Role of the vitamin D receptor in hair follicle biology

The vitamin D receptor (VDR) is expressed in numerous cells and tissues, including the skin. The critical requirement for cutaneous expression of the VDR has been proven by investigations in mice and humans lacking functional receptors. These studies demonstrate that absence of the VDR leads to the development of alopecia. The hair follicle is formed by reciprocal interactions between an epidermal placode, which gives rise to the hair follicle keratinocytes and the underlying mesoderm which gives rise to the dermal papilla. Hair follicle morphogenesis ends the second week of life in mice. Studies in VDR null mice have failed to demonstrate a cutaneous abnormality during this period of hair follicle morphogenesis. However, VDR null mice are unable to initiate a new hair cycle after the period of morphogenesis is complete, therefore, do not grow new hair. Investigations in transgenic mice have demonstrated that restricted expression of the VDR to keratinocytes is capable of preventing alopecia in the VDR null mice, thus demonstrating that the epidermal component of the hair follicle requires VDR expression to maintain normal hair follicle homeostasis. Studies were then performed to determine which regions of the VDR were required for these actions. Investigations in mice lacking the first zinc finger of the VDR have demonstrated that they express a truncated receptor containing an intact ligand binding and AF2 domain. These mice are a phenocopy of mice lacking the VDR, thus demonstrate the critical requirement of the DNA binding domain for hair follicle homeostasis. Transgenic mice expressing VDRs with mutations in either the ligand-binding domain or the AF2 domain were generated. These investigations demonstrated that mutant VDRs incapable of ligand-dependent transactivation were able to prevent alopecia. Investigations are currently underway to define the mechanism by which the unliganded VDR maintains hair follicle homeostasis.     

淋巴囊肿病毒中国株TNFR类似物的原核表达与结构分析

肿瘤坏死因子受体(TNFR)是细胞因子受体家族中的一员,在大DNA病毒的免疫逃避中起着重要的作用。 淋巴囊肿病毒中国株(LCDV-C)是一种大DNA病毒,属于虹彩病毒科。参照已知虹彩病毒TNFR基因设计引物: P1,5′GGATCCAAAACTATGATTAAAATAAAGA 3′;P2:5′ATTACTCGAGAATGTTAAAAATTAAGCTT 3′。以LCDV-C基因组DNA为模板,PCR扩增得到一个834bp的DNA片段,并对该片段进行测序。构建原核表 达重组质粒后,在大肠杆菌DE3中诱导表达,其产物经SDS-PAGE电泳后,显示为45kDa的融合蛋白带。对测序 结果进行计算机辅助分析的结果显示,LCDV-C TNFR类似物是一个含278个氨基酸的多肽,具有典型的半胱氨 酸富集区功能结构域,与宿主牙鲆TNFRII氨基酸同源性为34％。     

Nitric oxide synthase sequences in the marine fish Stenotomus chrysops and the sea urchin Arbacia punctulata, and phylogenetic analysis of nitric oxide synthase calmodulin-binding domains

The phylogenetic distribution and structural diversity of the nitric oxide synthases (NOS) remain important and issues that are little understood. We present sequence information, as well as phylogenetic analysis, for three NOS cDNAs identified in two non-mammalian species: the vertebrate marine teleost fish Stenotomus chrysops (scup) and the invertebrate echinoderm Arbacia punctulata (sea urchin). Partial gene sequences containing the well-conserved calmodulin (CaM)-binding domain were amplified by RT-PCR. Identical 375-bp cDNAs were amplified from scup brain, heart, liver and spleen; this sequence shares 82% nucleic acid and 91% predicted amino acid identity with the corresponding region of human neuronal NOS. A 387-bp cDNA was amplified from sea urchin ovary and testes; this sequence shares 72% nucleic acid identity and 65% deduced amino acid identity with human neuronal NOS. A second cDNA of 381 bp was amplified from sea urchin ovary and it shares 66% nucleic acid and 57% deduced amino acid identity with the first sea urchin sequence. Together with earlier reports of neuronal and inducible NOS sequences in fish, these data indicate that multiple NOS isoforms exist in non-mammalian species. Phylogenetic analysis of these sequences confirms the conserved nature of NOS, particularly of the calmodulin-binding domains.     

