Basement Membrane Adhesion Research Articles

P16 Fibroblast signalling crosstalk drives junctional epidermolysis bullosa disease progression

Abstract Introduction and aims Junctional epidermolysis bullosa (JEB) is a devastating hereditary skin disorder characterized by severe fragility of skin and mucus membrane, emerges as blistering with little or no trauma. The major classification of JEB includes JEB intermediate and JEB severe; the latter is the most common subtype, which is the result of loss of function mutations in the skin basement membrane protein laminin-332. Lacking this protein impairs basement membrane adhesion of epidermal keratinocytes and leads to deregulated epidermal–dermal crosstalk. The skin dermis is composed of multiple fibroblast subpopulations with different functions in skin homeostasis and repair; however, their specific involvement in skin diseases is largely unknown. Here, we investigate how fibroblast subpopulations, namely papillary and reticular fibroblasts, impact JEB disease progression and skin regeneration. Methods Three-dimensional (3D) organotypic (OT) combining healthy (control) and diseased (JEB) keratinocytes cells with distinct fibroblast subpopulations from different anatomical sites were developed and analysed by immunohistochemistry and RNA-Seq. Results JEB OTs with papillary fibroblasts showed increased epidermal growth, invagination of basal layer and deregulated differentiation compared with reticular fibroblasts. RNA-Seq analysis revealed altered activity in key signalling including immune, cytokine, gap junction, mitogen-activated protein kinase and remodelling of keratinization and ECM proteins, particularly in JEB OT cultured with papillary fibroblast. JEB OTs and cells also demonstrated variation in cell proliferation and activation of transforming growth factor-β1 and immune cell signalling. Currently, we are targeting identified signalling pathways in our two-dimensional and 3D cell culture models and identifying druggable targets, before testing selected candidates in our JEB mouse disease model for further preclinical validation. Conclusions By dissecting the pathological signalling interactions between keratinocytes and distinct fibroblast subpopulations, our goal is to identify new therapeutic target to improve the skin health and regeneration in patients with JEB.

417-P: Functional Characterization of a Rare, Pathogenic Missense Variant Implicates FRAS1 as a Novel Gene for Risk of Progression to End-Stage Kidney Disease in Diabetes

Progressive renal decline is the central manifestation of diabetic nephropathy (DN) that ultimately leads to end-stage kidney disease (ESKD). Leveraging an innovative family-based approach that integrates electronic medical record data, a unique population-based genealogy resource, and next-generation sequencing, we recently identified a rare missense variant (p.R2516C, allele frequency=0.00002) in FRAS1, which encodes an extracellular matrix protein that regulates epidermal basement membrane adhesion and mediates glomerular integrity, in a family with diabetes and ESKD. While this rare missense variant is predicted to be deleterious (CADD score=32), whether this impacts FRAS1 function is unknown; here, we set out to assess the functional impact of this variant. To do so, we used CRISPR-Cas9 gene editing technology to generate 3 novel HEK293 cell lines (2 FRAS1 knock-in cell lines (a synonymous p.H2520H control cell line and a missense p.R2516C variant cell line) and a FRAS1 knock-out cell line) and performed in vitro cell migration and cell adhesion assays. Relative to the control cell line, both the FRAS1 knock-out cell line and the missense variant cell line displayed significantly inhibited cell migration (p = 1.54X10-13 and p = 5.05X10-5, respectively). Similarly, cell adhesion was significantly inhibited in cells in which FRAS1 was knocked out and in those carrying the p.R2516C variant compared to the control cell line (p = 6.72X10-9 and p = 6.63X10-6, respectively). Together, these data suggest that the FRAS1 p.R2516C variant negatively impacts FRAS1 function and further implicates this gene as having a potential role in the progression of ESKD in individuals with diabetes. Disclosure T.Chen: None. M.B.Neeley: None. J.M.Lazaro-guevara: None. M.Pezzolesi: None. C.A.Simeone: None. A.Rodan: None. W.L.Holland: Research Support; SymbioCellTech, LLC. M.G.Pezzolesi: Research Support; Renalytix. Funding National Institutes of Health (R01DK128641)

Matrix metalloproteinase-induced cervical extracellular matrix remodelling in pregnancy and cervical cancer.

