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PubMed2004

A mouse model for the study of recurrent corneal epithelial erosions: alpha9beta1 integrin implicated in progression of the disease.

PURPOSE: To describe an in vivo mouse model for the study of recurrent corneal erosion syndrome (RCES) in mice and to characterize the changes in alpha9 integrin expression during wound healing. METHODS: Corneal epithelial debridement wounds of two sizes (1.5 and 2.5 mm) were made on the ocular surface of BALB/c mice and were evaluated at various times after wounding. Corneas were processed either as whole mounts and stained with propidium iodide and an antibody against alpha9 integrin or for bromodeoxyuridine analyses of cell proliferation. A separate study involved analyses of corneal wound healing over time in individual mice with large and small debridement wounds. Mice were anesthetized once per week and their corneas stained with fluorescein to assess the quality of the corneal epithelium. After 6 weeks, mice were killed and eyes processed for study by immunofluorescence in either whole mounts or frozen sections. RESULTS: Whole mount confocal microscopy showed open wounds on the ocular surface of mice at 1 and 2 weeks after large wounds were created, but not after small wounds. In addition, alpha9 integrin was upregulated during healing, and changes were observed in alpha9 integrin localization at the limbus with large wounds but not with small wounds. Although only 1 of 16 corneas with small wounds had erosions at 1 and 2 weeks, 11 of 16 with large wounds had erosions. However, by 6 weeks, 13 of 16 eyes showed signs of erosion whether wounds were small or large. With large wounds, RCES corneas frequently showed numerous goblet cells adjacent to a limbus lacking alpha9 integrin. Corneas from mice with documented RCES showed both retention of alpha9 integrin and tenascin-C expression at the anterior stromal-epithelial interface as well as impaired relocalization of alpha3beta1 integrin to the basement membrane zone. CONCLUSIONS: These data show that spontaneous recurrent corneal erosions occur in a mouse model after manual creation of a single wound by debridement. Differences between the healing of small (1.5 mm) and large (2.5 mm) wounds were observed. Large wounds often resulted in the presence of goblet cells on the central cornea and a loss of alpha9 integrin at the limbus. Small wounds never showed differences in the localization of alpha9 integrin at the limbus, and no goblet cells were observed in the central cornea. More studies are needed to understand the causes of erosions in these mice.

Animals↗

PubMed2003

Alpha 6 beta 4 integrin regulates keratinocyte chemotaxis through differential GTPase activation and antagonism of alpha 3 beta 1 integrin.

Growth factor-induced cell migration and proliferation are essential for epithelial wound repair. Cell migration during wound repair also depends upon expression of laminin-5, a ligand for alpha 6 beta 4 integrin. We investigated the role of alpha 6 beta 4 integrin in laminin-5-dependent keratinocyte migration by re-expressing normal or attachment-defective beta 4 integrin in beta 4 integrin null keratinocytes. We found that expression of beta 4 integrin in either a ligand bound or ligand unbound state was necessary and sufficient for EGF-induced cell migration. In a ligand bound state, beta 4 integrin supported EGF-induced cell migration though sustained activation of Rac1. In the absence of alpha 6 beta 4 integrin ligation, Rac1 activation became tempered and EGF chemotaxis proceeded through an alternate mechanism that depended upon alpha 3 beta 1 integrin and was characterized by cell scattering. alpha 3 beta 1 integrin also relocalated from cell-cell contacts to sites of basal clustering where it displayed increased conformational activation. The aberrant distribution and activation of alpha 3 beta 1 integrin in attachment-defective beta 4 cells could be reversed by the activation of Rac1. Conversely, in WT beta 4 cells the normal cell-cell localization of alpha 3 beta 1 integrin became aberrant after the inhibition of Rac1. These studies indicate that the extracellular domain of beta 4 integrin, through its ability to bind ligand, functions to integrate the divergent effects of growth factors on the cytoskeleton and adhesion receptors so that coordinated keratinocyte migration can be achieved.

Cell Adhesion Molecules↗

PubMed2004

Inhibition of basement membrane formation by a nidogen-binding laminin gamma1-chain fragment in human skin-organotypic cocultures.

