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PubMed1995

Activation of the p21 pathway of growth arrest and apoptosis by the beta 4 integrin cytoplasmic domain.

The integrin alpha 6 beta 4, a receptor for members of the laminin family of basement membrane components, contributes to the function of epithelial cells and their oncogenically transformed derivatives. In our efforts to study alpha 6 beta 4-mediated functions in more detail and to assess the contribution of the beta 4 cytoplasmic domain in such functions, we identified a rectal carcinoma cell line that lacks expression of the beta 4 integrin subunit. This cell line, termed RKO, expresses alpha 6 beta 1 but not alpha 6 beta 4, and it interacts with laminin-1 less avidly than similar cell lines that express alpha 6 beta 4. We expressed a full-length beta 4 cDNA, as well as a mutant cDNA that lacks the beta 4 cytoplasmic domain, in RKO cells and isolated stable subclones of these transfectants. In this study, we report that subclones that expressed the full-length beta 4 cDNA in association with endogenous alpha 6 exhibited partial G1 arrest and apoptosis, properties that were not evident in RKO cells transfected with either the cytoplasmic domain mutant or the expression vector alone. In an effort to define a mechanism for these observed changes in growth, we observed that expression of the alpha 6 beta 4 integrin induced expression of the p21 (WAF1; CiP1) protein, an inhibitor of cyclin-dependent kinases. These data suggest that the beta 4 integrin cytoplasmic domain is linked to a signaling pathway involved in cell cycle regulation in the beta 4 transfected RKO cells.

Antigens, CD↗
PubMed2000

Characterization of AMC-HN-9, a cell line established from an undifferentiated carcinoma of the parotid gland: expression of alpha6beta4 with the absence of BP180 and 230.

We recently reported the development of a cell line, AMC-HN-9, established from an undifferentiated carcinoma (UDC) of the parotid gland. AMC-HN-9 consists mostly of spindle-shaped cells, has poor in vitro adhesiveness and an in vitro appearance that is different from that of other epithelial cell lines. To test the hypothesis that structural or functional abnormalities of the hemidesmosomes might contribute to the morphological appearance and biology of UDCs, we studied the expression of hemidesmosomal proteins in AMC-HN-9. Flow cytometry, indirect immunofluorescence, immunoprecipitation, reverse transcriptase-polymerase chain reaction, and cytogenetic analysis were used. AMC-HN-9 cells express the alpha6 and beta4 integrin subunits at nearly the same intensity as head and neck squamous cell carcinoma cell lines. However, AMC-HN-9 does not express BP180 and BP230, although there is no gross deletion of the loci of the BP180 and BP230 genes, suggesting that a more subtle mechanism has silenced these genes. In conclusion, the failure to express certain hemidesmosomal proteins is a likely explanation for the functional and morphologic characteristics of UDC cells both in vivo and in vitro.

Antibodies, Monoclonal↗
PubMedDate not supplied

Mesothelial cells in suspension expose an enriched integrin repertoire capable of capturing soluble fibronectin and laminin.

Pleural cavities are lined by a polarized monolayer of mesothelial cells (MC). During pleuritis, MC are shed into effusions, and pleural obstruction may occur. Integrins are cell surface receptors mediating interactions with extracellular matrix (ECM) proteins. The distribution of beta 1-, beta 3-, beta 4-integrins and fibronectin and laminin in normal and chronically inflamed pleura and in/on MC from pleural effusions was examined by immunomorphology and flow cytometry. Adhesion assays of MC to fibronectin and laminin were performed. In situ, resting MC expressed beta 1-, beta 3-, and beta 4-, and alpha v-subunits. Activated MC were beta 1- and alpha v-positive and also expressed alpha 3 and alpha 6; beta 4 was confined to the basal surface of MC; beta 3 was absent. Floating MC from effusions neoexpressed alpha 5 and reexpressed beta 3. In vitro, MC surface expressed beta 1, beta 3, alpha 3, alpha 5, alpha 6, alpha v, and also alpha 1 and alpha 2. In normal pleura, fibronectin and laminin were components of the basement membrane. In pleuritis, the basement membrane was desintegrated. Instead, newly formed fibronectin/laminin containing fibrils extended into the submesothelial connective tissue. Floating MC freshly isolated from effusions carried fibronectin and laminin on their surface and showed specific binding to these ECM proteins. Binding was blocked by anti-beta 1 or anti-alpha 5 and anti-alpha 6 antibodies, respectively. MC incubated with fibronectin showed a clear shift to the S phase, while laminin had no effect. In conclusion, activated and detached MC progressively enrich their integrin repertoire. By capturing soluble fibronectin and laminin and by matrix-mediated bridging, readhering MC may contribute to pleural obstruction. Further, soluble fibronectin bound to alpha 5 beta 1 might be life-sustaining for floating MC by driving cells into cell cycle.

Antigens, CD↗
PubMed1995

A recombinant tail-less integrin beta 4 subunit disrupts hemidesmosomes, but does not suppress alpha 6 beta 4-mediated cell adhesion to laminins.

To examine the function of the alpha 6 beta 4 integrin we have determined its ligand-binding ability and overexpressed two potentially dominant negative mutant beta 4 subunits, lacking either the cytoplasmic or extracellular domain, in bladder epithelial 804G cells. The results of cell adhesion and radioligand-binding assays showed that alpha 6 beta 4 is a receptor for several laminin isoforms, including laminin 1, 2, 4, and 5. Overexpression of the tail-less or head-less mutant beta 4 subunit did not suppress alpha 6 beta 4-mediated adhesion to laminins, as both types of transfectants adhered to these ligands in the presence of blocking anti-beta 1 antibodies as well as the controls. However, immunofluorescence experiments indicated that the endogenous alpha 6 beta 4 integrin and other hemidesmosomal markers were not concentrated in hemidesmosomes in cells overexpressing tail-less beta 4, while the distribution of these molecules was not altered in cells overexpressing the head-less subunit. Electron microscopic studies confirmed that cells overexpressing tail-less beta 4 had a drastically reduced number of hemidesmosomes, while cells expressing the head-less subunit had a normal number of these structures. Thus, expression of a tail-less, but not a head-less mutant beta 4 subunit leads to a dominant negative effect on hemidesmosome assembly without suppressing initial adhesion to laminins. We conclude that the alpha 6 beta 4 integrin binds to several laminins and plays an essential role in the assembly and/or stability of hemidesmosomes, that alpha 6 beta 4-mediated adhesion and hemidesmosome assembly have distinct requirements, and that it is possible to use a dominant negative approach to selectively interfere with a specific function of an integrin.

Amino Acid Sequence↗
PubMed1997

Key role of the Cdx2 homeobox gene in extracellular matrix-mediated intestinal cell differentiation.

To explore the role of homeobox genes in the intestine, the human colon adenocarcinoma cell line Caco2-TC7 has been stably transfected with plasmids synthesizing Cdx1 and Cdx2 sense and antisense RNAs. Cdx1 overexpression or inhibition by antisense RNA does not markedly modify the cell differentiation markers analyzed in this study. In contrast, Cdx2 overexpression stimulates two typical markers of enterocytic differentiation: sucrase-isomaltase and lactase. Cells in which the endogenous expression of Cdx2 is reduced by antisense RNA attach poorly to the substratum. Conversely, Cdx2 overexpression modifies the expression of molecules involved in cell-cell and cell-substratum interactions and in transduction process: indeed, E-cadherin, integrin-beta4 subunit, laminin-gamma2 chain, hemidesmosomal protein, APC, and alpha-actinin are upregulated. Interestingly, most of these molecules are preferentially expressed in vivo in the differentiated villi enterocytes rather than in crypt cells. Cdx2 overexpression also results in the stimulation of HoxA-9 mRNA expression, an homeobox gene selectively expressed in the colon. In contrast, Cdx2-overexpressing cells display a decline of Cdx1 mRNA, which is mostly found in vivo in crypt cells. When implanted in nude mice, Cdx2-overexpressing cells produce larger tumors than control cells, and form glandular and villus-like structures. Laminin-1 is known to stimulate intestinal cell differentiation in vitro. In the present study, we demonstrate that the differentiating effect of laminin-1 coatings on Caco2-TC7 cells is accompanied by an upregulation of Cdx2. To further document this observation, we analyzed a series of Caco2 clones in which the production of laminin-alpha1 chain is differentially inhibited by antisense RNA. We found a positive correlation between the level of Cdx2 expression, that of endogenous laminin-alpha1 chain mRNA and that of sucrase-isomaltase expression in these cell lines. Taken together, these results suggest (a) that Cdx1 and Cdx2 homeobox genes play distinct roles in the intestinal epithelium, (b) that Cdx2 provokes pleiotropic effects triggering cells towards the phenotype of differentiated villus enterocytes, and (c) that Cdx2 expression is modulated by basement membrane components. Hence, we conclude that Cdx2 plays a key role in the extracellular matrix-mediated intestinal cell differentiation.

