Human Skin is a Steroidogenic Tissue: Steroidogenic Enzymes and Cofactors Are Expressed in Epidermis, Normal Sebocytes, and an Immortalized Sebocyte Cell.

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Human Skin is a Steroidogenic Tissue: Steroidogenic Enzymes and Cofactors Are Expressed in Epidermis, Normal Sebocytes, and an Immortalized Sebocyte Cell Line (SEB-1) Diane Thiboutot, Kathyrn Gilliland, Zhaoyuan Cong Journal of Investigative Dermatology Volume 120, Issue 6, Pages 905-914 (June 2003) DOI: 10.1046/j.1523-1747.2003.12244.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 SEB-1 sebocytes are stably transfected with SV40 large T antigen.Panel a: Negative control; SEB-1 sebocytes, no primary antibody. Panel b: Positive control; mouse brain tumor expresses SV40 antigen. Panel c: Human sebocytes prior to transfection do not express SV40 antigen (negative control). Panel d: SEB-1 sebocytes express SV40 antigen in the nucleus. Bar: 100 μM. Journal of Investigative Dermatology 2003 120, 905-914DOI: (10.1046/j.1523-1747.2003.12244.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 SEB-1 sebocytes express proteins characteristic of human sebaceous glands.Panel a: Negative control; SEB-1 sebocytes, no primary antibody. Panels b-e: SEB-1 sebocytes exhibit cytoplasmic expression of type 1 5α-reductase (panel b), cytokeratin 7 (panel c), cytokeratin 13 (panel d), and OM-1 sebaceous gland antigen (panel e). Panel f: SEB-1 sebocytes exhibit cytoplasmic lipid droplets with Oil Red O staining (arrow). Bar: 100 μM. Journal of Investigative Dermatology 2003 120, 905-914DOI: (10.1046/j.1523-1747.2003.12244.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 P450scc, P450c17, adrenodoxin reductase, and SF-1 are expressed in human facial skin. Antibodies were reacted with cryostat sections of human facial skin. Panel a: Negative control; human skin, no primary antibody. Panel b: Antibody to P450scc. Panel c: Antibody to P450c17. Panel d: Antibody to adrenodoxin reductase. Panel e: Antibody to SF-1. Localization of each antibody was noted in epidermis (short arrow), sebaceous duct (thin arrow), and sebaceous gland (long arrow). Bar: 100 μM. Journal of Investigative Dermatology 2003 120, 905-914DOI: (10.1046/j.1523-1747.2003.12244.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Secondary cultures of human sebocytes express P450scc, P450c17, adrenodoxin reductase, and SF-1. Antibodies were reacted with secondary cultures of human facial sebocytes. Panel a: Negative control; secondary sebocytes, no primary antibody. Panel b: Antibody to P450scc. Panel c: Antibody to P450c17. Panel d: Antibody to adrenodoxin reductase. Panel e: Antibody to SF-1. Cytoplasmic localization of P450scc, P450c17, and adrenodoxin was noted. Antibody to SF-1 (panel e) exhibited perinuclear localization. Bar: 100 μM. Journal of Investigative Dermatology 2003 120, 905-914DOI: (10.1046/j.1523-1747.2003.12244.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 SEB-1 sebocytes express P450scc, P450c17, adrenodoxin reductase, and SF-1. Antibodies were reacted with SEB-1 sebocytes. Panel a: Negative control; SEB-1 sebocytes, no primary antibody. Panel b: Antibody to P450scc. Panel c: Antibody to P450c17. Panel d: Antibody to adrenodoxin reductase. Panel e: Antibody to SF-1. Cytoplasmic localization of P450scc, P450c17, and adrenodoxin was noted. Antibody to SF-1 exhibited perinuclear and nuclear localization. Bar: 100 μM. Journal of Investigative Dermatology 2003 120, 905-914DOI: (10.1046/j.1523-1747.2003.12244.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Radioimmunoassay of SEB-1 sebocytes reveals activity of the P450scc and P450c17 enzymes and response to forskolin. SEB-1 sebocytes were incubated with 22-hydroxycholesterol in the presence (F+) and absence (F-) of 10 μM forskolin. Samples were assayed at time 0, 3 h, 10 h, and 48 h. Data were normalized to cell counts performed at each time point. Levels of 17-hydroxypregnenolone in forskolin-treated cells were on the order of 1.5–3.3-fold higher compared to untreated cells. Using a paired t test, the difference between forskolin-treated and untreated cells was significant at 48 h (p=0.03, α=0.05) indicating an increase in enzyme activity in response to increased levels of cyclic AMP resulting from treatment with forskolin. Journal of Investigative Dermatology 2003 120, 905-914DOI: (10.1046/j.1523-1747.2003.12244.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Western blot analyses of sebaceous gland and SEB-1 extracts confirm expression of P450scc, P450c17, and adrenodoxin reductase.Panel a: P450scc. Lane M, molecular weight markers; lanes A, B, 52 kDa bands corresponding to P450scc are noted in extracts of theca cells (lane A) and SEB-1 cells (lane B). Panel b: P450c17. Lane M, molecular weight markers; lanes A, B, 60 kDa bands corresponding to P450c17 are noted in extracts of theca cells (lane A) and SEB-1 cells (lane B). Panel c: SF-1. Lane M, molecular weight markers; lanes A, B, 53 kDa bands corresponding to SF-1 are noted in sebaceous glands (lane A) and JEG-3 cells (lane B). Panel d: Adrenodoxin reductase. Lane M, molecular weight markers; lanes A, B, bands of 48 kDa are noted in JEG-3 cells (lane A) and sebaceous glands (lane B). Journal of Investigative Dermatology 2003 120, 905-914DOI: (10.1046/j.1523-1747.2003.12244.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 QPCR reveals significant differences in message abundance for steroidogenic enzymes in SEB-1 sebocytes, sebaceous glands, and ovarian theca and granulosa cells. Reverse transcription was performed on one sample of SEB-1 sebocyte RNA, one sample of sebaceous gland RNA, three samples of theca cell RNA, and three samples of granulosa cell RNA. QPCR was performed in triplicate from each cDNA. The cycle thresholds for each reaction were determined and plotted against a standard curve derived using normal theca RNA where relative fluorescence units were assigned to each point on the standard curve. Relative fluorescence values were normalized for GAPDH and compared between tissues using an unpaired t test (α=0.05). The numbers above each bar represent the relative fluorescence units. Panel a: Relative abundance of mRNA for P450scc was determined. Levels in SEB-1 sebocytes are significantly greater than in other tissues (p< 0.05 for each comparison). Panel b: Relative abundance of P450c17 mRNA was determined. Levels in theca cells are significantly greater than those in SEB-1 sebocytes (p=0.009), sebaceous glands (p=0.007), and granulosa cells (p=0.006). Panel c: Relative abundance of mRNA for SF-1 was determined. Levels in SEB-1 sebocytes are significantly less than those in other cell types (p < 0.01). Journal of Investigative Dermatology 2003 120, 905-914DOI: (10.1046/j.1523-1747.2003.12244.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions