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PubMed2002

Beta 1-integrin protects hepatoma cells from chemotherapy induced apoptosis via a mitogen-activated protein kinase dependent pathway.

BACKGROUND: beta 1-integrin modulates cellular phenotype by mediating signals from the extracellular matrix (ECM). Although overexpression of integrin molecules in hepatocellular carcinoma (HCC) has been reported, the role of overexpressed beta 1-integrin in the disease process of HCC is not fully understood. The authors investigated the effects of beta 1-integrin on apoptosis in hepatoma cells. METHODS: Human hepatoma cell lines HepG2, Huh7, and HLE were stably transfected with full-length beta 1-integrin. Cells underwent apoptosis induced by chemotherapeutic reagents, including cis-platinum (II)-diammine dichloride, etoposide, and docetaxel. Cell survival and intracellular signaling pathways dependent on beta 1-integrin-mediated apoptosis effects were analyzed by treating cells with PD98059 (ERK inhibitor), SB203580 (p38MAP kinase inhibitor), wortmannin (phosphatidyl inositol-3-kinase inhibitor), and herbimycin A (tyrosine kinase inhibitor). RESULTS: All three hepatoma cell lines overexpressing beta 1-integrin were protected from apoptosis induced by chemotherapeutic reagents, whereas parental or mock transfected cells were not. Treatment with PD98059 or SB203580 abolished the protective effect on apoptosis in cells overexpressing beta 1-integrin. Neither herbimycin nor wortmannin blocked the protective effects of beta 1-integrin overexpression. CONCLUSIONS: These data suggest that overexpression of beta 1-integrin confers resistance to apoptosis in hepatoma cells via a MAP kinase dependent pathway. beta1-integrin mediated signaling from the ECM in HCC cells may contribute to chemotherapy resistance.

Antineoplastic Agents↗
PubMed1999

Focal adhesion motility revealed in stationary fibroblasts.

Focal adhesions (FAs) are clustered integrins and associated proteins that mediate cell adhesion and signaling. A green fluorescent protein-beta1 integrin chimera was used to label FAs in living cells. In stationary cells, FAs were highly motile, moving linearly for several plaque lengths toward the cell center. FA motility was independent of cell density and resulted from contraction of associated actin fibers. In migrating cells, FAs were stationary and only moved in the tail. FA motility in stationary cells suggests that cell movement may be regulated by a clutch-like mechanism by which the affinity of integrins to substrate may be altered in response to migratory cues.

3T3 Cells↗
PubMed2002

RACK1, an insulin-like growth factor I (IGF-I) receptor-interacting protein, modulates IGF-I-dependent integrin signaling and promotes cell spreading and contact with extracellular matrix.

The insulin-like growth factor I (IGF-I) receptor (IGF-IR) is known to regulate a variety of cellular processes including cell proliferation, cell survival, cell differentiation, and cell transformation. IRS-1 and Shc, substrates of the IGF-IR, are known to mediate IGF-IR signaling pathways such as those of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K), which are believed to play important roles in some of the IGF-IR-dependent biological functions. We used the cytoplasmic domain of IGF-IR in a yeast two-hybrid interaction trap to identify IGF-IR-interacting molecules that may potentially mediate IGF-IR-regulated functions. We identified RACK1, a WD repeat family member and a Gbeta homologue, and demonstrated that RACK1 interacts with the IGF-IR but not with the closely related insulin receptor (IR). In several types of mammalian cells, RACK1 interacted with IGF-IR, protein kinase C, and beta1 integrin in response to IGF-I and phorbol 12-myristate 13-acetate stimulation. Whereas most of RACK1 resides in the cytoskeletal compartment of the cytoplasm, transformation of fibroblasts and epithelial cells by v-Src, oncogenic IR or oncogenic IGF-IR, but not by Ros or Ras, resulted in a significantly increased association of RACK1 with the membrane. We examined the role of RACK1 in IGF-IR-mediated functions by stably overexpressing RACK1 in NIH 3T3 cells that expressed an elevated level of IGF-IR. RACK1 overexpression resulted in reduced IGF-I-induced cell growth in both anchorage-dependent and anchorage-independent conditions. Overexpression of RACK1 also led to enhanced cell spreading, increased stress fibers, and increased focal adhesions, which were accompanied by increased tyrosine phosphorylation of focal adhesion kinase and paxillin. While IGF-I-induced activation of IRS-1, Shc, PI3K, and MAPK pathways was unaffected, IGF-I-inducible beta1 integrin-associated kinase activity and association of Crk with p130(CAS) were significantly inhibited by RACK1 overexpression. In RACK1-overexpressing cells, delayed cell cycle progression in G(1) or G(1)/S was correlated with retinoblastoma protein hypophophorylation, increased levels of p21(Cip1/WAF1) and p27(Kip1), and reduced IGF-I-inducible Cdk2 activity. Reduction of RACK1 protein expression by antisense oligonucleotides prevented cell spreading and suppressed IGF-I-dependent monolayer growth. Our data suggest that RACK1 is a novel IGF-IR signaling molecule that functions as a positive mediator of cell spreading and contact with extracellular matrix, possibly through a novel IGF-IR signaling pathway involving integrin and focal adhesion signaling molecules.

Animals↗
PubMed1998

Localization and seizure-regulation of integrin beta 1 mRNA in adult rat brain.

Recent findings indicate that RGD-binding integrin receptors play a critical role in the maintenance of long-term potentiation but the identity and location of the integrin proteins involved are not known. The integrin beta1 is of particular interest in regard to synaptic plasticity because it is a component of many of the RGD-binding integrins and beta1-immunoreactivity has been localized within synaptic density fractions. The present study used in situ hybridization to evaluate the distribution of beta1 mRNA in adult rat brain and to determine if expression is altered by seizures. In untreated rats, beta1 mRNA is present at high levels in the ventricular epithelium and discrete neuronal groups including the magnocellular hypothalamic and efferent cranial nerve nuclei and the cerebellar Purkinje cells. Hybridization was less dense in the substantia nigra and hippocampal stratum pyramidale and low but present throughout the gray matter. Limbic seizures increased beta1 cRNA labeling of both neurons (e.g., hippocampal stratum pyramidale) and astroglial cells from 8 h through 48 h after seizure onset. These results indicate that in adult rat brain, beta1 mRNA is expressed by both neurons and glia; neuronal expression is highest in hypothalamic and peripherally projecting neurons capable of substantial morphological plasticity. Seizure effects demonstrate that beta1 is positively regulated by activity, and suggest that activity-dependent expression may play a role in synaptic plasticity in the adult brain.

Animals↗
PubMed1998

Beta1B integrin interferes with matrix assembly but not with confluent monolayer polarity, and alters some morphogenetic properties of FRT epithelial cells.

Beta1B is a beta1 integrin splice variant that differs from the ubiquitous beta1A in the terminal portion of the cytosolic tail. The expression of this variant in CHO cells results in reduced fibroblast adhesion and motility (Balzac, E et al., J. Cell Biol. 127, 557-565 (1994)). We have evaluated the phenotypic changes induced by the expression of beta1B in the FRT epithelial cell line. Stable transfectants of FRT cells expressing beta1B or beta1A human integrins were obtained. The transfected integrins associated with the endogenous alpha subunits and were delivered to the plasma membrane. Beta1B expressing cells attached less efficiently and spread less on fibronectin, laminin or type IV collagen coated dishes. A great reduction of fibronectin fibrils associated to the basal membrane of non-confluent beta1B transfected cells was observed. This was paralleled by the disappearance of microfilament bundles and loss of basally located focal adhesions. On the contrary, upon beta1A transfection, a higher amount of fibronectin fibrils, together with microfilament bundles and focal adhesions, was observed. Expression of beta1B did not significantly modify the ability to manifest the polarized phenotype when cells were grown to confluence on filters in two-chamber-systems. Beta1B-transfected cells showed reduced motile properties when embedded as aggregates in type I collagen gels. Moreover, formation of polarized cysts in suspension culture was impaired. The results show that beta1B, by interfering with focal adhesion organization, microfilament and fibronectin assembly, cell spreading and migration, affects some morphogenetic properties of FRT epithelial cells.

