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Basement membranes (BMs) are structured regions of the extracellular matrix that provide multiple functions including physical support and acting as a barrier, as a repository for nutrients and growth factors, and as biophysical signalling hubs. At the core of all BMs is the laminin (LM) family of proteins. These large heterotrimeric glycoproteins are essential for tissue integrity, and differences between LM family members represent a key nexus in dictating context and tissue-specific functions. These variations reflect genetic diversity within the family, which allows for multiple structurally and functionally distinct heterotrimers to be produced, each with different architectures and affinities for other matrix proteins and cell surface receptors. The ratios of these LM isoforms also influence the biophysical properties of a BM owing to differences in their relative ability to form polymers or networks. Intriguingly, the LM superfamily is further diversified through the related netrin family of proteins and through alternative splicing leading to the generation of non-LM short proteins known as the laminin N-terminus (LaNt) domain proteins. Both the netrins and LaNt proteins contain structural domains involved in LM-to-LM interaction and network assembly. Emerging findings indicate that one netrin and at least one LaNt protein can potently influence the structure and function of BMs, disrupting the networks, changing physical properties, and thereby influencing tissue function. These findings are altering the way that we think about LM polymerisation and, in the case of the LaNt proteins, suggest a hitherto unappreciated form of LM self-regulation.

Laminins (LMs) are essential components of all basement membranes where they regulate an extensive array of tissue functions. Alternative splicing from the laminin α3 gene produces a non-laminin but netrin-like protein, Laminin N terminus α31 (LaNt α31). LaNt α31 is widely expressed in intact tissue and is upregulated in epithelial cancers and during wound healing. In vitro functional studies have shown that LaNt α31 can influence numerous aspects of epithelial cell behavior via modifying matrix organization, suggesting a new model of laminin auto-regulation. However, the function of this protein has not been established in vivo. Here, a mouse transgenic line was generated using the ubiquitin C promoter to drive inducible expression of LaNt α31. When expression was induced at embryonic day 15.5, LaNt α31 transgenic animals were not viable at birth, exhibiting localized regions of erythema. Histologically, the most striking defect was widespread evidence of extravascular bleeding across multiple tissues. Additionally, LaNt α31 transgene expressing animals exhibited kidney epithelial detachment, tubular dilation, disruption of the epidermal basal cell layer and of the hair follicle outer root sheath, and ~50% reduction of cell numbers in the liver, associated with depletion of hematopoietic erythrocytic foci. These findings provide the first in vivo evidence that LaNt α31 can influence tissue morphogenesis.

Effective suturing remains key to achieving successful outcomes in corneal surgery, especially anterior lamellar keratoplasty and full thickness transplantation. Limitations in the technique may result in complications such as wound leak, infection, or high astigmatism post corneal graft. By using a systematic approach, this study reviews articles and conducts content analysis based on update 2020 PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses criteria). The aim of this paper is to summarize the state of the art of corneal suturing techniques for every type of corneal transplant and patient age and also their outcomes regarding astigmatism and complications. Future developments for corneal transplantation will be also discussed. This is important because especially the young surgeon must have knowledge of the implications of every suture performed in order to achieve consistent and predictable post-operative outcomes and also be aware of all the possible complications.

As the desire and popularity of a tanned appearance continues, the social effects of UV-free tanning are becoming more important. Dihydroxyacetone (DHA) has seen extensive use as the main tanning agent in sunless tanners. The DHA-induced tan is a result of brown melanoidins formed by a non-enzymatic Maillard reaction between DHA and amino acid species found in the stratum corneum. DHA, thereby, provides a safer route to a tanned appearance compared with exposure to ultraviolet radiation. However, DHA is a highly reactive molecule, posing a multitude of challenges for potential product formulations. With their increased use, the safety considerations of topically applied DHA tanners have been investigated. Many different vehicles have been used for topical delivery of DHA, and they are becoming increasingly multifunctional. This review provides a holistic overview of dihydroxyacetone sunless tanning products.

This study determined the effectiveness of three deidentification methods: use of a) a black box to obscure facial landmarks, b) a letterbox view to display restricted facial landmarks and c) a half letterbox view. Facial images of well-known celebrities were used to create a series of decreasingly deidentified images and displayed to participants in a structured interview session. 55.5% were recognised when all facial features were covered using a black box, leaving only the hair and neck exposed. The letterbox view proved more effective, reaching over 50% recognition only once the periorbital region, eyebrows, and forehead were visible. The half letterbox was the most effective, requiring the nose to be revealed before recognition reached over 50%, and should be the option of choice where appropriate. These findings provide valuable information for informed consent discussions, and we recommend consent to publish forms should stipulate the deidentification method that will be used.

Purpose To determine if sex is associated with corneal epithelial wound healing time in patients with persistent corneal epithelial defects (PCEDs). Methods Retrospective case series on patients with PCED from November 2014 to January 2019. Records of 127 patients with diagnosis of PCED were reviewed. Patients with an epithelial defect that lasted more than two weeks in the absence of an active corneal infection were included. Main outcome was corneal epithelial wound healing time. Results 55 patients (29 males) with a mean age of 65.3 ± 16.5 years were included. No difference was found between female and male patients in terms of risk factors, age, treatment strategies or intervals between visits (median of 15 days in females and 12 days in males; p = 0.24). Median duration of the PCED was 51 days (IQR 32-130), with a median number of 5 clinical visits (IQR 4-8). Female patients had significantly longer healing times (p = 0.004) and a corresponding increase in the number of clinical visits (median of 7 visits vs. 5 clinical visits in males, p = 0.012). Conclusion Results from this study suggest female patients with PCED might have a longer corneal epithelial wound healing duration and may therefore require earlier intervention.

