The Zinc Transporter SLC39A13/ZIP13 Is Required for Connective Tissue Development; Its Involvement in BMP/TGF-β Signaling Pathways

Background

Zinc (Zn) is an essential trace element and it is abundant in connective tissues, however biological roles of Zn and its transporters in those tissues and cells remain unknown.

Methodology/Principal Findings

Here we report that mice deficient in Zn transporter Slc39a13/Zip13 show changes in bone, teeth and connective tissue reminiscent of the clinical spectrum of human Ehlers-Danlos syndrome (EDS). The Slc39a13 knockout (Slc39a13-KO) mice show defects in the maturation of osteoblasts, chondrocytes, odontoblasts, and fibroblasts. In the corresponding tissues and cells, impairment in bone morphogenic protein (BMP) and TGF-β signaling were observed. Homozygosity for a SLC39A13 loss of function mutation was detected in sibs affected by a unique variant of EDS that recapitulates the phenotype observed in Slc39a13-KO mice.

Conclusions/Significance

Hence, our results reveal a crucial role of SLC39A13/ZIP13 in connective tissue development at least in part due to its involvement in the BMP/TGF-β signaling pathways. The Slc39a13-KO mouse represents a novel animal model linking zinc metabolism, BMP/TGF-β signaling and connective tissue dysfunction.     

Biliary excretion of excess iron in mice requires hepatocyte iron import by Slc39a14

Iron is essential for erythropoiesis and other biological processes, but is toxic in excess. Dietary absorption of iron is a highly regulated process and is a major determinant of body iron levels. Iron excretion, however, is considered a passive, unregulated process, and the underlying pathways are unknown. Here we investigated the role of metal transporters SLC39A14 and SLC30A10 in biliary iron excretion. While SLC39A14 imports manganese into the liver and other organs under physiological conditions, it imports iron under conditions of iron excess. SLC30A10 exports manganese from hepatocytes into the bile. We hypothesized that biliary excretion of excess iron would be impaired by SLC39A14 and SLC30A10 deficiency. We therefore analyzed biliary iron excretion in Slc39a14-and Slc30a10-deficient mice raised on iron-sufficient and -rich diets. Bile was collected surgically from the mice, then analyzed with nonheme iron assays, mass spectrometry, ELISAs, and an electrophoretic assay for iron-loaded ferritin. Our results support a model in which biliary excretion of excess iron requires iron import into hepatocytes by SLC39A14, followed by iron export into the bile predominantly as ferritin, with iron export occurring independently of SLC30A10. To our knowledge, this is the first report of a molecular determinant of mammalian iron excretion and can serve as basis for future investigations into mechanisms of iron excretion and relevance to iron homeostasis.     

Molecular Mechanism of SLC5A8 Inactivation in Breast Cancer

SLC5A8 is a putative tumor suppressor that is inactivated in more than 10 different types of cancer, but neither the oncogenic signaling responsible for SLC5A8 inactivation nor the functional relevance of SLC5A8 loss to tumor growth has been elucidated. Here, we identify oncogenic HRAS (HRAS^G12V) as a potent mediator of SLC5A8 silencing in human nontransformed normal mammary epithelial cell lines and in mouse mammary tumors through DNMT1. Further, we demonstrate that loss of Slc5a8 increases cancer-initiating stem cell formation and promotes mammary tumorigenesis and lung metastasis in an HRAS-driven murine model of mammary tumors. Mammary-gland-specific overexpression of Slc5a8 (mouse mammary tumor virus-Slc5a8 transgenic mice), as well as induction of endogenous Slc5a8 in mice with inhibitors of DNA methylation, protects against HRAS-driven mammary tumors. Collectively, our results provide the tumor-suppressive role of SLC5A8 and identify the oncogenic HRAS as a mediator of tumor-associated silencing of this tumor suppressor in mammary glands. These findings suggest that pharmacological approaches to reactivate SLC5A8 expression in tumor cells have potential as a novel therapeutic strategy for breast cancer treatment.     