Actin Cytoskeleton↗
PubMed1999

Vinculin, Talin, Integrin alpha6beta1 and laminin can serve as components of attachment complex mediating contraction force transmission from cardiomyocytes to extracellular matrix.

Recently, we reported that cardiomyocytes adhere to extracellular matrix at costameres, the striated distribution of vinculin between Z-lines and the sarcolemma, where transmission of contraction forces from myofibrils to the extracellular matrix occurs. To identify other molecules involved in force transmission at costameres, we examined adult rat and embryonic chick cardiomyocytes cultured on coverslips or flexible thin silicone rubber substrata. Immunolocalization of talin showed a costameric, striated distribution, which corresponded to dark contacts with interference reflection microscopy. The molecules involved in substrate adhesion were cross-linked with the non-penetrating cross-linking agent Bis(sulfosuccinimidyl)-suberate and detected by immunohistochemical staining with anti-alpha6, alpha3, alphav, or beta1 integrin antibodies. Both alpha6 and beta1 showed costameric distributions, but alpha3 and alpha(v) did not. The distribution of laminin after cross-linking and extraction also showed a costameric distribution. When anti-integrin beta1 antibody was added to live cardiomyocytes grown on the silicone rubber substratum, the transmission of contraction forces was inhibited. These findings suggest that vinculin, talin, integrin alpha6beta1 and laminin system can be involved in transmission of contraction force to the extracellular matrix.

Animals↗
PubMed1997

Urokinase-type plasminogen activator receptors associate with beta1 and beta3 integrins of fibrosarcoma cells: dependence on extracellular matrix components.

We have shown previously that the urokinase-type plasminogen activator receptor (uPAR) physically associates with beta2 integrins on human leukocyte membranes. We now report that uPAR associates with certain members of the beta1 and beta3 integrin families expressed by a nonhematopoietic fibrosarcoma cell line (HT1080) when adherent to certain extracellular matrix molecules. Flow cytometry studies indicated that HT1080 cells expressed uPAR and beta1 and beta3 integrins. Double staining immunofluorescence was used to label uPAR and beta1 and beta3 integrins. The staining patterns of uPAR and beta1 integrins were strikingly similar when attached to fibronectin, laminin, or vitronectin but not polylysine-coated substrates. Resonance energy transfer (RET) between uPAR and beta1 integrins was observed, especially at focal adhesion plaques; this indicates that these molecules are within about 7 nm of each other on these cell membranes. uPAR and beta3 integrin coclustering and RET were also observed on tumor cells adherent to vitronectin but not to fibronectin, laminin, or polylysine-coated surfaces. Because N-acetyl-D-glucosamine was found previously to inhibit beta2 integrin-uPAR association, we tested the effect of saccharides on the beta1-uPAR and beta3-uPAR colocalization and RET. Colocalization and RET between uPAR and beta1 or beta3 integrins were effectively inhibited by N-acetyl-D-glucosamine on extracellular matrix-coated surfaces. To better define which members of beta1 and beta3 integrin families associate with uPAR, we studied the association of several alpha subunits with uPAR on tumor cells. We found that: (a) alpha5 colocalizes with uPAR on cells attached to fibronectin-coated surfaces; (b) alpha5 and alpha(v) colocalize with uPAR on cells adherent to vitronectin; and (c) alpha3 and alpha6 associate with uPAR on cells attached to laminin. In further support of physical associations between integrins and uPAR on tumor cells, uPAR was found to coimmunoprecipitate with beta1 integrins in Brij-58 lysates of HT1080 cells (as detected by anti-uPAR Western blotting of material isolated from an anti-beta1 integrin immunoaffinity column). Thus, uPAR may laterally associate with integrins of tumor cells when attached to specific extracellular matrix elements to enable directional proteolysis for tumor cell migration and invasion.

Antibodies, Monoclonal↗
PubMed2002

Temporal gene expression profile of human precursor B leukemia cells induced by adhesion receptor: identification of pathways regulating B-cell survival.

