDKs. Regulation of cyclin D1 and by binding of growth factors to by the ERK pathway may provide a paradigm for their receptors. This regulation is mediated by G1 phase understanding how can determine cell cycle cyclin-dependent kinases (CDKs), which are downstream progression. of signaling pathways under the integrated control of both integrins and receptors. Recent Key words: Tyrosine , Signal transduction, Growth control, advances demonstrate a surprisingly diverse array of Cell adhesion

Introduction Integrin-dependent signaling pathways Proliferation of mammalian cells is tightly regulated by multiple Signal transduction by integrins is initiated by both occupancy environmental influences, primarily adhesion to the and crosslinking of integrins by ECM (Schwartz et al., extracellular matrix (ECM), cell-cell adhesion and soluble 1991; Kornberg et al., 1991; Miyamoto et al., 1995; Shankar factors (i.e. polypeptide growth factors or inhibitors, mitogenic et al., 1993). These events lead to changes in activity of most lipids, inflammatory and ). Of these if not all of the known growth regulatory pathways (reviewed environmental cues, soluble growth factors and integrin- by Giancotti and Ruoslahti, 1999; Cary et al., 1999). The list mediated adhesion are most crucial, loss of adhesion generally is long but includes tyrosine and serine/threonine resulting in complete G1 phase cell cycle arrest (Stoker et al., kinase cascades, small GTPases, inositol lipid pathways and 1968). For susceptible cell types, loss of adhesion leads to other phospholipid cascades. One potentially important aspect (reviewed in Giancotti and Ruoslahti, 1999), which of integrin signaling stems from the fact that stimulation occurs might be regarded as the extreme case in which cell numbers at defined contacts, in contrast to stimulation by soluble decrease rather than remain stable. Conversely, formation and factors, which can occur over the entire cell. Because integrins spread of tumors is closely associated with decreased also cooperate with growth factor receptors to regulate dependence on ECM for growth and survival. Thus, loss of downstream events, localized adhesions have the potential to tumor suppressors or constitutive activation of proto-oncogenes impose a measure of spatial specificity on otherwise global not only leads to elevated growth rates but also enables tumor growth-factor-induced events. This level of regulation is likely cells to colonize inappropriate environments (reviewed by to be important for or tissue organization, but to Schwartz, 1997). Not only are many different integrins required what extent it contributes to cell cycle progression remains to for survival of multicellular organisms, but genetic deletion of be determined. several of the major intracellular proteins that transduce integrin In the context of growth regulation, it is essential to signals also results in early embryonic lethality (Ilic et al., 1995; distinguish transient effects seen in replated cells from Honda et al., 1998). Both integrins and growth factor receptors sustained effects in stably adherent cells. Investigators have use multiple cytoplasmic signaling pathways to regulate G1 often studied integrin signaling by plating suspended cells on phase cyclins and associated kinases that determine cell cycle ECM proteins, which is analogous to acute stimulation of progression. The many cases where integrins potentiate starved cells with growth factors. This protocol initiates signaling by other receptors have contributed to the growing transient stimulation of many pathways, including Rac, Cdc42 recognition that signaling pathways are highly interwoven into and their downstream kinases; tyrosine kinases or substrates complex networks. Here, we focus on recent advances in our such as Src and Shc; Ras and JNK; and phosphoinoside 3- understanding of how integrins, in cooperation with tyrosine kinase (PI3K) and its downstream pathways (for reviews see kinase growth factor receptors (RTKs), regulate the signal Giancotti and Ruoslahti, 1999; Cary et al., 1999). These events transduction pathways that ultimately control the G1 phase are likely to be important in regulation of cell migration, cyclin-dependent kinases (CDKs). cytoskeletal organization or gene expression; however, 2554 JOURNAL OF CELL SCIENCE 114 (14) transient stimulation of pathways in early G1 is generally not synergy pathway by comparing adherent and suspended cells. sufficient to induce S-phase entry. For example, ERK and PI3K Finally, inhibition of protein kinase A delayed the decline in must be active in mid-G1 phase for cells to enter S phase, and both FAK phosphorylation and Raf activity after cell transient PI3K activity in early G1 phase is even dispensible detachment (Howe and Juliano, 2000). These results suggest for cell cycle progression (Meloche et al., 1992; Jones et al., that FAK is a multifunctional protein that plays a critical role 1999; Gille and Downward, 1999; Jones and Kazlauskas, in the integrin enhancement of mitogenic signaling. 2001). Similarly, Bohmer et al. reported that cell adhesion is A number of other RTK pathways are sensitive to cell required throughout most of G1 phase (Bohmer et al., 1996). adhesion. Growth factor induction of the Rac pathway is Among the integrin-dependent signals characterized to date, integrin dependent (del Pozo et al., 2000). In this context, stimulation of tyrosine phosphorylation of RTKs still activate Rac in suspended cells, but the activated kinase (FAK), p130cas and , PIP 5-kinase activity and Rac fails to target to the plasma membrane and interact with Na+/H+ antiporter activity are sustained as long as cells remain its effectors. Activation of PI3K and Akt in response to EGF adherent (reviewed by Schwartz et al., 1995). There are older is also attenuated in non-adherent cells (Khwaja et al., 1997), data suggesting that Na+/H+ exchange is important (Schwartz as is growth factor activation of JNK (Short et al., 1998). et al., 1995), but in most cases causal relationships between Finally, PDGF activation of protein kinase C and downstream these sustained integrin-dependent signals and the cell cycle events was decreased in non-adherent cells, despite equivalent machinery have yet to be established. FAK has been the most phosphorylation of phospholipase C (McNamee et al., 1993). studied, and the results of studies linking FAK to G1 phase cell In this system, detachment from the ECM led to a decline cycle progression are outlined below. in activity of PIP 5-kinase and cellular levels of Potentiation of growth factor signaling by integrins phosohatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), which also contributes critically to cell cycle progression. This is the substrate for phospholipase C. As PtdIns(4,5)P2 is a cooperation begins at the level of the receptors themselves. Cell major determinant of the and membrane adhesion increases the number of PDGF receptors by blocking targeting of pleckstrin homology domain proteins (Toker, their degradation by a ubiquitin-dependent pathway (Baron and 1998), a decrease in its levels in suspended cells could have Schwartz, 2001). Integrins also physically associate with widespread effects on signaling pathways. Interestingly, actin growth factor receptors and enhance receptor activation polymerization itself is a determinant of gene expression (Schneller et al., 1997; Jones et al., 1997; Moro et al., 1998). through the serum-response element, which is found in the Under many conditions, growth factor activation is promoters of many cytoskeletal and cell cycle regulatory genes substantial in non-adherent cells (McNamee et al., 1993; (Sotiropoulos et al., 1999). Integrins induce polymerization DeMali et al., 1999), in which cases integrins enhance and organization of actin through both physical protein-protein downstream signaling. The ERK MAP kinase cascade is interactions that anchor actin filaments at sites of adhesion and regulated by integrins at multiple points. One study showed through signaling pathways such as PtdIns(4,5)P2 and Rho that, although overall PDGFR activation was unaffected by family GTPases (reviewed by Schoenwaelder and Burridge, adhesion, phosphorylation at a single site and binding of 1999). Thus, actin itself could also contribute to integrin effects RasGAP to the PDGFR was decreased in cells plated on FN on cell cycle progression. These findings are summarized in compared to polylysine, leading to increased Ras and ERK Fig. 1. activation on FN (DeMali et al., 1999). In other studies, Finally, we note that, although the major model for integrin activation of Ras was unaffected by adhesion, but activation of signaling is positive regulation due to ligation and crosslinking Raf and subsequently ERK was strongly adhesion-dependent of integrins, several labs have observed that unoccupied (Lin et al., 1997; Howe and Juliano, 2000). Additionally, others integrin α5β1 in nonadherent cells has dominant effects that reported that activation of MEK by Raf requires integrin- inhibit growth or survival (Giancotti and Ruoslahti, 1990; mediated adhesion (Renshaw et al., 1997), or that sustained Symington, 1992; Varner et al., 1995). In an interesting twist, activation of ERK by growth factors requires cell adhesion the CDK inhibitor INK4a was found to sensitize cells to (Roovers et al., 1999). These discrepancies suggest that the detachment-induced apoptosis by increased transcription of ERK cascade has multiple adhesion-dependent steps whose integrin α5β1; the unoccupied integrin then promotes cell importance varies between cells or experimental conditions. death (Plath et al., 2000). Thus, an alternative model is that Nevertheless, it is agreed that sustained ERK activation unoccupied or unclustered integrins transmit signals that are requires cooperative signaling between RTKs and integrins. terminated by integrin occupancy or clustering. Accordingly, Interestingly, FAK participates in multiple growth regulatory the integrin β3 cytoplasmic domain was recently shown to events. Integrin-mediated effects on FAK control transcription interact with and activate caspase 8 to induce death of of the adapter protein IRS-1 (Lebrun et al., 2000). This effect endothelial cells, and clustering negatively regulated this appears to be mediated in part by effects on JNK activity, in interaction (Stupack and Cheresh, submitted). Thus, although general agreement with a previous study reporting that JNK many studies show integrin stimulation of cytoplasmic mediates FAK regulation of cell cycle progression (Oktay et pathways, these do not in principle distinguish direct activation al., 1999). Although proliferation was not assayed, FAK also from reversal of an inhibition. Both mechanisms probably mediates integrin enhancement of signaling by growth factors contribute to cell survival and growth to some extent. to activate MEK and ERK (Renshaw et al., 1999). A recent paper showed that FAK can mediate activation of the ERK cascade in cells replated on fibronectin (FN) via Rap1 and B- Regulation of cyclins and CDKs Raf rather than Ras and Raf-1 (Berberis et al., 2000). However, Regulation of G1 phase by CDKs is now fairly well understood this paper did not directly evaluate the integrin–growth-factor (reviewed by Weinberg, 1995; Sherr et al., 1999). Of the major Integrins and cell proliferation 2555

