Induction of SREBP-1c mRNA by Differentiation and LXR Ligand in Human Keratinocytes Ai Yokoyama, Makoto Makishima, Mihwa Choi, Yoshitake Cho, Shigeru.

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Induction of SREBP-1c mRNA by Differentiation and LXR Ligand in Human Keratinocytes Ai Yokoyama, Makoto Makishima, Mihwa Choi, Yoshitake Cho, Shigeru Nishida, Yuichi Hashimoto, Tadashi Terui Journal of Investigative Dermatology Volume 129, Issue 6, Pages 1395-1401 (June 2009) DOI: 10.1038/jid.2009.15 Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Confluency-dependent induction of LXR target genes in HaCaT cells. (a) HaCaT cells proliferate until reaching confluency at day 7. Bar=50μm. (b) Cell number reaches maximum at day 7. (c) Experimental procedure for T0901317 treatment in cells of different confluency. HaCaT cells were treated with vehicle control or T0901317 (1μM) at day 2, 4, 6, or 8; cultured for 24hours; and harvested for examination of mRNA (d) and protein expression (e). (d) mRNA expression of SREBP-1c, ABCA1, FAS, involucrin, LXRα, and LXRβ. *P<0.05; **P<0.01. (e) Nuclear expression of SREBP-1, LXRβ, and lamin B. Western blotting was repeated twice with similar results. Journal of Investigative Dermatology 2009 129, 1395-1401DOI: (10.1038/jid.2009.15) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Density-dependent expression of genes involved in lipid metabolism in HaCaT cells. (a) Experimental procedure for T0901317 treatment in HaCaT cells cultured at low density or high density. HaCaT cells were plated at 1 × 105 cells per well or at 4 × 105 cells per well in a 9.6cm2 per well plate and treated with vehicle control or 1μM T0901317 for 24hours. (b) mRNA expression of keratinocyte differentiation markers (involucrin and transglutaminase 1), genes involved in lipid metabolism (SREBP-1c, SCD-1, ABCA1, FAS, acetyl CoA carboxylase (ACC), SREBP-2, HMG-CoA synthase, HMG-CoA reductase), and LXRs. *P<0.05; **P<0.01. Journal of Investigative Dermatology 2009 129, 1395-1401DOI: (10.1038/jid.2009.15) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Differentiation of HaCaT cells in suspension culture increases expression of SREBP-1c. (a) mRNA expression of involucrin and SREBP-1c in a regular plate (non-coated) and in a poly(2-hydroxyethyl methacrylate)-coated plate for suspension (coated) for 1 (D1), 2 (D2), or 3 days (D3). (b) Effect of T0901317 on SREBP-1c expression in HaCaT cells in suspension culture. HaCaT cells were cultured in suspension for 3 days and treated with vehicle control or 1μM T0901317 for 24hours. *P<0.05; **P<0.01. Journal of Investigative Dermatology 2009 129, 1395-1401DOI: (10.1038/jid.2009.15) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Differentiation-associated expression of LXR target genes in epidermoid carcinoma A431 cells. (a) Number of A431 cells. A431 carcinoma cells proliferated until day 11. (b) mRNA expression of involucrin, SREBP-1c, FAS, and ABCA1 in A431 cells. Cells were cultured, treated with vehicle control or 1μM T0901317 for 24hours, and harvested at the indicated day as shown in Figure 1c. *P<0.05; **P<0.01. Journal of Investigative Dermatology 2009 129, 1395-1401DOI: (10.1038/jid.2009.15) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Expression of involucrin and SREBP-1c in primary NHEK. (a) Time course of mRNA expression of involucrin and SREBP-1c. **P<0.01 compared with expression at day 3. (b) Effect of T0901317 on expression of involucrin and SREBP-1c in NHEK. Cells were plated at low density or high density and treated with vehicle control or 1μM T0901317 for 24hours, as shown in Figure 2a. *P<0.05; **P<0.01. Journal of Investigative Dermatology 2009 129, 1395-1401DOI: (10.1038/jid.2009.15) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Effect of LXR antagonist on expression of SREBP-1c in HaCaT cells. (a) Experimental procedure for treatment with LXR agonist and antagonist in HaCaT cells. Cells were cultured for 6 days and treated with vehicle control or 10μM 5CPPSS-50 in the absence or presence of 0.3μM T0901317 for 24hours. (b) Effect of 5CPPSS-50 on confluency-dependent expression of ABCA1 and SREBP-1c. *P<0.05; **P<0.01. Journal of Investigative Dermatology 2009 129, 1395-1401DOI: (10.1038/jid.2009.15) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions