myospheroid

EVOLUTIONARY HOMOLOGS

Table of contents

Signaling downstream of integrins: Rho family GTPases and the cytoskeleton

The ligation of available alpha6beta1 integrin in adherent LOX melanoma cells by laminin G peptides and integrin stimulatory antibodies, induces cell invasiveness. This occurs independent of the adhesion activity of integrins that are pre-bound to extracellular matrix This induced invasion involves an increase in tyrosine phosphorylation of a 190-kDa GTPase-activating protein for Rho family members (p190(RhoGAP); p190: Drosophila homolog RhoGAP) and membrane-protrusive activities at invadopodia. This tyrosine phosphorylation does not occur when the adherent cells are treated with non-activating antibody against beta1 integrin, control laminin peptides, or tyrosine kinase inhibitors genistein and herbimycin A. Although p190 and F-actin co-distribute in all cell cortex extensions, tyrosine-phosphorylated proteins, including p190, appear to associate with F-actin specifically in invadopodia. In addition, the localized matrix degradation and membrane-protrusive activities are blocked by treatment of LOX cells with tyrosine kinase inhibitors as well as microinjection of antibodies directed against p190, but not by non-perturbing antibodies or control buffers. It is suggested that activation of the alpha6beta1 integrin signaling regulates the tyrosine phosphorylation state of p190, which in turn connects downstream signaling pathways through Rho family GTPases to actin cytoskeleton in invadopodia, thus promoting membrane-protrusive and degradative activities necessary for cell invasion (Nakahara, 1998).

FRT thyroid epithelial cells synthesize fibronectin and organize a network of fibronectin fibrils at the basal surface of the cells. Fibronectin fibril formation is enhanced by the overexpression of the ubiquitous beta1A integrin and is inhibited by the expression of the dominant-negative beta1B subunit. The hypothesis was tested that RhoA activity might mediate the integrin-dependent fibronectin fibrillogenesis and might counteract beta1B integrin inhibitory effect. FRT-beta1A cells were transfected with a vector carrying a dominant negative form of RhoA (RhoAN19) or treated with the C3 transferase exoenzyme. Both treatments inhibit fibronectin assembly and causes loss of actin microfilaments and adhesion plaques. FRT-beta1B cells were also transfected with the constitutively activated form of RhoA (RhoAV14) or treated with the E. coli cytotoxic necrotizing factor 1, which directly activates RhoA. Either treatment restores microfilament and adhesion plaque assembly and promotes fibronectin fibril organization. A great increase in fibronectin fibril assembly was also obtained by treatment of FRT-beta1B cells with TGF-beta. These data indicate that RhoA is required to promote fibronectin matrix assembly in FRT cells and that the activation of the signal transduction pathway downstream of RhoA can overcome the inhibitory effect of beta1B integrin (Cali, 1999).

Integrin-mediated adhesion is a critical regulator of cell migration. Integrin-mediated adhesion to high fibronectin concentrations induces a stop signal for cell migration by inhibiting cell polarization and protrusion. On fibronectin, the stop signal is generated through alpha5beta1 integrin-mediated signaling to the Rho family of GTPases. Specifically, Cdc42 and Rac1 activation exhibit a biphasic dependence on fibronectin concentration that parallels optimum cell polarization and protrusion. In contrast, RhoA activity increases with increasing substratum concentration. Cross talk between Cdc42 and Rac1 is required for substratum-stimulated protrusion, whereas RhoA activity is inhibitory. Cdc42 activity is inhibited by Rac1 activation, suggesting that Rac1 activity may down-regulate Cdc42 activity and promote the formation of stabilized rather than transient protrusion. Furthermore, expression of RhoA down-regulates Cdc42 and Rac1 activity, providing a mechanism whereby RhoA may inhibit cell polarization and protrusion. These findings implicate adhesion-dependent signaling as a mechanism to stop cell migration by regulating cell polarity and protrusion via the Rho family of GTPases (Cox, 2001).

