- 2006 -

The Role of Arnt gene in Perinatal Mouse Skin Neovascularization

Geng Songmei¹, Mezentsev Alexandre¹, Ishi Miho¹, Kalachikov Sergey M.² and Panteleyev Andrey A.¹

¹Dermatology, Columbia University, New York, NY USA.
²Genome Center, Columbia University, New York, NY USA

Abstract Body: As a subunit of hypoxia-induced factor HIF-1, Aryl hydrocarbon Receptor Nuclear Translocator (Arnt, HIF1b) plays a key role in control of anaerobic metabolism, erythropoesis, angiogenesis, and vasodilation. Angiogenesis is essential for skin biology, including thermoregulation, tumor growth, wound healing, immune response and hair-follicle cycling. Despite of it, the role of Arnt in skin vascularization is not yet explored. Targeted ablation of Arnt in mouse epidermis using Arnt-flox/KK14-Cre model resulted in epidermal-barrier failure and produced severe abnormalities in the dermal vasculature. In the skin of Arnt-null newborn mice, hypervascularization was accompanied by a dilation of blood vessels that had atrophic or even discontinuous walls. Mechanical stress resulted in noticeable intracutaneous bleeding. Immunohistochemistry revealed a significant decline of von Willebrand factor in the cutaneous vasculature. The microarray analysis showed that most of the genes affected by the knockout were assigned to hypoxia/angiogenic pathways. Egln3, which plays a key role in both: HIF-1a degradation and support of arterial-wall integrity was downregulated in 17.4 times. Some genes implicated in mediation of the hypoxic response and vascular remodeling were downregulated as well: Bnip- in 19.7, Btc- 8.7, Edg8- 6.5, Mt4- 6.2 and Aldoc- 4.1 times. Besides, other angiogenesis-related genes were upregulated: Procr- in 87.4, Mmp3- 34.3, Hs3st1- 31.8 and Lcn7- in 12.6 times. The prevalence of angiogenic factors on the epidermal microarray coupled with a prominent dermal vascular phenotype in Arnt-flox/K14-Cre mice, suggested the existence of Arnt-dependent epidermis-secreted hypoxia-sensoring pathway that controls dermal angiogenesis. Expression of Arnt in blood-vessel free mouse follicular papillae, along with an absence of Arnt in vascularized human papillae proposed the involvement of Arnt-dependent pathway in control of the skin hypoxic response.

Regulation of Epidermal Late- Differentiation Genes via Arnt-dependent Pathway in Primary Mouse Keratinocytes Requires Chromatin Remodeling

Mezentsev Alexandre, Kalachikov Sergey M. and Panteleyev Andrey A.

Dermatology, Columbia University, New York, NY USA.
Genome Center, Columbia University, New York, NY USA

Abstract Body: To evaluate a role of histone acetylation in the regulation of genes comprising the epidermal differentiation complex (EDC) and assess the contribution of Arnt to chromatin remodeling we performed a transfection of primary mouse keratinocytes with Arnt-expressing vector, pALTER-Arnt followed by the treatment of cells with trichostatin A (TSA), a specific inhibitor of histone deacetylasaes. Total RNA isolated from the harvested cells was converted to cDNA and analyzed for the expression of selected genes that are the members of EDC located on mouse Chromosome 3. Although cell transfection led to 10-fold increase of Arnt mRNA regardless of treatment with TSA, it had different consequences for gene expression in TSAAA-treated cells versus the untreated ones. Interestingly, the expression of Ivl and Lor, the most upregulated genes (25 and 60 fold, respectively) in the transfected and TSA-treated cells, did not appear in Arnt-transfected keratinocytes that had normal histone-deacetylases activity (no TSA treatment). The opposite effect, downregulation, although in a lower range was observed in Arnt-transfected TSA-treated cells for Sprr2i (20%) and Sprr2j (60%). In addition, S100a8, S100a9, and Sprr1a were faintly upregulated (~50%) in the transfected TSA-treated cells, and the expression of Sprrl1 was slightly upregulated in the transfected cells regardless of treatment them with TSA. The level of S100a10 was not affected. We conclude that function of Arnt in control of late-differentiation genes is not limited to the role of transcription factor and may also comprise participation in chromatin remodeling via an interaction as a cofactor with histone deacetylation complexes. This interaction is probably reflected in Arnt-induced downregulation of the selected genes even in the presence of the histone deacetylases inhibitor and strong upregulation of late-differentiation genes in Arnt-deficient keratinocytes.

The Patterns of Periderm Shedding in Mice and the Role of Arnt in Regulation of This Process

Mezentsev Alexandre, Kalachikov Sergey M. and Panteleyev Andrey A.

Dermatology, Columbia University, New York, NY USA.
Genome Center, Columbia University, New York, NY USA

Abstract Body: The periderm is a transitory epithelial sheet that forms an interface between the embryonic epidermis and the amniotic fluid. The functions and fate of the periderm in the murine fetal skin, as well as the molecular mechanisms that control its development and shedding have not been yet elucidated. It is a common view that shedding of the murine periderm occurs like in humans- prenatally and is a prerequisite of the formation of the epidermal barrier. However, it was also shown that the murine periderm is retained to postnatal days 4-5. Here we used EM, immunohistochemistry, qPCR and microarray hybridization to study perinatal epidermis in wild type and Arnt-flox/K14-Cre mice that die immediately after the birth due to barrier defects. In contrast to the human periderm, the mouse periderm is bilayered and is shed in two steps: the outer periderm (OP) is shed prenatally, whereas the glycogen-rich inner periderm (IP) is retained after the birth and likely provides an additional provisional barrier during the transition of the newborns to the terrestrial environment. The Arnt-null epidermis is characterized by an impeded development of the IP (abnormal peridermal granules) and atypical postnatal retention of the OP. On the molecular level, retention of both peridermal layers was reflected in upregulation of the periderm- specific keratins Krt2-6a and Krt-18. The neonatal Arnt-null epidermis also showed upregulation of Gjb2, Capn2, and Areg, that specifically appear at the earlier stages of the murine periderm development. EM revealed a retention of gap junctions in the superficial layers of Arnt-null epidermis. Being in the line with upregulation of Gjb2 as major component of gap junctions and negative regulator of periderm shedding this finding suggests possible pathomechanism of the OP retention in Arnt-null pups. Our results broaden the role of Arnt gene in the epidermal development and propose that deferral of the periderm development may contribute to the failure of the permeability barrier formation in Arnt-deficient fetuses.