Gene expression profile of the skin in the 'hairpoor' (Hr ^Hp ) mice by microarray analysis

With a complex and dynamic structure, hair is generated by hair-producing follicles and has a patterned cycle of growth and remodeling, which consists of growth (anagen), regression (catagen), and rest (telogen) stages. There are many genes involved in mature hair follicle (HF) regulation [1].

One of these genes, hairless (Hr), is expressed in skin, specifically in the suprabasal cell layer of the interfollicular epidermis and in the lower portion of the HF epithelium; its expression is dependent on the hair cycle. Hr encodes a 130 kDa protein (HR), which contains a zinc finger domain and is localized in the nucleus [2], and acts as a transcriptional corepressor that regulates transcription through directly binding to the thyroid hormone receptor [3, 4], vitamin D receptor [5], and retinoic acid-like orphan receptor α [6].

Various Hr mutant mice have been studied to understand the function of HR, and most Hr mutant mice are created by causing the loss of HR function in their cells, giving them a typical phenotype with a recessive inheritance mode [7–14]. Microarray analysis of the skin of Hr ^tm1Cct /Hr ^tm1Cct mice has revealed that loss of HR function results in specific changes in the expression of epidermal differentiation-associated genes such as keratin10, loricrin, filaggrin, keratinocyte differentiation-associated protein (Kdap), and calmodulin 4, among other such mouse genes [7]. These results suggest that HR also plays a role in keratinocyte terminal differentiation through regulation of gene transcription.

The new Hr mutant mouse called 'hairpoor' (Hr ^Hp ) that we reported recently has a phenotype that is inherited in an autosomal semidominant manner [15]. Therefore, the heterozygote shows poor hair distribution, whereas the homozygote displays total alopecia. The Hr ^Hp mouse genome harbors a T-to-A substitution at position 403 in the noncoding exon 2 of Hr, and this mutation confers overexpression of HR in the mutant mouse skin [15]. This clearly distinguishes Hr ^Hp mice from other Hr mutant mice with loss of function of Hr.

Marie Unna hereditary hypotrichosis (MUHH; OMIM-146550) is an autosomal dominant disorder that displays coarse and twisted hair development in early age in humans and progresses to alopecia as the patients grow older. Recently, the genetic cause of MUHH was found to be similar to the mutation found in Hr ^Hp , namely a mutation in the 5' UTR of the HR gene [16, 17]. This makes the Hr ^Hp mouse one of the valuable animal models for MUHH, and studying Hr ^Hp mice will facilitate the understanding of MUHH pathogenesis. Hr ^N mutant is another model for MUHH [15, 18]. We recently reported that overexpression of HR is associated with alterations in the morphology and expression of a number of genes in the skin of the Hr ^Hp mouse [16]. In the present study, we performed microarray analysis and compared Hr ^Hp /Hr ^Hp skin with that of age-matched wild-type mice to identify the genes whose expression was affected by the overexpression of HR. We catalogued the genes showing differential expression in the mutant skin and found some of them to be tightly regulated by HR, which we confirmed using a reporter expression system.

Recently, we reported the Hr ^Hp mouse generated by N-ethyl-N-nitrosourea mutagenesis as an animal model of human MUHH [15]. As an initial step to delineate the molecular basis of the underlying mechanism for the Hr ^Hp phenotype, we investigated the differential expression of genes in the skin immediately before morphological changes occurred in the Hr ^Hp /Hr ^Hp mouse. Microarray analysis revealed that various biological pathways and the expression of many genes were affected by overexpression of HR.

Other studies have reported systematic screening of differentially expressed genes in the skin of mice with the Hr mutation, including analyses of gene expression in the skin of Hr ^N mice, another Hr-overexpressing mutant, and Hr ^tm1Cct , an Hr-loss-of-function mutant [7, 18]. Hr ^N mutants harbor the mutation A402G, which abolishes the same uATG as in Hr ^Hp mutants, indicating that they have an identical defect [16]. A comparison of our microarray analysis results with those reported for the Hr ^N mutant did not show that the same genes displayed differential expression. This may be due to the difference in the developmental stage of the HF and epidermis (P0 vs. P7 or 5 weeks after birth) used in these analyses. However, many keratin-associated protein genes were detected in both mutants. At P0 in Hr ^Hp /Hr ^Hp mice, the expression of Krtap6-2, Krtap16-7, and Krtap16-3 increased, whereas the expression of Krtap5-1 and Krt71 decreased. Similarly, Krtap6-3, Krtap8-2, Krtap14, Krt1-1, and Krt1-3 were downregulated in Hr ^N /Hr ^N mice at P7 [18]. These results suggest that HR-regulated genes are associated with keratinocyte differentiation and/or hair-shaft structure. Furthermore, changes in the expression of keratin10, Kdap, and many epidermal differentiation-associated genes were also detected in Hr ^tm1Cct /Hr ^tm1Cct mice at P12 [7]. Results suggest that mutation of Hr causes the abnormal expression of many keratin-associated genes during HF morphogenesis and result in disruption of normal hair formation [7].