Adenocarcinoma↗
PubMed2006

Effect of fibroblasts on tracheal epithelial regeneration in vitro.

Several artificial grafts for covering deficient trachea have been produced through tissue engineering. Recently, our group clinically used an artificial trachea made from collagen sponge for patients with noncircumferential tracheal resection. However, the slowness of epithelial regeneration on the surface of the artificial trachea was confirmed as one particular problem. In this study, we co-cultured tracheal epithelial cells with fibroblasts and examined effects of fibroblasts on epithelial regeneration in vitro. Fibroblasts activated epithelial cell proliferation and migration. In co-culture with fibroblasts, epithelial cells reconstructed pseudostratified epithelium, which was composed of ciliated, goblet, and basal cells. Furthermore, a basement membrane was reconstructed between epithelial cells and fibroblasts, and integrin beta4 was also observed there. Fibroblasts rapidly increased mucin secretion by epithelial cells. These results indicate that stimulatory effects of fibroblasts on epithelial cell migration, proliferation, and differentiation would reduce the time required for covering of epithelial cells on the defect of luminal surface and hasten regeneration of morphologically and functionally normalized epithelium involving the reconstruction of basement membrane.

Animals↗
PubMed1999

Identification of the hemidesmosomal 500 kDa protein (HD1) as plectin.

HD1 is a 500 kDa hemidesmosomal plaque protein recognized by monoclonal antibody mAb-121. Recent research on inherited skin disease has suggested that it might be identical to plectin or an isoform. To cast light on this question, we have prepared several monoclonal antibodies that recognize a 500 kDa protein in the hemidesmosome fraction. Unexpectedly, some staining pattern heterogeneity was observed on immunofluorescence microscopy. Attention was focused on two monoclonal antibodies which gave different localization in bovine skin and retinal pigment epithelial cells. Determination of the amino-terminal sequence of an antigenic 100 kDa polypeptide fragment derived from the 500 kDa component of an insoluble fraction of bovine hepatocytes revealed it was identical to that of plectin. Using the two antibodies, we screened a cDNA library derived from BMGE+H, a bovine mammary gland epithelial cell line. The isolated cDNA clones corresponded to the rod domain of bovine plectin, with two separate epitope regions for each of the antibodies. From these results we conclude that the hemidesmosomal 500 kDa component HD1 is identical to plectin. As judged on rough estimation of molar ratios on this basis, hemidesmosomes are composed of plectin, BP230, the integrin beta4 subunit, and alpha6 in a 1:1:1:1 ratio.

Animals↗
PubMed2003

Gene expression patterns as potential molecular biomarkers for malignant transformation in human keratinocytes treated with MNNG, arsenic, or a metal mixture.