Graphical abstract The phenomenal extracellular matrix (ECM) remodelling of the cervix that precedes the myometrial contraction of labour at term or preterm appears to share some common mechanisms with the occurrence, growth, invasion and metastasis of cervical carcinoma. Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that are pivotal to the complex extracellular tissue modulation that includes degradation, remodelling and exchange of ECM components, which contribute to homeostasis under normal physiological conditions such as cervical remodelling during pregnancy and puerperium. However, in cancer such as that of the uterine cervix, this extensive network of extracellular tissue modulation is altered leading to disrupted cell–cell and cell–basement membrane adhesion, abnormal tissue growth, neovascularization and metastasis that disrupt homeostasis. Cervical ECM remodelling during pregnancy and puerperium could be a physiological albeit benign neoplasm. In this review, we examined the pathophysiologic differences and similarities in the role of MMPs in cervical remodelling and cervical carcinoma.Lay summaryDuring pregnancy and childbirth, the cervix, which is the barrel-shaped lower portion of the womb that connects to the vagina, gradually softens, shortens and opens to allow birth of the baby. This process requires structural and biochemical changes in the cervix that are stimulated by enzymes known as matrix metalloproteinases. Interestingly, these enzymes also affect the structural and biochemical framework of the cervix during cervical cancer, although cervical cancers usually occur after infection by human papillomavirus. This review is intended to identify and explain the similarities and differences between the structural and chemical changes in the cervix during pregnancy and childbirth and the changes seen in cervical cancer.

Cell-Matrix Interactions Contribute to Barrier Function in Human Colon Organoids.

The importance of cell-matrix adhesion to barrier control in the colon is unclear. The goals of the present study were to: (i) determine if disruption of colon epithelial cell interactions with the extracellular matrix alters permeability control measurement and (ii) determine if increasing the elaboration of protein components of cell-matrix adhesion complexes can mitigate the effects of cell-matrix disruption. Human colon organoids were interrogated for transepithelial electrical resistance (TEER) under control conditions and in the presence of Aquamin®, a multi-mineral product. A function-blocking antibody directed at the C-terminal region of the laminin α chain was used in parallel. The effects of Aquamin® on cell-matrix adhesion protein expression were determined in a proteomic screen and by Western blotting. Aquamin® increased the expression of multiple basement membrane, hemidesmosomal and focal adhesion proteins as well as keratin 8 and 18. TEER values were higher in the presence of Aquamin® than they were under control conditions. The blocking antibody reduced TEER values under both conditions but was most effective in the absence of Aquamin®, where expression of cell-matrix adhesion proteins was lower to begin with. These findings provide evidence that cell-matrix interactions contribute to barrier control in the colon.

Integrin: Basement membrane adhesion by corneal epithelial and endothelial cells

Integrins mediate adhesion of cells to substrates and maintain tissue integrity by facilitating mechanotransduction between cells, the extracellular matrix, and gene expression in the nucleus. Changes in integrin expression in corneal epithelial cells and corneal endothelial cells impacts their adhesion to the epithelial basement membrane (EpBM) and Descemet's membrane, respectively. Integrins also play roles in assembly of basement membranes by both activating TGFβ1 and other growth factors. Over the past two decades, this knowledge has been translated into methods to grow corneal epithelial and endothelial cells in vitro for transplantation in the clinic thereby transforming clinical practice and quality of life for patients. Current knowledge on the expression and function of the integrins that mediate adhesion to the basement membrane expressed by corneal epithelial and endothelial cells in health and disease is summarized. This is the first review to discuss similarities and differences in the integrins expressed by both cell types.

Esophageal extracellular matrix hydrogel mitigates metaplastic change in a dog model of Barrett's esophagus.

