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PubMed1998

A Neu differentiation factor (NDF) domain essential for proliferation and alterations in morphology of colonic epithelial cells in vitro.

The Neu Differentiation Factors (NDFs, also termed "heregulins") are a family of proteins that were first isolated as ligands for the HER2 (ergB2, or p185neu) receptor protein tyrosine kinase. Here we show that NDF acts to stimulate the proliferation and alter the cellular morphology of colonic epithelial cells in culture. Dramatic NDF-induced changes in cellular morphology were noted in the colonic epithelial cell line, LIM 1215. In addition, the expression of specific cell proteins, such as carcinoembryonic antigen and integrin beta 4, was induced in LIM 1215 cells by NDF. These effects were more pronounced with the beta isoform than with the alpha isoform of NDF. The EGF-homology domain of NDF beta was sufficient to stimulate the proliferation and alteration in cell morphology. The use of chemically synthesized chimeric NDF alpha and NDF beta proteins enabled use to identify a region of seven amino acids in the EGF-homology domain of NDF beta that is required for both activities. These in vitro experiments suggest that NDF may act as a regulator of growth and differentiation of colonic epithelial cells in vivo.

Amino Acid Sequence↗
PubMed2006

Functional genomics analysis of low concentration of ethanol in human hepatocellular carcinoma (HepG2) cells. Role of genes involved in transcriptional and translational processes.

We previously found that ethanol at millimolar level (1 mM) activates the expression of transcription factors with subsequent regulation of apoptotic genes in human hepatocellular carcinoma (HCC) HepG2 cells. However, the role of ethanol on the expression of genes implicated in transcriptional and translational processes remains unknown. Therefore, the aim of this study was to characterize the effect of low concentration of ethanol on gene expression profiling in HepG2 cells using cDNA microarrays with especial interest in genes with transcriptional and translational function. The gene expression pattern observed in the ethanol-treated HepG2 cells revealed a relatively similar pattern to that found in the untreated control cells. The pairwise comparison analysis demonstrated four significantly up-regulated (COBRA1, ITGB4, STAU2, and HMGN3) genes and one down-regulated (ANK3) gene. All these genes exert their function on transcriptional and translational processes and until now none of these genes have been associated with ethanol. This functional genomic analysis demonstrates the reported interaction between ethanol and ethanol-regulated genes. Moreover, it confirms the relationship between ethanol-regulated genes and various signaling pathways associated with ethanol-induced apoptosis. The data presented in this study represents an important contribution toward the understanding of the molecular mechanisms of ethanol at low concentration in HepG2 cells, a HCC-derived cell line.

Carcinoma, Hepatocellular↗
PubMed2000

Influence of intravenous immunoglobulin therapy on serum levels of anti-beta 4 antibodies in ocular cicatricial pemphigoid. A correlation with disease activity. A preliminary study.

PURPOSE: To determine any correlation between activity of ocular cicatricial pemphigoid and titer of anti-beta 4 antibodies, and any effect of intravenous immunoglobulin (IVIg) therapy on serum levels of anti-beta 4 antibodies followed over a 12 month period, using the specific immunoblot assay (IBA). PATIENTS AND METHODS: Eight patients diagnosed with OCP and treated with IVIg as monotherapy were included in the study. Each patient was treated with at least two immunosuppressive agents prior to the institution of IVIg. The presence of anti-beta 4 antibodies in the patients' sera was detected by IBA using bovine gingival lysate (BGL) or tumor cell line lysate (TCL) as substrates. The activity of OCP was graded based on the conjunctival injection using a scale of zero to four in increments of 0.5 at monthly intervals. To determine the correlation between serum levels of circulating autoantibody and the patients' conjunctival disease activity, the titer of anti-beta 4 antibodies was determined at monthly intervals during the course of IVIg therapy. Blood samples were drawn prior to administration of IVIg infusion. The titer was determined by IBA, using serial dilutions of the patients' sera. The highest dilution at which the binding was visible was considered the titer. The dose of IVIg administered was approximately 2-3 g/kg/cycle. The infusion cycles were initially given at monthly intervals, approximately 70 grams daily over four hours for three consecutive days. As clinical improvement was observed, the interval between the cycles was increased, but the dose of IVIg remained the same for each cycle. To study whether the IVIg has an effect on other antibodies, monthly serum levels of antibodies to tetanus toxoid were measured by ELISA. RESULTS: We observed a continuous decrease in mean monthly titer of circulating anti-beta 4 antibodies in the patients' sera during IVIg therapy. A decrease in conjunctival inflammation during the course of IVIg was documented by monthly examination in every patient and paralleled the decrease in titer of anti-beta 4 antibodies. Since the fifth month of IVIg therapy, the mean conjunctival inflammation remained less than 0.5, suggesting a clinical remission of OCP. Titers of antibodies to tetanus toxoid remained unchanged during the study period. CONCLUSIONS: This preliminary study demonstrates a correlation between serum titers of anti-beta 4 antibodies and clinical disease activity in patients with OCP. In addition, the study shows that the use of IVIg is associated with a decrease in the serum titer of anti-beta 4 antibodies.

