Associations between gastric histopathology and the occurrence of colonic polyps.

AIM: Compromise of the gastric acid barrier may facilitate bacterial invasion of the lower intestinal tract and promote the development of colonic neoplasia. Our study aimed to test the associations between histopathological abnormalities of the upper and lower gastrointestinal tract in patients undergoing bidirectional endoscopy. METHOD: The Inform Diagnostics database is a national electronic repository of histopathological records of patients distributed throughout the USA. A case-control study of 302 061 patients, 163 168 of whom had colonic polyps, evaluated whether the occurrence of colonic polyps was influenced by the presence of the following gastro-oesophageal diagnoses: gastric Helicobacter pylori infection, gastric intestinal metaplasia, fundic gland polyps and gastric hyperplastic polyps. The influence of individual diagnoses on the occurrence of colonic polyps was expressed as odds ratios with their 95% confidence intervals. RESULTS: The odds ratio for tubular adenomas being associated with gastric H. pylori was 1.53 (1.49-1.58), with intestinal metaplasia 1.65 (1.59-1.71), with fundic gland polyps 1.49 (1.45-1.54) and with gastric hyperplastic polyps 1.85 (1.75-1.96). The odds ratio for sessile serrated polyps being associated with gastric H. pylori was 1.03 (0.96-1.10), with intestinal metaplasia 1.21 (1.13-1.30), with fundic gland polyps 1.79 (1.69-1.89) and with gastric hyperplastic polyps 1.52 (1.35-1.71. CONCLUSION: A diminished gastric acid barrier function, which occurs in various upper gastrointestinal diseases associated with lowered gastric acid output, may promote the development of colonic neoplasia.

Screening of white-rot fungi for bioprocessing of wheat straw into ruminant feed.

AIM: In this study, the biological variation for improvement of the nutritive value of wheat straw by 12 Ceriporiopsis subvermispora, 10 Pleurotus eryngii and 10 Lentinula edodes strains was assessed. Screening of the best performing strains within each species was made based on the in vitro degradability of fungal-treated wheat straw. METHODS AND RESULTS: Wheat straw was inoculated with each strain for 7 weeks of solid state fermentation. Weekly samples were evaluated for in vitro gas production (IVGP) in buffered rumen fluid for 72 h. Out of the 32 fungal strains studied, 17 strains showed a significantly higher (P < 0·05) IVGP compared to the control after 7 weeks (227·7 ml g-1 OM). The three best Ceriporiopsis subvermispora strains showed a mean IVGP of 297·0 ml g-1 OM, while the three best P. eryngii and L. edodes strains showed a mean IVGP of 257·8 and 291·5 ml g-1 OM, respectively. CONCLUSION: Ceriporiopsis subvermispora strains show an overall high potential to improve the ruminal degradability of wheat straw, followed by L. edodes and P. eryngii strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Large variation exists within and among different fungal species in the valorization of wheat straw, which offers opportunities to improve the fungal genotype by breeding.

Decreased risk for microscopic colitis and inflammatory bowel disease among patients with reflux disease.

AIM: Previous studies have found an increased risk for microscopic colitis (MC) associated with proton pump inhibitors. In patients with ulcerative colitis (UC) or Crohn's disease (CD), proton pump inhibitors have been associated with an increased risk for IBD flares and impaired outcomes. The aim of this study was to test the epidemiological associations between gastro-oesophageal reflux disease (GERD) and MC, UC or CD in a large database. METHOD: The Miraca Life Sciences Database is a national electronic repository of histopathological records of patients distributed throughout the entire USA. A case-control study evaluated whether the presence of Barrett's metaplasia, erosive oesophagitis on endoscopy or histological signs of reflux oesophagitis, clinical diagnosis of GERD or any GERD type affected the occurrence of MC, UC or CD among 228 506 subjects undergoing bidirectional endoscopy. Multivariate logistic regression analyses were used to calculate ORs and their 95% CI for the risk of MC, UC or CD associated with various types of GERD and were adjusted for age, sex and presence of Helicobacter pylori. RESULTS: The analysis revealed an inverse relationship between GERD and different types of inflammatory bowel disease. The inverse relationships applied similarly to MC (mean = 0.62, 95% CI: 0.58-0.66), UC (mean = 0.89, 95% CI: 0.81-0.97) and CD (mean = 0.76, 95% CI: 0.69-0.85). It also applied to different forms of GERD, with a trend towards more pronounced inverse relationships associated with Barrett's metaplasia or erosive oesophagitis than clinical diagnosis of GERD. CONCLUSION: Gastro-oesophageal reflux disease is inversely associated with all forms of inflammatory bowel disease, such as MC, UC, or CD.

