CREB3L2 Regulates Hemidesmosome Formation during Epithelial Sealing.

The long-term success rate of dental implants can be improved by establishing a favorable biological sealing with a high-quality epithelial attachment. The application of mesenchymal stem cells (MSCs) holds promise for facilitating the soft tissue integration around implants, but the molecular mechanism is still unclear and the general application of MSC sheet for soft tissue integration is also relatively unexplored. We found that gingival tissue-derived MSC (GMSC) sheet treatment significantly promoted the expression of hemidesmosome (HD)-related genes and proteins in gingival epithelial cells (GECs). The formation of HDs played a key role in strengthening peri-implant epithelium (PIE) sealing. Further, high-throughput transcriptome sequencing showed that GMSC sheet significantly upregulated the PI3K/AKT pathway, confirming that cell adhesion and HD expression in GECs were regulated by GMSC sheet. We observed that the expression of transcription factor CREB3L2 in GECs was downregulated. After treatment with PI3K pathway inhibitor LY294002, CREB3L2 messenger RNA and protein expression levels were upregulated. Further experiments showed that overexpression or knockdown of CREB3L2 could significantly inhibit or promote HD-related genes and proteins, respectively. We confirmed that CREB3L2 was a transcription factor downstream of the PI3K/AKT pathway and participated in the formation of HDs regulated by GMSC sheet. Finally, through the establishment of early implant placement model in rats, we clarified the molecular function of CREB3L2 in PIE sealing as a mechanical transmission molecule in GECs. The application of GMSC sheet-implant complex could enhance the formation of HDs at the implant-PIE interface and decrease the penetration distance of horseradish peroxidase between the implant and PIE. Meanwhile, GMSC sheet reduced the length of CREB3L2 protein expression on PIE. These findings elucidate the potential function and molecular mechanism of MSC sheet regulating the epithelial sealing around implants, providing new insights and ideas for the application of stem cell therapy in regenerative medicine.

Mechanotransduction through hemidesmosomes during aging and longevity.

Hemidesmosomes are structural protein complexes localized at the interface of tissues with high mechanical demand and shear forces. Beyond tissue anchoring, hemidesmosomes have emerged as force-modulating structures important for translating mechanical cues into biochemical and transcriptional adaptation (i.e. mechanotransduction) across tissues. Here, we discuss the recent insights into the roles of hemidesmosomes in age-related tissue regeneration and aging in C. elegans, mice and humans. We highlight the emerging concept of preserved dynamic mechanoregulation of hemidesmosomes in tissue maintenance and healthy aging.

Keratinocyte Proliferation and Hemidesmosome Formation on Surfaces for Dental Implants: In Vitro Study.

PURPOSE: To demonstrate the likelihood of the polyetheretherketone (PEEK), zirconia (ZrO2), and titanium (Ti) disks to support proliferation and hemidesmosome formation of gingival cells. MATERIALS AND METHODS: Water contact angle was performed on each material, and surface roughness (Ra) was measured. Scanning electron microscopy and x-ray photoelectron spectroscopy were used. Later, oral keratinocyte cells were cultured on disks, and metabolic activity and expression of hemidesmosome markers, integrin α6 and ß4, in relation to the biomaterial disks at 1, 3, and 5 days of cell culture were quantified. Tissue culture polystyrene was used as the control. Statistical analysis was performed with analysis of variance (ANOVA) with Tukey post hoc comparison test. A P value of < .05 was considered statistically significant. RESULTS: The water contact angle ranged from 70.2 degrees (Ti) to a maximum of hydrophobicity of 93.3 degrees (PEEK). Ra was highest on ZrO2, followed by PEEK. Ti showed the most keratinocyte metabolic activity at 1, 3, and 5 culture periods. Contrarily, ZrO2 and PEEK disks had lower keratinocyte metabolic activity at all observation times, with no statistical differences between both groups. Integrin α6 and ß4 expression was highest on TCPS and ZrO2 compared to Ti and PEEK. CONCLUSION: Keratinocytes proliferated faster on Ti than on ZrO2 and PEEK substrates, and expression of hemidesmosome formation markers, integrin α6 and ß4, were higher on ZrO2 than either Ti or PEEK. Int J Oral Maxillofac Implants 2023;38:496-502. doi: 10.11607/jomi.9894.

Implant surface physicochemistry affects keratinocyte hemidesmosome formation.

Previous studies have shown hydrophilic/hydrophobic implant surfaces stimulate/hinder osseointegration. An analogous concept was applied here using common biological functional groups on a model surface to promote oral keratinocytes (OKs) proliferation and hemidesmosomes (HD) to extend implant lifespans through increased soft tissue attachment. However, it is unclear what physicochemistry stimulates HDs. Thus, common biological functional groups (NH2 , OH, and CH3 ) were functionalized on glass using silanization. Non-functionalized plasma-cleaned glass and H silanization were controls. Surface modifications were confirmed with X-ray photoelectron spectroscopy and water contact angle. The amount of bovine serum albumin (BSA) and fibrinogen, and BSA thickness, were assessed to understand how adsorbed protein properties were influenced by physicochemistry and may influence HDs. OKs proliferation was measured, and HDs were quantified with immunofluorescence for collagen XVII and integrin ß4. Plasma-cleaned surfaces were the most hydrophilic group overall, while CH3 was the most hydrophobic and OH was the most hydrophilic among functionalized groups. Modification with the OH chemical group showed the highest OKs proliferation and HD expression. The OKs response on OH surfaces appeared to not correlate to the amount or thickness of adsorbed model proteins. These results reveal relevant surface physicochemical features to favor HDs and improve implant soft tissue attachment.

SCD1 Sustains Homeostasis of Bulge Niche via Maintaining Hemidesmosomes in Basal Keratinocytes.

Niche for stem cells profoundly influences their maintenance and fate during tissue homeostasis and pathological disorders; however, the underlying mechanisms and tissue-specific features remain poorly understood. Here, it is reported that fatty acid desaturation catabolized by stearoyl-coenzyme A desaturase 1 (SCD1) regulates hair follicle stem cells (HFSCs) and hair growth by maintaining the bulge, niche for HFSCs. Scd1 deletion in mice results in abnormal hair growth, an effect exerted directly on keratin K14+ keratinocytes rather than on HFSCs. Mechanistically, Scd1 deficiency impairs the level of integrin α6ß4 complex and thus the assembly of hemidesmosomes (HDs). The disruption of HDs allows the aberrant activation of focal adhesion kinase and PI3K in K14+ keratinocytes and subsequently their differentiation and proliferation. The overgrowth of basal keratinocytes results in downward extension of the outer root sheath and interruption of bulge formation. Then, inhibition of PI3K signaling in Scd1-/- mice normalizes the bulge, HFSCs, and hair growth. Additionally, supplementation of oleic acid to Scd1-/- mice reestablishes HDs and the homeostasis of bulge niche, and restores hair growth. Thus, SCD1 is critical in regulating hair growth through stabilizing HDs in basal keratinocytes and thus sustaining bulge for HFSC residence and periodic activity.

Diagnostic and prognostic role of protein and ultrastructural alterations at cell-extracellular matrix junctions in neoplastic progression of human oral malignancy.

Despite advancements in technology and increase in favorable outcomes associated with oral cancer, early detection remains the most significant factor in limiting mortality. The current study aimed to develop early diagnostic and prognostic markers for oral tumorigenesis. Protein and ultrastructural alterations at cell-extracellular matrix (ECM) adhesion junctions were examined concurrently using immunohistochemistry (IHC) and transmission electron microscopy (TEM) on progressive grade of oral carcinomas (n = 285). The expression of hemidesmosome (HD) proteins-integrin ß4, BP180, and laminin-5 increased in hyperplasia as compared to normal, and significantly increased further, as the disease progressed. TEM analysis in parallel tissues revealed a significant decrease in HD number and increase in the length of basal lamina (BL) in hyperplasia. With cancer progression, the severity of ultrastructural alterations increased gradually and significantly. Overexpression of HD proteins, decrease in HD number and increase in BL length significantly correlated with nodal metastasis, local recurrence, and recurrence-free survival of patients. Concurrent use of IHC and TEM can add value to early recognition of neoplastic changes in primary carcinomas of oral cavity. In this regard, altered expression of integrin ß4 and laminin-5, loss of HDs, and increased BL length could offer criteria for early diagnosis and prognosis of oral malignancy.