Signaling downstream of integrins: PKC and the MAPK cascade

Adhesion of fibroblasts to extracellular matrices via integrin receptors is accompanied by extensive cytoskeletal rearrangements and intracellular signaling events. The protein kinase C (PKC) family of serine/threonine kinases has been implicated in several integrin-mediated events including focal adhesion formation, cell spreading, cell migration, and cytoskeletal rearrangements. However, the mechanism by which PKC regulates integrin function is not known. To characterize the role of PKC family kinases in mediating integrin-induced signaling, the effects of PKC inhibition on fibronectin-induced signaling events in Cos7 cells were monitored using pharmacological and genetic approaches. Inhibition of classical and novel isoforms of PKC by down-regulation with 12-0-tetradeconoyl-phorbol-13-acetate or overexpression of dominant-negative mutants of PKC significantly reduces extracellular regulated kinase 2 (Erk2) activation by fibronectin receptors in Cos7 cells. Furthermore, overexpression of constitutively active PKCalpha, PKCdelta, or PKCepsilon is sufficient to rescue 12-0-tetradeconoyl-phorbol-13-acetate-mediated down-regulation of Erk2 activation, and all three of these PKC isoforms are activated following adhesion. PKC is required for maximal activation of mitogen-activated kinase kinase 1, Raf-1, and Ras, tyrosine phosphorylation of Shc, and Shc association with Grb2. PKC inhibition does not appear to have a generalized effect on integrin signaling, because it does not block integrin-induced focal adhesion kinase or paxillin tyrosine phosphorylation. These results indicate that PKC activity enhances Erk2 activation in response to fibronectin by stimulating the Erk/mitogen-activated protein kinase pathway at an early step upstream of Shc (Miranti, 1999).

Integrin-mediated anchorage of NIH3T3 fibroblasts to the extracellular matrix component fibronectin permits efficient growth factor signaling to the p42 and p44 forms of mitogen-activated protein kinase (MAPK). Since integrins bridge the extracellular matrix to focal adhesion sites and to the actin cytoskeleton, the role of these integrin-associated structures in efficient growth factor activation of p42 and p44-MAPKs were analyzed. Use of specific reagents that disrupt actin stress fiber and focal adhesion formation demonstrate that upon readhesion of NIH3T3 cells to fibronectin, cells that are poorly spread and lack prominent focal adhesions but that form cortical actin structures, efficiently signal to p42 and p44-MAPKs upon EGF stimulation. In contrast, failure to form the cortical actin structures, despite attachment to fibronectin, precludes effective EGF signaling to p42 and p44-MAPKs. Actin cytoskeletal changes induced by expression of dominant-negative and constitutively active forms of Rho GTPases do not alter EGF activation of MAPK in adherent cells. However, active Cdc42, but not active Rac1 or RhoA, partially rescue EGF signaling to p44-MAPK in cells maintained in suspension. These data indicate that a limited degree of adhesion-mediated cytoskeletal organization and focal adhesion complex formation are required for efficient EGF activation of p42 and p44-MAPKs. These studies exclude a major role for the GTPases RhoA and Rac1 in the formation of cytoskeletal structures relevant for signaling, but indicate that structures regulated by Cdc42 enhance the ability of suspension cells to activate MAPK in response to growth factors (Aplin, 1999).

Tenascin-C is an extracellular matrix glycoprotein, the expression of which is upregulated in remodeling arteries. The presence of tenascin-C alters vascular smooth muscle cell shape and amplifies their proliferative response by promoting growth factor receptor clustering and phosphorylation. Denatured type I collagen induces smooth muscle cell tenascin-C protein production via beta3 integrins. The pathway by which beta3 integrins stimulate expression of tenascin-C has been examined, and a promoter sequence is defined that is critical for tenascin-C induction. On native collagen, A10 smooth muscle cells adopt a stellate morphology and produce low levels of tenascin-C mRNA and protein, whereas on denatured collagen they spread extensively and produce high levels of tenascin-C mRNA and protein, which is incorporated into an elaborate extracellular matrix. Increased tenascin-C synthesis on denatured collagen is associated with elevated protein tyrosine phosphorylation, including activation of extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2). beta3 integrin function-blocking antibodies attenuate ERK1/2 activation and tenascin-C protein synthesis. Consistent with these findings, treatment with the specific MEK inhibitor, PD 98059, results in suppression of tenascin-C protein synthesis. To investigate whether beta3 integrin-dependent activation of ERK1/2 regulates the tenascin-C promoter, A10 cells were transfected with a full-length (approx. 4 kb) mouse tenascin-C gene promoter-chloramphenicol acetyltransferse reporter construct. Relative to native collagen, the activity of the reporter construct is increased on denatured collagen. To identify regions of the promoter involved, a series of tenascin-C promoter constructs with 5' deletions were examined. Denatured collagen-dependent promoter activity is retained by a 122-base pair element, located -43 to -165 bp upstream of the RNA start site. Activation of this element is suppressed either by blocking beta3 integrins, or by preventing ERK1/2 activation. These observations demonstrate that smooth muscle cell binding to beta3 integrins activates the mitogen activated protein kinase pathway, which is required for the induction of tenascin-C gene expression via a potential extracellular matrix response element in the tenascin-C gene promoter. These data suggest a mechanism by which remodeling of type I collagen modulates tenascin-C gene expression via a beta3 integrin-mediated signaling pathway, and as such represents a paradigm for vascular development and disease whereby smooth muscle cells respond to perturbations in extracellular matrix composition by altering their phenotype and patterns of gene expression (Jones, 1999).

Protein kinase C (PKC) alpha has been implicated in ß1 integrin-mediated cell migration. Stable expression of PKCalpha is shown here to enhance wound closure. This PKC-driven migratory response directly correlates with increased C-terminal threonine phosphorylation of ezrin/moesin/radixin (ERM) at the wound edge. Both the wound migratory response and ERM phosphorylation are dependent upon the catalytic function of PKC and are susceptible to inhibition by phosphatidylinositol 3-kinase blockade. Upon phorbol 12,13-dibutyrate stimulation, green fluorescent protein-PKCalpha and ß1 integrins co-sediment with ERM proteins in low-density sucrose gradient fractions that are enriched in transferrin receptors. Using fluorescence lifetime imaging microscopy, PKCalpha has been shown to form a molecular complex with ezrin, and the PKC-co-precipitated endogenous ERM is hyperphosphorylated at the C-terminal threonine residue, i.e. activated. Electron microscopy shows an enrichment of both proteins in plasma membrane protrusions. Finally, overexpression of the C-terminal threonine phosphorylation site mutant of ezrin has a dominant inhibitory effect on PKCalpha-induced cell migration. This is the first evidence that PKCalpha or a PKCalpha-associated serine/threonine kinase can phosphorylate the ERM C-terminal threonine residue within a kinase-ezrin molecular complex in vivo (Ng, 2001).

The emerging evidence that stem cells develop in specialised niches highlights the potential role of environmental factors in their regulation. The role of ß1 integrin/extracellular matrix interactions has been examined in neural stem cells. High levels of ß1 integrin expression are found in the stem-cell containing regions of the embryonic CNS, with associated expression of the laminin alpha2 chain. Expression levels of laminin alpha2 are reduced in the postnatal CNS, but a population of cells expressing high levels of ß1 remains. Using neurospheres -- aggregate cultures, derived from single stem cells, that have a three-dimensional architecture that results in the localisation of the stem cell population around the edge of the sphere -- it has been shown directly that ß1 integrins are expressed at high levels on neural stem cells and can be used for their selection. MAPK (but not PI3K) signalling is required for neural stem cell maintenance, as assessed by neurosphere formation, and inhibition or genetic ablation of ß1 integrin using cre/lox technology reduces the level of MAPK activity. It is concluded that integrins are therefore an important part of the signalling mechanisms that control neural stem cell behaviour in specific areas of the CNS (Campos, 2004).

Netrin signaling downstream of integrins

Integrin expression and activity have been strongly correlated with developmental and pathological processes involving cell invasion through basement membranes. The role of integrins in mediating these invasions, however, remains unclear. Utilizing the genetically and visually accessible model of anchor cell (AC) invasion in C. elegans, it has been shown that netrin signaling orients a specialized invasive cell membrane domain toward the basement membrane. This study demonstrates that the integrin heterodimer INA-1/PAT-3 plays a crucial role in AC invasion, in part by targeting the netrin receptor UNC-40 (DCC) to the AC's plasma membrane. Analyses of the invasive membrane components phosphatidylinositol 4,5-bisphosphate, the Rac GTPase MIG-2, and F-actin further indicate that INA-1/PAT-3 plays a broad role in promoting the plasma membrane association of these molecules. Taken together, these studies reveal a role for integrin in regulating the plasma membrane targeting and netrin-dependent orientation of a specialized invasive membrane domain (Hagedorn, 2009).

Anchor cell invasion into the vulval epithelium in C. elegans is an in vivo model of invasive behavior that allows for genetic and single-cell visual analysis of invasion. During the mid-L3 larval stage, a basally derived invasive process from the AC crosses the gonadal and ventral epidermal BMs and then moves between the central 1°-fated vulval precursor cells (VPCs) to mediate uterine-vulval attachment. Recent studies have shown that the invasive cell membrane of the AC is a specialized subcellular domain that is polarized toward the BM by the action of the UNC-6 (netrin) pathway. Approximately 4 hr prior to invasion, expression of the secreted guidance cue UNC-6 (netrin) from the ventral nerve cord targets its receptor, UNC-40 (DCC), to the AC's invasive membrane. There, netrin signaling localizes a number of actin regulators that promote invasion, including the Rac GTPases MIG-2 and CED-10, the Ena/VASP ortholog UNC-34, and the phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). The proper orientation of these components at the basal membrane is required to generate robust protrusions that breach the BM in response to a later cue from the 1° VPCs that stimulates invasion. Although the molecular components of the invasive membrane are misoriented in unc-6 mutants, they still associate in a nonpolarized manner with the AC's plasma membrane, suggesting that a distinct mechanism exists for regulating their targeting to the cell membrane (Hagedorn, 2009).

Integrins are one of the major cell surface receptors used by metazoan cells to mediate direct cell-matrix interactions. All integrins are heterodimers composed of a single α and β subunit. In vertebrates, integrins have been implicated in regulating cell invasion during blastocyst implantation, angiogenesis, and leukocyte trafficking. Furthermore, the dysregulation of integrin expression and function has been associated with a number of metastatic cancers. Mammals utilize 18 α and 8 β subunits, which combine to form an array of different heterodimers. The complexity of the mammalian integrin receptor family, combined with the difficulty of in vivo analysis has hindered an understanding of the requirement and function of integrin receptors in mediating BM invasion. C. elegans possess only two predicted integrin receptors, composed of an α PAT-2 or α INA-1 subunit bound with the sole β subunit, PAT-3, providing a simplified genetic landscape for examining integrin function (Hagedorn, 2009).

An RNAi screen was conducted to identify additional pathways that regulate invasion, and this study reports that the C. elegans integrin heterodimer INA-1/PAT-3 is a crucial regulator of AC invasion. Cell biological and genetic analyses indicate that INA-1/PAT-3 functions within the AC to control the formation of invasive protrusions that breach the BM. This analysis identifies a key role for integrin in regulating the membrane association of components of the invasive cell membrane, including the netrin receptor UNC-40 (DCC). This work demonstrates an essential role for integrin in controlling BM invasion and reveals an integrin-netrin pathway interaction that mediates the membrane targeting and polarization of the molecular constituents of the AC's invasive membrane (Hagedorn, 2009).

Integrins and cell cycle progression

Cell adhesion to substratum has been shown to regulate cyclin A expression as well as cyclin D- and E-dependent kinases, the latter via the up-regulation of cyclin D1 and the down-regulation of cyclin-Cdk inhibitors p21 and p27, respectively. This adhesion-dependent regulation of cell cycle is thought to be mediated by integrins. Stable transfection and overexpression of the integrin-linked kinase (ILK), which interacts with the beta1 and beta3 integrin cytoplasmic domains, induces anchorage-independent cell cycle progression but not serum-independent growth of rat intestinal epithelial cells (IEC18). ILK overexpression results in increased expression of cyclin D1, activation of Cdk4 and cyclin E-associated kinases, and hyperphosphorylation of the retinoblastoma protein. In addition, ILK overexpression results in the expression of p21 and p27 Cdk inhibitors (See Drosophila Dacapo) with altered electrophoretic mobilities, with the p27 from ILK-overexpressing cells having reduced inhibitory activity. The transfer of serum-exposed IEC18 cells from adherent cultures to suspension cultures results in a rapid down-regulation of expression of cyclin D1 and cyclin A proteins, as well as in retinoblastoma protein dephosphorylation. In marked contrast, transfer of ILK-overexpressing cells from adherent to suspension cultures results in continued high levels of expression of cyclin D1 and cyclin A proteins, and a substantial proportion of the retinoblastoma protein remains in a hyperphosphorylated state. These results indicate that, when overexpressed, ILK induces signaling pathways resulting in the stimulation of G1/S cyclin-Cdk activities, which are normally regulated by cell adhesion and integrin engagement (Radeva, 1997).

At mitosis, focal adhesions disassemble and the signal transduction from focal adhesions is inactivated. Components of focal adhesions including focal adhesion kinase (FAK), paxillin, and p130(CAS) (CAS) are serine/threonine phosphorylated during mitosis when all three proteins are tyrosine dephosphorylated. Mitosis-specific phosphorylation continues past cytokinesis and is reversed during post-mitotic cell spreading. Two significant alterations in FAK-mediated signal transduction are found during mitosis: (1) the association of FAK with CAS or c-Src is greatly inhibited, with levels decreasing to 16% and 13% of the interphase levels, respectively; (2) mitotic FAK shows decreased binding to a peptide mimicking the cytoplasmic domain of beta-integrin when compared with FAK of interphase cells. Mitosis-specific phosphorylation is responsible for the disruption of FAK/CAS binding because dephosphorylation of mitotic FAK in vitro by protein serine/threonine phosphatase 1 restores the ability of FAK to associate with CAS, though not with c-Src. These results suggest that mitosis-specific modification of FAK uncouples signal transduction pathways involving integrin, CAS, and c-Src, and may maintain FAK in an inactive state until post-mitotic spreading (Yamakita, 1999).

The potential role and mechanisms of integrin signaling through FAK in cell cycle regulation has been analyzed by using tetracycline-regulated expression of exogenous FAK and mutants. Overexpression of wild-type FAK accelerates G1 to S phase transition. Conversely, overexpression of a dominant-negative FAK mutant DeltaC14 inhibits cell cycle progression at G1 phase and this inhibition requires the Y397 in DeltaC14. Biochemical analyses indicate that FAK mutant DeltaC14 is mislocalized and functions as a dominant-negative mutant by competing with endogenous FAK in focal contacts for binding signaling molecules such as Src and Fyn, resulting in a decrease of Erk activation in cell adhesion. Consistent with this, inhibition of BrdU incorporation and Erk activation by FAK Y397F mutant and FRNK, but not FRNKDeltaC14, is also observed in transient transfection assays using primary human foreskin fibroblasts. Finally, DeltaC14 blocks cyclin D1 upregulation and induces p21 expression, while wild-type FAK increases cyclin D1 expression and decreases p21 expression. Taken together, these results have identified FAK and its associated signaling pathways as a mediator of the cell cycle regulation by integrins (Zhao, 1998).

Adhesion to fibronectin through the alpha5beta1 integrin enables endothelial cells to proliferate in response to growth factors, whereas adhesion to laminin through alpha2beta1 results in growth arrest under the same conditions. On laminin, endothelial cells fail to translate Cyclin D1 mRNA and activate CDK4 and CDK6. Activated Rac, but not MEK1, PI-3K, or Akt, rescues biosynthesis of cyclin D1 and progression through the G1 phase. Conversely, dominant negative Rac prevents these events with culture on fibronectin. Mitogens promote activation of Rac when cells are grown on fibronectin but not laminin. This process is mediated by SOS and PI-3K and requires coordinate upstream signals through Shc and FAK. These results indicate that Rac is a crucial mediator of the integrin-specific control of cell cycle in endothelial cells (Mettouchi, 2001).

