SENP1 mediates zinc-induced ZnT6 deSUMOylation at Lys-409 involved in the regulation of zinc metabolism in Golgi apparatus.

Zinc (Zn) transporters contribute to the maintenance of intracellular Zn homeostasis in vertebrate, whose activity and function are modulated by post-translational modification. However, the function of small ubiquitin-like modifier (SUMOylation) in Zn metabolism remains elusive. Here, compared with low Zn group, a high-Zn diet significantly increases hepatic Zn content and upregulates the expression of metal-response element-binding transcription factor-1 (MTF-1), Zn transporter 6 (ZnT6) and deSUMOylation enzymes (SENP1, SENP2, and SENP6), but inhibits the expression of SUMO proteins and the E1, E2, and E3 enzymes. Mechanistically, Zn triggers the activation of the MTF-1/SENP1 pathway, resulting in the reduction of ZnT6 SUMOylation at Lys 409 by small ubiquitin-like modifier 1 (SUMO1), and promoting the deSUMOylation process mediated by SENP1. SUMOylation modification of ZnT6 has no influence on its localization but reduces its protein stability. Importantly, deSUMOylation of ZnT6 is crucial for controlling Zn export from the cytosols into the Golgi apparatus. In conclusion, for the first time, we elucidate a novel mechanism by which SUMO1-catalyzed SUMOylation and SENP1-mediated deSUMOylation of ZnT6 orchestrate the regulation of Zn metabolism within the Golgi apparatus.

LATS1 inhibitor and zinc supplement synergistically ameliorates contrast-induced acute kidney injury: Induction of Metallothionein-1 and suppression of tubular ferroptosis.

Contrast-induced acute kidney injury (CI-AKI) is a prevalent cause of renal dysfunction among hospitalized patients, yet the precise pathogenesis and effective therapeutic strategies remain elusive. In this study, we investigated the role of tubular ferroptosis in both experimental CI-AKI models and in primary tubular epithelial cells (PTECs) treated with ioversol. Using whole exome sequencing, we identified metallothioneins (MTs) as being among the most significantly downregulated genes following ioversol exposure. Our findings reveal that overexpression of Mt1 mitigates, whereas suppression of Mt-1 exacerbates, ioversol-induced tubular ferroptosis. Interestingly, the level of MTF1 (metal regulatory transcription factor 1), a principal regulator of Mt1, was found to increase in response to ioversol treatment. We further elucidated that ioversol activates LATS1 (Large tumor suppressor homolog 1), a kinase that promotes the phosphorylation and nuclear translocation of MTF1, thereby inhibiting its transcriptional activity for Mt1. Both genetic and pharmacological inhibition of LATS1 reversed the ioversol-induced suppression of Mt-1. From a therapeutic perspective, the LATS1 inhibitor TDI-011536, in combination with zinc acetate, was administered to a rodent model of CI-AKI. Our data indicate that this combination synergistically upregulates Mt1 expression and provides protection against contrast media-induced tubular ferroptosis. In summary, our study demonstrates that the reduction of Mt-1 contributes to tubular ferroptosis associated with CI-AKI. We show that contrast media activate LATS1, which in turn suppresses the transcriptional activity of MTF1 for Mt1. Herein, the combination of zinc acetate and a LATS1 inhibitor emerges as a potential therapeutic approach for the treatment of CI-AKI.

Expression and Functional Analysis of the Metallothionein and Metal-Responsive Transcription Factor 1 in Phascolosoma esculenta under Zn Stress.

Metallothioneins (MTs) are non-enzymatic metal-binding proteins widely found in animals, plants, and microorganisms and are regulated by metal-responsive transcription factor 1 (MTF1). MT and MTF1 play crucial roles in detoxification, antioxidation, and anti-apoptosis. Therefore, they are key factors allowing organisms to endure the toxicity of heavy metal pollution. Phascolosoma esculenta is a marine invertebrate that inhabits intertidal zones and has a high tolerance to heavy metal stress. In this study, we cloned and identified MT and MTF1 genes from P. esculenta (designated as PeMT and PeMTF1). PeMT and PeMTF1 were widely expressed in all tissues and highly expressed in the intestine. When exposed to 16.8, 33.6, and 84 mg/L of zinc ions, the expression levels of PeMT and PeMTF1 in the intestine increased first and then decreased, peaking at 12 and 6 h, respectively, indicating that both PeMT and PeMTF1 rapidly responded to Zn stress. The recombinant pGEX-6p-1-MT protein enhanced the Zn tolerance of Escherichia coli and showed a dose-dependent ABTS free radical scavenging ability. After RNA interference (RNAi) with PeMT and 24 h of Zn stress, the oxidative stress indices (MDA content, SOD activity, and GSH content) and the apoptosis indices (Caspase 3, Caspase 8, and Caspase 9 activities) were significantly increased, implying that PeMT plays an important role in Zn detoxification, antioxidation, and anti-apoptosis. Moreover, the expression level of PeMT in the intestine was significantly decreased after RNAi with PeMTF1 and 24 h of Zn stress, which preliminarily proved that PeMTF1 has a regulatory effect on PeMT. Our data suggest that PeMT and PeMTF1 play important roles in the resistance of P. esculenta to Zn stress and are the key factors allowing P. esculenta to endure the toxicity of Zn.

Metallothionein 3 Potentiates Pulmonary Artery Smooth Muscle Cell Proliferation by Promoting Zinc-MTF1-ATG5 Axis-mediated Autophagosome Formation.

Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is one of the critical pathological mechanisms of pulmonary hypertension (PH), and therefore is gradually being adopted as an important direction for the treatment of PH. Metallothioneins (MTs) have been reported to be associated with PH, but the underlying mechanisms are not fully understood. Here, we demonstrated that the expression level of metallothionein 3 (MT3) was significantly increased in pulmonary arterioles from PH patients and chronic hypoxia-induced rat and mouse PH models, as well as in hypoxia-treated human PASMCs. Knockdown of MT3 significantly inhibited the proliferation of human PASMCs by arresting the cell cycle in the G1 phase, while overexpression of MT3 had the opposite effect. Mechanistically, we found that MT3 increased the intracellular zinc (Zn2+) concentration to enhance the transcriptional activity of metal-regulated transcription factor 1 (MTF1), which promoted the expression of autophagy-related gene 5 (ATG5), facilitating autophagosome formation. More importantly, MT3-induced autophagy and proliferation of human PASMCs were largely prevented by knockdown of MTF1 and ATG5. Therefore, in this study, we identified MT3-Zinc-MTF1-ATG5 as a novel pathway that affects PASMC proliferation by regulating autophagosome formation, suggesting that MT3 may be a novel target for the treatment of PH.

MTF1 genetic variants are associated with lung cancer risk in the Chinese Han population.

BACKGROUND: Metal-regulatory transcription factor 1 (MTF1), a conserved metal-binding transcription factor in eukaryotes, regulates the proliferation of cancer cells by activating downstream target genes and then participates in the formation and progression of tumors, including lung cancer (LC). The expression level of MTF1 is down-regulated in LC, and high expression of MTF1 is associated with a good prognosis of LC. However, the association between MTF1 polymorphism and LC risk has not been explored. METHODS: The genotyping of MTF1 Single nucleotide polymorphisms (SNPs) including rs473279, rs28411034, rs28411352, and rs3748682 was identified by the Agena MassARRAY system among 670 healthy controls and 670 patients with LC. The odds ratio (OR) and 95% confidence intervals (CI) were calculated by logistics regression to assess the association of these SNPs with LC risk. RESULTS: MTF1 rs28411034 (OR 1.22, 95% CI 1.03-1.45, p = 0.024) and rs3748682 (OR 1.24, 95% CI 1.04-1.47, p = 0.014) were associated with higher LC susceptibility overall. Moreover, the effect of rs28411034 and rs3748682 on LC susceptibility was observed in males, subjects with body mass index (BMI) ≥ 24 kg/m2, smokers, drinkers, and patients with lung squamous carcinoma (OR and 95% CI > 1, p < 0.05). Besides, rs28411352 (OR 0.73, 95% CI 0.55-0.97, p = 0.028,) showed protective effect for reduced LC risk in drinkers. CONCLUSIONS: We were first who reported that rs28411034 and rs3748682 tended to be relevant to increased LC susceptibility among the Chinese Han population. These results of this study could help to recognize the pathogenic mechanisms of the MTF1 gene in LC progress.

Knocking-down long non-coding RNA LINC01094 prohibits chondrocyte apoptosis via regulating microRNA-577/metal-regulatory transcription factor 1 axis.

PURPOSE: The abnormal function and survival of chondrocytes result in articular cartilage failure, which may accelerate the onset and development of osteoarthritis (OA). This study is aimed to investigate the role of LINC01094 in chondrocyte apoptosis. METHODS: The viability and apoptosis of lipopolysaccharide (LPS)-induced chondrocytes were evaluated through CCK-8 assay and flow cytometry analysis, respectively. The expression levels of LINC01094, miR-577 and MTF1 were detected by qRT-PCR. Dual luciferase reporter tests were implemented for the verification of targeted relationships among them. Western blotting was employed to measure the levels of pro-apoptotic proteins (Caspase3 and Caspase9). RESULTS: The viability of LPS-induced chondrocytes was overtly promoted by loss of LINC01094 or miR-577 upregulation, but could be repressed via MTF1 overexpression. The opposite results were observed in apoptosis rate and the levels of Caspase3 and Caspase9. LINC01094 directly bound to miR-577, while MTF1 was verified to be modulated by miR-577. Both LINC01094 and MTF1 were at high levels, whereas miR-577 was at low level in OA synovial fluid and LPS-induced chondrocytes. Furthermore, the highly expressed miR-577 abolished the influences of MTF1 overexpression on LPS-induced chondrocytes. CONCLUSIONS: Silencing of LINC01094 represses the apoptosis of chondrocytes through upregulating miR-577 expression and downregulating MTF1 levels, providing a preliminary insight for the treatment of OA in the future.

Transcriptional regulation of Znt family members znt4, znt5 and znt10 and their function in zinc transport in yellow catfish (Pelteobagrus fulvidraco).

The study characterized the transcriptionally regulatory mechanism and functions of three zinc (Zn) transporters (znt4, znt5 and znt10) in Zn2+ metabolism in yellow catfish (Pelteobagrus fulvidraco), commonly freshwater fish in China and other countries. We cloned the sequences of znt4 promoter, spanning from -1217 bp to +80 bp relative to TSS (1297 bp); znt5, spanning from -1783 bp to +49 bp relative to TSS (1832 bp) and znt10, spanning from -1923 bp to +190 bp relative to TSS (2113 bp). In addition, after conducting the experiments of sequential deletion of promoter region and mutation of potential binding site, we found that the Nrf2 binding site (-607/-621 bp) and Klf4 binding site (-5/-14 bp) were required on znt4 promoter, the Mtf-1 binding site (-1674/-1687 bp) and Atf4 binding site (-444/-456 bp) were required on znt5 promoter and the Atf4 binding site (-905/-918 bp) was required on znt10 promoter. Then, according to EMSA and ChIP, we found that Zn2+ incubation increased DNA affinity of Atf4 to znt5 or znt10 promoter, but decreased DNA affinity of Nrf2 to znt4 promoter, Klf4 to znt4 promoter and Mtf-1 to znt5 promoter. Using fluorescent microscopy, it was revealed that Znt4 and Znt10 were located in the lysosome and Golgi, and Znt5 was located in the Golgi. Finally, we found that znt4 knockdown reduced the zinc content of lysosome and Golgi in the control and zinc-treated group; znt5 knockdown reduced the zinc content of Golgi in the control and zinc-treated group and znt10 knockdown reduced the zinc content of Golgi in the zinc-treated group. High dietary zinc supplement up-regulated Znt4 and Znt5 protein expression. Above all, for the first time, we revealed that Klf4 and Nrf2 transcriptionally regulated the activities of znt4 promoter; Mtf-1 and Atf4 transcriptionally regulated the activities of znt5 promoter and Atf4 transcriptionally regulated the activities of znt10 promoter, which provided innovative regulatory mechanism of zinc transporting in yellow catfish. Our study also elucidated their subcellular location, and regulatory role of zinc homeostasis in yellow catfish.

Developmental regulation of zinc homeostasis in differentiating oligodendrocytes.

Oligodendrocytes develop through sequential stages and understanding pathways regulating their differentiation remains an important area of investigation. Zinc is required for the function of enzymes, proteins and transcription factors, including those important in myelination and mitosis. Our previous studies using the ratiometric zinc sensor chromis-1 demonstrated a reduction in intracellular free zinc concentrations in mature MBP+ oligodendrocytes compared with earlier stages (Bourassa et al., 2018). We performed a more detailed developmental study to better understand the temporal course of zinc homeostasis across the oligodendrocyte lineage. Using chromis-1, we found a transient increase in free zinc after O4+,O1- pre-oligodendrocytes were switched from proliferation medium into terminal differentiation medium. To gather other evidence for dynamic regulation of free zinc during oligodendrocyte development, qPCR was used to evaluate mRNA expression of major zinc storage proteins metallothioneins (MTs) and metal regulatory transcription factor 1 (MTF1), which controls expression of MTs. MT1, MT2 and MTF1 mRNAs were increased several fold in mature oligodendrocytes compared to oligodendrocytes in proliferation medium. To assess the depth of the zinc buffer, we assayed zinc release from intracellular stores using the oxidizing thiol reagent 2,2'-dithiodipyridine (DTDP). Exposure to DTDP resulted in âˆ¼ 100% increase in free zinc in pre-oligodendrocytes but, paradoxically more modest âˆ¼ 60% increase in mature oligodendrocytes despite increased expression of MTs. These results suggest that zinc homeostasis is regulated during oligodendrocyte development, that oligodendrocytes are a useful model for studying zinc homeostasis in the central nervous system, and that regulation of zinc homeostasis may be important in oligodendrocyte differentiation.

The Role of Zinc on Liver Fibrosis by Modulating ZIP14 Expression Throughout Epigenetic Regulatory Mechanisms.

Zinc plays a pivotal role in tissue regeneration and maintenance being as a central cofactor in a plethora of enzymatic activities. Hypozincemia is commonly seen with chronic liver disease and is associated with an increased risk of liver fibrosis development and hepatocellular carcinoma. Previously favorable effects of zinc supplementation on liver fibrosis have been shown. However, the underlying mechanism of this effect is not elucidated. Liver fibrosis was induced in mice by using CCl4 injection, followed by treatment with zinc chloride (ZnCl2) both at fibrotic and sham groups, and their hepatocytes were isolated. Our results showed that the administration of ZnCl2 restored the depleted cytosolic zinc levels in the hepatocytes isolated from the fibrotic group. Also, alpha-smooth muscle actin (αSMA) expression in hepatocytes was decreased, indicating a reversal of the fibrotic process. Notably, ZIP14 expression significantly increased in the fibrotic group following ZnCl2 treatment, whereas in the sham group ZIP14 expression decreased. Chromatin immunoprecipitation (ChIP) experiments revealed an increased binding percentage of Metal-regulatory transcription factor 1 (MTF1) on ZIP14 promoter in the hepatocytes isolated from fibrotic mice compared to the sham group after ZnCl2 administration. In the same group, the binding percentage of the histone deacetylase HDAC4 on ZIP14 promoter decreased. Our results suggest that the ZnCl2 treatment ameliorates liver fibrosis by elevating intracellular zinc levels through MTF1-mediated regulation of ZIP14 expression and the reduction of ZIP14 deacetylation via HDAC4. The restoration of intracellular zinc concentrations and the modulation of ZIP14 expression by zinc orchestrated through MTF1 and HDAC4, appear to be essential determinants of the therapeutic response in hepatic fibrosis. These findings pave the way for potential novel interventions targeting zinc-related pathways for the treatment of liver fibrosis and associated conditions.

Genomic Redistribution of Metal-Response Transcription Factor-1 (MTF-1) in Cadmium Resistant Cells.

(1) Background: Metal homeostasis is an important part of cellular programs and is disrupted when cells are exposed to carcinogenic heavy metals. Metal response is mediated by the metal response element transcription factor MTF-1. However, where MTF-1 binds and how that binding changes in response to heavy metals, such as cadmium, remains unknown. (2) Methods: To investigate the effects of prolonged cadmium exposure on the genomic distribution of MTF-1, we performed MTF-1 CUT&RUN, RNA-seq and ATAC-seq on control and cadmium-resistant cells. (3) Results: Changes in MTF-1 binding primarily occur distal to the transcription start sight. Newly occupied MTF-1 sites are enriched for FOS/JUN DNA binding motifs, while regions that lose MTF-1 binding in cadmium are enriched for the FOX transcription factor family member DNA binding sites. (4) Conclusions: Relocalization of MTF-1 to new genomic loci does not alter the accessibility of these locations. Our results support a model whereby MTF-1 is relocalized to accessible FOS/JUN-bound genomic locations in response to cadmium.

lncRNA SND1-IT1 delivered via intracerebral hemorrhage-derived exosomes affect the growth of human microglia by regulating the miR-124-3p/MTF1 axis.

