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At least 541 records · Page 30Linked to original sources

PubMed2004

CD98hc (SLC3A2) mediates integrin signaling.

Integrins regulate cellular behaviors through signaling pathways, including Rho GTPases and kinases. CD98 heterodimers, comprised of a heavy chain (CD98hc, SLC3A2) and one of several light chains, interact with integrins through CD98hc. CD98hc overexpression leads to anchorage-independent cell growth and tumorigenesis in 3T3 fibroblasts and activates certain integrin-regulated signaling pathways. To establish the biological function of CD98hc, we disrupted the gene and analyzed CD98hc-null cells. Here we report that CD98hc contributes to integrin-dependent cell spreading, cell migration, and protection from apoptosis. Furthermore, CD98hc is required for efficient adhesion-induced activation of Akt and Rac GTPase, major contributors to the integrin-dependent signals involved in cell survival and cell migration. CD98 promotes amino acid transport through its light chains; however, a CD98hc mutant that interacts with beta1 integrins, but not CD98 light chains, restored integrin-dependent signaling and protection from apoptosis. beta1 integrins are involved in the pathogenesis of certain cancers. CD98hc deletion markedly impaired the ability of embryonic stem cells to form teratocarcinomas in mice; teratocarcinoma formation was reconstituted by reexpression of CD98hc or of the mutant that interacts exclusively with integrins. Thus, CD98hc is an integrin-associated protein that mediates integrin-dependent signals, which promote tumorigenesis.

Animals↗

PubMed1999

Activation of c-Raf-1 kinase signal transduction pathway in alpha(7) integrin-deficient mice.

Integrin alpha(7)-deficient mice develop a novel form of muscular dystrophy. Here we report that deficiency of alpha(7) integrin causes an activation of the c-Raf-1/mitogen-activated protein (MAP) 2 kinase signal transduction pathway in muscle cells. The observed activation of c-Raf-1/MAP2 kinases is a specific effect, because the alpha(7) integrin deficiency does not cause unspecific stress as determined by measurement of the Hsp72/73 level and activity of the JNK2 kinase. Because an increased level of activated FAK was found in muscle of alpha(7) integrin-deficient mice, the activation of c-Raf-1 kinase is triggered most likely by an integrin-dependent pathway. In accordance with this, in the integrin alpha(7)-deficient mice, part of the integrin beta(1D) variant in muscle is replaced by the beta(1A) variant, which permits the FAK activation. A recent report describes that integrin activity can be down-modulated by the c-Raf-1/MAP2 kinase pathway. Specific activation of the c-Raf-1/MAP2 kinases by cell-permeable peptides in skeletal muscle of rabbits causes degeneration of muscle fibers. Therefore, we conclude that in alpha(7) integrin-deficient mice, the continuous activation of c-Raf-1 kinase causes a permanent reduction of integrin activity diminishing integrin-dependent cell-matrix interactions and thereby contributing to the development of the dystrophic phenotype.

Animals↗

PubMed2000

Urokinase-type plasminogen activator receptor (CD87) is a ligand for integrins and mediates cell-cell interaction.

Binding of urokinase-type plasminogen activator (uPA) to its receptor (uPAR/CD87) regulates cellular adhesion, migration, and tumor cell invasion. However, it is unclear how glycosyl phosphatidylinositol-anchored uPAR, which lacks a transmembrane structure, mediates signal transduction. It has been proposed that uPAR forms cis-interactions with integrins as an associated protein and thereby transduces proliferative or migratory signals to cells upon binding of uPA. We provide evidence that soluble uPAR (suPAR) specifically binds to integrins alpha4beta1, alpha6beta1, alpha9beta1, and alphavbeta3 on Chinese hamster ovary cells in a cation-dependent manner. Anti-integrin and anti-uPAR antibodies effectively block binding of suPAR to these integrins. Binding of suPAR to alpha4beta1 and alphavbeta3 is blocked by known soluble ligands and by the integrin mutations that inhibit ligand binding. These results suggest that uPAR is an integrin ligand rather than, or in addition to, an integrin-associated protein. In addition, we demonstrate that glycosyl phosphatidylinositol-anchored uPAR on the cell surface specifically binds to integrins on the apposing cells, suggesting that uPAR-integrin interaction may mediate cell-cell interaction (trans-interaction). These previously unrecognized uPAR-integrin interactions may allow uPAR to transduce signals through the engaged integrin without a hypothetical transmembrane adapter and may provide a potential therapeutic target for control of inflammation and cancer.

Animals↗

PubMed2003

Modulation of Thr phosphorylation of integrin beta1 during muscle differentiation.

By using transient elevations of cytosolic free calcium levels triggered by integrin antibody or laminin (Kwon, M. S., Park, C. S., Choi, K., Park, C.-S., Ahnn, J., Kim, J. I., Eom, S. H., Kaufman, S. J., and Song, W. K. (2000) Mol. Biol. Cell 11, 1433-1443), we have demonstrated that protein phosphatase 2A (PP2A) is implicated in the regulation of reversible phosphorylation of integrin. In E63 skeletal myoblasts, the treatment of PP2A inhibitors such as okadaic acid and endothall induces an increase of phosphorylation of integrin beta1A and thereby inhibits integrin-induced elevation of cytosolic calcium level and formation of focal adhesions. None of these effects were in differentiated myotubes expressing the alternate beta1D isoform. In the presence of okadaic acid, PP2A in association with integrin beta1A was reduced on myoblasts, whereas beta1D on myotubes remained bound with PP2A. Both co-immunoprecipitation and in vitro phosphatase assays revealed that dephosphorylation of residues Thr788-Thr789 in the integrin beta1A cytoplasmic domain is dependent upon PP2A activity. Mutational analysis of the cytoplasmic domain and confocal microscopy experiments indicated that substitution of Thr788-Thr789 with Asn788-Asn789 is of critical importance for regulating the function of integrin beta1. These results suggest that PP2A may be a primary regulator of threonine phosphorylation of integrin beta1A and subsequent activation of downstream signaling molecules. Taken together, we propose that dephosphorylation of residues Thr788-Thr789 in the cytoplasmic domain of integrin beta1A may contribute to the linkage of integrins to focal adhesion sites and induce the association with cytoskeleton proteins. The switch of integrin beta1A to beta1D isoform in myotubes therefore may be a mechanism to escape from phospho-regulation by PP2A and promotes a more stable association of the cytoskeleton with the extracellular matrix.

Amino Acid Sequence↗

PubMed2004

Expression of the vacuolar H+-ATPase 16-kDa subunit results in the Triton X-100-insoluble aggregation of beta1 integrin and reduction of its cell surface expression.

Vacuolar H(+)-ATPase functions as a vacuolar proton pump and is responsible for acidification of intracellular compartments such as the endoplasmic reticulum, Golgi, lysosomes, and endosomes. Previous reports have demonstrated that a 16-kDa subunit (16K) of vacuolar H(+)-ATPase via one of its transmembrane domains, TMD4, strongly associates with beta(1) integrin, affecting beta(1) integrin N-linked glycosylation and inhibiting its function as a matrix adhesion receptor. Because of this dramatic inhibition of beta(1) integrin-mediated HEK-293 cell motility by 16K expression, we investigated the mechanism by which 16 kDa was having this effect. Using HT1080 cells whose alpha(5)beta(1) integrin-mediated adhesion to fibronectin has been extensively studied, the expression of 16 kDa also resulted in reduced cell spreading on fibronectin-coated substrates. A pulse-chase study of beta(1) integrin biosynthesis indicated that 16K expression down-regulated the level of the 110-kDa biosynthetic form of beta(1) integrin (premature form) and, consequently, the level of the 130-kDa form of beta(1) integrin (mature form). Further experiments showed that the normal levels of association between the premature beta(1) integrin form and calnexin were significantly decreased by the expression of either 16 kDa or TMD4. Expression of 16 kDa also resulted in a Triton X-100-insoluble aggregation of an unusual 87-kDa form of beta(1) integrin. Interestingly, both Western blotting and a pulse-chase experiment showed co-immunoprecipitation of calnexin and 16K. These results indicate that 16K expression inhibits beta(1) integrin surface expression and spreading on matrix by a novel mechanism that results in reduced levels of functional beta(1) integrin.