Nucleus

ERK Fig. 1. Integrin/RTK synergies that regulate cell cycle progression. Integrins enhance activation of RTKs by their growth factor (GF) ligands and MEK enhance transmission of the signal to ERK at Raf Actin multiple points. Integrins both directly activate and ERK enhance growth factor activation of Rac and Cdc42 through an effect on membrane targeting. Ras Akt K JNK Integrins contribute to activation of PI3K, possibly GF RT via FAK and Cdc42, and promote polymerization ILK PDK1 and organization of actin filaments through both Ras PAK Cas ? direct physical connections (that are not displayed) Membrane tar and a variety of signaling pathways. Integrin Integrins g PI3K eti Src interactions with and Shc also contribute ng FAK to cell cycle progression, although whether this is Shc Cav due to Ras/ERK or unknown pathways is unclear. ECM Rac Note that many signaling events such as Cdc42 Integrins Raf/MEK/ERK and Akt activation occur on the membrane even though they are drawn in the to avoid overcrowding. ECM components in the G1 phase CDK system (cyclins D and E, (Danilkovitch et al., 2000). Alternatively, PI3K is required for CDK4, CDK2 and their inhibitors), integrin/RTK signaling has the expression and stability of cyclin D1 (Gille and Downward, been most closely associated with the induction of cyclin D1 1999; Takuwa et al., 1999; Diehl et al., 1998). As discussed and the downregulation of the CDK inhibitors, p21cip1 and above, both integrins and RTKs contribute to PI3K activity. p27kip1 (reviewed by Roovers and Assoian, 2000). Cyclin D1 The relative importance of ERK signal duration, ERK nuclear binds to CDK4 or CDK6, which leads to their activation. translocation and PI3K activity in the induction of cyclin D1 p21cip1 and p27kip1 bind to cyclin-E–CDK2 and cyclin- among different cell types remains to be determined. A–CDK2 complexes, leading to their inhibition. Activated Integrin signaling also stimulates the translation of cyclin D1 CDK4/6 and CDK2 are required for phosphorylation of the mRNA, and this effect seems to be the major mechanism by retinoblastoma protein, an event that allows the induction of which integrins increase cyclin D1 levels in endothelial cells E2F-dependent genes and signifies the end of cells’ (Huang et al., 1998). Very recent studies indicate that activation dependence on extracellular stimuli. Thus, regulation of cyclin of Rac by integrins is important for the translation of cyclin D1 D1, p21cip1 and p27kip1 by growth factors and the ECM in endothelial cells (F. Giancotti, personal communication), ultimately commits the cell to division. again suggesting that regulation of cyclin D1 expression is The induction of cyclin D1 mRNA has most frequently been likely to involve more than MEK/ERK activation. Interestingly, attributed to the activation of ERKs (reviewed by Roovers and integrins mediate the translocation of activated Rac to the Assoian, 2000). In most cases, activation of the Ras-Raf-MEK- plasma membrane (see above) and the movement of mRNA ERK cascade induces cyclin D1 gene expression. Conversely, and ribosomes to focal adhesions (Chicurel et al., 1998b). inhibition of the Ras pathway inhibits cyclin D1 gene These complementary effects suggest that subcellular expression. Several studies have shown that the induction of compartmentalization by the ECM might integrate different cyclin D1 requires only moderate ERK activity, but the activity integrin-dependent events affecting cell proliferation. must be sustained for several hours (Weber et al., 1997). Overexpression of activated Rac also stimulates cyclin D1 gene Sustained ERK signaling in response to integrin and RTK expression, at least in fibroblasts (Gille and Downward, 1999; synergism (Roovers and Assoian, 2000) may therefore explain Joyce et al., 1999; Page et al., 1999). Rac may therefore have the joint growth factor plus ECM requirement for cyclin D1 cell-specific effects on the synthesis of cyclin D1 mRNA and expression. protein. Rac activity and function are regulated by integrins A sustained ERK signal, although necessary, is not sufficient (see above), which again suggests that regulation of cyclin D1 to induce cyclin D1 protein. Sustained ERK signaling increases expression is likely to involve more than MEK/ERK activation. cyclin D1 expression in adherent but not suspended CCL39 A few studies have assessed the upstream signaling cells (Le Gall et al., 1998). Thus, adhesion must make other mechanisms by which integrins might regulate cyclin D1. contributions to G1 progression. One such contribution is Overexpression of wild-type and dominant negative FAK evident from studies indicating that the transport of ERK from cDNAs showed that integrin-dependent phosphorylation of the cytoplasm to the nucleus is dependent upon cell adhesion FAK plays an important role in the phosphorylation of ERK 2556 JOURNAL OF CELL SCIENCE 114 (14) and induction of cyclin D1 (Zhao et al., 1998). The results with et al., 1998) and to induce calcium entry into cells (Schwartz FAK agree well with studies implicating FAK in integrin/RTK- and Denninghoff 1994). In several of these studies, other dependent ERK activation (Renshaw et al, 1999). Others have integrins failed to induce similar effects despite similar abilities shown that overexpression of ILK leads to the expression of to promote adhesion and cytoskeletal organization. cyclin D1 (Radeva et al., 1997). Given that ILK is downstream In addition to integrin αvβ3, integrin α5β1 (the classical of PI3K (Delcommenne et al., 1998), these results agree with fibronectin receptor), associates with caveolin and Shc (as studies implicating PI3K signaling in control of cyclin D1 mentioned above) and also promotes DNA synthesis (Wary et expression. al., 1996). In myoblasts, elevated α5β1 expression promotes In addition to the stimulation of cyclin D1 synthesis, integrin