Functional evolution of the vitamin D and pregnane X receptors

Background  

The vitamin D receptor (VDR) and pregnane X receptor (PXR) are nuclear hormone receptors of the NR1I subfamily that show contrasting patterns of cross-species variation. VDR and PXR are thought to have arisen from duplication of an ancestral gene, evident now as a single gene in the genome of the chordate invertebrate Ciona intestinalis (sea squirt). VDR genes have been detected in a wide range of vertebrates including jawless fish. To date, PXR genes have not been found in cartilaginous fish. In this study, the ligand selectivities of VDRs were compared in detail across a range of vertebrate species and compared with those of the Ciona VDR/PXR. In addition, several assays were used to search for evidence of PXR-mediated hepatic effects in three model non-mammalian species: sea lamprey (Petromyzon marinus), zebrafish (Danio rerio), and African clawed frog (Xenopus laevis).     

Immunocytology with microwave-fixed fibroblasts shows 1 alpha,25- dihydroxyvitamin D3-dependent rapid and estrogen-dependent slow reorganization of vitamin D receptors

Prior studies have given no evidence for regulation of vitamin D receptor (VDR) compartmentalization or subcellular organization. Microwave fixation (9-15 s) and an indirect immunodetection system of avidin-biotin enhancement and phycoerythrin fluorophore resulted in sufficient spatial and temporal resolution to allow analysis of these processes. We studied cultured fibroblasts from normals or from patients with four different types of hereditary defect compromising VDR function (mutant cells). Compartmentalization of VDRs in the absence of 1,25-dihydroxyvitamin D3 (calcitriol) was regulated by serum or estrogen. VDRs were mainly cytoplasmic in cells cultured without serum and phenol red, but VDRs were mainly intranuclear after addition of serum or an estrogen to cells for at least 18 h (slow regulation). Calcitriol initiated a rapid and multistep process (rapid regulation) of reorganization in a portion of VDRs: clumping within 15-45 s, alignment of clumps along fibrils within 30-45 s, perinuclear accumulation of clumps within 45-90 s, and intranuclear accumulation of clumps within 1-3 min. We found similar rapid effects of calcitriol on VDRs in various other types of cultured cells. These sequential VDR pattern changes showed calcitriol dose dependency and calcitriol analogue specificity characteristic for the VDR. In mutant fibroblasts VDR pattern changes after calcitriol were absent or severely disturbed at selected steps. Treatment of normal cells with wheat germ agglutinin, which blocks protein transport through nuclear pores, also blocked calcitriol-dependent translocation of VDRs. We conclude that immunocytology after microwave fixation provides evidence for regulation of VDR organization and localization.     

The hepcidin-binding site on ferroportin is evolutionarily conserved

Mammalian iron homeostasis is regulated by the interaction of the liver-produced peptide hepcidin and its receptor, the iron transporter ferroportin. Hepcidin binds to ferroportin resulting in degradation of ferroportin and decreased cellular iron export. We identify the hepcidin-binding domain (HBD) on ferroportin and show that a synthetic 19 amino acid peptide corresponding to the HBD recapitulates the characteristics and specificity of hepcidin binding to cell-surface ferroportin. The binding of mammalian hepcidin to ferroportin or the HBD shows an unusual temperature dependency with an increased rate of dissociation at temperatures below 15°C. The increased rate of dissociation is due to temperature- dependent changes in hepcidin structure. In contrast, hepcidin from poikilothermic vertebrates, such as fish or frogs, binds the HBD in a temperature-independent fashion. The affinity of hepcidin for the HBD permits a rapid, sensitive assay of hepcidin from all species and yields insights into the evolution of hepcidin.     

Identification of amino acid sequence in the hinge region of human vitamin D receptor that transfers a cytosolic protein to the nucleus

The localization of human vitamin D receptor (VDR) in the absence of its ligand 1,25-dihydroxyvitamin D(3) was investigated using chimera proteins fused to green fluorescent protein (GFP) at either the N or C terminus, and the nuclear localization signal (NLS) was identified. Plasmids carrying the fusion proteins were transiently or stably introduced into COS7 cells, and the subcellular distribution of the fusion proteins was examined. GFP-tagged wild-type VDRs were located predominantly in nuclei but with a significant cytoplasmic presence, while GFP alone was equally distributed throughout the cells. 10(-8) M 1,25-dihydroxyvitamin D(3) promoted the nuclear import of VDR in a few hours. To identify the NLS, we constructed several mutated VDRs fused to GFP. Mutant VDRs that did not bind to DNA were also localized predominantly in nuclei, while the deletion of the hinge region resulted in the loss of preference for nucleus. A short segment of 20 amino acids in the hinge region enabled cytoplasmic GFP-tagged alkaline phosphatase to translocate to nuclei. These results indicate that 1) VDR is located predominantly in nuclei with a significant presence in cytoplasm without the ligand and 2) an NLS consisting of 20 amino acids in the hinge region facilitates the transfer of VDR to the nucleus.     

