Fig 1.
Chemical structures of RXR and RAR ligands.
Kd values for 9-cis-RA and UAB30 are from Grubbs et al. [43] and for Targretin (Bexarotene) from Desphande et al. [44]. EC50 values are from Atigadda et al. [45].
Fig 2.
H&E staining of skin rafts treated with UAB30.
A, Human organotypic skin raft cultures were grown in the presence of 0–10 μM UAB30. B, Primary neonatal keratinocytes (PHKs) were infected with Moloney murine leukemia retroviral vector pBabe puro expressing RDH10 and selected with 1.5 μg/ml puromycin for 2 days as described previously [51]. Retrovirus-transduced PHKs were cultured at the medium-air interface to produce RDH10 transgenic rafts. Differently colored brackets demarcate the layers of epidermis: cornified (red), granular (orange), spinous (purple), and basal (gray). Note the similarly increased thickness of the basal layer (gray bracket) and reduced differentiation to cornified layer (red bracket) in rafts treated with 10 μM UAB30 as compared with RDH10-overexpresing rafts.
Fig 3.
QPCR analysis of gene expression in skin rafts treated with UAB30 or Bexarotene.
Three individual rafts were used for each treatment. QPCR analysis for each raft was done in triplicate. Data were normalized per peptidylprolyl isomerase A (PPIA, cyclophilin A) and represent average ± SEM. DHRS9, dehydrogenase/reductase member 9 (SDR9C4); DHRS3, dehydrogenase/reductase member 3 (SDR16C1, also known as retSDR1); RDH10, retinol dehydrogenase 10 (SDR16C4); RARγ, retinoic acid receptor γ; LRAT, lecithin retinol acyltransferase; STRA6, stimulated by retinoic acid gene 6; MUC21, mucin 21; GABRP, γ-aminobutyric acid (GABA) A receptor π; FLG, filaggrin; CYP26B1, cytochrome P450 26B1; MCM7, minichromosome maintenance complex component 7; CRBP1, cellular retinol binding protein type 1. Symbol * indicates statistically significant differences in expression of genes in UAB30-treated rafts versus vehicle-treated rafts. Symbol # indicates the differences in expression of genes in bexarotene-treated rafts versus UAB30-treated rafts. *and # indicate p values <0.05; ** and ## indicate p values <0.005.
Fig 4.
QPCR analysis of gene expression in skin rafts treated with different doses of UAB30.
QPCR analysis was performed as described in Fig 3. Error bars represent mean ± SEM of three independent rafts. *p<0.05; **p<0.01.
Fig 5.
HPLC analysis of retinyl esters in UAB30-treated skin rafts.
Retinyl esters were extracted from three samples each containing five pooled UAB30-treated or DMSO-treated skin rafts. Data represent mean ± SEM; **p<0.01.
Table 1.
ATRA concentration in samples of skin rafts.
Fig 6.
UAB30 dose-dependent induction of retinoid metabolic genes.
QPCR analysis was performed as described in Fig 3. Error bars represent mean ± SEM of three independent rafts. *p<0.05; **p<0.01.
Fig 7.
QPCR analysis of select RXR target genes in skin rafts treated with UAB30.
QPCR analysis was performed as described in Fig 3 using skin rafts prepared from two different batches (designated 1 and 2) of skin keratinocytes each pooled from 2–3 donors. Error bars represent mean ± SEM of three independent rafts. *p<0.05; **p<0.005.
Fig 8.
Expression levels of ATRA target genes in UVB irradiated mouse skin and in mouse models of UVB-induced SCC and BCC.
A. UVB irradiation. B. SCC. C. BCC. QPCR analysis was performed as described in Fig 3. Gene expression was normalized per the most stable gene in each model (Hprt for UVB; Gapdh for SCC; actin for BCC). Normal mice, n = 13; UVB-irradiated, n = 13; SCC and BCC, n = 7. *p<0.05; **p<0.005.
Fig 9.
QPCR analysis of gene expression in UVB-exposed mice pretreated with UAB30.
The mRNA from three individual animals was used for each condition. QPCR analysis for each mouse was performed in triplicates. Data were normalized per Gapdh and represent average ± SEM. Symbol * refers to expression of genes in epidermis of control versus UVB-exposed mice. Symbol # refers to expression of genes in epidermis of UVB-exposed mice versus UAB30-pretreated and UVB-exposed. *,#p <0.05; **,##p <0.01.