Aldo-Keto Reductase 1C3 Is Expressed inDifferentiated Human Epidermis, AffectsKeratinocyte Differentiation, and Is Upregulated inAtopic DermatitisAlon Mantel1,2, Amanda B. Carpenter-Mendini1, JoAnne B. VanBuskirk1, Anna De Benedetto1, Lisa A. Beck1

and Alice P. Pentland1

Aldo-keto reductase 1C3 (AKR1C3) has been shown to mediate the metabolism of sex hormones andprostaglandin D2 (PGD2), a lipid mediator that promotes skin inflammation in atopic dermatitis (AD). As bothhave a role in skin function and pathology, we first sought to investigate the expression pattern of AKR1C3 innormal human epidermis. Immunofluorescence revealed a strong expression of AKR1C3 in the differentiatedsuprabasal layers compared with the basal layer. Western blot analysis and quantitative PCR confirmed thatAKR1C3 expression was also upregulated in differentiation-induced primary human keratinocytes (PHKs). Toinvestigate the functional role of AKR1C3 during PHK differentiation, its expression and activity (measured asPGD2 reduction to 9a,11b-PGF2 by ELISA) were impaired by small interfering RNA or 20-hydroxyflavanone,respectively. Cytokeratin 10 (K10) and loricrin expression were then examined by western blot analysis, thusrevealing altered expression of these differentiation markers. Finally, following an observation that the AD-associated mediator, PGD2, upregulated AKR1C3 expression in PHKs, we used immunofluorescence to examineAKR1C3 expression in AD and psoriasis lesions. AKR1C3 was found to be upregulated in AD but not in psoriasislesions 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.

Journal of Investigative Dermatology (2012) 132, 1103–1110; doi:10.1038/jid.2011.412; published online 15 December 2011

INTRODUCTIONHuman skin is considered a steroidogenic organ because itlocally synthesizes and metabolizes various steroid hormonesand expresses their corresponding receptors (Thiboutot et al.,2003; Kanda and Watanabe, 2005; Zouboulis et al., 2007).Prostaglandins (PGs), a large family of arachidonic acid–derived lipid mediators, have also been shown to have a rolein skin function and pathology, including keratinocyteproliferation and differentiation, skin cancer, and allergicinflammation (Konger et al., 2009; Chun et al., 2010; Heet al., 2010; Surh et al., 2011). As specific enzymes

contribute to the regulation of local concentrations of steroidhormones and PGs in the skin, their distribution and activitymay indirectly affect skin function.

Several aldo-keto reductase 1C (AKR1C) enzymes havebeen shown to synthesize or metabolize steroid hormonesand/or PGs (Penning et al., 2003, 2006), and recent work hasalso characterized their expression in cultured humankeratinocytes and suggested a possible role for them inkeratinocyte survival (Marin et al., 2009). AKR1C enzymesare part of a larger AKR superfamily that uses NAD(P)(H) as acofactor to mediate the enzymatic reduction of aldehyde andketone groups of various substrates to their correspondingalcohols (Jez et al., 1997a, b). In humans, there are fourAKR1C enzymes, AKR1C1–AKR1C4, that share about 86%amino-acid sequence identity, yet vary in their substrate-binding specificities (Penning et al., 2000). AKR1C1 andAKR1C2 primarily convert specific potent steroid hormonesinto their less potent forms (Rizner et al., 2003, 2006). Incontrast, AKR1C3, also known as type 5 17b-hydroxysteroiddehydrogenase (17b-HSD; Lin et al., 1997) and prostaglandinF synthase (Matsuura et al., 1998; Suzuki-Yamamoto et al.,1999), converts the weak androgen D4-androstene-3,17-dioneto testosterone (potent androgen) and the weak estrogenestrone to its potent form, 17b-estradiol (Lin et al., 1997).

