Regulation of HMG-CoA Synthase and HMG-CoA Reductase by Insulin and Epidermal Growth Factor in HaCaT Keratinocytes  Ian R. Harris, Hendrik Höppner, Wilfried Siefken, Klaus-Peter Wittern  Journal of Investigative Dermatology  Volume 114, Issue 1, Pages 83-87 (January 2000) DOI: 10.1046/j.1523-1747.2000.00822.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 EGF or insulin increase HMG-CoA synthase mRNA levels. HMG-CoA synthase steady-state mRNA levels in preconfluent keratinocytes treated with 0.1 μg per ml EGF or 10 μg per ml insulin. Poly(A)+ RNA was prepared after 18 h of treatment and northern blotting was performed as described in Materials and Methods. The data shown (mean ± SEM) is from two independent experiments (n = 4) expressed as a percentage of control (100%) after correcting for loading using G3DPH mRNA levels. Statistical significance is calculated using the Student’s t test. Journal of Investigative Dermatology 2000 114, 83-87DOI: (10.1046/j.1523-1747.2000.00822.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 HMG-CoA synthase promoter activity is regulated by insulin or EGF. Luciferase activity in HaCaT keratinocytes transfected with a luciferase reporter construct containing the wild-type HMG-CoA synthase promoter. Keratinocytes were treated with 10 μg per ml insulin or 0.1 μg per ml EGF as described in Materials and Methods. The data shown (mean ± SEM) is from four independent experiments (n = 11–14) expressed as percentage of control (100%) after correcting for cell number using protein levels. Statistical significance is calculated using the Student’s t test. Journal of Investigative Dermatology 2000 114, 83-87DOI: (10.1046/j.1523-1747.2000.00822.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Mutation of an AP-1 binding site does not affect regulation of HMG-CoA synthase by insulin or EGF. Luciferase activity in HaCaT keratinocytes transfected with a luciferase reporter construct containing the HMG-CoA synthase promoter with mutated AP-1 binding site. Keratinocytes were treated with 10 μg per ml insulin or 0.1 μg per ml EGF as described in Materials and Methods. The data shown (mean ± SEM) is from four independent experiments (n = 11–14) expressed as a percentage of control (100%) after correcting for cell number using protein levels. Statistical significance is calculated using the Student’s t test. Journal of Investigative Dermatology 2000 114, 83-87DOI: (10.1046/j.1523-1747.2000.00822.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Mutation of an SRE site stops the increase in HMG-CoA synthase expression by insulin but not of EGF. Luciferase activity in HaCaT keratinocytes transfected with a luciferase reporter construct containing the HMG-CoA synthase promoter with mutated SRE. Keratinocytes were treated with 10 μg per ml insulin or 0.1 μg per ml EGF as described in Materials and Methods. The data shown (mean ± SEM) is from four independent experiments (n = 11–14) expressed as a percentage of control (100%) after correcting for cell number using protein levels. Statistical significance is calculated using the Student’s t test. Journal of Investigative Dermatology 2000 114, 83-87DOI: (10.1046/j.1523-1747.2000.00822.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 HMG-CoA synthase mRNA levels are increased by cycloheximide and not by TPA. Northern blots of poly(A)+ RNA from HaCaT keratinocytes treated with 100 ng per ml TPA and/or 10 μg per ml cycloheximide (CHX) for 3.5 h as described in Materials and Methods. The data shown (mean ± SEM, n = 2) is expressed as a percentage of control (100%) after correcting for loading using G3DPH mRNA levels. Journal of Investigative Dermatology 2000 114, 83-87DOI: (10.1046/j.1523-1747.2000.00822.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions