(alpha)v(beta)3 integrins and Pyk2 mediate insulin-like growth factor I activation of Src and mitogen-activated protein kinase in 3T3-L1 cells

IGF-I stimulates cell growth through interaction of the IGF receptor with multiprotein signaling complexes. However, the mechanisms of IGF-I receptor-mediated signaling are not completely understood. We have previously shown that IGF-I-stimulated 3T3-L1 cell proliferation is dependent on Src activation of the ERK-1/2 MAPK pathway. We hypothesized that IGF-I activation of the MAPK pathway is mediated through integrin activation of Src-containing signaling complexes. The disintegrin echistatin decreased IGF-I phosphorylation of Src and MAPK, and blocking antibodies to (alpha)v and beta3 integrin subunits inhibited IGF-I activation of MAPK, suggesting that (alpha)v(beta)3 integrins mediate IGF-I mitogenic signaling. IGF-I increased ligand binding to (alpha)v(beta)3 as detected by immunofluorescent staining of ligand-induced binding site antibody and stimulated phosphorylation of the beta3 subunit, consistent with inside-out activation of (alpha)v(beta)3 integrins. IGF-I increased tyrosine phosphorylation of the focal adhesion kinase (FAK) Pyk2 (calcium-dependent proline-rich tyrosine kinase-2) to a much greater extent than FAK, and increased association of Src with Pyk2 but not FAK. The intracellular calcium chelator BAPTA prevented IGF-I phosphorylation of Pyk2, Src, and MAPK, suggesting that IGF-I activation of Pyk2 is calcium dependent. Transient transfection with a dominant-negative Pyk2, which lacks the autophosphorylation and Src binding site, decreased IGF-I activation of MAPK, but no inhibition was seen with transfected wild-type Pyk2. These results indicate that IGF-I signaling to MAPK is dependent on inside-out activation of (alpha)v(beta)3 integrins and integrin-facilitated multiprotein complex formation involving Pyk2 activation and association with Src.     

FADD is required for multiple signaling events downstream of the receptor Fas.

To identify essential components of the Fas-induced apoptotic signaling pathway, Jurkat T lymphocytes were chemically mutagenized and selected for clones that were resistant to Fas-induced apoptosis. We obtained five cell lines that contain mutations in the adaptor FADD. All five cell lines did not express FADD by immunoblot analysis and were completely resistant to Fas-induced death. Complementation of the FADD mutant cell lines with wild-type FADD restored Fas-mediated apoptosis. Fas activation of caspase-2, caspase-3, caspase-7, and caspase-8 and the proteolytic cleavage of substrates such as BID, protein kinase Cdelta, and poly(ADP-ribose) polymerase were completely defective in the FADD mutant cell lines. In addition, Fas activation of the stress kinases p38 and c-Jun NH2 kinase and the generation of ceramide in response to Fas ligation were blocked in the FADD mutant cell lines. These data indicate that FADD is essential for multiple signaling events downstream of Fas.     

A tumor-associated beta 1 integrin mutation that abrogates epithelial differentiation control

SCC4 human keratinocytes are derived from a squamous cell carcinoma of the tongue and undergo very little spontaneous differentiation. Introduction of a wild-type beta 1 integrin subunit into SCC4 cells stimulates differentiation, suggesting either that the cells have a defect in the integrin signaling pathways that control differentiation or that the beta1 subunit itself is defective. Here we describe a heterozygous mutation in the SCC4 beta 1 subunit. The mutation, T188I, maps to the I-like domain. It results in constitutive activation of ligand binding, irrespective of the partner alpha subunit, in solid phase assays with recombinant protein and in living cells. The mutation promotes cell spreading, but not proliferation, motility, or invasiveness. It results in sustained activation of Erk MAPK independent of cell spreading. When introduced into SCC4 keratinocytes, the wild-type beta1 integrin stimulates differentiation, whereas the mutant is inactive. Activation of beta 1 integrins in normal keratinocytes also suppresses differentiation. These results establish, for the first time, mutation as a mechanism by which integrins can contribute to neoplasia, because the degree of differentiation in epithelial cancers is inversely correlated with prognosis. They also provide new insights into how integrins regulate keratinocyte differentiation.     

Human intestinal epithelial crypt cell survival and death: Complex modulations of Bcl-2 homologs by Fak, PI3-K/Akt-1, MEK/Erk, and p38 signaling pathways

To investigate the mechanisms responsible for survival and apoptosis/anoikis in normal human intestinal epithelial crypt cells, we analyzed the roles of various signaling pathways and cell adhesion on the expression of six Bcl-2 homologs (Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, Bad) in the well established HIEC-6 cell model. Pharmacological inhibitors and/or dominant-negative constructs were used to inhibit focal adhesion kinase (Fak) and p38 isoforms, as well as the phosphatidylinositol 3'-kinase (PI3-K)/Akt-1 and mitogen-activated protein kinase [MAPK] kinase (MEK)/extracellular regulated kinases (Erk) pathways. Cell adhesion was disrupted by antibody-inhibition of integrin binding or forced cell suspension. The activation levels of studied kinase pathways were also analyzed. Herein, we report that beta1 integrins, Fak, and the PI3-K/Akt-1 pathway, but not beta4 integrins or the MEK/Erk pathway, are crucial for the survival of HIEC-6 cells. Conversely, p38beta, but not p38alpha or gamma, is required for the induction of apoptosis/anoikis in HIEC-6 cells. However, each of the signaling molecules/pathways analyzed were found to affect distinctively the individual expression of the Bcl-2 homologs studied. For example, the inhibition of the PI3-K/Akt-1 pathway down-regulated Bcl-XL, Mcl-1, and Bad, while at the same time up-regulating Bax, whereas the inhibition of Fak up-regulated both Bax and Bak, down-regulated Bad, and did not affect the other Bcl-2 homologs analyzed. These results indicate that integrins, Fak, PI3-K/Akt-1, MEK/Erk, and p38 isoforms perform distinct roles in the regulation of HIEC-6 cell survival and/or death. In addition, our data show that the functions performed by these molecules/pathways in promoting cell survival or apoptosis/anoikis translate into complex, differential modulations of individual Bcl-2 homologs.     

beta1 integrins expression in adult rat ventricular myocytes and its role in the regulation of beta-adrenergic receptor-stimulated apoptosis

We have shown that the stimulation of beta-adrenergic receptors (beta-AR) increases apoptosis in adult rat ventricular myocytes (ARVMs). Integrins, a family of alphabeta-heterodimeric cell surface receptors, are postulated to play a role in ventricular remodeling. Here, we show that norepinephrine (NE) increases beta1 integrins expression in ARVMs via the stimulation of alpha1-AR, not beta-AR. Inhibition of ERK1/2 using PD 98059, an inhibitor of ERK1/2 pathway, inhibited alpha1-AR-stimulated increases in beta1 integrins expression. Activation of beta1 integrins signaling pathway using laminin (LN) inhibited beta-AR-stimulated apoptosis as measured by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL)-staining and flow cytometry. Likewise, ligation of beta1 integrins with anti-beta1 integrin antibodies prevented beta-AR-stimulated apoptosis. Treatment of cells using LN or anti-beta1 integrin antibodies activated ERK1/2 pathway. PD 98059 inhibited activation of ERK1/2 by LN, and prevented the anti-apoptotic effects of LN. Thus (1) stimulation of alpha1-AR regulates beta1 integrins expression via the activation of ERK1/2, (2) beta1 integrins signaling protects ARVMs from beta-AR-stimulated apoptosis, (3) activation of ERK1/2 plays a critical role in the anti-apoptotic effects of beta1-integrin signaling. These data suggest that beta1 integrin signaling protects ARVMs against beta-AR-stimulated apoptosis possibly via the involvement of ERK1/2.     

MEK1 activation rescues Jurkat T cells from Fas-induced apoptosis.

Although the protease cascade initiated by Fas (CD95, Apo-1) is well characterized, there remains little known about how kinase pathways may impact on Fas-mediated apoptosis. We recently observed that in T lymphocytes Fas strongly induced activation of JNK (c-Jun N-terminal kinase) but not of second messengers leading to activation of ERK (extracellular regulated kinase). Additionally, Fas-mediated apoptosis was significantly inhibited with PMA, a potent activator of the ERK signaling pathway. This suggested a model whereby activation of the ERK pathway might attenuate Fas-mediated apoptosis. This was confirmed in the current study by showing that activation of MEK1, the upstream regulator of ERK, reduces Fas-mediated apoptosis, whereas inhibition of MEK1 augments apoptosis by Fas. Furthermore, Fas-mediated apoptosis of Jurkat T cells is not affected by constitutively active or dominant negative variants that modulate the JNK pathway. These results demonstrate that Fas-induced JNK activation is not required for apoptosis by Jurkat T cells, but rather is more likely secondary to cell stress during the early phases of apoptosis. This is supported by the ability of the caspase blocker zVAD to inhibit both apoptosis and JNK activation by Fas.     