& 2012 The Society for Investigative Dermatology www.jidonline.org 1103

ORIGINAL ARTICLE

Received 5 August 2011; revised 10 October 2011; accepted 24 October2011; published online 15 December 2011

1Department of Dermatology, University of Rochester, Rochester, New York,USA and 2Department of Pharmacology and Physiology, University ofRochester, Rochester, New York, USA

Correspondence: Alice P. Pentland, Department of Dermatology, Universityof Rochester, 601 Elmwood Avenue, Box 697, Rochester, New York 14642,USA. E-mail: alice_pentland@urmc.rochester.edu

Abbreviations: AD, atopic dermatitis; AKR1C3, aldo-keto reductase family 1member C3; 17b-HSD, 17-beta-hydroxysteroid dehydrogenase; 15d-PGJ2,15-deoxy-D12,14-PGJ2; 2HFN, 20-hydroxyflavanone; K5, cytokeratin 5;K10, cytokeratin 10; PG, prostaglandin; PGD2, prostaglandins D2;PHK, primary human keratinocyte; PPAR-g, peroxisome proliferator-activatedreceptor gamma; siRNA, small interfering RNA

Unlike other AKR1C members, AKR1C3 can also synthesizePGF2a from the cyclooxygenase product PGH2 and mediateone of its most favorable catalytic activities, the reductionof prostaglandin D2 (PGD2) to 9a,11b-PGF2 (Lin et al., 1997;Matsuura et al., 1998; Suzuki-Yamamoto et al., 1999;Penning et al., 2000, 2006; Byrns et al., 2010).

Human skin is a major site for synthesis of PGD2,where it is synthesized primarily by immune cells suchas macrophages, Langerhans cells, and mast cells (Morrowet al., 1992; Shimura et al., 2010). In skin, PGD2 ismostly investigated in the context of allergic responses,particularly in supporting inflammation in atopic dermatitis(AD) lesions (Barr et al., 1988; Satoh et al., 2006; Shimuraet al., 2010).

The involvement of AKR1C3 in both PG and steroidhormone metabolism clearly situates this enzyme at theintersection of several very important physiological andpathological signaling pathways in the skin. We thereforecharacterized its expression pattern in normal humanepidermis and in the epidermis from AD and psoriasispatients, as well as examined its regulation and function incultured primary human keratinocytes (PHKs).

RESULTSAKR1C3 is expressed in differentiated human epidermis andcolocalizes with cytokeratin 10 (K10)

To define the expression pattern of AKR1C3 within thehuman epidermis, skin was evaluated by immunofluores-

cence using AKR1C3 antibody alone or in combination withlayer-specific epidermal markers. AKR1C3 staining intensityvaried within the epidermal layers, with the weakest intensityobserved in the basal layer and stronger expression notedin suprabasal layers, especially in the stratum spinosum(Figure 1a). On average, AKR1C3 expression was 2.3±0.07-fold higher in the suprabasal layers compared with the basallayer as determined by ImageJ analysis (Figure 1b). In doubleimmunolabeling experiments (Figure 1c) using the basal layermarker cytokeratin 5 (K5, green) or the early differentiationmarker K10 (green) together with AKR1C3 (red), AKR1C3colocalized with K10 (yellow), especially in the stratumspinosum, but did not appear to colocalize with K5 in thebasal layer.

AKR1C3 protein and mRNA are upregulated incalcium-induced PHK differentiation

As AKR1C3 showed stronger expression in differentiatedepidermis, its regulation in response to calcium-induceddifferentiation was evaluated in PHKs. Cultures treated with1.8 mM calcium chloride or with equal volume of phosphate-buffered saline were harvested at 24, 48, and 72 hours posttreatment, and AKR1C3 protein and mRNA levels wereassessed using western blot analysis or quantitative reversetranscription-PCR. Western blot analysis demonstrated atime-dependent upregulation of AKR1C3 protein in highcalcium-treated cells, and was similar to the expectedupregulation trend of the early differentiation marker K10

Suprabasal

Basal layer

AKR1C3 DAPI

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Figure 1. Aldo-keto reductase 1C3 (AKR1C3) is expressed in differentiated human epidermis and colocalizes with cytokeratin 10 (K10). (a) AKR1C3

expression patterns were evaluated in normal human epidermis by immunofluorescence staining using AKR1C3 antibody and (b) quantified with ImageJ.

Data are shown as the mean±SEM pixel intensity after background subtraction (n¼3, *Po0.05). (c) Normal human skin was subjected to double-label

immunofluorescence staining using AKR1C3 (red) and cytokeratin 10 (K10, green) or cytokeratin 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 colocalization of the two proteins.

Bar¼30 mm, n¼3. DAPI, 40-6-diamidino-2-phenylindole.

1104 Journal of Investigative Dermatology (2012), Volume 132

A Mantel et al.AKR1C3 in Normal Epidermis and Atopic Dermatitis

(Figure 2a). A 30- to 40-fold increase in AKR1C3 mRNAwas also observed in high calcium-treated cells (Figure 2c).Next, cells were treated with the indicated calcium dosesand harvested 48 hours post treatment. Western blotanalysis revealed a dose-dependent upregulation of AKR1C3protein, which again correlated with K10 expression(Figure 2b). A dose-dependent upregulation of AKR1C3mRNA of up to 28±20.5-fold over control was also noted(Figure 2d).

AKR1C3 small interfering RNA (siRNA) specifically attenuatescalcium-induced AKR1C3 expression

To investigate AKR1C3 function in keratinocytes, the effec-tiveness and specificity of an AKR1C3 siRNA constructwere evaluated. PHKs were transiently transfected with either10 nM of AKR1C3 or nonspecific siRNA, allowed to differ-entiate in 1.8 mM calcium for 48 hours, and then harvested forprotein and mRNA analysis. AKR1C3 protein expression,determined by western blot analysis, was markedly reducedin AKR1C3 siRNA–treated cells compared with controls,demonstrating the effectiveness of this treatment(Figure 3a). AKR1C2 is a closely related family member ofAKR1C3, which shares about 86% amino-acid sequencehomology (Jez et al., 1997a). To test the efficiency andspecificity of the AKR1C3 siRNA construct, mRNA samplesfrom AKR1C3 or control siRNA–treated cells werecollected, and the relative change in AKR1C3 and AKR1C2,compared with controls, was determined using reverse

transcription-PCR (Figure 3b). Although AKR1C3 mRNAlevels in AKR1C3 siRNA–treated cells were reduced46.6±19.1-fold over control siRNA, the reduction ofAKR1C2 was not significant.

AKR1C3 siRNA and 20-hydroxyflavanone (2HFN) attenuatedAKR1C3 enzymatic activity (measured as PGD2 reduction to9a,11b-PGF2) in PHKs

We next evaluated the effects of AKR1C3 protein reductionby siRNA or the specific AKR1C3 inhibitor 2HFN (Skarydovaet al., 2009) on AKR1C3 enzymatic activity in PHKs. Cellswere transfected with 10 nM AKR1C3 or control siRNA andinduced to differentiate for 48 hours by 1.8 mM calcium.Conversion of exogenously added PGD2 to 9a,11b-PGF2 wasthen assessed in the supernatants by ELISA. 9a,11b-PGF2

levels in supernatants from AKR1C3 siRNA–treated cells werelower at all time points (14.7±2.4 fold on average) comparedwith control cells (Figure 3c). Next, keratinocytes wereinduced to differentiate for 48 hours, and 9a,11b-PGF2

generation from PGD2 in the presence of the indicated dosesof 2HFN was assessed in supernatants 2 hours post treatment.The presence of 2HFN dose-dependently attenuated 9a,11b-PGF2 generation. The maximal 2HFN concentration tested(5 mM) reduced 9a,11b-PGF2 levels to 15.4±2% of thatmeasured in vehicle-treated cells (Figure 3d). These dataconfirmed that both AKR1C3 siRNA and 2HFN treatmentsresulted in the inhibition of this specific activity of AKR1C3in PHKs.

CaCl2 (mM) CaCl2 (mM)

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Figure 2. Aldo-keto reductase 1C3 (AKR1C3) protein and mRNA are

upregulated in response to calcium-induced primary human keratinocyte

(PHK) differentiation in a time- and dose-dependent manner. (a) Western

blot analysis demonstrating the regulation of AKR1C3 by PHK cultured

under 1.8 or 0.03 mM 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 three separate experiments

is shown). (c, d) Reverse transcription-PCR analysis demonstrating the

regulation of AKR1C3 mRNA by cells treated under the same conditions

described in a or b, respectively. Data are expressed as means±SEM fold

change over the baseline (n¼ 3, *Po0.015).

ConsiRNA

AKR1C3siRNA

AKR1C3 siRNAControl siRNA

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Figure 3. Aldo-keto reductase 1C3 (AKR1C3) small interfering RNA (siRNA)

and 20-hydroxyflavanone (2HFN) attenuate AKR1C3 enzymatic activity.

(a) Primary human keratinocytes (PHKs) transfected with 10 nM of AKR1C3

or nonspecific siRNA (Con siRNA) were allowed to differentiate for

48 hours and AKR1C3 protein was evaluated by western blot (n¼ 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 reverse transcription-PCR (means±SEM, n¼4, *Po0.05).

(c) PHKs were treated with AKR1C3 or control siRNA, allowed to differentiate

for 48 hours, and AKR1C3 enzymatic activity was evaluated at intervals by

assessing 9a,11b-PGF2 in supernatants using ELISA (means±SEM, n¼4,

*Po0.05, **Po0.01). (d) 2HFN dose dependently reduced AKR1C3

activity in PHKs, as determined by ELISA quantification of 9a,11b-PGF2

levels in supernatants, 2 hours post treatment (n¼ 4, *Po0.05,

**Po0.01).

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A Mantel et al.AKR1C3 in Normal Epidermis and Atopic Dermatitis

AKR1C3 siRNA and 2HFN treatment altered K10 and loricrinregulation during calcium-induced PHK differentiation

We postulated that AKR1C3 upregulation has a role indifferentiation-associated gene regulation during calcium-induced differentiation. To test this, AKR1C3 protein expressionor enzymatic activity was attenuated during differentiation,

and K10 and loricrin expressions were evaluated at intervalsby western blot analysis (Figure 4). PHKs were transfectedwith AKR1C3 or nonspecific siRNA and induced to differ-entiate for the indicated times (Figure 4a). AKR1C3 siRNAtreatment effectively reduced AKR1C3 protein expression atall tested time points, which also resulted in a markedreduction in K10 expression. Interestingly, in contrast to K10reduction, AKR1C3 siRNA transfectants demonstrated anearlier and stronger expression of loricrin. Furthermore, cellsthat were induced to differentiate by calcium in the presenceof 1 mM of the AKR1C3-specific inhibitor 2HFN also demon-strated the same abnormal expression pattern of K10 andloricrin (Figure 4b).

AKR1C3 is upregulated in response to PGD2 in PHKs

As our data suggested that PHKs use AKR1C3 to metabolizePGD2, we sought to further evaluate the effect of PGD2 onAKR1C3 expression. PHKs were incubated in variousconcentrations of PGD2 (under low calcium conditions) for24 hours and then harvested for analysis of AKR1C3protein and mRNA. A dose-dependent upregulation ofAKR1C3 in response to PGD2 was observed at the proteinlevel (Figure 5a), determined by western blot analysis, and atthe transcription level (Figure 5b), determined by reverse

con

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Figure 4. Treatment with aldo-keto reductase 1C3 (AKR1C3) siRNA

or the AKR1C3 inhibitor 20-hydroxyflavanone (2HFN) results in abnormal

cytokeratin 10 (K10) and loricrin regulation during primary human

keratinocytes (PHK) differentiation. (a) PHKs were treated with 10 nM of

control or AKR1C3 siRNA, or with (b) 1 mM 2HFN or an equal volume of

DMSO (con). Cells from both experiments were induced to differentiate

with 1.8 mM calcium for the indicated durations, and the expression

of K10 and loricrin was evaluated by western blot analysis. A representative

blot of three separate experiments is shown. K10 and loricrin are shown

on two separate blots in panel b.

Non

-lesi

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PGD2 (μM) 0 0.1 1 5Atopic dermatitis Psoriasis Normal skin

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Figure 5. Aldo-keto reductase 1C3 (AKR1C3) is upregulated in atopic dermatitis (AD) lesions and in primary human keratinocytes (PHKs) treated

with prostaglandin D2 (PGD2). (a) Dose-dependent upregulation of AKR1C3 protein and (b) mRNA by PHKs treated with various doses of PGD2 and

harvested after 24 hours, determined by western blot analysis (n¼ 3) and reverse transcription-PCR (means±SEM, n¼ 3, *Po0.01), respectively. (c) PHKs

were treated with 5 mM of the indicated prostaglandin for 24 hours, and the regulation of AKR1C3 was assessed by western blot analysis (n¼ 3).

(d) Immunofluorescence staining showing the relative expression of AKR1C3 in the epidermis of AD (n¼ 4) psoriasis (Pso, n¼ 3) and normal skin (n¼ 4).

One representative case is shown; images obtained at � 10 magnification. (e) ImageJ analysis of AKR1C3 staining intensity. Data are presented as the

mean±SEM of two separate experiments in which four normal skin samples, four AD, and three psoriasis cases were evaluated. L, lesion; NL, non-lesion.

*Po0.001, **P¼ 0.012.

