Thy-1 regulates fibroblast focal adhesions, cytoskeletal organization and migration through modulation of p190 RhoGAP and Rho GTPase activity

The precise biological role of Thy-1, a glycophosphatidyl-inositol (GPI)-linked cell surface glycoprotein in non-caveolar lipid raft microdomains, remains enigmatic. Evidence suggests that Thy-1 affects intracellular signaling through src-family protein kinases, and modulates adhesive and migratory events, such as thymocyte adhesion and neurite extension. Primary fibroblasts sorted based on presence or absence of cell surface Thy-1 display strikingly distinct morphologies and differ with respect to production of and response to cytokines and growth factors. It is unclear the extent to which Thy-1 mediates these differences. Findings reported here indicate a novel role for Thy-1 in regulating the activity of Rho GTPase, a critical regulator of cellular adhesion and cytoskeletal organization. Endogenous or heterologous Thy-1 expression promotes focal adhesion and stress fiber formation, characteristic of increased Rho GTPase activity, and inhibits migration. Immunoblotting following transfection of RFL6 fibroblasts with Thy-1 demonstrates that Thy-1 expression inhibits src-family protein tyrosine kinase (SFK) activation, resulting in decreased phosphorylation of p190 Rho GTPase-activating protein (GAP). This results in a net increase in active Rho, and increased stress fibers and focal adhesions. We therefore conclude that Thy-1 surface expression regulates fibroblast focal adhesions, cytoskeletal organization and migration by modulating the activity of p190 RhoGAP and Rho GTPase.     

Threonine 788 in integrin subunit beta1 regulates integrin activation

In the present study, the functional role of suggested phosphorylation of the conserved threonines in the cytoplasmic domain of integrin subunit beta1 was investigated. Mutants mimicking phosphorylated and unphosphorylated forms of beta1 were expressed in beta1 deficient GD25 cells. T788 in beta1 was identified as a site with major influence on integrin function. The mutation to A788 strongly reduced beta1-dependent cell attachment and exposure of the extracellular 9EG7 epitope, whereas replacement of T789 with alanine did not interfere with the ligand-binding ability. Talin has been shown to mediate integrin activation, but the talin head domain bound equally well to the wild-type beta1 and the mutants indicating that the T788A mutation caused defect integrin activation by another mechanism. The phosphorylation-mimicking mutation T788D was fully active in promoting cell adhesion. GD25 cells expressing beta1T788D accumulated increased number of focal contacts and migrated slowly compared to GD25 beta1 wild-type. An analogous phenotype is seen when focal adhesion kinase activation is abrogated. However, neither the beta1T788D nor the beta1T788A mutation failed to induce tyrosine phosphorylation of focal adhesion kinase. The results suggest that phosphorylation of T788 in integrin beta1 promotes inside-out receptor activation, as well as focal contact accumulation.     

Alpha7beta1-integrin regulates mechanotransduction and prevents skeletal muscle injury

Alpha7beta1-integrin links laminin in the extracellular matrix with the cell cytoskeleton and therein mediates transduction of mechanical forces into chemical signals. Muscle contraction and stretching ex vivo result in activation of intracellular signaling molecules that are integral to postexercise injury responses. Because alpha7beta1-integrin stabilizes muscle and provides communication between the matrix and cytoskeleton, the role of this integrin in exercise-induced cell signaling and skeletal muscle damage was assessed in wild-type and transgenic mice overexpressing the alpha7BX2 chain. We report here that increasing alpha7beta1-integrin inhibits phosphorylation of molecules associated with muscle damage, including the mitogen-activated protein kinases (JNK, p38, and ERK), following downhill running. Likewise, activation of molecules associated with hypertrophy (AKT, mTOR, and p70(S6k)) was diminished in mice overexpressing integrin. While exercise resulted in Evans blue dye-positive fibers, an index of muscle damage, increased integrin protected mice from injury. Moreover, exercise leads to an increase in alpha7beta1 protein. These experiments provide the first evidence that alpha7beta1-integrin is a negative regulator of mechanotransduction in vivo and provides resistance to exercise-induced muscle damage.     

IQGAP1 regulates hyaluronan-mediated fibroblast motility and proliferation

IQGAP1, an essential scaffolding protein, forms a complex with the hyaluronan receptor CD44. In this study, we have examined the importance of IQGAP1 for hyaluronan-mediated fibroblast migration and proliferation. Hyaluronan induced formation of F-actin fibers and focal adhesions, which was dependent on IQGAP1. IQGAP1 was required for hyaluronan- but not for platelet-derived growth factor (PDGF)-BB-induced cell migration, and was required for both hyaluronan- and PDGF-BB-mediated fibroblast proliferation, but not for proliferation induced by 10% fetal bovine serum. Depletion of IQGAP1 suppressed hyaluronan-induced activation of Rac1 and enhanced the activation of RhoA. Taken together, these findings indicate important roles for IQGAP1 in hyaluronan-stimulated migration and proliferation of fibroblasts.     

Signaling triggered by Thy-1 interaction with beta 3 integrin on astrocytes is an essential step towards unraveling neuronal Thy-1 function

Thy-1 is an abundant neuronal glycoprotein in mammals. Despite such prevalence, Thy-1 function remains largely obscure in the absence of a defined ligand. Recently described evidence that Thy-1 interacts with beta 3 integrin on astrocytes will be discussed. Thy-1 binding to beta 3 integrin triggers tyrosine phosphorylation of focal adhesion proteins in astrocytes, thereby promoting focal adhesion formation, cell attachment and spreading. Thy-1 has been reported to modulate neurite outgrowth by triggering a cellular response in neurons. However, our data indicate that Thy-1 can also initiate signaling events that promote adhesion of adjacent astrocytes to the underlying surface. Preliminary results suggest that morphological changes observed in the actin cytoskeleton of astrocytes as a consequence of Thy-1 binding is mediated by small GTPases from the Rho family. Our findings argue that Thy-1 functions in a bimodal fashion, as a receptor on neuronal cells and as a ligand for beta 3 integrin receptor on astrocytes. Since Thy-1 is implicated in the inhibition of neurite outgrowth, signaling events in astrocytes are likely to play an important role in this process.     

Direct Thy-1/alphaVbeta3 integrin interaction mediates neuron to astrocyte communication

Thy-1 is an abundant neuronal glycoprotein of poorly defined function. We recently provided evidence indicating that Thy-1 clusters a beta3-containing integrin in astrocytes to induce tyrosine phosphorylation, RhoA activation and the formation of focal adhesions and stress fibers. To date, the alpha subunit partner of beta3 integrin in DI TNC1 astrocytes is unknown. Similarly, the ability of neuronal, membrane-bound Thy-1 to trigger astrocyte signaling via integrin engagement remains speculation. Here, evidence that alphav forms an alphavbeta3 heterodimer in DI TNC1 astrocytes was obtained. In neuron-astrocyte association assays, the presence of either anti-alphav or anti-beta3 integrin antibodies reduced cell-cell interaction demonstrating the requirement of both integrin subunits for this association. Moreover, anti-Thy-1 antibodies blocked stimulation of astrocytes by neurons but not the binding of these two cell types. Thus, neuron-astrocyte association involved binding between molecular components in addition to the Thy-1-integrin; however, the signaling events leading to focal adhesion formation in astrocytes depended exclusively on the latter interaction. Additionally, wild-type (RLD) but not mutated (RLE) Thy-1 was shown to directly interact with alphavbeta3 integrin by Surface Plasmon Resonance analysis. This interaction was promoted by divalent cations and was species-independent. Together, these results demonstrate that the alphavbeta3 integrin heterodimer interacts directly with Thy-1 present on neuronal cells to stimulate astrocytes.     

Functional coupling of ion channels in cellular mechanotransduction

The major players in the processes of cellular mechanotransduction are considered to be mechanosensitive (MS) or mechano-gated ion channels. Non-selective Ca²⁺-permeable channels, whose activity is directly controlled by membrane stretch (stretch-activated channels, SACs) are ubiquitously present in mammalian cells of different origin. Ca²⁺ entry mediated by SACs presumably has a significant impact on various Ca²⁺-dependent intracellular and membrane processes. It was proposed that SACs could play a crucial role in the different cellular reactions and pathologies, including oncotransformation, increased metastatic activity and invasion of malignant cells. In the present work, coupling of ion channels in transformed fibroblasts in course of stretch activation was explored with the use of patch-clamp technique. The combination of cell-attached and inside-out single-current experiments showed that Ca²⁺ influx via SACs triggered the activity of Ca²⁺-sensitive K⁺ channels indicating functional compartmentalization of different channel types in plasma membrane. Importantly, the analysis of single channel behavior demonstrated that K⁺ currents could be activated by the rise of intracellular calcium but displayed no direct mechanosensitivity. Taken together, our data imply that local changes in Ca²⁺ concentration due to SAC activity may provide a functional link between various Ca²⁺-dependent molecules in the processes of cellular mechanotransduction.     

Conformational equilibria and intrinsic affinities define integrin activation

We show that the three conformational states of integrin α₅β₁ have discrete free energies and define activation by measuring intrinsic affinities for ligand of each state and the equilibria linking them. The 5,000‐fold higher affinity of the extended‐open state than the bent‐closed and extended‐closed states demonstrates profound regulation of affinity. Free energy requirements for activation are defined with protein fragments and intact α₅β₁. On the surface of K562 cells, α₅β₁ is 99.8% bent‐closed. Stabilization of the bent conformation by integrin transmembrane and cytoplasmic domains must be overcome by cellular energy input to stabilize extension. Following extension, headpiece opening is energetically favored. N‐glycans and leg domains in each subunit that connect the ligand‐binding head to the membrane repel or crowd one another and regulate conformational equilibria in favor of headpiece opening. The results suggest new principles for regulating signaling in the large class of receptors built from extracellular domains in tandem with single‐span transmembrane domains.     

Talin1 regulates integrin turnover to promote embryonic epithelial morphogenesis

Talin is a cytoskeletal protein that binds to integrin β cytoplasmic tails and regulates integrin activation. Talin1 ablation in mice disrupts gastrulation and causes embryonic lethality. However, the role of talin in mammalian epithelial morphogenesis is poorly understood. Here we demonstrate that embryoid bodies (EBs) differentiated from talin1-null embryonic stem cells are defective in integrin adhesion complex assembly, epiblast elongation, and lineage differentiation. These defects are accompanied by a significant reduction in integrin β1 protein levels due to accelerated degradation through an MG-132-sensitive proteasomal pathway. Overexpression of integrin β1 or MG-132 treatment in mutant EBs largely rescues the phenotype. In addition, epiblast cells isolated from talin1-null EBs exhibit impaired cell spreading and focal adhesion formation. Transfection of the mutant cells with green fluorescent protein (GFP)-tagged wild-type but not mutant talin1 that is defective in integrin binding normalizes integrin β1 protein levels and restores focal adhesion formation. Significantly, cell adhesion and spreading are also improved by overexpression of integrin β1. All together, these results suggest that talin1 binding to integrin promotes epiblast adhesion and morphogenesis in part by preventing integrin β1 degradation.     

Organization of the integrin LFA-1 in nanoclusters regulates its activity

The beta2-integrin LFA-1 facilitates extravasation of monocytes (MOs) into the underlying tissues, where MOs can differentiate into dendritic cells (DCs). Although DCs express LFA-1, unlike MOs, they cannot bind to ICAM-1. We hypothesized that an altered integrin organization on the DC plasma membrane might cause this effect and investigated the relationship between membrane organization and function of LFA-1 on MOs and DCs. High-resolution mapping of LFA-1 surface distribution revealed that on MOs LFA-1 function is associated with a distribution in well-defined nanoclusters (100-150-nm diameter). Interestingly, a fraction of these nanoclusters contains primed LFA-1 molecules expressing the specific activation-dependent L16-epitope. Live imaging of MO-T-cell conjugates showed that only these primed nanoclusters are dynamically recruited to the cellular interface forming micrometer-sized assemblies engaged in ligand binding and linked to talin. We conclude that besides affinity regulation, LFA-1 function is controlled by at least three different avidity patterns: random distributed inactive molecules, well-defined ligand-independent proactive nanoclusters, and ligand-triggered micrometer-sized macroclusters.     