Post-translational modifications to hemidesmosomes in human airway epithelial cells following diacetyl exposure.

Diacetyl (DA; 2,3-butanedione) is a highly reactive alpha (α)-diketone. Inhalation exposure to DA can cause significant airway epithelial cell injury, however, the mechanisms of toxicity remain poorly understood. The purpose of these experiments was to assess for changes in abundance and distribution of hemidesmosome-associated proteins following DA exposure that contribute to DA-induced epithelial toxicity. Human bronchial epithelial cells were grown in submerged cultures and exposed to three occupationally-relevant concentrations of DA (5.7, 8.6, or 11.4 mM) for 1 h. Following DA exposure, epithelial cells were cultured for 4 days to monitor for cell viability by MTT and WST-1 assays as well as for changes in cellular distribution and relative abundance of multiple hemidesmosome-associated proteins, including keratin 5 (KRT5), plectin (PLEC), integrin alpha 6 (ITGα6) and integrin beta 4 (ITGß4). Significant toxicity developed in airway epithelial cells exposed to DA at concentrations ≥ 8.6 mM. DA exposure resulted in post-translational modifications to hemidesmosome-associated proteins with KRT5 crosslinking and ITGß4 cleavage. Following DA exposure at 5.7 mM, these post-translational modifications to KRT5 resolved with time. Conversely, at DA concentrations ≥ 8.6 mM, modifications to KRT5 persisted in culture with decreased total abundance and perinuclear aggregation of hemidesmosome-associated proteins. Significant post-translational modifications to hemidesmosome-associated proteins develop in airway epithelial cells exposed to DA. At DA concentrations ≥ 8.6 mM, these hemidesmosome modifications persist in culture. Future work targeting hemidesmosome-associated protein modifications may prevent the development of lung disease following DA exposure.

Pancreatic ductal adenocarcinoma cells employ integrin α6ß4 to form hemidesmosomes and regulate cell proliferation.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis due to its aggressive progression, late detection and lack of druggable driver mutations, which often combine to result in unsuitability for surgical intervention. Together with activating mutations of the small GTPase KRas, which are found in over 90% of PDAC tumours, a contributory factor for PDAC tumour progression is formation of a rigid extracellular matrix (ECM) and associated desmoplasia. This response leads to aberrant integrin signalling, and accelerated proliferation and invasion. To identify the integrin adhesion systems that operate in PDAC, we analysed a range of pancreatic ductal epithelial cell models using 2D, 3D and organoid culture systems. Proteomic analysis of isolated integrin receptor complexes from human pancreatic ductal epithelial (HPDE) cells predominantly identified integrin α6ß4 and hemidesmosome components, rather than classical focal adhesion components. Electron microscopy, together with immunofluorescence, confirmed the formation of hemidesmosomes by HPDE cells, both in 2D and 3D culture systems. Similar results were obtained for the human PDAC cell line, SUIT-2. Analysis of HPDE cell secreted proteins and cell-derived matrices (CDM) demonstrated that HPDE cells secrete a range of laminin subunits and form a hemidesmosome-specific, laminin 332-enriched ECM. Expression of mutant KRas (G12V) did not affect hemidesmosome composition or formation by HPDE cells. Cell-ECM contacts formed by mouse and human PDAC organoids were also assessed by electron microscopy. Organoids generated from both the PDAC KPC mouse model and human patient-derived PDAC tissue displayed features of acinar-ductal cell polarity, and hemidesmosomes were visible proximal to prominent basement membranes. Furthermore, electron microscopy identified hemidesmosomes in normal human pancreas. Depletion of integrin ß4 reduced cell proliferation in both SUIT-2 and HPDE cells, reduced the number of SUIT-2 cells in S-phase, and induced G1 cell cycle arrest, suggesting a requirement for α6ß4-mediated adhesion for cell cycle progression and growth. Taken together, these data suggest that laminin-binding adhesion mechanisms in general, and hemidesmosome-mediated adhesion in particular, may be under-appreciated in the context of PDAC. Proteomic data are available via ProteomeXchange with the identifiers PXD027803, PXD027823 and PXD027827.

Protein Ligand Nanopattern Size Selects for Cellular Adhesion via Hemidesmosomes over Focal Adhesions.

Hemidesmosomes (HDs) are multiprotein complexes that firmly anchor epidermal cells to the basement membrane of skin through the interconnection of the cytoplasmic intermediate filaments with extracellular laminin 332 (Ln332). Considerably less attention has been paid to HDs compared to focal complexes/focal adhesions (FC/FAs) in mechanistic single-cell structures due to the lack of suitable in vitro model systems. Here nanopatterns of Ln332 (100-1000 nm) are created to direct and study the formation of HD in adherent HaCaT cells. It is observed that HaCaT cells at Ln 332 nanopatterns adhere via hemidesmosomes, in stark contrast to cells at homogeneous Ln332 surfaces that adhere via FC/FAs. Clustering of α6 integrin is observed at nanopatterned Ln332 of 300 nm patches and larger. Cells at 500 nm diameter patterns show strong colocalization of α6 integrin with ColXVII or pan-cytokeratin compared to 300 nm/1000 nm indicating a threshold for HD initiation >100 nm but a pattern size selection for maturation of HDs. It is demonstrated that the pattern of Ln332 can determine the cellular selection of adhesion types with a size-dependent initiation and maturation of HDs. The protein nanopatterning approach that is presented provides a new in vitro route to study the role of HDs in cell signaling and function.

Dual keratinocyte-attachment and anti-inflammatory coatings for soft tissue sealing around transmucosal oral implants.

Unlike the attachment of soft epithelial skin tissue to penetrating solid natural structures like fingernails and teeth, sealing around percutaneous/permucosal devices such as dental implants is hindered by inflammation and epidermal down growth. Here, we employed a dual keratinocyte-adhesive peptide and anti-inflammatory biomolecule coating on titanium to promote oral epithelial tissue attachment. For minimizing inflammation-triggered epidermal down growth, we coated pristine and oxygen plasma pre-treated polished titanium (pTi) with conjugated linoleic acid (CLA). Further, in order to aid in soft tissue attachment via the formation of hemidesmosomes, adhesive structures by oral keratinocytes, we coated the anionic linoleic acid (LA) adsorbed titanium with cationic cell adhesive peptides (CAP), LamLG3, a peptide derived from Laminin 332, the major extracellular matrix component of the basement membrane in skin tissue and Net1, derived from Netrin-1, a neural chemoattractant capable of epithelial cell attachment via α6ß4 integrins. The dual CLA-CAP coatings on pTi were characterized by X-ray photoelectron spectroscopy and dynamic water contact angle measurements. The proliferation of human oral keratinocytes (TERT-2/OKF6) was accelerated on the peptide coated titanium while also promoting the expression of Col XVII and ß-4 integrin, two markers for hemidesmosomes. Simultaneously, CLA coating suppressed the production of inducible nitric oxide synthase (anti-iNOS); a pro-inflammatory M1 marker expressed in lipopolysaccharide (LPS) stimulated murine macrophages (RAW 264.7) and elevated expression of anti-CD206, associated to an anti-inflammatory M2 macrophage phenotype. Taken together, the dual keratinocyte-adhesive peptide and anti-inflammatory biomolecule coating on titanium can help reduce inflammation and promote permucosal/peri-implant soft tissue sealing.

Wnt/ß-Catenin Signaling Stabilizes Hemidesmosomes in Keratinocytes.

