ABSTRACT
The main limitation of using topical corticosteroids in dermatology is their atrophic effects on the skin. We have previously proposed a molecular platform composed of CD44, EGFR, and hyaluronate synthase (HAS) that is functionally defective in dermatoporosis, a chronic cutaneous insufficiency/fragility syndrome. In this study, we explored the molecular mechanisms of the skin atrophy induced by corticosteroids. We observed an important skin atrophy and a significant decrease of hyaluronic acid (HA), its main cell surface receptor CD44, and F-actin in mouse skin treated with topical clobetasol propionate (CP). Human keratinocytes exposed to CP showed an impaired HA secretion and diminished expression of CD44 and HAS3. CP also abolished filopodia of keratinocytes exposed to CP together with a redistribution of CD44 and F-actin depolymerization. We also show that HA fragments of intermediary size (HAFi) induced keratinocyte filopodia and protected them against CP. Topical HAFi induced hyperplasia in mouse epidermis and prevented CP-induced atrophy. Our results suggest that a CD44/EGFR/HAS platform associated with F-actin and filopodia of keratinocytes is the target of corticosteroids for their atrophogenic effects. These observations may lead to the development of new treatment and prevention strategies for corticosteroid-induced skin atrophy.
Subject(s)
Clobetasol/pharmacology , Dermatologic Agents/pharmacology , Epidermis/drug effects , Hyaluronic Acid/pharmacology , Keratinocytes/drug effects , Animals , Atrophy/chemically induced , Atrophy/metabolism , Atrophy/pathology , Cell Line, Transformed , Drug Interactions , Epidermis/metabolism , Epidermis/pathology , Glucocorticoids/pharmacology , Glucuronosyltransferase/metabolism , Homeostasis/drug effects , Hyaluronan Receptors/metabolism , Hyaluronan Synthases , Hyaluronic Acid/chemistry , Hymecromone/analogs & derivatives , Hymecromone/pharmacology , Keratinocytes/metabolism , Keratinocytes/pathology , Mice , Mice, Hairless , Molecular Weight , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Pseudopodia/drug effects , Pseudopodia/metabolism , Pseudopodia/pathology , Skin Cream/pharmacology , Viscosupplements/chemistry , Viscosupplements/pharmacologyABSTRACT
BACKGROUND: Paraneoplastic pemphigus (PNP) is a devastating autoimmune blistering disease, involving mucocutaneous and internal organs, and associated with underlying neoplasms. PNP is characterized by the production of autoantibodies targeting proteins of the plakin and cadherin families involved in maintenance of cell architecture and tissue cohesion. Nevertheless, the identity of an antigen of Mr 170,000 (p170), thought to be critical in PNP pathogenesis, has remained unknown. METHODOLOGY/PRINCIPAL FINDINGS: Using an immunoprecipitation and mass spectrometry based approach, we identified p170 as alpha-2-macroglobuline-like-1, a broad range protease inhibitor expressed in stratified epithelia and other tissues damaged in the PNP disease course. We demonstrate that 10 PNP sera recognize alpha-2-macroglobuline-like-1 (A2ML1), while none of the control sera obtained from patients with bullous pemphigoid, pemphigus vulgaris, pemphigus foliaceus and normal subjects does. CONCLUSIONS/SIGNIFICANCE: Our study unravels a broad range protease inhibitor as a new class of target antigens in a paraneoplastic autoimmune multiorgan syndrome and opens a new challenging investigation avenue for a better understanding of PNP pathogenesis.
Subject(s)
Autoantibodies/immunology , Autoantigens/immunology , Autoimmune Diseases/immunology , Protease Inhibitors/immunology , alpha-Macroglobulins/immunology , Autoantigens/chemistry , Autoantigens/metabolism , Autoimmune Diseases/blood , Cell Line , Culture Media , Epidermis/metabolism , Gene Expression Regulation , Humans , Immunoprecipitation , Keratinocytes/cytology , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Protein Structure, Tertiary , Reducing Agents/pharmacology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Transfection , alpha-Macroglobulins/chemistry , alpha-Macroglobulins/metabolismABSTRACT
Desmosomes are intercellular adhesive complexes that anchor the intermediate filament cytoskeleton to the cell membrane in epithelia and cardiac muscle cells. The desmosomal component desmoplakin plays a key role in tethering various intermediate filament networks through its C-terminal plakin repeat domain. To gain better insight into the cytoskeletal organization of cardiomyocytes, we investigated the association of desmoplakin with desmin by cell transfection, yeast two-hybrid, and/or in vitro binding assays. The results indicate that the association of desmoplakin with desmin depends on sequences within the linker region and C-terminal extremity of desmoplakin, where the B and C subdomains contribute to efficient binding; a potentially phosphorylatable serine residue in the C-terminal extremity of desmoplakin affects its association with desmin; the interaction of desmoplakin with non-filamentous desmin requires sequences contained within the desmin C-terminal rod portion and tail domain in yeast, whereas in in vitro binding studies the desmin tail is dispensable for association; and mutations in either the C-terminus of desmoplakin or the desmin tail linked to inherited cardiomyopathy seem to impair desmoplakindesmin interaction. These studies increase our understanding of desmoplakin-intermediate filament interactions, which are important for maintenance of cytoarchitecture in cardiomyocytes, and give new insights into the molecular basis of desmoplakin- and desmin-related human diseases.
Subject(s)
Cardiomyopathies/genetics , Desmin/genetics , Desmoplakins/genetics , Animals , Animals, Newborn , Cells, Cultured , Desmin/metabolism , Desmoplakins/metabolism , Humans , Mutant Proteins/metabolism , Mutation , Myocytes, Cardiac/metabolism , Protein Binding , Protein Structure, Tertiary , Rats , Rats, Wistar , Sequence Analysis, Protein , Tissue Distribution , Transfection , Two-Hybrid System TechniquesABSTRACT
The bullous pemphigoid antigen 1 (BP230) and desmoplakin (DP) are members of the plakin protein family of cytolinkers. Despite their homology, their COOH termini selectively bind distinct intermediate filaments (IFs). We studied sequences within their COOH termini required for their interaction with the epidermal keratins K5/K14, the simple epithelial keratins K8/K18, and type III IF vimentin by yeast three-hybrid, cell transfection, and overlay assays. The results indicate that BP230 interacts with K5/K14 but not with K8/K18 or vimentin via a region encompassing both the B and C subdomains and the COOH extremity, including a COOH-terminal eight-amino-acid stretch. In contrast, the C subdomain with the COOH-terminal extremity of DP interacts with K5/K14 and K8/K18, and its linker region is able to associate with K8/K18 and vimentin. Furthermore, the potential of DP to interact with IF proteins in yeast seems to be regulated by phosphorylation of Ser 2849 within its COOH terminus. Strikingly, BP230 and DP interacted with cytokeratins only when both type I and type II keratins were present. The head and tail domains of K5/K14 keratins were dispensable for their interaction with BP230 or DP. On the basis of our findings, we postulate that (1) the binding specificity of plakins for various IF proteins depends on their linker region between the highly homologous B and C subdomains and their COOH extremity and (2) the association of DP and BP230 with both epidermal and simple keratins is critically affected by the tertiary structure induced by heterodimerization and involves recognition sites located primarily in the rod domain of these keratins.