A homologue of human placental protein,PP11, and mouse T cell-specific protein,Tcl-30, in exocrine pancreas of a teleost (Paralichthys olivaceus)

We cloned a 1401-bp cDNA encoding a novel pancreatic protein (PPSB) with two cysteine-rich somatomedin B-like domains from Japanese flounder (Paralichthys olivaceus). PPSB is predicted to be composed of 385 amino acids, including a signal sequence. The peptide sequence shares high homology with human placental protein 11 (PP11) and mouse T-cell specific protein (Tcl-30), which both contain a single somatomedin B-like domain. PPSB shares 47% and 44% identity with PP11 and Tcl-30, respectively. The unique point of PPSB is that an additional, somatomedin B-like domain is tandemly inserted. Unlike PP11 and Tcl-30, PPSB mRNA is specifically expressed by the exocrine pancreatic acinar cells, together with trypsinogen. Since PP11 has serine protease activity, it is predicted that its teleost homologue, PPSB, may function as a pancreatic digestive enzyme.     

Cloning of two members of the calcitonin-family receptors from stingray, Dasyatis akajei: possible physiological roles of the calcitonin family in osmoregulation

In cartilaginous fish, two cDNAs encoding calcitonin-family receptors were isolated for the first time from the stingray brain. The open reading frame of one receptor cDNA coded a 525-amino acid protein. The amino acid identity of this receptor to human calcitonin-receptor-like receptor (CRLR) is 64.5%, frog CRLR is 64.7%, and flounder CRLR is 61.2% and this was higher than to human calcitonin receptor (CTR) (46.1%), frog CTR (54.7%), and flounder CTR (48.9%). We strongly suggested that this receptor is a ray CRLR based on phylogenetic analysis. In case of the second receptor, amino acid identity among CRLRs (human 50.5%, frog 50.7%, flounder 48.0%) and CTRs (human 43.2%, frog 49.1%, flounder 41.8%) was similar. From phylogenetic analysis of both CRLRs and CTRs, we believe that this receptor is ray CTR. The expression of ray CRLR mRNA was predominantly detected in the nervous system (brain) and vascular system (atrium, ventricle, and gill), which reflects the similar localization of CGRP in the nervous and vascular systems as mammals. It was observed that the second receptor was expressed in several tissues, namely cartilage, brain, pituitary gland, gill, atrium, ventricle, pancreas, spleen, liver, gall bladder, intestine, rectal gland, kidney, testis and ovary. This localization pattern was very similar to flounder CTR. Both receptor mRNAs were strongly expressed in the gill. This suggests that the calcitonin-family members are involved in the osmoregulation of stingray as this fish is known to be euryhaline. When a stingray was transferred to diluted seawater (20% seawater), the expression of both receptors significantly decreased in the gill. Similar results were obtained in the kidney of the stingray. Thus, our cloning and isolation of both receptors in the stingray will be helpful for elucidation of their physiological role(s) such as osmoregulation including calcium metabolism of cartilaginous fish.     

Cloning of a calcitonin gene-related peptide from genomic DNA and its mRNA expression in flounder, Paralichthys olivaceus

A part of genomic DNA including the calcitonin gene-related peptide (CGRP) gene was cloned from flounder by the genome-walking method. The intron/exon boundary was predicted to occur exactly at the same position as in salmon. The 37-amino acid molecule coded by the region from the intron/exon boundary to the stop codon was preceded by a typical Lys-Arg cleavage signal and included a cleavage/amidation site common to the CGRP of other vertebrates. The predicted amino acid sequence of flounder CGRP had 78%, 78%, 78%, 81%, and 73-78% identity to that of salmon, cod, frog, chicken, and mammalian CGRPs, respectively. Among vertebrates, CGRP is more conserved than calcitonin (CT) because the identity of flounder CT to mammalian CTs is 31-50%. Expression analysis indicated that this hormone is synthesized in the brain, heart, intestine, testis, and ovary. Since we have previously shown that the CGRP receptor is expressed in these tissues, it is suggested that CGRP secreted from each tissue functions in a paracrine or autocrine manner.     