ERK activation and proliferation, whereas elevated levels of signals are important for the downregulation of CDK inhibitors integrin α6β1 (a receptor) inhibit ERK and promote cip1 of the p21 family. p21 is induced in early G1 phase. Its withdrawal from the cell cycle and differentiation (Sastry et al., induction requires strong ERK activity (reviewed by Roovers 1999). In astrocytoma cells, adhesion to or and Assoian, 2000), which is dependent upon synergistic fibronectin promotes activation of PI3K and its association signaling by growth factor receptors and integrins (discussed with FAK much better than does adhesion to (Ling cip1 kip1 above). In mid-late G1 phase, the levels of p21 and p27 et al., 1999). Integrins α5β1 and αvβ3 also promote are downregulated coincidently with activation of cyclin- responsiveness of vascular smooth muscle cells to -like E–CDK2. This downregulation is impaired when integrin growth factor by a distinct mechanism: secretion of IGF signaling is blocked (reviewed by Roovers and Assoian, 2000). binding protein-5 (IGFBP-5; Zheng et al., 1998). Adhesion of The mechanisms underlying these effects are poorly these cells to collagen or laminin promotes much understood, but ERK seems not to be not directly involved less well but the difference is eliminated by addition of soluble (Bottazzi et al., 1999; Olson et al., 1998). Several studies have IGFBP-5 implicated Rho in the downregulation of both p21cip1 and Different collagen receptors, however, have distinct growth p27kip1 (Weber et al., 1997; Olson et al., 1998; Adnane et al., regulatory and signaling properties. Integrin α1β1 in skin 1998; Laufs et al., 1999), but there is little evidence to support fibroblasts promotes cell proliferation, apparently by the a direct link between integrin signaling and Rho activation. caveolin-dependent signaling pathway discussed above (Pozzo Indeed, direct analysis of Rho-GTP levels shows that integrins et al., 1998). By contrast, stimulation of integrin α2β1 does not have complex effects on Rho activity and do not simply promote proliferation in endothelial cells and fibroblasts (Wary stimulate Rho activity throughout G1 phase (Ren et al., 1999). et al., 1996; Pozzo et al., 1998; Davey et al., 1999). Interestingly, integrin α2β1 activates p38 MAP kinase (Ivaska et al., 1999), and p38 is thought to inhibit transcription of the cyclin D1 Integrin- and matrix-specific effects promoter (Lavoie et al., 1996). However, the inhibitory effect of There are many indications that not all ECM-integrin integrin α2β1 on cell proliferation may be cell-type specific; interactions support cell cycle progression equally well. In α2β1 promotes mammary epithelial tube formation and some instances differences appear to be due to qualitative proliferation more effectively than does integrin α1β1 (Zutter et differences in signaling rather than to quantitative differences al., 1999). These findings are summarized in Fig. 2. in integrin expression, avidity or affinity. Expression of integrin αvβ3, which binds vitronectin, , fibronectin and other matrix proteins, is associated with growth or Organization of the extracellular matrix tumorigenesis of several cell types, as is deposition of In vivo, cells interact with ECM proteins in polymeric matrices vitronectin or osteopontin matrices that bind αvβ3 (reviewed whose macromolecular organization and mechanical properties by Varner and Cheresh, 1996). Integrin αvβ3 has been found can be important determinants of cell cycle progression. to specifically associate with tyrosine kinase growth factor Plating cells on a mutant FN, FN∆III1-7, that contains all of the receptors and the downstream adapter IRS-1 (Schneller et al., known cell-binding motifs but forms a structurally distinct 1997; Jones et al., 1997; Vuori and Ruoslahti, 1994; Soldi et matrix induces less efficient phosphorylation of FAK and al., 1999), and these effects correlate with enhanced progression through G1 phase compared with wild-type FN proliferation on αvβ3 ligands compared with collagen. (Sechler and Schwartzbauer, 1998). When cells were treated Interaction sites on the integrin appear to reside in the with a 76-residue fragment of FN (III1-C) that forms one of extracellular domain of integrin β3 for the PDGFR and the self-assembly sites and inhibits FN matrix assembly, the VEGFR2, although the latter also required integrin αv (Borges FN matrix was disassembled and the activation of cyclin- et al., 2000). E–cdk2 was blocked (Bourdoulous et al., 1998). Cell adhesion Integrin αv is among those α subunits that, along with α5 to FN that contains the alternatively spliced EDA segment and α1, associate with Shc and caveolin and promote DNA produced more efficient progression through G1 phase synthesis (Wary et al., 1996; Wary et al., 1998). These (assessed by phosphorylation of ERK, induction of cyclin D1, associations unexpectedly depend on sequences in the α phosphorylation of pRb and entry into S phase) than did FN subunit transmembrane and extracellular domains. Although lacking EDA (Manabe et al., 1999). However, at least some of the transient activation of ERK that occurs downstream of Shc these effects may be secondary to increased rates of cell appears to be too brief to account for transit through G1 phase, adhesion and spreading. Indeed, Huang et al. reported that cell the association could mediate other growth-promoting signals. spreading on FN, rather than cell adhesion per se, is the critical Integrin αvβ3 also shows a particular ability to cooperate with determinant underlying the adhesion dependency of cyclin D1 bFGF to promote long term ERK activation and mRNA translation and the downregulation of p27kip1 in human (Elcieri et al., 1998), to activate the NF-κB pathway (Scatena capillary endothelial cells (Huang and Ingber, 1999). Integrins and cell proliferation 2557

αvβ3 α5β1, α1β1 α2β1 Most integrins

Fig. 2. The hierarchy of integrin-specific signals. Integrin αvβ3 selectively associates with and enhances signaling by RTK receptors. It also activates several pathways κ κ Caveolin, p38 including NF- B, calcium influx and possibly NF- B, FAK, Malleable ECM, α β PI3K, others yet to be identified. Integrins v 3, calcium, ERK, low tension α5β1 and α1 β1 interact with caveolin and RTK assoc., others? Rho GTPases stimulate Shc phosphorylation and possibly others? other events. Integrins αvβ3 and α5β1 also activate PI3K. Integrin α2β1 stimulates p38 MAP kinase, which may inhibit cyclin D1 expression in some cells. Most integrins activate FAK and Rho family GTPases on rigid ECMs that resist tension and promote focal adhesion formation. The model is meant to emphasize general trends rather than the Cell cycle absolute specificity of each effect. progression

A few studies have examined the role of the collagen matrix embedded within gels that resist tensional forces, ERK is in cell proliferation. Aortic smooth muscle cells proliferate phosphorylated, cyclin D1 is expressed, p27 is downregulated poorly on polymerized collagen gels (conditions in which and the cells cycle when stimulated with growth factors. cell spreading is minimal), which correlated with poor Disruption of mechanical tension leads to the loss of actin stress downregulation of p27kip1 (Koyama et al., 1996). Conversely, fibers, the inactivation of ERKs, the loss of cyclin D1 and the monomeric collagen films supported efficient cell spreading, upregulation of p27kip1 (Rosenfeldt and Grinnell, 2000; H. kip1 p27 downregulation and G1 phase cell cycle progression. Rosenfeldt and F. Grinnell, personal communication). Several Subsequent studies in this system revealed that FN/α5β1 laboratories have used cytochalasin D to disrupt the actin binding mediates the proliferation of smooth muscle cells on cytoskeleton and cell spreading, and these experiments typically collagen films and that the inefficient proliferation of smooth result in an inhibition of integrin signaling and integrin- muscle cells on collagen gels reflects impaired formation of FN dependent cell cycle progression. However, when fibroblasts are fibrils and downregulation of integrin α5β1 (Raines et al., 2000; cultured in collagen gels, cell spreading can persist even when E. W. Raines et al., personal communication). These new results stress fibers have been disassembled. Using this approach, the fit nicely with studies indicating that cell spreading on collagen, Grinnell lab showed that cell spreading in the absence of actin in the absence of FN/α5β1 signaling, does not support cell stress fibers is not sufficient to support ERK activation, cyclin proliferation (Wary et al., 1996; Davey et al., 1999). D1 expression, p27kip1 downregulation or cell proliferation. In addition to chemical signaling, integrin ligation results in Others studies discussed above also indicate that cell spreading the organization of the actin cytoskeleton and generation of itself is not sufficient to support proliferation (Wary et al., 1996; tensional forces (mechanical signaling) that contribute to G1 Davey et al., 1999). Together, the results to date support the idea phase progression (reviewed by Huang and Ingber, 1999; that both chemical and mechanical signals play roles in Koyama et al., 1996; Raines et al., 2000; Chicurel et al., stimulating progression through G1 phase. 1998a). Early studies showed that when hepatocytes are plated Mechanical and chemical signaling appear to be linked. Cells on increasing amounts of fibronectin, attachment per se is respond to mechanical forces by activating a substantial range sufficient to promote progression through early G1 phase but of signaling pathways, including the ERK and JNK pathways cell spreading is required to complete progression into S phase (reviewed by Shyy and Chien, 1997). Among these effects, (Hansen et al., 1994). Subsequently, micropatterned matrices increasing mechanical tension stimulates FAK phosphorylation were used to demonstrate that the effect of cell spreading is (Yano et al., 1996; Tang et al., 1999). Cell-generated tension independent of the density of integrin clusters in capillary might therefore feed back on focal adhesions to influence the endothelial cells (Chen et al., 1997). More recently, these activity of FAK, which could promote cell proliferation through techniques were used to show that cell spreading is required the pathways discussed above. Effects of tension on ERK, PI3K for the translation of cyclin D1 and downregulation of p27kip1 and Rho family GTPases could also contribute. These results in endothelial cells (Huang et al., 1998). These results support may be relevant to the wide range of systems in which malleable the premise that the proliferative effect of cell spreading matrices inhibit cell proliferation but promote differentiation reflects the development of mechanical tension within the cell (reviewed by Adams and Watt, 1993). that is specifically dependent on the ligation of integrins (Huang et al., 1998; Chicurel et al., 1998). The Grinnell laboratory has studied effects of mechanical Conclusions and summary tension in fibroblasts within collagen gels. When cells are Anchorage dependence of growth was described in the 1960s, 2558 JOURNAL OF CELL SCIENCE 114 (14)

GgfF FN

α β

PI3K ERK Rac

Cyclin D1 mRNA

Nucleus

Cyclin D1 mRNA

Fig. 3. Regulation of cyclin D1. A working model depicts the cooperative effects of growth factor receptors and Cyclin D1 integrins on PI3K, ERK and Rac that regulate cyclin D1 levels. Each of these signaling components has the potential to stimulate the expression of cyclin D1 mRNA. Rac and PI3K are also involved in the translation and Stabilized Cyclin D1 stabilization of cyclin D1 protein, respectively. but as late as 1988 it was possible for a reviewer to write that References “the molecular mechanisms of these effects are completely Adams, J. C. and Watt, F. M. (1993). Regulation of development and unknown” (Ruoslahti, 1988). Immense progress over the past differentiation by the extracellular matrix. Development 117, 1183-1198. decade or so has revealed that integrins both transmit direct Adnane, J., Bizouarn, F. A., Qian, Y., Hamilton, A. D. and Sebti, S. M. (1998). p21(WAF1/CIP1) is upregulated by the geranylgeranyltransferase I signals and synergize with growth factor receptors to regulate inhibitor GGTI-298 through a transforming growth factor beta- and Sp1- the pathways that control the G1 cyclin-CDK machinery. responsive element: involvement of the small GTPase rhoA. Mol. Cell. Biol. Additionally, it is clear that a subset of integrin signals depends 18, 6962-6970. upon the organization and physical characteristics of the matrix Baron, V. and Schwartz, M. (2001). Cell adhesion regulates ubiquitin- and that these factors can also influence the cell cycle. Many mediated degradation of the derived growth factor β. J. Biol. Chem. (in press). distinct events involving multiple pathways have now been Berberis, L., Wary, K. K., Fiucci, G., Liu, F., Hirsch, E., Brancaccio, M., described, and it is likely that many of these events contribute ALtruda, F., Tarone, G. and Giancotti, F. G. (2000). Distinct roles for the to the complex networks that determine cell cycle progression. adapter protein Shc and focal adhesion kinase in integrin signaling to Erk. The best-characterized pathway involves cyclin D1, which J. Biol. Chem. (in press). many labs have found depends on both integrins and growth Bohmer, R. M., Scharf, E. and Assoian, R. K. (1996). Cytoskeletal integrity is required throughout the mitogen stimulation phase of the cell cycle and factors via several mechanisms to induce its expression. The mediates the anchorage-dependent expression of cyclin D1. Mol. Biol. Cell role of ERK has been especially well characterized and appears 7, 101-111. to be functionally important. We suggest that the integrin- Borges, E., Jan, Y. and Ruoslahti, E. (2000). Platelet derived growth factor ERK-cyclin pathway may therefore serve as a paradigm for receptor β and vascular endothelial 2 bind to the β understanding how integrins combine with growth factors to 3 integrin through its extracellular domain. J. Biol. Chem. 275, 39867- 39873. control the cell cycle (see Fig. 3). Bottazzi, M. E., Zhu, X., Bohmer, R. M. and Assoian, R. K. (1999). Many questions remain about the full range and mechanisms Regulation of p21(cip1) expression by growth factors and the extracellular of these effects; however, two areas of greatest ignorance stand matrix reveals a role for transient ERK activity in G1 phase. J. Cell Biol. out. First, despite extensive data on pathways controlled 146, 1255-1264. by integrins, the proximal mechanisms by which integrins Bourdoulous, S., Orend, G., MacKenna, D. A., Pasqualini, R. and Ruoslahti, E. (1998). matrix regulates activation of RHO and signal remain poorly defined. Second, the contribution of CDC42 GTPases and cell cycle progression. J. Cell Biol. 143, 267-276. mechanical effects to proliferation and the mechanisms of Cary, L. A., Han, D. C. and Guan, J. L. (1999). Integrin-mediated signal are very poorly understood. We transduction pathways. Histol. Histopathol. 14, 1001-1009. anticipate rapid progress in these areas and eagerly await the Chen, C. S., Mrksich, M., Huang, S., Whitesides, G. M. and Ingber, D. E. answers. (1997). Geometric control of cell and death. Science 276, 1425-1428. Chicurel, M. E., Chen, C. S. and Ingber, D. E. (1998a). Cellular control lies in the balance of forces. Curr. Opin. Cell Biol. 10, 232-239. We are grateful to Bette Cessna for expert secretarial assistance. Chicurel, M. E., Singer, R. H., Meyer, C. J. and Ingber, D. E. (1998b). The writing of this review was supported by USPHS grants to M.A.S. Integrin binding and mechanical tension induce movement of mRNA and and R.K.A. We thank the authors who have made unpublished ribosomes to focal adhesions. Nature 392, 730-733. material available for this article. Danilkovitch, A., Donley, S., Skeel, A. and Leonard, E. J. (2000). Two Integrins and cell proliferation 2559