Differences between two strains of Ceriporiopsis subvermispora on improving the nutritive value of wheat straw for ruminants.

AIM: This study evaluated differences between two strains of Ceriporiopsis subvermispora on improving the nutritive value and in vitro degradability of wheat straw. METHODS AND RESULTS: Wheat straw was treated with the fungi for 7 weeks. Weekly samples were analysed for ergosterol content, in vitro gas production (IVGP), chemical composition and lignin-degrading enzyme activity. Ergosterol data showed CS1 to have a faster initial growth than CS2 and reaching a stationary phase after 3 weeks. The IVGP of CS1-treated wheat straw exceeded the control earlier than CS2 (4 vs 5 weeks). CS1 showed a significantly higher (P < 0·001) selectivity in lignin degradation compared to CS2. Both strains showed peak activity of laccase and manganese peroxidase (MnP) at week 1. CS1 showed a significantly higher (P < 0·001) laccase activity, but lower (P = 0·008) MnP activity compared to CS2. CONCLUSION: Both CS strains improved the nutritive value of wheat straw. Variation between strains was clearly demonstrated by their growth pattern and enzyme activities. SIGNIFICANCE AND IMPACT OF THE STUDY: The differences among the two strains provide an opportunity for future selection and breeding programs in improving the extent and selectivity of lignin degradation in agricultural biomass.

The ethnic distribution of sessile serrated polyps in the United States is inversely associated with Helicobacter pylori prevalence.

AIM: Little is known about the epidemiology of sessile serrated polyps (SSP). Our study aimed to investigate the influence of Helicobacter pylori gastritis and patient demographic characteristics (age, gender, ethnicity) on the prevalence of SSP using a large national database of patients undergoing bi-directional endoscopy. METHOD: De-identified patient data were extracted from the Miraca Life Sciences electronic database of histopathological reports. Using multivariate logistic regression analysis, the influence of H. pylori gastritis and demographic characteristics on the occurrence of SSP were expressed as odds ratios (OR) with their 95% confidence intervals (CI). RESULTS: The total study population comprised 228 506 subjects, of whom 28 890 carried a diagnosis of H. pylori gastritis and 11 285 SSP. Age (OR 4.35, 95% CI: 3.82-4.96), female gender (0.92, 0.88-0.95) and H. pylori gastritis (0.94, 0.88-0.99) exerted the strongest influence on the occurrence of SSP. In comparison with the population comprising Caucasians and African Americans, SSP were less common among subjects of Hispanic (0.67, 0.62-0.73), East Asian (0.59, 0.50-0.69), Indian (0.43, 0.27-0.64) or Middle Eastern descent (0.61, 0.41-0.87). All these ethnic subgroups were also characterized by a higher prevalence of H. pylori than the comparison group. A low prevalence of H. pylori was significantly associated with a high prevalence of SSP (R2  = 0.82, P < 0.001). CONCLUSION: The prevalence of SSP within the United States is characterized by a marked ethnic variation. The inverse correlation between the prevalence of H. pylori and SSP suggests that gastric infection with H. pylori may be partly responsible for the observed ethnic distribution of SSP.

Differences in the socio-economic distribution of inflammatory bowel disease and microscopic colitis.

