Lymph node metastasis in RXRα^ep−/−/Cdk4^R24C/R24C mice. (A, B) Excised draining lymph nodes from Cdk4^R24C/R24C and RXRα^ep−/−/Cdk4^R24C/R24C mice after DMBA/TPA treatment. (C, D) Fontana-Masson staining of draining lymph nodes from Cdk4^R24C/R24C and RXRα^ep−/−/Cdk4^R24C/R24C mice after carcinogenic treatment. Melanin granules stain black and red corresponds to Nuclear Fast Stain. (E, F) IHC staining was performed using antibodies directed against Trp1 [green]. (G, H) IHC staining was performed using antibodies directed against HMB45 and MART-1 [red]. Scale bar (in yellow) = 33μm. Blue corresponds to DAPI staining of the nuclei.

Immunohistochemical (IHC) characterization of melanocytic tumors in Cdk4^R24C/R24C and RXRα^ep−/−/Cdk4^R24C/R24C bigenic mice. (A, B) Enhanced melanocyte proliferation in melanocytic tumors from RXRα^ep−/−/Cdk4^R24C/R24C mice as determined by co-labeling with anti-Trp1 [green] and anti-Pcna [red] antibodies. (C, D) IHC staining for malignant melanocytes using a cocktail of antibodies directed against melanoma antigens HMB45 and MART-1 [red]. (E, F) Increased vascularization was detected by anti-CD31 antibody [green] in melanocytic tumors from bigenic mice compared to Cdk4^R24C/R24 alone. E, epidermis; D, dermis. Scale bar (in yellow) = 33μm. White dashed lines, artificially added, indicate epidermal-dermal junction. Blue color corresponds to DAPI staining of the nuclei.

Reduced expression of keratinocytic RXRα during melanoma progression in human skin. Representative localization of RXRα protein within the epidermis of normal (A,B), tumor adjacent normal [TAN] (C,D), benign melanocytic nevi (E,F), in situ melanoma (G,H) and malignant melanoma [MM] sections (I, J). Brown staining represents RXRα expression (except for the dermal pigmentation present in malignant melanoma sections), counterstained with Harris hematoxylin. E, epidermis; D, dermis. Orange dashed line encompass nests of nevus cells, yellow dashed lines indicates epidermal-dermal junction. Scale bar = 33μm.

Formation of larger melanomas in RXRα^ep−/−/Cdk4^R24C/R24C bigenic mice. (A) Size distribution of melanocytic tumors in RXRα^L2/L2, RXRα^ep−/−, Cdk4^R24C/R24C and RXRα^ep−/−/Cdk4^R24C/R24C mice after DMBA/TPA treatment. The number of tumors, less than 2 mm, 2–4mm and greater than 4 mm in diameter, were determined in the four groups of mice, as indicated. Values are expressed as mean +/− SEM. (B) Histological analyses of melanocytic tumors from Cdk4^R24C/R24C and RXRα^ep−/−/Cdk4^R24C/R24C mice. Hematoxylin and eosin (H&E)-stained 5μm thick paraffin skin sections of biopsies taken after DMBA/TPA treatment. E, epidermis; D, dermis; HD, hypodermis. Scale bar = 62μm. (C, D) Increased epidermal thickness, and higher radial growth phase (RGP) and vertical growth phase (VGP) of melanocytic tumors in RXRα^ep−/−/Cdk4^R24C/R24C mice. Statistical significance was calculated using unpaired Student’s t-test with GraphPad Prism software. Statistical relevance indicated as follows; * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Nuclear receptor RXRα associates with target gene promoter encoding soluble growth factors in primary keratinocytes. (A) Putative RXREs identified upstream of the transcriptional start site of genes for specific soluble growth factors such as Et-1, Hgf, p53, Pomc and Scf. Arrows indicate position of primers designed to capture all potential binding sequences, asterisks (*) indicate location of results shown in figure. The position of transcriptional start site is indicated as +1, promoter diagrams not to scale. (B) Chromatin immunoprecipitation (ChIP) assay was performed on primary keratinocytes using antibody against RXRα protein and results were analyzed by qPCR using specific primers (indicated in A). RXRα association on consensus sequences for RXRα/NR heterodimers located on the promoters of Et-1, Hgf, p53, Pomc and Scf genes in wild type keratinocytes was compared to cells lacking RXRα protein or wild type cells immunoprecipitated with an irrelevant antibody. Endothelin 1, Et-1; proopiomelanocortin, Pomc; hepatocyte growth factor, Hgf; stem cell factor, Scf. Statistical analyses was done by Student’s unpaired T-test using GraphPad Prism software and significance level (*) were set at p < 0.05.

