Aryl hydrocarbon receptor is regulated via multiple mechanisms in human keratinocytes.

Aryl hydrocarbon receptor (AhR) is a basic helix-loop-helix transcription factor activated by polycyclic aromatic hydrocarbons of synthetic and natural origin. While a number of novel AhR ligands have been recently identified, little is known about their possible influence on AhR levels and stability. We used western blot, qRT-PCR and immunocytochemistry to determine the effects of AhR ligands on AhR expression in N-TERT (N-TERT1) immortalized human keratinocytes, and immunohistochemistry to assess patterns of AhR expression in human and mouse skin and skin appendages. While AhR was highly expressed in cultured keratinocytes and in the skin, it was found primarily in the cytoplasm, but not in the nucleus, suggesting its inactivity. At the same time, treatment of N-TERT cells with proteasomal inhibitor MG132 and eventual inhibition of AhR degradation resulted in nuclear AhR accumulation. Treatment of keratinocytes with AhR ligands such as TCDD, FICZ, caused near-complete disappearance of AhR, and treatment with I3C resulted in substantially diminished level of AhR possibly due to ligand-induced AhR degradation. The AhR decay was blocked by proteasome inhibition, indicating degradation-based mechanism of regulation. Additionally, AhR decay was blocked by ligand-selective AhR antagonist CH223191, implying substrate-induced mechanism of degradation. Furthermore, degradation of AhR was blocked in N-TERT cells with knockdown of AhR dimerization partner ARNT (HIF1ß), suggesting that ARNT is required for AhR proteolysis. However, addition of hypoxia mimetics (HIF1 pathway activators) CoCl2 and DMOG had only minor effects on degradation of AhR. Additionally, inhibition of HDACs with Trichostatin A resulted in enhanced expression of AhR in both untreated and ligand-treated cells. These results demonstrate that in immortalized epidermal keratinocytes AhR is primarily regulated post-translationally via proteasome-mediated degradation, and suggest potential means to manipulate AhR levels and signaling in the skin. Overall, the AhR is regulated via multiple mechanisms, including proteasomal ligand- and ARNT-dependent degradation, and transcriptional regulation by HDACs, implying complex system of balancing its expression and protein stability.

Recurrent missense mutations in the hair keratin gene hHb6 in monilethrix.

Monilethrix is an autosomal dominant hair disorder characterized by a beaded appearance of the hair resulting from periodic thinning of the shaft (MIM 158000). The phenotype shows variable penetrance and results in hair fragility and patchy dystrophic alopecia. Mutations of the helix-encoded region in two hair-specific keratins (hHb1 and hHb6) have been identified as responsible for this disorder. We investigated two unrelated families from Russia and Colombia with monilethrix and found two missense mutations in hHb6. In the Russian family, we found a G to A transition at the first base of codon 402, resulting in a lysine substitution (GAG to AAG), designated E402K. In the Colombian family, affected patients carried a missense mutation of codon 413, involving a transition from G to A causing a lysine substitution (GAG to AAG), designated E413K. These two mutations have been identified in other monilethrix families from Europe. Our findings extend the body of evidence implicating recurrent hHb6 and hHb1 mutations in monilethrix families from around the world.

Hair follicle predetermination.

Recent genetic and molecular studies of hair follicle (HF) biology have provided substantial insight; however, the molecular data, including expression patterns, cannot be properly appreciated without an understanding of the basic cellular rearrangements and interactions that underpin HF cyclic transformations. We present a novel interpretation of the major cellular processes that take place during HF cycling--the hypothesis of hair follicle predetermination. This hypothesis is an extension of previous models of HF cellular kinetics but has two critical modifications: the dual origin of the cycling portion of the HF, and the timing of the recruitment of stem cells. A compilation of evidence suggests that the ascending portion of the HF (hair shaft and inner root sheath) arises not from bulge-located HF stem cells that contribute to the formation of only the outer root sheath (ORS), but instead from the germinative cells localized in the secondary hair germ. In middle anagen, upon completion of the downward growth of the HF, cells derived from the bulge region migrate downward along the ORS to reside at the periphery of the HF bulb as a distinct, inactive cell population that has specific patterns of gene expression - 'the lateral disc'. These cells survive catagen-associated apoptosis and, under the direct influence of the follicular papilla (FP), transform into the hair germ and acquire the ability to respond to FP signaling and produce a new hair. Thus, we propose that the specific sensitivity of germ cells to FP signaling and their commitment to produce the ascending HF layers are predetermined by the previous hair cycle during the process of transformation of bulge-derived lateral disc cells into the secondary hair germ.