independent signaling pathways mediate the antiapoptotic action of Kornberg, L. J., Earp, H. S., Turner, C. E., Prockop, C. and Juliano, R. macrophage-stimulating protein on epithelial cells. Mol. Cell. Biol. 20, L. (1991). Signal transduction by integrins: increased protein tyrosine 2218-2227. phosphorylation caused by integrin clustering. Proc. Natl. Acad. Sci. USA Davey, G., Buzzai, M. and Assoian, R. K. (1999). Reduced expression of 88, 8392-8396. (alpha)5(beta)1 integrin prevents spreading-dependent cell proliferation. J. Koyama, H., Raines, E. W., Bornfeldt, K. E., Roberts, J. M. and Ross, R. Cell Sci. 112, 4663-4672. (1996). Fibrillar collagen inhibits arterial smooth muscle proliferation Delcommenne, M., Tan, A., Gray, V., Rue, L., Woodgett, J. and Dedhar, through regulation of Cdk2 inhibitors. Cell 87, 1069-1078. S. (1998). Phosphoinositide-3-OH kinase-dependent regulation of glycogen Laufs, U., Marra, D., Node, K. and Liao, J. K. (1999). 3-Hydroxy-3- synthase kinase 3 and protein kinase B/AKT by the integrin linked kinase. methylglutaryl-CoA reductase inhibitors attenuate vascular smooth muscle Proc. Natl. Acad. Sci. USA (in press). proliferation by preventing rho GTPase-induced down-regulation of Del Pozo, M. A., Price, L. S., Alderson, N., Ren, X. D. and Schwartz, M. p27(Kip1). J. Biol. Chem. 274, 21926-21931. A. (2000). Adhesion to the extracellular matrix regulates the coupling of the Lavoie, J. N., L’Allemain, G., Brunet, A., Muller, R. and Pouyssegur, J. small GTPase Rac to its effector Pak. EMBO J. 19, 2008-2014. (1996). Cyclin D1 expression is regulated positively by the p42/p44MAPK DeMali, K. A., Balciunaite, E. and Kazlauskas, A. (1999). Integrins enhance and negatively by the p38/HOGMAPK pathway. J. Biol. Chem. 271, 20608- platelet-derived growth factor (PDGF) dependent responses by altering the 20616. signal relay enzymes that are recruited to the PDGF β receptor. J. Biol. Le Gall, M., Grall, D., Chambard, J. C., Pouyssegur, J. and Van Chem. 274, 19551-19558. Obberghen-Schilling, E. (1998). An anchorage-dependent signal distinct Diehl, J. A., Cheng, M., Roussel, M. F. and Sherr, C. J. (1998). Glycogen from p42/44 MAP kinase activation is required for cell cycle progression. synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular Oncogene 17, 1271-1277. localization. Genes Dev. 12, 3499-3511. Lebrun, P., Baron, V., Hauck, C. R., Schlaepfer, D. D. and Obberghen, E. Elicieri, B. P., Klemke, R., Stromblad, S. and Cheresh, D. A. (1998). V. (2000). Cell adhesion and focal adhesion kinase regulate Integrin αvβ3 requirement for sustained mitogen-activated protein kinase substrate-1 expression. J. Biol. Chem. 275, 38371-38377. activity during angiogenesis. J. Cell Biol. 140, 1255-1263. Lin, T. H., Chen, Q., Howe, A. and Juliano, R. L. (1997). Cell anchorage Giancotti, F. G. and Ruoslahti, E. (1990). Elevated levels of the α5β1 permits efficient signal transduction between ras and its downstream fibronectin receptor suppress the transformed phenotype of CHO cells. Cell kinases. J. Biol. Chem. 272, 8849-8852. 60, 849-859. Ling, J., Liu, Z., Wang, D. and Gladson, C. L. (1999). Malignant Giancotti, F. G. and Ruoslahti, E. (1999). Integrin signaling. Science 285, astrocytoma cell attachment and migration to various matrix proteins is 1028-1032. differentially sensitive to phosphoinositide inhibitors. J. Cell. Biochem. 73, Gille, H. and Downward, J. (1999). Multiple ras effector pathways contribute 533-544. to G(1) cell cycle progression. J. Biol. Chem. 274, 22033-22040. Manabe, R., Oh-e, N. and Sekiguchi, K. (1999). Alternatively spliced EDA Hansen, L. K., Mooney, D. J., Vacanti, J. P. and Ingber, D. E. (1994). segment regulates fibronectin-dependent cell cycle progression and Integrin binding and cell spreading on extracellular matrix act at different mitogenic signal transduction. J. Biol. Chem. 274, 5919-5924. points in the cell cycle to promote hepatocyte growth. Mol. Biol. Cell 5, 967- McNamee, H. M., Ingber, D. E. and Schwartz, M. A. (1993). Adhesion to 975. fibronectin stimulates inositol lipid synthesis and enhances PDGF-induced Honda, H., Oda, H., Nakamoto, T., Honda, Z. I., Sakai, R., Suzuki, T., inositol lipid breakdown. J. Cell Biol. 121, 673-678. Saito, T., Nakamura, K., Nakao, K., Ishiwara, T. et al. (1998). Meloche, S., Seuwen, K., Pages, G. and Pouyssegur, J. (1992). Biphasic and Cardiovascular anomaly, impaired actin bundling and resistance to src- synergistic activation of p44mapk (ERK1) by growth factors: correlation induced transformation in mice lacking p130cas. Nat. Genet. 