MicroRNA-488 regulates zinc transporter SLC39A8/ZIP8 during pathogenesis of osteoarthritis

Background

Even though osteoarthritis (OA) is the most common musculoskeletal dysfunction, there are no effective pharmacological treatments to treat OA due to lack of understanding in OA pathology. To better understand the mechanism in OA pathogenesis and investigate its effective target, we analyzed miRNA profiles during OA pathogenesis and verify the role and its functional targets of miR-488.

Results

Human articular chondrocytes were obtained from cartilage of OA patients undergoing knee replacement surgery and biopsy samples of normal cartilage and the expression profile of miRNA was analyzed. From expression profile, most potent miR was selected and its target and functional role in OA pathogenesis were investigated using target validation system and OA animal model system. Among miRNAs tested, miR-488 was significantly decreased in OA chondrocytes Furthermore, we found that exposure of IL-1β was also suppressed whereas exposure of TGF-β3 induced the induction of miR-488 in human articular chondrocytes isolated from biopsy samples of normal cartilages. Target validation study showed that miR-488 targets ZIP8 and suppression of ZIP8 in OA animal model showed the reduced cartilage degradation. Target validation study showed that miR-488 targets ZIP8 and suppression of ZIP8 in OA animal model showed the reduced cartilage degradation.

Conclusions

miR-488 acts as a positive role for chondrocyte differentiation/cartilage development by inhibiting MMP-13 activity through targeting ZIP-8.     

The zinc transporter SLC39A14/ZIP14 controls G-protein coupled receptor-mediated signaling required for systemic growth

Aberrant zinc (Zn) homeostasis is associated with abnormal control of mammalian growth, although the molecular mechanisms of Zn's roles in regulating systemic growth remain to be clarified. Here we report that the cell membrane-localized Zn transporter SLC39A14 controls G-protein coupled receptor (GPCR)-mediated signaling. Mice lacking Slc39a14 (Slc39a14-KO mice) exhibit growth retardation and impaired gluconeogenesis, which are attributable to disrupted GPCR signaling in the growth plate, pituitary gland, and liver. The decreased signaling is a consequence of the reduced basal level of cyclic adenosine monophosphate (cAMP) caused by increased phosphodiesterase (PDE) activity in Slc39a14-KO cells. We conclude that SLC39A14 facilitates GPCR-mediated cAMP-CREB signaling by suppressing the basal PDE activity, and that this is one mechanism for Zn's involvement in systemic growth processes. Our data highlight SLC39A14 as an important novel player in GPCR-mediated signaling. In addition, the Slc39a14-KO mice may be useful for studying the GPCR-associated regulation of mammalian systemic growth.     

Slc39a1 to 3 (subfamily II) Zip genes in mice have unique cell-specific functions during adaptation to zinc deficiency

Subfamily II of the solute carrier (Slc)39a family contains three highly conserved members (ZIPs 1-3) that share a 12-amino acid signature sequence present in the putative fourth transmembrane domain and function as zinc transporters in transfected cells. The physiological significance of this genetic redundancy is unknown. Here we report that the complete elimination of all three of these Zip genes, by targeted mutagenesis and crossbreeding mice, causes no overt phenotypic effect. When mice were fed a zinc-adequate diet, several indicators of zinc status were indistinguishable between wild-type and triple-knockout mice, including embryonic morphogenesis and growth, alkaline phosphatase activity in the embryo, ZIP4 protein in the visceral yolk sac, and initial rates (30 min) of accumulation/retention of (67)Zn in liver and pancreas. When mice were fed a zinc-deficient diet, embryonic membrane-bound alkaline phosphatase activity was reduced to a much greater extent, and 80% of the embryos of the triple-knockout mice developed abnormally compared with 12% of the embryos of wild-type mice. During zinc deficiency, the accumulation/retention (3 h) of (67)Zn in the liver and pancreas of weanlings was significantly impaired in the triple-knockout mice compared with wild-type mice. Thus none of these three mammalian Zip genes apparently plays a critical role in zinc homeostasis when zinc is replete, but they play important, noncompensatory roles when this metal is deficient.     

Characterization of the organic cation transporter SLC22A16: a doxorubicin importer

Specific efflux transporters, such as P-glycoprotein, have been shown to confer drug resistance by decreasing the intracellular accumulation of anticancer drugs. Understanding influx transporters, as well as efflux transporters, is essential to overcome this resistance. We report the expression profile and pharmacological characterization of an organic cation transporter, SLC22A16. The results of our experiments indicate that SLC22A16 is a mediator of doxorubicin uptake in cancer cells. Quantitative real-time RT-PCR analyses show that SLC22A16 is expressed in primary samples taken from patients with acute leukemia. Xenopus oocytes injected with SLC22A16 cRNA import doxorubicin, a widely used anticancer drug for hematological malignancies, in a saturable and dose-dependent manner. The apparent Km value for doxorubicin import was 5.2+/-0.4 microM. In cytotoxic assays, stable transfectants of leukemic Jurkat cells overexpressing SLC22A16 cells became significantly more sensitive to doxorubicin (2 microM) treatment. Characterization of SLC22A16 will help in designing novel therapies targeting hematological malignancies.     

Spondylocheiro dysplastic form of the Ehlers-Danlos syndrome--an autosomal-recessive entity caused by mutations in the zinc transporter gene SLC39A13

We present clinical, radiological, biochemical, and genetic findings on six patients from two consanguineous families that show EDS-like features and radiological findings of a mild skeletal dysplasia. The EDS-like findings comprise hyperelastic, thin, and bruisable skin, hypermobility of the small joints with a tendency to contractures, protuberant eyes with bluish sclerae, hands with finely wrinkled palms, atrophy of the thenar muscles, and tapering fingers. The skeletal dysplasia comprises platyspondyly with moderate short stature, osteopenia, and widened metaphyses. Patients have an increased ratio of total urinary pyridinolines, lysyl pyridinoline/hydroxylysyl pyridinoline (LP/HP), of approximately 1 as opposed to approximately 6 in EDS VI or approximately 0.2 in controls. Lysyl and prolyl residues of collagens were underhydroxylated despite normal lysyl hydroxylase and prolyl 4-hydroxylase activities; underhydroxylation was a generalized process as shown by mass spectrometry of the alpha1(I)- and alpha2(I)-chain-derived peptides of collagen type I and involved at least collagen types I and II. A genome-wide SNP scan and sequence analyses identified in all patients a homozygous c.483_491 del9 SLC39A13 mutation that encodes for a membrane-bound zinc transporter SLC39A13. We hypothesize that an increased Zn(2+) content inside the endoplasmic reticulum competes with Fe(2+), a cofactor that is necessary for hydroxylation of lysyl and prolyl residues, and thus explains the biochemical findings. These data suggest an entity that we have designated "spondylocheiro dysplastic form of EDS (SCD-EDS)" to indicate a generalized skeletal dysplasia involving mainly the spine (spondylo) and striking clinical abnormalities of the hands (cheiro) in addition to the EDS-like features.     

A novel member of a zinc transporter family is defective in acrodermatitis enteropathica

The rare inherited condition acrodermatitis enteropathica (AE) results from a defect in the absorption of dietary zinc. Recently, we used homozygosity mapping in consanguineous Middle Eastern kindreds to localize the AE gene to an approximately 3.5-cM region on 8q24. In this article, we identify a gene, SLC39A4, located in the candidate region and, in patients with AE, document mutations that likely lead to the disease. The gene encodes a histidine-rich protein, which we refer to as "hZIP4," which is a member of a large family of transmembrane proteins, some of which are known to serve as zinc-uptake proteins. We show that Slc39A4 is abundantly expressed in mouse enterocytes and that the protein resides in the apical membrane of these cells. These findings suggest that the hZIP4 transporter is responsible for intestinal absorption of zinc.     

Molecular cloning of SLC26A7, a novel member of the SLC26 sulfate/anion transporter family, from high endothelial venules and kidney

A unique characteristic of endothelial cells from high endothelial venules (HEVEC) in lymphoid organs and chronically inflamed tissues is their capacity to incorporate large amounts of sulfate into sialomucin-type counter-receptors for the lymphocyte homing receptor L-selectin. We have previously shown that HEVEC express two functional classes of sulfate transporters: sodium/sulfate cotransporters and sulfate/anion exchangers. Here, we report the molecular cloning from human HEVEC of a 2.9-kb cDNA encoding SLC26A7, a novel member of the SLC26 (solute carrier 26) sulfate/anion exchanger family. SLC26A7 exhibits 30% identity with three known sulfate transporters from the SLC26 family: SLC26A2 (also known as DTDST), SLC26A1 (also known as SAT1), and SLC26A3 (also known as DRA). Northern blot analysis revealed specific expression of SLC26A7 mRNA in kidney. Alternative splicing and polyadenylation of SLC26A7 pre-mRNA in kidney suggest the existence of two protein isoforms, SLC26A7.1 and SLC26A7.2, differing in their carboxy termini.     