Biotinylation↗

PubMed2002

Ionizing radiation induces up-regulation of functional beta1-integrin in human lung tumour cell lines in vitro.

PURPOSE: Cell-matrix interactions are in part mediated through the beta1-integrin pathway regulating cell survival, proliferation, adhesion and migration. This study was performed to elucidate alterations of expression of the beta1-integrin and its co-localized protein kinase, integrin-linked kinase (ILK), after exposure to ionizing radiation in two lung carcinoma cell lines in the presence or absence of different beta1-integrin-dependent matrix proteins. MATERIALS AND METHODS: Exponentially growing A549 and SKMES1 cells grown on fibronectin, laminin, BSA or plastic were exposed to 2 Gy or 6 Gy. Besides colony formation assays (0.5-8 Gy) and immediate plating experiments, flow cytometry (for beta1-integrin) and immunoblotting (for beta1-integrin and ILK) were carried out to analyze the protein expression. The localization of both proteins plus filamentous (f-) actin was further examined by immunofluorescence staining and laser confocal scanning microscopy. Functionality of the beta1 receptor subunit after irradiation was investigated in adhesion assays. RESULTS: A549 and SKMES1 cells grown on fibronectin or laminin demonstrated a significant increase in cell survival after irradiation compared to cells grown on BSA or plastic. Immediate plating of cells after irradiation on fibronectin did not show an improved survival. Flow cytometric and Western blot data showed a dose- and matrix-dependent induction of beta1-integrin and ILK expression after irradiation within 48 h. Adhesion to fibronectin or laminin compared to BSA or plastic was increased by 10-fold after irradiation, demonstrating these specific cell surface receptors to be functional. The staining of beta1-integrin and ILK in A549 cells confirmed the radiation-induced up-regulation of both proteins. Additionally, beta1-integrin and ILK co-localized with accumulated actin fibers at the cytoplasmic face of the cell membrane at confined areas. CONCLUSIONS: Ionizing radiation strongly induced the expression of functional beta1-integrin and ILK in the two lung cancer cell lines, A549 and SKMES1, dependent on different matrices used. Additionally, the subcellular localization of both proteins was altered by irradiation, and the individual cellular radiosensitivity was reduced in the presence of an extracellular matrix. On the one hand, this may result in aggravated therapeutic approaches and on the other hand, cells could adhere more strongly in their environment by the increase in functional surface receptor density preventing metastasis. Concerning intravascular located tumour cells, beta1-integrin up-regulation might enable these cells to adhere to the endothelium, which represents a prerequisite for metastatic disease. Identification of such mechanisms will provide considerable insights into the understanding of tumorigenicity and metastatic phenotypes, possibly leading to new, optimized radiochemotherapeutic regimens.

Cell Adhesion↗

PubMed1994

Evidence that cadherins play a role in the downregulation of integrin expression that occurs during keratinocyte terminal differentiation.

In epidermis the onset of terminal differentiation normally coincides with inhibition of integrin function and expression, thereby ensuring that differentiating cells are selectively expelled from the basal layer. However, when stratification of cultured human epidermal keratinocytes is prevented by reducing the calcium concentration of the medium to 0.1 mM, keratinocytes initiate terminal differentiation while still attached to the culture substrate. We have examined the mechanism by which differentiating keratinocytes adhere to extracellular matrix proteins in low calcium medium and the consequences of inducing stratification by raising the calcium ion concentration to 1.8 mM (Standard Medium). In low calcium medium keratinocytes co-expressed integrins and terminal differentiation markers such as involucrin and peanut lectin-binding glycoproteins: differentiating cells contained integrin mRNA, synthesized integrin proteins de novo and expressed functional mature integrins. There were no differences in integrin synthesis, maturation or break down in low calcium or standard medium, although the level of beta 1 integrins on the surface of proliferating cells was higher in standard medium. Within 6 h of transfer from low calcium to standard medium integrin mRNA was no longer detectable in terminally differentiating cells, integrins were being lost from the cell surface, and selective migration out of the basal layer had begun. Antibodies to P- and E-cadherin, which block calcium-induced stratification, prevented the selective loss of integrin mRNA and protein from terminally differentiating cells. This suggests that cadherins may play a role in the down-regulation of integrin expression that is associated with terminal differentiation.

