An Immunocytochemical Approach to the Study of β‐Endorphin Production in Human Keratinocytes using Confocal Microscopy

ABSTRACT: Proopiomelanocortin (POMC) is a protein that is posttranslationally processed to yield POMC peptides. The main site of POMC expression is the anterior pituitary lobe but many other sources have been identified. There is evidence that the skin produces POMC peptides, although their roles have not yet been defined. In the skin, regulation of POMC gene expression is known to be hair‐cycle dependent, and it is localized to the sebaceous gland. In particular, β‐endorphin, a POMC peptide, has been shown to be modulated by TPA, IL‐1α, and ultraviolet radiation in keratinocytes. These results were obtained by examination of POMC mRNA levels using the Northern blot method; β‐endorphin protein production by the Western blot method on cultured cells; and immunocytochemistry for tissue preparations. This report represents an approach to use immunocytochemistry to quantify β‐endorphin production in cultured human keratinocytes. Additionally, we examined whether exposure to 20 mJ ultraviolet B radiation (UVB) and/or UVA could influence β‐endorphin production in these cells. Keratinocytes were grown in monolayers, in serum‐free medium, fixed, and incubated with antiserum to whole synthetic β‐endorphin. Fluorescence microscopy was performed with a confocal laser scanning microscope. The integrated level of fluorescence was evaluated in n= 18 ± 8 individual cells, and this was assumed to be proportional to β‐endorphin content. High variability was observed in the fluorescence intensity among cells. No significant differences between control and UVB‐ or UVA+UVB‐treated cells was found. Similar results were produced by using brefeldin A, a compound that disrupts the secretory pathway, eliminating the possibility that the absence of a difference between β‐endorphin content in the treated and control cells was due to secretion of the peptide into the medium. We conclude that: (1) β‐endorphin or β‐endorphin‐like peptides are produced in human keratinocytes and are readily detected by immunocytochemistry; (2) under the conditions tested, UVA and/or UVB did not increase β‐endorphin‐like immunoreactivity in these cells.