Inhibition of PAX3 by TGF-β Modulates Melanocyte Viability Guang Yang, Yitang Li, Emi K. Nishimura, Hong Xin, Anyu Zhou, Yinshi Guo, Liang Dong, Mitchell F. Denning, Brian J. Nickoloff, Rutao Cui  Molecular Cell  Volume 32, Issue 4, Pages 554-563 (November 2008) DOI: 10.1016/j.molcel.2008.11.002 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 PAX3 Is Repressed by TGF-β in HPM Cells (A) TGF-β is repressed by UV irradiation. Human primary keratinocytes (HPKs) were irradiated with UV as described. Protein was collected at time 0 and at different time points after stimulation. TGF-β protein levels, which were analyzed by western blot, are shown along with actin, which served as loading control. (B) HPKs were irradiated with UV as described. TGF-β levels were measured in culture medium. Secretion of TGF-β was repressed by UV irradiation. (C) Human primary melanocytes (HPMs) were stimulated with different doses of TGF-β as indicated. RNA and protein were collected at 24 hr after TGF-β stimulation. The left panels represent PAX3 RNA levels as measured by quantitative RT-PCR and normalized to GAPDH. Results are expressed as the mean of the experiment done in triplicate ± the standard error of the mean (SEM). Repression is calculated relative to PAX3 levels in vehicle (DMSO)-treated cells. Protein levels of PAX3 and its downstream gene, Mitf, which were analyzed by western blot, are shown on the right along with actin, which served as loading control. (D) HPMs were stimulated with TGF-β. RNA and protein were collected at time 0 and at different time points after stimulation. The left panels represent PAX3 RNA levels as measured by quantitative RT-PCR and normalized to GAPDH. Results are expressed as the mean of the experiment done in triplicate ± SEM. Induction is calculated relative to PAX3 levels in vehicle (DMSO)-treated cells. Protein levels of PAX3 and its downstream gene, Mitf, which were analyzed by western blot, are shown on the right along with actin, which served as loading control. Molecular Cell 2008 32, 554-563DOI: (10.1016/j.molcel.2008.11.002) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Melanocyte Activation Requires TGF-β Pathway Inhibition (A) Mitf and PAX3 induction by quantitative RT-PCR and western blot analysis of human primary melanocytes (HPMs) incubated (24 hr) with 48 hr conditioned supernatants from human primary keratinocytes either untreated (lanes 3–5) or irradiated with 100 J/m2 UVB (lane 2). Anti-TGF-β affinity chromatography (but not control anti-HA) induced PAX3 and Mitf expression. (B) Protein prepared from HPMs untreated or treated with 200 pM TGF-β for the indicated times was directly subjected to immunoblotting with antibodies against C-terminal phosphorylated Smad2 (p-Smad2). Treatment with TGF-β strongly induced Smad2 phosphorylation. (C) HPMs were treated with or without TGF-β for 24 hr and immunoprecipitated, and these precipitates were subjected to immunoblotting with the indicated antibodies. (D) HPMs were infected with Smad4 RNAi or control vector. After 24 hr, cells were incubated with 200 pM TGF-β for 24 hr. Samples were subjected to western blot assays with the indicated antibodies. (E) B16 Ski or SnoN knockdown cells were stimulated with TGF-β. The expression of PAX3 RNA is shown. Results of RNA levels are expressed as the mean of the experiment done in triplicate ± the SEM. As seen on the quantitative RT-PCR, knockdown of Ski, not SnoN, rescued TGF-β-induced PAX3 downregulation. (F) Knockdown of Ski or SnoN in B16 mouse melanoma cells. (G) In vivo association of Ski with the PAX3 promoter by chromatin immunoprecipitation (ChIP). Human primary melanocytes (HPM) were treated with 200 pM TGF-β or no additions for 24 hr and subjected to ChIP assays with the indicated antibodies and PCR primers. Actin served as a TGF-β unresponsive control. Molecular Cell 2008 32, 554-563DOI: (10.1016/j.molcel.2008.11.002) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 Smads Binds and Represses the PAX3 Promoter (A) Schematic representation of the human PAX3 locus, which indicates the location of a Smad-binding consensus element (SBE) that is highly conserved among human, mouse, rat, and monkey promoters. (B) Promoter activity was studied using reporter assays with different promoter portions (including mutated SBEs), which drive expression of firefly luciferase. Relative luciferase activity was normalized to empty pGL4-basic plasmid using the dual-luciferase assay system (Promega). Results are expressed as the mean of the experiment done in triplicate ± SEM. Analysis of PAX3 promoter transactivation capacity by α-MSH, after TGF-β stimulation of B16 cells transiently transfected with PAX3 reporter, employed mutations at Smad binding sites (Mut). (C) In vivo association of Smad4 with the PAX3 promoter by chromatin immunoprecipitation. Human primary melanocytes were treated with 200 pM TGF-β or no additions for 24 hr and subjected to chromatin immunoprecipitation assays with the indicated antibodies and PCR primers. Actin served as a TGF-β unresponsive control. Molecular Cell 2008 32, 554-563DOI: (10.1016/j.molcel.2008.11.002) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 Immunohistochemical Staining of TGF-β and PAX3 in Human Foreskins with or without UVB Irradiation Sections of human foreskin were irradiated and subjected to immunohistochemistry at different time points. Arrows reveal the first time point when the staining marker is positive. Molecular Cell 2008 32, 554-563DOI: (10.1016/j.molcel.2008.11.002) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 Transcription of TGF-β Was Repressed by UV Irradiation (A) Primary keratinocytes isolated from wild-type and p53 null mice were UV irradiated. RNA of TGF-β1 and protein of the TGF-β precursor are shown. Results of RNA levels are expressed as the mean of the experiment done in triplicate ± the SEM. As seen on the quantitative RT-PCR, basal TGF-β1 levels are lower in the absence of p53, whereas repression is p53 independent. (B) Human primary keratinocytes (HPK) were treated with specific c-jun N-terminal kinase inhibitor, SP600125, for 1 hr and then UV irradiated. UV-irradiated HPK or HPK treated with SP600125 were processed for RNA and protein isolation. UV-induced TGF-β repression is inhibited by treatment with SP600125. Results of RNA levels are expressed as the mean of the experiment done in triplicate ± the SEM. (C) PAM212 c-jun or c-fos knockdown cells were irradiated with UV. The expression of TGF-β RNA is shown. Results of RNA levels are expressed as the mean of the experiment done in triplicate ± the SEM. As seen on the quantitative RT-PCR, knockdown of c-jun, not c-fos, is required in UV-induced TGF-β repression. (D) Knockdown of c-jun or c-fos in PAM212 mouse keratinocytes. Molecular Cell 2008 32, 554-563DOI: (10.1016/j.molcel.2008.11.002) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 PAX3 Works in Synergy with SOX10 in a CRE/CREB-Dependent Manner to Regulate Mitf Transcription in Melanocytes (A) Schematic representation of the human Mitf locus. (B) Promoter activity was studied using reporter assays with different promoter portions, which drive expression of firefly luciferase. Relative luciferase activity was normalized to empty pGL4-basic plasmid using the dual-luciferase assay system (Promega). Results are expressed as the mean of the experiment done in triplicate ± SEM. Molecular Cell 2008 32, 554-563DOI: (10.1016/j.molcel.2008.11.002) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 7 Schematic Diagram of the UV-Induced Melanogenic Response Molecular Cell 2008 32, 554-563DOI: (10.1016/j.molcel.2008.11.002) Copyright © 2008 Elsevier Inc. Terms and Conditions