Basement membranes generally determine different tissue compartments in complex organs, such as skin, playing not only an important structural but also a regulatory role. We have previously demonstrated the formation of a regular basement membrane in organotypic three-dimensional (3D)-cocultures of human skin keratinocytes and fibroblasts by indirect immunofluorescence and transmission electron microscopy. In this assembly process, cross-linking of type IV collagen and the laminin gamma1 chain by nidogen is considered a crucial step. For a functional proof, we have now competitively inhibited nidogen binding to laminin in 3D-cocultures with a recombinant laminin gamma1 fragment (gamma1III3-5 module) spanning this binding site. Repeated treatment abolished the deposition of nidogen at the epithelial-matrix interface but also greatly perturbed the presence of other matrix constituents such as laminin and perlecan. This effect persisted over the entire observation period of 10 to 21 days. In contrast, some components of the basement membrane zone were only moderately affected, with the laminin-5 isoform (gamma2 chain), type IV collagen and integrin alpha6ss4 still showing a distinct staining at their regular position, when seen by light microscopy. Furthermore, epidermal morphology and differentiation remained largely normal as indicated by the regular location of keratins K1/K10 and also of late differentiation markers. Ultrastructural examination demonstrated that the gamma1 fragment completely suppressed any formation of basement membrane structures (lamina densa) and also of hemidesmosomal adhesion complexes. As a consequence of hemidesmosome deficiency, keratin filament bundles were not attached to the ventral basal cell aspect. These findings were further substantiated by immuno-electron microscopy, revealing either loss or drastic reduction and dislocation of basement membrane and hemidesmosomal components. Taken together, in this simplified human skin model (representing a 'closed system') a functional link has been demonstrated between compound structures of the extra- and intracellular space at the junctional zone providing a basis to interfere at distinct points and in a controlled fashion.

Basement Membrane↗

PubMed2004

Alpha 3 beta 1 integrin promotes keratinocyte cell survival through activation of a MEK/ERK signaling pathway.

Inadequate or inappropriate adhesion of epithelial cells to extracellular matrix leads to a form of apoptosis known as anoikis. During various tissue remodelling events, such as wound healing or carcinoma invasion, changes in the physical properties, and/or composition of the extracellular matrix, can lead to anoikis of epithelial cells that lack appropriate receptor-matrix interactions. Laminin-5 is the major ligand for keratinocyte adhesion in the epidermis, and it also promotes keratinocyte survival in vivo and in vitro. Integrins alpha 3 beta 1 and alpha 6 beta 4 are the major receptors for laminin-5; however, specific roles for these integrins in keratinocyte survival have not been determined. In the current study, we exploited keratinocyte cell lines derived from wild-type or alpha 3 integrin knockout mice to reveal a critical role for alpha 3 beta 1 in protecting keratinocytes from apoptosis upon serum withdrawal. We show that alpha 3 beta 1-mediated adhesion to laminin-5 extracellular matrix inhibits proteolytic activation of caspase-3 and TUNEL-staining, both hallmarks of apoptosis. We also show that alpha 3 beta1-mediated adhesion activates focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), and that inhibition of either FAK or ERK signaling leads to apoptosis of keratinocytes attached to laminin-5. alpha 6 beta 4-mediated adhesion to laminin-5 only partially protects cells from apoptosis in the absence of alpha 3 beta 1, and alpha 6 beta 4 is not necessary for cell survival in the presence of alpha 3 beta 1. These results suggest that alpha 3 beta 1 is necessary and sufficient for maximal keratinocyte survival on laminin-5. We propose a model to address the potential importance of alpha 3 beta 1-mediated survival for migrating keratinocytes at the leading edge of a cutaneous wound.

Animals↗

PubMed2006

Spatial and temporal control of laminin-332 (5) and -511 (10) expression during induction of anagen hair growth.

Basement membrane plays important roles in hair growth. We characterized changes in laminin isoform expression during hair cycling. At the mRNA level, laminin-511 (10) expression underwent a steady increase during anagen stages. In contrast, laminin-332 (5) expression was initially upregulated in outer root sheath (ORS) keratinocytes at anagen II and then transiently downregulated. Laminin-332 significantly increased coincident with the signal in inner root sheath and hair matrix cells after anagen IV. Levels of laminin-332 proteins were also upregulated at late anagen I-III but dropped after anagen IV. This decrease coincided with increased levels of mRNA encoding the two proteases, membrane type 1 metalloproteinase and bone morphogenetic protein 1, involved in laminin-332 processing. Immunohistochemistry demonstrated that laminin-332 and alpha6 beta4 integrin were well colocalized, but their signals were remarkably decreased in the lower half of follicles after anagen VI. Consistent with these data, ultrastructurally mature hemidesmosomes were seen in ORS keratinocytes at anagen II, whereas at anagen VI, only fragmental hemidesmosomes were present. In hair follicle culture, laminin-511 (10)/521 (11)-rich human placental laminin enhanced hair growth, whereas recombinant laminin-332 antagonized hair growth induced by laminin-511. Our results indicate a positive role for laminin-511 and a negative role for laminin-332 on hair growth.

Animals↗

PubMed2006

A hypomorphic mutation in the mouse laminin alpha5 gene causes polycystic kidney disease.

Extracellular matrix abnormalities have been found in both human and animal models of polycystic kidney disease (PKD). A new mouse PKD model has been produced through insertion of a PGKneo cassette in an intron of the gene that encodes laminin alpha5 (Lama5), a major tubular and glomerular basement membrane component that is important for glomerulogenesis and ureteric bud branching. Lama5neo represents a hypomorphic allele as a result of aberrant splicing. Lama5neo/neo mice exhibit PKD, proteinuria, and death from renal failure by 4 wk of age. This contrasts with mice that totally lack Lama5, which die in utero with multiple developmental defects. At 2 d of age, Lama5neo/neo mice exhibited mild proteinuria and microscopic cystic transformation. By 2 wk, cysts were grossly apparent in cortex and medulla, involving both nephron and collecting duct segments. Tubular basement membranes seemed to form normally, and early cyst basement membranes showed normal ultrastructure but developed marked thickening as cysts enlarged. Overall, Lama5 protein levels were severely reduced as a result of mRNA frameshift caused by exon skipping. This was accompanied by aberrant accumulation of laminin-332 (alpha3beta3gamma2; formerly called laminin-5) in some cysts, as also observed in human PKD. This constitutes the first evidence that a primary defect in an extracellular matrix component can cause PKD.

Alleles↗

PubMed2004

Mature human thymocytes migrate on laminin-5 with activation of metalloproteinase-14 and cleavage of CD44.

We have previously shown that laminin-5 is expressed in the human thymic medulla, in which mature thymocytes are located. We now report that laminin-5 promotes migration of mature medullary thymocytes, whereas it has no effect on cortical immature thymocytes. Migration was inhibited by blocking mAbs directed against laminin-5 integrin receptors and by inhibitors of metalloproteinases. Interactions of thymocytes with laminin-5 induced a strong up-regulation of active metalloproteinase-14. However, we found that thymocytes did not cleave the laminin-5 gamma(2) chain, suggesting that they do not use the same pathway as epithelial cells to migrate on laminin-5. Interactions of thymocytes with laminin-5 also induced the release of a soluble fragment of CD44 cell surface molecule. Moreover, CD44-rich supernatants induced thymocyte migration in contrast with supernatants depleted in CD44 by immunoadsorption. CD44 cleavage was recently reported to be due to metalloproteinase-14 activation and led to increased migration in cancer cells. Thus, in this study, we show that laminin-5 promotes human mature thymocyte migration in vitro via a multimolecular mechanism involving laminin-5 integrin receptors, metalloproteinase-14 and CD44. These data suggest that, in vivo, laminin-5 may function in the migration of mature thymocytes within the medulla and be part of the thymic emigration process.

Cell Adhesion Molecules↗

PubMed2003

[Integrin alpha6 beta4 and experimental allergic neuritis].

