Regulation of myoepithelial differentiation.

The salivary gland can be permanently impaired by radiation treatment for head and neck cancers. Efforts at tissue regeneration have focused on saliva-producing acinar cells. However, myoepithelial cells are also critical to gland function, but mechanisms that regulate their differentiation are poorly defined. To study myoepithelial differentiation, we employed mSG-PAC1 murine salivary gland epithelial cells. We demonstrate that mSG-PAC1 spheroids exhibit phenotypic plasticity between pro-acinar and myoepithelial cell fates. Increased expression of pro-acinar/acinar or myoepithelial RNAs was identified from spheroids cultured under different media conditions by microarray followed by gene-set enrichment analysis. Spheroids cultured with different medium components expressed proteins typical of either acinar or myoepithelial cells, as detected by immunocytochemistry. We demonstrate that the pattern of TAZ expression in the epithelial compartment of the differentiating murine salivary gland correlates with the expression of the myoepithelial marker alpha-SMA, as is the case for TAZ expression in mSG-PAC1 spheroids. Our analysis also indicates that YAP/TAZ target genes are upregulated together with myoepithelial markers. Importantly, siRNA targeting of TAZ expression in mSG-PAC1 spheroids diminished the expression of myoepithelial markers. Our results in this in vitro cell model implicate TAZ signaling in myoepithelial differentiation.

Contribution of Endothelial Laminin-Binding Integrins to Cellular Processes Associated with Angiogenesis.

Endothelial cells engage extracellular matrix and basement membrane components through integrin-mediated adhesion to promote angiogenesis. Angiogenesis involves the sprouting of endothelial cells from pre-existing vessels, their migration into surrounding tissue, the upregulation of angiogenesis-associated genes, and the formation of new endothelial tubes. To determine whether the endothelial laminin-binding integrins, α6ß4, and α3ß1 contribute to these processes, we employed RNAi technology in organotypic angiogenesis assays, as well in migration assays, in vitro. The endothelial depletion of either α6ß4 or α3ß1 inhibited endothelial sprouting, indicating that these integrins have non-redundant roles in this process. Interestingly, these phenotypes were accompanied by overlapping and distinct changes in the expression of angiogenesis-associated genes. Lastly, depletion of α6ß4, but not α3ß1, inhibited migration. Taken together, these results suggest that laminin-binding integrins regulate processes associated with angiogenesis by distinct and overlapping mechanisms.

Laminin-511 and α6 integrins regulate the expression of CXCR4 to promote endothelial morphogenesis.

During angiogenesis, endothelial cells engage components of the extracellular matrix through integrin-mediated adhesion. Endothelial expression of laminin-411 and laminin-511 is known to promote vessel stability. However, little is known about the contribution of these laminins to endothelial morphogenesis. We used two organotypic cell culture angiogenesis assays, in conjunction with RNAi approaches, to demonstrate that depletion of either the α4 chain of laminin-411 (LAMA4) or the α5 chain of laminin-511 (LAMA5) from endothelial cells inhibits sprouting and tube formation. Depletion of α6 (ITGA6) integrins resulted in similar phenotypes. Gene expression analysis indicated that loss of either laminin-511 or α6 integrins inhibited the expression of CXCR4, a gene previously associated with angiogenic endothelial cells. Pharmacological or RNAi-dependent inhibition of CXCR4 suppressed endothelial sprouting and morphogenesis. Importantly, expression of recombinant CXCR4 rescued endothelial morphogenesis when α6 integrin expression was inhibited. Additionally, the depletion of α6 integrins from established tubes resulted in the loss of tube integrity and laminin-511. Taken together, our results indicate that α6 integrins and laminin-511 can promote endothelial morphogenesis by regulating the expression of CXCR4 and suggest that the α6-dependent deposition of laminin-511 protects the integrity of established endothelial tubes.

Establishment of a Murine Pro-acinar Cell Line to Characterize Roles for FGF2 and α3ß1 Integrins in Regulating Pro-acinar Characteristics.