Apoptosis induced by protein phosphatase 2A (PP2A) inhibition in T leukemia cells is negatively regulated by PP2A-associated p38 mitogen-activated protein kinase

Serine/threonine phosphatase regulation of phosphorylation-mediated intracellular signaling controls a number of important processes in mammalian cells. In this study, we show that constitutively active protein phosphatase 2A (PP2A), which is a serine/threonine phosphatase, is essential for T leukemia cell survival. Jurkat and CCRF-CEM T leukemia cells treated with the PP2A-selective inhibitor okadaic acid (OA) showed a dose- and time-dependent induction of apoptosis, as indicated by loss of mitochondrial transmembrane potential (delta psi(m)), cleavage-induced activation of caspase-3, -8, and -9, and DNA fragmentation. In addition, caspase-8 or caspase-9 inhibition with z-IETD-fmk or z-LEHD-fmk, respectively, largely prevented OA-induced apoptosis. Although OA treatment did not affect constitutive Bcl-2 expression, overexpression of Bcl-2 prevented both OA-induced DNA fragmentation and dissipation of delta psi(m). Furthermore, inhibition of caspase-3, -8, or -9 partially protected against OA-induced loss of delta psi(m). In addition, caspase-9 and caspase-3 inhibition largely prevented procaspase-3 and procaspase-8 cleavage, respectively, while caspase-8 inhibition partially interfered with procaspase-9 cleavage in OA-treated T leukemia cells. Thus, PP2A inhibition triggered the intrinsic pathway of apoptosis, which was enhanced by a mitochondrial feedback amplification loop. PP2A has also been implicated in the regulation of p38 mitogen-activated protein kinase (MAPK). Co-immunoprecipitation analysis revealed a physical association between the catalytic subunit of PP2A and p38 MAPK in T leukemia cells. Moreover, OA treatment caused p38 MAPK to be phosphorylated in a dose- and time-dependent fashion, indicating that PP2A prevented p38 MAPK activation. Although p38 MAPK activation usually promotes apoptosis, pharmacologic inhibition of p38 MAPK exacerbated OA-induced DNA fragmentation and loss of delta psi(m) in T leukemia cells, suggesting that, in this instance, the p38 MAPK signaling pathway promoted cell survival. Collectively, these findings indicate that PP2A and p38 MAPK have coordinate effects on signaling pathways that regulate the survival of T leukemia cells.     

Phosphatidylinositol 3-kinase/Akt activation by integrin-tumor matrix interaction suppresses Fas-mediated apoptosis in T cells

It has recently become apparent that the microenvironment made up of the extracellular matrix may affect cell signaling. In this study, we evaluated Fas-triggered apoptosis in T cells in contact with tumor cells, which resembles the cell-to-cell interactions found in tumor regions. Jurkat cells were less susceptible to the Fas-mediated apoptosis when cocultured with U118, HeLa, A549, and Huh-7 tumor cells. This was indicated by less plasma membrane alteration, an amelioration of the loss of mitochondria membrane potential, a decrease in caspase-8 and caspase-3 activation, a decrease in DNA fragmentation factor-45/35 cleavage, and a reduction in the breakage of DNA when compared with Jurkat cells cultured alone. In contrast, the tumor cell lines MCF-7 and HepG2 produced no such protective effect. This protective event was independent of the expression of Fas ligand on the tumor cells. Interrupting the beta integrins-matrix interaction diminished the coculture effect. In Jurkat cells, cell matrix contact reduced the assembly of the Fas death-inducing signaling complex and Bcl-x(L) cleavage, but enhanced the phosphorylation of ERK1/2, p38 MAPK, and Akt. Only PI3K inhibitor, but not kinase inhibitors for MEK, ERK1/2, p38 MAPK, JNK, protein kinase C, and protein kinase A, completely abolished this tumor cell contact-associated protection and in parallel restored Fas-induced Bcl-x(L) cleavage as well as decreasing the phosphorylation of Bad at serine 136. Together, our results indicate that stimulation of the beta integrin signal of T cells by contact with tumor cells may trigger a novel protective signaling through the PI3K/Akt pathway of T cells against Fas-mediated apoptosis.     

