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PubMed1999

Integrin alpha1beta1 (VLA-1) mediates adhesion of activated intraepithelial lymphocytes to collagen.

Intraepithelial lymphocytes (IELs) from human intestinal epithelium are memory CD8+ T cells that bind to epithelial cells through human mycosal lymphocyte (HML)-1 and to mesenchymal cells through very late activation antigen-4 (VLA-4). Their binding of extracellular matrix proteins and the mechanism involved were tested. Activated 51Cr-labelled lymphocytes were incubated in protein-coated microwells with various additives. After washing, the adherent cells were detected by radioactivity. The percentages of activated IELs that bound to collagen types I and IV were 20 and 31%, respectively; fewer bound to fibronectin or laminin. Compared to interleukin-2-activated peripheral blood CD8+ T lymphocytes, more IELs bound collagen IV and fewer bound fibronectin. IEL adhesion to collagen (but not fibronectin or laminin) was up-regulated by antibody ligation of CD2 or by protein kinase C stimulation by phorbol ester; staurosporine reduced binding, while herbimycin, phytohaemagglutinin and CD3 ligation had no effect. Antibody-blocking of integrin VLA-1 subunits alpha1 (CD49a) and beta1 (CD18) inhibited adhesion to collagen type I by 82+/-6% and to type IV by 94+/-1% (P<0.001), implicating VLA-1 as the main collagen receptor for IELs. Cell adhesion was dependent on extracellular divalent cations, a characteristic event of VLA-1 never before shown for IELs: manganese and magnesium ions supported binding in a dose-dependent manner; calcium ions inhibited their effectiveness. Therefore, IELs bind collagen through integrin alpha1beta1 after protein kinase C activation. Adhesion is modulated by divalent cations.

CD8-Positive T-Lymphocytes↗
PubMed2002

Expression of beta1 integrins during periosteal chondrogenesis.

OBJECTIVE: The interactions between integrins and extracellular matrix proteins are known to modulate cell behavior, and may be involved in regulating cartilage formation and repair. The purpose of this study was to determine the patterns and localization of expression of the beta1 integrins during cartilage formation by periosteum, which is used to repair articular cartilage. DESIGN: Periosteal explants from 2-month-old rabbit medial proximal tibiae were cultured in agarose suspension for 0 to 6 weeks, with 10 ng/ml transforming growth factor-beta1 added for the first 2 days of culture. Integrin expressions were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and localized by immunohistochemistry. RESULTS: Normal periosteum expressed the alpha1, alpha3, alpha5, beta1 subunits at low levels, and the proteins for all but the alpha3 subunits were identified by immunohistochemistry in the periosteum. Significant two- to five-fold up-regulation of the mRNA expression of the alpha1, alpha3, alpha5 and beta1 integrin subunits during the early proliferative stage of chondrogenesis was observed. The initial change was a five-fold increase in alpha5 expression on day 2 and a two-fold increase in alpha3 expression. On day 5, alpha1 expression was up-regulated (four-fold). beta1 expression was broadly up-regulated (three to four-fold) from day 5 to 14. In the early stage of chondrocyte differentiation, after day 14, alpha1 expression was down-regulated, while there was upregulation of alpha3 (three-fold), alpha5 (three-fold) and beta1 (four-fold) expressions. Thereafter, alpha1 expression was down-regulated, while alpha3, alpha5 and beta1 expressions were up-regulated again during matrix synthesis. Immunohistochemistry confirmed this late decrease in alpha1 levels and increase in alpha3, alpha5 and beta1 levels in chondrocytes. CONCLUSIONS: These observations indicate that the beta1 integrins play an important role in the process of chondrogenesis in periosteum.

Animals↗
PubMed2002

Fibronectin fragments and blocking antibodies to alpha2beta1 and alpha5beta1 integrins stimulate mitogen-activated protein kinase signaling and increase collagenase 3 (matrix metalloproteinase 13) production by human articular chondrocytes.

OBJECTIVE: To determine if integrin-mediated signaling results in activation of chondrocyte mitogen-activated protein (MAP) kinases that lead to increased expression of matrix metalloproteinase 13 (MMP-13; collagenase 3), a potent mediator of cartilage matrix degradation. METHODS: Human articular chondrocytes isolated from normal ankle and knee cartilage obtained from tissue donors were cultured in monolayers. The cells were treated with a 120-kd fibronectin fragment (FN-f) that binds the alpha5beta1 integrin or with antibodies to specific integrin receptors. Activation of MAP kinases was determined by immunoblotting with phosphospecific antibodies. MMP production was measured by gelatin zymography, and MMP-13 production and activation were determined by immunoblotting and by a fluorogenic peptide assay. RESULTS: Human articular chondrocytes were found to respond to the 120-kd FN-f and to adhesion-blocking antibodies to the alpha2beta1 and alpha5beta1 integrins with increased phosphorylation of the extracellular signal-regulated kinase 1 (ERK1)/ERK2, c-Jun N-terminal kinase (JNK), and p38 MAP kinases. Intact FN and integrin-blocking antibodies to alpha1, alpha3, and alphaVbeta3 and a nonblocking alpha5 antibody had no effect. After MAP kinase activation, increased phosphorylation of c-Jun and the nuclear factor kappaB inhibitor was noted, followed by increased pro- and activated MMP-13 in the conditioned media. Inhibitors of mitogen-activated protein kinase kinase, p38, and JNK were each able to inhibit increased MMP-13 production, while the interleukin-1 receptor antagonist (IL-1Ra) protein did not. However, the IL-1Ra partially inhibited FN-f-induced activation of MMP-13. CONCLUSION: Integrin-mediated MAP kinase signaling stimulated by FN-f is associated with increased production and release of pro- and active MMP-13. Autocrine production of IL-1 appears to result in additional MMP-13 activation. These processes may play a key role in feedback loops responsible for progressive cartilage degradation in arthritis.

Antibodies↗
PubMed2006

Novel alpha1-adrenergic receptor signaling pathways: secreted factors and interactions with the extracellular matrix.

alpha1-Adrenergic receptor (alpha1-ARs) subtypes (alpha1A, alpha1B, and alpha1D) regulate multiple signal pathways, such as phospholipase C, protein kinase C (PKC), and mitogen-activated protein kinases. We employed oligonucleotide microarray technology to explore the effects of both short- (1 h) and long-term (18 h) activation of the alpha1A-AR to enable RNA changes to occur downstream of earlier well characterized signaling pathways, promoting novel couplings. Polymerase chain reaction (PCR) studies confirmed that PKC was a critical regulator of alpha1A-AR-mediated gene expression, and secreted interleukin (IL)-6 also contributed to gene expression alterations. We next focused on two novel signaling pathways that might be mediated through alpha1A-AR stimulation because of the clustering of gene expression changes for cell adhesion/motility (syndecan-4 and tenascin-C) and hyaluronan (HA) signaling. We confirmed that alpha1-ARs induced adhesion in three cell types to vitronectin, an interaction that was also integrin-, FGF7-, and PKC-dependent. alpha1-AR activation also inhibited cell migration, which was integrin- and PKC-independent but still required secretion of FGF7. alpha1-AR activation also increased the expression and deposition of HA, a glycosaminoglycan, which displayed two distinct structures: pericellular coats and long cable structures, as well as increasing expression of the HA receptor, CD44. Long cable structures of HA can bind leukocytes, which this suggests that alpha1-ARs may be involved in proinflammatory responses. Our results indicate alpha1-ARs induce the secretion of factors that interact with the extracellular matrix to regulate cell adhesion, motility and proinflammatory responses through novel signaling pathways.

Adrenergic alpha-1 Receptor Antagonists↗
PubMed2001

Cis-polyunsaturated fatty acids stimulate beta1 integrin-mediated adhesion of human breast carcinoma cells to type IV collagen by activating protein kinases C-epsilon and -mu.