Characterization of the desmosomal cadherin gene family: genomic organization of two desmoglein genes on human chromosome 18q12.

The human desmoglein genes, desmogleins 1--3, are members of the desmosomal cadherin superfamily, and encode critical components of the desmosome. These genes are tightly clustered within 150--200 kb of chromosome 18q12.1 and represent excellent candidate genes for genetic disorders of the epidermis linked to this region of the genome. Mutations in desmoglein 1 have already been implicated in the genetic disorder striate palmoplantar keratoderma. Similarly, a mutation in desmoglein 3 underlies the balding mouse phenotype, although no human mutations in desmoglein 3 have been identified to date. In this study, we have characterized the genomic organization of two of the three desmoglein genes mapped to chromosome 18q12. Comparison of their exon-intron structure reveals the high level of evolutionary conservation expected from these related genes. The identification of the genomic structure of the desmoglein genes will facilitate mutation detection in genodermatoses with desmosomal abnormalities resulting from underlying defects in these genes.

Structural analysis reflects the evolutionary relationship between the human desmocollin gene family members.

Desmocollins, members of the desmosomal cadherin family, are known to play an important role in desmosomal intercellular adhesion. The human desmosomal cadherin cluster is located on chromosome 18q12, and consists of three desmoglein and three desmocollin genes. The cDNAs of all six of these genes have been cloned and sequenced, however, the exon-intron organization was reported for only one human desmocollin gene, DSC2. We elucidated the exon-intron structures of the DSC1 and DSC3 genes using PCR amplification of genomic DNA and direct sequencing of BAC clones. The results suggest a strong evolutionary conservation between the genomic organization of the desmocollin genes.

The Charles River "hairless" rat mutation is distinct from the hairless mouse alleles.

The Charles River (CR) "hairless" rat is one of the autosomal recessive hypotrichotic animal models actively studied in pharmacologic and dermatologic research. Despite its widespread use, the molecular basis of this monogenic mutation remains unknown, and the skin histologic features of this phenotype have never been described. However, the designation "hairless" has been used as an extension of the hairless mouse (hr) nomenclature on the basis of the clinical absence of hairs in both phenotypes. We present a description of the histopathologic changes in heterozygous and homozygous CR hairless rat mutants during the first month of life. The postnatal homozygous rat skin was characterized by abnormal keratinization of the hair shaft and formation of a thick and dense layer of corneocytes in the lower portion of the epidermal stratum corneum. This layer prevented the improperly keratinized hair shaft from penetrating the skin surface. Starting from the latest stages of hair follicle (HF) development, obvious signs of HF degeneration were observed in homozygous skin. This process was extremely rapid, and by day 12, mainly atrophic HFs with abnormal or broken hairs were present in the skin. Therefore, the mutation in the CR rat abrogates cell proliferation in the hair matrix and affects keratinocyte differentiation in the HF and interfollicular epidermis, a phenotype that is completely distinct from hr/hr. To test whether the CR rat harbored a mutation in the hr gene, we analyzed the coding region of this gene and consensus intron splice site sequences in mutant rats and found no mutation, further supporting phenotypic evidence that the hairless phenotype in CR rats is not allelic with hairless. Finally, using intragenic polymorphisms, we were able to exclude homozygosity at the hairless locus by use of genotypic analysis. Thus, morphologic analysis of successive stages of phenotype development in the CR hairless rat, together with definitive molecular studies, indicate that this mutation may be unique among the other hypotrichotic rat mutations.

Clinical and molecular diagnostic criteria of congenital atrichia with papular lesions.