1106 Journal of Investigative Dermatology (2012), Volume 132

A Mantel et al.AKR1C3 in Normal Epidermis and Atopic Dermatitis

transcription-PCR. AKR1C3 mRNA levels were increasedby up to 58.5±18.1-fold in cells treated with 5 mM PGD2.To evaluate the specificity of this response, cells wereincubated in the presence of 5 mM of either PGD2, PGE2, or9a,11b-PGF2, and the expression of AKR1C3 wasevaluated by western blot analysis (Figure 5c). The expectedupregulation of AKR1C3 was observed in PGD2-treatedcells, but was absent in the other tested conditions,suggesting that this response was specific to PGD2. Inaddition, incubation of PHKs in the presence of 5 mM of D4-androstene-3,17-dione or estrone (other steroid hormonesubstrates of AKR1C3) did not alter the protein expression ofAKR1C3 (data not shown).

AKR1C3 expression is upregulated in skin lesions of ADbut not in psoriasis

PGD2 has a significant role in AD-associated inflammation(Satoh et al., 2006). Our data suggested that keratinocytesupregulate the expression of AKR1C3 in response to PGD2

and also use this enzyme to metabolize PGD2 to 9a,11b-PGF2. As the regulation of AKR1C3 in AD may have a role incontrolling the local concentration of this pro-inflammatoryPG, we investigated the expression of AKR1C3 in lesionaland non-lesional skin of AD and psoriasis patients byimmunofluorescence (Figure 5d). AKR1C3 was markedlyupregulated in AD lesions compared with non-lesionalmatching controls. In contrast, no differences in AKR1C3expression were observed in psoriasis lesions compared withnearby non-lesional skin. Quantification of AKR1C3 expres-sion by ImageJ confirmed a 2.8±0.4-fold increase inAKR1C3 expression in AD lesions over matching non-lesional skin, whereas no change in expression was notedin psoriasis samples (Figure 5e).

DISCUSSIONAKR1C3 is the only AKR1C family member that also mediates17b-HSD activity (Penning et al., 2001). Previous workinvestigating 17b-HSD activity in human skin sectionslocalized its peak activity around the basement layer of theepidermis (Hikima and Maibach, 2007), whereas anotherstudy also suggested a functional role for several AKR1Cisoforms in keratinocyte survival (Marin et al., 2009). Thecurrent work shows that AKR1C3 is differentially expressed inhuman epidermis, with the highest expression levels ob-served in mid-epidermis around the stratum spinosum. Otherwork documented multiple AKR1C3-associated 17b-HSDactivities in HaCaT cells, as well as in PHKs. Their datademonstrated that these enzymatic activities were relativelylow in basal keratinocytes, peaked during early stages ofdifferentiation, and returned to baseline in terminallydifferentiated cultures, suggesting that the expression ofsteroid metabolizing enzymes in keratinocytes is differentia-tion dependent (Gingras et al., 2003). Their work, however,did not evaluate the protein or transcriptional regulation ofany 17b-HSD enzymes. The current work provides furtherevidence that the 17b-HSD enzyme, AKR1C3, is markedlyupregulated in differentiated epidermis and in calcium-induced PHK differentiation.

AKR1C3 upregulation in differentiated PHK suggests adifferentiation-associated function for this enzyme. To testthis, a specific AKR1C3 siRNA and the specific AKR1C3inhibitor, 2HFN, were used. 2HFN has been shown tospecifically inhibit recombinant AKR1C3 with an IC50 of0.3 mM (Skarydova et al., 2009). Our work further demon-strates the capacity of this flavanoid derivative to inhibitcellular AKR1C3 ketoreduction activity with an IC50 between0.5 and 1 mM, suggesting a good bioavailability for this drugunder our experimental conditions.

The current work demonstrates a functional role forAKR1C3 in gene regulation during keratinocyte differentia-tion. Of interest is the observation that the interruption ofAKR1C3 expression or activity attenuates K10 expressionwhile resulting in the upregulation of loricrin. To investigatewhether the upregulation of loricrin was directly due toAKR1C3 inhibition or the subsequent decrease in K10expression, we attempted to attenuate K10 expression withsiRNA in PHK during differentiation; however, we wereunable to examine loricrin expression because of cytotoxicity(data not shown). On the basis of our current data showingearlier and more profound loricrin expression, it is reasonableto assume that attenuating AKR1C3 activity during differ-entiation disrupted this delicate process at its early stages,which hastened terminal differentiation. Future studies willbe needed to evaluate the regulation of other differentiation-associated markers such as profilaggrin and involucrin. Thesedata will provide further insights into whether altered loricrinexpression was a direct outcome of AKR1C3 attenuationduring keratinocyte differentiation, or was it just a part of anoverall hastened terminal differentiation.

As there is no evidence that AKR1C3 directly regulatesgene expression, this action is probably mediated indirectlyby its related PG and/or steroid metabolites, its contributionto the cellular redox state, or its action in ketoreduction of anunknown substrate. Keratinocytes synthesize and secretePGF2a and express its corresponding receptor FP (Pentlandand Needleman, 1986; Scott et al., 2005). AKR1C3 upregula-tion during keratinocyte differentiation could result inincreased PGF2a synthesis, which may affect differentiation-associated gene regulation. However, this mechanism is notsupported by our observations, as the addition of variousdoses of PGF2a to AKR1C3 siRNA–treated cells failed toretrieve normal K10 expression during keratinocyte differ-entiation (data not shown). AKR1C3 can also convert steroidhormone precursors to their active form, but as the culturemedium does not contain significant quantities of theseprecursors, this mechanism is unlikely. Increased expressionof AKR1C3 during differentiation suggests a requirement forits enzymatic activity. Our work did not identify a specificsubstrate; however, as the ketoreduction activity mediated byAKR1C3 is also NAD(P)(H) dependent, its increased enzy-matic activity may affect cellular redox state. This action mayindirectly alter the activity of redox-sensitive transcriptionfactors that regulate the expression of differentiation-asso-ciated genes during keratinocyte differentiation (Meyer et al.,1994; Nakamura et al., 2007). Taken together, AKR1C3 islikely involved in differentiation-associated gene regulation

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A Mantel et al.AKR1C3 in Normal Epidermis and Atopic Dermatitis

in a PGF2a- and hormone-independent manner. However,the precise mechanism remains to be definitively resolved byfuture work.

Local release of PGD2 is known to have a major role inregulating T-helper type 2 (Th2) chronic inflammation in AD,where it mainly serves as a chemoattractant for Th2-associated immune cells (Iwasaki et al., 2002; Satoh et al.,2006). Recent work has shown that, in addition to PGD2, itsstable metabolite, 9a,11b-PGF2, mediates the same Th2inflammatory responses, but with much lower potency(Sandig et al., 2006). This suggests that both PGD2 and theaccumulation of its stable metabolite 9a,11b-PGF2 maycontribute to inflammation in lesional sites of AD.

Our studies show that PHKs upregulate AKR1C3 specifi-cally in response to PGD2 and use this enzyme to reducePGD2 to 9a,11b-PGF2. In fact, our preliminary data alsosuggest that treating PHK with other steroidal substrates ofAKR1C3 (i.e., estrone and D4-androstene-3,17-dione) or withtheir corresponding reduced products (17b-estradiol andtestosterone) had no effect on AKR1C3 expression (data notshown). In addition, treatment with other pro-inflammatorycytokines such as IL-4, IFN-g, and tumor necrosis factor-ahad no effect on AKR1C3 expression by PHKs (data notshown). PGD2 is a relatively unstable PG that has beenshown to spontaneously dehydrate to 15-deoxy-D12,14-PGJ2(15d-PGJ2) in vivo and in vitro (Shibata et al., 2002). 15d-PGJ2 is an anti-inflammatory lipid that mostly mediates itsactions directly via activation of peroxisome proliferator-activated receptor g (PPAR-g) and/or inhibition of NF-kBsignaling in immune cells (Forman et al., 1995; Maggi et al.,2000; Straus et al., 2000; Watanabe et al., 2010). Previousdata have shown that PPAR-g activation attenuates allergen-induced inflammation in skin and lungs of mice (Ward et al.,2006; Dahten et al., 2008). This suggests that PPAR-gactivation by 15d-PGJ2 may have a role in suppressinginflammation in AD patients.

A specific role for AKR1C3 in AD is supported by ourobservation that AKR1C3 expression is markedly upregulatedin lesions of this skin condition, but is unchanged in the Th1-mediated inflammatory lesions of psoriasis (Schlaak et al.,1994). We propose a model (Figure 6) in which upregulationof AKR1C3 in AD lesions supports inflammation by directlycausing an increase in 9a,11b-PGF2 synthesis rates anddiverting the spontaneous generation of the potent anti-inflammatory mediator, 15d-PGJ2. This function of AKR1C3has been previously implicated in HL-60 cells (Desmondet al., 2003) and in MCF-7 cells (Byrns et al., 2010). Thus,AKR1C3 activity and expression in AD lesions coulddetermine the balance between pro- and anti-inflammatoryPG mediators. This work suggests that inhibition of AKR1C3may be a potential therapeutic target in AD-associatedinflammation.