Hemidesmosomes (HDs) are adhesion complexes that promote epithelial-stromal attachment in stratified and complex epithelia, including the epidermis. In various biological processes, such as differentiation and migration of epidermal keratinocytes during wound healing or carcinoma invasion, quick assembly and disassembly of HDs are prerequisites. In this study, we show that inhibition of Wnt/ß-catenin signaling disturbs HD organization in keratinocytes. Screening with inhibitors identified the depletion of HD components and HD-like structures through Wnt inhibition, but keratinocyte differentiation was not affected. Wnt inhibition significantly diminished plectin and type XVII collagen expression in the basal side of Wnt-inhibited cells and the dermo-epidermal junction of the Wnt-inactive murine basal epidermis. Similar to Wnt inhibition, PLEC-knockout cells or cells with plectin-type XVII collagen binding defects showed type XVII collagen reduction in the basal side of the cells, implying the possible involvement of Wnt/ß-catenin signaling in HD assembly. Atypical protein kinase C inhibition ameliorated the phenotypes of Wnt-inhibited cells. These findings show that Wnt/ß-catenin signaling regulates the localization of HD components in keratinocytes and that the atypical protein kinase C pathway is involved in Wnt inhibition‒induced HD disarrangement. Our study suggests that the Wnt signaling pathway could be a potential therapeutic target for treating HD-defective diseases, such as epidermolysis bullosa.

BP180/Collagen XVII: A Molecular View.

BP180 is a type II collagenous transmembrane protein and is best known as the major autoantigen in the blistering skin disease bullous pemphigoid (BP). The BP180 trimer is a central component in type I hemidesmosomes (HD), which cause the adhesion between epidermal keratinocytes and the basal lamina, but BP180 is also expressed in several non-HD locations, where its functions are poorly characterized. The immunological roles of intact and proteolytically processed BP180, relevant in BP, have been subject to intensive research, but novel functions in cell proliferation, differentiation, and aging have also recently been described. To better understand the multiple physiological functions of BP180, the focus should return to the protein itself. Here, we comprehensively review the properties of the BP180 molecule, present new data on the biochemical features of its intracellular domain, and discuss their significance with regard to BP180 folding and protein-protein interactions.

Regulation of hemidesmosome dynamics and cell signaling by integrin α6ß4.

Hemidesmosomes (HDs) are specialized multiprotein complexes that connect the keratin cytoskeleton of epithelial cells to the extracellular matrix (ECM). In the skin, these complexes provide stable adhesion of basal keratinocytes to the underlying basement membrane. Integrin α6ß4 is a receptor for laminins and plays a vital role in mediating cell adhesion by initiating the assembly of HDs. In addition, α6ß4 has been implicated in signal transduction events that regulate diverse cellular processes, including proliferation and survival. In this Review, we detail the role of α6ß4 in HD assembly and beyond, and we discuss the molecular mechanisms that regulate its function.

Peptide location fingerprinting reveals modification-associated biomarker candidates of ageing in human tissue proteomes.

Although dysfunctional protein homeostasis (proteostasis) is a key factor in many age-related diseases, the untargeted identification of structurally modified proteins remains challenging. Peptide location fingerprinting is a proteomic analysis technique capable of identifying structural modification-associated differences in mass spectrometry (MS) data sets of complex biological samples. A new webtool (Manchester Peptide Location Fingerprinter), applied to photoaged and intrinsically aged skin proteomes, can relatively quantify peptides and map statistically significant differences to regions within protein structures. New photoageing biomarker candidates were identified in multiple pathways including extracellular matrix organisation (collagens and proteoglycans), protein synthesis and folding (ribosomal proteins and TRiC complex subunits), cornification (keratins) and hemidesmosome assembly (plectin and integrin α6ß4). Crucially, peptide location fingerprinting uniquely identified 120 protein biomarker candidates in the dermis and 71 in the epidermis which were modified as a consequence of photoageing but did not differ significantly in relative abundance (measured by MS1 ion intensity). By applying peptide location fingerprinting to published MS data sets, (identifying biomarker candidates including collagen V and versican in ageing tendon) we demonstrate the potential of the MPLF webtool for biomarker discovery.

