garza 1..11

DOI: 10.1126/scitranslmed.3003122, 126ra34 (2012);4 Sci Transl Med

, et al.Luis A. GarzaMen with Androgenetic Alopecia

Inhibits Hair Growth and Is Elevated in Bald Scalp of2Prostaglandin D

Editor's Summary

. may have given patients with AGA a long-awaited choice: curly or straight?et al. By discovering such therapeutic targets for downstream drug development, such as a topical treatment, Garza 2

Garza and coauthors identified the receptor GPR44 to be responsible for mediating the negative effects of PGD

prostaglandin pathway and AGA.inhibited hair growth in both mice and human hair follicles, providing a crucial functional link between the

,2 and a related metabolite, 15-dPGJ2expression and hair follicle regression. The authors further found that PGD gene2. uncovered a temporal relationship between PGDet alwith synchronized hair follicle cycling, Garza

was also elevated in bald versus haired scalp. In mice2of 17 men, they confirmed that the synthase product PGD) synthase was elevated at the mRNA and protein levels in bald scalp only. In a larger group2 (PGD2prostaglandin D

The authors first examined bald and haired scalp from five men with AGA and showed that the enzyme

responsible for baldness.insight into the pathogenesis of AGA, in hopes of developing new therapeutics that target specific pathways disorder that affects both men and women, leading to hair thinning and loss. Here, Garza and colleagues provide newPatients with androgenetic alopecia (AGA), however, would take either one, as long as it meant having hair. AGA is a

Everybody wishes his or her hair was different; curly hair wants straight locks, straight hair desires some curl.

A Not-So-Hairy Situation

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R E S EARCH ART I C L E

HA IR LOSS

Prostaglandin D2 Inhibits Hair Growth and Is Elevatedin Bald Scalp of Men with Androgenetic AlopeciaLuis A. Garza,1* Yaping Liu,2 Zaixin Yang,1 Brinda Alagesan,1 John A. Lawson,3 Scott M. Norberg,1

Dorothy E. Loy,4 Tailun Zhao,1 Hanz B. Blatt,1 David C. Stanton,5 Lee Carrasco,5

Gurpreet Ahluwalia,6† Susan M. Fischer,7 Garret A. FitzGerald,3 George Cotsarelis1‡

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Testosterone is necessary for the development of male pattern baldness, known as androgenetic alopecia (AGA);yet, the mechanisms for decreased hair growth in this disorder are unclear. We show that prostaglandin D2 synthase(PTGDS) is elevated at the mRNA and protein levels in bald scalp compared to haired scalp of men with AGA. Theproduct of PTGDS enzyme activity, prostaglandin D2 (PGD2), is similarly elevated in bald scalp. During normal folliclecycling in mice, Ptgds and PGD2 levels increase immediately preceding the regression phase, suggesting an inhib-itory effect on hair growth. We show that PGD2 inhibits hair growth in explanted human hair follicles and whenapplied topically to mice. Hair growth inhibition requires the PGD2 receptor G protein (heterotrimeric guaninenucleotide)–coupled receptor 44 (GPR44), but not the PGD2 receptor 1 (PTGDR). Furthermore, we find that a transgenicmouse, K14-Ptgs2, which targets prostaglandin-endoperoxide synthase 2 expression to the skin, demonstrateselevated levels of PGD2 in the skin and develops alopecia, follicular miniaturization, and sebaceous gland hyper-plasia, which are all hallmarks of human AGA. These results define PGD2 as an inhibitor of hair growth in AGA andsuggest the PGD2-GPR44 pathway as a potential target for treatment.

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INTRODUCTION

Eighty percent of Caucasian men experience some degree of androge-netic alopecia (AGA) before age 70 (1). Testosterone is necessary forAGA to develop, and a genetic susceptibility locus in the androgen recep-tor is present in a minority of men with AGA; however, additional factorscontributing to this disorder remain unknown (2–4). Studies on AGAhave the potential to yield insights into other androgen-mediated diseases,such as benign prostatic hypertrophy and prostate cancer (5). Currentlegitimate treatments for AGA include finasteride, minoxidil, and hairtransplantation (6). Finasteride inhibits 5-a reductase 2 (SRD5A2), whichconverts testosterone to a more potent androgen, dihydrotestosterone. Al-though the active targets of minoxidil in AGA therapy are not known,suggested ones include potassium channels or prostaglandins. Specificexamples of the latter have not been conclusively identified (7).

In AGA, large “terminal” hair follicles forming thick hair shafts min-iaturize over time to small follicles that generate microscopic effete hairs(8). Follicle miniaturization is accompanied by a decrease in the dura-tion of the growing phase of the follicle (anagen), which normally lastsseveral years to produce hairmore than 1m long, butwhich decreases toonly days or weeks in AGA. This results in an increase in the percentageof resting (telogen) hair follicles containing microscopic hairs in baldscalp (4). In addition to these intrinsic changes to the hair follicle, infil-

1Department of Dermatology, Kligman Laboratories, University of Pennsylvania Schoolof Medicine, Philadelphia, PA 19104, USA. 2Screening and Protein Sciences, Merck &Company, North Wales, PA 19454, USA. 3Department of Pharmacology and the Institutefor Translational Medicine and Therapeutics, University of Pennsylvania School ofMedicine, Philadelphia, PA 19104, USA. 4Department of Dermatology, Johns HopkinsUniversity School of Medicine, Baltimore, MD 21231, USA. 5Department of Oral andMaxillofacial Surgery, University of Pennsylvania School of Medicine, Philadelphia, PA19104, USA. 6Gillette Corporation, Foxborough, MA 02035, USA. 7University of Texas M.D. Anderson Cancer Center, Science Park–Research Division, Smithville, TX 78957, USA.*Present address: Department of Dermatology, Johns Hopkins University School ofMedicine, Baltimore, MD 21231, USA.†Present address: Allergan Inc., Irvine, CA 92623, USA.‡To whom correspondence should be addressed. E-mail: [email protected]

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trating lymphocytes andmast cells have been identified around themin-iaturizing follicle (9), especially in the area of the stem cell–rich bulgearea (10). Sebaceous glands, which attach to each follicle, hypertrophyin bald scalp (8). In balding scalp, the number of hair follicle stem cellsremains intact, whereas the number of more actively proliferating pro-genitor cells markedly decreases (11). This suggests that balding scalpeither lacks an activator or has an inhibitor of hair follicle growth.

Here, we used a global gene expression approach to define differen-tially expressed genes in balding versus nonbalding scalp from the sameindividuals. We found elevated levels of prostaglandin D2 synthase(PTGDS) at the message and protein levels in balding versus hairedscalp from men with AGA. Given the described metabolism of prosta-glandins in the human hair follicle (12, 13), we focused on the synthase,both in mouse (Ptgds) and in human (PTGDS). The enzymatic productof PTGDS, prostaglandin D2 (PGD2), was also elevated in bald humanscalp tissue. We show a close temporal relationship between elevationsin both Ptgds mRNA and PGD2 levels in mice with hair follicle regres-sion during normal hair follicle cycling. We further provide functionaldata indicating that PGD2 and its nonenzymatic metabolite, 15-deoxy-D12,14-prostaglandin J2 (15-dPGJ2), inhibit hair growth in both mouseand human hair follicles. In mice and humans, the PGD2-mediated in-hibition of hair growth required the G protein (heterotrimeric guaninenucleotide–binding protein)–coupled receptor 44 (GPR44), but not theprostaglandin D2 receptor 1 (PTGDR). Finally, we describe a mousemodel (K14-Ptgs2) with elevated PGD2 levels in the skin that phenocopieshuman AGA (14). These results implicate PGD2 in the pathogenesis ofAGA and suggest new receptor targets for its treatment.

RESULTS

PTGDS and PGD2 are elevated in bald scalpTo assess global gene expression changes in the bald scalp of men withAGA, we performed gene expression microarrays comparing bald tohaired scalp in five men with AGA. Correlation coefficients among haired

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Term % Count P value Development 26 40 1.99E-10 Morphogenesis 24 37 4.01E-14 Organ development 20 31 4.48E-12 Organogenesis 20 31 4.72E-12 Histogenesis 9 15 1.07E-13 Ectoderm development 7 12 4.89E-12 Epidermis development 7 11 2.9E-11 Biological process unknown 5 9 4.6E-3 Cell adhesion 5 9 8.05E-2 Neurogenesis 5 8 3.14E-2 Cell motility 3 6 1.46E-2 Skeletal development 3 6 2.08E-3 Central nervous development

3 5 8.19E-3

Term % Count P value Response to biotic stimulus 22 16 2.02E-2 Defense response 21 15 2.67E-2 Immune response 21 15 1.81E-2 Response to stress 12 9 2.81E-2 Antigen presentation 11 8 4.36E-3 Antigen processing 11 8 4.36E-3 Response to external biotic stimulus

9 7 1.38E-2

Lipid metabolism 8 9 6.37E-2

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and among bald replicate samples ap-proached 1 (Fig. 1A). Correlation coeffici-ents comparing haired to bald scalp withinindividuals were also close to 1, indicatingthe advantage of paired samples as internalcontrols.

We identified 250 transcripts that weredifferentially expressed in human hairedversus bald scalp. As evidence for the va-lidity of these transcripts, a clustering algo-rithm sorted out samples as either hairedor bald, on the basis of the expression ofthese genes (Fig. 1B). Hair keratins wereup-regulated in haired scalp, as would beexpected. For unclear reasons, hemoglobin-related transcripts were elevated in baldscalp. In haired scalp, 169 genes wereelevated, and 81 genes were elevated inbald scalp, with the expected directions
of expression (Fig. 1C). We determined enriched gene ontology func-tional classifications for these genes and identified overlapping catego-ries. In the haired scalp, morphogenesis and developmental pathwaygenes were overexpressed, whereas in the bald scalp immune responsegenes were overexpressed (Fig. 1D), consistent with the known histol-ogy of AGA (9).
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Considering the clinical use of prostaglandin F2a (PGF2a)–relatedcompounds to promote hair growth (15), and the often antagonisticfunctions of prostaglandins (16), we were intrigued to find PTGDS asone of the most highly expressed transcripts in human male baldingscalp (Fig. 1, E and F). Of the three probe sets that covered PTGDS onthe microarray, all were elevated in balding scalp (Fig. 1E). Increased

Fig. 1. Summary of gene expression pro-files of haired versus bald scalp from fivemen with AGA. (A) Correlation coefficientsto compare the degree of difference be-tween all haired samples, between allbald samples, or between haired versusbald samples within individuals. Data aremeans ± SEM (n = 5). ***P < 0.001 com-pared to both hair only and bald only. (B)Gene clustering algorithm of 250 significantgenes in haired and bald scalps in five menlabeled A to E. (C) Discontinuous graphswith gene expression level on the y axisand samples grouped as pairs along the xaxis for the 250 most significant genesdivided into those higher in the haired (H)scalp (169; left) and those higher in the bald(B) scalp (81; right). In each pair, the firstsample (beginning of line) is the haired scalpand the second sample (end of line) is thebald scalp. (D) Gene ontology categories inhaired scalp (left) versus balding scalp(right). Percentages listed are numbers oftranscripts in each category out of total num-ber of unique gene transcripts. P valuesare modified Fisher exact EASE (ExpressionAnalysis Systematic Explorer) score for thesignificant enrichment of each category offunction. (E) Fold change in PTGDS expres-sion in bald scalp compared to haired scalp.Data are means ± SEM (n = 5). ***P < 0.0001between haired and bald scalp for eachprobe set, which covers distinct areas with-in the PTGDS gene. (F) Schematic of thePGD2 pathway.

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PTGDS expression and PTGDS proteinlevels were further confirmed in baldingversus haired human scalp by real-timequantitative polymerase chain reaction(qPCR) (Fig. 2A) and Western blotting(Fig. 2, B and C).

To better understand the role of PTGDSin balding, we measured levels of its syn-thase product, PGD2, in both balding andhaired human scalps. We discovered asignificant increase in PGD2 in the bald-ing scalp compared to haired scalp by im-munoassay (Fig. 2D). The absolute levelof PGD2 was 16.3 ng/g tissue in baldingscalp and 1.5 ng/g tissue in haired scalp.PGD2 levelswere thenmeasuredusingultra–high-performance liquid chromatography–mass spectrometry (UHPLC-MS) becauseof its reported superior accuracy in measur-ing prostaglandins compared to immuno-assay (17). In a larger series of pairedbald andhaired samples from17menwithAGA, we noted an increase in PGD2

in bald scalp compared to haired scalp(Fig. 2E).

Other prostaglandins have activity onhair follicle growth. We measured levels ofadditional prostaglandin species in bald-ing and haired scalps. The nonenzymaticmetabolite of PGD2, 15-dPGJ2, was alsoincreased in balding scalp compared tohaired scalp (Fig. 2E). However, the ab-solute level of 15-dPGJ2 (3.8 ng/g baldscalp, 1.3 ng/g haired scalp) was lower thanPGD2 (53.5 ng/g bald scalp, 25.6 ng/ghaired scalp) (Fig. 2F). UHPLC-MS diddetect higher levels of PGD2 in baldingscalp than immunoassay (53.5 versus16.3 ng/g tissue), although both methodsdemonstrated increased PGD2 mass inbald scalp. In contrast to PGD2, the levelof prostaglandin E2 (PGE2), which is syn-
thesized by PTGES rather than PTGDS, was more abundant in hairedscalp compared to balding scalp (Fig. 2E), with 3.1 ng/g balding scalpand 6.4 ng/g haired scalp as detected by UHPLC-MS (Fig. 2F).
Nonpermanent follicle mouse keratinocytes express Ptgdsat the end of anagenTo better understand the normal biological function of Ptgds andPGD2 on hair growth, we capitalized on the well-characterized, syn-chronized hair cycle of the mouse (18). Fluctuations in gene expres-sion and protein levels can be tracked to different stages of the hairfollicle cycle by examining skin at different ages or after depilationof the mouse hair, which induces a synchronized new hair folliclecycle. In the process of investigating the features of the synchronizedmouse hair cycle, we verified hair cycle stage relative to postnatal(PN) day in mouse skin biopsy samples. We confirmed that, as sug-gested by gross appearance, the histological features of mice at PN21

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were those of late telogen, PN35 of late anagen, and PN46 of catagen(fig. S1).

Ptgds mRNA, as measured by qPCR, peaked in the late stage ofanagen and was sevenfold higher compared to the resting phase (telogen)at PN20 (Fig. 3A). Thus, Ptgds expression was lowest during telogenand increased progressively through anagen to peak at late anagennear the time of transition to catagen, the phase of regression. To moreclosely examine the proximity of the Ptgds expression peak to thecatagen transition, we compared it to the expression of fibroblast growthfactor 5 (Fg f5), a known marker of catagen onset (19). Fg f5 expressionpeaked in late anagen compared to telogen (Fig. 3A). Together, Fg f5and Ptgds peak expression levels overlapped during late anagen, justbefore the onset of catagen.

We next sought to identify control genes expressed differently fromFg f5 and Ptgds. As a marker of early anagen, we measured the expres-sion of the receptor for PGF2a, which is known for its ability to induce

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Fig. 2. Increased PGD2 pathway activity in balding scalp of men with AGA. (A) Expression of lipocalin-typePTGDSmRNA in bald versus haired scalp, as tested by qPCR. Data are means ± SEM (n = 4). (B and C) The
amount of PTGDS protein in paired bald (B) and haired (H) scalps (n = 4), as shown by Westernblotting with Ponceau stain used for verification of equal loading (B) and its quantitation as normal-ized to haired scalp (C). Data are means ± SEM. (D) PGD2 production in bald scalp, as tested by ELISA.Data are means ± SEM (n = 3). (E and F) Fold change in PGD2 (n = 17), 15-dPGJ2) (n = 7), and PGE2 (n = 17)expression in bald scalp compared to haired scalp (E). Total prostaglandin content is quantified in (F).Data are means ± SEM. *P < 0.05; **P < 0.01. In (F), P value compares haired versus bald samples foreach prostaglandin.



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hair growth both in mice (20) and in humans (15). In contrast to the latepeak of Ptgds and Fgf5, prostaglandin F receptor (Ptgfr) mRNA peakedin early anagen or late telogen, with a 21-fold change versus second telo-gen (PN52) (Fig. 3A) and a 3-fold change versus first telogen (PN20).

To determine the temporal relationship between expression of Ptgdsand the production of PGD2, we measured PGD2 levels by HPLC-MSin identical mice from which mRNA data were determined in Fig. 3A.We discovered that PGD2 production peaked after the apex of Ptgdsexpression. During the catagen stage, at PN46, PGD2 reached a peaklevel of 87.6 ng/g tissue compared to a nadir in early anagen on PN24of 7.4 ng/g tissue (Fig. 3B). Thus, PtgdsmRNA is first expressed in theskin in late anagen, and subsequently, PGD2 is produced via Ptgdsprotein during catagen.

Although early hair follicle cycle stages in individual mice are nat-urally synchronized, animals of the same age and even litter can ex-hibit heterogeneity with respect to hair follicle cycling. Therefore, we

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repeated the above analysis using micethat had been depilated simultaneouslyto synchronize the hair cycle betweenanimals as well as within animals. Corrob-orating our results from the spontaneoushair cycle, Ptgds mRNA also fluctuatedwith the hair cycle in depilated animals.Ptgds mRNA peaked in late anagen,followed by the peak of PGD2 in catagen(Fig. 3C). PGD2 production peaked onday 19.5 (catagen), with 199.9 ng/g tissuecompared to a nadir of 27.6 ng/g tissue onday 37. We also noted a unique peak ofPGD2 production within hours of depila-tion, which was not seen in the spontane-ous hair cycle in Fig. 3B. This coincideswith degranulation of mast cells observedpreviously after depilation (21). The morenarrow peaks of markers in the depilatedhair cycle likely reflect tighter synchroni-zation of the hair follicle cycle after dep-ilation. To better define expression ofPtgds, we performed immunohistochem-istry on tissue sections from skin of theanimals used for the time course in Fig.3C. Ptgds was evident in late anagen inthe keratinocytes of the outer root sheathbelow the stem cell–rich bulge area markedby the arrector pili muscle (Fig. 3D) (10).This area of the follicle regresses duringcatagen.

To corroborate this pattern seen by im-munohistochemistry, we next examineddouble immunofluorescence labeling forboth Ptgds and keratin 15 (Krt15), a hairfollicle stem cell marker (22). At day 0 ofdepilation (telogen), Krt15 was present inthe bulge area at the most inferior perma-nent part of the hair follicle, which persistsinto telogen (Fig. 3E). We did not identifyPtgds, which was consistent with qPCRdata showing lack of expression of Ptgds

during telogen (Fig. 3A). Also consistent with qPCR, at day 17 (lateanagen), we found a detectable Ptgds pattern by immunostaining (Fig. 3,D and F). Lower outer root sheath cells—but not bulge cells—expressedPtgds. Furthermore, at day 19 after depilation, keratinocytes in catagenfollicles expressed Ptgds, without overlapping Krt15-positive keratino-cytes (Fig. 3G). These results demonstrate that Ptgds is abundant innonpermanent keratinocytes of the hair follicle in mouse.