Chronic inflammatory gastric reflux alters the esophageal microenvironment and induces metaplastic transformation of the epithelium, a precancerous condition termed Barrett's esophagus (BE). The microenvironmental niche, which includes the extracellular matrix (ECM), substantially influences cell phenotype. ECM harvested from normal porcine esophageal mucosa (eECM) was formulated as a mucoadhesive hydrogel, and shown to largely retain basement membrane and matrix-cell adhesion proteins. Dogs with BE were treated orally with eECM hydrogel and omeprazole (n = 6) or omeprazole alone (n = 2) for 30 days. eECM treatment resolved esophagitis, reverted metaplasia to a normal, squamous epithelium in four of six animals, and downregulated the pro-inflammatory tumor necrosis factor-α+ cell infiltrate compared to control animals. The metaplastic tissue in control animals (n = 2) did not regress. The results suggest that in vivo alteration of the microenvironment with a site-appropriate, mucoadhesive ECM hydrogel can mitigate the inflammatory and metaplastic response in a dog model of BE.

Hypertensive nephropathy. Moving from classic to emerging pathogenetic mechanisms.

Hypertensive kidney disease classically entails nephroangiosclerosis and hyalinosis with glomerular damage. However, in recent years, several evidences showed that high blood pressure also injures tubular cells, inducing epithelial-to-mesenchymal transition and tubulointerstitial fibrosis. Recently investigated mechanisms are also podocyte effacement and loss, which lead to denudation of the glomerular basement membrane and focal adhesion of the tufts to the Bowman's capsule, with reduced filtration and scars. Starting from the classic concept of nephroangiosclerosis, this review examines the recently emerged knowledge of new biochemical and molecular mechanisms underlying the kidney damage in hypertension and discusses how viable podocytes or podocyte-deriving proteins are promising tools for early diagnosis of renal remodelling in hypertension.

Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins.

Refined methods for maintaining specific functions of isolated hepatocytes under xeno-free and chemical defined conditions is of great importance for the development of hepatocyte research and regenerative therapy. Laminins, a large family of heterotrimeric basement membrane adhesion proteins, are highly cell and tissue type specific components of the extracellular matrix and strongly influence the behavior and function of associated cells and/or tissues. However, detailed biological functions of many laminin isoforms are still to be evaluated. In this study, we determined the distribution of laminin isoforms in human liver tissue and isolated primary human hepatocytes by western blot analysis, and investigated the efficacy of different human recombinant laminin isoforms on hepatic functions during culture. Protein expressions of laminin-chain α2, α3, α4, β1, β3, γ1, and γ2 were detected in both isolated human hepatocytes and liver tissue. No α1 and α5 expression could be detected in liver tissue or hepatocytes. Hepatocytes were isolated from five different individual livers, and cultured on human recombinant laminin isoforms -111, -211, -221, -332, -411, -421, -511, and -521 (Biolamina AB), matrigel (extracted from Engelbreth-Holm-Swarm sarcoma), or collagen type IV (Collagen). Hepatocytes cultured on laminin showed characteristic hexagonal shape in a flat cell monolayer. Viability, double stranded DNA concentration, and Ki67 expression for hepatocytes cultured for six days on laminin were comparable to those cultured on EHS and Collagen. Hepatocytes cultured on laminin also displayed production of human albumin, alpha-1-antitrypsin, bile acids, and gene expression of liver-enriched factors, such as hepatocyte nuclear factor 4 alpha, glucose-6-phosphate, cytochrome P450 3A4, and multidrug resistance-associated protein 2. We conclude that all forms of human recombinant laminin tested maintain cell viability and liver-specific functions of primary human hepatocytes, and that recombinant laminin is a promising xeno-free and chemical defined strategy for preservation of hepatocyte specific function in vitro.

Effect of tetramethylpyrazine on adhesion and invasion ability of cell PG in hypoxic environment

