Bioactive forms of vitamin D selectively stimulate the skin analog of the hypothalamus-pituitary-adrenal axis in human epidermal keratinocytes

Abstract

Ultraviolet radiation B stimulates both the production of vitamin D₃ in the skin and the activation of the skin analog of the hypothalamic-pituitary-adrenal axis (HPA) as well as the central HPA. Since the role of vitamin D₃ in the regulation of the HPA is largely unknown, we investigated the impact of 1,25(OH)₂D₃ and its noncalcemic analogs, 20(OH)D₃ and 21(OH)pD, on the expression of the local HPA in human epidermal keratinocytes. The noncalcemic analogs showed similar efficacy to 1,25(OH)₂D₃ in stimulating the expression of neuropeptides, CRF, urocortins and POMC, and their receptors, CRFR1, CRFR2, MC1R, MC2R, MC3R and MC4R. Interestingly, unlike other secosteroids, the activity of 21(OH)pD did not correlate with induction of differentiation, suggesting a separate but overlapping mechanism of action. Thus, biologically active forms of vitamin D can regulate different elements of the local equivalent of the HPA with implications for the systemic HPA.

Keywords: Calcium; Corticotropin releasing factor; HPA axis; Keratinocytes differentiation; Vitamin D(3); Vitamin D(3) analogs.

Fig. 1. 1,25(OH)₂D₃ and it analogs inhibit cell proliferation and stimulate differentiation of primary human epidermal keratinocytes (HPEKp cell line)

Inhibition of the growth of primary human epidermal keratinocyte by 1,25(OH)₂D₃ (A), 20(OH)D₃ (B) and 21(OH)pD (C) was measured at 48 h using the SRB protein assay. Concentration-response curves were plotted and the IC₅₀ value determined as the concentration of the secosteroid which caused a 50% decrease in cell proliferation, calculated using Graph Pad Prism 5. Real-time quantitative PCR analyses were carried out on the expression of key differentiation markers, cytokeratin 1 (KRT1, D), cytokeratin 14 (KRT14, E) and involucrin (IVN, F), in primary human epidermal keratinocytes stimulated with 0.1 µM 1,25(OH)₂D₃, 20(OH)D₃ or 21(OH)pD for 24 h. Immunofluorescence labeling for Ki67 in control and stimulated cells with 1,25(OH)₂D₃ (0.1 µM) indicated a decrease in number of cells which entered cell cycle (G). Data are presented as means ± SD. Involucrin immunofluorescent-stained primary human epidermal keratinocytes (H) displayed increases in cell differentiation between control and stimulated cells with 1,25(OH)₂D₃ (0.1 µM), Ca²⁺ (2.5 mM) or both. Magnification 400×. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control.

Fig. 2. 1,25(OH)₂D₃ and it analogs modulate the expression of vitamin D-related genes in primary human epidermal keratinocytes (HPEKp cell line)

Real-time quantitative PCR analyses were carried out on the expression of key vitamin D-related genes: VDR (A), PDIA3 (B), CYP24A1 (C), CYP2R1 (D), CYP3A4 (E) and CYP27B1 (F) in primary human epidermal keratinocytes stimulated with 0.1 µM 1,25(OH)₂D₃, 20(OH)D₃, or 21(OH)pD for 24 h. Data are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control.

Fig. 3. 1,25(OH)₂D₃ and calcium modulate the expression of neuropeptides in primary human epidermal keratinocytes (HPEKp cell line)

Real-time quantitative PCR analyses of neuropeptide gene expression were carried out on CRF (A), UCN1 (B), UCN2 (C), UCN3 (D), POMC (E), MC1R (F), MC2R (G), MC4R (H) and NR3C1 (I) in primary human epidermal keratinocytes stimulated with 1,25(OH)₂D₃ (0.1 µM), Ca²⁺ (2.5 mM), or both, for 4 and 24 h. Data are presented as means ± SD. *P < 0.05, **P < 0.01 compared to control.

Fig. 4. Western Blot analyses of key proteins of the sHPA axis

CRF, CRFR1 and MC2R protein levels were measured in primary human epidermal keratinocytes stimulated with 1,25(OH)₂D₃ (0.1 µM), Ca²⁺ (2.5 mM), or both, for 24 h. β-Actin levels were measured as a control.

Fig. 5. 1,25(OH)₂D₃ and calcium modulate the protein level of neuropeptides and their receptors in primary human epidermal keratinocytes (HPEKp cell line)

Involucrin (red) and CRFR1 (green) immunofluorescent-stained primary human epidermal keratinocytes demonstrated increases in CRFR1 (A) in cells stimulated with 1,25(OH)₂D₃ (0.1 µM), Ca²⁺ (2.5 mM), or both, compared to the control. Ki67 (red) and ACTH (green) immunofluorescent stained primary human epidermal keratinocytes (B) demonstrated increases in ACTH in cells stimulated with 1,25(OH)₂D₃ (0.1 µM), Ca²⁺ (2.5 mM), or both, compared to control. Ki67 (red) and MC2R (green) immunofluorescent-stained primary human epidermal keratinocytes (C) demonstrated increases in MC1R in cells stimulated with 1,25(OH)₂D₃ (0.1 µM), Ca²⁺ (2.5 mM), or both, compared to control. Fluorescence intensity, was evaluated with the image analysis software, ImageJ (graphs on the right). Magnification 400×. Data are presented as means ± SD.*P < 0.05, **P < 0.01, ***P < 0.001 compared to control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6. 1,25(OH)₂D₃ and it analogs modulate the expression of sHPA axis elements in primary human epidermal keratinocytes (HPEKp cell line)

Real-time quantitative PCR analyses of the expression of key genes of the HPA axis were carried out for CRF (A), UCN1 (B), UCN2 (C), UCN3 (D), CRFR1 (E), POMC (F), MC1R (G), MC2R (H), MC3R (I) and NR3C1 (J) in primary human epidermal keratinocytes stimulated with 0.1 µM 1,25(OH)₂D₃, 20(OH)D₃ or 21(OH)pD for 24 h. Data are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control.

Fig. 7. 1,25(OH)₂D₃ and it analogs selectively modulate the expression of ACTH and MC2R in differentiated and undifferentiated primary human epidermal keratinocytes (HPEKp cell line)

Flow cytometry analyses were performed on ACTH and MC2R in involucrin positive and negative primary human epidermal keratinocytes stimulated with 0.1 µM 1,25(OH)₂D₃, 20(OH)D₃ or 21(OH)pD for 24 h. Data are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control.