Cadherins↗

PubMed1995

Integrin function: molecular hierarchies of cytoskeletal and signaling molecules.

Integrin receptors play important roles in organizing the actin-containing cytoskeleton and in signal transduction from the extracellular matrix. The initial steps in integrin function can be analyzed experimentally using beads coated with ligands or anti-integrin antibodies to trigger rapid focal transmembrane responses. A hierarchy of transmembrane actions was identified in this study. Simple integrin aggregation triggered localized transmembrane accumulation of 20 signal transduction molecules, including RhoA, Rac1, Ras, Raf, MEK, ERK, and JNK. In contrast, out of eight cytoskeletal molecules tested, only tensin coaccumulated. Integrin aggregation alone was also sufficient to induce rapid activation of the JNK pathway, with kinetics of activation different from those of ERK. The tyrosine kinase inhibitors herbimycin A or genistein blocked both the accumulation of 19 out of 20 signal transduction molecules and JNK- and ERK-mediated signaling. Cytochalasin D had identical effects, whereas three other tyrosine kinase inhibitors did not. The sole exception among signaling molecules was the kinase pp125FAK which continued to coaggregate with alpha 5 beta 1 integrins even in the presence of these inhibitors. Tyrosine kinase inhibition also failed to block the ability of ligand occupancy plus integrin aggregation to trigger transmembrane accumulation of the three cytoskeletal molecules talin, alpha-actinin, and vinculin; these molecules accumulated even in the presence of cytochalasin D. However, it was necessary to fulfill all four conditions, i.e., integrin aggregation, integrin occupancy, tyrosine kinase activity, and actin cytoskeletal integrity, to achieve integrin-mediated focal accumulation of other cytoskeletal molecules including F-actin and paxillin. Integrins therefore mediate a transmembrane hierarchy of molecular responses.

Cell Adhesion↗

PubMed2006

Cell surface transglutaminase promotes RhoA activation via integrin clustering and suppression of the Src-p190RhoGAP signaling pathway.

Tissue transglutaminase (tTG) is a multifunctional protein that serves as cross-linking enzyme and integrin-binding adhesion coreceptor for fibronectin on the cell surface. Previous work showed activation of small GTPase RhoA via enzymatic transamidation by cytoplasmic tTG. Here, we report an alternative nonenzymatic mechanism of RhoA activation by cell surface tTG. Direct engagement of surface tTG with specific antibody or the fibronectin fragment containing modules I(6)II(1,2)I(7-9) increases RhoA-GTP levels. Integrin-dependent signaling to RhoA and its downstream target Rho-associated coiled-coil containing serine/threonine protein kinase (ROCK) is amplified by surface tTG. tTG expression on the cell surface elevates RhoA-GTP levels in nonadherent and adherent cells, delays maximal RhoA activation upon cell adhesion to fibronectin and accelerates a rise in RhoA activity after binding soluble integrin ligands. These data indicate that surface tTG induces integrin clustering regardless of integrin-ligand interactions. This notion is supported by visualization of integrin clusters, increased susceptibility of integrins to chemical cross-linking, and biochemical detection of large integrin complexes in cells expressing tTG. In turn, integrin aggregation by surface tTG inhibits Src kinase activity and decreases activation of the Src substrate p190RhoGAP. Moreover, pharmacological inhibition of Src kinase reveals inactivation of Src signaling as the primary cause of elevated RhoA activity in cells expressing tTG. Together, these findings show that surface tTG amplifies integrin-mediated signaling to RhoA/ROCK via integrin clustering and down-regulation of the Src-p190RhoGAP regulatory pathway.

Animals↗

PubMed2004

Integrins are not involved in the process of human sperm-oolemmal fusion.

BACKGROUND: We investigated whether integrins are required for the human sperm-oocyte binding and fusion processes. METHODS: The expression of several integrin subunits at the human oocyte plasma membrane was investigated using immunofluorescence microscopy, and the functional role of integrins expressed at the human oocyte surface in sperm-oocyte interaction was studied using a zona-free human oocyte binding and fusion assay. A total of 144 unfertilized oocytes were stained with anti-integrin antibodies and 147 zona-free unfertilized oocytes were inseminated in the presence of various anti-integrin antibodies that were expressed in oocyte plasma membrane. RESULTS: The antibodies of six alpha integrin subunits (alpha(2), alpha(3), alpha(5), alpha(6), alpha(V), alpha(M)) and six beta integrin subunits (beta(1), beta(2), beta(3), beta(4), beta(5), beta(6)) were bound to the surface of fixed unfertilized oocytes. In contrast, the presence of alpha(1) and alpha(4) subunits could not be verified. The human sperm-oocyte binding was only partially inhibited by blocking antibodies of alpha(2), alpha(3), alpha(5), alpha(6), alpha(V), alpha(M), beta(1), beta(2) and beta(3) with a maximum of 55% inhibition, but antibodies of beta(4), beta(5) and beta(6) showed no effect on sperm-oolemmal binding. A similar reduction of the number of fused sperm was observed. However, the ratio of fused sperm to total sperm (bound and fused) was not impaired by all integrin antibodies, suggesting that integrins had no role in the sperm-oolemmal fusion process. CONCLUSIONS: These results suggest that one of the binding mechanisms can be inhibited by integrin antibodies but that this mechanism does not play an essential role in the human sperm-oolemmal binding and fusion processes. The other mechanisms, insensitive to integrins, may involve both binding and fusion processes in human oocytes.

Antibodies↗

PubMed2000

Beta1 integrin expression during normal and low score normal avian myogenesis.

Skeletal muscle development is, in part, regulated by myoblast-extracellular matrix interactions mediated by the transmembrane integrin family of heterodimeric receptors. The avian genetic muscle weakness, low score normal (LSN), exhibits modified myotube and sarcomere structure that may be associated with altered integrin expression. Protein expression of the beta1 integrin subunit was measured during normal and LSN Pectoralis major muscle development at 14, 16, and 18 d of embryonic development, and 1 d and 1 and 6 wk posthatch. During embryonic development, integrin expression was downregulated. However, by 1 wk posthatch, integrin levels were upregulated and remained elevated through 6 wk posthatch. This pattern was observed in both normal and LSN muscle development. Overall, beta1 integrin levels were lower in the LSN P. major muscle. In normal and LSN satellite cell cultures, beta1 integrin expression was low during proliferation. In early differentiation, beta1 integrin expression increased and was then downregulated. As observed in the muscle extracts, LSN beta1 integrin expression was significantly lower during differentiation. These results suggest that the regulation of beta1 integrin expression is critical to the progression of myogenesis, and, during LSN myogenesis, decreased expression of beta1 integrin may be associated with modifications in muscle structure.

Animals↗

PubMed2004

High expression of integrin beta1 and p53 is a greater poor prognostic factor than clinical stage in small-cell lung cancer.

The purpose of this study was to investigate the possible association between expression of integrin beta1 and p53 and survival in patients with small-cell lung cancer (SCLC). One hundred four patients with SCLC who had received an initial course of a full-dose of combination chemotherapy between February 1989 and July 1999 were entered in the study. Transbronchial biopsy specimens of tumors obtained before chemotherapy were subjected to immunostaining for integrin beta1 and p53. Twenty-eight and 58 patients could not be evaluated for integrin beta1 and p53 immunostaining, respectively, because the tissue samples had been crushed during the biopsy. Fifty-four patients had tumors with less than or equal to 25% integrin beta1-positive cells and 22 patients had tumors with more than 25% integrin beta1-positive cells, whereas 19 and 27 patients had tumors with less than or equal to 50% and more than 50% p53-positive cells, respectively. By comparison, the overall survival of patients with high expression of integrin beta1 and p53 were significantly worse than those of individuals whose tumors had low expression (log-rank test, p = 0.046 and p = 0.0067, respectively). Moreover, the overall survival of patients with high expression of either integrin beta1 or p53 (n = 42) was significantly worse than that of other patients without high expression of integrin beta1 and p53 (n = 38; log-rank test, p = 0.0003; Wilcoxon test, p = 0.0026). The association between survival and prognostic factors, including gender, age, performance status, clinical stage, and integrin beta1/p53 expression was examined by the Cox proportional hazards model; only integrin beta1/p53 expression was found to be a significant independent factor (hazard ratio = 0.394, p = 0.0005). In conclusion, the high expression of integrin beta1 and p53 in tumor cells is a greater poor prognostic factor than clinical stage in patients with SCLC.

Aged↗

PubMed1999

Cellular entry of hantaviruses which cause hemorrhagic fever with renal syndrome is mediated by beta3 integrins.

Hantaviruses replicate primarily in the vascular endothelium and cause two human diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). In this report, we demonstrate that the cellular entry of HFRS-associated hantaviruses is facilitated by specific integrins expressed on platelets, endothelial cells, and macrophages. Infection of human umbilical vein endothelial cells and Vero E6 cells by the HFRS-causing hantaviruses Hantaan (HTN), Seoul (SEO), and Puumala (PUU) is inhibited by antibodies to alphavbeta3 integrins and by the integrin ligand vitronectin. The cellular entry of HTN, SEO, and PUU viruses, but not the nonpathogenic Prospect Hill (PH) hantavirus (i.e., a virus with no associated human disease), was also mediated by introducting recombinant alphaIIbbeta3 or alphavbeta3 integrins into beta3-integrin-deficient CHO cells. In addition, PH infectivity was not inhibited by alphavbeta3-specific sera or vitronectin but was blocked by alpha5beta1-specific sera and the integrin ligand fibronectin. RGD tripeptides, which are required for many integrin-ligand interactions, are absent from all hantavirus G1 and G2 surface glycoproteins, and GRGDSP peptides did not inhibit hantavirus infectivity. Further, a mouse-human hybrid beta3 integrin-specific Fab fragment, c7E3 (ReoPro), also inhibited the infectivity of HTN, SEO, and PUU as well as HPS-associated hantaviruses, Sin Nombre (SN) and New York-1 (NY-1). These findings indicate that pathogenic HPS- and HFRS-causing hantaviruses enter cells via beta3 integrins, which are present on the surfaces of platelets, endothelial cells, and macrophages. Since beta3 integrins regulate vascular permeability and platelet function, these findings also correlate beta3 integrin usage with common elements of hantavirus pathogenesis.

Abciximab↗

PubMed1996

Sp1 binding plays a critical role in Erb-B2- and v-ras-mediated downregulation of alpha2-integrin expression in human mammary epithelial cells.

The human alpha2-integrin gene is transcriptionally downregulated in a nontumorigenic human mammary epithelial cell line, MTSV1-7, and its clonal variant HB2, overexpressing the Erb-B2 oncogene. In this study, we have used deletion mutations within the alpha2-integrin promoter inserted 5' of the chloramphenicol acetyltransferase or luciferase reporter genes to identify the element that is responsible for the Erb-B2-mediated downregulation. The results of the transient-transfection assay showed that the Sp1 binding element located in the core region (positions --64 to +1) of the alpha2-integrin promoter plays an essential role in the alpha2-integrin promoter activity and its downregulation by Erb-B2. By gel shift assay, we have demonstrated that this element binds with a high degree of affinity not only to Sp1, but also to Sp3. The downregulation of the alpha2-integrin promoter activity could also be achieved by overexpression of v-Hras (v-ras), suggesting that the signals generated by Erb-B2, which lead to downregulation of the alpha2-integrin gene expression, may proceed through the ras pathway. Both the Erb-B2- and the v-ras-overexpressing cells exhibited a Sp1 DNA binding activity lower than that of the parental line, while the relative levels of Sp1 protein in these cells were not altered. The Erb-B2- and v-ras-mediated downregulation could be reversed by the overexpression of Sp1 and by a dominant negative variant of ras (rasN17), confirming the importance of Sp1 and the ras pathway. The inhibitory effects of Erb-B2 on transcriptional activity of the alpha2-integrin promoter were observed in transient-cotransfection assays using alpha2-integrin reporter plasmids and plasmids expressing the Erb-B2 or v-ras oncogene. The same effects were seen when an alpha2-integrin reporter gene construct was transfected into MTSV1-7 or HB2 cells permanently overexpressing Erb-B2 or v-ras. The effects of Erb-B2 or v-ras on the transcriptional activity of the alpha2-integrin promoter were observed in nontumorigenic luminal epithelial cell lines (MTSV1-7 and HB2) as well as in the breast cancer cell line T47D. These data suggest that in luminal epithelial cells and the breast cancers which develop from them, the Erb-B2 proto-oncogene signaling leads to inhibition of (alpha)2(beta)1-integrin gene expression and could contribute to the disruption of tissue architecture seen in breast cancers.

Antigens, CD↗

PubMed1998

Identification of a proline-rich sequence in the CD2 cytoplasmic domain critical for regulation of integrin-mediated adhesion and activation of phosphoinositide 3-kinase.

The CD2 molecule is one of several lymphocyte receptors that rapidly initiates signaling events regulating integrin-mediated cell adhesion. CD2 stimulation of resting human T cells results within minutes in an increase in beta1-integrin-mediated adhesion to fibronectin. We have utilized the HL60 cell line to map critical residues within the CD2 cytoplasmic domain involved in CD2 regulation of integrin function. A panel of CD2 cytoplasmic domain mutants was constructed and analyzed for their ability to upregulate integrin-mediated adhesion to fibronectin. Mutations in the CD2 cytoplasmic domain implicated in CD2-mediated interleukin-2 production or CD2 avidity do not affect CD2 regulation of integrin activity. A proline-rich sequence, K-G-P-P-L-P (amino acids 299 to 305), is essential for CD2-mediated regulation of beta1 integrin activity. CD2-induced increases in beta1 integrin activity could be blocked by two phosphoinositide 3-kinase (PI 3-K) inhibitors or by overexpression of a dominant negative form of the p85 subunit of PI 3-K. In addition, CD2 cytoplasmic domain mutations that abrogate CD2-induced increases in integrin-mediated adhesion also ablate CD2-induced increases in PI 3-K enzymatic activity. Surprisingly, CD2 cytoplasmic domain mutations that inhibit CD2 regulation of adhesion do not affect the constitutive association of the p85 subunit of PI 3-K association with CD2. Mutation of the proline residues in the K-G-P-P-L-P motif to alanines prevented CD2-mediated activation of integrin function and PI 3-K activity but not mitogen-activated protein (MAP) kinase activity. Furthermore, the MEK inhibitor PD 098059 blocked CD2-mediated activation of MAP kinase but had no effect on CD2-induced adhesion. These studies identify a proline-rich sequence in CD2 critical for PI 3-K-dependent regulation of beta1 integrin adhesion by CD2. In addition, these studies suggest that CD2-mediated activation of MAP kinase is not involved in CD2 regulation of integrin adhesion.

Amino Acid Sequence↗

PubMed2006

The characterization of alpha5-integrin expression on tubular epithelium during renal injury.

