The significance of CYP11A1 expression in skin physiology and pathology.

CYP11A1, a member of the cytochrome P450 family, plays several key roles in the human body. It catalyzes the first and rate-limiting step in steroidogenesis, converting cholesterol to pregnenolone. Aside from the classical steroidogenic tissues such as the adrenals, gonads and placenta, CYP11A1 has also been found in the brain, gastrointestinal tract, immune systems, and finally the skin. CYP11A1 activity in the skin is regulated predominately by StAR protein and hence cholesterol levels in the mitochondria. However, UVB, UVC, CRH, ACTH, cAMP, and cytokines IL-1, IL-6 and TNFα can also regulate its expression and activity. Indeed, CYP11A1 plays several critical roles in the skin through its initiation of local steroidogenesis and specific metabolism of vitamin D, lumisterol, and 7-dehydrocholesterol. Products of these pathways regulate the protective barrier and skin immune functions in a context-dependent fashion through interactions with a number of receptors. Disturbances in CYP11A1 activity can lead to skin pathology.

Oncostatic effects of the indole melatonin and expression of its cytosolic and nuclear receptors in cultured human melanoma cell lines.

Melatonin has been shown to have oncostatic effects on malignant melanoma in vitro and in vivo. We studied the growth suppressive effects of melatonin over a wide range of concentrations in four melanoma cell lines (SBCE2, WM-98, WM-164 and SKMEL-188) representative for different growth stages and phenotype. Melanoma cells were incubated with melatonin 10(-12)-10(-3) M, and proliferation and clonogenicity was assessed at 12 h and 14 days, respectively. We also determined the expression of cytosolic quinone oxidoreductases NQO1, NQO2 (known as MT3 receptor) and nuclear receptor RORalpha by RT-PCR. Melatonin at pharmacological concentrations (10(-3)-10(-7) M) suppressed proliferation in all melanoma cell lines. In SKMEL-188 cells cultured in serum-free media, melatonin at low concentrations (10(-12)-10(-10) M) also slightly attenuated the proliferation. The effects of pharmacological doses of melatonin were confirmed in the clonogenic assay. Expression of NQO1 was detected in all cell lines, whereas NQO2 and nuclear receptor RORalpha including its isoform RORalpha4 were present only in SBCE2, WM-164 and WM-98. Thus, melatonin differentially suppressed proliferation in melanoma cell lines of different behaviour. The intensity of the oncostatic response to melatonin could be related to the cell-line specific pattern of melatonin cellular receptors and cytosolic binding protein expression.

CRH functions as a growth factor/cytokine in the skin.

We tested the effect of CRH and related peptides in a large panel of human skin cells for growth factor/cytokine activities. In skin cells CRH action is mediated by CRH-R1, a subject to posttranslational modification with expression of alternatively spliced isoforms. Activation of CRH-R1 induced generation of both cAMP and IP3 in the majority of epidermal and dermal cells (except for normal keratinocytes and one melanoma line), indicating cell type-dependent coupling to signal transduction pathways. Phenotypic effects on cell proliferation were however dependent on both cell type and nutrition conditions. Specifically, CRH stimulated dermal fibroblasts proliferation, by increasing transition from G1/0 to the S phase, while in keratinocytes CRH inhibited cell proliferation. In normal and immortalized melanocytes CRH effect showed dichotomy and thus, it inhibited melanocyte proliferation in serum-containing medium CRH through G2 arrest, while serum free media led instead to CRH enhanced DNA synthesis (through increased transition from G1/G0 to S phase and decreased subG1 signal, indicating DNA degradation). CRH also induced inhibition of early and late apoptosis in the same cells, demonstrated by analysis with the annexin V stains. Thus, CRH acts on epidermal melanocytes as a survival factor under the stress of starvation (anti-apoptotic) as well as inhibitor of growth factors induced cell proliferation. In conclusion, CRH and related peptides can couple CRH-R1 to any of diverse signal transduction pathways; they also regulate cell viability and proliferation in cell type and growth condition-dependent manners.

Corticotropin-releasing hormone triggers differentiation in HaCaT keratinocytes.

BACKGROUND: Corticotropin-releasing hormone (CRH) is proposed to be involved in the regulation of the proliferative capacity of keratinocytes, based on its significant actions in the skin. These are mediated by CRH-R1alpha and represented by adenylate cyclase activation, Ca2+ influx, inhibition of cell proliferation and modifications in intracellular signal transduction by NF-kappaB. OBJECTIVES: To define CRH action in the cell cycle we investigated its effects on the differentiation programme using the HaCaT keratinocytes model. METHODS: HaCaT keratinocytes were incubated with CRH in Dulbecco's modified Eagles's medium (containing 1.8 mmol L(-1) calcium) or EpiLife (containing 0.06 mmol L(-1) calcium) medium. Cell proliferation was assessed with the MTT assay. Flow cytometry was used for the measurement of DNA content, cell size and granularity and the expression of cytokeratin 14, cytokeratin 1 and involucrin. The electrophoretic mobility shift assay was used to determine DNA binding activity by AP-1 transcription factor. Expression of cytokeratin 1 was also assessed with immunofluorescence microscopy. RESULTS: CRH did produce inhibition of proliferation, which was dose-dependent; the shape of the inhibition curve was determined by the media calcium concentration. CRH action was pinpointed at inhibition of the G0/1 to the S phase transition of the cell cycle. CRH also increased AP-1 binding activity, cell granularity, cytokeratin 1 and involucrin expression, and inhibited cytokeratin 14 expression. CONCLUSIONS: These results are consistent with CRH induction of the keratinocyte differentiation programme. Thus, the overall CRH cutaneous actions connote protective functions for the epidermis, that appear to include the triggering or acceleration of the differentiation programme.