Laminin N-terminus α31 (LaNt α31) is an alternative splice isoform derived from the laminin α3 gene. The LaNt α31 protein is enriched around the terminal duct lobular units in normal breast tissue. In the skin and cornea the protein influences epithelial cell migration and tissue remodelling. However, LaNt α31 has never been investigated in a tumour environment. Here we analysed LaNt α31 in invasive ductal carcinoma and determined its contribution to breast carcinoma invasion. LaNt α31 expression and distribution were analysed by immunohistochemistry in human breast tissue biopsy sections and tissue microarrays covering 232 breast cancer samples. This analysis revealed LaNt α31 to be upregulated in 56% of invasive ductal carcinoma specimens compared with matched normal tissue, and further increased in nodal metastasis compared with the tumour mass in 45% of samples. 65.8% of triple negative cases displayed medium to high LaNt α31 expression. To study LaNt α31 function, an adenoviral system was used to induce expression in MCF-7 and MDA-MB-231 cells. 2D cell migration and invasion into collagen hydrogels were not significantly different between LaNt α31 overexpressing cells and control treated cells. However, LaNt α31 overexpression reduced the proliferation rate of MCF-7 and MDA-MB-231 cells. Moreover, LaNt α31 overexpressing MDA-MB-231 cells displayed a striking change in their mode of invasion into laminin-containing Matrigel; changing from multicellular streaming to individual cellular-invasion. In agreement with these results, 66.7% of the tumours with the highest LaNt α31 expression were non-cohesive. Together these findings indicate that breast cancer-associated changes in LaNt α31 expression could contribute to the processes involved in tumour invasion and may represent a new therapeutic target.

The conjunctiva can be damaged by numerous diseases with scarring, loss of tissue and dysfunction. Depending on extent of damage, restoration of function may require a conjunctival graft. A wide variety of biological and synthetic substrates have been tested in the search for optimal conditions for ex vivo culture of conjunctival epithelial cells as a route toward tissue grafts. Each substrate has specific advantages but also disadvantages related to their unique physical and biological characteristics, and identification and development of an improved substrate remains a priority. To achieve the goal of mimicking and restoring a biological material, requires information from the material. Specifically, extracellular matrix (ECM) derived from conjunctival tissue. Knowledge of the composition and structure of native ECM and identifying contributions of individual components to its function would enable using or mimicking those components to develop improved biological substrates. ECM is comprised of two components: basement membrane secreted predominantly by epithelial cells containing laminins and type IV collagens, which directly support epithelial and goblet cell adhesion differentiation and growth and, interstitial matrix secreted by fibroblasts in lamina propria, which provides mechanical and structural support. This review presents current knowledge on anatomy, composition of conjunctival ECM and related conjunctival disorders. Requirements of potential substrates for conjunctival tissue engineering and transplantation are discussed. Biological and synthetic substrates and their components are described in an accompanying review.

The conjunctiva is the largest component of the ocular surface. It can be damaged by various pathological processes leading to scarring, loss of tissue and dysfunction. Depending on the amount of damage, restoration of function may require a conjunctival graft. Numerous studies have investigated biological and synthetic substrates in the search for optimal conditions for the ex vivo culture of conjunctival epithelial cells that can be used as tissue grafts for transplantation. These substrates have advantages and disadvantages that are specific to the characteristics of each material; the development of an improved material remains a priority. This review is the second of a two-part review in The Ocular Surface. In the first review, the structure and function of the conjunctiva was evaluated with a focus on the extracellular matrix and the basement membrane, and biological and mechanical characteristics of the ideal substrate with recommendations for further studies. In this review the types of biological and synthetic substrates used for conjunctival transplantation are discussed including substrates based on the extracellular matrix. .

Laminins are essential components of all basement membranes where they regulate an extensive array of tissue functions. Alternative splicing from the laminin α3 gene produces a non-laminin but netrin-like protein, Laminin N terminus α31 (LaNt α31). LaNt α31 is widely expressed in intact tissue and is upregulated in epithelial cancers and during wound healing. In vitro functional studies have shown that LaNt α31 can influence numerous aspects of epithelial cell behaviour via modifying matrix organisation, suggesting a new model of laminin auto-regulation. However, the function of this protein has not been established beyond the epithelium and it has never been studied in vivo. Here, a mouse transgenic line was generated using the ubiquitin C promoter to drive inducible expression of LaNt α31. When expression was induced at embryonic day 15.5, LaNt α31 transgenic animals were not viable at birth, exhibiting localised regions of erythema. Numerous striking defects were apparent histologically, including extra-vascular erythrocytes in multiple tissues, kidney epithelial detachment, tubular dilation, interstitial bleeding, and thickening of tubule basement membranes, disruption of the epidermal basal cell layer and of the hair follicle outer root sheath, and ~50% reduction of cell numbers in the liver associated with depletion of hematopoietic erythrocytic foci. These findings demonstrate that LaNt α31 can influence tissue morphogenesis during development. More broadly, these data provide the first in vivo evidence to support an emerging model of laminin self-regulation and provide a valuable model for onward investigation into this important area.Summary StatementExpression during development of Laminin N-terminus α31, a netrin-like laminin splice isoform, caused defects indicating basement membrane disruption in multiple tissues; providing the first in vivo evidence for laminin self-regulation.

Laminin N-terminus alpha 31 (LaNt alpha 31) is a netrin-like protein derived from alternative splicing of the laminin alpha 3 gene. Although LaNt alpha 31 has been demonstrated to influence corneal and skin epithelial cell function, its expression has not been investigated beyond these tissues. In this study, we used immunohistochemistry to characterise the distribution of this protein in a wide-array of human tissue sections in comparison to laminin alpha 3. The data revealed widespread LaNt alpha 31 expression. In epithelial tissue, LaNt alpha 31 was present in the basal layer of the epidermis, throughout the epithelium of the digestive tract, and in much of the epithelium of the reproductive system. LaNt alpha 31 was also found throughout the vasculature of most tissues, with enrichment in reticular-like fibres in the extracellular matrix surrounding large vessels. A similar matrix pattern was observed around the terminal ducts in the breast and around the alveolar epithelium in the lung, where basement membrane staining was also evident. Specific enrichment of LaNt alpha 31 was identified in sub-populations of cells of the kidney, liver, pancreas, and spleen, with variations in intensity between different cell types in the collecting ducts and glomeruli of the kidney. Intriguingly, LaNt alpha 31 immunoreactivity was also evident in neurons of the central nervous system, in the cerebellum, cerebral cortex, and spinal cord. Together these findings suggest that LaNt alpha 31 may be functionally relevant in a wider range of tissue contexts than previously anticipated, and the data provides a valuable basis for investigation into this interesting protein.

Laminin N-terminus α31 (LaNt α31), a member of the laminin superfamily, expressed at low levels in intact epithelium but upregulated during wound repair. Increased expression of LaNt α31 reduced migration rate of corneal keratinocytes through an unknown mechanism. Here, we investigated whether LaNt α31 influences cell behaviour through modulating laminin-mediated processes. Adenoviral delivery of LaNt α31 into corneal epithelial cells led to reduced migration speed and increased cell spreading and changed laminin 332 organisation from diffuse arcs to tight clusters. Enhanced recruitment of collagen XVII and bullous pemphigoid antigen 1e to β4 integrin, indicating early maturation of hemidesmosomes, and changed focal adhesion distribution were also identified. LaNt α31 and laminin β3 co-immunoprecipitated from doubly transduced cells and were deposited together in live imaging experiment. Moreover, LaNt α31 expression led to increased matrix metalloproteinase (MMP) activity and proteolytic processing of laminin α3, and the inhibition of MMP activity rescued the laminin and hemidesmosome phenotypes. Provision of cell-derived extracellular matrix rescued the cell spreading and motility effects. These findings reveal LaNt α31 as a new player in regulating cell-to-matrix adhesion through its ability to influence laminin organisation and proteolytic processing.

Purpose To determine the composition of extracellular matrix (ECM) proteins secreted by a conjunctival epithelial cell line and to identify components that aid conjunctival epithelial cell culture. Methods Human conjunctival epithelial cell line (HCjE-Gi) cells were cultured in serum-free media and their ECM isolated using ammonium hydroxide. Growth characteristics were evaluated for fresh HCjE-Gi cells plated onto ECMs obtained from 3- to 28-day cell cultures. Mass spectrometry was used to characterize the ECM composition over 42 culture days. Cell adhesion and growth on pre-adsorbed fibronectin and α-2-HS-glycoprotein (α-2-HS-GP) were investigated. Results Day 3 ECM provided the best substrate for cell growth compared to ECM obtained from 5- to 28-day cell cultures. Mass spectrometry identified a predominantly laminin 332 matrix throughout the time course, with progressive changes to matrix composition over time: proportional decreases in matrix-bound growth factors and increases in proteases. Fibronectin and α-2-HS-GP were 5- and 200-fold enriched as a proportion of the early ECM relative to the late ECM, respectively. Experiments on these proteins in isolation demonstrated that fibronectin supported rapid cell adhesion, whereas fibronectin and α-2-HS-GP both supported enhanced cell growth compared to tissue culture polystyrene. Conclusions These data reveal α-2-HS-GP as a candidate protein to enhance the growth of conjunctival epithelial cells and raise the possibility of exploiting these findings for targeted improvement to synthetic tissue engineered conjunctival substrates.

PURPOSE: To determine the relationship between mechanical behavior in cross-linked corneas and changes in the corneal ultrastructure after corneal cross-linking (CXL). METHODS: Porcine corneas were treated following the “Dresden” protocol, the current gold standard for clinical treatment, consisting of dropwise application of 0.1% riboflavin in 20% dextran followed by 30 minutes of ultraviolet-A (UVA) irradiation. The effect of CXL was assessed using uniaxial tensile testing, transmission electron microscopy, and Fourier transform infrared spectroscopy, with results compared against corneas treated with each of the treatment solution components individually. RESULTS: UVA/riboflavin cross-linked corneas displayed 28% ± 17% increase in the material tangent modulus compared with dextran treatment alone, and altered collagen architecture within the first 300 µ m of stromal depth consisting of 5% increase in the thickness of collagen fibrils, no significant changes to interfibrillar spacing, and an 8% to 12% decrease in number of fibrils per unit area. Fourier transform infrared spectroscopy confirmed formation of interfibrillar bonds ( P = .012) induced by UVA-mediated CXL. CONCLUSIONS: The data support a model wherein collagen fibril diameter and structural density are fundamental parameters in defining tissue stiffening following UVA/riboflavin CXL and provide benchmarks against which modifications to the Dresden CXL protocol can be evaluated. [ J Refract Surg. 2018;34(4):264–272.]

Significance: Laminins are complex extracellular macromolecules that are major players in the control of a variety of core cell processes, including regulating rates of cell proliferation, differentiation, adhesion, and migration. Laminins, and related extracellular matrix components, have essential roles in tissue homeostasis; however, during wound healing, the same proteins are critical players in re-epithelialization and angiogenesis. Understanding how these proteins influence cell behavior in these different conditions holds great potential in identifying new strategies to enhance normal wound closure or to treat chronic/nonhealing wounds. Recent Advances: Laminin-derived bioactive peptides and, more recently, laminin-peptide conjugated scaffolds, have been designed to improve tissue regeneration after injuries. These peptides have been shown to be effective in decreasing inflammation and granulation tissue, and in promoting re-epithelialization, angiogenesis, and cell migration. Critical Issues: Alth...

Significance: During wound healing of the skin, keratinocytes should move over while still adhering to their underlying matrix. Thus, mechanistic insights into the wound-healing process require an understanding of the forms and functions of keratinocyte matrix adhesions, specifically focal contacts and hemidesmosomes, and their components. Recent Advances: Although the structure and composition of focal contacts and hemidesmosomes are relatively well defined, the functions of their components are only now being delineated using mouse genetic models and knockdown approaches in cell culture systems. Remarkably, both focal contact and hemidesmosomal proteins appear involved in determining the speed and directional migration of epidermal cells by modulating several signal transduction pathways. Critical Issues: Although many publications are centered on focal contacts, their existence in tissues such as the skin is controversial. Nonetheless, focal contact proteins are central to mechanisms that regulate skin cell motility. Conversely, hemidesmosomes have been identified in intact skin but whether hemidesmosomal components play a positive regulatory function in keratinocyte motility remains debated in the field. Future Directions: Defective wound healing is a developing problem in the aged, hospitalized and diabetic populations. Hence, deriving new insights into the molecular roles of matrix adhesion proteins in wound healing is a prerequisite to the development of novel therapeutics to enhance tissue repair and regeneration.

The repair of the bronchiolar epithelium damaged by cell-mediated, physical, or chemical insult requires epithelial cell migration over a provisional matrix composed of complexes of extracellular matrix molecules, including fibronectin and laminin. These matrix molecules support migration and enhance cell adhesion. When cells adhere too tightly to their matrix they fail to move; but if they adhere too little, they are unable to develop the traction force necessary for motility. Thus, we investigated the relative contributions of laminin and fibronectin to bronchiolar cell adhesion and migration using the immortalized bronchial lung epithelial cell line (BEP2D) and normal human bronchial epithelial (NHBE) cells, both of which assemble a laminin α3β3γ2 (LM332)/fibronectin-rich matrix. Intriguingly, BEP2D and NHBE cells migrate significantly faster on an LM332-rich matrix than on fibronectin. Moreover, addition of fibronectin to LM332 matrix suppresses motility of both cell types. Finally, fibronectin enhances the adhesion of both BEP2D and NHBE cells to LM332-coated surfaces. These results suggest that fibronectin fine tunes LM332-mediated migration by boosting bronchiolar cell adhesion to substrate. We suggest that, during epithelial wound healing of the injured airway, fibronectin plays an important adhesive role for laminin-driven epithelial cell motility by promoting a stable cellular interaction with the provisional matrix.

Objectives/Hypothesis Chronic Rhinosinusitis (CRS) is accompanied by evidence of a vigorous adaptive immune response, and emerging studies demonstrate that some nasal polyps manifest a polyclonal autoantibody response. We previously found that antibodies against BP180, a component of the hemidesmosome complex and the dominant epitope in autoimmune bullous pemphigoid, were found at elevated levels in nasal polyp tissue. Given the critical role of hemidesmosomes in maintaining epithelial integrity, we sought to investigate the distribution of BP180 in nasal tissue and evaluate for evidence of systemic autoimmunity against this antigen in CRS. Study Design Case-control experimental study. Methods The expression and distribution of BP180 in cultured nasal epithelial cells and normal nasal tissue were confirmed using real-time polymerase chain reaction (PCR), Western immunoblotting, immunofluorescence and immunohistochemistry. Sera were collected from three groups: control, CRSsNP, and CRSwNP. A commercially available ELISA was utilized to compare anti-BP180 autoantibody levels in sera. Results BP180 is expressed in nasal epithelium, but is not confined to the basement membrane as it is in human skin. In cultured nasal epithelial cells, confocal immunofluorescence showed a punctate distribution of BP180 along the basal surface, consistent with its distribution in epithelial keratinocytes. There are significantly higher levels of circulating nonpathologic anti-BP180 autoantibodies in CRS patients compared with normal controls (P