PTGDS is expressed in nonpermanent keratinocytes of thehuman hair follicleIn normal terminal human hair follicles, we found a similar presenceof PTGDS, primarily in the nonpermanent hair follicle below the ar-rector pili muscle (Fig. 4A). However, staining was variable with manynormal human follicles exhibiting little or no staining (Fig. 4B). This isconsistent with lower levels of PTGDS mRNA in haired versus baldscalp and likely also reflects the lack of synchronicity in human hair

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Fig. 3. An increase in Ptgds expression precedes elevation of PGD2 levels in the murine hair follicle duringcatagen. (A and B) At PN18 to PN53, Ptgds (n = 4), Ptgfr (n = 4), and Fgf5 (n = 4) mRNA (A and B) and PGD
2
lipid (n = 3) (B) were measured at different phases of hair follicle cycling. Letters correspond to hair cyclestages: A, anagen; C, catagen; T, telogen. Data are means ± SEM. In (A), **P < 0.01 for Ptgds and Fgf5 lateanagen versus first telogen and for Ptgfr first telogen versus second telogen. In (B), *P < 0.05 for PGD2

catagen versus early anagen. (C) Expression of Ptgds (n = 3) and production of PGD2 (n = 3) in murine skinduring the depilation-induced hair follicle cycle. Data are means ± SEM. *P < 0.05 for Ptgds on day 17versus day 0; **P < 0.01 for PGD2 day 19.5 versus day 37. (D) Murine outer root sheath keratinocytes belowthe stem cell–rich bulge area were stained for Ptgds (brown) 17 days after depilation. Dotted line deline-ates a full hair follicle. Scale bar, 100 mm. (E to G) Krt15 (red) and Ptgds (green) in permanent and non-permanent compartments of the hair follicle on day 0 (anagen) (E), day 17 (late anagen) (F), and day 19(catagen) (G) after depilation. Scale bars, 100 mm.

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follicle cycling. In miniaturized hair follicles in balding human scalp,sebaceous gland and hair follicle keratinocytes outside of the bulge(Fig. 4, C and E), and in some cases within the suprabasal bulge (Fig.4D), expressed PTGDS. We also detected PTGDS outside of the hairfollicle epithelium, indicating potential sources of PGD2 in the dermis(Fig. 4, E and F). In hair follicles undergoing catagen, PTGDS was presentin mast cells within the fibrous streamer, which is the former site ofthe regressed follicle (Fig. 4E). These results in mouse and human dem-onstrate that the lipocalin PTGDS and its product PGD2 are predomi-nantly expressed in the transient portion of the follicle at a time whenthe follicle begins regressing. These findings are in line with our hypoth-esis that the PGD2 pathway inhibits hair follicle growth.

Transgenic mice overexpressing Ptgs2 in the epidermisphenocopy AGAGiven the correlation of increased levels of PGD2 with balding scalp inhumans and the presumptive inhibitory role of PGD2 on the mousefollicle, we hypothesized that mice with high levels of PGD2 in the skin

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might develop features of AGA. BecausePtgs2 (cyclooxygenase 2, prostaglandinG/H synthase) is the enzyme upstreamto Ptgds, we further hypothesized thatmice overexpressing Ptgs2 would haveelevated PGD2 levels. Transgenic micethat overexpress Ptgs2 in the epidermishad been developed previously for car-cinogenesis studies. The hair follicles inthese K14-Ptgs2 transgenic mice werenoted to enter catagen prematurely, andthese mice reportedly developed alope-cia and sebaceous gland enlargement(14, 23).

We further analyzed the skin and hairfollicles of the K14-Ptgs2 mouse. Thesemice developed alopecia, which was evi-dent as a decrease in the normal murinepelage coat compared to control (Fig. 5,A and B). By histology, we also detectedsebaceous gland hyperplasia as indicatedby enlarged sebocytes clustered aroundthe hair follicle (Fig. 5, C and D). The hairfollicles in the K14-Ptgs2 mice were min-iaturized compared to controls (Fig. 5, Cand D), and these follicles bore a markedresemblance to the miniaturized folliclesin human bald scalp.

To determine the prostaglandin con-tent in the alopecic skin of the K14-Ptgs2mice, we measured prostaglandin levels byHPLC-MS during the anagen phase of thehair follicle cycle. PGE2 was elevated in theK14-Ptgs2 mice compared to age-matchedwild-type controls, as was previously shownusing immunoassays measuring PGE2 andPGF2a content in biopsied mouse skin(14, 24). PGD2 was also elevated and wasmore abundant than PGE2 in both wild-type and K14-Ptgs2 mice. 15-dPGJ2 was

elevated in K14-Ptgs2 mice compared to controls and demonstratedthe largest fold increase (~14-fold), although baseline values werelow (5.7 ng/g tissue) (Fig. 5E). We found low levels (18.4 ng/g tissue)of PGF2a, and an absence of prostacyclin (6k-PGF1a) and thrombox-ane (TxB2) (Fig. 5E), which are not known to be present in normalskin. Together, the balding phenotype in these mice is likely a result ofthe overwhelming PGD2 and 15-dPGJ2 inhibitory effects on the hairfollicle, despite the presence of PGE2, a known promoter of hairgrowth (20).

15-dPGJ2 and PGD2 inhibit hair growth in mouse and humanhair folliclesGiven the temporal peak of PGD2 before the apoptotic catagen stage,the published ability of its metabolite 15-dPGJ2 to induce apoptosis inother cell types, we tested the effects of the prostaglandins on primarycell culture of keratinocytes isolated from neonatal foreskin. 15-dPGJ2induces apoptosis (fig. S2A), as evidenced by plasma membrane bleb-bing and cell retraction/shrinkage. 15-dPGJ2 also decreased cell

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Fig. 4. PTGDS is expressed in the nonpermanent portion of select human hair follicles in haired scalp,and more variably in bald scalp. (A to C) PTGDS immunostaining (brown) with blue nuclear counter-
stain in normal human hair follicles (A and B) and bald scalp hair follicle (C). (A) A single follicle inhaired scalp with staining for PTGDS inferior to the isthmus. (B) Most anagen follicles of haired scalphave little staining for PTGDS. (C) Miniaturized hair follicle with PTGDS staining in sebocytes and hairfollicle keratinocytes. Scale bars, 100 mm. (D to F) Immunofluorescence staining for PTGDS (green),nuclei (blue), and either KRT15 (red) (D) or tryptase (red) (E and F) in the hair follicle and adjoiningsebaceous gland in bald scalp. Catagen follicle with some PTGDS-positive cells also expressed themast cell marker tryptase (E). Perifollicular cells show distinct populations of PTGDS- and tryptase-positive cells with overlapping expression (F). Scale bars, 100 mm.



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density, cell division, and live-cell numbers (fig. S2, B to D). Perhaps be-cause the origin of these keratinocytes was not the hair follicle, PGD2

had no such effect on the cells. However, 15-dPGJ2 did increase sub-G1 DNA quantities and activated caspase 3 in human keratinocytes,which are features of apoptotic cell death (fig. S2, E to G). We there-fore hypothesized that at least 15-dPGJ2, if not also PGD2, could di-rectly inhibit hair growth in vivo.