Radiation therapy for head and neck cancers results in permanent damage to the saliva producing acinar compartment of the salivary gland. To date, a pure pro-acinar cell line to study underlying mechanisms of acinar cell differentiation in culture has not been described. Here, we report the establishment of a pro-acinar (mSG-PAC1) and ductal (mSG-DUC1) cell line, from the murine submandibular salivary gland (SMG), which recapitulate developmental milestones in differentiation. mSG-DUC1 cells express the ductal markers, keratin-7 and keratin-19, and form lumenized spheroids. mSG-PAC1 cells express the pro-acinar markers SOX10 and aquaporin-5. Using the mSG-PAC1 cell line, we demonstrate that FGF2 regulates specific steps during acinar cell maturation. FGF2 up-regulates aquaporin-5 and the expression of the α3 and α6 subunits of the α3ß1 and α6ß1 integrins that are known to promote SMG morphogenesis and differentiation. mSG-DUC1 and mSG-PAC1 cells were derived from genetically modified mice, homozygous for floxed alleles of the integrin α3 subunit. Similar to SMGs from α3-null mice, deletion of α3 alleles in mSG-PAC1 cells results in the up-regulation of E-cadherin and the down-regulation of CDC42. Our data indicate that mSG-DUC1 and mSG-PAC1 cells will serve as important tools to gain mechanistic insight into salivary gland morphogenesis and differentiation.

Integrin and microtubule crosstalk in the regulation of cellular processes.

Integrins engage components of the extracellular matrix, and in collaboration with other receptors, regulate signaling cascades that impact cell behavior in part by modulating the cell's cytoskeleton. Integrins have long been known to function together with the actin cytoskeleton to promote cell adhesion, migration, and invasion, and with the intermediate filament cytoskeleton to mediate the strong adhesion needed for the maintenance and integrity of epithelial tissues. Recent studies have shed light on the crosstalk between integrin and the microtubule cytoskeleton. Integrins promote microtubule nucleation, growth, and stabilization at the cell cortex, whereas microtubules regulate integrin activity and remodeling of adhesion sites. Integrin-dependent stabilization of microtubules at the cell cortex is critical to the establishment of apical-basal polarity required for the formation of epithelial tissues. During cell migration, integrin-dependent microtubule stabilization contributes to front-rear polarity, whereas microtubules promote the turnover of integrin-mediated adhesions. This review focuses on this interdependent relationship and its impact on cell behavior and function.

Matrix compliance and the regulation of cytokinesis.

Integrin-mediated cell adhesion to the ECM regulates many physiological processes in part by controlling cell proliferation. It is well established that many normal cells require integrin-mediated adhesion to enter S phase of the cell cycle. Recent evidence indicates that integrins also regulate cytokinesis. Mechanical properties of the ECM can dictate entry into S phase; however, it is not known whether they also can affect the successful completion of cell division. To address this issue, we modulated substrate compliance using fibronectin-coated acrylamide-based hydrogels. Soft and hard substrates were generated with approximate elastic moduli of 1600 and 34,000 Pascals (Pa) respectively. Our results indicate that dermal fibroblasts successfully complete cytokinesis on hard substrates, whereas on soft substrates, a significant number fail and become binucleated. Cytokinesis failure occurs at a step following the formation of the intercellular bridge connecting presumptive daughter cells, suggesting a defect in abscission. Like dermal fibroblasts, mesenchymal stem cells require cell-matrix adhesion for successful cytokinesis. However, in contrast to dermal fibroblasts, they are able to complete cytokinesis on both hard and soft substrates. These results indicate that matrix stiffness regulates the successful completion of cytokinesis, and does so in a cell-type specific manner. To our knowledge, our study is the first to demonstrate that matrix stiffness can affect cytokinesis. Understanding the cell-type specific contribution of matrix compliance to the regulation of cytokinesis will provide new insights important for development, as well as tissue homeostasis and regeneration.

Integrins promote cytokinesis through the RSK signaling axis.

Cytokinesis is the final stage in cell division. Although integrins can regulate cytokinesis, the mechanisms involved are not fully understood. In this study, we demonstrate that integrin-regulated ERK (extracellular signal-related kinase) and RSK (p90 ribosomal S6 kinase) signaling promotes successful cytokinesis. Inhibiting the activation of ERK and RSK in CHO cells by a mutation in the integrin ß1 cytoplasmic tail or with pharmacological inhibitors results in the accumulation of cells with midbodies and the formation of binucleated cells. Activation of ERK and RSK signaling by the expression of constitutively active RAF1 suppresses the mutant phenotype in a RSK-dependent manner. Constitutively active RSK2 also restores cytokinesis inhibited by the mutant integrin. Importantly, the regulatory role of the RSK pathway is not specific to CHO cells. MCF-10A human mammary epithelial cells and HPNE human pancreatic ductal epithelial cells exhibit a similar dependence on RSK for successful cytokinesis. In addition, depriving mitotic MCF10A cells of integrin-mediated adhesion by incubating them in suspension suppressed ERK and RSK activation and resulted in a failure of cytokinesis. Furthermore, inhibition of RSK or integrins within the 3D context of a developing salivary gland organ explant also leads to an accumulation of epithelial cells with midbodies, suggesting a similar defect in cytokinesis. Interestingly, neither ERK nor RSK regulates cytokinesis in human fibroblasts, suggesting cell-type specificity. Taken together, our results identify the integrin-RSK signaling axis as an important regulator of cytokinesis in epithelial cells. We propose that the proper interaction of cells with their microenvironment through integrins contributes to the maintenance of genomic stability by promoting the successful completion of cytokinesis.

Dynamic Regulation of Integrin α6ß4 During Angiogenesis: Potential Implications for Pathogenic Wound Healing.

OBJECTIVE: Angiogenesis is an essential component of normal cutaneous wound repair, but is altered in pathogenic forms of wound healing, such as chronic wounds and fibrosis. We previously reported that endothelial expression of integrin α6ß4 is developmentally regulated, with α6ß4 expression correlating with tissue maturation and further showed that endothelial α6ß4 is downregulated in explant angiogenesis assays. These data support the hypothesis that dynamic regulation of α6ß4 may play an important role during new vessel formation in healing wounds. APPROACH: To test this hypothesis, we examined the endothelial expression of α6ß4 using a murine model of cutaneous wound healing and in vitro cultures of primary human dermal microvascular endothelial cells (HDMECs). RESULTS: Expression of α6ß4 is downregulated during early stages of wound healing; angiogenic vessels in day 7 wounds do not express α6ß4. Endothelial expression of α6ß4 is resumed in day 14 wounds. Moreover, explanted HDMECs do not express α6ß4, but expression is induced by treatment with histone deacetylase inhibitors. INNOVATION: We provide in vivo data supporting a role for the dynamic regulation of α6ß4 during vessel formation and remodeling during cutaneous wound repair and in vitro findings that suggest endothelial ß4 expression is regulated transcriptionally, providing an important foundation for future studies to understand the transcriptional mechanisms involved in endothelial cell maturation during normal wound repair. CONCLUSION: Our data indicate that α6ß4 is dynamically regulated during angiogenesis and vessel maturation and suggest that disruption of this regulation may contribute to defective angiogenesis associated with diabetic wounds or cutaneous fibrosis.

Integrins regulate microtubule nucleating activity of centrosome through mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling.

Microtubule nucleation is an essential step in the formation of the microtubule cytoskeleton. We recently showed that androgen and Src promote microtubule nucleation and γ-tubulin accumulation at the centrosome. Here, we explore the mechanisms by which androgen and Src regulate these processes and ask whether integrins play a role. We perturb integrin function by a tyrosine-to-alanine substitution in membrane-proximal NPIY motif in the integrin ß1 tail and show that this mutant substantially decreases microtubule nucleation and γ-tubulin accumulation at the centrosome. Because androgen stimulation promotes the interaction of the androgen receptor with Src, resulting in PI3K/AKT and MEK/ERK signaling, we asked whether these pathways are inhibited by the mutant integrin and whether they regulate microtubule nucleation. Our results indicate that the formation of the androgen receptor-Src complex and the activation of downstream pathways are significantly suppressed when cells are adhered by the mutant integrin. Inhibitor studies indicate that microtubule nucleation requires MEK/ERK but not PI3K/AKT signaling. Importantly, the expression of activated RAF-1 is sufficient to rescue microtubule nucleation inhibited by the mutant integrin by promoting the centrosomal accumulation of γ-tubulin. Our data define a novel paradigm of integrin signaling, where integrins regulate microtubule nucleation by promoting the formation of androgen receptor-Src signaling complexes to activate the MEK/ERK signaling pathway.

Androgen and Src signaling regulate centrosome activity.

Microtubules nucleated from gamma-tubulin ring complexes located at the centrosome regulate the localization of organelles, promote vesicular transport and direct cell migration. Although several signaling mechanisms have been identified that regulate microtubule dynamics during interphase, signaling pathways that promote microtubule nucleation remain elusive. We assayed microtubule regrowth following nocodazole washout in human fibroblasts and CHO-K1 cells adhered to fibronectin in either normal serum-free medium or the serum-free, growth-promoting medium, CCM1, which contains IGF1 and androgen, as well as other nutrients. The results indicate that integrin-mediated adhesion is not sufficient to promote rapid microtubule regrowth in either cell type. The addition of androgen, but not IGF1, for 5 minutes was sufficient to promote rapid regrowth and this occurred by a mechanism requiring the androgen receptor. Since Src is a component of the cytoplasmic androgen-receptor-signaling complex, we examined its role using Src siRNA, the Src kinase inhibitor SU6656, and the expression of a constitutively active Src mutant. The data show that Src signaling is both required and sufficient to promote rapid microtubule regrowth in cells adhered to fibronectin. Measurement of the density of microtubules close to the centrosome and the rates of GFP-EB1-labeled microtubules emanating from the centrosome indicated that Src signaling promotes microtubule nucleation. Furthermore, recovery of GFP-gamma-tubulin at the centrosome following photobleaching and measurements of endogenous gamma-tubulin levels at the centrosome showed that androgen and Src signaling regulate the levels of centrosomal gamma-tubulin. Thus, we propose that androgen and Src signaling regulate microtubule nucleation during interphase by promoting the centrosomal localization of gamma-tubulin.

The NPIY motif in the integrin beta1 tail dictates the requirement for talin-1 in outside-in signaling.

Protein interactions with the integrin beta-subunit cytoplasmic domain (beta-tail) are essential for adhesion-dependent processes, including cell spreading and the connection of integrins with actin filaments at adhesion sites. Talin-1 binds to the conserved membrane-proximal NPxY motif of beta-tails (NPIY in beta1 integrin) promoting the inside-out activation of integrins and providing a linkage between integrins and the actin cytoskeleton. Here, we characterize the role of interactions between talin-1 and beta-tail downstream of integrin activation, in the context of recombinant integrins containing either the wild type (WT) or the (YA) mutant beta1A tail, with a tyrosine to alanine substitution in the NPIY motif. In addition to inhibiting integrin activation, the YA mutation suppresses cell spreading, integrin signaling, focal adhesion and stress-fiber formation, as well as microtubule assembly. Constitutive activation of the mutant integrin restores these integrin-dependent processes, bringing into question the importance of the NPIY motif downstream of integrin activation. Depletion of talin-1 using TLN1 siRNA demonstrated that talin-1 is required for cell spreading, focal adhesion and stress-fiber formation, as well as microtubule assembly, even when cells are adhered by constitutively activated WT integrins. Depletion of talin-1 does not inhibit these processes when cells are adhered by constitutively activated mutant integrins, suggesting that the binding of an inhibitory protein to the NPIY motif negatively regulates integrin function when talin-1 is depleted. We identified filamin A (FLNa) as this inhibitory protein; it binds to the beta1A tail in an NPIY-dependent manner and inhibition of FLNa expression in talin-1-depleted cells restores integrin function when cells are adhered by constitutively activated WT integrins. FLNa binds FilGAP, which is a negative regulator of Rac activation. Expression of the dominant inhibitory mutant, FilGAP(DeltaGAP), which lacks GAP activity restores spreading in cells adhered by constitutively activated integrins containing the beta1A tail, but not by integrins containing the beta1D tail, which is known to bind poorly to FLNa. Together, these results suggest that the binding of talin-1 to the NPIY motif is required downstream of integrin activation to promote cell spreading by preventing the inappropriate recruitment of FLNa and FilGAP to the beta1A tail. Our studies emphasize the importance of understanding the mechanisms that regulate the differential binding FLNa and talin-1 to the beta1 tail downstream of integrin activation in promoting integrin function.

Integrins as regulators of the mitotic machinery.

Mitotic spindle bipolarity defines a unique division plane that promotes the successful transmission of genetic material during cytokinesis. The positioning and orientation of the spindle determines the symmetry of cell division and the relative location of daughter cells, which regulate cell fate decisions that contribute to embryonic development and tissue differentiation. Recent studies have identified integrins as regulators of spindle positioning and orientation, as well as spindle bipolarity and cytokinesis. This review summarizes and discusses the current effort focused on understanding how integrins regulate these mitotic events.

Tac-beta1 inhibits FAK activation and Src signaling.

The binding of integrins to extracellular matrix triggers signals that promote cell spreading. We previously demonstrated that expression of the integrin beta1 cytoplasmic domain in the context of a chimeric transmembrane receptor with the Tac subunit of the interleukin-2 receptor (Tac-beta1) inhibits cell spreading. To study the mechanism whereby Tac-beta1 inhibits cell spreading, we examined the effect of Tac-beta1 on early signaling events following integrin engagement namely FAK and Src signaling. We infected primary fibroblasts with adenoviruses expressing Tac or Tac-beta1 and found that Tac-beta1 prevented FAK activation by inhibiting the phosphorylation of FAK at Tyr-397. In contrast, Src activation was maintained, as phosphorylation of Src at Tyr-419 and Tyr-530 were not responsive to expression of Tac-beta1. Importantly, adhesion-induced tyrosine phosphorylation of the Src substrates p130Cas and paxillin was inhibited, indicating that Src signaling was blocked by Tac-beta1. These Src-dependent signaling events were found to require FAK signaling. Our results suggest that Tac-beta1 inhibits cell spreading, at least in part, by preventing the phosphorylation of FAK at Tyr-397 and the assembly of signaling complexes necessary for phosphorylation of p130Cas and other downstream effectors.

Perturbing integrin function inhibits microtubule growth from centrosomes, spindle assembly, and cytokinesis.

In many mammalian cell types, integrin-mediated cell-matrix adhesion is required for the G1-S transition of the cell cycle. As cells approach mitosis, a dramatic remodeling of their cytoskeleton accompanies dynamic changes in matrix adhesion, suggesting a mechanistic link. However, the role of integrins in cell division remains mostly unexplored. Using two cellular systems, we demonstrate that a point mutation in the beta1 cytoplasmic domain (beta1 tail) known to decrease integrin activity supports entry into mitosis but inhibits the assembly of a radial microtubule array focused at the centrosome during interphase, the formation of a bipolar spindle at mitosis and cytokinesis. These events are restored by externally activating the mutant integrin with specific antibodies. This is the first demonstration that the integrin beta1 tail can regulate centrosome function, the assembly of the mitotic spindle, and cytokinesis.

Regulation of the catalytic activity of PTP1B: roles for cell adhesion, tyrosine residue 66, and proline residues 309 and 310.

The reversible phosphorylation of proteins on tyrosine residues is fundamental to a variety of intracellular signaling pathways and is controlled by the actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). While much progress has been made in understanding the regulation of PTKs, there is still relatively little known concerning the regulation of PTPs. Using immune complex phosphatase assays, we demonstrated that the enzymatic activity of the nonreceptor type PTP, PTP1B, is regulated by cell adhesion. Placing primary human foreskin fibroblasts (HFFs) in suspension leads to a distinct increase in PTP1B activity, whereas the readhesion of suspended HFFs onto fibronectin or collagen I inhibited activity. To gain insight into the mechanisms involved, we analyzed recombinant forms of PTP1B mutated at potential regulatory sites. Our results indicated that tyrosine residue 66 is essential for maintaining activity at 37 degrees C. We also found that the C-terminal region of PTP1B and localization to the endoplasmic reticulum are not required for the inhibition of activity by cell adhesion. However, analysis of PA-PTP1B, in which alanines are substituted for prolines 309 and 310, revealed an important role for these residues as the catalytic activity of this mutant did not decrease following readhesion onto collagen I. Since the binding of p130cas and Src to PTP1B is dependent upon these proline residues, we assayed the regulation of PTP1B in mouse embryo fibroblasts deficient in these proteins. We found that neither p130cas nor Src is required for the inhibition of PTP1B activity by adhesion to extracellular matrix proteins. Additionally, pretreatment with cytochalasin D did not prevent the reduction of PTP1B activity when cells adhered to collagen I, indicating that cell spreading is not required for this regulation. The control of the catalytic activity of PTP1B by cell adhesion demonstrated in this study is likely to have important implications for growth factor and insulin signaling.

Cell-spreading assays.

A method is provided to quantitate the extent of cell spreading as a function of the expression level of transfected recombinant proteins. This chapter contains protocols for 1) replating and staining transfected cells for immunofluorescence microscopy, 2) optimizing image acquisition so that fluorescence intensity can be measured independent of cell morphology, and 3) quantitating cell area and expression levels of recombinant proteins for individual transfected cells using ImagePro-Plus software. This method can be used to further our understanding of intracellular signals and protein interactions that regulate cell spreading.

Endothelial expression of the alpha6beta4 integrin is negatively regulated during angiogenesis.

Development and homeostasis of the vascular system requires integrin-facilitated cellular adhesion, migration, proliferation and survival. A specific role for the alpha6beta4 integrin in the vasculature, however, has not been identified. Using immunohistochemistry, we observed alpha6beta4 expression on the dermal microvasculature of human foreskin. Analysis of individual cells isolated from trypsin-disrupted foreskin tissue indicated that alpha6beta4 was expressed by a subset of epithelial and endothelial cells, and not by smooth muscle cells. Expression of alpha6beta4 was also analyzed during new vessel growth using explants of human saphenous vein cultured in fibrinogen gels. The results indicate that alpha6beta4 is not expressed by outgrowing endothelial cells, and is downregulated by the original alpha6beta4-positive endothelial cells of the explant. To determine whether alpha6beta4 is expressed during angiogenesis in vivo, the expression of the beta4 subunit was analyzed during the development of the mouse mystacial (whisker) pad. Immunohistochemical staining of the whisker pad indicates that beta4 is expressed by the adult vasculature. To identify when and where beta4 is turned on in the vasculature, we examined the whisker pads from the developing embryo (E19.5 pc), and from postnatal days zero (P0), three (P3) and seven (P7) pups. The expression of alpha6beta4 was found to be turned on spatially and temporally from caudal to rostral regions and from the deep to superficial vasculature, correlating with the maturation of the whisker pad and its corresponding vasculature. Together, these findings suggest a potential role for alpha6beta4 as a negative component of the angiogenic switch, whereas expression of alpha6beta4 on the adult vasculature may indicate regions requiring additional adhesive mechanisms.

Regulation of the association of alpha 6 beta 4 with vimentin intermediate filaments in endothelial cells.

The adhesion of microvascular endothelial cells to their underlying basement membrane is important for the maintenance of vascular integrity. Most integrins function in endothelial cell adhesion by forming a transmembrane link between their basement membrane ligand and the actin microfilament cytoskeleton. The alpha 6 beta 4 laminin-binding integrin, however, associates with vimentin intermediate filaments (IFs) in microvascular endothelial cells and therefore is likely to uniquely contribute to the barrier function of the endothelium. In this study, we examined the regulation of alpha 6 beta 4-vimentin IF association. We first tested the requirement for alpha 6 beta 4-laminin interactions and actin microfilament assembly. We found that alpha 6 beta 4 associated with vimentin IFs when cells were adherent to either laminin 5 or fibronectin, indicating that this association can occur independent of alpha 6 beta 4-ligand interactions. Additionally, we found that alpha 6 beta 4 was associated with vimentin IFs prior to cell spreading, indicating that changes in the microfilament cytoskeleton associated with changes in cell shape are also not required. Thus, although the association of alpha 6 beta 4 with vimentin IFs may strengthen cell adhesion by providing endothelial cells with an additional transmembrane linkage between the basement membrane and the cytoskeleton, this association is not itself regulated by alpha 6 beta 4-mediated adhesion. Finally, we tested the role of plectin in the association of alpha 6 beta 4 with vimentin IFs. Plectin is known to bind in vitro to both IFs and the beta 4 cytoplasmic domain (beta 4 tail), suggesting that it may be important for this linkage. Therefore, we generated deletion mutants of the beta 4 tail and compared the ability of alpha 6 beta 4 containing these deletions to associate with vimentin IFs. We targeted the two regions of the beta 4 tail known to bind to plectin IN VITRO: the N-terminal and C-terminal plectin binding sites. We found that deletion of the N-terminal binding site inhibited the association of alpha 6 beta 4 with vimentin IFs. Thus, plectin-beta 4 tail interactions may play an important role in connecting alpha 6 beta 4 with vimentin IFs and may prove to be important targets in the regulation of this association in endothelial cells.