Objective To investigate the Effect of Tetramethylpyrazine on adhesion and invasion ability of Cell PG in Hypoxic Environment. Methods The revival cell PG was divided into three dosages（high, medium and low）group of Tetramethylpyrazine, DDP group and negative control group. All of five groups were cultured separately in the normal and hypoxic environment. The adhesion ability of Cell PG were detected by MTT. The invasion ability of Cell PG were detected by Transwell chamber. Results Observed on the reconstruction of the basement membrane adhesion in PG cells by MTT, in normoxic environment the mean absorbance of the TMP high, medium and low concentrations, cisplatin group, control group were （0.556±0.040）,（0.519±0.056）, （0.517±0.061）,（0.333±0.057）,（0.300±0.066）, P〈0.01. In hypoxic environment the mean absorbance of the TMP high , medium and low concentrations, cisplatin group, control group were （0.48±0.032）,（0.452±0.059）,（0.274±0.093）,（0.464±0.099）,（0.155±0.042）, P〈0.01. Promotion of cell adhesion with Tetramethylpyrazine of high, medium and low concentration group and DDP group in hypoxic environment is greater than in normoxic environment（P〈0.01）. Observed the reconstruction of the invasion of basement membrane of PG cells by Transwell chamber, in normoxic environment the invasive cells of the TMP high, medium and low concentrations, cisplatin group, control group were ：（28.00±17.00）,（91.67±30.37）,（105.67±39.58）,（85.00±8.19）,（152.33±24.71）,＆amp;nbsp;P<0.05. In hypoxic environment the invasive cells of the TMP high , medium and low concentrations , cisplatin group, control group were （20.33±15.31）, （22.33±17.62）,（56.00±11.53）,（57.67±35.95）, （76.33±18.01）, P<0.05. The invasive cells of normoxic control group was significantly more than hypoxic control group（P<0.05）. Conclusion In hypoxic environment , adhesion and invasion ability of Cell PG significantly decreased compared with normoxic environment ; In normoxia and hypoxia environment, Tetramethylpyrazine and cisplatin could promote the adhesion ability and inhibit the invasion ability of PG cells. Role in promoting of Tetramethylpyrazine on cell adhesion of PG cells was stronger in hypoxic environment. Key words: PG cell; Tetramethylpyrazine; Adhesion; Invasion

Tumor growth in complex, evolving microenvironmental geometries: A diffuse domain approach

We develop a mathematical model of tumor growth in complex, dynamic microenvironments with active, deformable membranes. Using a diffuse domain approach, the complex domain is captured implicitly using an auxiliary function and the governing equations are appropriately modified, extended and solved in a larger, regular domain. The diffuse domain method enables us to develop an efficient numerical implementation that does not depend on the space dimension or the microenvironmental geometry. We model homotypic cell-cell adhesion and heterotypic cell-basement membrane (BM) adhesion with the latter being implemented via a membrane energy that models cell-BM interactions. We incorporate simple models of elastic forces and the degradation of the BM and ECM by tumor-secreted matrix degrading enzymes. We investigate tumor progression and BM response as a function of cell-BM adhesion and the stiffness of the BM. We find tumor sizes tend to be positively correlated with cell-BM adhesion since increasing cell-BM adhesion results in thinner, more elongated tumors. Prior to invasion of the tumor into the stroma, we find a negative correlation between tumor size and BM stiffness as the elastic restoring forces tend to inhibit tumor growth. In order to model tumor invasion of the stroma, we find it necessary to downregulate cell-BM adhesiveness, which is consistent with experimental observations. A stiff BM promotes invasiveness because at early stages the opening in the BM created by MDE degradation from tumor cells tends to be narrower when the BM is stiffer. This requires invading cells to squeeze through the narrow opening and thus promotes fragmentation that then leads to enhanced growth and invasion. In three dimensions, the opening in the BM was found to increase in size even when the BM is stiff because of pressure induced by growing tumor clusters. A larger opening in the BM can increase the potential for further invasiveness by increasing the possibility that additional tumor cells could invade the stroma.

Peroxidasin is essential for eye development in the mouse.

Mutations in Peroxidasin (PXDN) cause severe inherited eye disorders in humans, such as congenital cataract, corneal opacity and developmental glaucoma. The role of peroxidasin during eye development is poorly understood. Here, we describe the first Pxdn mouse mutant which was induced by ENU (N-ethyl-N-nitrosourea) and led to a recessive phenotype. Sequence analysis of cDNA revealed a T3816A mutation resulting in a premature stop codon (Cys1272X) in the peroxidase domain. This mutation causes severe anterior segment dysgenesis and microphthalmia resembling the manifestations in patients with PXDN mutations. The proliferation and differentiation of the lens is disrupted in association with aberrant expression of transcription factor genes (Pax6 and Foxe3) in mutant eyes. Additionally, Pxdn is involved in the consolidation of the basement membrane and lens epithelium adhesion in the ocular lens. Lens material including γ-crystallin is extruded into the anterior and posterior chamber due to local loss of structural integrity of the lens capsule as a secondary damage to the anterior segment development leading to congenital ocular inflammation. Moreover, Pxdn mutants exhibited an early-onset glaucoma and progressive retinal dysgenesis. Transcriptome profiling revealed that peroxidasin affects the transcription of developmental and eye disease-related genes at early eye development. These findings suggest that peroxidasin is necessary for cell proliferation and differentiation and for basement membrane consolidation during eye development. Our studies provide pathogenic mechanisms of PXDN mutation-induced congenital eye diseases.

Long-term follow-up of patients with Herlitz-type junctional epidermolysis bullosa

Junctional epidermolysis bullosa, type Herlitz (JEB-H) is a rare, autosomal recessive disease caused by absence of the epidermal basement membrane adhesion protein laminin-332. It is characterized by extensive and devastating blistering of the skin and mucous membranes, leading to death in early childhood. To present the results of the long-term follow-up of a cohort of patients with JEB-H, and to provide guidelines for prognosis, treatment and care. All patients with JEB-H included in the Dutch Epidermolysis Bullosa (EB) Registry between 1988 and 2011 were followed longitudinally by our EB team. Diagnosis was established using immunofluorescence antigen mapping, electron microscopy and DNA analysis. In total, we included 22 patients with JEB-H over a 23-year period. Their average age at death was 5.8 months (range 0.5-32.6 months). The causes of death were, in order of frequency: failure to thrive, respiratory failure, pneumonia, dehydration, anaemia, sepsis and euthanasia. The pattern of initial weight gain was a predictor of lifespan in these patients. Invasive treatments to extend life did not promote survival in our patients. It is important to diagnose JEB-H as soon as possible after birth so that the management can be shifted from life-saving to comfort care. The palliative end-of-life care can take place in hospital, but is also safe in the home setting. Suffering in patients with JEB-H can become so unbearable that in some patients who do not respond to adequate analgesic and sedative treatment, newborn euthanasia, performed according to the Groningen protocol, is legally permitted in the Netherlands.

Effect of Phoneutria nigriventer Venom on the Expression of Junctional Protein and P-gp Efflux Pump Function in the Blood–Brain Barrier

Phoneutria nigriventer spider venom (PNV) contains Ca(2+), K(+) and Na(+) channel-acting peptides that affect neurotransmitter release and causes excitotoxicity in PNS and CNS. It has been demonstrated that PNV causes blood-brain barrier (BBB) breakdown of hippocampal microvessels time-dependently through enhanced microtubule-mediated vesicular transport. Herein, it is hypothesized that PNV can cause BBB breakdown in the hippocampus and cerebellum time-dependently through other molecular mechanisms. The BBB integrity was assessed through the analysis of expression of Poly-glycoprotein (P-gp) efflux transporter protein, laminin from basement membrane and endothelial tight junctional and adhesion junctional (TJ/AJ) proteins. Phosphatase and tensin homolog (PTEN) and protein phosphatase 2A (PP2A) expression, which are known to have a role in the phosphorylation of junctional proteins and BBB opening, were also investigated. Astrocytes P-gp activity was determined by flow cytometry. The study demonstrated temporary decreased expression of laminin, TJ and AJ proteins (ZO1//occludin//claudin-5//beta-catenin) and P-gp (more prominently in hippocampus), which was completely or partially resolved between 2 and 5h (and more quickly for cerebellum). PNV inhibited P-gp activity in astrocytes. PP2A phosphorylation, which inhibits the enzyme activity, was increased in both regions (15-45min); however the phosphorylation level returned to baseline after 2h. In conclusion, PNV disrupts paracellular transport in the BBB and possesses substrates for the active P-gp efflux transporter located in the BBB complex. Further studies into cellular mechanisms of astrocyte/endothelial interactions, using PNV as tool, may identify how astrocytes regulate the BBB, a characteristic that may be useful for the temporary opening of the BBB.