15-dPGJ2 was applied topically to dorsal back skin of C57BL/6mice that had been depilated to synchronize the hair follicle cycle.Starting on day 8 after depilation and continuing every other day,we applied 10 mg of 15-dPGJ2 or acetone vehicle. Hair length wasmeasured on days 4, 12, 14, and 16 after depilation. On days 12 to 16,hair at the site of treatment was shorter than in vehicle-treated animals(Fig. 6A). To determine a minimal effective dose, we tested the appli-cation of 1 mg of both PGD2 and 15-dPGJ2 as above and measuredhair length on day 20 after depilation. PGD2 inhibited hair growth,

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but to a lesser extent than 15-dPGJ2 (Fig.6B). We found no evidence of changes inhair follicle cycling grossly or by histo-logic examination.

Having demonstrated PGD2-mediatedinhibition of hair growth, we next soughtto determine which receptor was respon-sible for this effect. The two canonical re-ceptors for PGD2 are PTGDR (also knownas DP-1) and GPR44 (DP-2). Both DP-1and DP-2 have been reported to be ex-pressed by outer root sheath keratino-cytes in the hair follicle, among othersites (12). We therefore tested the ca-pacity for PGD2 to inhibit hair growthin both Ptgdr null mice (25) and Gpr44null mice (26) using Ptgds null mice(27) as a control. Whereas Ptgds andPtgdr knockout mice were both suscep-tible to the inhibition of hair lengthen-ing, Gpr44 null mice were resistant tothe inhibitory effect of PGD2 (Fig. 6C).These data suggest that GPR44, ratherthan PTGDR, is the receptor for PGD2-mediated inhibition of hair lengtheningand could therefore be a therapeutic tar-get for AGA.

To test the effect of PGD2 on humanhair growth, we used explanted humanhair follicles maintained in culture for7 days.We added increasing amounts (from0 to 10 mM) of PGD2, 15-dPGJ2, or vehicleto the culture medium and measured hairlength (Fig. 6D). Starting at 5 mM, PGD2

and 15-dPGJ2 significantly inhibited hairgrowth. At 10 mM, PGD2-treated hairwas 62 ± 5% shorter than vehicle, whereas10 mM 15-dPGJ2 completely inhibited allhair growth. We tested a variety of otherPGD2 analogs and found them to be capa-ble of inhibiting hair lengthening. Agonismfor GPR44 correlated with the ability to

inhibit hair lengthening (fig. S3). For example, the weak agonist 11-deoxy-11-methylene PGD2 (28) had the lowest activity (hair growthinhibition) of the compounds tested. Note that GPR44 has nanomolaraffinities for ligands, but as is typical for this type of experiment, higherconcentrations were required to overcome tissue penetration and com-pound lysis. Thus, in both mouse and human, PGD2 and 15-dPGJ2inhibit hair growth, likely through the GPR44 receptor.

DISCUSSION

Recent evidence has illustrated a role for prostaglandins in regulatinghair growth. For example, the PGF2a analog latanoprost is Food andDrug Administration (FDA)–approved and routinely used clinicallyto enhance hair growth of human eyelashes (15). PGE2 has been pro-posed to protect from radiation-induced hair loss in mice (29), and both

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Fig. 5. K14-Ptgs2 transgenic mouse skin phenoco-piesAGAwith alopecia, sebaceousglandhyperplasia,and elevated PGD2 levels in skin. (A andB) K14-Ptgs2animals develop alopecia (B) compared to wild-type(WT) controls (A). (C and D) Hematoxylin and eosin–stained skin from WT (C) and K14-Ptgs2 animals (D)shows sebaceous gland (yellow arrow) and hair folli-cle (black arrow)morphology. Scale bars, 100 mm. (E)Amount of different prostaglandins present in skintissue fromWTmice (n=5) andK14-Ptgs2 transgenicmice (n = 3). Data aremeans ± SEM. **P < 0.01 com-paring WT to K14-Ptgs2.




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PGE2 and PGF2a have been shown to enhance hair growth in mice(20). Our studies show that prostaglandins are dysregulated in AGA,the most common type of hair loss in men. Specifically, PGD2 inhibitshair growth and thus represents a negative counterbalance to the pos-itive effects on hair growth shown for PGE2 and PGF2a.

There is precedence for the opposing functions of individual pros-taglandins that are downstream from the PTGS enzymes. For exam-ple, in the lung, PGE2 causes relaxation, whereas PGD2 causescontraction of bronchial muscle tone (16). Our results suggest thatin mouse and human skin, a balance between PGE2 and PGD2

controls hair growth. This model predicts then that efforts to reversealopecia should optimally focus on both enhancing PGE2 andinhibiting PGD2 signaling. This model also explains why agents suchas aspirin, which inhibit upstream prostaglandin synthesis enzymes(PTGS1 and PTGS2), have minimal effects on hair growth becauseof likely equally decreased production of PGE2 and PGD2.

Given our current report that PGD2 inhibits hair growth, prosta-glandins represent an underappreciated network for controlling therate of hair lengthening. Evidence to support the PGD2 pathway’s di-rect induction of the apoptotic catagen stage includes the temporalexpression of Ptgds with catagen onset, in vitro studies of 15-dPGJ2showing inhibition of hair growth, and the Ptgs2-overexpressingmouse model, which develops alopecia (23). These results areconsistent with the notion that PTGDS elevation in balding scalpleads to increased levels of PGD2 and 15-dPGJ2, which then promotethe onset of catagen and decrease hair lengthening, leading to the in-crease in telogen follicles and miniaturization of the hair folliclecharacteristic of AGA. PGD2 may also cause sebaceous hyperplasiaseen in AGA.

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Although we did not elicit premature catagen in mice treated topi-cally with PGD2, we observed a negative effect on hair growth (Fig. 6).This could be explained because of inappropriate prostaglandin delivery,frequency, or concentration. Other explanations include potential cofac-tors that are required with PGD2 to more directly affect hair cycling.Nevertheless, our results do suggest that prostaglandins directly modu-late speed of hair growth during anagen. Current explanations of thedifferences in hair length by species focus mostly on duration of hairfollicle cycling rather than accompanying potential differences in hair-lengthening rates during anagen (4). Our results suggest that greaterattention should be paid to the possibility that prostaglandins modulatehair growth speed and that compounds increasing hair growth speedmay benefit patients with AGA.

Intriguingly, Ptgds is a highly testosterone-responsive transcript(30, 31), which further suggests its importance in AGA. PGD2 is thoughtto play a central role inmale gonadal sex determination (32) and is highlyexpressed in male genitalia (32, 33). Similarly, Ptgds expression in theheart is regulated by estrogen (34). Estrogen leads to increases in 15-dPGJ2levels in the uropygial gland (35). Recent evidence also suggests that pros-taglandins induce virilization of the mouse brain through estrogen (36).Given the androgens are aromatized into estrogens, these results may berelevant to hair growth and alopecia inbothmenandwomen.Thus, theseor similar pathways might be conserved in the skin and suggest that sexhormone regulation ofPtgdsmay contribute to the pathogenesis of AGA.

Additional evidence that prostaglandins control hair follicle cyclingand can be used therapeutically to treat AGA arises from findings onthe possible mechanism of the AGA drug minoxidil. Although min-oxidil alters potassium channel kinetics (7), it is also known to increaseproduction of PGE2 (37). Given the decreased amount of PGE2 presentin bald scalp versus haired scalp (Fig. 2E), minoxidil may normalizePGE2 levels. Future studies should address whether minoxidil can con-comitantly decrease PGD2 levels and thus normalize multiple prosta-glandin species as a mechanism to improve AGA.

The lower absolute amount of 15-dPGJ2 compared to PGE2 andPGD2 is particularly relevant; although this eicosanoid has been hy-pothesized to be a natural ligand for the nuclear hormone transcriptionfactor peroxisome proliferator–activated receptor g (PPARg), themeasured concentration of 15-dPGJ2 is often lower than the bindingconstant for PPARg (17). Thus, although it is attractive to speculate thatthe sebaceous gland hypertrophy and elevated levels of 15-dPGJ2 ob-served in both human AGA and mouse K14-Ptgds model might be re-lated, the causal connection is unclear. Despite this, a growing literaturecorrelates elevated 15-dPGJ2 levels with sebaceous hyperplasia, such asin acne (38). Given the common requirement of circulating sex hor-mones and common histology of sebaceous hyperplasia, acne andAGA may have overlapping pathogeneses.

Another report has linked altered lipidmetabolism and alopecia (39).In a distinct type of human alopecia called lichen planopilaris, expressionof PPARg is decreased with accompanying altered lipid metabolism.Thesewere discovered through an approach similar to ours usingmicro-array analysis of affected and unaffected scalp. The most up-regulatedtranscript from affected subjects, cytochrome P450 family 1 member A1(CYP1A1), was up-regulated 1020-fold. The authors hypothesized that axenobiotic, such as dioxin, might up-regulate CYP1A1 expression andtrigger lichen planopilaris. However, cytochrome P450s also have rolesin eicosanoid biology, and theymay alter prostaglandinmetabolism (40).Thus, altered prostaglandin metabolism might contribute to more thanone type of human alopecia, although additional studies are needed.

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Fig. 6. PGD2 inhibits mouse and human hair growth through GPR44. (A)Hair growth over the course of 16 days after depilation. 15-dPGJ2 (10 mg) or
vehicle was applied topically to mouse skin starting on day 8 after depilation.Data are means ± SEM (n = 3 per treatment group). *P < 0.05 compared tocontrol. (B) Hair length 10 days after topical PGD2 (1 mg; n = 3), 15-dPGJ2(1 mg; n = 3), or vehicle (n = 3) treatment. Data are means ± SEM. *P < 0.05;**P < 0.01. (C) Hair growth in Ptgdr (n = 8), Ptgds (n = 6), and Gpr44 (n = 11)knockout (KO) mice in response to PGD2. Data are means ± SEM and arerepresentative of three independent experiments per mouse. *P < 0.05compared to vehicle. (D) In vitro growth of explanted human hair folliclesover the course of 7 days in culture with PGD2 (n = 3), 15-dPGJ2 (n = 3), orvehicle (n = 3). Data are means ± SEM. ***P < 0.001 compared to vehicle.



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Our findings should lead directly to new treatments for the mostcommon cause of hair loss in men, AGA. Given its suspected role inallergic diseases, at least 10 antagonists of the GPR44 (DP-2) receptorhave been identified (41) and several are in clinical trials (42). Thepotential for developing these compounds into topical formulationsfor treating AGA should elicit great interest moving forward. The ques-tion of whether similar changes in PGD2 levels are found in the affectedscalp of women with AGA also needs to be addressed. AGA in womenmay not be androgen-mediated, but if prostaglandins represent a finalcommon pathway, targeting prostaglandins should benefit women withAGA as well.

Our findings also suggest that supplemental PGE2 could be ther-apeutic. By correcting its deficiency and increasing its level in baldscalp, the inhibitory effects of PGD2 may be overcome. Analogs ofPGF2a, which are already FDA-approved to promote eyelash growth,should also have similar effects on the scalp and are currently in clin-ical trials for this indication. Once issues of delivery, dosing, and safetyare addressed, additional agonists and antagonists of prostaglandinpathways should become available. The K14-Ptgs2 transgenic mousemodel, which phenocopies AGA, may assist in screening novel ther-apeutic agents. Ultimately, multiple mechanisms may be responsiblefor hair loss in AGA. Inhibiting PGD2 may prevent miniaturizationand provide benefit to those in the process of balding; however, it isunclear whether men who are already bald will regrow hair.

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MATERIALS AND METHODS

Human tissue samplesThis study used only normally discarded human scalp obtained anon-ymously and was approved by Penn’s Institutional Review Board of-fice as an exempt protocol. Normally discarded human scalp wasobtained during hair transplantation as previously described (11). Tis-sues originated from adult Caucasian males age 40 to 65 who were nottaking finasteride or minoxidil. All analyses used unique scalp samplesnot used in other experiments. To calculate average fold differencesbetween haired and bald scalp, we created ratios for each patientand then averaged them. Microarrays were performed with publishedmethods (11). Specifically, Affymetrix HG-U133A chip was used. Colorsof lines and cells in graphics indicate intensity relative to that gene’smedian expression across all samples. For hair-lengthening experiments,discarded tissue from face lifts, which contain terminal hair, was used.

AnimalsAll animal protocols were approved by the University of PennsylvaniaInstitutional Animal Care and Use Committee. Wild-type C57BL/6Jfemale mice were purchased from Jackson Laboratory under requestfor mice of identical weights and birth dates for time-course studies.K14-Cox2 transgenic mice (14) and Ptgdr (25), Gpr44 (26), and Ptgds(27) knockout mice were obtained from original sources. To decreaseeffects of endogenous testosterone, we used only female mice. Depila-tion to synchronize the hair cycle was performed as described (21). Ifan animal was found to be in a transitional state on the day of biopsy,the later-stage sample was labeled to be 12 hours more advanced.

Real-time qPCRMouse dorsal back skin was removed with scissors and forceps. Hu-man tissue was cut with scissors. Tissue (25 to 35 mg) was solubilized

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in 300 ml of Qiagen RLT buffer. Human scalp and mouse tissues werehomogenized with a tissue rotor, and RNA was collected andconverted to complementary DNA (cDNA) per the manufacturer’s in-structions with the Fibrous RNA Extraction Kit (Qiagen) and theHigh-Capacity cDNA kit (Applied Biosystems). cDNA (50 ng) wasused in real-time qPCRs with Fast Mix Quantitative RT-PCR MasterMix and measurements on the Step One Plus System (Applied Biosys-tems). b-Actin and 18S RNA probe standards were found to be equiv-alent to one another and did not fluctuate between samples of equalloading amounts. Fold change was calculated with the DDCT method.Primers used were inventoried TaqMan primers (Applied Biosystems):PTGDS (human), Ptgds (mouse), Fgf5 (mouse), Ptgfr (mouse), b-actin(human), b-actin (mouse), and 18S (human and mouse).

Western blotHuman scalp tissue was homogenized, and protein amount was quan-titated with a BCA kit (Pierce). Equal amounts for each discardedtissue lysate (15 to 35 mg) were loaded for SDS–polyacrylamide gelelectrophoresis. Polyvinylidene difluoride blots were probed withmouse monoclonal anti-PTGDS (Cayman Chemical), followed byanti-mouse secondary antibody conjugated to HRP (Vector). Theblot was developed with electrochemiluminescence (Amersham), ex-posed to autoradiography, and quantified with ImageJ (NationalInstitutes of Health), all per the manufacturers’ instructions. Blotswere subsequently stained with Ponceau Red (Sigma) to verify equalloading between all lanes tested.

PGD2 enzyme-linked immunosorbent assayHuman scalp was obtained from hair transplants to measure theamount of PGD2. Immediately after surgery, samples were placed at4°C in Dulbecco’s modified Eagle’s medium with 10% fetal bovineserum (FBS) and antibiotics/antimycotics, or they were snap-frozen.Tissue sample weights varied from 0.14 to 0.94 g. All values were nor-malized to the starting tissue weight. Within 1 to 2 days, sampleswere flash-frozen in liquid nitrogen and stored at −70°C for at least24 hours. After thawing, samples were diluted in 1 ml of acetone andhomogenized. Samples were centrifuged at 5000g for 10 min, super-natant was removed, and a second extraction was performed on thepellet. The combined supernatants were dried in a SpeedVac centri-fuge and resuspended in 100 ml of enzyme immunoassay bufferprovided in the Cayman Chemical Prostaglandin D2-MOX ELISA(enzyme-linked immunosorbent assay) kit. Manufacturer’s instruc-tions were followed for determination of PGD2 concentration relativeto a standard curve. Values were normalized to haired scalp.

Ultra–high-performance liquid chromatographyHuman tissue was prepared as above for ELISA. To maximize pros-taglandin yield in mice, we snap-froze the samples and kept thembelow 4°C at all times. Tissue from K14-Ptgs2 mice and Friend virusB-type (FVB) strain-matched animal controls was obtained at day 24of life. For mouse tissues, excised epidermis and dermis were snap-frozen in acetone, minced, and homogenized with an Omni TH 115Vwith metal tip in acetone for less than 2 min. Homogenized tissue wasthen centrifuged at 16,000g for 10 min, with the supernatant saved.The acetone was dried under a gentle stream of nitrogen gas, afterwhich the sample was redissolved in 1 ml of 5% acetonitrile in water,acidified with 20 ml of formic acid, and applied to a conditionedStrata-X SPE cartridge (Phenomenex). The SPE protocol included




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preconditioning with 1 ml of H2O followed by 1 ml of acetonitrile,sample application, washing with 1 ml of H2O, drying by applicationof house vacuum for 15 min, and elution with 1 ml of 10% acetonitrilein ethyl acetate. The eluate was then dried with a stream of nitrogen,redissolved in 200 ml of 30% acetonitrile in H2O, and filtered through0.2-mm Nylon Spin-X filters (Corning) before 100 ml was injected intothe instrument for UHPLC analysis.

The UHPLC consisted of an Accela solvent-delivery system (Thermo)and Hypersil GOLD C18, 200 mm × 1 mm, 1.9-mm particle-size col-umns (Thermo). The mobile phase consisted of water (solvent A) andacetonitrile/methanol (95:5, solvent B), both with 0.005% acetic acidadjusted to pH 5.7 using ammonium hydroxide. The mobile phase gra-dient started at 20% B and increased in a linear manner to 35% at 10min, followed by a linear increase to 80% B at 20 min, and held at 80% Bfor 3 min. The flow rate was 350 ml/min.

Mass spectrometryA TSQ Quantum Ultra instrument (Thermo) equipped with a heatedelectrospray ionization (ESI) source and triple quadrupole analyzerwas used. The ESI source used nitrogen for sheath and auxiliarygas, set to 70 and 5 arbitrary units, respectively. The mass spectrom-eter was operated in the negative ion mode with a capillary tempera-ture of 350°C and a spray voltage of 0 kV. The source offset was set to6 V. The analyzer was operated in the selected reaction monitoringmode. The transitions monitored for the first 16 min were as follows:mass/charge ratio (m/z) 351→271 (collision energy, 15 V) for endog-enous PGD2 and PGE2, m/z 355→275 (15 V) for tetradeuteratedPGD2 and PGE2, m/z 353→193 (24 V) for endogenous PGF2a, andm/z 357→197 (24 V) for tetradeuterated PGF2a. For the last 7 min,m/z 315→271 (15 V) for endogenous 15-dPGJ2 and m/z 319→275(15 V) for the tetradeuterated analog were monitored. All eicosanoidswere purchased from Cayman Chemical.

Immunohistology and immunofluorescenceMouse-back or human-scalp skin was fixed in 4% paraformaldehyde,embedded in parafilm, and sectioned at 5 to 10 mm. Non-immunoslideswere stained for hematoxylin and eosin (Fisher Scientific). For immu-nostaining, polyclonal rabbit anti-PTGDS (Cayman Chemical) ormonoclonal mouse anti-KRT15 (Lab Vision, clone LHKRT15) was ap-plied followed by biotinylated anti-rabbit or anti-mouse, respectively.ABC kit (Vector labs) was used with horseradish peroxidase and dia-zoaminobenzene for color development. Methyl green (Vector Labs)was used to counterstain the tissue for immunohistochemistry; antigenstaining appears brown and counterstain appears bluish green. Stainingwas visualized in a traditional light microscope (Nikon).

Skin was treated with the same primary antibodies as describedfor immunohistochemistry. Anti-rabbit Alexa Fluor 488 (Invitrogen)and anti-mouse rhodamine (Vector Labs) were used as secondaryantibodies. Tissues were subsequently mounted in 4′,6-diamidino-2-phenylindole (DAPI) medium (Vector Labs). Staining was visual-ized in a traditional fluorescence microscope with red, green, and bluefilter cubes (Leica).