The integrin beta tail is required and sufficient to regulate adhesion signaling to Rac1.

Rac1 is a small Rho family GTPase that regulates changes in cell morphology associated with cell spreading and migration. Integrin-mediated adhesion is known to activate Rac1 and to regulate the interaction of Rac1 with downstream effectors. Currently, it is not clear how integrins signal Rac1 activation following cell adhesion. Integrin beta cytoplasmic domains (beta-tails) are known to be required for integrin-mediated cell spreading, and isolated beta tails expressed as tac-beta tail chimeras can inhibit cell spreading indicating that protein interactions with beta tails can regulate this process. Our recent studies demonstrated that the expression of constitutively activated Rac1 can restore cell spreading inhibited by tac beta tail chimeras, suggesting a role for Rac1 in the regulation of cell spreading by beta tails. Hence, we examined the role of beta tails in integrin activation of Rac1. By using recombinant wild-type and mutant integrin heterodimers, we demonstrate that integrin beta tails are required for adhesion to increase Rac1-GTP loading. We demonstrate that clustering tac-beta tail chimeras, on the surface of cells in suspension, activates Rac1. Thus, beta tails are not only required, but also sufficient for integrin-triggered Rac1 activation. Our findings indicate that integrin beta-tails are an important link between integrin engagement and Rac1 signaling, and that protein interactions initiated at beta tails are sufficient for integrins to regulate Rac1 activity.

TNF-alpha disruption of lung endothelial integrity: reduced integrin mediated adhesion to fibronectin.

Tumor necrosis factor-alpha (TNF-alpha) causes an increase in transendothelial protein permeability of confluent monolayers of calf pulmonary artery endothelial (CPAE) cells, and the addition of plasma fibronectin (pFn) to the culture medium can attenuate this increase in permeability. We determined if reduced integrin function had a role in decreased endothelial cell adhesion to immobilized Fn after exposure of the endothelial monolayers to TNF-alpha. TNF-alpha also causes a reorganization of the subendothelial Fn rich matrix and a significant loss in RGD-dependent adhesion of TNF-alpha treated CPAE cells to pFn coated surfaces. However, flow cytometry revealed no decrease in alpha(5)beta(1) or total beta(1) integrin expression on the surface of the CPAE cells after TNF-alpha. Reduced CPAE adhesion to immobilized Fn was, in part, due to a loss of beta(1)-integrin function since the beta(1)-integrin blocking antibody mAb 13 significantly (P < 0.05) prevented the adhesion of normal control CPAE cells but did not further reduce the adhesion of TNF-alpha-treated cells. In addition, antibodies which activate beta(1) integrins restored (P < 0.05) adhesion of TNF-alpha-treated cells to immobilized pFn but did not alter the adhesion of control cells. Despite reduced ability to adhere to immobilized Fn, TNF-alpha-treated CPAE monolayers demonstrated increased binding and incorporation of fluid-phase pFn into the subendothelial extracellular matrix (ECM) as measured by the analysis of the deoxycholate (DOC) detergent insoluble pool of (125)I-Fn in the cell layer. In contrast to the RGD-mediated adhesion of CPAE cells to matrix Fn, the increased binding of soluble pFn after TNF-alpha was not inhibited by RGD peptides or mAb 13. Thus reduced integrin-dependent adhesion of the CPAE cells to matrix Fn as well as disruption of the Fn matrix may contribute to the increased protein permeability of previously confluent endothelial monolayer after TNF-alpha. In addition, increased ability for the monolayer to incorporate fluid-phase Fn into the ECM after TNF-alpha via a non-beta(1)- integrin dependent mechanism may be a compensatory response to stabilize the Fn matrix and the endothelial barrier.