Skeletal muscle represents the largest pool of body zinc, however, little is known about muscle zinc homeostasis or muscle-specific zinc functions. Zip14 (Slc39a14) was the most highly expressed zinc transporter in skeletal muscle of mice in response to LPS-induced inflammation. We compared metabolic parameters of skeletal muscle from global Zip14 knockout (KO) and wild-type mice (WT). At basal steady state Zip14 Ko mice exhibited a phenotype that included muscle wasting and metabolic endotoxemia. Microarray and qpcR analysis of gastrocnemius muscle RnA revealed that ablation of Zip14 produced increased muscle p-Mef2c, Hspb7 and miR-675-5p expression and increased p38 activation. chip assays showed enhanced binding of nf-κβ to the Mef2c promoter. in contrast, LpSinduced systemic inflammation enhanced Zip14-dependent zinc uptake by muscle, increased expression of Atrogin1 and MuRF1 and markedly reduced MyoD. these signatures of muscle atrophy and cachexia were not influenced by Zip14 ablation, however. LpS-induced miR-675-3p and-5p expression was Zip14-dependent. Collectively, these results with an integrative model are consistent with a Zip14 function in skeletal muscle at steady state that supports myogenesis through suppression of metabolic endotoxemia and that Zip14 ablation coincides with sustained activity of phosphorylated components of signaling pathways including p-Mef2c, which causes Hspb7-dependent muscle wasting.
Impaired manganese (Mn) homeostasis can result in excess Mn accumulation in specific brain regions and neuropathology. Maintaining Mn homeostasis and detoxification is dependent on effective Mn elimination. Specific metal transporters control Mn homeostasis. Human carriers of mutations in the metal transporter ZIP14 and whole body Zip14-knockout (WB-KO) mice display similar phenotypes, including spontaneous systemic and brain Mn overload and motor dysfunction. Initially, it was believed that Mn accumulation due to ZIP14 mutations was caused by impaired hepatobiliary Mn elimination. However, liver-specific Zip14-KO mice did not show systemic Mn accumulation or motor deficits. ZIP14 is highly expressed in the small intestine and is localized to the basolateral surface of enterocytes. Thus, we hypothesized that basolaterally localized ZIP14 in enterocytes provides another route for the elimination of Mn. Using wild-type and intestine-specific Zip14-KO (I-KO) mice, we have shown that ablation of intestinal Zip14 is sufficient to cause systemic and brain Mn accumulation. The lack of intestinal ZIP14-mediated Mn excretion was compensated for by the hepatobiliary system; however, it was not sufficient to maintain Mn homeostasis. When supplemented with extra dietary Mn, I-KO mice displayed some motor dysfunctions and brain Mn accumulation based on both MRI imaging and chemical analysis, thus demonstrating the importance of intestinal ZIP14 as a route of Mn excretion. A defect in intestinal Zip14 expresssion likely could contribute to the Parkinson-like Mn accumulation of manganism. NEW & NOTEWORTHY Mn-induced parkinsonism is recognized as rising in frequency because of both environmental factors and genetic vulnerability; yet currently, there is no cure. We provide evidence in an integrative animal model that basolaterally localized ZIP14 regulates Mn excretion and detoxification and that deletion of intestinal ZIP14 leads to systemic and brain Mn accumulation, providing robust evidence for the indispensable role of intestinal ZIP14 in Mn excretion.
Impaired manganese (Mn) homeostasis can result in excess Mn accumulation in specific brain regions and neuropathology. Maintaining Mn homeostasis and detoxification is dependent on effective Mn elimination. Specific metal transporters control Mn homeostasis. Human carriers of mutations in the metal transporter ZIP14 and whole-body Zip14 KO (WB-KO) mice display similar phenotypes, including spontaneous systemic and brain Mn overload, and motor dysfunction. Initially, it was believed that Mn accumulation due to ZIP14 mutations caused by impaired hepatobiliary Mn elimination. However, liver-specific Zip14 KO mice (L-KO) did not show systemic Mn accumulation or motor deficits. ZIP14 is highly expressed in the small intestine and is localized to the basolateral surface of enterocytes. Thus we hypothesized that basolaterally-localized ZIP14 in enterocytes provides another route for elimination of Mn. Using wild type and intestine-specific ZIP14 KO (I-KO) mice, we have shown that ablation of intestinal Zip14 is sufficient to cause systemic and brain Mn accumulation. The lack of intestinal ZIP14- mediated Mn excretion was compensated for by the hepatobiliary system; however, it was not sufficient to maintain Mn homeostasis. When supplemented with extra dietary Mn, I-KO mice displayed some motor dysfunctions, brain Mn accumulation based on both MRI imaging and chemical analysis, thus demonstrating the importance of intestinal ZIP14 as a route of Mn excretion. A defect in intestinal Zip14 expresssion likely could contribute to the Parkinson-like Mn accumulation of manganism.New & NoteworthyMn-induced parkinsonism is recognized as rising in frequency due to both environmental factors and genetic vulnerability, yet currently, there is no cure. We provide evidence in an integrative animal model that basolaterally localized ZIP14 regulates Mn excretion and detoxification and that deletion of intestinal ZIP14 leads to systemic and brain Mn accumulation, providing robust evidence for the indispensable role of intestinal ZIP14 on Mn excretion.
Zinc, an essential micronutrient, has anti-inflammatory properties employing mechanisms that are unclear. Zip14 (Slc39a14) is a zinc transporter induced by proinflammatory stimuli and is highly expressed at the basolateral membrane of intestinal epithelial cells (IECs). Enterocyte-specific Zip14 ablation (Zip14ΔIEC) in mice was developed to study the role of this transporter in enterocytes. This gene deletion led to increased intestinal permeability, increased IL-6 and IFNγ expression, mild endotoxemia and intestinal dysbiosis. Tissues and intestinal epithelial cells were obtained from anesthetized mice. RNA sequencing was used for transcriptome profiling. These analyses revealed differential expression of specific intestinal proinflammatory and tight junction (TJ) genes. Binding of transcription factors, including NF-ĸβ, STAT3 and CDX2, to appropriate sites of promoters of these genes support the differential expression was shown with chromatin immunoprecipitation assays. Total HDAC and specifically HDAC3 activities were markedly reduced with Zip14 ablation. Intestinal organoids derived from ΔIEC mice display TJ and cytokine gene dysregulation compared to control mice. Differential expression of specific genes was reversed with zinc supplementation of the organoids. We conclude that zinc-dependent HDAC enzymes acquire zinc ions via Zip14-mediated transport and that intestinal integrity is controlled in part through epigenetic modifications. These studies potentially represent a new health-promoting role for this micronutrient.
The mechanisms by which Zip14 ablation mediates mild intestinal inflammation and decreased barrier function are not completely understood. We used RNA sequencing to profile the transcriptome of intestinal epithelial cells (IEC) isolated from Zip14 Cre/flox and control flox/flox mice. RNA‐seq analysis identified 1,345 differentially expressed genes in Zip14 Cre/flox mice compared with control mice. In‐silico analyses revealed that Zip14ablation promoted dysregulation of specific proinflammatory genes (e.g. IL‐6 and Ifn‐γ) and genes influencing intestinal barrier homeostasis (specifically, Claudins 1‐and‐2). Highly dysregulated genes were selected for confirmation by qPCR, and proteins by western analysis, using extracts from isolated IECs from mice of the two genotypes. Highly upregulated genes in the Zip14 Cre/flox mice included Fabp6, Cldn2and Cldn8, while downregulated genes included Cldn1. To explore the possibility that Zip14ablation may alter transcriptional activity, ChIP assays of selected promoters were conducted. Increased binding of Stat3, FXR and NF‐kB was observed in Cldn2, FABP6, and Cldn8promoters, respectively, with chromatin from the Zip14 Cre/floxmice compared to control mice. By contrast, Cdx2 binding was decreased for the Cldn1promoter site of the knockout mice. Zip14 ablation also altered expression levels of long noncoding RNAs (lncRNAs). Specifically, the lncRNAs H19, U90926, and Meg3 were highly upregulated, and Gata4 was downregulated as validated by qPCR. Using organoids derived from intestines of mice of both genotypes, it was demonstrated that growth was not impaired with Zip14 ablation. Other biochemical markers of Zip14ablation were recapitulated in organoid cultures. This included indices of enhanced Zn2+ trafficking and differential expression of specific tight junction genes. Decreased HDAC3 activity in IECs from the Zip14 knockout mice suggest that epigenetic modifications account in part for the genomic changes observed. We conclude zinc transported by Zip14 controls intestinal permeability through epigenetic modifications, thus representing a novel health‐promoting role for this micronutrient.
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