The Expression of the Endogenous mTORC1 Inhibitor Sestrin 2 Is Induced by UVB and Balanced with the Expression Level of Sestrin 1.

Sestrin 2 (SESN2) is an evolutionarily conserved regulator of mechanistic target of rapamycin complex 1 (mTORC1) which controls central cellular processes such as protein translation and autophagy. Previous studies have suggested that SESN2 itself is subjected to regulation at multiple levels. Here, we investigated the expression of SESN2 in the skin and in isolated skin cells. SESN2 was detected by immunofluorescence analysis in fibroblasts and keratinocytes of human skin. Differentiation of epidermal keratinocytes was not associated with altered SESN2 expression and siRNA-mediated knockdown of SESN2 did not impair stratum corneum formation in vitro. However, SESN2 was increased in both cell types when the expression of its paralog SESN1 was blocked by siRNA-mediated knock down, indicating a compensatory mechanism for the control of expression. Irradiation with UVB but not with UVA significantly increased SESN2 expression in both keratinocytes and fibroblasts. Upregulation of SESN2 expression could be completely blocked by suppression of p53. These results suggest that SESN2 is dispensable for normal epidermal keratinization but involved in the UVB stress response of skin cells

Compensatory upregulation of SESN2 upon knockdown of SESN1 in fibroblasts.

<p>Human primary fibroblasts were cultured in triplicates and transfected with siRNAs directed against SESN1 or SESN2. 48 h after the transfection, cells were harvested, RNA was extracted, transcribed into cDNA, and subjected to quantitative PCRs for SESN1 <b>(A)</b> as well as SESN2 <b>(B)</b>. Arbitrary units (a.u.) were calculated by normalizing the mRNA levels of SESN1 <b>(A)</b> or SESN2 <b>(B)</b> to B2M levels. Values relative to the expression levels in untreated cells are shown. Error bars indicate standard deviations. Student’s t-test was performed for comparisons between each treatment and the control siRNA treatment. *p < 0.05, **p < 0.01, ***p < 0.001. SESN2 protein expression was determined by Western blot analysis <b>(C)</b>. The band intensities, normalized to the intensities of the GAPDH bands on the same blot and relative to the control siRNA-treated samples, are indicated below the blot. The results are representative for three independent experiments using cells from different donors.</p

UVB induces SESN2 protein in isolated keratinocytes and skin explants.

<p>Human skin explants <b>(A)</b> and cultured keratinocytes (KC) <b>(B)</b> were irradiated with the indicated doses of UVA (J/cm²) or UVB (mJ/cm²) or treated with UVA-induced oxidized phospholipids (UVA-oxPL, μg/ml). 24 h after treatment, cells were lyzed and Western blots for SESN2 and GAPDH were performed. Bands at the predicted sizes of SESN2 and GAPDH are indicated. An asterisk marks an unidentified band immunoreactive with anti-SESN2. The expression of SESN2 relative to GAPDH (lower panels) was normalized to the non-irradiated control. One of three independent experiments with similar results is shown.</p

Expression of SESN2 in the skin.

<p>Human skin sections <b>(A, B)</b> or <i>in vitro</i> cultured primary human dermal fibroblasts <b>(C, D)</b> were immunolabeled with anti-SESN2 (red) either without <b>(A, C)</b> or with <b>(B, D)</b> preabsorption of the antibody with the antigen. Inhibition of labeling by antigen preabsorption is a negative control reaction to confirm the specificity of the antibody. Insets in A and B show higher magnification of dermal cells from the boxed areas of the sections. The dermo-epidermal junction and the surface of the epidermis are indicated by dotted lines. Bars: A and B, 100 μm; C and D, 50 μm.</p