Congenital atrichia with papular lesions is a rare, autosomal recessive form of total alopecia and mutations in the hairless (hir) gene have been implicated in this disorder. Published estimates of the prevalence of this disorder remain surprisingly low considering pathogenetic mutations in hir have been found in distinct ethnicities around the world. Therefore, it is likely that congenital atrichia with papular lesions is far more common than previously thought and is often mistaken for its phenocopy, the putative autoimmune form of alopecia universalis. To clarify this discrepancy, we propose criteria for the clinical diagnosis of congenital atrichia with papular lesions. Among these is the novel report of the consistent observation of hypopigmented whitish streaks on the scalp surface of affected individuals. Additionally, we report the identification of a novel missense mutation in hir from a family of Arab Palestinian origin that exhibits the pathognomonic features of atrichia with papular lesions. Collectively, we anticipate that an increased recognition of this disorder will result in more accurate diagnosis and the sparing of unnecessarily treatment to patients.

Hoxa4 expression in developing mouse hair follicles and skin.

We have examined the expression of the Hoxa4 gene in embryonic vibrissae and developing and cycling postnatal pelage hair follicles by digoxigenin-based in situ hybridization. Hoxa4 expression is first seen in E13.5 vibrissae throughout the follicle placode. From E15.5 to E18.5 its expression is restricted to Henle's layer of the inner root sheath. Postnatally, Hoxa4 expression is observed at all stages of developing pelage follicles, from P0 to P4. Sites of expression include both inner and outer root sheaths, matrix cells, and the interfollicular epidermis. Hoxa4 is not expressed in hair follicles after P4. Hoxb4, however, is expressed both in developing follicles at P2 and in catagen at P19, suggesting differential expression of these two paralogous genes in the hair follicle cycle.

Patterns of hairless (hr) gene expression in mouse hair follicle morphogenesis and cycling.

The hr (hairless) gene encodes a putative transcription factor with restricted expression in the skin and brain. Mutations in the hr locus cause papular atrichia in humans and complete hair loss in mice and other mammals. To further elucidate the role of hr in skin biology, and to identify potential target cells for hr regulation, we studied hr mRNA localization during hair follicle (HF) morphogenesis and cycling in normal C57BL/6J mice. In situ hybridization revealed that hr expression was present in the suprabasal cell layers of the epidermis, whereas the basal and highly differentiated keratinocytes of the granular layer were hr-negative. During the early stages of HF morphogenesis, hr mRNA was detected in the developing hair peg. Later, it became concentrated in the HF infundibulum, in the HF matrix, and in the inner root sheath (IRS), whereas the dermal papilla (DP) and outer root sheath were consistently hr mRNA-negative. During catagen, hr gene expression gradually declined in the regressing IRS, shortly but dramatically increased in the zone of developing club hair, and became up-regulated in the epithelial cells adjacent to the DP. The co-localization of hr mRNA with the site of the morphological defects in mutant skin implicates hr as a key factor in regulating basic cellular processes during catagen, including club hair formation, maintenance of DP-epithelial integrity, IRS disintegration, and keratinocyte apoptosis in the HF matrix.

Ornithine decarboxylase transgenic mice as a model for human atrichia with papular lesions.

The hair follicle is characterized by cyclic transformations from active growth and hair fiber production through regression into a resting phase. The growth phase, known as anagen, is associated with rapid rates of cell turnover, and variations in the rate of DNA synthesis in mouse skin throughout the hair cycle are accompanied by changes in the activity of ornithine decarboxylase (ODC), a key enzyme in the synthesis of polyamines, which are actively involved in regulation of normal cell division, differentiation, and growth. Previously, a transgenic mouse was created that overexpressed ODC in the skin using a K6 promoter. The first hair cycle in neonatal transgenic mice appeared to be normal, but by the third week of postnatal life transgenic pups begin to progressively lose hair. The lower portion of the hair follicle was progressively replaced with enlarging cystic structures located in the deep dermis, and the transgenic mice exhibited excessive growth of skin mass resulting in pronounced wrinkling and folding. Interestingly, these findings bore striking resemblance to the rhino mouse phenotype and to human patients with papular atrichia, a rare congenital ectodermal disorder characterized by progressive and irreversible hair loss in early childhood. The similarities in phenotype between transgenic mice and human atrichia with papular lesions suggest that ODC transgenics may represent a useful model for studying this disorder. It appears that ODC plays a functionally important, yet still obscure role in a complex metabolic pathway that is critical in hair follicle function not only in mice, but in humans as well.