19, 361-365. between late phase activation and mitogenicity. Mol. Endocrinol. 6, 845- Howe, A. K. and Juliano, R. L. (2000). Regulation of anchorage-dependent 854. signal transduction by protein kinase A and p21-activated kinase. Nat. Cell Miyamoto, S., Akiyama, S. K. and Yamada, K. M. (1995). Synergistic roles Biol. 2, 593-600. for receptor occupancy and aggregation in integrin transmembrane function. Huang, S. and Ingber, D. E. (1999). The structural and mechanical Science 267, 883-885. complexity of cell-growth control. Nat. Cell Biol. 1, E131-E138. Moro, L., Venturino, M., Bozzo, C., Silengo, L., Altruda, F., Beguinot, L. Huang, S., Chen, C. S. and Ingber, D. E. (1998). Control of cyclin D1, and Tarone, G. (1998). Integrins induce activation of the EGF receptor: role p27(Kip1), and cell cycle progression in human capillary endothelial cells in MAP kinase induction and adhesion-dependent cell survival. EMBO J. by cell shape and cytoskeletal tension. Mol. Biol. Cell 9, 3179-3193. 17, 6622-6632. Ilic, D., Furuta, Y., Kanazawa, S., Takeda, N., Sobue, K., Nakatsuji, N., Oktay, M., Wary, K. K., Dans, M., Bilge, R. B. and Giancotti, F. G. (1999). Nomura, S., Fujimoto, J., Okada, M., Yamamoto, T. et al. (1995). Integrin-mediated activation of focal adhesion kinase is required for Reduced cell motility and enhanced focal adhesion contact formation in signaling to Jun NH2-terminal kinase and progression through the G1 phase cells from FAK-deficient mice. Nature 377, 539-544. of the cell cycle. J. Cell Biol. 145, 1461-1469. Ivaska, J., Reunanen, H., Westermarck, J., Koivisto, L., Kahari, V. M. and Olson, M. F., Paterson, H. F. and Marshall, C. J. (1998). Signals from Ras Heino, J. (1999). Integrin α2β1 mediates isoform-specific activation of p38 and Rho GTPases interact to regulate expression of p21Waf1/Cip1. Nature and upregulation of collagen gene transcription by a mechanism involving 394, 295-299. the α2 cytoplasmic tail. J. Cell Biol. 147, 401-415. Page, K., Li, J., Hodge, J. A., Liu, P. T., Vanden Hoek, T. L., Becker, L. Jones, P. L., Crack, J. and Rabinovitch, M. (1997). Regulation of tenascin B., Pestell, R. G., Rosner, M. R. and Hershenson, M. B. (1999). C, a vascular smooth muscle cell survival factor that interacts with the αvβ3 Characterization of a Rac1 signaling pathway to (1) expression in integrin to promote receptor phosphorylation and airway smooth muscle cells. J. Biol. Chem. 274, 22065-22071. growth. J. Cell Biol. 139, 279-293. Plath, T., Detjen, K., Welzel, M., von Marsha, Z., Murphy, D., Schirner, Jones, S. M. and Kazlauskas, A. (2001). Growth-factor-dependent M., Weidenmann, B. and Rosewicz, S. (2000). A novel function for the mitogenesis requires two distinct phases of signalling. Nat. Cell Biol. 3, 165- tumor suppressor p16INK4a: induction of anoikis via upregulation of the 172. α5β1 fibronectin receptor. J. Cell Biol. 150, 1467-1477. Jones, S. M., Klinghoffer, R., Prestwich, G. D., Toker, A. and Kazlauskas, Pozzo, A., Wary, K. K., Giancotti, F. G. and Gardner, H. A. (1998). Integrin A. (1999). PDGF induces an early and a late wave of PI3-kinase activity, α1β1 mediates a unique collagen-dependent proliferation pathway in vivo. and only the late wave is required for progression through G1. Curr. Biol. J. Cell Biol. 142, 587-594. 9, 512-521. Radeva, G., Petrocelli, T., Behrend, E., Leung-Hagesteijn, C., Filmus, J., Joyce, D., Bouzahzah, B., Fu, M., Albanese, C., D’Amico, M., Steer, J., Slingerland, J. and Dedhar, S. (1997). Overexpression of the integrin- Klein, J. U., Lee, R. J., Segall, J. E., Westwick, J. K. et al. (1999). linked kinase promotes anchorage-independent cell cycle progression. J. Integration of Rac-dependent regulation of cyclin D1 transcription through Biol. Chem. 272, 13937-13944. a nuclear factor-kappaB-dependent pathway. J. Biol. Chem. 274, 25245- Raines, E. W., Koyama, H. and Carragher, N. O. (2000). The extracellular 25249. matrix dynamically regulates smooth muscle cell responsiveness to PDGF. Khwaja, A., Rodriguez-Viciana, P., Wennstrom, S., Warne, P. H. and Ann. New York Acad. Sci. 902, 39-51. Downward, J. (1997). Matrix adhesion and Ras transformation both Ren, X. D., Kiosses, W. B. and Schwartz, M. A. (1999). Regulation of the activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular small GTP-binding protein Rho by cell adhesion and the cytoskeleton. survival pathway. EMBO J. 16, 2783-2793. EMBO J. 18, 578-585. 2560 JOURNAL OF CELL SCIENCE 114 (14)