Identification and characterization of human Golgi nucleotide sugar transporter SLC35D2, a novel member of the SLC35 nucleotide sugar transporter family

We report the molecular cloning of SLC35D2, a novel member of the SLC35 nucleotide sugar transporter family. The gene SLC35D2 maps to chromosome 9q22.33. SLC35D2 cDNA codes for a hydrophobic protein consisting of 337 amino acid residues with 10 putative transmembrane helices. Northern blot analysis revealed the SLC35D2 mRNA as a single major band corresponding to 2.0 kb in length. SLC35D2 was localized in the Golgi membrane and exhibited around 50% similarity with three nucleotide sugar transporters: human SLC35D1 (UDP-glucuronic acid/UDP-N-acetylgalactosamine transporter), fruitfly fringe connection (frc) transporter, and nematode SQV-7 transporter, the latter two being involved in developmental and organogenetic processes. Heterologous expression of SLC35D2 protein in yeast indicated that UDP-N-acetylglucosamine is a candidate for the substrate(s) of the transporter. The sequence similarity, subcellular localization, and transporting substrate suggest that SLC35D2 is a good candidate for the ortholog of frc transporter, which is involved in the Notch signaling system by providing the fringe N-acetylglucosaminyltransferase with the substrate. We also describe the identification and categorization of the human SLC35 gene family.     

Restraint stress up-regulates expression of zinc transporter Zip14 mRNA in mouse liver

The zinc concentration in the liver was significantly higher in mice at 12 h after the onset of restraint stress than that without stress. The IL-6 protein level in the serum was transiently elevated at 3 h after the onset of restraint stress, and the IL-6-responsive zinc transporter Zip14 mRNA in the liver was expressed markedly at 6 h. These results suggest that Zip14 plays a major role in the mechanism responsible for accumulation of zinc in the liver under restraint stress.     

Characterization of a member of the AN subfamily, IAN, from Ipomoea nil

ANGUSTIFOLIA (AN) is the first C-terminal binding protein (CtBP) gene from plants and controls leaf width and pattern of trichome branching in Arabidopsis thaliana (L.) Heynh. We characterized an ortholog of AN from Ipomoea nil (L.) Roth (Japanese morning glory) and designated it Ipomoea nil's AN (IAN). IAN is a single-copy gene in the genome and is expressed ubiquitously in various organs of I. nil. IAN contains not only a D2-HDH motif, which is highly conserved within the CtBP family, but also LXCXE, NLS and PEST motifs, which are specific to the AN subfamily. The expression of IAN cDNA driven by the cauliflower mosaic virus 35S promoter restored a defect in leaf expansion in the leaf width direction in the angustifolia-1 (an-1) mutant of Arabidopsis, suggesting that IAN retains a common function with AN. In contrast, the complementation by IAN of a defect in the trichome branching pattern on the leaf surface of the an-1 mutant was less effective than that observed for leaf shape. These results suggest that the mechanisms by which AN regulates leaf width and trichome branching are separable.     

The mammalian Zip5 protein is a zinc transporter that localizes to the basolateral surface of polarized cells

The mouse and human Zip5 proteins are members of the ZIP family of metal ion transporters. In this study, we present evidence that mouse Zip5 is a zinc uptake transporter that is specific for Zn(II) over other potential metal ion substrates. We also show that, unlike many other mammalian ZIP proteins, the endocytic removal of mZip5 from the plasma membrane is not triggered by zinc treatment. Thus, the activity of mZip5 does not appear to be down-regulated by zinc repletion. Zip5 expression is restricted to many tissues important for zinc homeostasis, including the intestine, pancreas, liver, and kidney. Zip5 is similar in sequence to the Zip4 protein, which is involved in the uptake of dietary zinc. Co-expression of Zip4 and Zip5 in the intestine led to the hypothesis that these proteins play overlapping roles in the uptake of dietary zinc across the apical membrane of intestinal enterocytes. Surprisingly, however, we found that mZip5 localizes specifically to the basolateral membrane of polarized Madin-Darby canine kidney cells. These observations suggest that Zip5 plays a novel role in polarized cells by carrying out serosal-to-mucosal zinc transport. Furthermore, given its expression in tissues important to zinc homeostasis, we propose that Zip5 plays a central role in controlling organismal zinc status.     

