Microarray analysis of androgenetic and senescent alopecia: Comparison of gene expression shows two distinct profiles

Androgenetic alopecia (AGA) is characterized by androgenmediated miniaturization of the hair follicle in susceptible individuals. Senescent alopecia (SA) is the diffuse scalp hair thinning that is seen with advanced age even in individuals without a family history of hair loss. Differences in follicular counts, anagen/telogen percentages, and terminal/vellus hair ratios have been reported [1]. In a pilot study, older males showed nearly a two-fold decrease in levels of androgen receptors, 5-alpha reductase 1 and 2, and aromatase compared to young males with AGA [2]. However, the concept of whether SA is a definable entity distinct from AGA remains controversial. Here, we used microarray analysis to compare gene expression profiles in AGA and SA in order to characterize novel aspects of their pathology and to identify new gene targets. The three groups of men in this study were age-matched and included: Group 1-Controls had no visible hair thinning. Group 2-AGA had male pattern hair thinning that was established to have occurred prior to age 30 and Group 3-SA had diffuse hair thinning that had its onset after the age of 60. RNA from scalp biopsies was isolated from each group (N = 10, pooled) and the gene expression was assessed on Affymetrix GeneChip Human U133Plus 2.0 microarrays as described [3]. Genes with fold changes of 2 and false discovery rate (FDR) <0.05 were considered to be part of the expression profiles. A total of 1200 differentially expressed genes (DEGs) in AGA and 1360 in SA were identified compared to controls. Of these, 442 genes were unique to AGA, 602 genes were unique to SA and 758 genes were common to both AGA and SA. Hair/skin development and function is the most significant physiological function altered in both AGA and SA, however, the DEGs in this category differed in the two diseases. Table 1 shows the 34 genes in this category that are differentially regulated in AGA that contribute to hair follicle development, morphology and cycling (BARX2, EGFR, INHBA, MSX2, OVOL1, KRTs, KRTAPs, RUNX3 and TIMP3). Many of these genes required for hair follicle homeostasis are significantly under expressed in AGA but not in SA compared to normal scalp tissue (Table 1 and Figure S1). Our data (Table 1 & Figure S1) showed that the Androgen Receptor (AR) is up regulated in AGA, but not in SA. Previous studies [4] have shown that genetic variability in AR is a prerequisite for the development of early-onset AGA. A novel AGA susceptibility locus has been identified at 17q21.31 [5]. In our dataset, the DEAD box polypeptide 5 (DDX5), a transcriptional regulator of AR [6] is down regulated in AGA and maps to this locus. Table 1 Genes and pathways altered in Androgenetic Alopecia (AGA). The most significant pathway altered in AGA is Notch Signaling which consists of 29 genes (Table 1) including HES1, Notch2, Notch4 and PROX1 that are known to play a role in cell fate determination [7]. The down regulated genes in this pathway in AGA include CNTN1, JAG1, NOTCH2 and PSEN1 and the genes that are up regulated include DTX3, HES and NOTCH4. The expression patterns of Notch signaling pathway genes including Notch 2 and JAG1 were validated by real-time PCR (Figure S1). Jagged1 (JAG1) gene which encodes a ligand for Notch receptor maps to chromosome 20p a susceptibility locus for male-pattern baldness [8]. A reciprocal negative feedback regulation exists between Notch and AR-dependent pathways in the prostate [9]. The activation of AR and the concomitant loss of Notch signaling may be contributing factors to hair follicle miniaturization and may serve as the mechanistic link between prostate cancer and AGA. Thus, modulating the Notch signaling pathway in AGA may lead to future therapies. In contrast to AGA, the 15 genes unique to SA (Table 2) (CALML5, CCND1, COL7A1, CTGF, GLI2, KRT15, KRT2, MYC, NAB1, POU2F3, FOS, FYN, JUNB, ID2, PPARA) participate in skin and epidermal development, keratinocyte proliferation, differentiation and cell cycle regulation. A number of transcription (FOS, FYN, JUN, JUNB, MYC, NAB1) and growth factors (CTGF, TGFα) are significantly decreased in SA. The expression patterns of c-MYC, CTGF and TGFα were validated by real-time PCR in independent sets of AGA and SA scalp biopsies (Figure S2). Table 2 Genes and pathways altered in Senescent Alopecia (SA). The most significant canonical pathway altered in SA is Neuregulin signaling (Table 2). Neuregulins (NRG) belong to the epidermal growth factor (EGF) family of growth factors and are ligands of the ErbB receptors. The 21 genes in this pathway that are significantly down-regulated include EGFR signaling pathway ligands (AREG, BTC, EREG, TGFα), transcription factors (EGR1, FOS, JUN, MYC) and associated genes (ADAM17, HSP90AB1, ERBB2IP, PTPN11, DCN, PSEN1, ITGA2, ITGB1). The neuregulin pathway genes that are up-regulated include kinases (PDPK1, AKT2, AKT3) and apoptotic genes (BAD, RAF, SOS1). The altered expression of neuregulin pathway genes in SA was further confirmed in independent scalp biopsies by real-time PCR (Figure S2). Although the role of neuregulins are not fully understood, previous studies have shown an important role for EGFR signaling pathway in the differentiation of the hair follicle and normal hair development [10]. Recent studies have implicated neuregulin and EGF signaling pathways with longevity and lifespan. Thus, loss of these signaling pathways may contribute to hair aging and senescent alopecia. The differences in gene expression profiles suggest that AGA and SA may represent two independent hair disorders and that non-androgen pathways may also contribute to hair loss. This study provides novel therapeutic targets for the prevention or treatment of two common hair disorders.