OBJECTIVE: To explore the relationship between integrins alpha6,beta4 and the process of the injury and repair of the myelin, their expressions in experimental allergic neuritis were analyzed. METHODS: Experimental allergic neuritis (EAN) models were induced in Lewis rats. The expressions of integrins alpha6 and beta 4 and their receptor laminin, both in the acute phase and recovery phase of EANs, were analyzed by in situ hybridization and immunohistochemistry. RESULTS: The expression of beta4 decreased in the acute phase P<0.05 and showed no significant changes in the recovery phase(P=0.6840). There were no significant changes of the expression of alpha6 and laminin both in the acute phase and recovery phase of EANs by immunohistochemistry (alpha6 P=0.0751;laminin P=0.2047). The similar results were obtained by in situ hybridization (beta4 in acute phase P<0.05;beta4 in recovery phase P=0.823;alpha6 P= 0.81). CONCLUSION: The expressions of integrins of Schwann cells were affected by inflammation injury, and their patterns of expression were similar to those of integrins alpha6, beta1 and beta4 during embryogenesis. It is inferred that integrin beta4 and the reformation of myelin have a more imitate relationship. The expression changes of alpha6 and beta4 correlate with the injury and repair of myelin during EAN.

Animals↗

PubMed2004

[Expression of the integrin alpha 6 beta 4 and effects of integrin alpha 6 beta 4 on adhesive and invasive behaviors of human laryngeal carcinoma cell lines].

OBJECTIVE: To study the expression of integrin alpha 6 beta 4 in human laryngeal carcinoma cell lines Hep-2 and AMC-HN8. To explore the mechanism and the effects of integrin alpha 6 beta 4 in human laryngeal carcinoma cell lines. METHOD: Flowcytometry was used to exame the surface expression of integrin alpha 6 beta 4 in human laryngeal carcinoma cell lines. Anti-alpha 6 and anti-beta 4 specific monoclonal antibody were used in adherence inhibition assay and in vitro invasion assay. RESULT: Overexpression of integrin alpha 6 beta 4 were present in both Hep-2 and AMC-HN8 cell lines. Anti-alpha 6 and anti-beta 4 specific monoclonal antibody together blocked the alpha 6 beta 4 on the surface of the cells resulting in low adherence on the extracellular matrix and weak invasion through the extracellular matrix. CONCLUSION: Integrin alpha 6 beta 4 play a vitral role in the adhension and invasion of laryngeal carcinoma. Anti-alpha 6 and anti-beta 4 specific monoclonal antibody together can block the function of integrin alpha 6 beta 4. So treatment with anti-alpha 6 and anti-beta 4 specific monoclonal antibody may reduce the development of laryngeal carcinoma.

Antibodies, Monoclonal↗

PubMed1988

Monoclonal antibody to human carcinoma-associated protein complex: quantitation in normal and tumor tissue.

We have identified and quantitated a tumor protein complex, TSP-180, on murine carcinomas with two monoclonal antibodies (MoAbs) (Cancer Res., 46: 707-712, 1986). One of the two MoAbs, 135-13C, recognizes a TSP-180-like protein complex on several human carcinomas in culture. MoAb 135-13C has been used to purify the human TSP-180 complex from A431 cells and the purified material used to immunize F344 rats to produce another MoAb, 439-9B, to the human TSP-180 complex. This MoAb does not precipitate the murine TSP-180 or bind to murine cells. Both MoAb 135-13C and 439-9B precipitated the same proteins from A431 cells but did not compete with each other for binding sites, indicating that they recognize different epitopes on the same protein. The two MoAbs have been used in a two-site assay to quantitate TSP-180 proteins on human cells and tissues. Carcinoma cell lines A431, SW948, and A549 all give high values (46 to 443 ng/mg of protein) while murine tumors, a human melanoma, and human fibroblasts are negative (less than 10 ng/mg of protein). Most tissues from autopsy of 2 normal individuals are negative for human TSP-180 at the levels tested (less than 10 ng/mg of protein). Some organs have intermediate range expression: spleen, 5 to 111 ng/ml of protein; colon, 24 to 111; and small intestine, 39 to 99. One primary colon and one larynx tumor were positive (144 to 372 ng/mg of protein) while 5 breast carcinomas, a stomach tumor, a metastatic melanoma, and a kidney tumor were negative. These data indicate that human TSP-180 may be preferentially expressed in certain malignant carcinomas of diverse origin. The potential for TSP-180 as a tumor marker requires further study.