In previous studies, treatment with 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) enhanced malignant transformation of immortal human epidermal (RHEK-1) keratinocytes. In contrast, arsenic (As) alone or in a mixture of As, cadmium (Cd), chromium (Cr), and lead (Pb) inhibited this process. Microarray analysis showed unique gene expression patterns in RHEK-1 exposed to MNNG, As, or the metal mixture. From this analysis, we have selected 16 genes potentially involved in the enhancement or inhibition of transformation. These 16 genes, nine (IFN inducible protein 9-27, MAA A32, CCLB protein, integrin beta4, XRCC1, K8, K18, MT3, MAPKK6) of which were altered in a chemical-specific manner and seven (MIC1, bikunin, MTS1, BMP4, RAD23A, DOC2, vimentin) of which were commonly affected by the MNNG and As or mixture treatments, were examined for expression in detail by real-time RT-PCR. Qualitatively, both microarray and real-time RT-PCR analyses gave comparable results for 15 of 16 genes, i.e., genes were consistently induced or suppressed under the different treatment regimens when measured by either technique. Of the seven genes altered in their expression by multiple chemical treatments, five showed patterns consistent with a role in the transformation process, i.e., they were oppositely regulated in MNNG-transformed RHEK-1 cells (designated as OM3) as compared to the nonmalignant As- and mixture-exposed cells. Through time-course studies, we also identified markers whose expression correlates with acquisition of transformation-associated characteristics in OM3. Identification of a battery of genes altered during progressive transformation of RHEK-1 should aid in developing a mechanistic understanding of this process, as well as strengthening the utility of these genes as biomarkers.

Animals↗
PubMed1994

Junctional epidermolysis bullosis: defects in expression of epiligrin/nicein/kalinin and integrin beta 4 that inhibit hemidesmosome formation.

Junctional epidermolysis bullosis (JEB) is a heterogeneous inherited blistering disorder of human epithelial basement membranes (BMs). Characteristically, the epidermis detaches from the BM between the basal cells and the lamina lucida due to reduced numbers of hemidesmosomes (HDs). Attempts to identify a candidate gene for JEB led to the characterization of nicein, a protein complex in normal BMs that is absent from BMs of patients with JEB gravis. In independent research, two related BM glycoproteins, epiligrin and kalinin, were identified as functional adhesion components of HDs. Epiligrin was characterized as a BM ligand for basal cell adhesion via integrins alpha 3 beta 1 in focal adhesions and alpha 6 beta 4 in HDs. Kalinin was characterized as an adhesive ligand and a component of anchoring filaments. Recent antibody and sequence studies on epiligrin/nicein/kalinin have identified limited homologies with laminin. Ongoing studies in multiple laboratories seek to identify mutations in one or more of the three subunits of epiligrin that are causal in JEB gravis. Consistent with the genetic heterogeneity of JEB, we have identified a patient with a variant form of JEB that is associated with pyloric atresia. This patient has negligible HDs, normal epiligrin, but reduced expression of integrin beta 4. A defect in the beta 4 expression may define a subset of JEB cases that also present with pyloric atresia. These results testify to the dual requirements for epiligrin in the BM and integrin beta 4 in the plasma membrane in regulating function of HDs in epithelium.

Cell Adhesion↗
PubMed2004

Integrin expression in cells of the intervertebral disc.

In this study, we investigated the profile of integrin expression in human and porcine intervertebral disc tissue. Differences in extracellular matrix composition between anulus fibrosus (AF) and nucleus pulposus (NP) regions of the disc, as well as differences in cellular responses to environmental stimuli, suggest a role for integrins in presenting matrix signals that may mediate these responses. Human disc tissue and porcine AF and NP tissue were stained with antibodies to alpha integrin subunits 1-6, V and IIb, and beta integrin subunits 1-6 and graded for evidence of positive staining on a scale from 0 (no staining) to 3 (high incidence of staining). Human tissue expressed alpha and beta integrin subunits shown to be present in articular cartilage, including alpha(1), alpha(5) and alpha(V). Porcine AF tissue expressed similar integrin subunits to human disc, with both expressing alpha(1), alpha(5), beta(1), beta(3) and beta(5) subunits, whereas porcine NP tissue expressed higher levels of alpha(6), beta(1) and beta(4) than AF tissue. The expressed subunits are known to interact with proteins including collagens, fibronectin and laminin; however, additional studies will be required to characterize the interactions of the integrin subunits with specific matrix constituents, as well as their specific involvement in regulating environmental stimuli.

Animals↗
PubMed2005

Plectin gene mutations can cause epidermolysis bullosa with pyloric atresia.

Epidermolysis bullosa with pyloric atresia (EB-PA), manifesting with neonatal blistering and gastric anomalies, is known to be caused by mutations in the hemidesmosomal genes ITGA6 and ITGB4, which encode the alpha6 and beta4 integrin polypeptides, respectively. As part of our molecular diagnostics program, we have now encountered four families with EB-PA in which no mutations could be identified in these two genes. Instead, PCR amplification followed by heteroduplex scanning and/or direct nucleotide sequencing revealed homozygous mutations in the plectin gene (PLEC1), encoding another hemidesmosomal protein previously linked to EB with muscular dystrophy. Our findings provide evidence for additional molecular heterogeneity in EB, and emphasize the importance of screening EB-PA patients not only for alpha6beta4 integrin but also for plectin deficiency.

Adult↗
PubMed2005

Disruption of ERBB2IP is not associated with dystrophic epidermolysis bullosa in both father and son carrying a balanced 5;13 translocation.

Mutations in the type VII collagen gene (COL7A1) cause autosomal recessive and autosomal dominant inherited dystrophic epidermolysis bullosa (DEB). We report a family with three individuals who present blistering, scarring, hypo- and hyperpigmentation, and nail dystrophy suggestive for DEB. Whereas father and son carry a 5;13 translocation, the daughter shows a normal karyotype. Segregation analysis revealed that all affected family members inherited the same COL7A1 allele. Mutation analysis disclosed a heterozygous missense mutation, c.6227G > A (p.G2076D), in COL7A1 in all affected individuals. Delineation of the translocation breakpoints showed that the ERBB2IP (erbb2 interacting protein or Erbin) gene is disrupted in 5q13.1 and GPC6 in 13q32. GPC6 encodes glypican 6 belonging to a family of cell surface heparan sulfate proteoglycans. The binding partners of Erbin, BP230 (BPAG1) and the integrin beta4 subunit, both involved in hemidesmosome (HD) function, and the presence of Erbin in HD suggested that it plays a role in establishment and maintenance of cell-basement membrane adhesions. However, loss of function of one ERBB2IP copy or expression of a putative novel ERBB2IP fusion protein did not apparently modulate the DEB phenotype in both translocation patients. Nonetheless, one cannot yet exclude that ERBB2IP is a candidate for human blistering disorders such as epidermolysis bullosa.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Progress in epidermolysis bullosa: the phenotypic spectrum of plectin mutations.

Plectin, a large multidomain adhesive protein with versatile binding functions, is expressed in a number of tissues and cell types. In the skin, plectin is a critical component of hemidesmosomes, interacting with keratin intermediate filaments and beta4 integrin. Mutations in the plectin gene (PLEC1) result in fragility of skin, demonstrating blister formation at the level of hemidesmosomes. These blistering disorders belong to the spectrum of epidermolysis bullosa (EB) phenotypes, and three distinct variants because of plectin mutations have been identified. First, EB with muscular dystrophy, an autosomal recessive syndrome, is frequently caused by premature termination codon-causing mutations leading to the absence of plectin both in the skin and in the muscle. Second, a heterozygous missense mutation (R2110W) in PLEC1 has been documented in patients with EB simplex of the Ogna type, a rare autosomal dominant disorder. Finally, recent studies have disclosed plectin mutations in patients with EB with pyloric atresia, an autosomal recessive syndrome, frequently with lethal consequences. Collectively, these observations attest to the phenotypic spectrum of plectin mutations, and provide the basis for accurate genetic counselling with prognostic implications, as well as for prenatal diagnosis in families at the risk of recurrence of the disease.

Animals↗
PubMed2004

Gene expression fingerprints in human tubulointerstitial inflammation and fibrosis as prognostic markers of disease progression.