Aged↗
PubMedDate not supplied

Functional suppression of integrin beta 4-mediated adhesion caused by in vivo sequential selection for cancer cell intravasation.

Intravasation is essential for hematogenous metastasis in cancer cells, but its cellular determinants have not been well elucidated because of a lack of suitable experimental cell systems. Int-3LL was originally developed by in vivo sequential selection for intravasation from Lewis lung carcinoma (3LL) cells. Here, we found that these variant cells showed a highly penetrating ability in vitro as well as an augmented intravasating potential in vivo. In three-dimensional collagen-gel, Int-3LL cells formed diffusive colonies with less plating efficiency than their parental cells. Despite these properties, Int-3LL cells showed an ability of invasive migration in vitro similar to parental cells. On the other hand, a reduced adhesiveness and less spreading on extracellular matrices were revealed in Int-3LL cells. Analyses using anti-integrin antibodies indicated that the dysadhesion phenotype in Int-3LL cells was associated with integrin beta 4 dysfunction, which is known to produce epithelial detachment. Also, the types and the levels of integrins were not indistinguishable between Int-3LL and parental 3LL cells. Thus, the impaired function of integrin beta 4-mediated adhesion is considered to be an important factor in intravasation during metastasis.

Animals↗
PubMedDate not supplied

CD104.

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Amino Acid Sequence↗
PubMed2003

[Beta-1 and beta-4 integrins, and laminin 67 kDa receptors in interaction between A431 cells and laminin isoforms ].

Laminins, as basal membrane glycoproteins, are able to stimulate cell adhesion and migration, and to influence gene expression. The laminin molecule has a set of bioactive sites that interact with different integrin and nonintegrin receptors, and, as a result, the reaction of the same cell type to different laminin isoforms may be different. The aim of this study was to determine the contributions of both integrins with beta 1 and beta 4 chains and 67 kDa laminin receptor in the interaction of A431 cells with two laminin isoforms: laminin-1 and laminin-2/4. The obtained data show that integrin alpha 6 beta 4 is more specific for interaction with laminin-2/4 than with laminin-1 and takes part in the stage of attachment of A431 cells to laminin. 67 kDa receptor promotes cell spreading on laminin-2/4 and inhibits cell spreading on laminin-1. An assumption was made about the complex action of receptors for interaction of A431 cells with laminins ("integrin alpha 6 beta 4--67 kDa receptors" complex).

Basement Membrane↗
PubMed1996

Expression of alpha 6 and beta 4 integrins in serous ovarian carcinoma correlates with expression of the basement membrane protein laminin.

The surface of a normal ovary is covered by a monolayer of epithelial cells that rest on a basement membrane. The glycoprotein laminin is the major noncollagenous protein present in the basement membrane. The integrins alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 6 beta 1, and alpha 6 beta 4 serve as cell surface receptors for laminin. During the progression of serous ovarian carcinoma, tumor cells are frequently exfoliated from the surface of the ovary, thereby losing contact with the basement membrane. This study was designed to determine whether alterations in integrin expression may be associated with the malignant phenotype of the primary ovarian tumor and exfoliated ovarian carcinoma cells in the ascites fluid. By immunohistochemical staining, the entire surface of epithelial cells of normal ovaries stained positively for beta 1, alpha 2, and alpha 3 integrins, whereas only the basal surface of the epithelial cells, where they are in contact with laminin, stained positively for alpha 6 and beta 4. The entire surface of epithelial cells of solid tumors from patients with serous ovarian carcinoma stained positively for beta 1, alpha 2, and alpha 3 integrins. In most cases, no intact basement membrane surrounded the tumor nests, and staining for alpha 6 and beta 4 was irregular. When present, the basement membrane stained positively for laminin, and the basal surface of the epithelial cells stained positively for alpha 6 and beta 4. Ovarian carcinoma ascites cells exhibited a distinct phenotype, with a significant decrease in expression of the alpha 6 and beta 4 integrin subunits. As alpha 6 and beta 4 integrin subunits are present at the basal surface of many epithelial cells and serve as receptors for laminin, it is possible that ovarian carcinoma epithelial cells may be released from the basement membrane of the ovary due to their deficit of alpha 6 and beta 4 integrin subunits.