Beta 3- and alpha1-adrenergic Erk1/2 activation is Src- but not Gi-mediated in Brown adipocytes

A novel signaling pathway for mediation of beta(3)-adrenergic activation of the mitogen-activated protein kinases Erk1/2 (associated with proliferation, differentiation, and apoptosis) has recently been proposed, which implies mediation via constitutively coupled G(i)-proteins and Gbetagamma-subunits, distinct from the classical cAMP pathway of beta-adrenergic stimulation. To verify the significance of this pathway in cells in primary cultures that entopically express beta(3)-adrenoreceptors, we examined the functionality of this pathway in cultured brown adipocytes. Norepinephrine activated Erk1/2 via both beta(3) receptors and alpha(1) receptors but not via alpha(2) receptors. Forskolin induced Erk1/2 activation similarly to beta(3) activation, indicating cAMP-mediation; this induction could be inhibited with H89, implying protein kinase A mediation. The G(i)-pathway was functional in these cells, as pertussis toxin increased agonist-induced cAMP accumulation. However, pertussis toxin was unable to affect adrenergically induced Erk1/2 activation. Also, wortmannin was without effect, implying that Gbetagamma activation of the phosphatidylinositol 3-kinase pathway was not involved. PP1/2, which inhibits Src, abolished both beta(3)- and alpha(1)-induced Erk1/2 activation. Thus, the proposed novel G(i) pathway for beta(3) mediation is not universal, because it is not functional in the untransformed primary cell culture system with entopically expressed beta(3) receptors examined here. Here, the beta(3) signal is mediated classically via cAMP/protein kinase A. beta(3) and alpha(1) signals converge at Src, which thus mediates Erk1/2 activation in both pathways.     

Membrane type-1 matrix metalloproteinase (MT1-MMP) processing of pro-alphav integrin regulates cross-talk between alphavbeta3 and alpha2beta1 integrins in breast carcinoma cells

We have recently demonstrated that in breast carcinoma MCF7 cells MT1-MMP processes the alphav, alpha3, and alpha5 integrin precursors generating the respective mature S-S-linked heavy and light alpha-chains. The precursor of alpha2 integrin subunit was found resistant to MT1-MMP proteolysis. The processing of the alphav subunit by MT1-MMP facilitated alphavbeta3-dependent adhesion, activation of FAK signaling pathway, and migration of MCF7 cells on vitronectin. To elucidate further the effects of MT1-MMP on cellular integrins, we examined the functional activity of alpha5beta1 and alpha2beta1 integrins in MCF7 cells expressing MT1-MMP. Either expression of MT1-MMP alone or its coexpression with alphavbeta3 failed to affect the functionality of alpha5beta1 integrin, and adhesion of cells to fibronectin. MT1-MMP, however, profoundly affected the cross-talk involving alphavbeta3 and alpha2beta1 integrins. In MT1-MMP-deficient cells, integrin alphavbeta3 suppressed the functional activity of the collagen-binding alpha2beta1 integrin receptor and diminished cell adhesion to type I collagen. Coexpression of MT1-MMP with integrin alphavbeta3 restored the functionality of alpha2beta1 integrin and, consequently, the ability of MCF7 cells to adhere efficiently to collagen. We conclude that the MT1-MMP-controlled cross-talk between alphavbeta3 and alpha2beta1 integrins supports binding of aggressive, MT1-MMP-, and alphavbeta3 integrin-expressing malignant cells on type I collagen, the most common substratum of the extracellular matrix.     

Selective up-regulation of phosphatidylinositol 3'-kinase activity in Th2 cells inhibits caspase-8 cleavage at the death-inducing complex: a mechanism for Th2 resistance from Fas-mediated apoptosis.

In this study the mechanism of differential sensitivity of CD3-activated Th1- and Th2-type cells to Fas-mediated apoptosis was explored. We show that the Fas-associated death domain protein (FADD)/caspase-8 pathway is differentially regulated by CD3 activation in the two subsets. The apoptosis resistance of activated Th2-type cells is due to an incomplete processing of caspase-8 at the death-inducing signaling complex (DISC) whereas recruitment of caspase-8 to the DISC of Th1- and Th2-like cells is comparable. Activation of phosphatidylinositol 3'-kinase upon ligation of CD3 in Th2-type cells blocked caspase-8 cleavage to its active fragments at the DISC, thereby preventing induction of apoptosis. This study offers a new pathway for phosphatidylinositol 3'-kinase in mediating protection from Fas-induced apoptosis.     

Matrix attachment regulates Fas-induced apoptosis in endothelial cells: a role for c-flip and implications for anoikis

Survival of endothelial cells is critical for cellular processes such as angiogenesis. Cell attachment to extracellular matrix inhibits apoptosis in endothelial cells both in vitro and in vivo, but the molecular mechanisms underlying matrix-induced survival signals or detachment-induced apoptotic signals are unknown. We demonstrate here that matrix attachment is an efficient regulator of Fas-mediated apoptosis in endothelial cells. Thus, matrix attachment protects cells from Fas-induced apoptosis, whereas matrix detachment results in susceptibility to Fas-mediated cell death. Matrix attachment modulates Fas-mediated apoptosis at two different levels: by regulating the expression level of Fas, and by regulating the expression level of c-Flip, an endogenous antagonist of caspase-8. The extracellular signal-regulated kinase (Erk) cascade functions as a survival pathway in adherent cells by regulating c-Flip expression. We further show that detachment-induced cell death, or anoikis, itself results from activation of the Fas pathway by its ligand, Fas-L. Fas-L/Fas interaction, Fas-FADD complex formation, and caspase-8 activation precede the bulk of anoikis in endothelial cells, and inhibition of any of these events blocks anoikis. These studies identify matrix attachment as a survival factor against death receptor-mediated apoptosis and provide a molecular mechanism for anoikis and previously observed Fas resistance in endothelial cells.     

Effects of I domain deletion on the function of the beta2 integrin lymphocyte function-associated antigen-1

A subset of integrin alpha subunits contain an I domain, which is important for ligand binding. We have deleted the I domain from the beta2 integrin lymphocyte function-asssociated antigen-1 (LFA-1) and expressed the resulting non-I domain-containing integrin (DeltaI-LFA-1) in an LFA-1-deficient T cell line. DeltaI-LFA-1 showed no recognition of LFA-1 ligands, confirming the essential role of the I domain in ligand binding. Except for I domain monoclonal antibodies (mAbs), DeltaI-LFA-1 was recognized by a panel of anti-LFA-1 mAbs similarly to wild-type LFA-1. However, DeltaI-LFA-1 had enhanced expression of seven mAb epitopes that are associated with beta2 integrin activation, suggesting that it exhibited an "active" conformation. In keeping with this characteristic, DeltaI-LFA-1 induced constitutive activation of alpha4beta1 and alpha5beta1, suggesting intracellular signaling to these integrins. This "cross-talk" was not due to an effect on beta1 integrin affinity. However, the enhanced activity was susceptible to inhibition by cytochalasin D, indicating a role for the cytoskeleton, and also correlated with clustering of beta1 integrins. Thus, removal of the I domain from LFA-1 created an integrin with the hallmarks of a constitutively active receptor mediating signals into the cell. These findings suggest a key role for the I domain in controlling integrin activity.     

Regulation of interleukin-1alpha expression by integrins and epidermal growth factor receptor in keratinocytes from a mouse model of inflammatory skin disease

Transgenic mice expressing beta1 integrins in the suprabasal epidermal layers have sporadic skin hyperproliferation and inflammation correlated with activation of extracellular signal-regulated kinase (Erk) mitogen-activated protein kinase and increased interleukin (IL)-1alpha production. We investigated the link between aberrant integrin expression, Erk activation, and expression of IL-1alpha. Transgenic keratinocytes had higher basal Erk activity and IL-1alpha levels than nontransgenic controls and were more sensitive to stimulation of Erk activity and IL-1alpha production by IL-1alpha, 12-O-tetradecanoylphorbol-13-acetate (TPA), epidermal growth factor (EGF), and serum. Inhibition of Erk in transgenic keratinocytes reduced basal IL-1alpha levels and the stimulation of IL-1alpha production by serum or phorbol ester, demonstrating that Erk could regulate IL-1alpha expression. TPA or IL-1alpha treatment resulted in rapid down-regulation of the EGF receptor in transgenic cells, indicative of transactivation. Inhibition of transactivation blocked basal and TPA or IL-1alpha induced Erk activation, but not IkappaBalpha degradation, and abolished increased IL-1alpha production in transgenic cells. In transgene-negative cells, constitutive activation of IL-1-dependent signaling by wild type or kinase-dead IRAK1 stimulated IL-1alpha production independent of Erk. We conclude that suprabasal integrin expression leads to Erk activation and increased IL-1alpha expression by potentiating activation of the EGF receptor. These results provide a mechanism by which aberrant integrin expression triggers epidermal hyperproliferation and inflammation.     