AIM: Inflammatory bowel disease (IBD) and microscopic colitis are characterized by different geographical distributions across the USA. In this cross-sectional study we utilized demographic and socio-economic information associated with individual ZIP codes to further delineate the epidemiological characteristics of the two diseases. METHOD: A total of 813 057 patients who underwent colonoscopy between 2008 and 2014 were extracted from an electronic database of histopathology reports. The prevalence of patients with IBD or microscopic colitis was expressed as percentage of the population associated with specific demographic (age, sex, ethnicity) and socio-economic characteristics (population size, housing value, annual income, tertiary education). RESULTS: Both diseases were more common among subjects from ZIP codes with predominantly White residents and less common among subjects from ZIP codes with predominantly non-White residents such as Black, Hispanic and Asian. These ethnic variations were more pronounced in microscopic colitis than IBD. Markers of affluence, such as average residential house value and annual income, were positively associated with IBD and negatively with microscopic colitis. The prevalence of both diseases was positively correlated with tertiary education. CONCLUSION: The occurrence of both IBD and microscopic colitis is influenced by environmental risk factors. The differences in the demographic, ethnic and socio-economic distributions of the two diseases suggest that different sets of risk factors affect the two diseases and that their aetiology is unrelated.

The stomach in health and disease.

The stomach is traditionally regarded as a hollow muscular sac that initiates the second phase of digestion. Yet this simple view ignores the fact that it is the most sophisticated endocrine organ with unique physiology, biochemistry, immunology and microbiology. All ingested materials, including our nutrition, have to negotiate this organ first, and as such, the stomach is arguably the most important segment within the GI tract. The unique biological function of gastric acid secretion not only initiates the digestive process but also acts as a first line of defence against food-borne microbes. Normal gastric physiology and morphology may be disrupted by Helicobacter pylori infection, the most common chronic bacterial infection in the world and the aetiological agent for most peptic ulcers and gastric cancer. In this state-of-the-art review, the most relevant new aspects of the stomach in health and disease are addressed. Topics include gastric physiology and the role of gastric dysmotility in dyspepsia and gastroparesis; the stomach in appetite control and obesity; there is an update on the immunology of the stomach and the emerging field of the gastric microbiome. H. pylori-induced gastritis and its associated diseases including peptic ulcers and gastric cancer are addressed together with advances in diagnosis. The conclusions provide a future approach to gastric diseases underpinned by the concept that a healthy stomach is the gateway to a healthy and balanced host. This philosophy should reinforce any public health efforts designed to eradicate major gastric diseases, including stomach cancer.

Epithelial detachment due to absence of hemidesmosomes in integrin beta 4 null mice.

Integrins are heterodimeric transmembrane glycoproteins which are engaged in a variety of cellular functions, such as adhesion, migration and differentiation1. The integrin alpha 6 beta 4 is expressed on squamous epithelia, on subsets of endothelial cells, immature thymocytes and on Schwann cells and fibroblasts in the peripheral nervous system. In stratified epithelia, alpha 6 beta 4 is concentrated in specialised adhesion structures, called hemidesmosomes, which are implicated in the stable attachment of the basal cells to the underlying basement membrane by connecting the intermediate filaments with the extracellular matrix. The nature of the interactions between the various hemidesmosomal proteins, that lead to the formation of hemidesmosome is poorly understood. To study the contribution of the integrin alpha 6 beta 4 in hemidesmosome formation and their anchoring properties, we inactivated the beta 4 gene in mice by targeted gene disruption. Homozygous beta 4 null mice died shortly after birth and displayed extensive detachment of the epidermis and other squamous epithelia. The dramatically reduced adhesive properties of the skin was accompanied by the absence of hemidesmosomes at the basal surface of keratinocytes. No evidence was found for impaired T-cell development, nor for defects in myelination in the peripheral nervous system.

Test sequence in the management of gastrointestinal bleeding.

BACKGROUND AND STUDY AIM: A large variety of test procedures is available to diagnose and treat patients with suspected gastrointestinal bleeding. The aim of the study was to investigate which test sequence should be utilized in managing gastrointestinal bleeding. METHODS: For each endoscopic, radiologic, or laboratory test procedure, professional fees and facility costs were estimated based on payments allowed by the US Centers for Medicare and Medicaid Services during the fiscal year 2010. A threshold analysis was used to compare the costs associated with different test sequences of varying clinical scenarios. RESULTS: A threshold represents the lowest expected probability of success, for which a test would still be indicated. In a work-up including all possible management options, the threshold associated with laboratory tests and gastric lavage was 1 %, esophagogastroduodenoscopy (EGD) 8 %, colonoscopy 9 %, nuclear scan 9 %, enteroscopy 11 %, computed tomography (CT) angiography 14 %, capsule endoscopy 23 %, and angiography with transcatheter embolization 25 %. Varying sets of thresholds were calculated for different clinical scenarios. The thresholds of EGD and colonoscopy remained low in most scenarios. In sensitivity analysis, rising risk of complications or costs of a procedure also lead to rising threshold values for it, potentially rendering the particular procedure untenable. CONCLUSIONS: A low threshold indicated a preferred management option that should be used early rather than late in a sequence of multiple possible test procedures to work up instances of gastrointestinal bleeding.

