The 105-kDa Basement Membrane Autoantigen p105 Is N-Terminally Homologous to a Tumor-Associated Antigen

Certain constitutive skin basement membrane components, such as bullous pemphigoid antigens and epidermolysis bullosa acquisita antigen, were discovered because they were targeted by an autoimmune reaction. We aimed to purify and characterize a 105-kDa skin basement membrane protein termed p105 recognized by autoantibodies (anti-p105) from patients with a unique immune-mediated subepidermal blistering skin disease. A simian virus 40-transformed human fibroblast cell line that synthesizes and secretes p105 was utilized as the protein source. p105 was partially purified by salt-gradient fractionation of serum-free conditioned medium through a Mono Q anion-exchange column and by examining each fraction with protein staining and immunoblotting against anti-p105. p105 was isolated from polyacrylamide gel electrophoresis gels, blotted onto polyvinylidene difluoride membrane, and subjected to protein microsequencing. The 20 microsequenced N-terminal amino acids exhibited no homology to known basement membrane proteins but exhibited a 70% homology to a 90-kDa tumor-associated antigen. Antibodies raised against a peptide generated from these amino acid sequences reacted to a 105-kDa western-blotted keratinocyte and fibroblast protein and a basement membrane component. p105 resisted digestion by glycosidases chondroitinase ABC, neuraminidase, and N-glycosidase F but was cleaved by protease V8 to antigenic fragments of 22kDa and 14kDa. The synthesis of p105 was inhibited by cycloheximide. We conclude that p105 is a unique basement membrane component produced by both keratinocytes and fibroblasts

Dibutyryl Cyclic AMP Modulates Keratinocyte Migration Without Alteration on Integrin Expression

Cyclic adenosine monophosphate (cAMP) has long been regarded as a second messenger and a regulator of human keratinocyte proliferation. It has been demonstrated that cAMP inhibits keratinocyte proliferation when used at high concentrations. Nevertheless, new recent reports have demonstrated that cAMP may stimulate or inhibit keratinocyte growth depending upon the concentration used. Studies to examine the influence of cAMP upon the migration of other cell types have been contradictory. To determine the direct effect of dibutyryl cAMP (DBcAMP) upon human keratinocyte migration, we used a quantitative locomotion assay using a wide range of DBcAMP concentrations. We found a bi-phasic effect of DBcAMP on keratinocyte migration across connective tissue matrices. Keratinocyte locomotion on the matrices was promoted at 10-5 M and 10-6 M of DBcAMP, but not at higher or lower concentrations. Timecourse experiments demonstrated that the effect of DBcAMP on keratinocyte locomotion and proliferation occurred independently. Fluorescence-activated cell sorter analysis demonstrated that the effect of DBcAMP on the migration of human keratinocytes was independent from the modulation of integrin receptors. Although the cellular mechanisms by which DBcAMP promotes keratinocyte migration is unclear, the addition of DBcAMP or TPA to keratinocyte cultures enhanced the synthesis of a 92-kDa metalloproteinase in association with enhanced cellular migration. These observations suggest a possible link between metalloproteinase expression and cellular migration

NC1 Domain of Type VII Collagen Binds to the β3 Chain of Laminin 5 Via a Unique Subdomain Within the Fibronectin-Like Repeats

Type VII collagen, the major component of anchoring fibrils, consists of a central collagenous triple-helical domain flanked by two noncollagenous, globular domains, NC1 and NC2. Approximately 50% of the molecular mass of the molecule is consumed by the NC1 domain. We previously demonstrated that NC1 binds to various extracellular matrix components including a complex of laminin 5 and laminin 6 (Chen et al. 1997a). In this study, we examined the interaction of NC1 with laminin 5 (a component of anchoring filaments). Both authentic and purified recombinant NC1 bound to human and rat laminin 5 as measured by enzyme-linked immunosorbant assay and by binding of 125I-radiolabeled NC1 to laminin 5-coated wells, but not to laminin 1 or albumin. NC1 bound predominantly to the β3 chain of laminin 5, but also to the γ2 chain when examined by a protein overlay assay. The binding of 125I-NC1 to laminin 5 was inhibited by a 50-fold excess of unlabeled NC1 or de-glycosylated NC1, as well as a polyclonal antibody to laminin 5 or a monoclonal antibody to the β3 chain. In contrast, the NC1–laminin 5 interaction was not affected by a monoclonal antibody to the α3 chain. Using NC1 deletion mutant recombinant proteins, a 285 AA (residues 760–1045) subdomain of NC1 was identified as the binding site for laminin 5. IgG from an epidermolysis bullosa acquisita serum containing autoantibodies to epitopes within NC1 that colocalized with the laminin 5 binding site inhibited the binding of NC1 to laminin 5. Thus, perturbation of the NC1–laminin 5 interaction may contribute to the pathogenesis of epidermolysis bullosa acquisita