p38 mitogen-activated protein kinase mediates the Fas-induced mitochondrial death pathway in CD8+ T cells

The p38 mitogen-activated protein kinase (MAPK) signaling pathway can be activated by a variety of stress stimuli such as UV radiation and osmotic stress. The regulation and role of this pathway in death receptor-induced apoptosis remain unclear and may depend on the specific death receptor and cell type. Here we show that binding of Fas ligand to Fas activates p38 MAPK in CD8+ T cells and that activation of this pathway is required for Fas-mediated CD8+ T-cell death. Active p38 MAPK phosphorylates Bcl-xL and Bcl-2 and prevents the accumulation of these antiapoptotic molecules within the mitochondria. Consequently, a loss of mitochondrial membrane potential and the release of cytochrome c lead to the activation of caspase 9 and, subsequently, caspase 3. Therefore, the activation of p38 MAPK is a critical link between Fas and the mitochondrial death pathway and is required for the Fas-induced apoptosis of CD8+ T cells.     

Overexpressed alpha3beta1 and constitutively activated extracellular signal-regulated kinase modulate the angiogenic properties of ECV304 cells.

ECV304, a spontaneously transformed cell line derived from the human umbilical vein endothelial cell (HUVEC) (Takahashi et al., 1990), has been developed as an in vitro angiogenesis model. In the present study, we further characterized the angiogenic properties of this cell line. Compared to HUVEC, ECV304 cells showed distinct features including a higher activity of cellular adhesion, slower but reproducible progression of angiogenesis on Matrigel, and resistance to apoptosis. Thus, the expression of integrin and activation of extracellular-signal regulated kinase 1/2 (Erk1/2), a downstream effector of the integrin pathway, were examined. Flow cytometry revealed that alpha3beta1 integrin was markedly upregulated in ECV304 cells, while alpha(v)beta1 and alpha5beta1 integrins were slightly downregulated. Consistent with this, the binding activity to collagen type IV and laminin, major extracellular matrices of Matrigel, was increased 1.4- and 1.9-fold in ECV304 cells, respectively. This tight binding may retard the initial stage of sprouting and migration in the angiogenesis of ECV304 cells. It has been further demonstrated that Erk1/2 is constitutively active in ECV304 cells, rendering them resistent to the inhibitory effect of PD98059 on proliferation. However, migration of both HUVEC and ECV304 cells was inhibited to a similar extent by PD98059 in a dose-dependent manner. Up to 50 microM of PD98059, no significant changes in cell binding and tubulogenesis on Matrigel was observed in ECV304 cells. In contrast, the tubulogenesis of HUVEC was severely impaired by PD98059. Elevated Erk1/2 activity in ECV304 cells was suppressed by dominant negative H-Ras, but not by cytochalasin D. These results suggest that the overexpression of alpha3beta1 integrin and the constitutive activation of Erk1/2 play a key role in the alteration of the angiogenic properties of ECV304 cells.     

Inhibition of integrin-mediated crosstalk with epidermal growth factor receptor/Erk or Src signaling pathways in autophagic prostate epithelial cells induces caspase-independent death

In vivo in the prostate gland, basal epithelial cells adhere to laminin 5 (LM5) via alpha3beta1 and alpha6beta4 integrins. When placed in culture primary prostate basal epithelial cells secrete and adhere to their own LM5-rich matrix. Adhesion to LM5 is required for cell survival that is dependent on integrin-mediated, ligand-independent activation of the epidermal growth factor receptor (EGFR) and the cytoplasmic tyrosine kinase Src, but not PI-3K. Integrin-mediated adhesion via alpha3beta1, but not alpha6beta4 integrin, supports cell survival through EGFR by signaling downstream to Erk. PC3 cells, which do not activate EGFR or Erk on LM5-rich matrices, are not dependent on this pathway for survival. PC3 cells are dependent on PI-3K for survival and undergo caspase-dependent death when PI-3K is inhibited. The death induced by inhibition of EGFR or Src in normal primary prostate cells is not mediated through or dependent on caspase activation, but depends on the induction of reactive oxygen species. In addition the presence of an autophagic pathway, maintained by adhesion to matrix through alpha3beta1 and alpha6beta4, prevents the induction of caspases when EGFR or Src is inhibited. Suppression of autophagy is sufficient to induce caspase activation and apoptosis in LM5-adherent primary prostate epithelial cells.