MATERIALS AND METHODSCell culture

Defatted skin, obtained from breast reduction or panniculectamies,

was placed in 0.25% trypsin in phosphate-buffered saline for 5 hours

at 37 1C and 5% CO2. The epidermis was separated and epidermal

cell suspension placed in T-75 flasks, pre-coated with 1:5 Purecol

(Advanced Biomatrix, San Diego, CA) in keratinocyte growth media

(Invitrogen, Grand Island, NY), and supplemented with 5 ng ml�1

epidermal growth factor, 20 mg ml�1 bovine pituitary extract, and

antibiotics. The medium was routinely changed every 3–4 days and cells

were passed at B90% confluence. Cells were used at passages 3–6.

Reagents

Monoclonal mouse anti-human AKR1C3 (ab49680), monoclonal

rabbit anti-human keratin 5 (ab52635), and polyclonal rabbit anti-

human b-actin were obtained from Abcam (Cambridge, MA).

Polyclonal rabbit anti-human keratin 10 (PRB-159p-100) and rabbit

anti-human loricrin (PRB-145p) were purchased from Covance

(Emeryville, CA). 20-Hydroxyflavanone, PGE2, PGD2, and 9a,11b-

PGF2 were purchased from Sigma-Aldrich (St Louis, MO) and

dissolved in DMSO as 10 mM stocks.

Immunofluorescence

Biopsies were fixed in 10% formalin and then embedded in paraffin.

Sections were deparaffinized in xylene for 10 minutes, followed by

graded rehydration in EtOH (100, 95, 85, 70, and 50%) for 5 minutes

each. Sections were then incubated in antigen retrieval buffer (10 mM

Tris, 1 mM EDTA, 0.05% Tween-20, pH 9.0) at 95 1C for 10 minutes.

Samples were incubated in 1:50 anti-AKR1C3, 1:2,000 K10, or

1:2,000 keratin 5 antibodies alone or in mixtures as indicated

Stimulus/scratch

Mast cells/Langerhans cells

PGD2

9α11β-PGF2

15d-PGJ2

AKR1C3

KeratinocyteTh2-associatedimmune cell

Pro-inflammatoryresponse

PPARγNF-κB

CRTH2 Stimulatoryeffect

Inhibitoryeffect

Figure 6. A role for aldo-keto reductase 1C3 (AKR1C3) in promoting

inflammation in atopic dermatitis (AD; a suggested model). Pruritus-induced

scratching causes mast cell degranulation and rapid prostaglandin D2

(PGD2) synthesis. PGD2 attracts CRTH2 (chemoattractant receptor-

homologous molecule expressed on Th2 cells)-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 use this enzyme to metabolize

PGD2 to 9a,11b-PGF2, a weaker but very stable pro-inflammatory mediator.

This activity of AKR1C3 competes with the spontaneous conversion of PGD2

to 15-deoxy-D12,14-PGJ2 (15d-PGJ2), an anti-inflammatory/pro-apoptotic

mediator. Upregulation of 9a,11b-PGF2 synthesis along with decreased

formation of 15d-PGJ2, an agonist for peroxisome proliferator-activated

receptor g (PPAR-g) and an inhibitor of NF-kB, potentially situates AKR1C3

as an indirect pro-inflammatory factor in AD.

1108 Journal of Investigative Dermatology (2012), Volume 132

A Mantel et al.AKR1C3 in Normal Epidermis and Atopic Dermatitis

overnight at 4 1C. Nonspecific mouse or rabbit IgG controls were

performed in parallel. Samples were then incubated in 1:400

Fluorescein-conjugated goat anti-rabbit IgG, 1:200 Texas Red goat

anti-mouse IgG, or a mixture of both, as required, for 1 hour at room

temperature. Samples were visualized using a fluorescence micro-

scope (Nikon Eclipse E800 equipped with a Spot RT3 camera,

Nikon, Melville, NY). Pictures were obtained using the Spot advan-

ced software (Diagnostic Instruments, Sterling Heights, MI).

ImageJ analysis

ImageJ version 1.440 (NIH, Bethesda, MD) was used. The averages

of the fluorescent pixel intensity of 10 random fields per sample

(rectangular tool) from the site of interest (i.e., the epidermis or a

specific epidermal area) minus the averages of nonspecific

fluorescence (background) were recorded and averaged.

Western blot analysis

Cells were extracted in RIPA buffer containing 1:100 Protease

Inhibitor Cocktail (Santa Cruz Biotechnology, Santa Cruz, CA).

Protein (50mg) was separated on 10% SDS-PAGE and transferred to

nitrocellulose. The membrane was blocked, and then incubated in

the presence of either 1:1,000 anti-AKR1C3, 1:2,000 anti-keratin 10,

1:2,000 anti-loricrin, or 1:4,000 anti-b-actin antibodies at 4 1C

overnight. Matching secondary horseradish peroxidase-conjugated

IgG at 1:2,000 was applied and immunoreactive protein bands

were detected using Western Blot Luminal Reagent (Santa Cruz

Biotechnology).

AKR1C3 siRNA transient transfection

Transfection was performed on cultures at 50% confluence,

according to the manufacturer’s protocol, using 10 nM AKR1C3

Stealth siRNA (oligo ID: HSS112670, Invitrogen) or nonspecific

siRNA (cat# 12935-200, Invitrogen). Transfection medium was

aspirated after 6 hours and replaced by conditioned keratinocyte

growth media as indicated.

Evaluation of AKR1C3 enzymatic activity (PGD2 to 9a,11b-PGF2 conversion) in PHKs

Cells were cultured in six-well dishes as indicated. Keratinocyte

growth media without supplements (500 ml per well), containing

1mM PGD2, was added to appropriate wells, followed by incubation

at 37 1C for the indicated durations. 9a,11b-PGF2 from each

condition was evaluated in the supernatant by a specific 9a,

11b-PGF2 kit (catalog: 516521, Cayman Chemical, Ann Arbor, MI)

according to manufacturer’s protocol, and results were normalized

to protein content. 9a,11b-PGF2 was detected in 1 mM PGD2-

containing media only after it had been incubated with cells,

whereas no signal was detected in PGD2-containing media alone or

in media exposed to cells that did not contain PGD2 (not shown).

Total RNA isolation and real-time PCR

Total RNA was extracted using Trizol reagent (Invitrogen) and

complementary DNA was synthesized using the SuperScript III

First Strand complementary DNA synthesis kit (Invitrogen). Real-

time PCR primers for AKR1C3 (fwd: 50-GAGAAGTAAAGCTTTGGAG

GTCACA-30, rev: 50-CAACCTGCTCCTCATTATTGTATAAATGA-30)

AKR1C2 (fwd: 50-CCTAAAAGTAAAGCTCTAGAGGCCGT-30, rev:

50-GAAAATGAATAAGATAGAGGTCAACATAG-30) and GAPDH

(fwd: 50-CCACCCATGGCAAATTCC-30, rev: 50-TGGGATTTCCATTG

ATGACAAG-30) were designed using the ABI Primer Express Software

(Applied Biosystems, Foster City, CA). The PCR products were

amplified and detected using SYBR Green in an iCycler iQ real-time

PCR system (Bio-Rad, Hercules, CA). Relative complementary DNA

amounts were calculated on the basis of the threshold cycle (CT)

value and were normalized to the amount of GAPDH by means of

the 2�DDCT method.

AD, psoriasis, and normal skin samples

All studies were approved by the Research Subject Review Boards at

the University of Rochester Medical Center and/or by the Research

Subject Review Boards at the Johns Hopkins University. All subjects

gave written informed consent. The diagnosis of AD was made using

the US consensus conference criteria (Eichenfield, 2004). All subjects

underwent a 5-mm punch biopsy from a non-lesional site and an

additional 5 mm biopsy from a lesional site. For the expression of

AKR1C3 in normal skin, 6 mm biopsies were obtained from the

buttock area of random volunteers who all gave written informed

consent. All experiments were conducted in accordance with

the Declaration of Helsinki Principles (hematoxylin–eosin stains of

psoriasis and AD are shown in Supplementary Figure S1 online).

Statistical analysis

The results are presented as means±SEM. The appropriate t-test was

calculated using Microsoft Excel or GraphPad Prism. A P-value

o0.05 was considered statistically significant.

CONFLICT OF INTERESTThe authors state no conflict of interest.

ACKNOWLEDGMENTSWe thank Dr Glynis Scott (Department of Dermatology, University ofRochester) for inspiring discussions and George Liu for providing editorialsupport for the manuscript. This work was supported by the NIH grant 5RO1CA117821.

SUPPLEMENTARY MATERIAL

Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

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