Inactivation of the mitochondrial carrier SLC25A25 (ATP-Mg2+/Pi transporter) reduces physical endurance and metabolic efficiency in mice

An ATP-Mg(2+/)P(i) inner mitochondrial membrane solute transporter (SLC25A25), which is induced during adaptation to cold stress in the skeletal muscle of mice with defective UCP1/brown adipose tissue thermogenesis, has been evaluated for its role in metabolic efficiency. SLC25A25 is thought to control ATP homeostasis by functioning as a Ca(2+)-regulated shuttle of ATP-Mg(2+) and P(i) across the inner mitochondrial membrane. Mice with an inactivated Slc25a25 gene have reduced metabolic efficiency as evidenced by enhanced resistance to diet-induced obesity and impaired exercise performance on a treadmill. Mouse embryo fibroblasts from Slc25a25(-/-) mice have reduced Ca(2+) flux across the endoplasmic reticulum, basal mitochondrial respiration, and ATP content. Although Slc25a25(-/-) mice are metabolically inefficient, the source of the inefficiency is not from a primary function in thermogenesis, because Slc25a25(-/-) mice maintain body temperature upon acute exposure to the cold (4 °C). Rather, the role of SLC25A25 in metabolic efficiency is most likely linked to muscle function as evidenced from the physical endurance test of mutant mice on a treadmill. Consequently, in the absence of SLC25A25 the efficiency of ATP production required for skeletal muscle function is diminished with secondary effects on adiposity. However, in the absence of UCP1-based thermogenesis, induction of Slc25a25 in mice with an intact gene may contribute to an alternative thermogenic pathway for the maintenance of body temperature during cold stress.     

Murine chromosome 16 telomeric region, homologous with human chromosome 21q22, contains the osmoregulatory Na(+)/myo-inositol cotransporter (SLC5A3) gene

The murine Na(+)/myo-inositol cotransporter (SLC5A3) gene (Slc5a3) was cloned, the restriction sites mapped, and the coding region sequenced. Similar to other mammalian counterparts, including human, the gene has a single coding exon, with an open reading frame of 2.2 kb. The predicted protein of 718 amino acids is also highly conserved, compared to other mammalian homologs. Using fluorescence in situ hybridization, Slc5a3 was localized to the telomeric region of mouse chromosome 16, which is syntenic to human chromosome 21q22. An increased Slc5a3 copy number may explain the increased levels of myo-inositol in the brains of trisomy 16 mice and the increased rate of transport of myo-inositol into cultured neurons derived from trisomy 16 mice.     

Connexin-32 acts as a downregulator of growth of thyroid gland

Thyroid epithelial cells communicate through gap junctions formed from connexin (Cx)32, Cx43, and Cx26. We previously reported that reexpression of Cx32 in "gap junction-deficient" FRTL-5 and FRT thyroid cell lines induces a reduction of cell proliferation rate and an activation of expression of cell differentiation. The present study aimed at determining whether Cx32 could exert similar regulatory functions in vivo. We investigated morphological and functional characteristics of thyroid gland of Cx32-deficient mice (Cx32-KO), mice overexpressing Cx32 selectively in the thyroid (Cx32-T+), and Cx32-KO mice with a thyroid-selective Cx32 complementation obtained by crossing Cx32-KO and Cx32-T+ mice. In basal conditions, Cx32-KO mice did not present any detectable thyroid alteration, whereas Cx32-T+ mice showed a thyroid hypoplasia (20% reduction) associated with a slight increase in thyroid functional activity. Under thyrotropin stimulation (following sodium perchlorate treatment), Cx32-KO mice developed a larger goiter (< or =65% increase) than wild-type littermates, whereas Cx32-T+ mice exhibited the same thyroid hyperplasia as wild-type mice. Restoration of Cx32 expression in the thyroid of Cx32-KO mice abrogated the thyroid growth increase related to Cx32 deficiency. All together, these data show that Cx32 acts as a downregulator of growth of thyroid gland; an excess of Cx32 limits growth of thyroid cells in the basal state, whereas a lack of Cx32 confers an additional growth potential to TSH-stimulated thyroid cells.     

Slc35c2 Promotes Notch1 Fucosylation and Is Required for Optimal Notch Signaling in Mammalian Cells

Mammalian Notch receptors require modification by fucose on epidermal growth factor-like (EGF) repeats of their extracellular domain to respond optimally to signal induction by canonical Notch ligands. Inactivation of the Golgi GDP-fucose transporter Slc35c1 in mouse or human does not cause marked defects in Notch signaling during development, and shows milder fucosylation defects than those observed in mice unable to synthesize GDP-fucose, indicating the existence of another mechanism for GDP-fucose transport into the secretory pathway. We show here that fibroblasts from mice or humans lacking Slc35c1 exhibit robust Notch signaling in co-culture signaling assays. A potential candidate for a second GDP-fucose transporter is the related gene Slc35c2. Overexpression of Slc35c2 reduces expression of the fucosylated epitopes Lewis X and sialylated Lewis X in CHO cells, indicating competition with Slc35c1. The fucosylation of a Notch1 EGF repeat fragment that occurs in the endoplasmic reticulum was increased in CHO transfectants overexpressing Slc35c2. In CHO cells with low levels of Slc35c2, both Delta1- and Jagged1-induced Notch signaling were reduced, and the fucosylation of a Notch1 fragment was also decreased. Immunofluorescence microscopy of rat intestinal epithelial cells and HeLa cells, and analysis of rat liver membrane fractions showed that Slc35c2 is primarily colocalized with markers of the cis-Golgi network and endoplasmic reticulum-Golgi intermediate compartment (ERGIC). The combined results suggest that Slc35c2 is either a GDP-fucose transporter that competes with Slc35c1 for GDP-fucose, or a factor that otherwise enhances the fucosylation of Notch and is required for optimal Notch signaling in mammalian cells.     

Targeting G protein-coupled receptor signaling in asthma

The complex disease asthma, an obstructive lung disease in which excessive airway smooth muscle (ASM) contraction as well as increased ASM mass reduces airway lumen size and limits airflow, can be viewed as a consequence of aberrant airway G protein-coupled receptor (GPCR) function. The central role of GPCRs in determining airway resistance is underscored by the fact that almost every drug used in the treatment of asthma directly or indirectly targets either GPCR–ligand interaction, GPCR signaling, or processes that produce GPCR agonists. Although many airway cells contribute to the regulation of airway resistance and architecture, ASM properties and functions have the greatest impact on airway homeostasis. The theme of this review is that GPCR-mediated regulation of ASM tone and ASM growth is a major determinant of the acute and chronic features of asthma, and multiple strategies targeting GPCR signaling may be employed to prevent or manage these features.     

Regulator of G-Protein Signaling-5 Is a Marker of Hepatic Stellate Cells and Expression Mediates Response to Liver Injury

Liver fibrosis is mediated by hepatic stellate cells (HSCs), which respond to a variety of cytokine and growth factors to moderate the response to injury and create extracellular matrix at the site of injury. G-protein coupled receptor (GPCR)-mediated signaling, via endothelin-1 (ET-1) and angiotensin II (AngII), increases HSC contraction, migration and fibrogenesis. Regulator of G-protein signaling-5 (RGS5), an inhibitor of vasoactive GPCR agonists, functions to control GPCR-mediated contraction and hypertrophy in pericytes and smooth muscle cells (SMCs). Therefore we hypothesized that RGS5 controls GPCR signaling in activated HSCs in the context of liver injury. In this study, we localize RGS5 to the HSCs and demonstrate that Rgs5 expression is regulated during carbon tetrachloride (CCl₄)-induced acute and chronic liver injury in Rgs5^LacZ/LacZ reporter mice. Furthermore, CCl₄ treated RGS5-null mice develop increased hepatocyte damage and fibrosis in response to CCl₄ and have increased expression of markers of HSC activation. Knockdown of Rgs5 enhances ET-1-mediated signaling in HSCs in vitro. Taken together, we demonstrate that RGS5 is a critical regulator of GPCR signaling in HSCs and regulates HSC activation and fibrogenesis in liver injury.     

Expression profiles of SLC39A/ZIP7, ZIP8 and ZIP14 in response to exercise-induced skeletal muscle damage

BackgroundZinc transporters are thought to facilitate the mobilization of zinc (Zn) and the role of Zn as a signaling mediator during cellular events. Little is known about the response of Zn movement and zinc transporters during muscle proliferation and differentiation processes after damage.MethodsAfter rats were subjected to one 90-min session of downhill running to cause muscle damage, the gastrocnemius muscles were harvested to assess the expression of zinc transporters SLC39A/ZIP7, ZIP8, ZIP14 and myogenic regulatory factors at the 0 h, 6 h, 12 h, 1 d, 2 d, 3 d, 1 w and 2 w time points after exercise.ResultsSLC39A/ZIP7, ZIP8 and ZIP14 had translocated to different compartments of the cell following damage, and they exhibited differential expression profiles after eccentric exercise. The results regarding the myogenetic regulators showed that nf-κb was upregulated 2 d after exercise, and STAT3 and Akt1 mRNA levels were mostly expressed 2 w after exercise. The upregulation of phosphatidylinositol 3-kinase, catalytic subunit gamma (pik3cg), erk1 and erk2 mostly occurred at the early stage (6 h or 12 h) after exercise. In addition, we found that zip7, zip8 and zip14 expression was moderately correlated with certain markers of muscle regeneration.ConclusionThe zinc transporters SLC39A/ZIP7, ZIP8 and ZIP14 have differential expression profiles upon eccentric exercise, and they might regulate muscle proliferation or differentiation processes through different cellular pathways after exercise-induced muscle damage.     

Mice lacking myotubularin-related protein 14 show accelerated high-fat diet-induced lipid accumulation and inflammation

The phosphoinositide phosphatase, myotubularin-related protein 14 (MTMR14), has been reported to play an important role in the regulation of muscle performance, autophagy, and aging in mice. We previously showed that MTMR14-knockout (KO) mice gain weight earlier than their wild-type (WT) littermates even on a normal chow diet (NCD), suggesting that this gene might also be involved in regulating metabolism. In the present study, we evaluated the effect of MTMR14 deficiency on high-fat diet (HFD)-induced obesity, lipid accumulation, metabolic disorders, and inflammation in WT and MTMR14-KO mice fed with NCD or HFD. To this end, MTMR14-KO mice fed with HFD showed significantly increased body weight, blood glucose levels, serum triglyceride (TG) levels, and total cholesterol (TC) levels as compared to their age-matched WT control. Additionally, lipid accumulation also increased in the KO mice. Simultaneously, the expression of metabolism-associated genes (Glut4, adiponectin, and leptin) was different in the liver, muscle, and fatty tissue of MTMR14-KO mice fed with HFD. More importantly, the expression of several inflammation-associated genes (TNF-α, IL-6, IL-1β, and MCP-1) dramatically increased in the liver, muscle, and fatty tissue of MTMR14-KO mice relative to control. Taken together, these results suggest that MTMR14 deficiency accelerates HFD-induced metabolic dysfunction and inflammation. Furthermore, the results showed that exacerbated metabolic dysfunction and inflammation may be regulated via the PI3K/Akt and ERK signaling pathways.     

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.     

Establishment of a knock-in mouse model with the SLC26A4 c.919-2A>G mutation and characterization of its pathology

Recessive mutations in the SLC26A4 gene are a common cause of hereditary hearing impairment worldwide. Previous studies have demonstrated that different SLC26A4 mutations may have different pathogenetic mechanisms. In the present study, we established a knock-in mouse model (i.e., Slc26a4(tm1Dontuh/tm1Dontuh) mice) homozygous for the c.919-2A>G mutation, which is a common mutation in East Asians. Mice were then subjected to audiologic assessment, a battery of vestibular evaluations, and inner ear morphological studies. All Slc26a4(tm1Dontuh/tm1Dontuh) mice revealed profound hearing loss, whereas 46% mice demonstrated pronounced head tilting and circling behaviors. There was a significant difference in the vestibular performance between wild-type and Slc26a4(tm1Dontuh/tm1Dontuh) mice, especially those exhibiting circling behavior. Inner ear morphological examination of Slc26a4(tm1Dontuh/tm1Dontuh) mice revealed an enlarged endolymphatic duct, vestibular aqueduct and sac, atrophy of stria vascularis, deformity of otoconia in the vestibular organs, consistent degeneration of cochlear hair cells, and variable degeneration of vestibular hair cells. Audiologic and inner ear morphological features of Slc26a4(tm1Dontuh/tm1Dontuh) mice were reminiscent of those observed in humans. These features were also similar to those previously reported in both knock-out Slc26a4(-/-) mice and Slc26a4(loop/loop) mice with the Slc26a4 p.S408F mutation, albeit the severity of vestibular hair cell degeneration appeared different among the three mouse strains.     

Muscarinic supersensitivity and impaired receptor desensitization in G protein-coupled receptor kinase 5-deficient mice

G protein-coupled receptor kinase 5 (GRK5) is a member of a family of enzymes that phosphorylate activated G protein-coupled receptors (GPCR). To address the physiological importance of GRK5-mediated regulation of GPCRs, mice bearing targeted deletion of the GRK5 gene (GRK5-KO) were generated. GRK5-KO mice exhibited mild spontaneous hypothermia as well as pronounced behavioral supersensitivity upon challenge with the nonselective muscarinic agonist oxotremorine. Classical cholinergic responses such as hypothermia, hypoactivity, tremor, and salivation were enhanced in GRK5-KO animals. The antinociceptive effect of oxotremorine was also potentiated and prolonged. Muscarinic receptors in brains from GRK5-KO mice resisted oxotremorine-induced desensitization, as assessed by oxotremorine-stimulated [5S]GTPgammaS binding. These data demonstrate that elimination of GRK5 results in cholinergic supersensitivity and impaired muscarinic receptor desensitization and suggest that a deficit of GPCR desensitization may be an underlying cause of behavioral supersensitivity.     

Impaired nutrient signaling and body weight control in a Na+ neutral amino acid cotransporter (Slc6a19)-deficient mouse

Amino acid uptake in the intestine and kidney is mediated by a variety of amino acid transporters. To understand the role of epithelial neutral amino acid uptake in whole body homeostasis, we analyzed mice lacking the apical broad-spectrum neutral (0) amino acid transporter B(0)AT1 (Slc6a19). A general neutral aminoaciduria was observed similar to human Hartnup disorder which is caused by mutations in SLC6A19. Na(+)-dependent uptake of neutral amino acids into the intestine and renal brush-border membrane vesicles was abolished. No compensatory increase of peptide transport or other neutral amino acid transporters was detected. Mice lacking B(0)AT1 showed a reduced body weight. When adapted to a standard 20% protein diet, B(0)AT1-deficient mice lost body weight rapidly on diets containing 6 or 40% protein. Secretion of insulin in response to food ingestion after fasting was blunted. In the intestine, amino acid signaling to the mammalian target of rapamycin (mTOR) pathway was reduced, whereas the GCN2/ATF4 stress response pathway was activated, indicating amino acid deprivation in epithelial cells. The results demonstrate that epithelial amino acid uptake is essential for optimal growth and body weight regulation.     

Golgi GDP-fucose transporter-deficient mice mimic congenital disorder of glycosylation IIc/leukocyte adhesion deficiency II

Modification of glycoproteins by the attachment of fucose residues is widely distributed in nature. The importance of fucosylation has recently been underlined by identification of the monogenetic inherited human disease "congenital disorder of glycosylation IIc," also termed "leukocyte adhesion deficiency II." Due to defective Golgi GDP-fucose transporter (SLC35C1) activity, patients show a hypofucosylation of glycoproteins and present clinically with mental and growth retardation, persistent leukocytosis, and severe infections. To investigate effects induced by the loss of fucosylated structures in different organs, we generated a mouse model for the disease by inactivating the Golgi GDP-transporter gene (Slc35c1). Lectin binding studies revealed a tremendous reduction of fucosylated glycoconjugates in tissues and isolated cells from Slc35c1(-/-) mice. Fucose treatment of cells from different organs led to partial normalization of the fucosylation state of glycoproteins, thereby indicating an alternative GDP-fucose transport mechanism. Slc35c1-deficient mice presented with severe growth retardation, elevated postnatal mortality rate, dilatation of lung alveoles, and hypocellular lymph nodes. In vitro and in vivo leukocyte adhesion and rolling assays revealed a severe impairment of P-, E-, and L-selectin ligand function. The diversity of these phenotypic aspects demonstrates the broad general impact of fucosylation in the mammalian organism.