BACKGROUND: Gene expression profiling of nephropathies may facilitate development of diagnostic strategies for complex renal diseases as well as provide insight into the molecular pathogenesis of kidney diseases. To test molecular based renal disease categorization, differential gene expression profiles were compared between control and hydronephrotic kidneys showing varying degrees of inflammation and fibrosis. METHODS: RNA expression profiles from 9 hydronephrotic and 3 control kidneys were analyzed using small macroarrays dedicated to genes involved in cell-cell contact, matrix turnover, and inflammation. In parallel, the degree of tubulointerstitial inflammation, fibrosis, and tubular atrophy using light microscopy and quantitative immunohistochemical parameters was determined. RESULTS: Hierarchic clustering and self-organizing maps led to a gene expression dendrogram with three distinct nodes representing the control group, four kidneys with high inflammation, and five kidneys giving high fibrosis scores. To evaluate the clinical applicability of the marker set, the expression of nine genes (6Ckine, IL-8, MMP-9, MMP-3, MMP-7, urokinase R, CXCR5, integrin-beta4, and pleiotrophin) was tested in tubulointerstitial samples from routine renal biopsies. Seven mRNA markers showed differential regulation in inflammation and fibrosis in the biopsy population. Clinical follow-up revealed stringent correlation between gene expression data and progression of renal disease, and allowed segregation of the biopsies into progressive or stable disease course based on gene expression profiles. CONCLUSION: This study suggests the feasibility of gene expression-based disease categorization in human nephropathies based on the extraction of marker gene sets.

Adult↗
PubMed1996

Distribution of alpha 6 and beta 4 integrins following epithelial abrasion in the rabbit cornea.

Integrin complex alpha 6 beta 4 is a component of the hemidesmosome. In the unwounded cornea both the integrin subunits face the laminin-containing basement membrane, but the alpha 6 subunit is also located between the basal cells. While the migrating epithelium is known to be without hemidesmosomes, we investigated the distribution of alpha 6 beta 4 during epithelial healing. Epithelial abrasion 7.5 mm in diameter was mechanically created. The rabbits were killed 1-24 h or 2, 3, or 7 days later. Monoclonal antibodies against alpha 6, beta 4, and laminin A were used to detect their distributions by immunohistochemistry. Positive immunostaining for laminin A on the surface of the unepithelialized stroma indicated that basement membrane was intact after the epithelial abrasion. Three hours after corneal wounding, alpha 6, was detectable around the entire cell up to the leading edge of the migrating epithelium. In the peripheral wound, alpha 6, was also prominently present around the basal and suprabasal cells with only the superficial cell layers being negative. The beta 4 subunit showed a dissimilar distribution; it was not detectable subjacent to the leading edge. After 1 h the immunoreaction for the beta 4 subunit had faded 15 - 20 microns peripheral to the wound margin. Thereafter the subepithelial band was segmentally reassembled, starting from the periphery and progressing toward the central area of the wound. One week after epithelial wounding, immunolabeling for both integrin subunits was indistinguishable from that of the control cornea. Our results indicate that in addition to the beta 4 in the rabbit cornea, the alpha 6 subunit is also complex with another beta subunit during the epithelial healing phase. The results also suggest that basal cells 15 - 20 microns peripheral to the wound margin disassemble their HDs prior to the migration process.

Animals↗
PubMed1998

Effects of topical calcipotriol on the expression of adhesion molecules in psoriasis.

Seven patients with chronic plaque psoriasis were treated with topical calcipotriol for 8 to 24 weeks; the lesions improved in 5 patients. Immunohistochemistry was performed on frozen sections, to evaluate the expression of adhesion molecules and extracellular matrix components before and after therapy. Changes in expression and topography of beta1 and beta4 integrins were found on psoriatic lesions before therapy and a reduction in the expression of tenascin was detected as well. Moreover, several activation markers such as ICAM-1, HLA-DR, CD26 were focally positive, with a diffuse cytoplasmic reactivity, in basal and suprabasal layers in untreated lesions. In the 5 patients in whom lesions regressed after topical calcipotriol treatment, we observed a histological normalization of the epidermis and the inflammatory infiltrate was reduced. Moreover, not only was there a normalization in the expression and topography of adhesion molecules, but also the integrin pattern observed after therapy was superimposable to that of normal skin.