The physical interactions between B cells and stromal cells from the lymphoid tissue microenvironment are critical to the survival of normal and malignant B cells. They are principally mediated by integrins expressed on B cells and counterreceptors on stromal cells. Specifically, alpha4beta1 integrin engagement rescues B cells from physiological or drug-induced apoptosis. Therefore, in order to understand the mechanisms by which integrins prevent apoptosis in leukemia B cells, we compared the temporal gene expression profiles induced by beta1-integrin ligation with fibronectin (Fn) or adhesion by poly-L-Lysine in serum-starved precursor B leukemia cells. Among the 38 selected differentially expressed genes, 6 genes involved in adhesion (VAV2, EPB41L1, CORO1A), proliferation (FRAP1, CCT4), and intercellular communication (GJB3) were validated by real-time quantitative polymerase chain reaction (RT-Q-PCR). Gene expression modulation could also be validated at the protein level for 5 other genes. We show that integrin stimulation up-regulated FBI-1 expression but inhibited CD79a, Requiem, c-Fos, and caspase 7 induction when the cells underwent apoptosis. We further demonstrate that Fn stimulation also inhibits caspase 3 activation but increases XIAP and survivin expression. Moreover, integrin stimulation also prevents caspase activation induced by doxorubicin. Therefore, we identified genes modulated by adhesion of human precursor B leukemia cells that regulate proliferation and apoptosis, highlighting new pathways that might provide insights into future therapy aiming at targeting apoptosis of leukemia cells.

Apoptosis↗
PubMed1998

Involvement of beta1 and beta2 integrin stimulation in RNA synthesis in an eosinophilic cell line (EoL-1).

In allergic inflammatory disease, especially in bronchial asthma, eosinophils play important roles as essential inflammatory cells. In the accumulation of eosinophils in airway inflammation, eosinophils receive very diverse stimuli. In this study, we investigated the influences of the signal between beta2 integrin/intercellular adhesion molecule-1 (ICAM-1) or beta1 integrin/fibronectin (FN) and RNA synthesis on proteins in eosinophilic cell line-1 (EoL-1), using recombinant soluble ICAM-1 (r-sICAM-1) as a global response of eosinophils. 3H-thymidine incorporation and 3H-uridine incorporation were used as indices for DNA synthesis and RNA synthesis, respectively. In a comparison of the RNA/DNA ratio with various durations of stimulation of 1 microg/ml of r-sICAM-1 and 100 microg/ml of FN, a time-dependent increase was observed, but the increase induced by FN rose slower than that induced by r-sICAM-1. From this result, a beta2 integrin/ICAM-1 signal induced an increase in the RNA/DNA ratio in EoL-1, implying that the signal promotes RNA synthesis, which suggests that various types of protein synthesis, such as the synthesis of various cytokines, are induced by the beta2 integrin/ICAM-1 signal. Similar results were obtained with a beta1 integrin signal using FN, but there was a difference in the time course between beta2 integrin/ICAM-1 and beta1 integrin/FN signals. This experimental method may be useful for understanding these manifestations as a global response of eosinophils.

Asthma↗
PubMed2005

Changes in distribution patterns of integrins in endometrium in copper T380 intrauterine device users.

Intrauterine contraception is the most cost-effective reversible method of contraception today, but its mechanism of action is not well understood. Our objective was to investigate immunohistochemical distribution patterns of alphav, alpha3, beta1 integrins in women using a copper T380 intrauterine device (IUD) for different periods of time to obtain insight into the role of integrins in intrauterine contraception. Endometrial biopsies were obtained from patients using T Cu380A IUD in follicular and luteal phases and in menopausal women grouped according to the period of time of IUD use (group 1: <3 year, and group 2:>or=3 years). Each group consisted of 10 patients, with a total number of 60 patients. Labelling intensity of all integrins, except for beta1 which increased in the follicular phase, were decreased in women who used IUD for>or=3 years when compared with group 1 in the follicular and luteal phases and in the menopause. We conclude that long-term use of IUD affects integrin expression in endometrium not only in follicular and luteal phases of premenopausal women but also in postmenopausal women. Copper IUD can inhibit binding of integrins to the extracellular matrix and it may cause inhibition of the implantation stage, which is crucial for pregnancy.

Endometrium↗
PubMed2003

Knock-in of integrin beta 1D affects primary but not secondary myogenesis in mice.

Integrins are extracellular matrix receptors composed of alpha and beta subunits involved in cell adhesion, migration and signal transduction. The beta1 subunit has two isoforms, beta 1A ubiquitously expressed and beta 1D restricted to striated muscle. They are not functionally equivalent. Replacement of beta 1A by beta 1D (beta 1D knock-in) in the mouse leads to midgestation lethality on a 50% Ola/50% FVB background [Baudoin, C., Goumans, M. J., Mummery, C. and Sonnenberg, A. (1998). Genes Dev. 12, 1202-1216]. We crossed the beta 1D knock-in line into a less penetrant genetic background. This led to an attenuation of the midgestation lethality and revealed a second period of lethality around birth. Midgestation death was apparently not caused by failure in cell migration, but rather by abnormal placentation. The beta 1D knock-in embryos that survived midgestation developed until birth, but exhibited severely reduced skeletal muscle mass. Quantification of myotube numbers showed that substitution of beta 1A with beta 1D impairs primary myogenesis with no direct effect on secondary myogenesis. Furthermore, long-term primary myotube survival was affected in beta 1D knock-in embryos. Finally, overexpression of beta 1D in C2C12 cells impaired myotube formation while overexpression of beta 1A primarily affected myotube maturation. Together these results demonstrate for the first time distinct roles for beta1 integrins in primary versus secondary myogenesis and that the beta 1A and beta 1D variants are not functionally equivalent in this process.

Animals↗
PubMed1999

Effect of gel re-organization and tensional forces on alpha2beta1 integrin levels in dermal fibroblasts.

Mechanical forces are known to play an important role in regulating cell function in a wide range of biological systems. This is of particular relevance to dermal fibroblast function, given that the skin is known to be held under an intrinsic natural tension. To understand more about the generation of force by dermal fibroblasts and their ability to respond to changes in it, we have studied the role of the beta1 integrin receptors expressed by dermal fibroblasts in their ability to generate tensional forces within a collagen type I matrix and the effect of altered tensional force on integrin expression by dermal fibroblasts. Using a purpose-built culture force monitor, function-blocking antibodies directed towards the beta1 receptors dramatically reduced the tensional forces generated by dermal fibroblasts in a 3D collagen I matrix. However, the specific involvement of alpha1 or alpha2 subunits could not be demonstrated. Analysis of cellular response demonstrated that cells isolated from contracting collagen gels expressed fourfold higher levels of alpha2 mRNA than cells isolated from fully restrained gels. The levels of beta1 messenger RNA were relatively unaffected by reductions in force. Cells exposed to single reductions in force, however, did not exhibit alterations in either alpha1 or beta1 mRNA levels. We propose, therefore that alpha2beta1 integrin receptor levels in dermal fibroblasts are not altered in response to single reductions of gel tension, but do change following a continual change in force and associated matrix re-organization

Antigens, CD↗
PubMed2006

Expression of collagen-binding integrin receptors in the mammalian sclera and their regulation during the development of myopia.

PURPOSE: The sclera has a collagen-rich extracellular matrix that undergoes significant biochemical and biomechanical remodeling during myopic eye growth. The integrin family of cell surface receptors play critical roles in extracellular matrix and biomechanical remodeling in connective tissues. This study identified the major collagen-binding integrin receptors in the mammalian sclera and investigated their mRNA expression during the development of and recovery from experimental myopia. METHODS: The presence of the alpha1, alpha2, and beta1 integrin subunits was examined by using tree-shrew-specific primers and RT-PCR. Scleral expression of alpha1beta1 and alpha2beta1 receptor proteins was further investigated by using Western blot analysis and immunocytochemistry. Myopia was induced monocularly by occluding pattern vision and scleral tissue collected after 24 hours and 5 days. In a subset of the 5-day treatment group, vision was restored for 24 hours before tissue was isolated. Total RNA was extracted, and integrin subunit expression levels were assessed with quantitative real-time PCR. RESULTS: The presence of the major collagen-binding integrin subunits alpha1, alpha2, and beta1 was confirmed by RT-PCR in both scleral tissue and cultured scleral fibroblasts. Both the alpha1 and alpha2 integrin subunit proteins were identified in tree shrew scleral tissues, and integrin receptor expression was localized to scleral fibroblast focal adhesions. After only 24 hours of myopia induction, a time when no structural elongation has occurred, significant decreases were observed in the expression of the alpha1 (-36%) and beta1 (-44%) integrin subunits. After 5 days of myopia induction, alpha1 integrin expression had returned to baseline levels, whereas the alpha2 subunit showed a significant decrease in expression (-52%). The 5-day integrin profiles were maintained during recovery from the induced myopia, with only alpha2 integrin showing a statistically significant relative decrease in expression (-41%). CONCLUSIONS: The mammalian sclera expresses the major collagen-binding integrin subunits. The alpha1 and beta1 subunit expression was decreased early during the development of myopia, whereas the regulation of alpha2 integrin occurred at a later time point. The differential regulation of alpha1beta1 and alpha2beta1 during the development of myopia may reflect specific roles for these receptors in the scleral extracellular matrix and biomechanical remodeling that accompanies myopic eye growth.