Adult↗
PubMed1995

Expression of cell surface adhesion molecules on the epithelium of Organ-cultured skin.

Human neonatal foreskin was maintained in organ culture under serum-free, growth-factor-free conditions or in the presence of a combination of growth factors that are known to stimulate keratinocyte proliferation in monolayer culture. Previously, we have shown that normal histology is maintained when growth-factor-free conditions are used but that the epithelium undergoes a hyperproliferative response and invades the dermis in the presence of the exogenous growth factors. In the present study, the tissue was examined by immunofluorescence for expression of alpha 6 and beta 4 integrin components and for E-cadherin. Under growth factor-free conditions, both alpha 6 and beta 4 were localized to the basal surface of epithelial cells in contact with the basement membrane. In contrast, both epitopes were diffusely distributed throughout the basal epithelium in the presence of growth factors. E-cadherin expression was rapidly lost from the tissue in organ culture. This occurred in both the presence and absence of exogenous growth factors. On the basis of these immunochemical results, we conclude that the same changes in alpha 6 and beta 4 expression that are seen in rapidly proliferating keratinocytes and squamous epithelial cell tumors can be seen in the epidermis of organ-cultured skin when it is maintained in the presence of epithelial growth factors. The observed loss of E-cadherin, in contrast, appears to be a consequence of incubation in organ culture.

Antigens, CD↗
PubMed1997

Beta 4 integrin transfection of UM-UC-2 (human bladder carcinoma) cells: stable expression of a spontaneous cytoplasmic truncation mutant with rapid loss of clones expressing intact beta 4.

The alpha 6 beta 4 integrin is a component of the hemidesmosome, the anchoring structure in the basal membrane of epithelial cells. alpha 6 beta 4 expression is frequently altered in neoplastic cells. It is sometimes lost and sometimes overexpressed, which suggests that disruption of normal function is involved in neoplastic transformation. To examine the effect of this integrin on the growth and behavior of malignant cells that have lost beta 4, we transfected a full-length beta 4 cDNA into the UM-UC-2 cell line that expresses alpha 6 but not beta 4. Although large numbers of clones were obtained when a control vector was used in the transfection, only 12 clones could be isolated that expressed beta 4. Of these, only two beta 4-positive clones, clones 8 and 11, persisted long enough for further study. Clone 8 cells initially expressed beta 4, but within 2 weeks, all positive cells were lost from the culture. Clone 11 persisted in culture and retained strong surface expression of alpha 6 beta 4. Biochemical analysis and Western blotting revealed that this clone contained a truncated form of beta 4 that had lost the distal cytoplasmic domain. We conclude that expression of wild-type beta 4 in UM-UC-2 inhibits cell growth, presumably by an integrin-mediated signaling pathway. Clone 11 escaped from normal signaling because the cytoplasmic domain, a region essential for basal polar localization, was lost. The alpha 6 beta 4 integrin appears to have tumor suppressor activity in epithelial tumors.

Antigens, CD↗
PubMed1997

Beta 4 integrin subunit expression is downregulated in low metastatic carcinoma variants.

Organization and expression of the gene coding for beta 4 integrin subunit were investigated in murine carcinoma lines endowed with low and high metastatic potential maintained both in vivo and in vitro. Probes corresponding to either the extracellular or the cytoplasmic domains of the protein were generated and employed for Southern and Northern blot hybridization experiments. Southern blot analysis demonstrates a restriction fragment-length polymorphism between BALB/c and C57BI/6J DNAs. The different genomic organization of the beta 4 gene apparently does not generate variation in mRNA length. Northern blot analysis reveals a 7-kb transcript encoding full-length beta 4 protein and shorter transcripts that apparently correspond to nonfunctional mRNAs. In carcinoma cells endowed with low metastatic capacity, the 7-kb mRNA cannot be revealed in spite of the presence of shorter transcripts. In contrast, the 7-kb transcript is always present in tumor cells endowed with high metastatic capacity. Accordingly, the beta 4 protein is detectable, either by immunofluorescence or by Western blot analyses, only in highly metastatic carcinoma cells. In conclusion, the data presented show that in these murine carcinoma cell lines (i) the 7-kb mRNA is associated with a more invasive phenotype, and (ii) missing expression of the beta 4 integrin subunit in low metastatic carcinoma cells depends, at least in part, on mechanisms acting at a post-transcriptional level. The possibility that beta 4 subunit expression is a consequence of specific differentiation stages of lung carcinoma cells is discussed.