We have investigated the effects of various fatty acids (FAs) on integrin-mediated MDA-MB-435 breast carcinoma cell adhesion to type IV collagen (collagen IV) in vitro. Arachidonic acid (AA) and linoleic acid both induced a dose-dependent increase in cell adhesion to collagen IV with no significant increase in nonspecific adhesion to polylysine and BSA. Oleic acid (a monounsaturated FA), AA methyl ester, and linoelaidic acid (a trans-isomer of linoleic acid) failed to stimulate adhesion to collagen IV, suggesting that these effects required cis-polyunsaturation and a free carboxylic moiety and that they were not due to membrane perturbations. Calphostin C, a protein kinase C (PKC) inhibitor, blocked cis-polyunsaturated FA (cis-PUFA)-induced cell adhesion in a dose-dependent manner, suggesting a role for a calcium-dependent PKC in this signal transduction pathway. Immunoblotting revealed that cis-PUFAs induced the translocation of PKCepsilon and PKCmu, two of the novel PKC isozymes, from the cytosol to the membrane. In contrast, a conventional PKC isozyme, PKCalpha, as well as the atypical isozymes, PKCzeta and PKCiota, did not translocate after cis-PUFA treatment. Function-blocking antibodies specific for alpha1, alpha2, and beta1, integrin subunits inhibited cell adhesion to collagen IV, whereas antibodies to alpha3 and alpha5 did not. No increase in the expression of these integrins on the cell surface was detected after the incubation of cells with cis-PUFAs, suggesting that there is an increase in the activity, but not in the amount, of these beta1, integrins. Altogether, these data suggest that cis-PUFAs enhance human breast cancer cell adhesion to collagen IV by selectively activating specific PKC isozymes, which leads to the activation of beta1 integrins.

Adenocarcinoma↗
PubMed2003

Angiogenesis in collagen I requires alpha2beta1 ligation of a GFP*GER sequence and possibly p38 MAPK activation and focal adhesion disassembly.

Angiogenesis depends on proper collagen biosynthesis and cross-linking, and type I collagen is an ideal angiogenic scaffold, although its mechanism is unknown. We examined angiogenesis using an assay wherein confluent monolayers of human umbilical vein endothelial cells were overlain with collagen in a serum-free defined medium. Small spaces formed in the cell layer by 2 h, and cells formed net-like arrays by 6-8 h and capillary-like lumens by 24 h. Blocking of alpha2beta1, but not alpha1 or alpha(v)beta3 integrin function halted morphogenesis. We found that a triple-helical, homotrimeric peptide mimetic of a putative alpha2beta1 binding site: alpha1(I)496-507 GARGERGFP*GER (where single-letter amino acid nomenclature is used, P* = hydroxyproline) inhibited tube formation, whereas a peptide carrying another putative site: alpha1(I)127-138 GLP*GERGRP*GAP* or control peptides did not. A chemical inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), SB202190, blocked tube formation, and p38 MAPK activity was increased in collagen-treated cultures, whereas targeting MAPK kinase (MEK), focal adhesion kinase (FAK), or phosphatidylinositol 3-kinase (PI3K) had little effect. Collagen-treated cells had fewer focal adhesions and 3- to 5-fold less activated FAK. Thus capillary morphogenesis requires endothelial alpha2beta1 integrin engagement of a single type I collagen integrin-binding site, possibly signaling via p38 MAPK and focal adhesion disassembly/FAK inactivation.

Binding Sites↗
PubMed2005

The effect of combined cyclic mechanical stretching and microgrooved surface topography on the behavior of fibroblasts.

Under the influence of mechanical stress, cultured fibroblasts have a tendency to orient themselves perpendicular to the stress direction. Similar cell alignment can be induced by guiding cells along topographical clues, like microgrooves. The aim of this study was to evaluate cell behavior on microgrooved substrates, exposed to cyclic stretching. We hypothesized that cellular shape is mainly determined by topographical clues. On basis of earlier studies, a 10-microm wide square groove, and a 40-microm wide V-shaped groove pattern were used. Smooth substrates served as controls. Onto all substrates fibroblasts were cultured and 1-Hz cyclic stretching was applied (0, 4, or 8%) for 3-24 h. Cells were prepared for scanning electron microscopy, immunostaining of filamentous actin, alignment measurements, and PCR (collagen-I, fibronectin, alpha1- and beta1-integrins). Results showed that cells aligned on all grooved surfaces, and fluorescence microscopy showed similar orientation of intracellular actin filaments. After 3 h of stretch, cellular orientation started to commence, and after 24 h the cells had aligned themselves almost entirely. Image analysis showed better orientation with increasing groove depth. Statistical testing proved that the parameters groove type, groove orientation, and time all were significant, but the variation of stretch force was not. Substrates with microgrooves perpendicular to the stretch direction elicit a better cell alignment. The expression of beta1-integrin and collagen-I was higher in the stretched samples. In conclusion, we can maintain our hypothesis, as microgrooved topography was most effective in applying strains relative to the long axis of the cell, and only secondary effects of stretch force were present.

Animals↗
PubMed2002

Collagen suppresses the proliferative phenotype of allylamine-injured vascular smooth muscle cells.

Repeated cycles of oxidative injury by allylamine induce proliferative rat vascular smooth muscle cell (vSMC) phenotypes characterized by enhanced secretion of osteopontin (OPN). The present study was designed to evaluate the role of extracellular matrix (ECM) interactions in the induction of proliferative phenotypes in this model of oxidant injury. Because OPN is involved in ECM/integrin signaling, and may participate in proliferative control, the proliferation profiles of control and allylamine vSMCs seeded on different matrices were compared. Allylamine cells exhibited a proliferative advantage over controls when seeded on plastic, Pronectin, or fibronectin, but not type I collagen. Addition of GRGDS peptide selectively enhanced [3H]-thymidine incorporation in allylamine vSMCs, while anti-OPN antibodies nullified their proliferative advantage. Allylamine cells exhibited altered expression of alpha1, alpha5 and beta3 integrin subunits and enhanced downstream integrin-coupled increases in focal adhesion kinase, AP-1 and NF-kappaB binding activity. Inhibition of NF-kappaB by pyrrolidine dithiocarbamate selectively compromised proliferation of allylamine vSMCs, while seeding on a non-permissive collagen matrix ablated enhancement of NF-kappaB inducibility. These results implicate ECM interactions in the deregulation of vSMC proliferation following repeated cycles of oxidative chemical injury.

Allylamine↗
PubMed2001

Schwann cell type V collagen inhibits axonal outgrowth and promotes Schwann cell migration via distinct adhesive activities of the collagen and noncollagen domains.

Previously, we reported the cloning of alpha4 type V collagen, a novel member of the collagen type V gene family that is expressed by Schwann cells in developing peripheral nerves (Chernousov et al., 2000). The present study was performed to investigate the effects of this collagen on the adhesion and migration of premyelinating Schwann cells and neurite outgrowth from embryonic dorsal root ganglion neurons. Purified alpha4(V)-containing collagen isolated from Schwann cell conditioned medium (collagen type V(SC)) promoted migration of Schwann cells but inhibited outgrowth of axons from rat embryo dorsal root ganglia. Collagen type V(SC) blocked axonal outgrowth in the presence of otherwise active substrates such as collagen type IV, indicative of active inhibition. The noncollagen N-terminal domain of alpha4(V) promoted Schwann cell adhesion, spreading, and migration. These processes were inhibited by soluble heparin but not by function-blocking antibodies against alpha1- and alpha2-integrins. The collagen domain of pepsin-digested collagen type V was poorly adhesive for Schwann cells. The type V collagen domain but not the alpha4(V) N-terminal domain blocked neurite outgrowth from dorsal root ganglion neurons. In cocultures of dorsal root ganglion neurons and Schwann cells, collagen type V(SC) promoted axon fasciculation and association of axons with Schwann cells. These results suggest that in embryonic peripheral nerves, collagen type V(SC) plays a dual role in regulating cell migration. This represents a heretofore unrecognized function of peripheral nerve collagen fibrils in regulating patterns of peripheral nerve growth during development.

Animals↗
PubMed1999

Secondary reduction of alpha7B integrin in laminin alpha2 deficient congenital muscular dystrophy supports an additional transmembrane link in skeletal muscle.