During wound repair, epidermal cells at the edge of an injury establish front-rear polarity through orchestrated changes in their cytoskeleton and adhesion structures. The polarity and directed migration of such cells is determined by the assembly, extension, and stabilization of a lamellipodium. Actinin-4 associates with lamellipodia and has been implicated in regulating lamellipodial structure, function and assembly. To study the functions of actinin-4 in human keratinocytes, we used shRNA to generate knockdown cells and compared their motility behavior and matrix adhesion assembly to scrambled shRNA treated control keratinocytes. Actinin-4 knockdown keratinocytes lack polarity, assemble multiple lamellipodia with a 2× increased area over controls, display reduced activity of the actin remodeling protein cofilin, and fail to migrate in a directional manner. This motility defect is rescued by plating knockdown cells on preformed laminin-332 matrix. In actinin-4-knockdown keratinocytes, focal contact area is increased by 25%, and hemidesmosome proteins are mislocalized. Specifically, α6β4 integrin localizes to large lamellipodial extensions, displays reduced dynamics, and fails to recruit its bullous pemphigoid antigen binding partners. Together, our data indicate a role for actinin-4 in regulating the steering mechanism of keratinocytes via profound effects on their matrix adhesion sites.—Hamill, K. J., Hopkinson, S. B., Skalli, O., Jones, J. C. R. Actinin-4 in keratinocytes regulates motility via an effect on lamellipodia stability and matrix adhesions.

Mouse keratinocytes migrate significantly slower than their human counterparts in vitro on uncoated surfaces. We tested the hypothesis that this is a consequence of differences in the extracellular matrix (ECM) that cells deposit. In support of this, human keratinocyte motility was markedly reduced when plated onto the ECM of mouse skin cells, whereas the latter cells migrated faster when plated onto human keratinocyte ECM. The ECM of mouse and human keratinocytes contained similar levels of the α3 laminin subunit of laminin-332. However, mouse skin cells expressed significantly more fibronectin (FN) than human cells. To assess whether FN is a motility regulator, we used small interfering RNA (siRNA) to reduce the expression of FN in mouse keratinocytes. The treated mouse keratinocytes moved significantly more rapidly than wild-type mouse skin cells. Moreover, the FN-depleted mouse cell ECM supported increased migration of both mouse and human keratinocytes. Furthermore, the motility of human keratinocytes was slowed when plated onto FN-coated substrates or human keratinocyte ECM supplemented with FN in a dose-dependent manner. Consistent with these findings, the ECM of α3 integrin-null keratinocytes, which also migrated faster than wild-type cells, was FN deficient. Our results provide evidence that FN is a brake to skin cell migration supported by laminin-332-rich matrices.

The laminins are a family of heterotrimeric basement membrane proteins that play roles in cellular adhesion, migration, and tissue morphogenesis. Through in silico analysis of the laminin-encoding genes, we identified a novel family of alternate splice isoforms derived from the 5'-end of the LAMA3 and LAMA5 genes. These isoforms resemble the netrins in that they contain a laminin N-terminal domain followed by a short stretch of laminin-type epidermal growth factor-like repeats. We suggest the terms LaNt (laminin N terminus) alpha3 and LaNt alpha5, for the predicted protein products of these mRNAs. RT-PCR confirmed the presence of these transcripts at the mRNA level. Moreover, they exhibit differential, tissue-specific, expression profiles. To confirm the existence of LaNt alpha3 protein, we generated an antibody to a unique domain within the putative polypeptide. This antibody recognizes a protein at the predicted molecular mass of 64 kDa by immunoblotting. Furthermore, immunofluorescence analyses revealed a basement membrane staining in epithelial tissue for LaNt alpha3 and LaNt alpha3 localized along the substratum-associated surface of cultured keratinocytes. We have also tested the functionality LaNt alpha3 through RNAi-mediated knockdown. Keratinocytes exhibiting specific knockdown of LaNt alpha3 displayed impaired adhesion, stress resistance, and reduced ability to close scratch wounds in vitro.

Recent analyses of collagen, elastin and fibronectin matrix assembly, organization and remodeling have been facilitated by the use of tagged proteins that can be visualized without the need for antibody labeling. Here, we report the generation of C-terminal tagged, full-length and "processed" (alpha3DeltaLG4-5) human alpha3 as well as C-terminal tagged, full-length human beta3 laminin subunits in adenoviral vectors. Human epidermal keratinocytes (HEKs) and human bronchial epithelial (BEP2D) cells, which assemble laminin-332-rich matrices, as well as primary rat lung alveolar type II (ATII) cells, which elaborate a fibrous network rich in laminin-311, were infected with adenovirus encoding the tagged human laminin subunits. In HEKs and BEP2D cells, tagged, full-length alpha3, alpha3DeltaLG4-5 and beta3 laminin subunits incorporate into arrays of matrix organized into patterns that are comparable to those observed when such cells are stained using laminin-332 subunit antibody probes. Moreover, HEKs and BEP2Ds move over these tagged, laminin-332-rich matrix arrays. We have also used the tagged beta3 laminin subunit-containing matrices to demonstrate that assembled laminin-332 arrays influence laminin matrix secretion and/or assembly. In the case of rat ATII cells, although tagged alpha3 laminin subunits are not detected in the matrix of rat ATII cells infected with virus encoding full-length human alpha3 laminin protein, processed human alpha3 laminin subunits are incorporated into an extracellular fibrous array. We discuss how these novel laminin reagents can be used to study the organization, processing and assembly of laminin matrices and how they provide new insights into the potential functional importance of laminin fragments.

Laminins are heterotrimeric proteins that are secreted by the alveolar epithelium into the basement membrane, and their expression is altered in extracellular matrices from patients with pulmonary fibrosis. In a small number of patients with pulmonary fibrosis, we found that the normal basement membrane distribution of the α3 laminin subunit was lost in fibrotic regions of the lung. To determine if these changes play a causal role in the development of fibrosis, we generated mice lacking the α3 laminin subunit specifically in the lung epithelium by crossing mice expressing Cre recombinase driven by the surfactant protein C promoter (SPC-Cre) with mice expressing floxed alleles encoding the α3 laminin gene (Lama3(fl/fl)). These mice exhibited no developmental abnormalities in the lungs up to 6 months of age, but, compared with control mice, had worsened mortality, increased inflammation, and increased fibrosis after the intratracheal administration of bleomycin. Similarly, the severity of fibrosis induced by an adenovirus encoding an active form of transforming growth factor-β was worse in mice deficient in α3 laminin in the lung. Taken together, our results suggest that the loss of α3 laminin in the lung epithelium does not affect lung development, but plays a causal role in the development of fibrosis in response to bleomycin or adenovirally delivered transforming growth factor-β. Thus, we speculate that the loss of the normal basement membrane organization of α3 laminin that we observe in fibrotic regions from the lungs of patients with pulmonary fibrosis contributes to their disease progression.

Item does not contain fulltext Laryngo-onycho-cutaneous (LOC or Shabbir) syndrome (OMIM 245660) is an autosomal recessive epithelial disorder confined to the Punjabi Muslim population. The condition is characterized by cutaneous erosions, nail dystrophy and exuberant vascular granulation tissue in certain epithelia, especially conjunctiva and larynx. Genome-wide homozygosity mapping localized the gene to a 2 Mb region on chromosome 18q11.2 with an LOD score of 19.8 at theta=0. This region includes the laminin alpha3 gene (LAMA3), in which loss-of-expression mutations cause the lethal skin blistering disorder Herlitz junctional epidermolysis bullosa. Detailed investigation showed that this gene possesses a further 38 exons (76 exons in total) spanning 318 kb of genomic DNA, and encodes three distinct proteins, designated laminin alpha3a, alpha3b1 and alpha3b2. The causative mutation in 15 families was a frameshift mutation 151insG predicting a stop codon 7 bp downstream in an exon that is specific to laminin alpha3a. This protein is secreted only by the basal keratinocytes of stratified epithelia, implying that LOC is caused by dysfunction of keratinocyte-mesenchymal communication. Surprisingly, the 151insG mutation does not result in nonsense-mediated mRNA decay due to rescue of the transcript by an alternative translation start site 6 exons downstream. The resultant N-terminal deletion of laminin alpha3a was confirmed by immunoprecipitation of secreted proteins from LOC keratinocytes. These studies show that the laminin alpha3a N-terminal domain is a key regulator of the granulation tissue response, with important implications not only in LOC but in a range of other clinical conditions associated with abnormal wound healing.

Chronic Rhinosinusitis (CRS) is accompanied by evidence of a vigorous adaptive immune response, and emerging studies demonstrate that some nasal polyps manifest a polyclonal autoantibody response. We previously found that antibodies against BP180, a component of the hemidesmosome complex and the dominant epitope in autoimmune bullous pemphigoid, were found at elevated levels in nasal polyp tissue. Given the critical role of hemidesmosomes in maintaining epithelial integrity, we sought to investigate the distribution of BP180 in nasal tissue and evaluate for evidence of systemic autoimmunity against this antigen in CRS.Case-control experimental study.The expression and distribution of BP180 in cultured nasal epithelial cells and normal nasal tissue were confirmed using real-time polymerase chain reaction (PCR), Western immunoblotting, immunofluorescence and immunohistochemistry. Sera were collected from three groups: control, CRSsNP, and CRSwNP. A commercially available ELISA was utilized to compare anti-BP180 autoantibody levels in sera.BP180 is expressed in nasal epithelium, but is not confined to the basement membrane as it is in human skin. In cultured nasal epithelial cells, confocal immunofluorescence showed a punctate distribution of BP180 along the basal surface, consistent with its distribution in epithelial keratinocytes. There are significantly higher levels of circulating nonpathologic anti-BP180 autoantibodies in CRS patients compared with normal controls (P0.05).BP180 is more widely expressed in nasal epithelium versus skin, although it appears to play a similar role in the formation of hemidesmosomes along the basement membrane. Further investigations are ongoing to characterize the pathogenicity of the anti-epithelial antibody response in CRS.

Rac1 activity, polarity, lamellipodial dynamics, and directed motility are defective in keratinocytes exhibiting deficiency in beta 4 integrin or knockdown of the plakin protein Bullous Pemphigoid Antigen 1e (BPAG1e). The activity of Rac, formation of stable lamellipodia, and directed migration are restored in beta 4 integrin-deficient cells by inducing expression of a truncated form of beta 4 integrin, which lacks binding sites for BPAG1e and plectin. In these same cells, BPAG1e, the truncated beta 4 integrin, and type XVII collagen (Col XVII), a transmembrane BPAG1e-binding protein, but not plectin, colocalize along the substratum-attached surface. This finding suggested to us that Col XVII mediates the association of BPAG1e and alpha 6 beta 4 integrin containing the truncated beta 4 subunit and supports directed migration. To test these possibilities, we knocked down Col XVII expression in keratinocytes expressing both full-length and truncated beta 4 integrin proteins. Col XVII-knockdown keratinocytes exhibit a loss in BPAG1e-alpha 6 beta 4 integrin interaction, a reduction in lamellipodial stability, an impairment in directional motility, and a decrease in Rac1 activity. These defects are rescued by a mutant Col XVII protein truncated at its carboxyl terminus. In summary, our results suggest that in motile cells Col XVII recruits BPAG1e to alpha 6 beta 4 integrin and is necessary for activation of signaling pathways, motile behavior, and lamellipodial stability.

The outer most layer of the skin, the epidermis, is attached to the dermis via a sheet of extracellular matrix proteins termed the basement membrane zone (BMZ). In the intact skin, adhesion of the keratinocytes in the basal layer of the epidermis to the BMZ is facilitated primarily by hemidesmosomes which associate with the keratin cytoskeleton. Cultured keratinocytes do not assemble bona fide hemidesmosomes although hemidesmosome protein clusters (stable anchoring contacts) are found along the substrate-attached surface of the cells and towards the leading edge of keratinocytes repopulating scratch wounds. Actin cytoskeleton-associated matrix adhesion devices termed focal contacts are not thought to play an important role in the adhesion of keratinocytes to the BMZ in intact skin but are prominent in cultured keratinocytes where they are believed to regulate cell migration. We review the molecular components, functions, dynamics and cross-talk of hemidesmosomes and focal contacts in keratinocytes. In addition, we briefly describe what is known about their role in autoimmune and genetic blistering diseases of the skin. We also discuss recent publications which indicate, contrary to expectation, that certain focal contact proteins retard keratinocyte migration while hemidesmosomal proteins regulate directed keratinocyte motility during wound healing.

The laminins are a secreted family of heterotrimeric molecules essential for basement membrane formation, structure, and function. It is now well established that the alpha3 subunit of laminins-332, -321, and -311 plays an important role in mediating epidermal-dermal integrity and is essential for the skin to withstand mechanical stresses. These laminins also regulate cell migration and mechanosignal transduction. This article provides an overview of the gene, transcripts, and protein structures of laminin alpha3. Also discussed are the proposed functions for the alpha3 subunit-containing laminins.

Laminins are structural components of basement membranes. In addition, they are key extracellular-matrix regulators of cell adhesion, migration, differentiation and proliferation. This Commentary focuses on a relatively understudied aspect of laminin biology: how is laminin deposited into the extracellular matrix? This topic has fascinated researchers for some time, particularly considering the diversity of patterns of laminin that can be visualized in the matrix of cultured cells. We discuss current ideas of how laminin matrices are assembled, the role of matrix receptors in this process and how laminin-associated proteins modulate matrix deposition. We speculate on the role of signaling pathways that are involved in laminin-matrix deposition and on how laminin patterns might play an important role in specifying cell behaviors, especially directed migration. We conclude with a description of new developments in the way that laminin deposition is being studied, including the use of tagged laminin subunits that should allow the visualization of laminin-matrix deposition and assembly by living cells.

The laminins are a family of heterotrimeric basement membrane proteins that play roles in cellular adhesion, migration, and tissue morphogenesis. Through in silico analysis of the laminin-encoding genes, we identified a novel family of alternate splice isoforms derived from the 5'-end of the LAMA3 and LAMA5 genes. These isoforms resemble the netrins in that they contain a laminin N-terminal domain followed by a short stretch of laminin-type epidermal growth factor-like repeats. We suggest the terms LaNt (laminin N terminus) alpha3 and LaNt alpha5, for the predicted protein products of these mRNAs. RT-PCR confirmed the presence of these transcripts at the mRNA level. Moreover, they exhibit differential, tissue-specific, expression profiles. To confirm the existence of LaNt alpha3 protein, we generated an antibody to a unique domain within the putative polypeptide. This antibody recognizes a protein at the predicted molecular mass of 64 kDa by immunoblotting. Furthermore, immunofluorescence analyses revealed a basement membrane staining in epithelial tissue for LaNt alpha3 and LaNt alpha3 localized along the substratum-associated surface of cultured keratinocytes. We have also tested the functionality LaNt alpha3 through RNAi-mediated knockdown. Keratinocytes exhibiting specific knockdown of LaNt alpha3 displayed impaired adhesion, stress resistance, and reduced ability to close scratch wounds in vitro.

α6β4 integrin, a component of hemidesmosomes, also plays a role in keratinocyte migration via signaling through Rac1 to the actin-severing protein cofilin. Here, we tested the hypothesis that the β4 integrin-associated plakin protein, bullous pemphigoid antigen 1e (BPAG1e) functions as a scaffold for Rac1/cofilin signal transduction. We generated keratinocyte lines exhibiting a stable knockdown in BPAG1e expression. Knockdown of BPAG1e does not affect expression levels of other hemidesmosomal proteins, nor the amount of β4 integrin expressed at the cell surface. However, the amount of Rac1 associating with β4 integrin and the activity of both Rac1 and cofilin are significantly lower in BPAG1e-deficient cells compared with wild-type keratinocytes. In addition, keratinocytes deficient in BPAG1e exhibit loss of front-to-rear polarity and display aberrant motility. These defects are rescued by inducing expression of constitutively active Rac1 or active cofilin. These data indicate that the BPAG1e is required for efficient regulation of keratinocyte polarity and migration by determining the activation of Rac1.

Laminin 5 (kalinin/epiligrin/nicein) is an essential structural component of the dermal-epidermal junction, composed of three polypeptide subunits: laminin alpha3, beta3 and gamma2. Studies of the inherited skin fragility disorder junctional epidermolysis bullosa (JEB) have suggested that the major role of this heterotrimeric protein is to act as an adhesive ligand essential for binding the epidermis to the underlying dermis and thus maintaining the integrity of the skin. Protein interaction studies have shown that the C terminus of the alpha3 subunit binds to a range of integrin complexes depending on the motility status of keratinocytes. This allows laminin 5 to interact with either hemidesmosomes or the actin cytoskeleton. Recently we have reported that the absence of the N-terminal region of laminin alpha3a in laryngo-onchyo-cutaneous syndrome causes excessive granulation tissue production at wound sites. As granulation tissue production is also a problem in JEB, this implicates laminin 5 in control of this wound healing response.

In 1994, the molecular basis of pachyonychia congenita (PC) was elucidated. Four keratin genes are associated with the major subtypes of PC: K6a or K16 defects cause PC-1; and mutations in K6b or K17 cause PC-2. Mutations in keratins, the epithelial-specific intermediate filament proteins, result in aberrant cytoskeletal networks which present clinically as a variety of epithelial fragility phenotypes. To date, mutations in 20 keratin genes are associated with human disorders. Here, we review the genetic basis of PC and report 30 new PC mutations. Of these, 25 mutations were found in PC-1 families and five mutations were identified in PC-2 kindreds. All mutations identified were heterozygous amino acid substitutions or small in-frame deletion mutations with the exception of an unusual mutation in a sporadic case of PC-1. The latter carried a 117 bp duplication resulting in a 39 amino acid insertion in the 2B domain of K6a. Also of note was mutation L388P in K17, which is the first genetic defect identified in the helix termination motif of this protein. Understanding the genetic basis of these disorders allows better counseling for patients and paves the way for therapy development.

Laminins are heterotrimeric glycoproteins of the extracellular matrix that are secreted by epithelial cells and which are crucial for the normal structure and function of the basement membrane. We have generated a mouse harboring a conditional knockout of α3 laminin (Lama3fl/fl), one of the main laminin subunits in the lung basement membrane. At 60 days after intratracheal treatment of adult Lama3fl/fl mice with an adenovirus encoding Cre recombinase (Ad-Cre), the protein abundance of α3 laminin in whole lung homogenates was more than 50% lower than that in control-treated mice, suggesting a relatively long half-life for the protein in the lung. Upon exposure to an injurious ventilation strategy (tidal volume of 35 ml per kg of body weight for 2 hours), the mice with a knockdown of the α3 laminin subunit had less severe injury, as shown by lung mechanics, histology, alveolar capillary permeability and survival when compared with Ad-Null-treated mice. Knockdown of the α3 laminin subunit resulted in evidence of lung inflammation. However, this did not account for their resistance to mechanical ventilation. Rather, the loss of α3 laminin was associated with a significant increase in the collagen content of the lungs. We conclude that the loss of α3 laminin in the alveolar epithelium results in an increase in lung collagen, which confers resistance to mechanical injury.

Laminin N-terminus α31 (LaNt α31) is an alternative splice isoform derived from the laminin α3 gene. The LaNt α31 protein is enriched around the terminal duct lobular units in normal breast tissue. In the skin and cornea the protein influences epithelial cell migration and tissue remodelling. However, LaNt α31 has never been investigated in a tumour environment. Here we analysed LaNt α31 in invasive ductal carcinoma and determined its contribution to breast carcinoma invasion. LaNt α31 expression and distribution were analysed by immunohistochemistry in human breast tissue biopsy sections and tissue microarrays covering 232 breast cancer samples. This analysis revealed LaNt α31 to be upregulated in 56 % of invasive ductal carcinoma specimens compared with matched normal tissue, and further increased in nodal metastasis compared with the tumour mass in 45 % of samples. 65.8 % of triple negative cases displayed medium to high LaNt α31 expression. To study LaNt α31 function, an adenoviral system was used to induce expression in MCF-7 and MDA-MB-231 cells. Metabolic activity, 2D cell migration, and invasion into collagen hydrogels were not significantly different between LaNt α31 overexpressing cells and control treated cells. However, LaNt α31 overexpressing MDA-MB-231 cells displayed a striking change in their mode of invasion into laminin-containing Matrigel; changing from multicellular streaming to individual cellular-invasion. In agreement with these results, 66.7% of the tumours with the highest LaNt α31 expression were non-cohesive. Together these findings indicate that breast cancer-associated changes in LaNt α31 expression could directly contribute to tumour invasiveness, and that this little-studied protein may become a therapeutic target.