Hemidesmosome-Related Keratin Filament Bundling and Nucleation.

The epithelial cytoskeleton encompasses actin filaments, microtubules, and keratin intermediate filaments. They are interconnected and attached to the extracellular matrix via focal adhesions and hemidesmosomes. To study their interplay, we inhibited actin and tubulin polymerization in the human keratinocyte cell line HaCaT by latrunculin B and nocodazole, respectively. Using immunocytochemistry and time-lapse imaging of living cells, we found that inhibition of actin and tubulin polymerization alone or in combination induced keratin network re-organization albeit differently in each situation. Keratin filament network retraction towards the nucleus and formation of bundled and radial keratin filaments was most pronounced in latrunculin-B treated cells but less in doubly-treated cells and not detectable in the presence of nocodazole alone. Hemidesmosomal keratin filament anchorage was maintained in each instance, whereas focal adhesions were disassembled in the absence of actin filaments. Simultaneous inhibition of actin and tubulin polymerization, therefore, allowed us to dissect hemidesmosome-specific functions for keratin network properties. These included not only anchorage of keratin filament bundles but also nucleation of keratin filaments, which was also observed in migrating cells. The findings highlight the fundamental role of hemidesmosomal adhesion for keratin network formation and organization independent of other cytoskeletal filaments pointing to a unique mechanobiological function.

Efficacy of Long-term Use of 0.3% Sodium Hyaluronate Eye Drops for Traumatic Corneal Abrasion: A Randomized Controlled, Pilot Trial.

PURPOSE: Traumatic corneal abrasion (TCA) causes damage to both corneal epithelium and the underlying hemidesmosomal junctions. Delayed recovery of hemidesmosomal junctions causes symptomatic episodes. However, there is no recommended treatment for recovery of hemidesmosomal junctions, indicating that a blank period exists in TCA treatment. In this study, the efficacy of long-term use of sodium hyaluronate on recovery of hemidesmosomal junctions during the blank period in TCA healing was investigated. METHODS: In this prospective, randomized control pilot study, 60 patients with TCA were enrolled. The patients were randomized 1:1 to receive 0.3% sodium hyaluronate eye drops for 3 months (HA group) or observation alone (control group) after complete corneal epithelium recovery. The primary and secondary outcomes were the cumulative incidence of major and minor symptomatic episodes during a 12-month follow-up, respectively. RESULTS: Fifty-six subjects (29 in the HA group and 27 in the control group) completed the 12-month follow-up. The 12-month cumulative incidence rate of major symptomatic episodes was 20.7% in the HA group and 18.5% in the control group. No significant difference was found between the 2 groups (P = 0.838). The 12-month cumulative incidence rate of minor symptomatic episodes was 48.3% and 37.0% in the HA and control groups, respectively, with no significant difference (P = 0.397). CONCLUSIONS: Approximately one-fifth of patients with TCA experience major symptomatic episodes again within their 1-year follow-up. Long-term use of sodium hyaluronate in the period of recovery of hemidesmosomal junctions has no benefit to it.

A Hemidesmosome-to-Cytoplasm Translocation of Small Heat Shock Proteins Provides Immediate Protection against Heat Stress.

Small heat shock proteins (sHSPs) are important regulators for maintaining protein homeostasis in response to stresses. However, the strategies used by constitutively expressed sHSPs to control their activities in normal versus stressed conditions are still not fully understood. Here we show that the constitutively expressed HSP-43 in the C. elegans epidermis is stored within the basal C. elegans hemidesmosomes (CeHDs) under normal conditions and is rapidly released into the cytoplasm to exert protective functions upon heat stress. The association with CeHDs protects HSP-43 from degradation or toxic cytoplasmic aggregation in unstressed situations. Our study reveals a rapid and specific translocation-based heat shock response of the sHSPs working through hemidesmosomes. It refreshes our knowledge about the stress-resistant functions of stable cellular adhesions and provides insight into the activity-control strategies of sHSPs. It also underlines the importance of structural integrity of the cells on stress resistance and damage control.