[DNA marking of some quantitative trait loci in the cultivated edible mushroom Pleurotus ostratus (Fr.) Kumm].

Fungi of the genus Pleurotus, in particular, species Pleurotus ostreatus (common oyster mushroom) are among most cultivated fungi in the world. Due to intense rates of development of studies in this field, efficient breeding programs are highly required in the search for new P. ostreatus strains. The principal traits used worldwide for selecting strains are intensity of fruitbearing, fruit body cap color (for some consumptive markets), and mycelium growth rate. In this connection, the objective of this work was to study these quantitative traits and to find molecular markers, which could be employed to accomplish breeding programs. In general, we found 12 genomic loci (quantitative trait loci, QTLs) controlling mycelium growth rate of oyster and six QTLs responsible for the fruit body cap color. The genetic map of P. ostreatus was constructed, and all markers of quantitative traits found by us were located on this genetic map. The obtained linkage map can be a useful tool for the accomplishment of breeding programs to improve economically important traits of oyster mushroom.

Hemidesmosomes: roles in adhesion, signaling and human diseases.

Our understanding of the role of hemidesmosomes in cell-substratum adhesion has greatly improved both as a result of targeted gene mutation experiments and by means of observations of several blistering disorders of the skin in which the absence or defects of hemidesmosomal proteins have been demonstrated. Functionally important domains within the proteins that constitute hemidesmosomes have recently been identified by transfection and mutagenesis studies. These multiprotein complexes appear not only to mediate cell adhesion, but also to transduce signals from the extracellular matrix to the cell interior that may profoundly modulate cell behavior.

When to stop the search for an elusive source of gastrointestinal bleeding.

BACKGROUND AND STUDY AIMS: This analysis investigates the clinical parameters that should drive decisions about when to continue or stop the search for an elusive source of gastrointestinal bleeding. PATIENTS AND METHODS: The number of endoscopies necessary to find a source of bleeding was estimated using the geometric distribution. A threshold analysis was used to develop a stop rule for the search for a site of bleeding. Bayes' formula served to estimate changes in the probability of achieving a diagnosis associated with a series of consecutive endoscopic tests. RESULTS: With decreasing probability of diagnostic success associated with an individual endoscopic procedure, such as P = 50%, 33%, or 25%, the mean (standard deviation [SD]) number of procedures needed to find the source of bleeding increases to 2 (1.41), 3 (2.45), or 4 (3.46), respectively. The threshold analysis suggests that work-up should be discontinued if the expected rise in diagnostic probability does not exceed the ratio of work-up cost to bleeding cost, that is, Δ P < work-up cost/bleeding cost. For instance, a 10-fold higher cost of bleeding than endoscopy would justify continued work-up if it can improve diagnostic probability by 10%. Bayesian analysis shows that after three negative tests the diagnostic probability drops below such a threshold. CONCLUSIONS: The analysis suggests the following basic rules. The search for a site of gastrointestinal bleeding will take on average 2 procedures with a range of 1 - 4. The search should be continued as long as the diagnostic probability is expected to rise by more than 10 %, which is unlikely after three consecutive negative tests.

Extracellular matrix formation after transplantation of human embryonic stem cell-derived cardiomyocytes.

Transplantation of human embryonic stem cell-derived cardiomyocytes (hESC-CM) for cardiac regeneration is hampered by the formation of fibrotic tissue around the grafts, preventing electrophysiological coupling. Investigating this process, we found that: (1) beating hESC-CM in vitro are embedded in collagens, laminin and fibronectin, which they bind via appropriate integrins; (2) after transplantation into the mouse heart, hESC-CM continue to secrete collagen IV, XVIII and fibronectin; (3) integrin expression on hESC-CM largely matches the matrix type they encounter or secrete in vivo; (4) co-transplantation of hESC-derived endothelial cells and/or cardiac progenitors with hESC-CM results in the formation of functional capillaries; and (5) transplanted hESC-CM survive and mature in vivo for at least 24 weeks. These results form the basis of future developments aiming to reduce the adverse fibrotic reaction that currently complicates cell-based therapies for cardiac disease, and to provide an additional clue towards successful engraftment of cardiomyocytes by co-transplanting endothelial cells.