The integrins are a large family of heterodimeric transmembrane cellular receptors which mediate the association between the extracellular matrix (ECM) and cytoskeletal proteins. The alpha7beta1 integrin is a major laminin binding integrin in skeletal and cardiac muscle and is thought to be involved in myogenic differentiation and migration processes. The main binding partners of the alpha7 integrin are laminin-1 (alpha1-beta1-gamma1), laminin-2 (alpha2-beta1-gamma1) and laminin-4 (alpha2-beta2-gamma1). Targeted deletion of the gene for the alpha7 integrin subunit (ITGA7) in mice leads to a novel form of muscular dystrophy. In the present study we have investigated the expression of two alternative splice variants, the alpha7B and beta1D integrin subunits, in normal human skeletal muscle, as well as in various forms of muscular dystrophy. In normal human skeletal muscle the expression of the alpha7 integrin subunit appeared to be developmentally regulated: it was first detected at 2 years of age. In contrast, the beta1D integrin could be detected in immature and mature muscle in the sarcolemma of normal fetal skeletal muscle at 18 weeks gestation. The expression of alpha7B integrin was significantly reduced at the sarcolemma in six patients with laminin alpha2 chain deficient congenital muscular dystrophy (CMD) (age >2 years). However, this reduction was not correlated with the amount of laminin alpha2 chain expressed. In contrast, the expression of the laminin alpha2 chain was not altered in the skeletal muscle of the alpha7 knock-out mice. These data argue in favor that there is not a tight correlation between the expression of the alpha7 integrin subunit and that of the laminin alpha2 chain in either human or murine dystrophic muscle. Interestingly, in dystrophinopathies (Duchenne and Becker muscular dystrophy; DMD/BMD) expression of alpha7B was upregulated irrespective of the level of dystrophin expression as shown by a strong sarcolemmal staining pattern even in young boys (age <2 years). The expression of the beta1D integrin subunit was not altered in any of our patients with different types of muscular dystrophy. In contrast, sarcolemmal expression of beta1D integrin was significantly reduced in the alpha7 integrin knock-out mice, whereas the expression of the components of the DGC was not altered. The secondary loss of alpha7B in laminin alpha2 chain deficiency defines a biochemical change in the composition of the plasma membrane resulting from a primary protein deficiency in the basal lamina. These findings, in addition to the occurrence of a muscular dystrophy in alpha7 deficient mice, implies that the alpha7B integrin is an important laminin receptor within the plasma membrane which plays a significant role in skeletal muscle function and stability.

Adolescent↗
PubMed1999

An IKLLI-containing peptide derived from the laminin alpha1 chain mediating heparin-binding, cell adhesion, neurite outgrowth and proliferation, represents a binding site for integrin alpha3beta1 and heparan sulphate proteoglycan.

We synthesized and characterized several peptides containing the IKLLI sequence in the alpha1 chain of laminin-1. The IKLLI-containing peptides, such as LA4 (CSRNLSEIKLLISRARK), LA5 (EIKLLIS) and LA5L (SEIKLLIS), were found to mediate heparin binding and cell adhesion, while also promoting neurite outgrowth in PC12 cells. Furthermore, peptides LA4 and LA5 also mediated proliferation. However, a scrambled peptide, LA5S (ILEKSLI), did not show any of these activities. Anti-LA4 antibodies inhibited laminin- and LA5-mediated cell adhesion and neurite outgrowth, and anti-(integrin alpha3) and anti-(integrin beta1) antibodies inhibited LA5-mediated cell adhesion and neurite outgrowth. Heparin and heparan sulphate inhibited LA5-mediated heparin binding and PC12 cell adhesion in a dose- dependent manner. The IC50 for inhibition of heparin binding and cell adhesion was observed with 9 microM and 8 microM heparin/heparan sulphate respectively. Furthermore, heparan sulphate proteoglycan also inhibited LA5-mediated PC12 cell adhesion with an IC50 of 100 micrograms/ml. However, chondroitin sulphate (dermatan sulphate) did not inhibit cell adhesion. These data suggest that an IKLLI-containing peptide derived from the laminin alpha1 chain may be an active site of laminin and that its cell adhesion may thus interact with both integrin alpha3beta1 and cell- surface heparan sulphate proteoglycan.

Amino Acid Sequence↗
PubMed2002

Transforming growth factor-beta1 modulates expression of adhesion and cytoskeletal proteins in human peritoneal fibroblasts.