PKCδ Clustering at the Leading Edge and Mediating Growth Factor-Enhanced, but not ECM-Initiated, Dermal Fibroblast Migration

We have previously shown that the immobilized extracellular matrices (ECMs) initiate cell migration and soluble growth factors (GFs) further enhance ECM-initiated cell migration. GFs alone cannot initiate cell migration. To further investigate the specificity of the two signaling mechanisms, we focused on the protein kinase C (PKC) family genes in primary human dermal fibroblasts (DFs). We here show that platelet-derived growth factor-BB (PDGF-BB) strongly stimulates membrane translocation and leading edge clustering of protein kinase Cδ (PKCδ). In contrast, attachment to collagen matrix alone does not cause the translocation. Although the kinase function of PKCδ is dispensable for initial membrane translocation, it is critical for its sustained presence at the cells's leading edge. Blockade of endogenous PKCδ signaling with dominant-negative kinase-defective PKC (PKCδ-KD) or PKCδ-small interfering RNA (siRNA) completely inhibited PDGF-BB-stimulated DF migration. In contrast, neither PKCδ-KD nor PKCδ-siRNA affected collagen-induced initiation of DF migration. Overexpression of a constitutively activated PKCδ (PKCδ-R144/145A) partially mimics the effect of PDGF-BB. However, PKCδ-KD, PKCδ-siRNA, or PKCδ-R144/145A does not affect PDGF-BB-stimulated activation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase1/2, or c-Jun N-terminal kinase. Instead, inhibition of PKCδ blocks PDGF-BB-stimulated activation of signal transducer and activator of transcription 3 (Stat3). This study unveiled the specificity of PKCδ in the control of DF migration

Evidence that Anti-Type VII Collagen Antibodies Are Pathogenic and Responsible for the Clinical, Histological, and Immunological Features of Epidermolysis Bullosa Acquisita

Epidermolysis bullosa acquisita (EBA) is an autoimmune blistering disease characterized by autoantibodies to type VII (anchoring fibril) collagen. Therefore, it is a prototypic autoimmune disease defined by a well-known autoantigen and autoantibody. In this study, we injected hairless immune competent mice with purified immunoglobulin G (IgG) fraction of serum from rabbits immunized with the non-collagenous amino-terminal domain (NC1) of human type VII collagen, the domain known to contain immunodominant epitopes. As a control, identical mice were injected with the IgG fraction of serum from non-immunized rabbits. Mice injected with immune IgG developed subepidermal skin blisters and erosions, IgG deposits at the epidermal-dermal junction of their skin, and circulating anti-NC1 antibodies in their serum-all features reminiscent of patients with EBA. Similar concentrations of control IgG purified from normal rabbits did not induce disease in the mice. These findings strongly suggest that autoantibodies that recognize human type VII collagen in EBA are pathogenic. This murine model, with features similar to the clinical, histological, and immunological features of EBA, will be useful for the fine dissection of immunopathogenic mechanisms in EBA and for the development of new therapeutic interventions

Laminin-6 and Laminin-5 Are Recognized by Autoantibodies in a Subset of Cicatricial Pemphigoid

We characterized basement membrane zone (BMZ) autoantigens targeted by autoantibodies (AAb) from patients with cicatricial pemphigoid. Serum from a patient with severe oral cicatricial pemphigoid contained IgG anti-BMZ AAb. The AAb labeled a lower BMZ component on salt-split skin and localized to the lower lamina lucida/lamina densa by direct and indirect immunoelectron microscopy (HEM) but did not label blood vessels. The AAb did not react with EHS laminin-1 and type IV collagen, pepsinized human type IV collagen, recombinant entactin, or NC1 domain of type VII collagen by dot blotting and western blotting. We focused our studies on the laminin family, as laminin-5 was identified as an autoantigen in cicatricial pemphigoid. Culture-conditioned media from normal keratinocytes (containing laminin-6 and laminin-5) and JEB keratinocytes (containing laminin-6 but not laminin-5) were studied by western blotting. Under nonreducing conditions, the patient's AAb recognized a 600-kDa protein (laminin-6) intensely and a 400-kDa protein (laminin-5) weakly in normal keratinocyte medium even though abundant laminin-5 was present. In JEB keratinocyte medium, however, the 600-kDa protein (laminin-6) alone was recognized by the patient's AAb. The AAb also immunolabeled BMZ of JEB skin that lacked laminin-5. The AAb from this patient and two other patients with anti-laminin-5 cicatricial pemphigoid immunoprecipitated both laminin-6 an4 laminin-5. Taken together, the results of IEM, non-reducing western blotting, immunoprecipitation, and JEB skin BMZ immunolabeling indicate that laminin-6, as well as laminin-5, is identified by the AAb from a subset of cicatricial pemphigoid patients. We propose the name “anti-laminin cicatricial pemphigoid” for this subset

Increased Frequency of HLA-DR2 in Patients with Autoantibodies to Epidermolysis Bullosa Acquisita Antigen: Evidence that the Expression of Autoimmunity to Type VII Collagen Is HLA Class II Allele Associated

Epidermolysis bullosa acquisita (EBA) is a chronic blistering disease characterized by circulating and tissue bound IgG auto-antibodies to the basement membrane zone (BMZ) of stratified squamous epithelium. Recent studies have shown that antibodies recognize epitopes present in the noncollagenous carboxyl-terminal domain of type VII collagen, a BMZ matrix protein. Antibodies with identical specificity also have been detected in patients with the rare blistering disease, bullous systemic lupus erythematosus (bullous SLE), suggesting EBA and bullous SLE are immunologically related diseases. In this study we determined the major histo-compatibility antigen types of 29 EBA patients and 6 patients with bullous SLE. Analysis of the results showed HLA-DR2 was significantly increased in both black EBA patients, P = 0.013 (corrected, RR = 4.8) and whit EBA patients, P = 0.0008 (corrected, RR = 13.1). Five of the six bullous SLE patients also were positive for the DR2 antigen, P = 0.009. These results show the expression of autoimmunity to type VII collagen is HLA class II allele associated and that EBA and bullous SLE are immunogentically related diseases

Gentamicin induces LAMB3 nonsense mutation readthrough and restores functional laminin 332 in junctional epidermolysis bullosa

Herlitz junctional epidermolysis bullosa (H-JEB) is an incurable, devastating, and mostly fatal inherited skin disease for which there is only supportive care. H-JEB is caused by loss-of-function mutations in LAMA3, LAMB3, or LAMC2, leading to complete loss of laminin 332, the major component of anchoring filaments, which mediate epidermal-dermal adherence. LAMB3 (laminin \u3b23) mutations account for 80% of patients with H-JEB, and 3c95% of H-JEB\u2013associated LAMB3 mutations are nonsense mutations leading to premature termination codons (PTCs). In this study, we evaluated the ability of gentamicin to induce PTC readthrough in H-JEB laminin \u3b23-null keratinocytes transfected with expression vectors encoding eight different LAMB3 nonsense mutations. We found that gentamicin induced PTC readthrough in all eight nonsense mutations tested. We next used lentiviral vectors to generate stably transduced H-JEB cells with the R635X and C290X nonsense mutations. Incubation of these cell lines with various concentrations of gentamicin resulted in the synthesis and secretion of full-length laminin \u3b23 in a dose-dependent and sustained manner. Importantly, the gentamicin-induced laminin \u3b23 led to the restoration of laminin 332 assembly, secretion, and deposition within the dermal/epidermal junction, as well as proper polarization of \u3b16\u3b24 integrin in basal keratinocytes, as assessed by immunoblot analysis, immunofluorescent microscopy, and an in vitro 3D skin equivalent model. Finally, newly restored laminin 332 corrected the abnormal cellular phenotype of H-JEB cells by reversing abnormal cell morphology, poor growth potential, poor cell-substratum adhesion, and hypermotility. Therefore, gentamicin may offer a therapy for H-JEB and other inherited skin diseases caused by PTC mutations