Adenocarcinoma, Bronchiolo-Alveolar↗
PubMed1998

Alterations in the expression of alpha6beta4 integrin and p21/WAF1/Cip1 in N-nitrosomethylbenzylamine-induced rat esophageal tumorigenesis.

Integrin alpha6beta4 is altered in many neoplastic cells, but no data exist to show this happens in esophageal neoplasms. To examine the expression of this integrin in rat esophageal tumorigenesis induced by N-nitrosomethylbenzylamine (NMBA), (alpha6 and beta4 expression was evaluated in normal esophageal epithelium, in NMBA-induced preneoplastic lesions, and in papillomas by quantitative reverse transcription (RT)-polymerase chain reaction (PCR) and immunohistochemical analysis. Because the 34 subunit of this integrin has been found to cause cell-cycle arrest by the induction of p21/WAF1/Cip1, the expression of p21/WAF1/Cip1 was also analyzed by RT-PCR. Compared with the levels in normal epithelium, the alpha6A, alpha6B, and beta4 integrin levels in esophageal papillomas were 1.9-, 2.2-, and 2.1-fold lower, respectively. RT-PCR analysis showed no significant differences in integrin levels between preneoplastic and normal samples, and northern blot analysis of the beta4 integrin produced results in agreement with the RT-PCR results. The p21/WAF1/Cip1 level was decreased 1.6-fold in preneoplastic tissues and 3.1-fold in papilloma samples when compared with the mRNA levels in normal epithelium. Immunostaining showed that alpha6beta4 integrin was localized at the basolateral surface of the basal cells in normal esophageal epithelium. In preneoplastic lesions, however, the expression of this integrin was not polarized and was expressed in basal cells as well as in suprabasal cells. Beta4 expression was significantly reduced and alpha6A expression was decreased and delocalized in papillomas. These findings suggest that alteration in alpha6beta4 integrin and p21/WAF1/Cip1 expression may be an important biomarker for tumor progression in NMBA-induced rat esophageal tumorigenesis.

Animals↗
PubMed2000

Re-epithelialization rate and protein expression in the suction-induced wound model: comparison between intact blisters, open wounds and calcipotriol-pretreated open wounds.

We have investigated re-epithelialization following induction of suction blisters in humans in intact blisters, open wounds, i.e. blister roofs removed immediately after blister induction, and calcipotriol-pretreated open wounds. Intact blisters simulate blister healing in bullous disease, while open wounds simulate re-epithelialization during wound healing. Re-epithelialization was clearly faster in open wounds than in intact blisters, and was not affected by calcipotriol pretreatment. Bullous pemphigoid antigen 2 (BP180), bullous pemphigoid antigen 1 (BP230), plectin/hemidesmosomal 1 protein (HD1), laminin 5, laminin alpha5, laminin beta1, type VII collagen, tenascin-C, beta4, alphavbeta5, alpha5 and alpha9 integrins were studied in intact blisters and open wounds by immunohistochemistry. Hemidesmosomal plaque proteins BP230 and plectin/HD1, which connect the keratin cytoskeleton to the hemidesmosome, appeared earlier at the leading edge in intact blisters than in open wounds. Band-like immunostaining in the basement membrane for laminin 5, alpha5 and beta1 chains was continuous in blister bases, but partially discontinuous in open wound bases. The other antigens studied showed similar expression in intact blisters and open wounds. BP180, BP230, plectin/HD1, beta4 integrin, laminin 5 and tenascin-C expression were further studied in calcipotriol-pretreated open wounds. Calcipotriol did not affect the expression of these antigens. The immunohistochemical results suggest that the keratin cytoskeleton is linked to the basal plasma membrane of migrating basal cells via BP230 and plectin/HD1 earlier in the more slowly re-epithelializing blisters than in open wounds. An intact laminin sheath may inhibit keratinocyte migration in intact blisters.

Adult↗
PubMed2003

[Expression and significance of integrin alpha6beta4 in experimental allergic neuritis].

OBJECTIVE: To investigate the expression of the integrin alpha6beta4 in experimental allergic neuritis (EAN) and the relationship of the integrin alpha6beta4 with functional states of Schwann cells (Sc) as well as the injury and repair of the myelin during EAN. METHODS: EAN was induced in Lewis rats and sciatic nerves were resected in 18 EAN and 3 normal rats. The expression of tissue integrin alpha6beta4 was analyzed during the course of EAN induction and in controls by in situ hybridization and semi-quantitative RT-PCR. RESULTS: The detection of integrin alpha6 and beta4 subunit by hybridization in situ demonstrated that expression of alpha6 subunit present no significant changes during the course of EAN, while expression of beta4 declined in the early phase, showing less positive signals than those of the control, and restored its expression in the later or recovery phase. The changes of expression of integrin alpha6 and beta4 in EAN were confirmed by semi-quantitative PT-PCR, using GAPDH as the internal standard. CONCLUSIONS: The degeneration and injury of Sc caused by inflammation affect the expression of integrin, which shows similar changes in Sc during embryogenesis, indicating alpha6beta4 may be a marker of Sc differentiation and at least an important molecule to mark the course of EAN. The expression of alpha6beta4 correlate with the injury and repair of myelin during EAN.

Animals↗
PubMed2000

Expression of integrin subunits correlates with differentiation of epithelial cell lineages in developing human gastric mucosa.

Laminins may play important roles in gastric gland development due to the differential localization of their alpha chains in human fetal fundic (oxyntic) mucosa. To extend this hypothesis, the current investigation was undertaken to compare the anatomical and cellular distribution of epithelial integrin subunits with those of laminin alpha chains in the human stomach at different ages (8-22 weeks of gestation) using indirect immunofluorescence. In the body, fundus and antrum regions, the beta1 and alpha6 subunits were associated with the entire epithelium at all developmental stages in the same way as laminin chains (alpha1/alpha5) detected with 4C7 monoclonal antibody. By contrast, the alpha3 and beta4 subunits of alpha3beta1 and alpha6beta4 integrins together with the laminin alpha3-chain were concentrated in the surface and foveolus compartments composed of differentiating mucous cells. Most importantly, the alpha2 integrin subunit was expressed in a rather complex pattern: (1) it was located at the base and at cell-cell boundaries of surface/foveolar epithelium, (2) was specifically repressed in differentiating parietal cells, and (3) its expression increased in maturing glands, where it became concentrated at the basal pole of epithelial cells simultaneously exhibiting a strong reactivity for laminin-2 (alpha2-chain). Taken altogether, our observations provide new evidence for the implication of different laminins and their receptors in the development of all human gastric epithelial lineages, surface mucous cells or glandular cells. The coordinated expression of alpha2 and alpha3 integrin subunits as well as the cellular re-distribution of alpha2beta1 integrin likely represent key events for the differentiation of glandular secretory cell types, especially maturing chief cells responsible for the synthesis/secretion of gastric digestive enzymes in fundic-type glands.

Antigens, CD↗
PubMed1999

Integrin alpha6beta1 role in metastatic behavior of human pancreatic carcinoma cells.

The factors that determine the metastatic behavior of pancreatic tumor cells are incompletely understood. In this study, we first demonstrate differences in adhesion properties, integrin expression and in vivo integrin function in the metastatic tumor cell line PaTu 8988s compared with the non-metastatic cell line PaTu 8988t. Both cell lines were derived from the same original tumor and exhibit identical genetic fingerprints. Using in vitro adhesion assays performed on purified extracellular matrix components, adhesion of PaTu 8988s cells was significantly increased on the basal membrane component laminin and decreased on the interstitial matrix protein fibronectin compared to PaTu 8988t cells. By immunocytochemistry and flow cytometry, and in correspondence with their adhesive properties, the metastatic PaTu 8988s cells did express a distinct pattern of integrin subunits. Laminin-binding integrins alpha6 and beta4 were overexpressed in PaTu 8988s cells. Fibronectin-binding alpha5 integrins were present at higher levels in the non-metastatic PaTu 8988t cells, whereas the beta1 subunit expression did not differ. Adhesion to laminin or fibronectin was specific and was mediated via integrins alpha6beta1 and alpha5beta1, respectively. In addition, metastasis formation in vivo after injection of cells into the tail vein of nude mice was inhibited by preincubation of PaTu 8988s cells with antibodies directed against the integrin alpha6 or beta1. We conclude that alpha6beta1 integrins are overexpressed and functionally active in metastatic human pancreatic carcinoma cells, and participate in metastasis formation probably through binding to the basal membrane component laminin.