OBJECTIVE: To determine the effects of TGF-beta1 on the expression of the alpha1, alpha2, alpha5, alpha(v), and alpha6 integrin subunits and on vinculin, and F-actin in human peritoneal fibroblasts. DESIGN: Descriptive study using cell culture, reverse transcriptase-polymerase chain reaction (RT-PCR), and immunofluorescent and confocal microscopy. SETTING: Academic medical center. PATIENT(S): Gynecological surgery patients. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Effects of TGF-beta1 on the steady state levels of alpha5, alpha(v), and alpha6 integrin transcripts were examined in the normal peritoneal fibroblasts using RT-PCR. Expression levels of the alpha1, alpha2, alpha5, alpha(v), and alpha6 integrin subunits and of F-actin were measured by immunofluorescent microscopy. The distribution pattern of the integrin subunits, vinculin, and F-actin were examined using confocal microscopy. RESULT(S): TGF-beta1 significantly up-regulated the expression levels of the alpha5, alpha(v), and alpha6 integrin subunits and modulated their expression pattern, resulting in relatively higher levels of these subunits in the focal contacts of peritoneal fibroblasts. It allocated vinculin expression primarily to the focal contacts of cells and caused distortion of F-actin structure. The transcript levels of the alpha5, alpha(v), and alpha6 integrin subunits were not altered by TGF-beta1 treatment. CONCLUSION(S): TGF-beta1 may promote postoperative adhesion formation by inducing the migration of peritoneal fibroblasts by altering the expression levels and patterns of specific integrin subunits, vinculin, and F-actin.

Actins↗
PubMed1997

Immunolocalization of integrins in the normal lung and in pulmonary carcinomas.

Cryosections of normal adult lung (n = 7) and pulmonary epithelial tumors, including squamous (n = 8), adeno (n = 8), bronchioloalveolar (n = 5), and large cell (n = 4) carcinomas (SCC, ACC, BAC, LCC), carcinoids (Cd, n = 7), and neuroendocrine carcinomas (NEC) of variable grades (n = 14) were immunostained by the avidin-biotin peroxidase (ABC) method with monoclonal antibodies to the alpha1-6 and alpha(v) and the beta1-4 integrin subunits. Normal adult alveolar septae showed variably intense immunoreactivity for alpha1,3,6 and beta1, whereas reactions for alpha5 and alpha(v) were weaker and uneven; the remaining integrin subunits were not detected. Bronchial and bronchiolar epithelium showed variably intense staining for alpha2.3,6,v and beta1,4. Reactions were often, though not invariably, basally polarized. SCC, ADC, and LCC showed variably intense reactions for alpha2.3,6,v and beta1,4. BAC were strongly and uniformly stained for alpha1.3 and beta1. In Cd, alpha1,2,3,v and beta1 reactions were noted, whereas in NEC, weak alpha1,3 and beta1 staining was detected with only traces of alpha6 and alpha(v). We conclude that alveolar epithelial cells do not express the hemidesmosome-associated, laminin-binding integrin alpha6beta4 of the bronchial epithelium but rather the alpha1beta1 and alpha3beta1, collagen IV, and laminin receptors, respectively. SCC, ADC, and sampled LCC express an integrin repertory qualitatively similar to that of the bronchial epithelium. Distinct from the latter, the integrin repertory of BAC parallels that of the alveolar epithelium by its strong expression of the multipotential alpha1beta1 and alpha3beta1 integrins. NEC tumors do not display the laminin receptors alpha6beta4 and alpha6beta1 shown by SCC and ADC but express instead alpha1beta1, a collagen IV-laminin receptor rarely found in epithelial neoplasms except for BAC. In NEC tumors, integrins, especially alpha2, decrease with dedifferentiation. Notably distinct from epithelial mesotheliomas, the major fibronectin-binding integrin alpha5beta1 was not found in any type of lung carcinoma.

Adenocarcinoma↗
PubMed1999

Alpha8 integrin in glomerular mesangial cells and in experimental glomerulonephritis.