Characterization of OSR1, a member of the mammalian Ste20p/germinal center kinase subfamily

In examining the protein kinase components of mitogen-activated protein (MAP) kinase (MAPK) cascades that regulate the c-Jun N-terminal kinase (JNK) in Drosophila S2 cells, we previously found that distinct upstream kinases were involved in responses to sorbitol and lipopolysaccharide. Here we have extended that analysis to the possible MAPK kinase kinase kinases (MAP4Ks) in the JNK pathway. Fray, a putative Drosophila MAP4K, provided a major contribution to JNK activation by sorbitol. To explore the possible link to JNK in mammalian cells, we isolated and characterized OSR1 (oxidative stress-responsive 1), one of two human Fray homologs. OSR1 is a 58-kDa protein of 527 amino acids that is widely expressed in mammalian tissues and cell lines. Of potential regulators surveyed, endogenous OSR1 is activated only by osmotic stresses, notably sorbitol and to a lesser extent NaCl. However, OSR1 did not increase the activity of coexpressed JNK, nor did it activate three other MAPKs, p38, ERK2, and ERK5. A two-hybrid screen implicated another Ste20p family member, the p21-activated protein kinase PAK1, as an OSR1 target. OSR1 phosphorylated threonine 84 in the N-terminal regulatory domain of PAK1. Replacement of threonine 84 with glutamate reduced the activation of PAK1 by an active form of the small G protein Cdc42, suggesting that phosphorylation by OSR1 modulates the G protein sensitivity of PAK isoforms.     

Structure, function, and regulation of a subfamily of mouse zinc transporter genes

Zinc is an essential metal for all eukaryotes, and cells have evolved a complex system of proteins to maintain the precise balance of zinc uptake, intracellular storage, and efflux. In mammals, zinc uptake appears to be mediated by members of the Zrt/Irt-like protein (ZIP) superfamily of metal ion transporters. Herein, we have studied a subfamily of zip genes (zip1, zip2, and zip3) that is conserved in mice and humans. These eight-transmembrane domain proteins contain a conserved 12-amino acid signature sequence within the fourth transmembrane domain. All three of these mouse ZIP proteins function to specifically increase the uptake of zinc in transfected cultured cells, similar to the previously demonstrated functions of human ZIP1 and ZIP2 (Gaither, L. A., and Eide, D. J. (2000) J. Biol. Chem. 275, 5560-5564; Gaither, L. A., and Eide, D. J. (2001) J. Biol. Chem. 276, 22258-22264). No ZIP3 orthologs have been previously studied. Furthermore, this first systematic comparative study of the in vivo expression and dietary zinc regulation of this subfamily of zip genes revealed that 1) zip1 mRNA is abundant in many mouse tissues, whereas zip2 and zip3 mRNAs are very rare or moderately rare, respectively, and tissue-restricted in their accumulation; and 2) unlike mouse metallothionein I and zip4 mRNAs (Dufner-Beattie, J., Wang, F., Kuo, Y.-M., Gitschier, J., Eide, D., and Andrews, G. K. (2003) J. Biol. Chem. 278, 33474-33481), the abundance of zip1, zip2, and zip3 mRNAs is not regulated by dietary zinc in the intestine and visceral endoderm, tissues involved in nutrient absorption. These studies suggest that all three of these ZIP proteins may play cell-specific roles in zinc homeostasis rather than primary roles in the acquisition of dietary zinc.     

Membrane topology of the cyanobacterial bicarbonate transporter,BicA, a member of the SulP (SLC26A) family

Abstract

We have completed the first comprehensive transmembrane topology determination for a member of the ubiquitous and important SulP/SLC26 family of coupled anion transporters found in eukaryotes and prokaryotes. The prokaryotic member that we have mapped, namely BicA from Synechococcus PCC7002, is an important Na⁺-dependent bicarbonate transporter that is likely to play a major role in global primary productivity via the CO₂ concentrating mechanism in cyanobacteria. We experimentally determined the topology based on phoA-lacZ topology mapping combined with reference to a range of predictive models based on hydropathy analysis and positive charge distribution. The 12-TMH structure for BicA is characterized by tight turns between several pairs of TMH and it features a prominent cytoplasmically-located STAS domain that is characteristic of the SulP family. A key difference from previous predicted models is that we identify a cytoplasmic loop between helices 8 and 9 where previous models suggested a TMH. This region includes a highly conserved motif that defines the SulP family. The identification of this region as cytoplasmic, rather than transmembrane, has implications for the function and perhaps regulation of SulP family members. This finding is used to reinterpret mutagenesis data relating to highly conserved residues in this region from both plant and human SulP transporters.