Of four genes with consistent expression patterns with respect to HR overexpression, two genes, Sfrp1 and Wif1, belong to Wnt inhibitor families. Both inhibitors interfere with Wnt signaling transduction by binding directly to the WNT protein [28, 29], but though they function in a similar fashion in the Wnt signaling pathway, they displayed completely different expression patterns in Hr ^Hp /Hr ^Hp skin compared with age-matched +/+ skin. Surprisingly, Sfrp1 was downregulated by overexpression of the HR protein, whereas Wif1 was upregulated. Although we cannot rule out the possibility that this result may be caused by different locations of Sfrp1 and Wif1 expression in the skin, this difference is more likely to result from the differential transcriptional regulation of these genes, as seen in keratinocyte cells with HR overexpression (Figure 4). The Hr overexpression in keratinocyte cells results in suppression of Sfrp1 by 39% and activation of Wif1 by 85%, suggesting that these promoters respond to HR differently. It is not known whether HR functions as an activator for Wif1 transcription, and further study is required to understand its mechanism of action. Regulation of the Wnt pathway by HR through transcriptional regulation of Wise, Soggy and Sfrp2 is reported to be important for proper HF cycling. While expression of Wise and Soggy mRNAs were upregulated in the Hr ^tm1Cct /Hr ^tm1Cct skin. Their expression levels were reduced in the skin of HR over-expressing transgenic mouse (2-fold). Sfrp2 was also shown to be downregulated in the Hr ^Hp /Hr ^Hp skin. [16, 21, 30]. We may include two more Wnt inhibitors, Sfrp1 and Wif1, for being regulated by HR based on this work. Further study is required to understand their function(s) in HF development and/or cycling.

Casp14 is another gene upregulated by overexpression of HR, and is expressed in IRS and corneous cells of the outer root sheath in HFs [31]. It plays a role in the formation of the stratum corneum and terminal differentiation of keratinocytes [22, 31]. The higher levels of Casp14 mRNA suggest that an increase in terminal differentiation of keratinocytes occurs in the mutant skin. This result is comparable with our observation that the epidermis of the mutant skin shows increased differentiation in three-week-old mutant mice compared with age-matched wild-type mice [16]. Interestingly, Casp14 is also highly expressed in Hr ^tm1Cct /Hr ^tm1Cct mice, which display the hairless phenotype and show an increase in terminal differentiation of the epidermis with loss of function of Hr [7]. Furthermore its striking up-regualtion is age specific, which closely resembles its expression pattern in the Hr ^Hp /Hr ^Hp mice. Thus, despite having the opposite molecular defect, Hr ^Hp /Hr ^Hp and Hr ^tm1Cct /Hr ^tm1Cct mice exhibit increased expression of Casp14 and displayed a similar increase in the terminal differentiation of keratinocytes, suggesting that, Casp14 may play an important role in proper differentiation of ketatinocyte. Thus, although it is not clear how HR regulates Casp14 expression, both loss of Hr expression and Hr overexpression lead to alopecia through expression of Casp14 expression. Krt71 is also expressed in the IRS, specifically in Henle's and Huxley's layers; gene knockout mice show irregularly formed Henle's and Huxley's layers compared with the wild-type mice, indicating that Krt71 plays an important role in the formation of linear IRS intermediate filaments [25]. Results show that the downregulation of Krt71 expression in the skin of Hr ^Hp /Hr ^Hp mice is comparable with that in Krt71 knockout mice in that IRSs in Hr ^Hp /Hr ^Hp mice were also abnormal.

Wif1 and Casp14 were found to be upregulated, whereas Krt71 and Sfrp1 were downregulated by overexpression of HR. These results suggest that HR may transcriptionally regulate these genes. Clearly, further studies are required to delineate the molecular mechanisms underlying how HR regulates its target genes and to elucidate the role of Hr in HF development, leading to a better understanding of MUHH.