BACKGROUND: Mesangial cell (MC) proliferation and extracellular matrix accumulation are typical responses of renal glomeruli to injury. Extracellular matrix components are known to affect MC behavior, which is mediated primarily via integrin receptors of the beta1 family. In addition to alpha1, alpha3, alpha5, and alpha6 chains of beta1 integrins, recent studies have shown the alpha8 chain to be expressed in glomeruli and renal vasculature. alpha8beta1 can serve as a receptor for fibronectin, which is abundant in the mesangium. We investigated the glomerular expression pattern of the alpha8 chain in renal tissues of mouse, rat, and humans as well as in cultured MCs. In addition, the regulation of alpha8 expression in MCs was studied in culture and in nephritic rats. METHODS: The expression of alpha8 protein in kidney tissue and cultured MCs was investigated by immunohistochemistry, immunocytochemistry, and Western blotting. The effects of TGF-beta1 on alpha8 mRNA levels in MCs were studied by Northern blot analysis. In addition, time course studies of glomerular abundance and localization of alpha8 were performed in rats with mesangioproliferative anti-Thy1.1 nephritis. RESULTS: In tissue sections of normal human, rat, and mouse kidney, we found strong immunohistochemical staining for alpha8 in the mesangium and in the media of renal arterioles. Double staining for alpha8 and Thy1.1, a surface antigen of rat MCs, showed alpha8 to be specifically expressed in MCs but not in glomerular endothelial and epithelial cells. In anti-Thy1.1 nephritis of rats, the glomerular abundance of alpha8 protein was reduced in the early mesangiolytic phase but was increased greatly with subsequent MC proliferation, peaking at day 6 of disease. At later stages of this reversible form of nephritis, the number of MCs and the extent mesangial alpha8 staining declined to control levels. Cell culture experiments revealed that freshly plated MCs organize alpha8 into focal contacts within one hour after attachment to fibronectin and vitronectin substrata, showing colocalization with focal contact proteins vinculin and talin. Stimulation of MCs with transforming growth factor-beta1 led to increases of alpha8 mRNA and protein levels. CONCLUSIONS: These results show that in human, rat, and mouse glomeruli, alpha8 integrin is strongly and exclusively expressed in MCs. Gene expression of alpha8 is regulated in cultured MCs, and alpha8 protein abundance is regulated in vivo and in MC culture. It is currently unclear what functional properties this integrin receptor protein has with regard to MC anchorage to extracellular matrix and modulation of the MC phenotype in normal and diseased glomeruli. However, in view of its abundance in the mesangium, alpha8beta1 integrin could be an important MC receptor of matrix ligands and may play a role in the embryology, physiology, and pathophysiology of the glomerular capillary tuft.

Animals↗
PubMed2003

Studies of the local conformational properties of the cell-adhesion domain of collagen type IV in synthetic heterotrimeric peptides.

Collagen type IV is a specialized form of collagen that is found only in basement membranes. It is involved in integrin-mediated cell-adhesion processes, and the responsible binding sites for the alpha1beta1 integrin cell receptor have been identified as Asp461 of the two alpha1 chains and Arg461 of the alpha2 chain. In the most plausible stagger of native collagen type IV the alpha2 chain is the tailing one. This has recently been confirmed by the differentiated binding affinities of synthetic heterotrimeric collagen peptides in which the chains were staggered in this native register as well as in the less plausible alpha1alpha2alpha1' register with an artificial cystine knot. In the present work, two heterotrimeric collagen peptides with chain registers identical to the previous ones were synthesized for fluorescence resonance energy transfer and emission anisotropy measurements, exploiting the native Phe464 in the alpha2 chain as donor and an Ile467Tyr mutation in the alpha1' chain as acceptor fluorophore. This fluorophore pair allowed extraction of more detailed information on the conformational properties of the cell-adhesion epitope incorporated into the central part of the trimeric collagen model peptides. A comparison of the experimentally derived values of the interfluorophore distance and of the orientation factor kappa(2) with the values extracted from the molecular model of the trimer in the native stagger confirmed a triple-helical structure of the adhesion-site portion at low temperature. The thermal unfolding of this central domain was specifically monitored by emission anisotropy, allowing unambiguous assignment of the three structural domains of the trimeric collagen molecules detected by microcalorimetry, with the integrin binding site as the portion of weakest triple-helical stability flanked by two more stable triple-helical regions. The results are consistent with the picture of a conformational microheterogeneity as the responsible property for selective recognition of collagens by interacting proteins.

Amino Acid Sequence↗
PubMed1998

alpha2beta1 integrin signaling via the mitogen activated protein kinase pathway modulates retinoic acid-dependent tumor cell invasion and transcriptional downregulation of matrix metalloproteinase 9 activity.

