Regulation of poorly characterized skin-associated gene expression in primary human keratinocytes.

<p>Semi-quantitative PCR analysis of expression of seven of the eight selected SAGs in primary human keratinocytes incubated with either cytokines (as indicated) or medium controls for 24 hours relative (RU) to control gene levels (18S RNA). Data shown are the mean ± SD. *p<0.05, **p<0.01 (Student’s t-test). SAGs tested were: (A) MUCL1, mucin-like 1; (B) WFDC5, WAP four-disulfide core domain 5; (C) TMEM45A, transmembrane protein 45A; (D) GPR115, G protein-coupled receptor 115; (E) CDHR1, cadherin-related family member 1; (F) SERPINB7, serpin peptidase inhibitor, clade B (ovalbumin), member 7; (G) GPR87, G protein-coupled receptor 87. C5orf46 expression was not detected in keratinocytes.</p

Detection of selected poorly characterized SAG encoded proteins in normal human skin and primary keratinocytes.

<p>Panels A–F; formalin-fixed paraffin embedded normal human skin sections were stained for DNA with DAPI and also with either SAG protein-specific (A, C, E), or isotype control antibodies (B, D, F) and visualized by immunofluorescent microscopy, 40× magnification. WFDC5, TMEM45A and GPR115 protein expression compared to beta actin was also detected in primary cultured human keratinocyte lysates using Western blotting (panels G, H and I respectively).</p

Top 100 skin-associated genes (SAGs) ranked by fold change of expression compared to the mean of the remaining 104 adult human tissue and cell types in the BIGE.

<p>Top 100 skin-associated genes (SAGs) ranked by fold change of expression compared to the mean of the remaining 104 adult human tissue and cell types in the BIGE.</p

Semi-quantitative PCR confirmation of the specificity of expression of poorly characterized skin-associated genes (SAGs) in human total RNAs.

<p>Expression levels of three control genes: (A) ALDOB, aldolase B fructose-biphosphate (not expressed in skin); (B) DCD, dermcidin; and (C) KRT1, keratin 1; and eight selected SAGs: (D) MUCL1, mucin-like 1; (E) WFDC5, WAP four-disulfide core domain 5; (F) TMEM45A, transmembrane protein 45A; (G) GPR115, G protein-coupled receptor 115; (H) CDHR1, cadherin-related family member 1; (I) SERPINB7, serpin peptidase inhibitor, clade B (ovalbumin), member 7; (J) C5orf46, chromosome 5 open reading frame 46; (K) GPR87, G protein-coupled receptor 87, were measured in healthy skin (eleven independent samples) and pooled total RNAs from spleen, kidney, brain and liver measured relative to control gene levels (18S RNA) and plotted as individual ratios, including, for skin samples, mean ratio values shown by the horizontal bar.</p

Expression profiles of top human skin-associated genes in the body index of gene expression (BIGE).

<p>Affymetrix GeneChip data for the top 10 SAGs are shown as mean normalized relative expression (RU) (+) Standard deviation (y axis), plotted against the sample IDs from 105 human tissue and cell types grouped by system (x axis), as listed in Panel A. CNS, central nervous system; PNS, peripheral nervous system. Asterisk marks skin sample in each profile.</p

Expression of poorly characterized skin-associated genes in human skin-derived cell lines.

<p>Semi-quantitative PCR analysis of expression of three control genes: (A) ALDOB, aldolase B fructose-biphosphate (not expressed in skin); (B) DCD, dermcidin; and (C) KRT1, keratin 1; and eight selected SAGs: (D) MUCL1, mucin-like 1; (E) WFDC5, WAP four-disulfide core domain 5; (F) TMEM45A, transmembrane protein 45A; (G) GPR115, G protein-coupled receptor 115; (H) CDHR1, cadherin-related family member 1; (I) SERPINB7, serpin peptidase inhibitor, clade B (ovalbumin), member 7; (J) C5orf46, chromosome 5 open reading frame 46; (K) GPR87, G protein-coupled receptor 87, were measured in low passage number primary human cells relative to control gene levels (18S RNA) and plotted as individual and mean ratios (horizontal bar).</p

Relationship between HVRP1 and other VSD-containing proteins.