Animals↗
PubMed2000

Skin and hair follicle integrity is crucially dependent on beta 1 integrin expression on keratinocytes.

beta 1 integrins are ubiquitously expressed receptors that mediate cell-cell and cell-extracellular matrix interactions. To analyze the function of beta1 integrin in skin we generated mice with a keratinocyte-restricted deletion of the beta 1 integrin gene using the cre-loxP system. Mutant mice developed severe hair loss due to a reduced proliferation of hair matrix cells and severe hair follicle abnormalities. Eventually, the malformed hair follicles were removed by infiltrating macrophages. The epidermis of the back skin became hyperthickened, the basal keratinocytes showed reduced expression of alpha 6 beta 4 integrin, and the number of hemidesmosomes decreased. Basement membrane components were atypically deposited and, at least in the case of laminin-5, improperly processed, leading to disruption of the basement membrane and blister formation at the dermal-epidermal junction. In contrast, the integrity of the basement membrane surrounding the beta 1-deficient hair follicle was not affected. Finally, the dermis became fibrotic. These results demonstrate an important role of beta 1 integrins in hair follicle morphogenesis, in the processing of basement membrane components, in the maintenance of some, but not all basement membranes, in keratinocyte differentiation and proliferation, and in the formation and/or maintenance of hemidesmosomes.

Animals↗
PubMed2003

Structural and compositional analysis of the keratinocyte migration track.

Slowly migrating cells such as fibroblasts leave behind a "migration track," which has been assumed not to occur in fast-moving cells such as keratinocytes. Here we show that keratinocytes left behind "migration tracks" of cellular remnants consisting of membranous patches or macroaggregates that were anchored to a meshwork of extracellular matrix proteins consisting of collagen type IV, fibronectin, laminin, and laminin 5. According to their origin and localisation, two types of macroaggregates could be distinguished : (1) Spherical and elongated tubular structures (diameter about 50-110 nm) both of which were arranged like "pearls on a string" and that apparently derived from fragmentation of retracting fibres. (2) Spherical structures (diameter about 50 nm) left behind in the gaps between the retracting fibres and presumably derived from former focal adhesion sites. Both types of macroaggregates did not contain cytoplasmic proteins but carried on their surface adhesion proteins, particularly high amounts of integrins : type 1 macroaggregates contained alpha3beta1-integrins, whereas type 2 macroaggregates contained other types of integrins such as alpha6beta4-integrins. Modulation of keratinocyte adhesion by using poly-L-lysine coated cover slips resulted in an increased application of inhibitory beta1-antibodies and slightly reduced migration velocity and track formation. Within 24 h of migration, we observed a migration velocity-dependent loss of cellular beta1-integrin by macroaggregate formation of about 11% for fast and about 4% for slowly migrating keratinocytes. The physiological role of the migration track is unclear. However, with its multiple adhesion sites it may serve as a provisional basement membrane during reepithelialization of epidermal wounds.

Amyloid beta-Protein Precursor↗
PubMed2006

Integrin alpha3 subunit participates in myoblast adhesion and fusion in vitro.