Mutations in AEC syndrome skin reveal a role for p63 in basement membrane adhesion, skin barrier integrity and hair follicle biology

AEC (ankyloblepharon-ectodermal defects-clefting) syndrome is an autosomal dominant ectodermal dysplasia disorder caused by mutations in the transcription factor p63. Clinically, the skin is dry and often fragile; other features can include partial eyelid fusion (ankyloblepharon), hypodontia, orofacial clefting, sparse hair or alopecia, and nail dystrophy. To investigate how p63 gene mutations affect gene and protein expression in AEC syndrome skin. We performed microarray analysis on samples of intact and eroded AEC syndrome skin compared with control skin. Changes were verified by quantitative real-time reverse transcription-polymerase chain reaction and, for basal keratinocyte-associated genes, by immunohistochemistry and analysis of microdissected skin. We identified significant upregulation of six genes and downregulation of 69 genes in AEC syndrome skin, with the main changes in genes implicated in epidermal adhesion, skin barrier formation and hair follicle biology. There was reduced expression of genes encoding the basement membrane proteins FRAS1 and collagen VII, as well as the skin barrier-associated small proline-rich proteins 1A and 4, late cornified envelope protein 5A, hornerin, and lipid transporters including ALOX15B. Reduced expression of the hair-associated keratins 25, 27, 31, 33B, 34, 35, 81 and 85 was also noted. We also confirmed similar alterations in gene expression for 26 of the 75 genes in eroded AEC scalp skin. This study identifies specific changes in skin structural biology and signalling pathways that result from mutant p63 and provides new molecular insight into the AEC syndrome phenotype.

Dapsone in the Management of Autoimmune Bullous Diseases

Dapsone is used in the treatment of autoimmune bullous diseases (AIBD), a group of disorders resulting from autoimmunity directed against basement membrane and/or intercellular adhesion molecules on cutaneous and mucosal surfaces. This review summarizes the limited published data evaluating dapsone as a therapy for AIBD.

Integrative multicellular biological modeling: a case study of 3D epidermal development using GPU algorithms

BackgroundSimulation of sophisticated biological models requires considerable computational power. These models typically integrate together numerous biological phenomena such as spatially-explicit heterogeneous cells, cell-cell interactions, cell-environment interactions and intracellular gene networks. The recent advent of programming for graphical processing units (GPU) opens up the possibility of developing more integrative, detailed and predictive biological models while at the same time decreasing the computational cost to simulate those models.ResultsWe construct a 3D model of epidermal development and provide a set of GPU algorithms that executes significantly faster than sequential central processing unit (CPU) code. We provide a parallel implementation of the subcellular element method for individual cells residing in a lattice-free spatial environment. Each cell in our epidermal model includes an internal gene network, which integrates cellular interaction of Notch signaling together with environmental interaction of basement membrane adhesion, to specify cellular state and behaviors such as growth and division. We take a pedagogical approach to describing how modeling methods are efficiently implemented on the GPU including memory layout of data structures and functional decomposition. We discuss various programmatic issues and provide a set of design guidelines for GPU programming that are instructive to avoid common pitfalls as well as to extract performance from the GPU architecture.ConclusionsWe demonstrate that GPU algorithms represent a significant technological advance for the simulation of complex biological models. We further demonstrate with our epidermal model that the integration of multiple complex modeling methods for heterogeneous multicellular biological processes is both feasible and computationally tractable using this new technology. We hope that the provided algorithms and source code will be a starting point for modelers to develop their own GPU implementations, and encourage others to implement their modeling methods on the GPU and to make that code available to the wider community.

Caenorhabditis elegansTeneurin,ten-1, Is Required for Gonadal and Pharyngeal Basement Membrane Integrity and Acts Redundantly with Integrinina-1and Dystroglycandgn-1

The Caenorhabditis elegans teneurin ortholog, ten-1, plays an important role in gonad and pharynx development. We found that lack of TEN-1 does not affect germline proliferation but leads to local basement membrane deficiency and early gonad disruption. Teneurin is expressed in the somatic precursor cells of the gonad that appear to be crucial for gonad epithelialization and basement membrane integrity. Ten-1 null mutants also arrest as L1 larvae with malformed pharynges and disorganized pharyngeal basement membranes. The pleiotropic phenotype of ten-1 mutant worms is similar to defects found in basement membrane receptor mutants ina-1 and dgn-1 as well as in the mutants of the extracellular matrix component laminin, epi-1. We show that the ten-1 mutation is synthetic lethal with mutations of genes encoding basement membrane components and receptors due to pharyngeal or hypodermal defects. This indicates that TEN-1 could act redundantly with integrin INA-1, dystroglycan DGN-1, and laminin EPI-1 in C. elegans development. Moreover, ten-1 deletion sensitizes worms to loss of nidogen nid-1 causing a pharynx unattached phenotype in ten-1;nid-1 double mutants. We conclude that TEN-1 is important for basement membrane maintenance and/or adhesion in particular organs and affects the function of somatic gonad precursor cells.

