all-trans-3,4-didehydroretinoic acid

Introduction

The aim of this study was to examine whether the two endogenous retinoids atRA and ddRA affect the mRNA expression profile differently in cultured human primary keratinocytes using oligonucleotide arrays. All genes causing ARCI were analyzed in detail. The expression profiles after retinoid treatment were examined both in proliferating and differentiated keratinocytes, as representatives for basal and suprabasal epidermal keratinocytes, respectively.

Materials

atRA and ddRA were obtained from Sigma-Aldrich (Stockholm, Sweden) and Roche AG (Basle, Switzerland), respectively. The compounds were dissolved in ethanol and the concentration and purity were determined by UV absorbance and HPLC analysis, respectively. Stock solutions of 1 mM were prepared. The purity of atRA and ddRA was 99.1 and 88.3 %, respectively. The two major impurities in ddRA were 13cRA (1.5 %) and atRA (10.2 %). All handling of retinoids was performed under yellow reduced light.

Cell culture and retinoid treatment

Primary keratinocytes from a lightly pigmented female donor (42 years) were used at passage 3 (Life Technologies, Stockholm, Sweden). The cells were maintained in EpiLife serum-free keratinocyte medium containing growth supplement (HKGS or HKGS kit) and gentamicin/amphotericin (all from Life Technologies). Proliferating cells in 6-well plates at 30–40 % confluency were switched to EpiLife medium without EGF for 5 h prior to starting treatment with retinoids. Keratinocytes at 80 % confluency were switched to EpiLife− supplemented with 1.5 mM CaCl2 for 4 days with the purpose to induce cellular differentiation. Cells were subsequently exposed to retinoids as above in EpiLife medium without rhEGF. Both proliferating and differentiated cells were exposed to 1 µM of atRA, ddRA or vehicle (ethanol) for 4 and 24 h. All treatments were performed in triplicates. The cells were washed with PBS twice and frozen at −70 °C until further processing.

atRA and ddRA regulate gene expression most profoundly in differentiating keratinocytes

We compared the transcriptional profiles in atRA- and ddRA-treated keratinocytes with vehicle-treated keratinocytes. In cells exposed to one of the two retinoids, the affected RNA transcripts were few in numbers in proliferating keratinocytes as compared to differentiated cells (see Table ). The genes altered in response to atRA exposure is shown as a heat map (Online Resource 4). However, the number of altered transcripts was in the same range after treatments with both retinoids, and most of the transcripts were significantly altered by both retinoids. The top ten-induced and -suppressed annotated transcripts after atRA exposure are shown in (Online Resource 5). The expression of some genes in atRA- vs. vehicle-treated differentiating cells was verified by qPCR (Fig. b–i).

Gene expression was not regulated differently by atRA and ddRA

Although the major number of retinoid-regulated transcripts was induced or suppressed equally efficiently by atRA and ddRA, there were also transcripts that were significantly altered by only one of the retinoids when comparing to vehicle-treated controls. However, when comparing the expression profiles generated by the two retinoids with each other, no genes were significantly altered (data not shown).