Protein phosphatase 1 associates with the integrin alphaIIb subunit and regulates signaling

Regulation of integrin activation occurs by specific interactions among cytoplasmic proteins and integrin alpha and beta cytoplasmic tails. We report that the catalytic subunit of protein phosphatase 1 (PP1c) constitutively associates with the prototypic integrin alphaIIbbeta3 in platelets and in cell lines overexpressing the integrin. PP1c binds directly to the cytoplasmic domain of integrin alphaIIb subunit containing a conserved PP1c binding motif 989KVGF992. Anchored PP1c is inactive, while thrombin-induced platelet aggregation or fibrinogen-alphaIIbbeta3 engagement caused PP1c dissociation and concomitant activation as revealed by dephosphorylation of PP1c substrate, myosin light chain. Inhibition of ligand binding to activated alphaIIbbeta3 blocks PP1c dissociation and represses PP1c activation. These studies reveal a previously unrecognized role for integrins whereby the alpha subunit cytoplasmic tail localizes the machinery for initiating and temporally maintaining the regulatory signaling activity of a phosphatase.     

Thy-1 binds to integrin beta(3) on astrocytes and triggers formation of focal contact sites

BACKGROUND: Thy-1 is an abundant neuronal glycoprotein in mammals. Despite such prevalence, Thy-1 function remains largely obscure in the absence of a defined ligand. Astrocytes, ubiquitous cells of the brain, express a putative Thy-1 ligand that prevents neurite outgrowth. In this paper, a ligand molecule for Thy-1 was identified, and the consequences of Thy-1 binding for astrocyte function were investigated. RESULTS: Thy-1 has been implicated in cell adhesion and, indeed, all known Thy-1 sequences were found to contain an integrin binding, RGD-like sequence. Thy-1 interaction with beta3 integrin on astrocytes was demonstrated in an adhesion assay using a thymoma line (EL-4) expressing high levels of Thy-1. EL-4 cells bound to astrocytes five times more readily than EL-4(-f), control cells lacking Thy-1. Binding was blocked by either anti-Thy-1 or anti-beta3 antibodies, by RGD-related peptides, or by soluble Thy-1-Fc chimeras. However, neither RGE/RLE peptides nor Thy-1(RLE)-Fc fusion protein inhibited the interaction. Immobilized Thy-1-Fc, but not Thy-1(RLE)-Fc fusion protein supported the attachment and spreading of astrocytes in a Mn(2+)-dependent manner. Binding to Thy-1-Fc was inhibited by RGD peptides. Moreover, vitronectin, fibrinogen, denatured collagen (dcollagen), and a kistrin-derived peptide, but not fibronectin, also mediated Mn(2+)-dependent adhesion, suggesting the involvement of beta3 integrin. The addition of Thy-1 to matrix-bound astrocytes induced recruitment of paxillin, vinculin, and focal adhesion kinase (FAK) to focal contacts and increased tyrosine phosphorylation of proteins such as p130(Cas) and FAK. Furthermore, astrocyte binding to immobilized Thy-1-Fc alone was sufficient to promote focal adhesion formation and phosphorylation on tyrosine. CONCLUSIONS: Thy-1 binds to beta3 integrin and triggers tyrosine phosphorylation of focal adhesion proteins in astrocytes, thereby promoting focal adhesion formation, cell attachment, and spreading.     

DNA methylation alters chromatin structure and regulates Thy-1 expression in EL-4 T cells