A novel missense mutation (C622G) in the zinc-finger domain of the human hairless gene associated with congenital atrichia with papular lesions.

Congenital atrichia with papular lesions is a rare, recessively inherited form of hair loss characterized by a complete absence of all body hair shortly after birth. Mutations in the human ortholog of the mouse hairless (hr) gene have been implicated in the pathogenesis of this disorder. In this study, we screened, by direct sequence analysis, the hairless gene in a family of Polish descent and identified a novel missense mutation (C622G). The mutation alters the third of four invariant cysteins in the zinc-finger domain, which has high homology to the C-X-X-C-(X)17-C-X-X-C structure of the zinc-fingers of the GATA family of transcription factors. The human hairless gene encodes a putative transcription factor with restricted expression in the brain and skin, which is involved in the regulation of apoptosis during catagen remodeling in the hair cycle.

The role of the hairless (hr) gene in the regulation of hair follicle catagen transformation.

Mice that carry a mutation at the hairless (hr) locus develop seemingly normal hair follicles (HF) but shed their hairs completely soon after birth. Histologically, their HFs degenerate into characteristic utriculi and dermal cysts shortly after the entry of the HF into the first regression phase (catagen), during the initiation of HF cycling. Here, we show that at least nine distinct stages of HF disintegration can be distinguished in hr/hr mice. Toward the end of HF morphogenesis (day 15 postpartum) the proximal hair bulb in hr/hr skin undergoes premature and massive apoptosis. This is associated with a dyscoordination of cell proliferation in defined HF compartments, malpositioning of the proximal inner root sheath, striking atrophy of outer root sheath, and failure of trichilemmal keratinization in the developing club hair. Rather than undergoing their normal catagen-associated involution, the hair bulb and central outer root sheath disintegrate into separate cell clusters, thus disrupting all epithelial contact with the dermal papilla. Dermal papilla fibroblasts fail to migrate upward, and break up into clusters of shrunken cells stranded in the reticular dermis as dermal cyst precursors, while the upper HF epithelium transforms into utriculi. Some dermal papilla cells, which normally never undergo apoptosis, also become TUNEL+ in hr/hr skin, and their normally high expression of a key adhesion molecule, neural cell adhesion molecule, declines. Thus, loss of a functional hr gene product (a putative zinc finger transcription factor) initiates a premature, highly dysregulated catagen, which results in the destruction of the normal HF architecture and abrogates the HF's ability to cycle. This provides new insights into the pathobiology of the hr mutation, and suggests that the normal hr gene product is a crucial element of catagen control.

Genomic organization of the human hairless gene (HR) and identification of a mutation underlying congenital atrichia in an Arab Palestinian family.

Congenital atrichia is a rare form of hereditary human hair loss, characterized by the complete shedding of hair shortly after birth, together with the formation of papular lesions on the skin. Recently, we cloned the human homolog of the mouse hairless gene and identified pathogenic mutations in several families with inherited congenital atrichia. Here, we present the genomic organization of the human hairless gene (HGMW-approved symbol HR), which spans over 14 kb on chromosome 8p12 and is organized into 19 exons. In addition, we report the identification of a 22-bp deletion mutation in exon 3 of the hairless gene in a large consanguineous Arab Palestinian family from a village near Jerusalem, Israel. These findings extend the body of evidence implicating mutations in the hairless gene as an underlying cause of congenital atrichia in humans.

The molecular basis of congenital atrichia in humans and mice: mutations in the hairless gene.

Congenital atrichia is a form of total alopecia inherited in an autosomal recessive pattern. In individuals affected with this form of hair loss, hairs are typically absent from the scalp, and patients are nearly completely devoid of eyebrows, eyelashes, axillary and pubic hair, following shedding of the natural hair shortly after birth. We have recently linked this disorder to the chromosomal region 8p12, and cloned the human hairless gene, which resides within this interval. We have identified several mutations in the hairless gene in atrichia families from around the world. In hairless mice, the hair matrix cells appear to undergo a premature and massive apoptosis, together with a concomitant decline in Bcl-2 expression, a loss of NCAM positivity, and a disconnection with the overlying epithelial sheath essential for the movement of the dermal papilla. As a consequence, the hair bulb and dermal papilla remain stranded in the dermis, and indispensible messages between the dermal papilla and stem cells in the bulge are not transmitted, so no further hair growth occurs. These findings suggest that the hairless gene product may play a crucial role in maintaining the delicate balance between cell proliferation, differentiation and apoptosis in the hair follicle, as well as in the interfollicular epidermis.

Molecular and functional aspects of the hairless (hr) gene in laboratory rodents and humans.

For many years, hairless and rhino mouse mutants have provided a useful and extensively exploited model for studying different aspects of skin physiology, including skin aging, pharmacokinetic evaluation of drug activity and cutaneous absorption, skin carcinogenesis, and skin toxicology. Interestingly, however, hairless and rhino mice have rarely been studied for their primary cellular defect - hairlessness - and thus, the hairless gene itself and its physiological functions have been largely overlooked for decades. The recent identification of the human homolog of the hairless gene on human Chromosome 8p12 confirmed the clinical significance of the phenomenon of "hairlessness" in humans, which was predicted on the basis of similarities between hairless mice and a congenital hair disorder characterized by atrichia with papules. Mutations in the hairless gene of mice provide instructive models for further studies of hr gene function, and may facilitate insights into the pathophysiology of different human disorders associated with the disruption of hr gene activity. We provide an overview of current data on the structure and expression patterns of the hr gene, and of mutations at the hairless locus in mice and humans, including the genetic basis of different alleles, the pathology of hairlessness, reproductive and immunological defects, and susceptibility to dioxin toxicity. On the basis of our current understanding of hairlessness, we speculate on the putative functions of the hr gene product in skin physiology, and particularly, in hair follicle biology.

Molecular basis of a novel rhino (hr(rhChr)) phenotype: a nonsense mutation in the mouse hairless gene.

The hairless and rhino mutations are autosomal recessive allelic mutations that map to mouse Chromosome 14. In general, the rhino phenotype is a more severe manifestation of the hairless phenotype. In both hairless and rhino mice, the hair begins shedding in a cephalocaudal pattern within 7 days after birth, and never regrows due to a series of irreversible cellular events. The hairless mutation closely resembles the human disease known as papular atrichia (MIM 209500). Recently, this disease was linked to Chromosome 8p12, the human homolog of hairless was cloned and mapped to the same locus, and mutations have been identified in several different families. In order to gain insight into the pathophysiology of disease in papular atrichia, we sought to utilize mouse mutations as in vivo model systems. In this study, we report the identification of a homozygous nonsense mutation in the coding region of the hr gene in a hairless mouse captured on a chicken farm in the Midwestern United States. To reflect the place of identification of this new mutation at the hr locus, we have designated this allele hr(rhChr) using the laboratory code Chr (Christiano).

Molecular basis for the rhino Yurlovo (hr(rhY)) phenotype: severe skin abnormalities and female reproductive defects associated with an insertion in the hairless gene.

In 1989, mice bearing mutations at the hr (hairless) locus were first proposed as a model for the human hair growth disorder papular atrichia, since in both these mice and in corresponding patients, a complete hair loss develops due to disintegration of the normal follicle structure into dermal cysts and so-called utriculi. Recently, the human hairless gene was characterized, and pathogenetic mutations were found to be associated with a recessively inherited form atrichia with papular lesions; however, the functions of hr gene remain unclear. Allelic mutations in the murine hairless gene represent a potentially powerful tool to elucidate the role of the hairless gene protein product in hair follicle physiology. In 1980, several naked animals were discovered in a breeding colony of B10.R109/Y mice maintained in the Laboratory of Experimental Biological Models (L.E.B.M., Yurlovo, Moscow District, Russia). By cross breeding with hairless HRS/J hr/hr mice, this mutation was shown to be allelic with hairless. Here, we describe the molecular basis of the hr(rhY) mutation in mice, which consists of a 13 bp insertion in exon 16 of the hr gene. Histological evaluation of Yurlovo mouse skin revealed some differences as compared to the hairless and rhino mutations, with the formation of dermal megacysts being the most specific peculiarity of the Yurlovo mutation. These results, together with previous studies of hr(rhY)/hr(rhY) mutant mice, suggest that the rhino Yurlovo (hr(rhY)) mutation represents a third and potentially more severe variation of the hairless phenotype.