Animals↗

PubMed1989

Ligand-induced phosphorylation of a murine tumor surface protein (TSP-180) associated with metastatic phenotype.

A tumor surface protein (TSP-180) has been identified on murine lung carcinomas using two monoclonal antibodies (MoAbs) (135-13C and 346-11A). Quantitative analysis of TSP-180 on 3LL variants maintained either in vitro or in vivo indicates that TSP-180 is highly expressed in highly malignant metastatic cells. In reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns of TSP-180 obtained with MoAb 135-13C from cell lysates of 3LL metastatic cells show three proteins migrating to Mr 204,000, 134,000, and 116,000. In the same experimental conditions MoAb 135-13C precipitates from low metastasizing ones only one band, corresponding to the lower molecular weight (Mr 116,000). All bands of TSP-180 observed in 3LL variants are labeled by lactoperoxidase-catalyzed radioiodination of viable cells, incorporate 32PO4, and contain carbohydrates, as judged by binding to wheat germ agglutinin. These results indicate that all proteins have external exposure on the cell surface and that at least some of TSP-180 proteins could be differentially regulated in different tumor cells (highly metastatic versus low metastatic). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns and immunoblots obtained from cell lysates of 3LL variants by using a monoclonal antibody to phosphotyrosine (IG-2) indicate that this MoAb recognizes proteins migrating with molecular weights identical to those reported for TSP-180. Moreover, the immunoblots of solubilized immunocomplex, obtained from cell lysates of 3LL variants by using MoAb 135-13C, demonstrate that MoAb IG-2 specifically reacts with TSP-180 proteins. Experiments undertaken in order to assess if some or all of TSP-180 proteins have tyrosine kinase activity demonstrate that MoAb 135-13C binding to the cell surface induces specific phosphorylation of the Mr 204,000 protein of TSP-180. Phosphoaminoacid analysis of the ligand-induced phosphorylated protein (pp204) demonstrates that this protein is phosphorylated at serine and tyrosine. Results reported lead us to hypothesize that TSP-180 is involved in growth-regulation mechanisms and that its high expression on cells with more malignant phenotype could be responsible for a proliferative advantage of such tumor clones.

Amino Acids↗

PubMed1993

The integrin alpha 6 beta 4 on TA3/Ha mammary carcinoma cells is involved in adhesion to hepatocytes.

TA3/Ha mammary carcinoma cells form liver metastases upon intraportal injection. We have studied the interaction between these cells and hepatocytes that is likely to be important for liver metastasis formation. We show that the integrin alpha 6 beta 4, which is highly expressed on TA3 cells, is involved in this interaction. Fab fragments, generated from a polyclonal serum against TA3 cells, inhibited TA3-hepatocyte adhesion. By affinity purification on purified alpha 6 beta 4, we isolated alpha 6 beta 4-specific Fab fragments from this anti-TA3 serum. These antibodies were highly specific for alpha 6 beta 4, as demonstrated by Western blot analysis and immunoprecipitation, and inhibited TA3-hepatocyte adhesion. This shows that alpha 6 beta 4, which thus far has only been implicated in cell-matrix adhesion, can also mediate interactions with cell surfaces. Our results suggest that the high levels of alpha 6 beta 4 often expressed by metastatic carcinoma cells contribute to liver metastasis formation.

Animals↗

PubMed1993

The suprabasal expression of alpha 6 beta 4 integrin is associated with a high risk for malignant progression in mouse skin carcinogenesis.