Dual stimulation of Ras/mitogen-activated protein kinase and RhoA by cell adhesion to fibronectin supports growth factor-stimulated cell cycle progression.

In cellular transformation, activated forms of the small GTPases Ras and RhoA can cooperate to drive cells through the G1 phase of the cell cycle. Here, we show that a similar but substrate-regulated mechanism is involved in the anchorage-dependent proliferation of untransformed NIH-3T3 cells. Among several extracellular matrix components tested, only fibronectin supported growth factor-induced, E2F-dependent S phase entry. Although all substrates supported the mitogen-activated protein kinase (MAPK) response to growth factors, RhoA activity was specifically enhanced on fibronectin. Moreover, induction of cyclin D1 and suppression of p21(Cip/Waf) occurred specifically, in a Rho-dependent fashion, in cells attached to fibronectin. This ability of fibronectin to stimulate both Ras/MAPK- and RhoA-dependent signaling can explain its potent cooperation with growth factors in the stimulation of cell cycle progression.

Recognition of the laminin E8 cell-binding site by an integrin possessing the alpha 6 subunit is essential for epithelial polarization in developing kidney tubules.

It has been previously shown that A-chain and domain(E8)-specific antibodies to laminin that inhibit cell adhesion also interfere with the establishment of epithelial cell polarity during kidney tubule development (Klein, G., M. Langegger, R. Timpl, and P. Ekblom. 1988. Cell. 55:331-341). A monoclonal antibody specific for the integrin alpha 6 subunit, which selectively blocks cell binding to E8, was used to study the receptors involved. Immunofluorescence staining of embryonic kidneys and of organ cultures of metanephric mesenchyme demonstrated coappearance of the integrin alpha 6 subunit and the laminin A-chain in regions where nonpolarized mesenchymal cells convert into polarized epithelial cells. Both epitopes showed marked colocalization in basal areas of tubules, while an exclusive immunostaining for alpha 6 was observed in lateral and apical cell surfaces of the tubular epithelial cells. Organ culture studies demonstrated a consistent inhibition of kidney epithelium development by antibodies against the alpha 6 subunit. The data suggest that the recognition of E8 cell-binding site of laminin by a specific integrin is crucial for the formation of kidney tubule epithelium from undifferentiated mesenchymal stem cells. In some other cell types (endothelium, some ureter cells) an exclusive expression of alpha 6 with no apparent colocalization of laminin A-chain in the corresponding basement membrane was seen. Thus, in these cells, integrins possessing the alpha 6 subunit may bind to laminin isoforms that differ from those synthesized by developing tubules.

Episialin (MUC1) overexpression inhibits integrin-mediated cell adhesion to extracellular matrix components.

Episialin (MUC1) is a transmembrane molecule with a large mucin-like extracellular domain protruding high above the cell surface. The molecule is located at the apical side of most glandular epithelial cells, whereas in carcinoma cells it is often present at the entire surface and it is frequently expressed in abnormally large quantities. We have previously shown that overexpression of episialin reduces cell-cell interactions. Here we show that the integrin-mediated adhesion to extracellular matrix of transfectants of a melanoma cell line (A375), a transformed epithelial cell line (MDCK-ras-e) and a human breast epithelial cell line (HBL-100) is reduced by high levels of episialin. This reduction can be reversed by inducing high avidity of the beta 1 integrins by mAb TS2/16 (at least for beta 1-mediated adhesion). The adhesion can also be restored by redistribution of episialin on the cell surface by monoclonal antibodies into patches or caps. Similarly, capping of episialin on ZR-75-1 breast carcinoma cells, growing in suspension, caused adherence and spreading of these cells. We propose that there is a delicate balance between adhesion and anti-adhesion forces in episialin expressing cells, which can be shifted towards adhesion by strengthening the integrin-mediated adhesion, or towards anti-adhesion by increasing the level of expression of episialin.

