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A monoclonal antikeratin antibody, EKH4, was produced from a hybridoma cell line which was established by fusing P3X63SAg8 mouse myeloma cells with spleen cells of mice immunized with human trichilemmoma cells. Immunoblot analysis showed that EKH4 antibody reacts predominantly with 50 kilodalton keratin polypeptide in normal epidermis. By indirect immunofluorescence and immunoperoxidase techniques, EKH4 antibody reacted with the lower 2-3 cell layers of the epidermis as well as most cells of pilosebaceous follicle of human and animal skin. Tumor cells of human basal cell epitheliomas and squamous cell carcinomas were also stained with this antibody. The staining was much more regular and intense compared with an available monoclonal antikeratin antibody, AE1. In the lesion of epidermal proliferative disorders, such as psoriasis and actinic keratosis, the entire epidermis instead of the lower layers was stained with EKH4 antibody. Normal skin overlying or adjacent to epithelial tumors also showed positive staining in the entire epidermis. By using indirect immunoperoxidase technique, EKH4 also stained alcohol-fixed, paraffin-embedded tissue sections.

The transglutaminases of cultured normal and transformed human keratinocytes (line SV-K14) are characterized. Both cell types display two forms of the enzyme, one of which is cytosoluble (TGc) and the other which is associated with the plasma membrane (TGm). Normal keratinocytes contain predominantly TGm, and SV-K14 cells mainly TGc. The ratio of TGm to TGc can be modulated by the culture conditions and correlates with the competence of the cells to form a cornified envelope. TGm and TGc differ in their biochemical and immunological properties. SDS electrophoresis reveals apparent molecular weights of 92 and 85 kD, respectively. Only the activity of TGc is inhibited in the presence of guanosine 5'-triphosphate. Their response to Ca2+ is different: TGc exhibits a sigmoidal activation kinetics with an A50 value of about 200 microM, whereas the kinetics for TGm is hyperbolic with an A50 value of 75 microM. TGm reacts with a monoclonal antibody raised against epidermal "particulate" transglutaminase, and TGc with a polyclonal antibody raised against guinea pig liver transglutaminase. These reactions are very specific and no cross-reaction occurs. The coappearance of TGm with a proteolytic fragment (Mr 82,000) in the cytosol and intracellular particulate fraction of normal human keratinocytes is probably a preparation artifact.

Terminal differentiation of the keratinocytes (cornification) has been linked to a restricted supply of retinol. Retinol is distributed to target cells by the retinol-binding protein (RBP), which circulates in the plasma in complex with transthyretin (TTR). In this study we have addressed the question of retinol delivery to the epidermis via RBP. Retinol radiobinding assays, affinity chromatography with TTR coupled to Sepharose beads, polyacrylamide gel electrophoresis, and immunoblotting techniques were used to show that epidermal extracts contain retinol binding sites with no affinity for TTR. Immunoreactive RBP was detected in the epidermal extracts. The RBP in the epidermis was in the apoform (without retinol) in contrast to serum where the majority of RBP is in the holoform (with retinol). Epidermal RBP was converted in vitro into the holoform only after addition of 20 times more retinol, which was needed to reconstitute holoforms of RBP in dermal extracts, human buccal mucosal extracts, and delipidized normal serum or purified delipidized RBP. Moreover, several immunoreactive RBP bands (possibly degradation products) were identified in the epidermal but not in dermal extracts. Retinol-binding protein from nonkeratinizing human oral mucosa showed different immunoblotting patterns when compared to epidermal RBP. These results suggest that degradation of RBP within the epidermis may result in a decreased retinol supply to the keratinocytes, and may lead to the cornification of the epidermis.

The epidermolysis bullosa acquisita antigen is a major constituent of the basement membrane zone beneath stratified squamous epithelium. The antigen which is recognized in extracts of skin basement membrane by Western blot analysis with polyclonal or monoclonal antiepidermolysis bullosa acquisita antigen antibodies as 2 chains (a major chain of 290,000 and a minor chain of 145,000) has a native molecular weight over 800,000. Both epidermolysis bullosa acquisita antigen chains contain carbohydrate and the 290K chain is sensitive to collagenase.