Proteolytic Processing of the Laminin α3 G Domain Mediates Assembly of Hemidesmosomes but Has No Role on Keratinocyte Migration

Laminin-5 (Lm5), the major adhesion ligand of basal epithelial cells, undergoes complex extracellular proteolytic processing that influences cell adhesion and migration. In tumor cell lines, the proteolytic truncation of the C-terminal G domain of the Lm alpha3 chain induces assembly of hemidesmosomes and downregulates cell migration. To define the biological functions of the alpha3 G domain processing in physiological conditions, we have expressed a series of mutant alpha3 complementary DNA in human primary alpha3-null keratinocytes immortalized by human papillomavirus E6E7 (HKalpha3 cells). Using monolayer and organotypic cell cultures we show that: (1) the hinge region between subdomains G3 and G4 carries the proteolytic cleavage sites; (2) nucleation of the hemidesmosomal proteins is independent of the proteolytic maturation of the alpha3 G domain, whereas formation of mature hemidesmosomes relies on proteolytic cleavage of alpha3; and (3) the proteolytic processing plays no role in cell migration, which suggests that nucleation of hemidesmosomal structures in culture does not reflect the migratory potential of the epithelial cells. Our results also demonstrate that HKalpha3 cells are a unique model system, which will be useful to dissect the functions and molecular interactions of Lm5.

Subepidermal blistering disease with autoantibodies to both the p200 autoantigen and the α3 chain of laminin 5

Anti-p200 pemphigoid and anti-laminin 5 mucous membrane pemphigoid are two distinct autoimmune blistering skin diseases. Patients with anti-p200 pemphigoid demonstrate circulating autoantibodies to an unknown 200-kd acidic noncollagenous glycoprotein of the lower lamina lucida, whereas anti-laminin 5 mucous membrane pemphigoid is characterized by an autoimmune response against the major basement membrane adhesion molecule laminin 5. In this report, we describe a patient who developed a recurrent nonscarring blistering eruption, affecting both skin and mucous membranes. Immunoblotting of dermal extracts and extracellular matrix of cultured keratinocytes revealed circulating autoantibodies directed to the p200 antigen and the alpha3 chain of laminin 5, respectively. The unusual clinical and immunologic profile of this case suggests that molecular specificity of circulating autoantibodies affects the clinical presentation of autoimmune subepidermal blistering skin diseases.

The extracellular matrix gene Frem1 is essential for the normal adhesion of the embryonic epidermis.

Fraser syndrome is a rare recessive disorder characterized by cryptophthalmos, syndactyly, renal defects, and a range of other developmental abnormalities. Because of their extensive phenotypic overlap, the mouse blebbing mutants have been considered models of this disorder, and the recent isolation of mutations in Fras1 in both the blebbed mouse and human Fraser patients confirms this hypothesis. Here we report the identification of mutations in an extracellular matrix gene Fras1-related extracellular matrix gene 1 (Frem1) in both the classic head blebs mutant and in an N-ethyl-N-nitrosourea-induced allele. We show that inactivation of the gene results in the formation of in utero epidermal blisters beneath the lamina densa of the basement membrane and also in renal agenesis. Frem1 is expressed widely in the developing embryo in regions of epithelial/mesenchymal interaction and epidermal remodeling. Furthermore, Frem1 appears to act as a dermal mediator of basement membrane adhesion, apparently independently of the other known "blebs" proteins Fras1 and Grip1. Unlike both Fras1 and Grip1 mutants, collagen VI and Fras1 deposition in the basement membrane is normal, indicating that the protein plays an independent role in epidermal differentiation and is required for epidermal adhesion during embryonic development.