Multiple essential MT1-MMP functions in tooth root formation, dentinogenesis, and tooth eruption.

Membrane-type matrix metalloproteinase 1 (MT1-MMP) is a transmembrane zinc-endopeptidase that breaks down extracellular matrix components, including several collagens, during tissue development and physiological remodeling. MT1-MMP-deficient mice (MT1-MMP(-/-)) feature severe defects in connective tissues, such as impaired growth, osteopenia, fibrosis, and conspicuous loss of molar tooth eruption and root formation. In order to define the functions of MT1-MMP during root formation and tooth eruption, we analyzed the development of teeth and surrounding tissues in the absence of MT1-MMP. In situ hybridization showed that MT1-MMP was widely expressed in cells associated with teeth and surrounding connective tissues during development. Multiple defects in dentoalveolar tissues were associated with loss of MT1-MMP. Root formation was inhibited by defective structure and function of Hertwig's epithelial root sheath (HERS). However, no defect was found in creation of the eruption pathway, suggesting that tooth eruption was hampered by lack of alveolar bone modeling/remodeling coincident with reduced periodontal ligament (PDL) formation and integration with the alveolar bone. Additionally, we identified a significant defect in dentin formation and mineralization associated with the loss of MT1-MMP. To segregate these multiple defects and trace their cellular origin, conditional ablation of MT1-MMP was performed in epithelia and mesenchyme. Mice featuring selective loss of MT1-MMP activity in the epithelium were indistinguishable from wild type mice, and importantly, featured a normal HERS structure and molar eruption. In contrast, selective knock-out of MT1-MMP in Osterix-expressing mesenchymal cells, including osteoblasts and odontoblasts, recapitulated major defects from the global knock-out including altered HERS structure, short roots, defective dentin formation and mineralization, and reduced alveolar bone formation, although molars were able to erupt. These data indicate that MT1-MMP activity in the dental mesenchyme, and not in epithelial-derived HERS, is essential for proper tooth root formation and eruption. In summary, our studies point to an indispensable role for MT1-MMP-mediated matrix remodeling in tooth eruption through effects on bone formation, soft tissue remodeling and organization of the follicle/PDL region.

Clinicians' prescribing practices for bariatric surgery patients: is there an issue?

WHAT IS KNOWN AND OBJECTIVES: Patients undergoing Roux-en-Y gastric bariatric (RYGB) surgery present a reduced absorption site, and special attention should therefore be taken when prescribing oral-dosage forms. This study was carried out to investigate the extent to which non-bariatric clinicians are aware of this issue when prescribing medicines for this population, and what type of information is available to aid them in their decision-making. METHODS: Two questionnaires were created, one for non-bariatric clinicians and another for their patients who had undergone RYGB surgery, to gather information about the prescription practices for this population. Additionally, a literature search of pharmacokinetic studies on bariatric patients and recommended prescription practices was carried out. RESULTS AND DISCUSSION: Of the 62 non-bariatric clinicians surveyed, 50% believed RYGB surgery interferes with drug absorption; however, 68% still prescribed tablets as the first choice form of dosage. Young clinicians (35%) were less likely to believe that RYGB surgery could affect drug absorption than experienced clinicians (43%). The main reasons for changing dosage forms were patient complaints about efficacy or difficulty in swallowing tablets. Of the 73 patients, 43 were taking drugs in tablet form after the surgery, 24 of whom had health issues unrelated to the surgery. None of the journals read by the clinicians contained pharmacokinetics (PK) studies involving bariatric surgery patients or presented recommendations for the prescription of oral-dosage forms for this population. The literature search revealed a total of 22 drugs that had undergone PK studies in RYGB patients. Fifteen of them were reported to have decreased effects, 12 of which were administered as tablets. WHAT IS NEW AND CONCLUSION: There is still a relative lack of clinical evidence to guide clinicians when prescribing medicines for bariatric patients. It is therefore recommended that pharmacists should have greater participation in the prescription process to advise non-bariatric clinicians and educate RYGB surgery patients to help avoid therapeutic failure.

Effects of drospirenone/estradiol on steroid receptors and Bcl-2 in the postmenopausal endometrium.

OBJECTIVE: To evaluate the effect of drospirenone with 17ß-estradiol on the histology and expression of estrogen and progesterone receptors and of Bcl-2 protein, in endometrium of postmenopausal women. METHOD: Forty postmenopausal women, including controls, participated in this study evaluating oral hormone replacement treatment combining 2 mg/day of drospirenone with 1 mg/day of 17ß-estradiol administered for a 24-week period. The effect on the endometrium was assessed by histology and the apoptosis marker Bcl-2. The immunoexpression of estrogen (ER) and progesterone (PR) receptors in the endometrium was also measured. RESULTS: No increase in endometrial thickness was evident after either treatment, although endometrial histology was atrophic in most biopsies. The drospirenone/estradiol group showed higher expression of ER and PR in glandular epithelium compared to stroma, but the Bcl-2 protein was more immunoreactive in stroma than in glandular epithelium. Compared to controls, drospirenone/estradiol users showed higher immunoexpression of ER, PR and Bcl-2 in both glandular epithelium and endometrial stroma. CONCLUSION: A 24-week course of drospirenone with 17ß-estradiol resulted in low proliferation and was shown to lead to atrophic endometrium. The novel progestogen drospirenone seems to have favorable effects on the endometrium of postmenopausal women due to its pro-apoptotic action in glandular epithelium.

MT1-MMP is required for efficient tumor dissemination in experimental metastatic disease.

Membrane-type I matrix metalloproteinase (MT1-MMP) is associated with multiple forms of cancer including mammary cancer. To directly evaluate the significance of MT1-MMP expression in tumor progression and metastasis using a genetically induced cancer model, we crossed MT1-MMP-deficient mice to MMTV-polyoma virus middle T-antigen (PyMT) mice. Expression of PyMT in the MT1-MMP-deficient background consistently resulted in hyperplasia of the mammary gland as seen in wild-type PyMT littermates. Following orthotopic transplantation of PyMT+ glands into the cleared mammary fat pad of syngeneic recipient mice, MT1-MMP-deficient tumors were palpable earlier than wild-type tumors. Moreover, MT1-MMP-deficient tumors grew to the experimental end point size quicker than control tumors, but demonstrated markedly reduced ability to metastasize to the lungs of recipient mice. Accordingly, MT1-MMP-deficient mice displayed an overall reduction in metastasis count of 50%. MT1-MMP was expressed solely in the stroma of PyMT-induced tumors and those metastatic nodules that formed in the lungs were devoid of MT1-MMP expression. Stromal fibroblasts isolated from MT1-MMP-deficient tumors did not degrade type I collagen suggesting that efficient dissemination of tumor cells is dependent on stromal cell remodeling of the tumor environment. The data demonstrate directly that MT1-MMP-mediated proteolysis by stromal cells is important in the metastatic process.

Effect of salt intake and inhibitor dose on arterial hypertension and renal injury induced by chronic nitric oxide blockade.

Long-term nitric oxide blockade by N omega -nitro-L-arginine methyl ester (L-NAME) leads to severe and progressive hypertension. The role of salt intake in this model is unclear. To verify whether salt dependence in this model is related to the extent of nitric oxide inhibition, we gave adult male Munich-Wistar rats a low salt, standard salt, or high salt diet and oral L-NAME treatment at either 3 or 25 mg/kg per day. At 10 to 15 days of treatment, the slope of the pressure-natriuresis line was decreased in rats receiving low-dose L-NAME compared with untreated controls. In rats treated with the higher dose, the line was shifted to the right but remained parallel to that obtained in untreated controls. Renal vascular resistance was moderately increased in rats receiving low-dose L-NAME, whereas high-dose L-NAME induced a marked vasoconstriction that was aggravated by salt overload. Low-dose L-NAME treatment induced hypertension only when associated with sodium overload. In rats receiving high-dose L-NAME, hypertension was aggravated by sodium excess but was not ameliorated by sodium restriction. Long-term (6 weeks) L-NAME treatment was associated with progressive hypertension, which was aggravated by salt overload, and with the development of albuminuria, focal glomerular collapse, glomerulosclerosis, and renal interstitial expansion. These abnormalities were worsened by salt overload and largely prevented by salt restriction. In the model of chronic nitric oxide blockade, salt dependence is a function of the inhibitor dose, and renal injury varies directly with the level of salt intake.

HIV-1 infection alters monocyte interactions with human microvascular endothelial cells.

HIV infection of monocytes resulted in twofold elevation of adhesion molecule LFA-1 (both alpha L/CD11a and beta 2/CD18 subunits) and LFA-3 (CD58), with no apparent increase in LFA-2 (CD2) or various beta 1-integrins. Homotypic aggregation of monocytes was evident 2 h after exposure to virus and was inhibited by mAbs to both the alpha L- and beta 2-subunits of LFA-1. HIV-infected monocytes also showed a marked increase in adherence to human capillary endothelial cell monolayers derived from brain, lung, and skin. This adherence was inhibited by mAb to either LFA-1 subunit and by mAb to the counter-receptor intercellular adhesion molecule-1. Cocultivation of HIV-infected monocytes with endothelial cells increased permeability of endothelial cell monolayers to 125I albumin in transwell assay systems. The increased endothelial permeability induced by HIV-infected monocytes was associated with a substantial disruption of the endothelial cell monolayer. Morphologic disruption was not a direct toxic effect on endothelial cells, but appeared to be secondary to changes in endothelial cell-cell or cell-matrix interactions. Northern blot analysis showed increased expression of gelatinase B (92-kDa gelatinase), tissue inhibitor of metalloproteinase TIMP-1, and TIMP-2 in the HIV-infected monocytes. Consistent with these Northern analyses, secretion of gelatinase activity in culture fluids of HIV-infected monocytes was also increased and was dependent on the stage of virus replication. Incubation of HIV-infected monocytes with the proteinase inhibitors TIMP-1 and TIMP-2 inhibited the increased permeability of endothelial cell monolayers to 125I albumin. These results suggest possible mechanisms for extravasation of HIV-infected monocytes through vascular endothelium into tissue in early stages of HIV disease.

Single subunit chimeric integrins as mimics and inhibitors of endogenous integrin functions in receptor localization, cell spreading and migration, and matrix assembly.

The ability of single subunit chimeric receptors containing various integrin beta intracellular domains to mimic and/or inhibit endogenous integrin function was examined. Chimeric receptors consisting of the extracellular and transmembrane domains of the small subunit of the human interleukin-2 receptor connected to either the beta 1, beta 3, beta 3B, or beta 5 intracellular domain were transiently expressed in normal human fibroblasts. When expressed at relatively low levels, the beta 3 and beta 5 chimeras mimicked endogenous ligand-occupied integrins and, like the beta 1 chimera (LaFlamme, S. E., S. K. Akiyama, and K. M. Yamada. 1992. J. Cell Biol. 117:437), concentrated with endogenous integrins in focal adhesions and sites of fibronectin fibril formation. In contrast, the chimeric receptor containing the beta 3B intracellular domain (a beta 3 intracellular domain modified by alternative splicing) was expressed diffusely on the cell surface, indicating that alternative splicing can regulate integrin receptor distribution by an intracellular mechanism. Furthermore, when expressed at higher levels, the beta 1 and beta 3 chimeric receptors functioned as dominant negative mutants and inhibited endogenous integrin function in localization to fibronectin fibrils, fibronectin matrix assembly, cell spreading, and cell migration. The beta 5 chimera was a less effective inhibitor, and the beta 3B chimera and the reporter lacking an intracellular domain did not inhibit endogenous integrin function. Comparison of the relative levels of expression of the transfected beta 1 chimera and the endogenous beta 1 subunit indicated that in 10 to 15 h assays, the beta 1 chimera can inhibit cell spreading when expressed at levels approximately equal to the endogenous beta 1 subunit. Levels of chimeric receptor expression that inhibited cell spreading also inhibited cell migration, whereas lower levels were able to inhibit alpha 5 beta 1 localization to fibrils and matrix assembly. Our results indicate that single subunit chimeric integrins can mimic and/or inhibit endogenous integrin receptor function, presumably by interacting with cytoplasmic components critical for endogenous integrin function. Our results also demonstrate that beta intracellular domains, expressed in this context, display specificity in their abilities to mimic and inhibit endogenous integrin function. Furthermore, the approach that we have used permits the analysis of intracellular domain function in the processes of cell spreading, migration and extracellular matrix assembly independent of effects due to the rest of integrin dimers. This approach should prove valuable in the further analysis of integrin intracellular domain function in these and other integrin-mediated processes requiring the interaction of integrins with cytoplasmic components.

Transmembrane signal transduction by integrin cytoplasmic domains expressed in single-subunit chimeras.