The hallmark of progressive chronic kidney disease is the deposition of extracellular matrix proteins and tubulointerstitial fibrosis. Integrins mediate cell-extracellular matrix interaction and may play a role tubular epithelial injury. Murine primary tubular epithelial cells (TECs) express alpha(5)-integrin, a fibroblast marker and the natural receptor for fibronectin. Microscopy localized alpha(5)-integrin on E-cadherin-positive cells, confirming epithelial expression. The expression of alpha(5)-integrin increased in TECs grown on fibronectin and occurred in parallel with an upregulation of alpha-smooth muscle actin (alphaSMA), a marker of epithelial-mesenchymal transition (EMT). Exposure of TECs to transforming growth factor (TGF)-beta also increased TEC alpha(5)-integrin expression in association with alphaSMA and EMT. Knock-down of alpha5-integrin expression with short interfering RNA attenuated the TGF-beta induction of alphaSMA but did not alter morphologic EMT. Rather, alpha5-integrin was necessary for epithelial cell migration on fibronectin but not type IV collagen during cell spreading and epithelial wound healing in vitro. Immunohistochemistry revealed basolateral tubular epithelial alpha(5)-integrin expression in mouse kidneys after unilateral ureteric obstruction but not in contralateral control kidneys. In patient biopsies of nondiabetic kidney disease, alpha(5)-integrin expression was increased significantly in the renal interstitium. Focal basolateral staining was also detected in injured, but not in normal, tubular epithelium. In summary, these data show that TECs are induced to express alpha(5)-integrin during EMT and tubular epithelial injury in vitro and in vivo. These results increase our understanding of the biology of integrins during EMT and tubular injury in chronic kidney disease.

Animals↗

PubMed2005

The glycosphingolipid, lactosylceramide, regulates beta1-integrin clustering and endocytosis.

Glycosphingolipids are known to play roles in integrin-mediated cell adhesion and migration; however, the mechanisms by which glycosphingolipids affect integrins are unknown. Here, we show that addition of the glycosphingolipid, C8-lactosylceramide (C8-LacCer), or free cholesterol to human fibroblasts at 10 degrees C causes the formation of glycosphingolipid-enriched plasma membrane domains as shown by visualizing a fluorescent glycosphingolipid probe, BODIPY-LacCer, incorporated into the plasma membrane of living cells. Addition of C8-LacCer or cholesterol to cells initiated the clustering of beta1-integrins within these glycosphingolipid-enriched domains and the activation of the beta1-integrins as assessed using a HUTS antibody that only binds activated integrin. On warming to 37 degrees C, beta1-integrins were rapidly internalized via caveolar endocytosis in cells treated with C8-LacCer or cholesterol, whereas little beta1-integrin was endocytosed in untreated fibroblasts. Incubation of cells with C8-LacCer or cholesterol followed by warm-up caused src activation, a reorganization of the actin cytoskeleton, translocation of RhoA GTPase away from the plasma membrane as visualized using total internal reflection fluorescence microscopy, and transient cell detachment. These studies show that LacCer can regulate integrin function both by modulating integrin clustering in microdomains and by regulating integrin endocytosis via caveolae. Our findings suggest the possibility that aberrant levels of glycosphingolipids found in cancer cells may influence cell attachment events by direct effects on integrin clustering and internalization.

Caveolae↗

PubMedDate not supplied

Integrins as unique receptors for vascular control.

Cells within the vascular wall connect their cytoskeleton to the extracellular matrix (ECM) through a family of cell surface receptors known as integrins. The ability of integrins to act as a link between the extracellular and intracellular environments allows transmission of inside-out and outside-in signals capable of modulating diverse vascular phenomena. In this review we summarize what is currently known about the involvement of integrins in the control of vascular tone, permeability and remodeling. We discuss the capacity of integrins to act as detectors of injury-generated molecules derived from ECM proteins, as well as the putative role of integrins as mechanosensors for shear and tension. Particular attention is given to the mechanisms responsible for linking integrins to the control of vascular tone, and we review the intracellular signaling pathways involved in effecting the vascular responses elicited by integrin activation. Finally, the involvement of integrins in vascular remodeling and vascular disease is analyzed. Considerable evidence strongly indicates that integrins are involved in both acute and chronic vascular control. Understanding the elements and the sequence of events linking integrins with vasoregulation is important for deciphering phenomena such as the pressure-dependent myogenic response, flow-dependent changes in vascular diameter, and vascular remodeling as they occur in physiological and pathological conditions. Further understanding of the role of integrins in vascular control holds promise as new avenues for prophylactic and therapeutic manipulation of vascular phenomena.

Animals↗