Pak1 maintains epidermal stem cells by regulating Langerhans cells and is required for skin carcinogenesis.

Pak1 (serine/threonine p21-activated kinases) was previously reported to have oncogenic activity in several cancers. However, its roles in the cancer microenvironment are poorly understood. We demonstrated that Pak1 expression in Langerhans cells (LCs) is essential for the maintenance of epidermal stem cells and skin tumor development. We found that PAK1 is localized in LCs by immunohistochemistry. Furthermore, the number of LCs significantly decreased in MSM/Ms Pak1 homozygous knockout mice (MSM/Ms-Pak1-/-). F1 hybrid (FVB/N×MSM/Ms) Pak1 heterozygous knockout mice (F1-Pak1+/-) had increased numbers of Th17 cells in the skin. Therefore, Pak1 knockdown cells were prepared using LC-derived XS52 cells (XS52-Pak1KD) and co-cultured with keratinocyte-derived C5N cells. As a result, XS52-Pak1KD cell supernatants promoted C5N cell proliferation. We then carried out DMBA/TPA skin carcinogenesis experiments using F1-Pak1+/- mice. Of note, F1-Pak1+/- mice exhibited stronger resistance to skin tumors than control mice. F1-Pak1+/- mice had fewer epidermal stem cells in the skin bulge. Our study suggested that Pak1 regulates the epidermal stem cell number by changing the properties of LCs and functions in skin carcinogenesis. We clarified a novel role of Pak1 in regulating LCs as a potential therapeutic target in skin immune disease and carcinogenesis.

The parathyroid hormone regulates skin tumour susceptibility in mice.

Using a forward genetics approach to map loci in a mouse skin cancer model, we previously identified a genetic locus, Skin tumour modifier of MSM 1 (Stmm1) on chromosome 7, conferring strong tumour resistance. Sub-congenic mapping localized Parathyroid hormone (Pth) in Stmm1b. Here, we report that serum intact-PTH (iPTH) and a genetic polymorphism in Pth are important for skin tumour resistance. We identified higher iPTH levels in sera from cancer-resistant MSM/Ms mice compared with susceptible FVB/NJ mice. Therefore, we performed skin carcinogenesis experiments with MSM-BAC transgenic mice (Pth MSM-Tg) and Pth knockout heterozygous mice (Pth +/-). As a result, the higher amounts of iPTH in sera conferred stronger resistance to skin tumours. Furthermore, we found that the coding SNP (rs51104087, Val28Met) localizes in the mouse Pro-PTH encoding region, which is linked to processing efficacy and increased PTH secretion. Finally, we report that PTH increases intracellular calcium in keratinocytes and promotes their terminal differentiation. Taken together, our data suggest that Pth is one of the genes responsible for Stmm1, and serum iPTH could serve as a prevention marker of skin cancer and a target for new therapies.

CENP-R acts bilaterally as a tumor suppressor and as an oncogene in the two-stage skin carcinogenesis model.

CENP-R is a component of the CENP-O complex, including CENP-O, CENP-P, CENP-Q, CENP-R, and CENP-U and is constitutively localized to kinetochores throughout the cell cycle in vertebrates. CENP-R-deficient chicken DT40 cells are viable and show a very minor effect on mitosis. To investigate the functional roles of CENP-R in vivo, we generated CENP-R-deficient mice (Cenp-r-/- ). Mice heterozygous or homozygous for Cenp-r null mutation are viable and healthy, with no apparent defect in growth and morphology, indicating Cenp-r is not essential for normal development. Accordingly, to investigate the role of the Cenp-r gene in skin carcinogenesis, we subjected Cenp-r-/- mice to the 7,12-dimethylbenz(a)anthracene (DMBA)/TPA chemical carcinogenesis protocol and monitored tumor development. As a result, Cenp-r-/- mice initially developed significantly more papillomas than control wild-type mice. However, papillomas in Cenp-r-/- mice showed a decrease of proliferative cells and an increase of apoptotic cells. As a result, they did not grow bigger and some papillomas showed substantial regression. Furthermore, papillomas in Cenp-r-/- mice showed lower frequency of malignant conversion to squamous cell carcinomas. These results indicate Cenp-r functions bilaterally in cancer development: during early developmental stages, Cenp-r functions as a tumor suppressor, but during the expansion and progression of papillomas it functions as a tumor-promoting factor.

A 1-bp deletion in Fgf5 causes male-dominant long hair in the Syrian hamster.

Hair length in mammals is generally regulated by the hair cycle, and its disruption leads to abnormal hair morphogenesis in several species. FGF5, one of the hair cycle regulators, has a role in inducing catagen, and that mutation causes abnormal hair length in both sexes in humans, mice, dogs, and cats. Male-dominant long-haired coat (MALC) is an inbred strain of Syrian hamster exhibiting spontaneous long hair in males. After castration, MALC exhibited significantly shorter hair than the control individuals, but testosterone administration to castrated MALC showed reversion to the original phenotype. Moreover, flutamide administration led to MALC phenotype repression. Histological analysis revealed that hair follicle regression was shown in the wild-type 4 weeks after depilation, but that of MALC remained in the anagen phase. We detected a c.546delG of Fgf5 in MALC (Fgf5malc) that might lead to truncation resulting from a frame shift in FGF5 (p.Arg184GlyfsX6). Additionally, homozygous Fgf5malc was only detected in long-haired (Slc:Syrian×MALC)F2 and (J-2-Nn×MALC)F2 progenies, and all homozygous wild and heterozygous Fgf5malc individuals showed normal hair length. Thus, Fgf5malc leads to male-dominant long hair via a prolonged anagen phase which is affected by testosterone in hamsters. To our knowledge, this report is the first to present the sexual dimorphism of hair length caused by the Fgf5 mutation.

Oncogenic Lmo3 cooperates with Hen2 to induce hydrocephalus in mice.

We previously reported that LMO3 and HEN2 act as oncogenes in neuroblastoma development through up-regulating MASH1 transcription by interfering with HES1. To confirm these results in vivo, we generated transgenic mice of these genes. Lmo3 or Hen2 was expressed under the control of Wnt1 promoter, which is expressed in the central nervous system and neural crest of the sympathoadrenal lineage from which neuroblastoma develops. Heterozygous Lmo3 and Hen2 transgenic mice (Tg (Lmo3) and Tg (Hen2)) developed hydrocephalus at higher frequency than for the wild type mice, and all heterozygous double-transgenic mice (Tg (Lmo3; Hen2)) developed hydrocephalus. Therefore, Lmo3 and Hen2 may be involved in and have synergistic effects on hydrocephalus development. Although aqueduct stenosis occurred in all genotypes, it was mild in Tg (Lmo3; Hen2) mice. Furthermore, hydrocephalus was detected at E18.5 in Tg (Lmo3; Hen2). These results suggest that the causes of hydrocephalus are not only aqueduct stenosis but also disorder of neocortical development. A similar phenotype was reported in Robo1/2(-/-) mice, in which Hes1 expression level was decreased in ventricular zone progenitors. Thus, it is suggested that the expression levels of Lmo3 and/or Hen2 could determine the fate of stem cells by inhibiting Hes1 function during nervous system development and might be a trigger of aberrant neurogenesis in vivo.

Expression of truncated PITX3 in the developing lens leads to microphthalmia and aphakia in mice.

Microphthalmia is a severe ocular disorder, and this condition is typically caused by mutations in transcription factors that are involved in eye development. Mice carrying mutations in these transcription factors would be useful tools for defining the mechanisms underlying developmental eye disorders. We discovered a new spontaneous recessive microphthalmos mouse mutant in the Japanese wild-derived inbred strain KOR1/Stm. The homozygous mutant mice were histologically characterized as microphthalmic by the absence of crystallin in the lens, a condition referred to as aphakia. By positional cloning, we identified the nonsense mutation c.444C>A outside the genomic region that encodes the homeodomain of the paired-like homeodomain transcription factor 3 gene (Pitx3) as the mutation responsible for the microphthalmia and aphakia. We examined Pitx3 mRNA expression of mutant mice during embryonic stages using RT-PCR and found that the expression levels are higher than in wild-type mice. Pitx3 over-expression in the lens during developmental stages was also confirmed at the protein level in the microphthalmos mutants via immunohistochemical analyses. Although lens fiber differentiation was not observed in the mutants, strong PITX3 protein signals were observed in the lens vesicles of the mutant lens. Thus, we speculated that abnormal PITX3, which lacks the C-terminus (including the OAR domain) as a result of the nonsense mutation, is expressed in mutant lenses. We showed that the expression of the downstream genes Foxe3, Prox1, and Mip was altered because of the Pitx3 mutation, with large reductions in the lens vesicles in the mutants. Similar profiles were observed by immunohistochemical analysis of these proteins. The expression profiles of crystallins were also altered in the mutants. Therefore, we speculated that the microphthalmos/aphakia in this mutant is caused by the expression of truncated PITX3, resulting in the abnormal expression of downstream targets and lens fiber proteins.