Satellite cells are myogenic precursor cells, participating in growth, and regeneration of skeletal muscles. The proteins that play a role in myogenesis are integrins. In this report, we show that the integrin alpha3 subunit is expressed in quiescent satellite cells and activated myoblasts. We also find that in myoblasts the integrin alpha3 subunit is localized at cell-cell and cell-extracellular matrix contacts. We notice that increase in protein and mRNA encoding the integrin alpha3 subunit accompanies myoblast differentiation. Using double immunofluorescence and immunoprecipitation experiments, we demonstrate that the integrin alpha3 subunit co-localizes with actin, and binds the integrin beta1 subunit and ADAM12, suggesting that the complex alpha3beta1/ADAM12 is probably involved in myoblast fusion. Importantly, overexpression of the full-length integrin alpha3 subunit increases myoblast fusion whereas an antibody against its extracellular domain inhibits fusion. These data demonstrate that the integrin alpha3 subunit may contribute to satellite cell activation and then myoblast adhesion and fusion.

ADAM Proteins↗
PubMed1998

Initiation of skin basement membrane formation at the epidermo-dermal interface involves assembly of laminins through binding to cell membrane receptors.

To study the mechanism of basement membrane formation, we determined by immunochemistry temporal and spatial expression of laminin-5 (Ln-5), laminin-1 (Ln-1) and their integrin receptors during early skin morphogenesis. A 3-dimensional skin culture was used that allows the study of the sequential molecular events of basement membrane formation at the epidermodermal interface. During early anchorage of keratinocytes to the extracellular matrix there is expression of Ln-5, BP-230 antigen and alpha3, beta1 integrin subunits. During epidermal stratification and prior to the formation of the lamina densa there is assembly of Ln-5, Ln-1, collagen IV and nidogen accompanied by keratinocyte basal clustering of alpha2, alpha3, alpha6, beta1, and beta4+ integrin subunits. The assembly pattern of Ln-1 and Ln-5 can be disturbed with functional antibodies against the beta1 (AIIB2) and alpha6 (GoH3) integrin subunits. Ln-1 assembly can also be disturbed with antibodies against its E8 domain and by competitive inhibition with a synthetic peptide (AG-73) derived from its G-4 domain. Quantitative RT-PCR showed that the dermis contributes about 80% of the laminin gamma)1 chain mRNA while 20% is produced by the epidermis which emphasizes its dual tissue origin and the major contribution of the mesenchyma in laminin production. The laminin gamma2 chain mRNA, present in Ln-5, was mostly of epidermal origin. This study presents evidence that during the initiation of basement membrane formation, laminins bind to keratinocyte plasma membrane receptors and thus may serve as nucleation sites for further polymerization of these compounds by a self-assembly process.

Actin Cytoskeleton↗
PubMed1996

Expression of the human alpha2 integrin subunit in mouse melanoma cells confers the ability to undergo collagen-directed adhesion, migration and matrix reorganization.

Previous studies have shown that cultured human and other mammalian cell require the alpha2 beta1 integrin receptor to reorganize and contract 3-dimensional extracellular matrix lattices containing collagen type I. This function is of prime importance for the later phases of wound healing, in which fibroblasts reorganize and contract extracellular matrix components newly deposited in the granulation tissue. It is also known that highly aggressive human melanoma cells have acquired this function, possibly enhancing their invasive potential. To further study alpha2 beta1-mediated functions, we expressed the human alpha2 and beta1 chains of integrins in mouse melanoma cells (BULT). The parental cell line was unable to exert known alpha2 beta1-mediated functions: The cells did not adhere, spread, or migrate on collagen type I, and they did not reorganize 3-dimensional collagen I lattices. Transfection of the human alpha2 chain was sufficient to confer not only specific adhesion to collagen type I in static adhesion assays, but also the ability to exert complex functions such as migration on collagen and efficient reorganization of collagen-I-containing matrices. Coexpression of the human beta1 chain did not further enhance these functions in BULT melanoma cells. This was underscored by the observation that alpha1-specific monoclonal antibodies were able to completely block the newly introduced functions, whereas beta1-specific antibodies had no such effect. Moreover, in transfectants expressing both the human alpha2 and beta1 chains, the human alpha2 chain did not preferentially associate with the human beta1 chain as compared with mouse beta-chains. This may indicate that the molecular structures guiding the physical association of the human alpha2 chain with beta chains are extremely highly conserved between the species.

Animals↗