Abstract
In order to isolate genes that are upregulated in human keratinocytes upon loss of cell/matrix contact, a subtractive cDNA library was constructed from dispase-treated versus untreated keratinocytes. Among the cloned cDNAs one was pKe#192 having an open reading frame of 411 bp. By database analysis pKe#192 was found to be identical with the gene "MAP17" previously isolated from human kidney. Kyte-Doolittle hydrophobicity analyzes showed a hydrophobic amino terminus of 13 amino acids, a transmembrane region and a 61 amino acid hydrophilic carboxy-terminus and two potential phosphorylation sites. In order to study regulation of pKe#192/MAP17 expression, RNA was extracted from resting human keratinocytes and from keratinocytes stimulated by dispase-induced detachment from the growth substratum. Reverse transcription polymerase chain reaction did not reveal specific mRNA in resting keratinocytes, whereas mRNA was detectable after detachment. For further characterization poly- and monoclonal antibodies were generated against a recombinant fusion protein. Immunohistologic studies using the mono- and polyclonal antibodies showed staining of the upper layers of the stratum granulosum in normal human epidermis. The staining was colocalized with involucrin. Immunhistologic staining of frozen sections derived from lesional skin of bullous pemphigoid und pemphigus vulgaris indicated that pKe#192/MAP17 was upregulated in the epidermis adjacent to the blister. Taken together, the data demonstrate that pKe#192/MAP17 is expressed in keratinocytes and may be involved in epidermal physiology and pathology. bullous diseases/differentiation.
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