Renshaw, M. W., Ren, X. D. and Schwartz, M. A. (1997). Activation of the Soldi, R., Mitola, S., Strasly, M., Defilippi, P., Tarone, G. and Bussolino, MAP kinase pathway by growth factors requires integrin-mediated cell F. (1999). Role of alphavbeta3 integrin in the activation of vascular adhesion. EMBO J. 16, 5592-5599. endothelial growth factor receptor-2. EMBO J. 18, 882-892. Renshaw, M. W., Price, L. S. and Schwartz, M. A. (1999). Focal adhesion Sotiropoulos, A., Gineitis, D., Copeland, J. and Treisman, R. (1999). Signal kinase mediates the integrin signaling requirement for growth factor regulated activation of serum response factor is mediated by changes in actin activation of MAP kinase. J. Cell Biol. 147, 611-618. dynamics. Cell 98, 159-169. Roovers, K. and Assoian, R. K. (2000). Integrating the MAP kinase signal Stoker, M., O’Neill, C., Berryman, S. and Waxman, V. (1968). Anchorage into the G1 phase cell cycle machinery. BioEssays 22, 818-826. and growth regulation in normal and -transformed cells. Int. J. Cancer Roovers, K., Davey, G., Zhu, X., Bottazzi, M. E. and Assoian, R. K. (1999). 3, 683-693. α5β1 integrin controls cyclin D1 expression by sustaining mitogen activated Symington, B. E. (1992). Fibronectin receptor modulates cyclin-dependent protein kinase activity in growth factor treated cells. Mol. Biol. Cell 10, kinase activity. J. Biol. Chem. 267, 25744-25747. 3197-3204. Takuwa, N., Fukui, Y. and Takuwa, Y. (1999). Cyclin D1 expression Rosenfeldt, H. and Grinnell, F. (2000). Fibroblast quiescence and the mediated by phosphatidylinositol 3-kinase through mTOR-p70(S6K)- disruption of ERK signaling in mechanically unloaded collagen matrices. J. independent signaling in growth factor-stimulated NIH 3T3 fibroblasts. Mol. Biol. Chem. 275, 3088-3092. Cell. Biol. 19, 1346-1358. Ruoslahti, E. (1988). Fibronectin and its receptors. Annu. Rev. Biochem. 57, Tang, D., Mehta, D. and Gunst, S. J. (1999). Mechanosensitive tyrosine 375-413. phosphorylation of paxillin and focal adhesion kinase in tracheal smooth Sastry, S. K., Lakonishok, M., Wu, S., Truong, T. Q., Huttenlocher, A., muscle. Am. J. Physiol. 276, C250-C258. Turner, C. E. and Horwitz, A. F. (1999). Quantitative changes in integrin Toker, A. (1998). The synthesis and cellular roles of phosphatidylinositol 4,5- and focal adhesion signaling regulate myoblast cell cycle withdrawal. J. Cell bisphosphate. Curr. Opin. Cell Biol. 10, 254-261. Biol. 144, 1295-1309. Varner, J. A. and Cheresh, D. A. (1996). Integrins and cancer. Curr. Opin. Scatena, M., Almeida, M., Chaisson, M. L., Fausto, N., Nicosia, R. F. and Cell Biol. 8, 724-730. Giachelli, C. M. (1998). NF-κB mediates αvβ3 integrin-induced cell Varner, J. A., Emerson, D. A. and Juliano, R. L. (1995). survival. J. Cell Biol. 141, 1083-1093. beta 1 expression negatively regulates cell growth: reversal by attachment Schneller, M., Vuori, K. and Ruoslahti, E. (1997). αvβ3 integrin associates to fibronectin. Mol. Biol. Cell 6, 725-740. with activated insulin and PDGFβ receptors and potentiates the biological Vuori, K. and Ruoslahti, E. (1994). Association of insulin receptor substrate- activity of PDGF. EMBO J. 16, 5600-5607. 1 with integrins. Science 266, 1576-1578. Schoenwaelder, S. M. and Burridge, K. (1999). Bidirectional signaling Wary, K. K., Maneiro, F., Isakoff, S. J., Marcantonio, E. E. and Giancotti, between the cytoskeleton and integrins. Curr. Opin. Cell Biol. 11, 274-286. F. G. (1996). The adapter protein Shc couples a class of integrins to the Schwartz, M. A. (1997). Integrins, oncogenes and anchorage independence. control of cell cycle progression. Cell 87, 733-743. J. Cell Biol. 139, 575-578. Wary, K. K., Mariotti, A., Zurzolo, C. and Giancotti, F. G. (1998). A Schwartz, M. A. and Denninghoff, K. (1994). αv integrins mediate the rise requirement for caveolin-1 and associated kinase fyn in integrin-dependent in intracellular calcium in endothelial cells on fibronectin even though they signaling and anchorage-dependent cell growth. Cell 94, 625-634. play a minor role in adhesion. J. Biol. Chem. 269, 11133-11137. Weber, J. D., Hu, W., Jefcoat, S. C., Jr, Raben, D. M. and Baldassare, J. Schwartz, M. A., Lechene, C. and Ingber, D. E. (1991). Insoluble fibronectin J. (1997). Ras-stimulated extracellular signal-related kinase 1 and RhoA activates the Na/H antiporter by clustering and immobilizing integrin α5β1, activities coordinate platelet-derived growth factor-induced G1 progression independent of cell shape. Proc. Natl. Acad. Sci. USA 88, 7849-7853. through the independent regulation of cyclin D1 and p27. J. Biol. Chem. Schwartz, M. A., Schaller, M. D. and Ginsberg, M. H. (1995). Integrins: 272, 32966-32971. emerging paradigms of signal transduction. Annu. Rev. Cell Biol. 11, 549- Weinberg, R. A. (1995). The retinoblastoma protein and cell cycle control. 599. Cell 81, 323-330. Sechler, J. L. and Schwarzbauer, J. E. (1998). Control of cell cycle Yano, Y., Geibel, J. and Sumpio, B. E. (1996). Tyrosine phosphorylation of progression by fibronectin matrix architecture. J. Biol. Chem. 273, 25533- pp125FAK and paxillin in aortic endothelial cells induced by mechanical 25536. strain. Am. J. Physiol. 271, C635-C649. Shankar, S., Davison, I., Mason, W. T. and Horton, M. A. (1993). Integrin Zhao, J. H., Reiske, H. and Guan, J. L. (1998). Regulation of the cell cycle receptor mediated mobilization of intranuclear calcium in rat osteoclasts. J. by focal adhesion kinase. J. Cell Biol. 143, 1997-2008. Cell Sci. 105, 61-68. Zheng, B., Duan, C. and Clemmons, D. R. (1998). The effect of extracellular Sherr, C. J. and Roberts, J. M. (1999). CDK inhibitors: positive and negative matrix proteins on porcine smooth muscle cell insulin like growth factor regulators of G1 phase progression. Genes Dev. 13, 1501-1512. (IGF) binding protein-5 synthesis and responsiveness to IGF-I. J. Biol. Short, S. M., Talbott, G. A. and Juliano, R. L. (1998). Integrin-mediated Chem. 273, 8994-9000. signaling events in human endothelial cells. Mol. Biol. Cell 9, 1969-1980. Zutter, M. M., Santoro, S. A., Wu, J. E., Wakatsuki, T., Dickeson, S. K. Shyy, J. Y. and Chien, S. (1997). Role of integrins in cellular responses to and Elson, E. L. (1999). Collagen receptor control of epithelial mechanical stress and adhesion. Curr. Opin. Cell Biol. 9, 707-713. and cell cycle progression. Am. J. Pathol. 155, 927-940.