Thy-1 exhibits marked differences in expression in various tissues in many species; therefore, it is of interest to define possible mechanisms that may regulate Thy-1 expression. We produced Thy-1 negative variants of the murine T cell lymphoma EL-4 by mutagenesis with ethylmethanesulfonate (EMS), negative selection with anti-Thy-1 monoclonal antibodies (mAb) plus complement, and fluorescence-activated cell sorting (FACS). Thy-1 surface negative (Thy-1-) mutants produced in this manner were shown to produce no detectable Thy-1 mRNA, but contained an intact Thy-1 gene as determined by Southern blotting. 5' CG sequences, which had been demethylated in the parent EL-4 clone, were completely methylated in the EMS-induced Thy-1-variant. In addition, a DNase I hypersensitive site that mapped to the 5' end of the Thy-1 gene in EL-4 was absent in the Thy-1- variant. Treatment of this Thy-1- clone with 5-azadeoxycytidine (5-dAZA) resulted in re-expression of surface Thy-1, demethylation of the 5' CG sequences, and regeneration of the DNase I hypersensitive site. These studies indicate that methylation of certain critical DNA sequences in the 5' region of the Thy-1 gene can alter local chromatin structure and regulate expression of this gene.     

SUR1 regulates PKA-independent cAMP-induced granule priming in mouse pancreatic B-cells

Measurements of membrane capacitance were applied to dissect the cellular mechanisms underlying PKA-dependent and -independent stimulation of insulin secretion by cyclic AMP. Whereas the PKA-independent (Rp-cAMPS-insensitive) component correlated with a rapid increase in membrane capacitance of approximately 80 fF that plateaued within approximately 200 ms, the PKA-dependent component became prominent during depolarizations >450 ms. The PKA-dependent and -independent components of cAMP-stimulated exocytosis differed with regard to cAMP concentration dependence; the K(d) values were 6 and 29 micro M for the PKA-dependent and -independent mechanisms, respectively. The ability of cAMP to elicit exocytosis independently of PKA activation was mimicked by the selective cAMP-GEFII agonist 8CPT-2Me-cAMP. Moreover, treatment of B-cells with antisense oligodeoxynucleotides against cAMP-GEFII resulted in partial (50%) suppression of PKA-independent exocytosis. Surprisingly, B-cells in islets isolated from SUR1-deficient mice (SUR1(-/-) mice) lacked the PKA-independent component of exocytosis. Measurements of insulin release in response to GLP-1 stimulation in isolated islets from SUR1(-/-) mice confirmed the complete loss of the PKA-independent component. This was not attributable to a reduced capacity of GLP-1 to elevate intracellular cAMP but instead associated with the inability of cAMP to stimulate influx of Cl(-) into the granules, a step important for granule priming. We conclude that the role of SUR1 in the B cell extends beyond being a subunit of the plasma membrane K(ATP)-channel and that it also plays an unexpected but important role in the cAMP-dependent regulation of Ca(2+)-induced exocytosis.     

beta 1 integrin regulates fibroblast viability during collagen matrix contraction through a phosphatidylinositol 3-kinase/Akt/protein kinase B signaling pathway

Integrins regulate cell viability through their interaction with the extracellular matrix. Integrins can sense mechanical forces arising from the matrix and convert these stimuli to chemical signals capable of modulating intracellular signal transduction. The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is a major regulator of cell survival. It is not known, however, whether integrins, acting as mechanoreceptors, regulate cell survival via the PI3K/Akt pathway. Here, we show that in response to a matrix-derived mechanical stimulus, beta1 integrin regulated cell viability by regulating Akt activity in a PI3K-dependent fashion. To accomplish this, we employed fibroblasts cultured in collagen gels. During contraction of collagen matrices, fibroblasts underwent apoptosis. We demonstrate that ligation of beta1 integrin with anti-beta1 integrin antibodies protected fibroblasts from apoptosis. The nature of the survival signal activated by beta1 integrin engagement with antibody was mediated by PI3K acting through Akt/protein kinase B. We show that Akt phosphorylation decreased during collagen contraction and that this decrease correlated precisely with the onset of fibroblast apoptosis. Fibroblasts transfected with constitutively active PI3K displayed increased Akt phosphorylation and were protected from anoikis and collagen gel contraction-induced apoptosis. Our data identify a novel role for beta1 integrin in regulating fibroblast viability through a PI3K/Akt/protein kinase B signaling pathway in response to a matrix-derived mechanical stimulus.     

Resveratrol regulates circadian clock genes in Rat-1 fibroblast cells

Circadian clocks, especially peripheral clocks, can be strongly entrained by daily feedings, but few papers have reported the effects of food components on circadian rhythm. The effects of resveratrol, a natural polyphenol, on circadian clocks of Rat-1 cells were analyzed. A dose of 100 muM resveratrol, which did not show cytotoxicity, regulated the expression of clock genes Per1, Per2, and Bmal1.     

IGF-1 regulates cardiac fibroblast apoptosis induced by osmotic stress

In this study we have determined the ability of IGF-1 to protect cardiac fibroblasts against osmotic-induced apoptosis and investigated the potential mechanism(s) underlying this protection. Treatment with IGF-1 (1-100 ng/ml) promoted a dose dependent increase in cell survival against osmotic cell death. Both Akt and ERK1/2 were rapidly phosphorylated by IGF-1 and blocked by wortmannin and PD98059, inhibitors of their upstream activators respectively. However, IGF-1-induced protection was mediated via a wortmannin-dependent but PD98059-independent pathway as determined by cell survival assay suggesting a role of PI3-K/Akt. Furthermore, IGF-1 appeared to reduce the activation of a number of early components in the apoptotic pathway in a wortmannin dependent manner including the osmotic stress-induced perturbation in mitochondrial membrane potential, cleavage and activation of caspase-3 and DNA fragmentation. Thus, the results suggest that IGF-1 regulates osmotic stress-induced apoptosis via the activation of the PI3-K/Akt pathway at a point upstream of the mitochondria and caspase-3.     

The role of focal adhesion complexes in fibroblast mechanotransduction during scar formation

Historically, great efforts have been made to elucidate the biochemical pathways that direct the complex process of wound healing; however only recently has there been recognition of the importance that mechanical signals play in the process of tissue repair and scar formation. The body's physiologic response to injury involves a dynamic interplay between mechanical forces and biochemical cues which directs a cascade of signals leading ultimately to the formation of fibrotic scar. Fibroblasts are a highly mechanosensitive cell type and are also largely responsible for the generation of the fibrotic matrix during scar formation and are thus a critical player in the process of mechanotransduction during tissue repair. Mechanotransduction is initiated at the interface between the cell membrane and the extracellular matrix where mechanical signals are first translated into a biochemical response. Focal adhesions are dynamic multi-protein complexes through which the extracellular matrix links to the intracellular cytoskeleton. These focal adhesion complexes play an integral role in the propagation of this initial mechanical cue into an extensive network of biochemical signals leading to widespread downstream effects including the influx of inflammatory cells, stimulation of angiogenesis, keratinocyte migration, fibroblast proliferation and collagen synthesis. Increasing evidence has demonstrated the importance of the biomechanical milieu in healing wounds and suggests that an integrated approach to the discovery of targets to decrease scar formation may prove more clinically efficacious than previous purely biochemical strategies.     

Akt1 signaling regulates integrin activation, matrix recognition, and fibronectin assembly

Akt, a serine-threonine kinase, regulates multiple cellular processes in vascular cells. We have previously documented that Akt activates integrins and Akt1 deficiency results in matrix abnormalities in skin and blood vessels in vivo. Based on these observations, we hypothesized that Akt1 is necessary for integrin activation and matrix assembly by fibroblasts. In this study, using various cell systems, we show that Akt1 is essential for the inside-out activation of integrins in endothelial cells and fibroblasts, which in turn, mediates matrix assembly. Fibronectin is a major extracellular matrix component of the skin and the vascular basement membrane, which possesses binding sites for many integrins and extracellular matrix proteins. Akt1(-/-) fibroblasts and NIH fibroblasts expressing dominant negative Akt1 (K179M-Akt1) showed impaired fibronectin assembly compared with control fibroblasts. In contrast, expression of constitutively active Akt1 (myrAkt1) resulted in enhanced fibronectin assembly. Although increased fibronectin assembly by myrAkt1-expressing human foreskin fibroblasts was abolished by treatment with anti-integrin beta(1) blocking antibodies, treatment with beta(1)-stimulating antibodies rescued the impaired fibronectin assembly that was due to lack of Akt activity. Finally, expression of myrAkt1 corrected the phenotype of Akt1(-/-) fibroblasts thus showing that Akt1 regulates fibronectin assembly through activation of integrin alpha(5)beta(1).