Molecular cloning of stanniocalcin 1 and its extracorpuscular regulation by salinity and Ca2+ in the Japanese flounder

Stanniocalcin 1 (Stc1) was originally identified as an anti-hypercalcemic hormone produced by the corpuscles of Stannius (CS) associated with the kidney in teleosts. While the stc1 gene is expressed in various tissues in fishes, its role and regulation in extra-CS tissues are unexplored. In the present study, we characterized a cDNA of stc1 in a euryhaline fish, the Japanese flounder (Paralichyhus olivaceus), and examined its expression in peripheral tissues in response to different salinities and Ca2+ ion concentrations. The Japanese flounder stc1 cDNA (1331 bp) encodes a preprohormone of 251 amino acids (aa), with a signal peptide of 17 aa and a pro-sequence peptide of 15 aa followed by the mature protein of 219 aa. The deduced aa sequence of Japanese flounder stc1 showed highest sequence identity (94.0%) with the European flounder Stc1 among fish and mammalian species, but lower identity to zebrafish, pufferfish, and human STC2 (23.1-25.4%). Lowered environmental salinity resulted in a decrease in stc1 mRNA expression in vivo in the gills, kidney, intestine, and CS glands of the Japanese flounder. Furthermore, we found that extracellular Ca2+ increased steady-state stc1 mRNA levels in gill and kidney cells as well as in the CS cells. Our findings suggest that Stc1 synthesis in the ionregulatory tissues is responsive to environmental salinity and Ca2+ level.     

Molecular cloning of Quek 1 and 2, two quail vascular endothelial growth factor (VEGF) receptor-like molecules

We have previously reported the cloning of two partial cDNAs corresponding to two quail (Coturnix coturnix japonica) receptor tyrosine kinases (RTKs), named Quek 1 and Quek 2, and their expression in endothelial cells of the early avian embryo. We here report the cloning of the full-size cDNAs for both molecules. Sequence comparison shows that Quek 1 and 2 share an overall amino acid (aa) identity of 49%. They both comprise seven extracellular immunoglobulin-like (Ig-like) domains, a single transmembrane domain, and an intracellular kinase domain split into two by a 70 aa insertion. These structural characteristics are shared by the members of the recently discovered VEGF receptor (VEGFR) family. We have compared the sequences of Quek 1 and 2 to the other VEGFRs. At the aa level, Quek 1 is most closely related to KDR/flk-1 (VEGFR 2) (aa identity of 69% and 71%, respectively). Quek 2 shows a similar degree of aa identity to flt-4 (VEGFR 3). Quek 1 and 2 display a lower homology to flt-1 (VEGFR 1) (about 45% aa identity). These data suggest that Quek 1 and 2 are the avian homologues of VEGFRs 2 and 3, respectively.     

Molecular Characterization and Expression Analysis of ATP-Gated P2X7 Receptor Involved in Japanese Flounder (Paralichthys olivaceus) Innate Immune Response

ATP-gated P2X7 receptor (P2RX7) channel is a key component for purinergic signaling and plays important roles in the innate immune response in mammals. However, the expression, molecular properties and immune significances of P2RX7 in lower vertebrates are still very limited. Here we identified and characterized a novel bony fish P2RX7 homologue cDNA, termed poP2RX7, in Japanese flounder (Paralichthys olivaceus). PoP2RX7 protein shares about 60–88% sequence similarity and 45–78% sequence identity with known vertebrate P2RX7 proteins. Phylogenetic analysis placed poP2RX7 and other P2RX7 proteins within their own cluster apart from other P2RX members. While the functional poP2RX7 channel shares structural features in common with known P2RX7 homologs, electrophysiological studies revealed that BzATP, the more potent agonist for known mammalian and fish P2RX7s, shows similar potency to ATP in poP2RX7 activation. poP2RX7 mRNA constitutively expressed in all examined tissues from unstimulated healthy Japanese flounder with dominant expression in hepatopancreas and the lowest expression in head kidney, trunk kidney, spleen and gill. poP2RX7 mRNA expression, however, was significantly induced in Japanese flounder head kidney primary cells by Poly(I:C) and bacterial endotoxin LPS stimulations. In vivo experiments further revealed that poP2RX7 gene expression was substantially up-regulated by immune challenge with infectious bacteria Edwardsiella tarda and Vibrio anguillarum. Moreover, activation of poP2RX7 results in an increased gene expression of multifunctional cytokines IL-1β and IL-6 in the head kidney primary cells. Collectively, we identified and characterized a novel fish P2RX7 homolog which is engaged in Japanese flounder innate immune response probably through modulation of pro-inflammatory cytokines expression.     