Enhanced expression of the alpha 6 beta 4 integrin complex has been linked to malignant progression in mouse skin carcinogenesis. To determine if alpha 6 beta 4 expression can predict risk for malignant conversion among populations of benign skin tumors, we analyzed the distribution of alpha 6 beta 4 and other markers of progression in papillomas at high and low risk for malignant progression. After initiation with 7,12-dimethylbenz[a]anthracene, mice were promoted with 12-O-tetradecanoylphorbol-13-acetate to induce predominantly low risk tumors or promoted with mezerein to produce predominantly high risk tumors. When tumors first appeared at 8 weeks after promotion, high risk papillomas demonstrated basal and suprabasal alpha 6 beta 4 expression, loss of keratin 1, and aberrant expression of keratin 13. In these tumors alpha 6 beta 4 expression coincided with an expansion of the proliferating compartment as indicated by suprabasal bromodeoxyuridine labeling. In contrast, alpha 6 beta 4 immunostaining was confined to basal cells in low risk tumors, keratin 1 was abundant, and keratin 13 was absent in the majority of this group, while proliferating cells were largely in the basal compartment. By 33 weeks, alpha 6 beta 4 suprabasal expression continued to distinguish groups at higher risk for malignant conversion, but keratin 13 was expressed in all groups. At this time, high risk tumors displayed focal expression of keratin 8 and gamma-glutamyltranspeptidase, markers also found in chemically induced carcinomas. Keratin 8 and gamma-glutamyltranspeptidase were expressed only in alpha 6 beta 4 positive cells. These results indicate that expression of alpha 6 beta 4 integrin in suprabasal strata serves as an early predictive marker to identify benign squamous tumors at high risk for malignant progression.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1993

Specific internalization of basal membrane domains containing the integrin alpha 6 beta 4 in dispase-detached cultured human keratinocytes.

The integrin alpha 6 beta 4 is polarized towards the basal side of basal keratinocytes and helps anchor these cells to the basement membrane components. We have found that cultured human epidermal keratinocytes, when detached from their culture substratum, as for grafting, using the enzyme dispase, rapidly internalize the basal membrane domains containing the integrin alpha 6 beta 4, while integrins of the very late antigen subtype remain on the cell surface. Detachment and incubation at 4 degrees C prevent this internalization, as well as the contraction of the detached sheet area. Subsequent incubation at 37 degrees C initializes this contraction and allows the basal integrin alpha 6 beta 4 to be internalized. We took advantage of this blockage to label upon detachment using immunogold techniques, the alpha 6 subunit present on the basal cell surface; then we studied its internalization with the electron microscope. This internalization pathway differs from classical receptor-mediated endocytosis, and intermediate filaments might possibly play a role in this process. Interestingly, 1 h after their internalization from the basal membrane, a third of the gold particles labeling the alpha 6 subunit was found between lateral membranes of basal cells, strongly suggesting that the integrin alpha 6 beta 4 can be partly recycled to the cell surface in these conditions.

Antigens, Surface↗

PubMed2003

Phenotypic study of a case receiving a keratolimbal allograft and amniotic membrane for total limbal stem cell deficiency.

PURPOSE: To report the expression pattern of key molecules by the reconstructed corneal epithelium after a keratolimbal allograft (KLAL) and amniotic membrane transplantation (AMT) for total limbal stem cell deficiency. DESIGN: Interventional case report. METHOD: A 50-year-old woman with severe chemical burns in both eyes received an AMT as a temporary patch at the acute stage, and a KLAL with AMT as a graft at the chronic stage for total limbal stem cell deficiency. The corneal button removed during subsequent corneal transplantation was submitted for immunofluorescence staining with monoclonal antibodies against keratin K3, MUC5AC, connexin 43, integrins alpha3beta1 and alpha6beta4, and laminin 5 for comparison with a normal cornea. RESULTS: Histologically, a normal stratified corneal epithelium has five to six cell layers that lay on the thick amniotic membrane basement membrane. The phenotype was of a corneal origin, based on expression of positive keratin K3, negative MUC5AC, and positive connexin 43. Furthermore, intact basement membrane complexes were present, evidenced by positive staining to integrins alpha3beta1 and alpha6beta4 and to laminin 5. CONCLUSIONS: A normal corneal epithelial phenotype with normal basement membrane complexes was restored after a KLAL and AMT in a case with total limbal stem cell deficiency.

Amnion↗

PubMed1997

alpha3beta1 Integrin is required for normal development of the epidermal basement membrane.

