Expression pattern of Nav2 in the murine CNS with development.

Neuron navigator 2 (NAV2, RAINB1, POMFIL2, HELAD1, unc53H2) is essential for nervous system development. In the present study the spatial distribution of Nav2 transcript in mouse CNS during embryonic, postnatal and adult life is examined. Because multiple NAV2 proteins are predicted based on alternate promoter usage and RNA splicing, in situ hybridization was performed using probes designed to the 5' and 3' ends of the Nav2 transcript, and PCR products using primer sets spanning the length of the mRNA were also examined by real time PCR (qPCR). These studies support full-length Nav2 transcript as the predominant form in the wild-type mouse CNS. The developing cortex, hippocampus, thalamus, olfactory bulb, and granule cells (GC) within the cerebellum show the highest expression, with a similar staining pattern using either the 5'Nav2 or 3'Nav2 probe. Nav2 is expressed in GC precursors migrating over the cerebellar primordium as well as in the postmitotic premigratory cells of the external granule cell layer (EGL). It is expressed in the cornu ammonis (CA) and dentate gyrus (DG) throughout hippocampal development. In situ hybridization was combined with immunohistochemistry for Ki67, CTIP2 and Nissl staining to follow Nav2 transcript location during cortical development, where it is observed in neuroepithelial cells exiting the germinal compartments, as well as later in the cortical plate (CP) and developing cortical layers. The highest levels of Nav2 in all brain regions studied are observed in late gestation and early postnatal life which coincides with times when neurons are migrating and differentiating. A hypomorphic mouse that lacks the full-length transcript but expresses shorter transcript shows little staining in the CNS with either probe set except at the base of the cerebellum, where a shorter Nav2 transcript is detected. Using dual fluorescent probe in situ hybridization studies, these cells are identified as oligodendrocytes and are detected using both Olig1 and the 3'Nav2 probe. The identification of full-length Nav2 as the primary transcript in numerous brain regions suggests NAV2 could play a role in CNS development beyond that of its well-established role in the cerebellum.

Differential activity of 2-methylene-19-nor vitamin D analogs on growth factor gene expression in rhino mouse skin and comparison to all-trans retinoic acid.

While all 2-methylene-19-nor analogs of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) tested produce an increase in epidermal thickness in the rhino mouse, only a subset reduce utricle size (comedolysis). All-trans retinoic acid (atRA) also causes epidermal thickening and a reduction in utricle size in the rhino mouse. We now report that 2-methylene-19-nor-(20S)-1α-hydroxybishomopregnacalciferol (2MbisP), a comedolytic analog, increases epidermal thickening more rapidly than does atRA, while both reduce utricle area at an equal rate. Whereas unlike atRA, 2MbisP does not alter the epidermal growth factor receptor ligand, heparin-binding epidermal growth factor-like growth factor, it does increase the expression of both amphiregulin and epigen mRNA, even after a single dose. In situ hybridization reveals an increase in these transcripts throughout the closing utricle as well as in the interfollicular epidermis. The mRNAs for other EGFR ligands including betacellulin and transforming growth factor-α, as well as the epidermal growth factor receptor are largely unaffected by 2MbisP. Another analog, 2-methylene-19-nor-(20S)-26,27-dimethylene-1α,25-dihydroxyvitamin D3 (CAGE-3), produces epidermal thickening but fails to reduce utricle size or increase AREG mRNA levels. CAGE-3 modestly increases epigen mRNA levels, but only after 5 days of dosing. Thus, 2-MbisP produces unique changes in epidermal growth factor receptor ligand mRNAs that may be responsible for both epidermal proliferation and a reduction in utricle size.

Identification of a unique subset of 2-methylene-19-nor analogs of vitamin D with comedolytic activity in the rhino mouse.

The active metabolite of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), and a series of 2-methylene-19-nor analogs of 1,25(OH)(2)D(3) were evaluated for their ability to reduce the size of utricles (comedolytic activity) in a rhino mouse model of acne. All analogs tested, as well as the native hormone, increased the skin epidermal thickness. In contrast, only a subset of analogs that lacked a full side chain and 25-hydroxyl group were found to possess comedolytic activity. A reduction in comedone area could be achieved without adversely affecting serum calcium levels. Although all compounds that contained a side chain ranging from 2 to 5 carbons in length had similar potency as comedolytic agents, increasing the length of the side chain resulted in a progressive increase in calcemic liability. Dose-response studies of the comedolytic analogs showed that an increase in epidermal thickness was achieved at a lower dose than that needed to induce comedolysis. Thus, we have identified a unique subset of vitamin D analogs that produce comedolysis in the absence of hypercalcemia. Further, the activity of vitamin D analogs in causing epidermal hyperproliferation has been distinguished from that resulting in a reduction in utricle size.

2-Methylene-19-nor-1alpha-hydroxyvitamin D3 analogs inhibit adipocyte differentiation and PPARgamma2 gene transcription.

The hormonally active form of vitamin D3, 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3), prevents adipogenesis in the 3T3-L1 preadipocyte cell line. In this paper, both a shortened side-chain non-calcemic analog, (20S)-1alpha-hydroxy-2-methylene-19-nor-bishomopregnacalciferol (2MbisP), as well as a highly bone-selective compound, 2-methylene-19-nor-(20S)-1alpha-hydroxyvitamin D3 (2MD), are shown to completely prevent adipocyte differentiation in this cell line. 2MbisP is slightly less potent than 1,25(OH)2D3, whereas 2MD is approximately two orders of magnitude more potent that the parent hormone in preventing adipocyte differentiation. The ability to block differentiation requires binding to the vitamin D receptor. Both 1,25(OH)2D3 and the analogs prevent the induction of the C/EBPalpha and PPARgamma2 transcription factors, which is necessary for terminal differentiation. In contrast, the normal increase in C/EBPbeta protein that occurs shortly after the induction of differentiation occurs both in the presence and absence of vitamin D compounds, and the C/EBPbeta protein appears functional with respect to DNA binding and nuclear localization. Transient transfection studies show that 1,25(OH)2D3 prevents transactivation through the 5' region of the PPARgamma2 promoter, and thus acts at the transcriptional level to inhibit the normal upregulation of PPARgamma2 that occurs during the course of 3T3-L1 cell differentiation.

All-trans retinoic acid-responsive genes identified in the human SH-SY5Y neuroblastoma cell line and their regulated expression in the nervous system of early embryos.

The vitamin A metabolite, all-trans retinoic acid (atRA), is required for embryonic development. atRA binds to the nuclear retinoic acid receptors and regulates the transcription of specific target genes. In order to identify atRA-induced genes that play a role in neural development, a subtractive library was created from SH-SY5Y neuroblastoma cells, a human cell line that exhibits changes in cell adhesion and neurite outgrowth after exposure to the vitamin A acid. We report here the identification of 14 genes that are rapidly induced by atRA (retinoic acid induced in neuroblastoma or RAINB), eight of which were previously not known to be atRA responsive (BTBD11, calmin, cyclin M2, ephrin B2, HOXD10, NEDD9, RAINB6 and tenascin R). mRNA regulation by atRA was confirmed in SH-SY5Y cells by Northern blotting, and gene regulation was studied in additional human cell lines using the quantitative polymerase chain reaction. The majority of the atRA-responsive clones revealed in this screen are highly expressed in the nervous system of developing rat embryos. Further, the expression of several of these genes is perturbed in developing rat embryos exposed to excess atRA or conversely, deprived of sufficient retinoid during early development. We propose that a subset of these genes lie downstream of atRA and its receptors in the regulation of neurite outgrowth and cell adhesion in both neural and non-neural tissues within the developing embryo.