<p><b>A</b>) Modular organization of HVRP1 compared to known voltage sensing proteins. The Shaker potassium channel was chosen as an example of protein containing both a VSD and a pore domain. C2D: C2 domain, CCD: coiled-coil domain, PhosD: phosphatase domain, PD: pore domain, TD: tetramerization domain. <b>B</b>) Phylogram showing amino acid sequence relationship between full-length VSP/TPTE/TPTE2 phosphatases, Hv1 channels, and HVRP1 proteins. See Methods for details. Ap: <i>Anas platyrhynchos</i>, Bt: <i>Bos taurus</i>, Ci: <i>Ciona intestinalis</i>, Dr: <i>Danio rerio</i>, Gg: <i>Gallus gallus</i>, Hs: <i>Homo sapiens</i>, Mb: <i>Myotis brandtii</i>, Mo: <i>Metaseiulus occidentalis</i>, Oa: <i>Ornithorhynchus anatinus</i>, Oo: <i>Orcinus orca</i>, Ps: <i>Pelodiscus sinensis</i>, Sp: <i>Strongylocentrotus purpuratus</i>, Tt: <i>Tursiops truncates</i>, Xt: <i>Xenopus tropicalis</i>. <b>C</b>) Hydrophobicity plot (generated with TopPred, Institute Pasteur, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105926#pone.0105926-vonHeijne1" target="_blank">[53]</a>), comparing human Hv1 and HVRP1 and showing relative positions of transmembrane helices S1–S4 (identified with SOSUI, Nagoya University, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105926#pone.0105926-Hirokawa1" target="_blank">[54]</a>). <b>D</b>) Sequence alignment of human Hv1 and HVRP1. Only regions containing segments S1 through S4 are shown.</p

Tissue distribution of HVRP1 transcript.

<p><b>A</b>) HVRP1 expression in human tissues assessed by Affymetrix microarray analysis. HVRP1 tissue distribution was compared to the distributions of human Hv1 (HVCN1) and TPTE. Red and green signals indicate up-regulated and down-regulated expression, respectively. Struct.: skeletal muscle, adipose tissue, skin. CVS: heart and blood vessels. Resp.: respiratory system. Endo.: endocrine organs. Urinary & Repro.: urinary & reproductive systems (male and female). Immun.: immune tissues. Leuk.: peripheral white blood cells. Embr.: embryonic tissues (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105926#pone.0105926.s003" target="_blank">Table S1</a>). <b>B</b>) Levels of HVRP1 transcript in total RNA extracts from the indicated human tissues measured by qRT-PCR. Levels are reported as relative expression units (RU) in relation to the housekeeping control gene beta-actin. Water was used as negative control. Error bars are S.E.M., n = 3. <b>C</b>) <i>In situ</i> hybridization on a 20-µm thick sagittal section of an adult mouse brain cerebellar region, using a 600-bp riboprobe targeting the HVRP1 mRNA. Positively stained regions are dark purple. Left panel: antisense probe. Right panel: control sense probe. Scale bars: 1 mm.</p

Cellular and subcellular localization of the HVRP1 protein.

<p><b>A</b>) Immunohistochemical analysis of HVRP1 distribution in cerebellar tissue. Cerebellar cortical section from a wild-type rat stained with polyclonal antibody raised against HVRP1 (red). GC: granule cell layer, PC: Purkinje cell layer, ML: molecular layer, scale bar: 50 µm. <b>B</b>) Higher magnification of granule neurons expressing HVRP1, from (A). Scale bar is 10 µm. <b>C</b>) Ultrastructural localization of HVRP1 in the rat cerebellar GC layer visualized with the pre-embedding immunogold method. Positive labeling is in black, in the glomeruli, at the dendritic claws of granule cells surrounding mossy fiber terminals. Scale bar is 0.1 µm. <b>D</b>) Basic circuit diagram of the cerebellar cortex showing the location of HVRP1 in the dendrites and cell bodies of GCs. GO: Golgi cell, GR: glomerulus, MF: mossy fiber, PC: Purkinje cell. <b>E</b>) Confocal images of cultured mouse cerebellar granule neurons fixed and immunostained at 8 DIV. HVRP1 signal is shown in red (antibody dilution 1∶1000), DAPI signal is in blue. <b>F</b>) Staining as in (E) but with anti-HVRP1 antibody at dilution 1∶500 pre-incubated with HVRP1 antigen peptide (see Methods). Scale bars are 10 µm. <b>G</b>) Confocal image of live HEK293A cells expressing recombinant EGFP-tagged human HVRP1 (green) and labeled with the plasma membrane marker FM-464 (red). Scale bar is 10 µm. <b>H</b>) Western blot of total protein extracts from HEK293A cells transfected with hHVRP1 (H) and non transfected (N). AP indicates pre-incubation of anti-HVRP1 antibody with antigen peptide.</p

Consequences of increasing VSD similarity between Hv1 and HVRP1.

<p><b>A</b>) Sequence alignments of transmembrane segments S1 through S4 of human HVRP1 and Hv1 channels from three different species. Residues of the same category (see text) are shown in gray background. Category mismatches have white backgrounds. Mutated residues in HVRP1* are shown in red. <b>B</b>) Quantification of test currents measured at +120 mV in oocytes expressing the indicated proteins (conditions as in Fig. 4B–C). Hv1 and HVRP1 are positive and negative controls, respectively. Box indicates median ± S.D., whisker shows range. Individual measurements are shown as gray circles, n = 5–12. <b>C</b>) Confocal image of live HEK293A cells expressing recombinant EGFP-tagged HVRP1* (green) and labeled with the plasma membrane marker FM-464 (red). Scale bar is 10 µm.</p