Aldo-keto reductase 1C3 is expressed in differentiated human epidermis, affects keratinocyte differentiation, and is upregulated in atopic dermatitis

Abstract

Aldo-keto reductase 1C3 (AKR1C3) has been shown to mediate the metabolism of sex hormones and prostaglandin D(2) (PGD(2)), a lipid mediator that promotes skin inflammation in atopic dermatitis (AD). As both have a role in skin function and pathology, we first sought to investigate the expression pattern of AKR1C3 in normal human epidermis. Immunofluorescence revealed a strong expression of AKR1C3 in the differentiated suprabasal layers compared with the basal layer. Western blot analysis and quantitative PCR confirmed that AKR1C3 expression was also upregulated in differentiation-induced primary human keratinocytes (PHKs). To investigate the functional role of AKR1C3 during PHK differentiation, its expression and activity (measured as PGD(2) reduction to 9α,11β-PGF(2) by ELISA) were impaired by small interfering RNA or 2'-hydroxyflavanone, respectively. Cytokeratin 10 (K10) and loricrin expression were then examined by western blot analysis, thus revealing altered expression of these differentiation markers. Finally, following an observation that the AD-associated mediator, PGD(2), upregulated AKR1C3 expression in PHKs, we used immunofluorescence to examine AKR1C3 expression in AD and psoriasis lesions. AKR1C3 was found to be upregulated in AD but not in psoriasis lesions compared with non-lesional skin. Our work demonstrates a function for AKR1C3 in differentiation-associated gene regulation and also suggests a role in supporting inflammation in AD.

Figure 1. AKR1C3 is expressed in differentiated human epidermis and co-localizes with K10

(a) AKR1C3 expression patterns were evaluated in normal human epidermis by immunofluorescence staining using AKR1C3 antibody and (b) quantified with ImageJ. Data is shown as the mean ±SEM pixel intensity after background subtraction (n=3, *P<0.05). (c) Normal human skin was subjected to double label immunofluorescence staining using AKR1C3 (red) and keratin 10 (K10, green) or keratin 5 (K5, green) antibodies. Labeling of each protein is shown separately in the middle and left panels. Yellow color in the merged pictures (right panels) suggests a co-localization of the 2 proteins. Bar=30μm, n=3.

Figure 2. AKR1C3 protein and mRNA are upregulated in response to calcium-induced PHK differentiation in a time and dose dependent manner

(a) Western blot analysis demonstrating the regulation of AKR1C3 by PHK cultured under 1.8 mM or 0.03mM calcium conditions and harvested at intervals or (b) by cells treated with the indicated dose of calcium and harvested 48 hours post treatment (representative results of 3 separate experiments is shown). (c, d) RT-PCR analysis demonstrating the regulation of AKR1C3 mRNA by cells treated under the same conditions described in a or b, respectively. Data is expressed as means ± SEM fold change over the baseline (n=3, *P<0.015).

Figure 3. AKR1C3 siRNA and 2HFN attenuate AKR1C3 enzymatic activity

(a) PHK transfected with 10nM of AKR1C3 or non-specific siRNA (Con siRNA) were allowed to differentiate for 48 hours and AKR1C3 protein was evaluated by western blot (n=3). (b) To examine AKR1C3 siRNA specificity, cells were treated as described in (a), mRNA was extracted and the transcription of AKR1C3 and AKR1C2 was evaluated by RT-PCR (means ± SEM, n=4, *P<0.05). (c) PHK were treated with AKR1C3 or control siRNA, allowed to differentiate for 48 hours and AKR1C3 enzymatic activity was evaluated at intervals by assessing 9α,11β-PGF₂ in supernatants using ELISA (means ± SEM, n=4, *P<0.05, **P<0.01). (d) 2HFN dose-dependently reduced AKR1C3 activity in PHK, as determined by ELISA quantification of 9α,11β-PGF₂ levels in supernatants, 2 hours post treatment (n=4, *P<0.05, **P<0.01).

Figure 4. Treatment with AKR1C3 siRNA or the AKR1C3 inhibitor 2HFN results in abnormal K10 and loricrin regulation during PHK differentiation

(a) PHK were treated with 10nM of control or AKR1C3 siRNA, or with (b) 1μM 2HFN or equal volume of DMSO (con). Cells from both experiments were induced to differentiate with 1.8mM calcium for the indicated times and the expression of K10 and loricrin was evaluated by western blot. A representative blot of 3 separate experiments is shown. K10 and loricrin are shown on 2 separate blots in panel b.

Figure 5. AKR1C3 is upregulated in AD lesions and in PHK treated with PGD₂

(a) Dose-dependent upregulation of AKR1C3 protein and (b) mRNA by PHK treated with various doses of PGD₂ and harvested after 24 hours, determined by western blot (n=3) and RT-PCR (means ± SEM, n=3, *P<0.01), respectively. (c) PHK were treated with 5μM of the indicated prostaglandin for 24 hours and the regulation of AKR1C3 was assessed by western blot (n=3). (d) Immunofluorescence staining showing the relative expression of AKR1C3 in epidermis of AD (n=4) psoriasis (n=3) and normal skin (n=4). One representative case is shown, images obtained at 10x magnification. (e) ImageJ analysis of AKR1C3 staining intensity. Data is presented as the mean ±SEM of 2 separate experiments where 4 normal skin samples, 4 AD and 3 psoriasis cases were evaluated. L=lesion, NL=non-lesion, *P<0.001, **P=0.012.

Figure 6. A role for AKR1C3 in promoting inflammation in AD (a suggested model)

Pruritus-induced scratching causes mast cell degranulation and rapid PGD₂ synthesis. PGD₂ attracts CRTH2-expressing immune cells which in turn can amplify its signaling by synthesizing and secreting more of this prostaglandin. Keratinocytes respond to high levels of PGD₂ by upregulating AKR1C3 expression and utilize this enzyme to metabolize PGD₂ to 9α,11β-PGF₂, a weaker but very stable pro-inflammatory mediator. This activity of AKR1C3 competes with the spontaneous conversion of PGD₂ to 15d-PGJ₂, an anti-inflammatory/pro-apoptotic mediator. Upregulation of 9α,11β-PGF₂ synthesis along with decreased formation of 15d-PGJ₂, an agonist for PPARγ and an inhibitor of NF-κB, potentially situates AKR1C3 as an indirect pro-inflammatory player in AD.