The skin produces urocortin

Since the skin produces POMC peptides, in the present work we investigated local production of urocortin, a peptide related to CRH, the normal endogen ous stimulant for POMC. Urocortin immunoreactivity was detected by direct R IA in extracts of human skin, mouse skin (C57BL-6 strain), cultured cells f rom established lines of human melanoma and squamous cell carcinoma, human keratinocytes (HaCaT), and hamster melanomas. Addition of a reverse phase h igh performance liquid chromatography step before the RIA. confirmed the pr esence of urocortin, as the immunoreactivity eluted at the same retention t ime as urocortin standard in extracts from HaCaT keratinocytes and mouse sk in. Using the tandem technique of liquid chromatography-mass spectrometry, we identified a peptide with the same mass and retention time as the urocor tin standard in human skin extracts. The urocortin antigen could be immunol ocalized to normal keratinocytes of the epidermis and hair follicle, epithe lium of sweat and sebaceous glands, dermal skeletal muscle, and nevocytes; it was also detected in melanoma and basal cell carcinoma cells. RT-PCR amp lification of ribonucleic acid from human skin, cultured keratinocytes, and melanoma cells showed a 145-kb fragment fi om the coding region of exon 2 of the urocortin gene in all of the tested sources. Lastly, sequencing of t he amplified fragment confirmed 100% homology with the known sequence of th e urocortin gene. In conclusion, we now demonstrate that human skin and mou se skin as well as cultured keratinocytes and melanoma cells exhibit functi onal expression of the urocortin gene with actual production of urocortin p eptide.