Altered expressions of genes in laser capture microdissected (LCM) melanocytic tumors. (A) Relative fold change differences ≥ 2 of RXRα^ep−/−/Cdk4^R24C/R24C mRNA transcripts taken from LCM isolated melanocytes compared to Cdk4^R24C/R24C mice. Amplified cDNA was analyzed using a RT² Nano PreAMP cDNA Synthesis Kit and evaluated using a Mouse Cancer PathwayFinder PCR Array System. Gene expression was normalized to a panel of housekeeping genes (B2m, Hprt1, Rpl13a, Gapdh, Actb). (B) Validation of PCR array results using relative mRNA transcript levels of Fas, Birc5, Plaur, Cdh1 and Pten from LCM captured melanocytes of Cdk4^R24C/R24C and RXRα^ep−/−/Cdk4^R24C/R24C mice treated with DMBA/TPA for 25 weeks. (C) Validation of PCR array results using western blot detection for protein levels in both RXRα^ep−/−/Cdk4^R24C/R24C [MT] and Cdk4^R24C/R24C [CT] melanocytic tumors using antibodies against Fas, Plaur, Cdh1 and Pten. All experiments were done using a minimum of three biological replicates from each group of mice. Data represents three individual experiments and in all cases are expressed as mean +/− SEM. Statistical significance was calculated using unpaired Student’s t-test with GraphPad Prism software. Statistical relevance indicated as follows; * = p < 0.05, ** = p < 0.01.

Increased expression of paracrine factors and regulatory proteins in the skin of RXRα^ep−/−/Cdk4^R24C/R24C mice. (A) Enhanced expression of mitogenic paracrine factors and regulatory proteins in skin of RXRα^ep−/−/Cdk4^R24C/R24C mice compared to Cdk4^R24C/R24C mice after 25 weeks of DMBA/TPA treatment. Relative mRNA transcript levels of endothelin 1 (Et-1), hepatocyte growth factor (Hgf), stem cell factor (Scf), proopiomelanocortin (Pomc), fibroblast growth factor 2 (Fgf2) and microphtalmia-associated transcription factor (Mitf) were measured. (B) Enhanced expression of autocrine factors in skin of RXRα^ep−/−/Cdk4^R24C/R24C mice compared to Cdk4^R24C/R24C 25 weeks after DMBA/TPA treatment. Relative mRNA transcript levels of heparin-binding EGF-like growth factor (HbEgf), interleukin 1-alpha (Il1α) and cyclooxygenase 2 (Cox2) were measured. (C) Western blot analysis of different proteins in the mitogenic MAPK pathway on skin extracts from RXRα^ep−/−/Cdk4^R24C/R24C [MT] and Cdk4^R24C/R24C [CT] mice 25 weeks after DMBA/TPA treatment. Antibodies used were against p38, phospho-p38, Jnk, phospho-Jnk. Data (A and B) represents mean ± SEM. All reactions were performed in triplicates using skin samples collected from a minimum of three different mice in each group. Statistical analyses were done using GraphPad Prism software and significance level (*) were set at p < 0.05. # = Not significant.