Integrins are heterodimeric, transmembrane cell adhesion receptors that have recently been shown to function in transmembrane signal transduction. To examine the specific role of integrin intracellular domains in signal transduction, chimeric receptors containing various integrin intracellular domains coupled to a reporter consisting of the transmembrane and extracellular domains of the small, non-signaling subunit of the interleukin-2 receptor were expressed in cultured human fibroblasts and assayed for their ability to trigger tyrosine phosphorylation of the 125-kDa cytoplasmic tyrosine kinase, pp125FAK. Tyrosine phosphorylation of pp125FAK was induced in cultured fibroblasts that transiently expressed chimeric receptors containing either the beta 1, beta 3, or beta 5 integrin intracellular domain and were selected by magnetic bead sorting. However, expression of chimeric receptors containing either the alpha 5 or an alternatively spliced form of the beta 3 intracellular domain (beta 3B), as well as those lacking an intracellular domain, failed to induce tyrosine phosphorylation of pp125FAK. These results indicate that information contained in the beta 1, beta 3, or beta 5 integrin intracellular domain is sufficient to stimulate integrin-mediated tyrosine phosphorylation of specific intracellular proteins and that integrin extracellular and transmembrane domains are not required for inducing tyrosine phosphorylation. Our results also indicate that alternative splicing can regulate the ability of beta integrin intracellular domains to participate in signal transduction, and they further suggest that the carboxyl-terminal region of specific beta integrins may play a role in the signal transduction pathway involving extracellular matrix molecules.

The complete primary structure of type XII collagen shows a chimeric molecule with reiterated fibronectin type III motifs, von Willebrand factor A motifs, a domain homologous to a noncollagenous region of type IX collagen, and short collagenous domains with an Arg-Gly-Asp site.

Extracellular matrix molecules are generally categorized as collagens, elastin, proteoglycans, or other noncollagenous structural/cell interaction proteins. Many of these extracellular proteins contain distinctive repetitive modules, which can sometimes be found in other proteins. We describe the complete primary structure of an alpha 1 chain of type XII collagen from chick embryonic fibroblasts. This large, structurally chimeric molecule identified by cDNA analysis combines previously unrelated molecular domains into a single large protein 3,124 residues long (approximately 340 kD). The deduced chicken type XII collagen sequence starts at the amino terminus with one unit of the type III motif of fibronectin, which is followed by one unit homologous to the von Willebrand factor A domain, then one more fibronectin type III module, a second A domain from von Willebrand factor, 6 units of type III motif and a third A domain, 10 consecutive units of type III motif and a fourth A domain, a domain homologous to the NC4 domain peptide of type IX collagen, and finally two short collagenous regions previously described as part of the partially sequenced collagen type XII molecule; an Arg-Gly-Asp potential cell adhesive recognition sequence is present in a hydrophilic region at the terminus of one collagenous domain. Antibodies raised to type XII collagen synthesized in a bacterial expression system recognized not only previously reported bands (220 kD et cetera) in tendons, but also bands with apparently different molecular sizes in fibroblasts and 4-d embryos. The antibodies stained a wide variety of extracellular matrices in embryos in patterns distinct from those of fibronectin or interstitial collagens. They prominently stained extracellular matrix associated with certain neuronal tissues, such as axons from dorsal root ganglia and neural tube. These studies identify a novel chimeric type of molecule that contains both adhesion molecule and collagen motifs in one protein. Its structure blurs current classification schemes for extracellular proteins and underscores the potentially large diversity possible in these molecules.

Monoclonal antibody characterization of two distant sites required for function of the central cell-binding domain of fibronectin in cell adhesion, cell migration, and matrix assembly.

Site-directed mutagenesis studies have suggested that additional peptide information in the central cell-binding domain of fibronectin besides the minimal Arg-Gly-Asp (RGD) sequence is required for its full adhesive activity. The nature of this second, synergistic site was analyzed further by protein chemical and immunological approaches using biological assays for adhesion, migration, and matrix assembly. Fragments derived from the cell-binding domain were coupled covalently to plates, and their specific molar activities in mediating BHK cell spreading were compared with that of intact fibronectin. A 37-kD fragment purified from chymotryptic digests of human plasma fibronectin had essentially the same specific molar activity as intact fibronectin. In contrast, other fragments such as an 11.5-kD fragment lacking NH2-terminal sequences of the 37-kD fragment had only poor spreading activity on a molar basis. Furthermore, in competitive inhibition assays of fibronectin-mediated cell spreading, the 37-kD fragment was approximately 325-fold more active than the GRGDS synthetic peptide on a molar basis. mAbs were produced using the 37-kD protein as an immunogen and their epitopes were characterized. Two separate mAbs, one binding close to the RGD site and the other to a site approximately 15 kD distant from the RGD site, individually inhibited BHK cell spreading on fibronectin by greater than 90%. In contrast, an antibody that bound between these two sites had minimal inhibitory activity. The antibodies found to be inhibitory in cell spreading assays for BHK cells also inhibited both fibronectin-mediated cell spreading and migration of human HT-1080 cells, functions which were also dependent on function of the alpha 5 beta 1 integrin (fibronectin receptor). Assembly of endogenously synthesized fibronectin into an extracellular matrix was not significantly inhibited by most of the anti-37-kD mAbs, but was strongly inhibited only by the antibodies binding close to the RGD site or the putative synergy site. These results indicate that a second site distant from the RGD site on fibronectin is crucial for its full biological activity in diverse functions dependent on the alpha 5 beta 1 fibronectin receptor. This site is mapped by mAbs closer to the RGD site than previously expected.