Tumor cell invasion and metastasis requires precise coordination of adherence to the extracellular matrix (ECM) and controlled degradation of its components. lnvasive cells secrete proteolytic enzymes known as matrix metalloproteinases which degrade specific basement membrane molecules. Expression of these enzymes is regulated by multiple signaling mechanisms, including attachment to the extracellular matrix via integrins. All-trans retinoic acid can inhibit tumor cell invasion of ECM by regulating matrix metalloproteinase expression. Using a series of squamous cell carcinoma lines, we investigated the interactions between integrin and retinoic acid signaling in these cells. In a cell line sensitive to RA-mediated inhibition of invasion, this ligand downregulated MMP-9 activity in cells grown on specific ECM molecules but not on plastic. Inhibition of integrin signaling with anti-alpha1 antibodies or MAPK pathway inhibitors abrogated RA mediated down-regulation of MMP-9 activity and invasion. The effects of RA and MAPK signaling on MMP-9 activity was mediated at the transcriptional level. These data indicate that crosstalk between RA- and integrin dependent signaling pathways regulate MMP activity and invasion in squamous cell carcinoma lines.

Antineoplastic Agents↗
PubMed1998

Interactions of type IV collagen and its domains with human mesangial cells.

Type IV collagen (COL-IV) interacts with a variety of cell types. We present evidence that human mesangial cells (HMC) bind directly to COL-IV, its major triple helical domain, and the main non-collagenous, NC1 domain. A synthetic peptide, HEP-III, and its triple helical counterpart (THP-III), previously reported to be a heparin-binding domain, also promoted approximately 15% adhesion of HMC. HMC bound to solid-phase-immobilized, intact COL-IV (approximately 75%), isolated NC1 domain (approximately 15%), and a pepsin-derived triple helical fragment,which lacks Hep-III (approximately 65%). We further examined inhibition of HMC adhesion to COL-IV and its domains by using anti-integrin antibodies. Blocking monoclonal antibodies against the alpha2 integrin resulted in 70% inhibition of adhesion to COL-IV and 80% inhibition to HEP-III. Moderate inhibition was observed on the NC1 and triple helical fragments. Anti-alpha1 antibodies inhibited the binding of HMC to COL-IV, the NC1, and triple helical domains, but not to peptide HEP-III. Anti-beta1 antibodies inhibited almost completely (>95%) the adhesion to COL-IV, the NC1, and triple helical fragments; inhibition on HEP-III was approximately 30%. Affinity chromatography studies with solid-phase HEP-III and mesangial cell lysate also demonstrated the presence of integrin alpha2 beta1 along with alpha3 beta1. We conclude that alpha2 beta1 and alpha1 beta1 integrins mediate HMC adhesion to COL-IV. Peptide HEP-III is a major, specific site for alpha2 integrin-mediated binding of mesangial cells to COL-IV. Both the alpha1 beta1 and alpha2 beta1 integrins interact with the NC1 and triple helical fragments of COL-IV. Therefore, we demonstrate that several sites for integrin-mediated interactions exist on several collagenous and non-collagenous domains of COL-IV.

Antibodies, Monoclonal↗
PubMed2002

Deleted in colorectal carcinoma and differentially expressed integrins mediate the directional migration of neural precursors in the rostral migratory stream.

Precursors of the olfactory interneurons migrate from the subventricular zone via the rostral migratory stream (RMS). To investigate the molecular mechanisms by which RMS cells migrate, we used a slice preparation, which allows the migrating cells to be imaged at very high temporal and spatial resolution in the presence of added inhibitors. Using immunohistochemistry, we first determined that the alpha1-, beta8-, and beta1-integrin subunits and the alpha5- and gamma1-laminin subunits are expressed during embryonic day 16 to the early postnatal stage. During early postnatal days, alpha(v)- and beta6-integrins appeared, and their expression persisted throughout adulthood. The migrating cells also expressed the netrin receptors neogenin and Deleted in Colorectal Carcinoma (DCC). Netrin-1 is expressed in olfactory mitral cells. Anti-integrin antibodies inhibited the production of protrusions as well as cellular translocation. In contrast, anti-DCC antibodies primarily altered the direction of the protrusions; consequently, the migration was no longer unidirectional, and the speed was reduced. Thus, the interaction of DCC, possibly through an interaction with netrin-1, contributes to the direction of migration by regulating the formation of directed protrusions. In contrast, the integrins function in production of protrusions and cellular translocation, with different integrins participating at different developmental stages.

Aging↗