Keratinocyte-Specific Peptide-Based Surfaces for Hemidesmosome Upregulation and Prevention of Bacterial Colonization.

Percutaneous devices like orthopedic prosthetic implants for amputees, catheters, and dental implants suffer from high infection rates. A critical aspect mediating peri-implant infection of dental implants is the lack of a structural barrier between the soft tissue and the implant surface which could impede bacteria access and colonization of exposed implant surfaces. Parafunctional soft tissue regeneration around dental implants is marked by a lack of hemidesmosome formation and thereby weakened mechanical attachment. In response to this healthcare burden, a simultaneously hemidesmosome-inducing, antimicrobial, multifunctional implant surface was engineered. A designer antimicrobial peptide, GL13K, and a laminin-derived peptide, LamLG3, were coimmobilized with two different surface fractional areas. The coimmobilized peptide surfaces showed antibiofilm activity against Streptococcus gordonii while enhancing proliferation, hemidesmosome formation, and mechanical attachment of orally derived keratinocytes. Notably, the coatings demonstrated specific activation of keratinocytes: the coatings showed no effects on gingival fibroblasts which are known to impede the quality of soft tissue attachment to dental implants. These coatings demonstrated stability and retained activity against mechanical and thermochemical challenges, suggesting their intraoral durability. Overall, these multifunctional surfaces may be able to reduce peri-implantitis rates and enhance the success rates of all percutaneous devices via strong antimicrobial activity and enhanced soft tissue attachment to implants.

Composition of hemidesmosomes in basal keratinocytes of normal buccal mucosa and oral lichen planus.

Oral lichen planus (OLP) is a chronic inflammatory disease displaying ultrastructural disturbances in epithelial hemidesmosomes. The expression of several key hemidesmosomal components in OLP as well as in normal buccal mucosa is, however, unknown. The aim of the study was therefore to examine intracellular and extracellular components involved in hemidesmosomal attachment, in OLP (n = 20) and in normal buccal mucosa (n = 10), by immunofluorescence. In normal buccal mucosa, laminin-α3γ2, integrin-α6ß4, CD151, collagen α-1(XVII) chain, and dystonin showed linear expression along the basal membrane, indicating the presence of type I hemidesmosomes. Plectin stained most epithelial cell membranes and remained unphosphorylated at S4642. In OLP, most hemidesmosomal molecules examined showed disturbed expression consisting of discontinuous increases, apicolateral location, and/or intracellular accumulation. Plectin showed S4642-phosphorylation at the basement membrane, and deposits of laminin-α3 and laminin-γ2 were found within the connective tissue. The disturbed expression of hemidesmosomal proteins in OLP indicates deficient attachment of the basal cell layer, which can contribute to detachment and cell death of basal keratinocytes seen in the disease.

Comparative interactomics analysis reveals potential regulators of α6ß4 distribution in keratinocytes.

The integrin α6ß4 and cytoskeletal adaptor plectin are essential components of type I and type II hemidesmosomes (HDs). We recently identified an alternative type II HD adhesion complex that also contains CD151 and the integrin α3ß1. Here, we have taken a BioID proximity labeling approach to define the proximity protein environment for α6ß4 in keratinocytes. We identified 37 proteins that interacted with both α6 and ß4, while 20 and 78 proteins specifically interacted with the α6 and ß4 subunits, respectively. Many of the proximity interactors of α6ß4 are components of focal adhesions (FAs) and the cortical microtubule stabilizing complex (CMSC). Though the close association of CMSCs with α6ß4 in HDs was confirmed by immunofluorescence analysis, CMSCs have no role in the assembly of HDs. Analysis of the ß4 interactome in the presence or absence of CD151 revealed that they are strikingly similar; only 11 different interactors were identified. One of these was the integrin α3ß1, which interacted with α6ß4 more strongly in the presence of CD151 than in its absence. These findings indicate that CD151 does not significantly contribute to the interactome of α6ß4, but suggest a role of CD151 in linking α3ß1 and α6ß4 together in tetraspanin adhesion structures.