The minimal essential sequence for a major cell type-specific adhesion site (CS1) within the alternatively spliced type III connecting segment domain of fibronectin is leucine-aspartic acid-valine.

Fibronectin contains at least two major domains that support cell adhesion. One is the central cell-binding domain that is recognized by a variety of cell types via the integrin alpha 5 beta 1. The second, originally identified by its ability to support melanoma cell adhesion, is located in the alternatively spliced type III connecting segment (IIICS). A dominant cell type-specific adhesion site within the IIICS has been localized to a peptide designated as CS1 comprising its amino-terminal 25 residues. The receptor for CS1 is the integrin alpha 4 beta 1. We have synthesized a variety of peptides with overlapping sequences in order to identify the minimum active amino acid sequence of this major cell adhesion site. A peptide comprising the carboxyl-terminal 8 amino acids of CS1, EILDVPST, was found to support melanoma cell spreading, while all peptides without this sequence had little or no activity. Two smaller overlapping pentapeptides, EILDV and LDVPS, were also active, whereas EILEV, containing a conservative substitution of Glu for Asp, was inactive. These data suggested that the minimum sequence for cell adhesion activity is Leu-Asp-Val, the tripeptide sequence common to both active peptides. This prediction was confirmed by the observed ability of the Leu-Asp-Val peptide itself to block spreading on fibronectin, whereas Leu-Glu-Val was inactive. Interspecies amino acid sequence comparison also supports the importance of the LDV sequence, since it is completely conserved in the IIICS regions of human, rat, bovine, and avian fibronectins.

Neural crest cell locomotion induced by antibodies to beta 1 integrins. A tool for studying the roles of substratum molecular avidity and density in migration.

Migration of neural crest cells depends on direct, transient interactions between fibronectin molecules and their corresponding Arg-Gly-Asp integrin receptors. We have previously suggested that the moderate-activity interaction between integrin receptors and fibronectin may be critical for the transient association of the cells with their substratum. In order to test this hypothesis, we have examined the in vitro locomotory behavior of neural crest cells on substrata of differing apparent avidities for integrin receptors. As substrata, we used a variety of monoclonal and polyclonal antibodies to the integrin beta 1 subunit that were characterized for their respective relative apparent avidities for the receptor. Neural crest cells were able to migrate on these antibodies and exhibited an organization of substratum-adhesion sites and of cytoskeletal elements virtually identical to that observed on fibronectin, indicating that they can at least partially mimic the migration-promoting activity of fibronectin. However, the number of migrating cells as well as their morphology and their speed of locomotion varied significantly with both the concentration of the antibody substratum and its relative avidity for the receptor. Thus, on high-avidity monoclonal antibodies and on polyclonal divalent antibodies at high concentrations only a limited number of cells escaped from the neural tube, and the rate of their migration was reduced compared to that on fibronectin (23 +/- 5 microns h-1 versus 65 +/- 10 microns h-1). In addition, cells were unusually flattened and cohesive. Time-lapse videomicroscopy revealed that, on high-avidity substrata, neural crest cells were able to extend cell processes that adhered to the substratum, but showed a dramatically reduced capability of breaking pre-existing substratum contacts. In contrast, the same antibodies at low concentrations produced neural crest cell migration at rates very similar to those on fibronectin at the same concentrations. Low-avidity monoclonal antibodies and polyclonal monovalent antibodies at all concentrations tested permitted extensive migration of neural crest cells, which exhibited the same morphology and locomotory behavior as on fibronectin. These results indicate that both the avidity of receptors for the substratum and the number of receptors bound to the substratum are critical in regulating the locomotory behavior of neural crest cells in vitro, and therefore might help to regulate the directionality of migration and final localization pattern of neural crest cells in vivo.

Monoclonal antibody and synthetic peptide inhibitors of human tumor cell migration.

The processes of migration and invasion by human tumor cells are likely to involve specific cell surface receptors, such as receptors for the extracellular matrix molecules fibronectin, laminin, and collagen. We have examined the roles of several of these receptors using a set of monoclonal antibodies directed against the beta 1 integrin family, as well as a series of synthetic peptides reported to inhibit various interactions of each of these proteins with the cell surface. The most general inhibitor of tumor cell migration was found to be the anti-beta 1 monoclonal antibody 13, which inhibited the migration of human HT-1080 fibrosarcoma cells, 5637 bladder carcinoma cells, VA13 viral transformants, and HCT 116 colon carcinoma cells when fibronectin was the migration substrate. Moreover, this antibody was particularly effective in blocking cell migration on laminin, as well as migration within 3-dimensional collagen gels. It also inhibited in vitro invasiveness in a reconstituted basement membrane invasion assay (Matrigel assay) at concentrations as low as 1 microgram/ml. Integrins of the beta 1 class thus appear to play a central role in several types of migration by a variety of human tumor cell lines. Anti-alpha 5 fibronectin receptor monoclonal antibody 16 also significantly inhibited migration on fibronectin, but not on other substrates, in 3 of the 4 cell lines. Conversely, anti-alpha 2 monoclonal antibody F17 strikingly inhibited migration in 3-dimensional collagen gels, but not on other substrates, implicating the alpha 2 beta 1 integrin system in migration of tumor cells within collagenous matrices. A series of synthetic peptides previously reported to inhibit interactions of normal cells with fibronectin, laminin, and collagen were also tested as inhibitors of tumor cell migration. Peptides containing the Arg-Gly-Asp adhesive recognition signal were partially inhibitory, but with occasional exceptions, most other peptides had no effects on migration. Our results indicate the central importance of several specific beta 1 integrins in human tumor cell migration and show the effectiveness of monoclonal antibody treatment in blocking this process in vitro.

Novel function for beta 1 integrins in keratinocyte cell-cell interactions.

We have examined the expression, localization, and function of beta 1 integrins on cultured human epidermal keratinocytes using polyclonal and monoclonal antibodies against the beta 1, alpha 2, alpha 3, and alpha 5 integrin subunits. The beta 1 polypeptide, common to all class 1 integrins, was localized primarily in areas of cell-cell contacts of cultured keratinocytes, as were alpha 2 and alpha 3 polypeptides, suggesting a possible role in cell-cell adhesion for these integrin polypeptides. In contrast, the fibronectin receptor alpha 5 subunit showed no such accumulations in regions of cell-cell contact but was more diffusely distributed in the keratinocyte plasma membrane, consistent with the absence of fibronectin at cell-cell contact sites. Colonies of cultured keratinocytes could be dissociated by treatment with monoclonal antibody specific to the beta 1 polypeptide. Such dissociation of cell-cell contacts also occurred under conditions where the monoclonal antibody had no effect on cell-substrate adhesion. Therefore, beta 1 integrin-dependent cell-cell adhesion can be inhibited without affecting other cell-adhesive interactions. Antibody treatment of keratinocytes maintained in either low (0.15 mM) or high (1.2 mM) CaCl2 also resulted in the loss of organization of intracellular F-actin filaments and beta 1 integrins, even when the anti-beta 1 monoclonal antibody had no dissociating effect on keratinocyte colonies at the higher calcium concentration. Our results indicate that beta 1 integrins play roles in the maintenance of cell-cell contacts between keratinocytes and in the organization of intracellular microfilaments. They suggest that in epithelial cells integrins can function in cell-cell interactions as well as in cell-substrate adhesion.

Localization of integrin receptors for fibronectin, collagen, and laminin in human skin. Variable expression in basal and squamous cell carcinomas.

VLA integrins in human skin were examined by indirect immunofluorescence utilizing antibodies recognizing the beta 1, alpha 2, alpha 3, or alpha 5 subunits. Staining of fetal, newborn, or adult skin with antibodies to beta 1, alpha 2, or alpha 3 subunits gave essentially similar staining patterns: intense staining was associated with the basal layer of the epidermis, hair follicles, and blood vessel walls. The alpha 5 subunit could be detected only in epidermis and the inner root sheath of hair follicles in fetal skin. In epidermis, the staining reaction for the beta 1 subunit was not only found in sites interfacing with the basement membrane zone, but also around the entire periphery of these cells. We speculate that these receptors might have previously unrecognized functions in cell-cell interactions or that these findings may suggest the presence of previously unrecognized ligands in the intercellular spaces of keratinocytes. Examination of nine nodular basal cell carcinomas revealed a prominent staining reaction with anti-beta 1 and anti-alpha 3 antibodies at the periphery of the tumor islands. In contrast, staining of five squamous cell carcinomas revealed either the absence of integrins or altered and variable expression. Thus, matrix components and their receptors may participate in modulation of growth, development, and organization of human skin.

Analysis of the role of glycosylation of the human fibronectin receptor.

1-Deoxymannojirimycin (MNJ), an inhibitor of Golgi alpha-mannosidase IA and IB, was used to assess the possible roles of asparagine-linked oligosaccharides in the structure and function of the integrin fibronectin receptor from cultured human fibroblasts. These cells normally attach well to fibronectin substrates and have only mature forms of the fibronectin receptor on their surfaces. MNJ inhibits the intracellular trimming of high mannose oligosaccharides, and cells treated with 0.2 mg/ml MNJ synthesize only immature precursor forms of both the alpha and beta subunits of the fibronectin receptor. The immature receptor polypeptides were found to be nonfunctional by two criteria: 1) cells treated with MNJ attached poorly to fibronectin substrates; and 2) receptor from the treated cells was defective in binding to fibronectin affinity columns. The precursor forms of the fibronectin receptor subunits were found on the surfaces of cells treated with MNJ, demonstrating that processing of receptor carbohydrates to mature forms was not necessary for receptor insertion into the plasma membrane. A monoclonal antibody that specifically bound the alpha subunit of the fibronectin receptor immunoprecipitated both alpha and beta subunit polypeptides from both control cells and cells treated with MNJ. Similarly, a monoclonal antibody that specifically bound only the beta subunit also immunoprecipitated both alpha and beta subunit polypeptides of the receptor from extracts of both control and MNJ-treated cells. These results indicate that receptor assembly can occur in the absence of complete oligosaccharide processing. Thus, oligosaccharide processing to the mature form of the fibronectin receptor is important for its binding function but not for receptor assembly or insertion into the plasma membrane.

Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization.

We have developed two rat mAbs that recognize different subunits of the human fibroblast fibronectin receptor complex and have used them to probe the function of this cell surface heterodimer. mAb 13 recognizes the integrin class 1 beta polypeptide and mAb 16 recognizes the fibronectin receptor alpha polypeptide. We tested these mAbs for their inhibitory activities in cell adhesion, spreading, migration, and matrix assembly assays using WI38 human lung fibroblasts. mAb 13 inhibited the initial attachment as well as the spreading of WI38 cells on fibronectin and laminin substrates but not on vitronectin. Laminin-mediated adhesion was particularly sensitive to mAb 13. In contrast, mAb 16 inhibited initial cell attachment to fibronectin substrates but had no effect on attachment to either laminin or vitronectin substrates. When coated on plastic, both mAbs promoted WI38 cell spreading. However, mAb 13 (but not mAb 16) inhibited the radial outgrowth of cells from an explant on fibronectin substrates. mAb 16 also did not inhibit the motility of individual fibroblasts on fibronectin in low density culture and, in fact, substantially accelerated migration rates. In assays of the assembly of an extracellular fibronectin matrix by WI38 fibroblasts, both mAbs produced substantial inhibition in a concentration-dependent manner. The inhibition of matrix assembly resulted from impaired retention of fibronectin on the cell surface. Treatment of cells with mAb 16 also resulted in a striking redistribution of cell surface fibronectin receptors from a streak-like pattern to a relatively diffuse distribution. Concomitant morphological changes included decreases in thick microfilament bundle formation and reduced adhesive contacts of the streak-like and focal contact type. Our results indicate that the fibroblast fibronectin receptor (a) functions in initial fibroblast attachment and in certain types of adhesive contact, but not in the later steps of cell spreading; (b) is not required for fibroblast motility but instead retards migration; and (c) is critically involved in fibronectin retention and matrix assembly. These findings suggest a central role for the fibronectin receptor in regulating cell adhesion and migration.

Electron microscopy and structural model of human fibronectin receptor.

Highly-purified human fibronectin receptor (a heterodimer of two distinct subunits, alpha and beta) was studied using electron microscopy and a variety of preparative procedures. It was found that the receptor consists of a globular head approximately 80 by 120 A and two tails about 20 A thick and 180-200 A long. The whole complex is approximately 280 A long. At low concentrations of detergent the receptor forms doublets, triplets or rosettes associated with the tails which possess the transmembrane portion of the molecule. Computer-assisted structure prediction using the published amino acid sequence of both subunits showed differences in the secondary structure of the tails, the alpha-tail being rich in beta-strands, the beta-tail having five cysteine-rich repeats analogous to the EGF-like repeats of laminin. Estimates of the length of the tails from the predicted structure conformed well with the dimensions obtained from electron micrographs.