Keratinocyte cell cultureHuman foreskin keratinocytes were isolated from neonatal foreskins,as described previously (43). Equal densities of keratinocytes wereplated (50,000 cells/cm2) between passages 2 and 3 after initiation intosix-well culture dishes. Cells were maintained in 2 ml of EpiLife me-

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dium (Cascade Biologics) and treated for 12 to 48 hours with stocksof PGD2, PGE2, or 15-dPGJ2 (Cayman Chemical) that had beendissolved in acetone. Unless specified otherwise, cells were treatedwith 10 mM prostaglandins for 20 hours. Acetone alone was usedas a control.

Flow cytometryDuring culture, human keratinocytes were pulsed with 0.2 mM bro-modeoxyuridine (BrdU) for the final 3 hours of incubation with pros-taglandins. Cells were typsinized to release cells into suspension,counted, fixed, and permeabilized (Caltech). Cells were stained with20 ml of anti–activated caspase 3 (Becton Dickinson, clone CPP32)or anti-BrdU fluorescein isothiocyanate (Becton Dickinson, cloneB44) per 106 cells. Cells were resuspended in phosphate-buffered sa-line (PBS) with 5% FBS and DAPI (5 mg/ml) before being run on anLSRII flow cytometer (Becton Dickinson). Sub-G1 cells were gated asthose containing less than diploid amounts of DNA.

Mouse hair-lengthening studiesTo compare the effect of vehicle, PGD2, and 15-dPGJ2 on the hair cycleamong wild-type and knockout mice, we applied 1 mg of the listedprostaglandin in 200 ml of acetone or acetone alone to the central backon days 8, 10, 12, 14, 16, and 18 after depilation, with measurementof hair length performed on day 20. However, to test the time courseof hair lengthening on 15-dPGJ2 versus vehicle, we used 10 mg in-stead. Hair length was measured as described (44), with the selectionof only awl (non-zigzag) hairs measured. For testing mutant mousestrains, the experiment was repeated on three separate litters.

Human hair follicle cultureThe hair follicle organ culture model was performed as described (45).Briefly, human hair follicles in growth phase (anagen) were isolatedfrom face and brow-lift tissue obtained from plastic surgeons. At leastthree different human donors (a mixture of men and women) wereused for each compound tested. All subjects were in the age rangeof 40 to 60 years. The skin was sliced into thin strips of 1 to 2 mm,exposing two to three rows of follicles that could readily be dissected.Follicles were placed into 0.5 ml of William’s E medium (Life Tech-nologies) supplemented with L-glutamine (2 mM), insulin (10 mg/ml),hydrocortisone (10 ng/ml), penicillin (100 U), streptomycin (0.1 mg/ml), and amphotericin B (0.25 mg/ml). The follicles were incubated in24-well plates (one follicle per well) at 37°C in an atmosphere of 5%CO2 and 95% air. PGD2, PGD2 metabolites, and PGD2 analogs(Cayman Chemical) were dissolved into dimethyl sulfoxide (DMSO)as 100× stock solutions (for example, 0.5 mM for 5 mM treatmentsand 1 mM for 10 mM treatments). DMSO vehicle controls were usedat 1% (v/v). Hair follicles were treated with DMSO only as a control.Follicles were imaged typically on day 0 (the day follicles were placedin culture) and again on day 7. The length of the hair fiber wasassessed with Vision Builder (National Instruments). The growth ofthe hair fiber was calculated by subtracting the length on day 0 fromthat determined on day 7. Each measurement reported is the averageof the results from 14 to 20 follicles.

Statistical analysisFor all P value calculations, paired Student’s t test with a one-taileddistribution was calculated and P < 0.05 was considered to be sig-nificant. All data are means ± SEM. Analysis of variance (ANOVA)




test was used to rank genes from microarray as primarily differentwith respect to site (haired versus bald) and irrespective of person,date of biopsy, or date of microarray. The top 250 genes of the mostsignificant P values for this ANOVA analysis were used for furtheranalysis. A paired t test was performed on array intensities.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/4/126/126ra34/DC1Fig. S1. Verification of hair cycle stage in wild-type mice.Fig. S2. 15-Deoxy-D12,14-prostaglandin J2 (15-dPGJ2) induces apoptosis in cultured humankeratinocytes.Fig. S3. Analogs of PGD2 inhibit hair lengthening according to their relative agonism of GPR44.

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REFERENCES AND NOTES

1. A. Alsantali, J. Shapiro, Androgens and hair loss. Curr. Opin. Endocrinol. Diabetes Obes. 16,246–253 (2009).

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Acknowledgments: We gratefully acknowledge the assistance of J. Tobias and D. Baldwin ofthe Penn Microarray Facility core, L. Ash in the histology core, and the Penn Human Cooper-ative Tissue Network. Funding: NIH grants R01-AR46837, R01-AR055309, P30-AR057217, and

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K08AR055666; Skin Disease Research Center grant 5-P30-AR-057217-02; Pennsylvania Depart-ment of Health; Edwin and Fannie Gray Hall Center for Human Appearance at University ofPennsylvania Medical Center; American Skin Association; Dermatology Foundation; and L’Oreal.Author contributions: L.A.G. and G.C. designed the studies, analyzed and interpreted the results,and co-wrote the paper. Y.L. and Z.Y. performed the microarrays. L.A.G. and G.C. selected PTGDSfor further analysis. L.A.G. performed reverse transcription–PCR, ELISA, Western blotting, lipid ex-tractions, histology, and mouse work, with assistance from B.A., S.M.N., T.Z., H.B.B., and D.E.L. J.A.L.performed mass spectrometry and interpretations. G.A.F. supervised mass spectrometry andaided with interpretations and design. D.C.S. and L.C. provided expertise in male pattern bald-ness and discarded tissue samples. A.G. performed and supervised human hair-lengtheningexperiments. S.M.F. provided the K14-Ptgs2 mice and assisted with interpretation and design.Competing interests: A.G. is an inventor of a patent owned by the Gillette Corporation to usePGD2 to inhibit hair growth. L.A.G. and G.C. are co-inventors of a patent owned by the Universityof Pennsylvania describing the PGD2 pathway as a target for inhibiting hair loss, among other

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claims. The Pennsylvania Department of Health specifically disclaims responsibility for any analy-ses, interpretations, or conclusions. Data and materials availability: The K14-Ptgs2 mouse isunder materials transfer agreement from the University of Texas M. D. Anderson Cancer Center.

Submitted 24 August 2011Accepted 3 February 2012Published 21 March 201210.1126/scitranslmed.3003122

Citation: L. A. Garza, Y. Liu, Z. Yang, B. Alagesan, J. A. Lawson, S. M. Norberg, D. E. Loy, T. Zhao,H. B. Blatt, D. C. Stanton, L. Carrasco, G. Ahluwalia, S. M. Fischer, G. A. FitzGerald, G. Cotsarelis,Prostaglandin D2 inhibits hair growth and is elevated in bald scalp of men with androgeneticalopecia. Sci. Transl. Med. 4, 126ra34 (2012).

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