The mucin epiglycanin on TA3/Ha carcinoma cells prevents alpha 6 beta 4-mediated adhesion to laminin and kalinin and E-cadherin-mediated cell-cell interaction.

TA3/Ha murine mammary carcinoma cells grow in suspension, do not adhere to extracellular matrix molecules, but do adhere to hepatocytes and form liver metastases upon intraportal injection. Recently we showed that the integrin alpha 6 beta 4 on the TA3/Ha cells is involved in adhesion to hepatocytes. However, despite high cell surface levels of alpha 6 beta 4, TA3/Ha cells do not adhere to the alpha 6 beta 4 ligands laminin and kalinin. Here we show that this is due to the mucin epiglycanin that is highly expressed on TA3/Ha cells. Some monoclonal antibodies generated against epiglycanin induced capping of most of the epiglycanin molecules. TA3/Ha cells treated with these mAb did adhere to laminin and kalinin, and an epithelial monolayer was formed on kalinin, with alpha 6 beta 4 localized in HD1-containing hemidesmosome-like structures and E-cadherin at the cell-cell contact sites. Similar results were obtained after treatment of TA3/Ha cells with O-sialoglycoprotein endopeptidase which removes all epiglycanin. In addition, the enzyme induced E-cadherin-mediated cell-cell aggregation. Both treatments also enhanced the adhesion to hepatocytes, but given the potent antiadhesive effect of epiglycanin it is remarkable that nontreated TA3/Ha cells adhere to hepatocytes at all. We found that during this interaction, epiglycanin was redistributed. We conclude that epiglycanin can completely prevent both intercellular and matrix adhesion, but that this effect can be overcome in certain intercellular interactions because of the induced redistribution of the mucin.

Cre-loxP-mediated inactivation of the alpha6A integrin splice variant in vivo: evidence for a specific functional role of alpha6A in lymphocyte migration but not in heart development.

Two splice variants of the alpha6 integrin subunit, alpha6A and alpha6B, with different cytoplasmic domains, have previously been described. While alpha6B is expressed throughout the development of the mouse, the expression of alpha6A begins at 8.5 days post coitum and is initially restricted to the myocardium. Later in ontogeny, alpha6A is found in various epithelia and in certain cells of the immune system. In this study, we have investigated the function of alpha6A in vivo by generating knockout mice deficient for this splice variant. The Cre- loxP system of the bacteriophage P1 was used to specifically remove the exon encoding the cytoplasmic domain of alpha6A in embryonic stem cells, and the deletion resulted in the expression of alpha6B in all tissues that normally express alpha6A. We show that alpha6A-/- mice develop normally and are fertile. The substitution of alpha6A by alpha6B does not impair the development and function of the heart, hemidesmosome formation in the epidermis, or keratinocyte migration. Furthermore, T cells differentiated normally in alpha6A-/- mice. However, the substitution of alpha6A by alpha6B leads to a decrease in the migration of lymphocytes through laminin-coated Transwell filters and to a reduction of the number of T cells isolated from the peripheral and mesenteric lymph nodes. Lymphocyte homing to the lymph nodes, which involves various types of integrin-ligand interactions, was not affected in the alpha6A knockout mice, indicating that the reduced number of lymph node cells could not be directly attributed to defects in lymphocyte trafficking. Nevertheless, the expression of alpha6A might be necessary for optimal lymphocyte migration on laminin in certain pathological conditions.

Biochemical characterization and tissue distribution of the A and B variants of the integrin alpha 6 subunit.