What is the mechanism by which growth factor receptors and specific integrins jointly activate Rac? This process requires the DH domain of SOS as well as PI-3K. Structural and functional studies indicate that the PH domain of SOS exerts an allosteric inhibition on the adjacent DH domain. Upon interaction of the PH domain with PIP-3 in the plasma membrane, SOS is thought to undergo a conformational transition that exposes the DH domain and allows it to activate Rac. It is proposed that alpha5beta1 and other Shc-linked integrins cooperate with growth factor receptors to recruit the Grb2/SOS complex at sites of integrin-mediated adhesion, where FAK-PI3K signaling increases the local concentration of PIP-3 and activates the exchange activity of SOS toward Rac. This model accounts for the ability of both dominant negative Shc and FAK to suppress activation of Rac. The mechanism by which PI-3K is activated upon recruitment by FAK remains to be examined, but the ability of dominant negative Ras to inhibit activation of Rac suggests an involvement of Ras (Mettouchi, 2001).

Growth factor dependent activation of integrins

Integrin activation is a multifaceted phenomenon leading to increased affinity and avidity for matrix ligands. To investigate whether cytokines produced during stromal infiltration of carcinoma cells activate nonfunctional epithelial integrins, a cellular system of human thyroid clones derived from normal glands (HTU-5) and papillary carcinomas (HTU-34) was employed. In HTU-5 cells, alphavbeta3 integrin is diffused all over the membrane, disconnected from the cytoskeleton, and unable to mediate adhesion. Conversely, in HTU-34 cells, alphavbeta3 is clustered at focal contacts (FCs) and mediates firm attachment and spreading. alphavbeta3 recruitment at FCs and ligand-binding activity, essentially identical to that of HTU-34, occurs in HTU-5 cells upon treatment with hepatocyte growth factor/scatter factor (HGF/SF). The HTU-34 clone secretes HGF/SF and its receptor is constitutively tyrosine phosphorylated suggesting an autocrine loop responsible for alphavbeta3 activated state. Antibody-mediated inhibition of HGF/SF function in HTU-34 cells disruptes alphavbeta3 enrichment at FCs and impaires adhesion. Accordingly, activation of alphavbeta3 in normal cells is produced by HTU-34 conditioned medium on the basis of its HGF/SF content. These results provide the first example of a growth factor-driven integrin activation mechanism in normal epithelial cells and uncover the importance of cytokine-based autocrine loops for the physiological control of integrin activation (Trusolino, 1998).

Integrins and Calcium Currents

Vasoactive effects of soluble matrix proteins and integrin-binding peptides on arterioles are mediated by alphavbeta3 and alpha5beta1 integrins. To examine the underlying mechanisms, L-type Ca2+ channel current was examined in arteriolar smooth muscle cells in response to integrin ligands. Whole-cell, inward Ba2+ currents were inhibited after application of soluble cyclic RGD peptide, vitronectin (VN), fibronectin (FN), either of two anti-beta3 integrin antibodies, or monovalent beta3 antibody. With VN or beta3 antibody coated onto microbeads and presented as an insoluble ligand, current is also inhibited. In contrast, beads coated with FN or alpha5 antibody produce significant enhancement of current after bead attachment. Soluble alpha5 antibody has no effect on current but blocks the increase in current evoked by FN-coated beads and enhances current when applied in combination with an appropriate IgG. The data suggest that alphavbeta3 and alpha5beta1 integrins are differentially linked through intracellular signaling pathways to the L-type Ca2+ channel and thereby alter control of Ca2+ influx in vascular smooth muscle. This would account for the vasoactive effects of integrin ligands on arterioles and provide a potential mechanism for wound recognition during tissue injury (Wu, 1998).

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