Increased apoptosis of periprostatic adipose tissue in VDR null mice

The vitamin D receptor (VDR) is a member of the steroid/retinoid receptor superfamily of nuclear receptors that controls mineral ion homeostatis and has potential tumor-suppressive functions for various cancer types, specifically prostate cancer. A VDR ablated transgenic animal model (VDDRII, vitamin D-dependent rickets type II) has been developed and the animals typically have various diseases including, hypocalcemia, hyperparathyroidism, rickets, osteomalacia, and alopecia. This transgenic mouse system provides us with a model to decipher the influences of the VDR on prostatic growth and function. VDRs are abundant both in prostatic epithelial and stromal cells, and vitamin D signaling can be studied in this model. Although, there were no gross differences between the prostate tissue of the experimental and control groups, VDR null mice showed fat necrosis and individual cell apoptosis in the periprostatic adipose tissue. This indicates a possible role of VDR in the signaling pathways resulting the prostate. This may be particularly attractive for VDR targets for the inhibition of cancer progression using VD(3) and its analogs as potential chemo-preventive agents.     

Cloning and characterisation of a cDNA encoding Japanese flounder Paralichthys olivaceus IgD

A cDNA containing the gene for Japanese flounder IgD consisted of 3240 bp encoding 998 amino acid residues. The amino acid sequence of the constant region of Japanese flounder IgD shares 38-80% identity with the sequences of previously reported teleost IgDs. The structure of the constant region of Japanese flounder IgD, which contains the micro1, delta1, delta2, delta3, delta4, delta5, delta6, delta7, and TM regions, is similar to the structures of the constant regions of the IgDs of channel catfish and Atlantic salmon. Southern blot hybridisation showed that the Japanese flounder IgD gene exists as a single locus. The Japanese flounder IgD gene was mainly detected in peripheral blood leucocytes (PBLs) and small amounts were detected in the spleen, head and trunk kidney, although IgM mRNA was detected in similar amounts in PBLs, the head kidney, and spleen. The copy number of IgM mRNA in Japanese flounder PBL was 56-fold higher than that of IgD.     

Expression and initial characterization of a recombinant human thrombospondin heparin binding domain

Thrombospondin (TSP) is a trimeric glycoprotein of Mr 420,000. It was originally described as a major component of human platelet alpha granules and is essential for the secondary phase of platelet aggregation. TSP is also synthesized and secreted by a variety of nucleated cells where it functions in processes involving growth and adhesion of cells to the extracellular matrix. Many of these processes are heparin-inhibitable and are mediated by a proteolytic fragment of TSP called the heparin binding domain (HBD). In order to facilitate the analysis of the structure and function(s) of this domain, we have expressed this molecule in Escherichia coli. A fragment of a TSP cDNA that encodes the heparin binding domain was inserted into the prokaryotic expression vector pJBL6. In bacterial cells grown at 42 degrees C, this vector directs the synthesis of a 24,000-Da polypeptide. Milligram quantities of this protein were purified to homogeneity from E. coli lysates. The structure of the recombinant HBD was confirmed by protein sequencing. The protein was further characterized by analysis of its conformation and function. The recombinant HBD binds [3H]heparin with a Kd of 71 nM, almost identical to that of TSP-derived HBD (80 nM). Additionally, the recombinant HBD is able to compete for TSP binding to 11B carcinoma cells. These studies indicate that the recombinant HBD is synthesized and purified in a native configuration and is functionally equivalent to thrombospondin-derived HBD. They further indicate that glycosylation of the thrombospondin HBD is not necessary for its interaction with heparin and that sequences essential to this interaction reside within the first 229 amino acids of secreted thrombospondin.