Two cytoplasmic variants of the alpha 6 integrin, alpha 6A and alpha 6B, have been identified previously (Hogervorst, F., I. Kuikman, A. G. van Kessel, and A. Sonnenberg. 1991. Eur. J. Biochem. 199:425-433; Cooper, H. M., R. N. Tamura, and V. Quaranta. 1991. J. Cell Biol. 115:843-850). Using synthetic peptides, containing sequences of their cytoplasmic domains, we have produced mAbs specific for either of the variants. These antibodies reacted with a variety of different epithelial tissues. In some tissues (e.g., salivary gland) both variants could be detected while in others only one of the variants was found (e.g., alpha 6A in epidermis and alpha 6B in kidney). Among nonepithelial cells and tissues, perineural fibroblasts and Schwann cells in peripheral nerves and platelets reacted with anti-alpha 6A, while microvascular endothelia reacted with both anti-alpha 6A and anti-alpha 6B. From our immunohistochemical results there is not evidence that combination with beta 1 or beta 4 is restricted to one of the two variants of alpha 6. This was confirmed by immunoprecipitation studies which showed that both beta 1 and beta 4 were coprecipitated by both anti-alpha 6A or anti-alpha 6B antibodies from cells. Also, the distribution of alpha 6A and alpha 6B subunits associated with beta 1 on cells attached to laminin was similar: both were found in focal contacts colocalizing with vinculin. In contrast, the alpha 6A subunit, associated with beta 4 in cultures of a squamous cell carcinoma cell line, was found to codistribute with bullous pemphigoid antigen 230 in hemidesmosomal-like structures. The alpha 6A and alpha 6B variants, immunoprecipitated from various cell lines, exhibited slightly different electrophoretic mobilities. Analysis of the antigens under reducing conditions showed that the mobility of the light chains, but not of the heavy chains, is different. In addition, in some cells the light chains of alpha 6A and alpha 6B, each are of two different sizes. Treatment with N-glycanase showed that these two light chain variants of alpha 6A and alpha 6B are not due to differences in N-linked glycosylation, and may therefore represent alternative proteolytic products of the alpha 6 precursor. We further demonstrate that alpha 6A, but not alpha 6B, is a major target for PMA-induced phosphorylation. Phosphorylated alpha 6A contained phosphoserine and a small amount of phosphotyrosine. There are also two variants of the integrin alpha 3 subunit with different cytoplasmic domains, but in the cell lines examined only alpha 3A could be demonstrated by RT-PCR.(ABSTRACT TRUNCATED AT 400 WORDS)

The tetraspan molecule CD151, a novel constituent of hemidesmosomes, associates with the integrin alpha6beta4 and may regulate the spatial organization of hemidesmosomes.

CD151 is a cell surface protein that belongs to the tetraspan superfamily. It associates with other tetraspan molecules and certain integrins to form large complexes at the cell surface. CD151 is expressed by a variety of epithelia and mesenchymal cells. We demonstrate here that in human skin CD151 is codistributed with alpha3beta1 and alpha6beta4 at the basolateral surface of basal keratinocytes. Immunoelectron microscopy showed that CD151 is concentrated in hemidesmosomes. By immunoprecipitation from transfected K562 cells, we established that CD151 associates with alpha3beta1 and alpha6beta4. In beta4-deficient pyloric atresia associated with junctional epidermolysis bullosa (PA-JEB) keratinocytes, CD151 and alpha3beta1 are clustered together at the basal cell surface in association with patches of laminin-5. Focal adhesions are present at the periphery of these clusters, connected with actin filaments, and they contain both CD151 and alpha3beta1. Transient transfection studies of PA-JEB cells with beta4 revealed that the integrin alpha6beta4 becomes incorporated into the alpha3beta1-CD151 clusters where it induces the formation of hemidesmosomes. As a result, the amount of alpha3beta1 in the clusters diminishes and the protein becomes restricted to the peripheral focal adhesions. Furthermore, CD151 becomes predominantly associated with alpha6beta4 in hemidesmosomes, whereas its codistribution with alpha3beta1 in focal adhesions becomes partial. The localization of alpha6beta4 in the pre-hemidesmosomal clusters is accompanied by a strong upregulation of CD151, which is at least partly due to increased cell surface expression. Using beta4 chimeras containing the extracellular and transmembrane domain of the IL-2 receptor and the cytoplasmic domain of beta4, we found that for recruitment of CD151 into hemidesmosomes, the beta4 subunit must be associated with alpha6, confirming that integrins associate with tetraspans via their alpha subunits. CD151 is the only tetraspan identified in hemidesmosomal structures. Others, such as CD9 and CD81, remain diffusely distributed at the cell surface. In conclusion, we show that CD151 is a major component of (pre)-hemidesmosomal structures and that its recruitment into hemidesmosomes is regulated by the integrin alpha6beta4. We suggest that CD151 plays a role in the formation and stability of hemidesmosomes by providing a framework for the spatial organization of the different hemidesmosomal components.