Characterization and modulation of sex steroid metabolizing activity in normal human keratinocytes in primary culture and HaCaT cells.

Skin, the largest organ of the human body, synthesizes active sex steroids from adrenal C19 precursor steroids. Normal human breast epidermal keratinocytes in primary culture were used to evaluate the enzymatic activities responsible for the formation and degradation of active androgens and estrogens during keratinocyte differentiation. Enzymatic activities, including 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase (3beta-HSD), 17beta-hydroxysteroid dehydrogenase (17beta-HSD), 5alpha-reductase and 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) were measured using [3H] steroids as substrates. After 10-60 days in culture, no 3beta-HSD activity was detected, but all other activities were measured, demonstrating the ability of keratinocytes to convert androstenedione (4-DIONE) into the potent androgen dihydrotestosterone (DHT). Furthermore, marked changes in enzymatic activity were observed during cell differentiation: 17beta-HSD was first detected during the third week of culture, the level of activity reaching a peak during the fourth week. This peak was followed by a progressive decrease during keratinization. On the other hand, 5alpha-reductase and 3alpha-HSD activities were first detected during the fourth week of culture. The enzymatic activities involved in the formation and degradation of sex steroids were also characterized in the immortalized human keratinocyte cell line HaCaT. It was then found that HaCaT cells possess a pattern of steroid metabolizing enzymes similar to that of human epidermal keratinocytes in culture. Since glucocorticoids are known to exert potent pharmacological effects on the skin, the effect of dexamethasone (DEX) on cell proliferation and enzymatic activities was determined using HaCaT cells. DEX causes a 55% decrease in HaCaT cell proliferation (IC50: 10nM) whereas DEX caused a three- to five-fold stimulation of oxidative 17beta-HSD activity in intact cells in culture (ED50: 30 nM) and this stimulatory effect was competitively blocked by the glucocorticoid antagonist RU486. A four-fold increase in type 2 17beta-HSD mRNA levels was also observed as measured by real-time PCR, correlating with the increase in oxidative activity. No effect of DEX on the other enzymatic activities (3beta-HSD, 5alpha-reductase, and 3alpha-HSD) was observed. Since increased levels of inflammatory cytokines have been detected in some skin diseases then these cytokines might play a role in the differentiation of keratinocytes. In this regard, we found that interleukin-4 (IL-4) induced the expression of 3beta-HSD in HaCaT cells, thus allowing the cells to produce a different set of sex steroids from adrenal C19 precursors. The present data thus indicate that HaCaT cells are a useful model to further study the regulation of the enzymes involved in the metabolism of sex steroids in keratinocytes.