Animals↗
PubMed2003

Pathological adhesion of primary human schwannoma cells is dependent on altered expression of integrins.

Mutations in the tumor suppressor gene coding for merlin cause Neurofibromatosis type 2 (NF2), all spontaneous schwannomas, and a majority of meningiomas. Merlin links transmembrane proteins to the cytoskeleton. Accordingly, primary human schwannoma cells lacking merlin show an increased number of lamellipodia and filopodia as well as increased cell spreading. We show enhanced adhesion in primary human schwannoma cells and present evidence that this is dependent on the integrin chains alpha6beta1 and alpha6beta4. We further demonstrate that the integrin chains beta1 and beta4 are upregulated in schwannomas using different complementary methods, and report higher expression of these integrins per schwannoma cell by fluorescence assisted cell sorting (FACS). Finally we report clustering of the integrin chains alpha6, beta1, and beta4 on schwannoma cells. Our findings fit well into recent data on the role of merlin in signaling cascades connected to integrins and help explain pathological ensheathment of extracellular matrix or pseudomesaxon formation which is a hallmark of schwannoma histopathology.

Blotting, Western↗
PubMed2004

PTH-related protein enhances MCF-7 breast cancer cell adhesion, migration, and invasion via an intracrine pathway.

Breast cancer is the most common carcinoma that metastasizes to the bone. Parathyroid hormone-related protein (PTHrP), a known stimulator of osteoclastic bone resorption, is a major mediator of the osteolytic process in breast cancer. PTHrP overexpression increases mitogenesis and decreases apoptosis in the human breast cancer cell line MCF-7. In this study, MCF-7 cells were used as a model system to study the effects of PTHrP on breast cancer cell adhesion, migration, and invasion. Clones of MCF-7 cells were established that overexpress wild-type PTHrP or PTHrP mutated in the nuclear localization sequence (NLS). Wild-type PTHrP-overexpressing cells showed significantly higher laminin adhesion and migration, and Matrigel invasion than empty vector-transfectants or cells overexpressing NLS-mutated PTHrP. Wild-type PTHrP also increased the cell surface expression of the pro-invasive integrins alpha6 and beta4; deletion of the NLS negated these effects. Exogenous PTHrP (1-34), (67-86), (107-139), and (140-173) had no effect on integrin expression, or on cell adhesion, migration, and invasion. These results indicate that PTHrP exerts its effects on cell adhesion, migration, invasion, and integrin expression via an intracrine pathway. PTHrP may play a role in breast cancer metastasis by upregulating proinvasive integrin expression, and controlling PTHrP production in breast cancer may provide therapeutic benefit.

Breast Neoplasms↗
PubMed2005

Laminin alpha5 is required for dental epithelium growth and polarity and the development of tooth bud and shape.

In tooth development, the oral ectoderm and mesenchyme coordinately and reciprocally interact through the basement membrane for their growth and differentiation to form the proper shape and size of the tooth. Laminin alpha5 subunit-containing laminin-10/11 (LM-511/521) is the major laminin in the tooth germ basement membrane. Here, we have examined the role of laminin alpha5 (Lama5) in tooth development using laminin alpha5-null mouse primary dental epithelium and tooth germ organ cultures. Lama5-null mice develop a small tooth germ with defective cusp formation and have reduced proliferation of dental epithelium. Also, cell polarity and formation of the monolayer of the inner dental epithelium are disturbed. The enamel knot, a signaling center for tooth germ development, is defective, and there is a significant reduction of Shh and Fgf4 expression in the dental epithelium. In the absence of laminin alpha5, the basement membrane in the inner dental epithelium becomes discontinuous. In normal mice, integrin alpha6beta4, a receptor for laminin alpha5, is strongly localized at the basal layer of the epithelium, whereas in mutant mice, integrin alpha6beta4 is expressed around the cell surface. In primary dental epithelium culture, laminin-10/11 promotes cell growth, spreading, and filopodia-like microspike formation. This promotion is inhibited by anti-integrin alpha6 and beta4 antibodies and by phosphatidylinositol 3-kinase inhibitors and dominant negative Rho-GTPase family proteins Cdc42 and Rac. In organ culture, anti-integrin alpha6 antibody and wortmannin reduce tooth germ size and shape. Our studies demonstrate that laminin alpha5 is required for the proliferation and polarity of basal epithelial cells and suggest that the interaction between laminin-10/11-integrin alpha6beta4 and the phosphatidylinositol 3-kinase-Cdc42/Rac pathways play an important role in determining the size and shape of tooth germ.

Animals↗