Transforming growth factor beta stimulates the expression of fibronectin and of both subunits of the human fibronectin receptor by cultured human lung fibroblasts.

Transforming growth factors of the beta-class (TGFs-beta) stimulate extracellular matrix synthesis and have been implicated in embryogenesis, wound healing, and fibroproliferative responses to tissue injury. Because cells communicate with several extracellular matrix components via specific cell membrane receptors, we hypothesized that TGFs-beta may also regulate the expression of such receptors. We confirmed that TGF-beta 1 increases the expression of fibronectin, an adhesive glycoprotein expressed during embryogenesis and tissue remodeling. Based upon the 48-72-h period required for a maximal fibroproliferative response to dermal injections of TGF-beta 1, we exposed human fetal lung fibroblasts (IMR-90) to TGF-beta 1 for periods up to 48 h in vitro. We observed as much as 6-fold increases in fibronectin synthesis by 24 h as previously reported for fibroblastic cells (Ignotz, R. A., and Massagué, J. (1986) J. Biol. Chem. 261, 4337-4345; Ignotz, R. A., Endo, T., and Massagué, J. (1987) J. Biol. Chem. 262, 6443-6446; Raghow, R., Postlethwaithe, A. E., Keski-Oja, J., Moses, H. L., and Kang, A. H. (1987) J. Clin. Invest. 79, 1285-1288), but up to 30-fold increases by 48 h. These increases are accompanied by similar increases in fibronectin mRNA levels which are prevented by actinomycin D treatment. Using a monospecific antibody raised to the human placental fibronectin receptor complex, we found that TGF-beta 1 stimulated fibronectin receptor synthesis up to 20-40-fold and increases mRNA levels encoding both the alpha- and beta-subunits up to 3-fold, compared to control IMR-90 in serum-free medium. Actinomycin D blocks TGF-beta 1-mediated increases in receptor mRNA levels. The earliest detectable TGF-beta 1-mediated increases in fibronectin receptor complex protein synthesis and mRNA levels occur at 8 h, whereas the earliest increases in fibronectin protein synthesis and mRNA levels occur at 12 h. These results demonstrate that TGF-beta 1 stimulates fibronectin receptor synthesis, extending the diverse stimulatory activities of this polypeptide to matrix receptors. In addition, because fibronectin matrix assembly may involve the fibronectin cell adhesive receptor complex, increased receptor expression may help drive fibronectin deposition into matrix.

Transmembrane orientation of the fibronectin receptor complex (integrin) demonstrated directly by a combination of immunocytochemical approaches.

The avian 140-KD cell adhesion receptor or "integrin," a complex of three glycoproteins with molecular masses averaging 140 KD, interacts with extracellular fibronectin and forms a linkage complex that co-localizes with intracellular actin. To probe the molecular interactions involved in this linkage complex, we used monoclonal antibodies and a combination of immunolocalization approaches to determine whether any component was transmembrane. Immunoadsorption and immunoblotting experiments demonstrated that anti-120-KD Mabs recognized the band 3 component of integrin isolated from chicken embryo fibroblasts (CEF) by JG22E immunoaffinity chromatography, and they co-localize with anti-fibronectin and polyclonal anti-integrin at cell contact sites in double-labeling experiments. Immunofluorescence experiments involved comparisons of double-labeled intact cells or substrate-attached, ventral plasma membrane "rip-off" fragments, using anti-fibronectin and each of the anti-120-KD Mabs. The extracellular faces of living intact cells were strongly labeled by a majority (approximately 70%) of the anti-120-KD Mabs at fibronectin-membrane attachment sites. The remainder (approximately 30%) labeled intact cells weakly or not at all. However, although the anti-120-KD Mab ES186 did not stain living cells, it did demonstrate positive staining above substratum contact sites over entire isolated rip-off membranes. In contrast, Mabs directed against putative extracellular epitopes and anti-fibronectin antibodies did not label these sites at the center of rip-offs unless the membranes were detergent permeabilized. Proteolysis experiments suggested that the ES186 epitope was located at one end of the molecule, since removal of short fragments from integrin band 3 concomitantly removed or destroyed the ES186 epitope, whereas the extracellular epitopes still remained. These experiments directly demonstrate that integrin band 3 is a transmembrane polypeptide with at least one epitope recognized by anti-120-KD Mabs on the cytoplasmic side of the plasma membrane and at least one epitope on the extracellular cell surface.