Integrins alpha3beta1 and alpha6beta4 are abundant receptors on keratinocytes for laminin-5, a major component of the basement membrane between the epidermis and the dermis in skin. These integrins are recruited to distinct adhesion structures within keratinocytes; alpha6beta4 is present in hemidesmosomes, while alpha3beta1 is recruited into focal contacts in cultured cells. To determine whether differences in localization reflect distinct functions of these integrins in the epidermis, we studied skin development in alpha3beta1-deficient mice. Examination of extracellular matrix by immunofluorescence microscopy and electron microscopy revealed regions of disorganized basement membrane in alpha3beta1-deficient skin. Disorganized matrix was first detected by day 15.5 of embryonic development and became progressively more extensive as development proceeded. In neonatal skin, matrix disorganization was frequently accompanied by blistering at the dermal-epidermal junction. Laminin-5 and other matrix proteins remained associated with both the dermal and epidermal sides of blisters, suggesting rupture of the basement membrane itself, rather than detachment of the epidermis from the basement membrane as occurs in some blistering disorders such as epidermolysis bullosa. Consistent with this notion, primary keratinocytes from alpha3beta1-deficient skin adhered to laminin-5 through alpha6 integrins. However, alpha3beta1-deficient keratinocytes spread poorly compared with wild-type cells on laminin-5, demonstrating a postattachment requirement for alpha3beta1 and indicating distinct roles for alpha3beta1 and alpha6beta4. Our findings support a novel role for alpha3beta1 in establishment and/or maintenance of basement membrane integrity, while alpha6beta4 is required for stable adhesion of the epidermis to the basement membrane through hemidesmosomes.

Animals↗

PubMed1999

Synergistic activities of alpha3 and alpha6 integrins are required during apical ectodermal ridge formation and organogenesis in the mouse.

Integrins alpha6beta1 and alpha6beta4 are cell surface receptors for laminins. Integrin alpha6-null mice die at birth with severe skin blistering and defects in the cerebral cortex and in the retina. Integrin alpha3beta1 can associate with laminins and other ligands. Integrin alpha3-null mice also die at birth, with kidney and lung defects at late stages of development, and moderate skin blistering. To investigate possible overlapping functions between alpha3 and alpha6 integrins, we analyzed the phenotype of compound alpha3-/-/alpha6-/- mutant embryos. Double homozygous mutant embryos were growth-retarded and displayed several developmental defects not observed in the single mutant animals. First, limb abnormalities characterized by an absence of digit separation and the fusion of preskeletal elements were observed. Further analyses indicated a defect in the apical ectodermal ridge, an essential limb organizing center. In the double mutant, the ridge appeared flattened, and ridge cells did not show a columnar morphology. A strong reduction in ridge cell proliferation and alterations of the basal lamina underlying the ectoderm were observed. These results suggest that alpha3 and alpha6 integrins are required for the organization or compaction of presumptive apical ectodermal ridge cells into a distinct differentiated structure. Additional defects were present: an absence of neural tube closure, bilateral lung hypoplasia, and several abnormalities in the urogenital tract. Finally, an aggravation of brain and eye lamination defects was observed. The presence of novel phenotypes in double mutant embryos demonstrates the synergism between alpha3 and alpha6 integrins and their essential roles in multiple processes during embryogenesis.

Abnormalities, Multiple↗

PubMed2005

A secreted form of ADAM9 promotes carcinoma invasion through tumor-stromal interactions.

Tumor cell invasion is a process regulated by integrins, matrix-degrading enzymes, and interactions with host tissue stromal cells. The ADAM family of proteins plays an important role in modulating various cellular responses. Here, we show that an alternatively spliced variant of ADAM9 is secreted by hepatic stellate cells and promotes carcinoma invasion. ADAM9-S induced a highly invasive phenotype in several human tumor cell lines in Matrigel assays, and the protease activity of ADAM9-S was required for invasion. ADAM9-S binds directly to alpha6beta4 and alpha2beta1 integrins on the surface of colon carcinoma cells through the disintegrin domain. ADAM9-S was also able to cleave laminin and promote invasion. Analysis of human liver metastases revealed that ADAM9 is expressed by stromal liver myofibroblasts, particularly those that are localized within the tumor stroma at the invasive front. These results emphasize the importance of tumor-stromal interactions in invasion and suggest that ADAM9-S can be an important determinant in the ability of cancer cells to invade and colonize the liver.

ADAM Proteins↗