Administration, Topical↗
PubMed2006

Effects of minocycline and doxycycline on cell survival and gene expression in human gingival and periodontal ligament cells.

OBJECTIVES AND BACKGROUND: Periodontitis is an infectious disease in the gingival crevice caused by periodontopathic bacteria, including Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans, and Tennerella forsythensis, and antibacterial agents are directly administered to the site of infection to treat it. To maximize the therapeutic effects while reducing the adverse effects, the antibacterial agents should be administered at concentrations greater than their MIC(90) doses required to inhibit the growth of 90% of periodontopathic bacteria and the administration should not damage the periodontal tissue. One approach for estimating cellular damage in the periodontal tissue caused by the administration is to assay cytological damages following exposures of cultured human cells derived from periodontal tissues to antibacterial agents. In the present study, we investigated the cytotoxic effect of minocycline (MINO) and doxycycline (DOX) by using a human gingival fibroblast cell line, a human gingival epithelial cell line, and a human periodontal ligament fibroblast cell line. We also used these cell lines to study the effect of MINO or DOX on the mRNA and protein expressions of genes associated with the differentiation of fibroblasts and the proliferation, differentiation, or cellular adhesion important to the epithelial regeneration of the periodontal attachment. METHODS: The cytotoxic effect of MINO or DOX was measured as a decrease in cell survivals. The effects of these antibiotics on the mRNA and protein expressions in the cell lines were studied by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analyses, respectively. RESULTS: The maximum concentration of MINO or DOX that has little effect on the cell survivals and the mRNA and protein expressions of genes for alkaline phosphatase, type I procollagen, keratinocyte growth factor receptor, keratin 18 or 8/18, integrin beta1, integrin beta4, and laminin 5gamma2 was 10 or 30 microm, respectively, which are greater than their MIC(90) doses against periodontopathic bacteria described above. CONCLUSIONS: These findings suggest that little, if any, cellular damage would be expected with topical administration of MINO or DOX to the periodontal pocket at a dose equivalent to the MIC(90). It is important to note, however, that the extrapolation of these findings to in vivo conditions has yet to be undertaken.

Administration, Topical↗
PubMed1995

Changes in the expression of integrins and basement membrane proteins in benign mucous membrane pemphigoid.

OBJECTIVE: To determine the location of the subepithelial split in benign mucous membrane pemphigoid (BMMP) and its relationship to the anchoring filaments and their receptors. MATERIALS AND METHODS: Frozen sections of lesional and perilesional oral mucosa from 10 cases of BMMP were stained, using an immunofluorescence method, for the beta-1, beta-4, alpha-3 and alpha-6 integrin subunits and for their ligands, laminin I and laminin V (kalinin). In all cases the diagnosis was confirmed by the demonstration of linear staining for IgG at the basement membrane zone. Six specimens of normal mucosa were stained for comparison. RESULTS: Staining for integrins, laminin and kalinin in perilesional mucosa was similar to normals, although one case showed loss of alpha-6 and beta-4 . In lesional mucosa, laminin and kalinin showed strong linear staining localised to the floor of the bullae. The alpha-6 and beta-4 subunits were expressed only on the roof of the bullae but staining was weak and patchy with areas of loss. In some sections alpha-6 showed a punctate intracellular distribution similar to IgG. The distribution of alpha-3 and beta-1 was similar to that seen in normals. CONCLUSIONS: In all cases kalinin was found on the connective tissue side of the lesions and alpha-6 beta-4 localised to the epithelial side. This shows that the split occurs at a location which separates anchoring filaments from the hemidesmosomes. Loss of the alpha-6 beta-4 integrin in the lesions and the similar intracellular staining of alpha-6 and IgG, suggest that disruption of hemidesmosomes may be a key event in the immunopathogenesis of the lesions and that the alpha-6 integrin subunit is a potential antigen in oral mucosal BMMP.

Actin Cytoskeleton↗