In this study, we investigated the effects of long noncoding RNA (lncRNA) SND1-IT1 on human microglia (HMC3 cells) delivered by intracerebral hemorrhage (ICH)-derived exosomes (ICH-exos) as well as a competitive endogenous RNA (ceRNA) network. Exosomes obtained from ICH plasma were characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blot. RNA sequencing was performed to study the lncRNA transcriptome from ICH-exos and the healthy control-derived exosomes (HC-exos) and differentially expressed lncRNAs (DE-lncRNAs) were identified. HMC3 cells were treated with ICH-exos or transfected with pcDNA3.1-SND1-IT1, and then cell viability and apoptosis were measured. The ceRNA network (lncRNA SND1-IT1/miR-124-3p/messenger RNA MTF1) was chosen for further investigation. NTA, TEM, and western blot showed that exosomes were successfully separated and could be absorbed by HMC3 cells. The expression of lncRNA SND1-IT1 in ICH-exos was significantly higher than that of HC-exos (p < 0.05). In addition, lncRNA SND1-IT1 overexpression and ICH-exos significantly inhibited cell viability and enhanced apoptosis. A total of 162 DE-lncRNAs were identified by sequencing, and a ceRNA network was constructed. The dual-luciferase reporter gene indicated that lncRNA SND1-IT1, miR-124-3p, and MTF1 interacted with each other. Cell experiments showed that lncRNA SND1-IT1 affected the growth of HMC3 cells through miR-124-3p/MTF1. In conclusion, ICH-exos delivered lncRNA SND1-IT1 to HMC3 cells, and exosomal lncRNA SND1-IT1 can regulate cell viability and apoptosis to influence HMC3 cell growth via the SND1-IT1/miR-124-3p/MTF1 axis.

miRNA-22 Upregulates Mtf1 in Dorsal Horn Neurons and Is Essential for Inflammatory Pain.

Chronic inflammatory pain seriously affects patients' quality of life because of a paucity of effective clinical treatments caused, at least in part, by lack of full understanding of the underlying mechanisms. miRNAs are known to be involved in inflammatory pain via silencing or degrading of target mRNA in the cytoplasm. The present study provides a novel mechanism by which miRNA-22 positively regulates metal-regulatory transcription factor 1 (Mtf1) in the nuclei of neurons in the dorsal horn of the spinal cord. We found that miRNA-22 was significantly increased in the dorsal horn of mice with either inflammatory pain induced by plantar injection of complete Freund's adjuvant (CFA) or neuropathic pain induced by unilateral sciatic nerve chronic constrictive injury (CCI). Knocking down or blocking miRNA-22 alleviated CFA-induced mechanical allodynia and heat hyperalgesia, whereas overexpressing miRNA-22 produced pain-like behaviors. Mechanistically, the increased miRNA-22 binds directly to the Mtf1 promoter to recruit RNA polymerase II and elevate Mtf1 expression. The increased Mtf1 subsequently enhances spinal central sensitization, as evidenced by increased expression of p-ERK1/2, GFAP, and c-Fos in the dorsal horn. Our findings suggest that the miRNA-22-Mtf1 signaling axis in the dorsal horn plays a critical role in the induction and maintenance of inflammatory pain. This signaling pathway may be a promising therapeutic target in inflammatory pain.

The Hippo pathway kinases LATS1 and LATS2 attenuate cellular responses to heavy metals through phosphorylating MTF1.

Heavy metals are both integral parts of cells and environmental toxicants, and their deregulation is associated with severe cellular dysfunction and various diseases. Here we show that the Hippo pathway plays a critical role in regulating heavy metal homeostasis. Hippo signalling deficiency promotes the transcription of heavy metal response genes and protects cells from heavy metal-induced toxicity, a process independent of its classic downstream effectors YAP and TAZ. Mechanistically, the Hippo pathway kinase LATS phosphorylates and inhibits MTF1, an essential transcription factor in the heavy metal response, resulting in the loss of heavy metal response gene transcription and cellular protection. Moreover, LATS activity is inhibited following heavy metal treatment, where accumulated zinc directly binds and inhibits LATS. Together, our study reveals an interplay between the Hippo pathway and heavy metals, providing insights into this growth-related pathway in tissue homeostasis and stress response.

A circuit of COCH neurons encodes social-stress-induced anxiety via MTF1 activation of Cacna1h.

The hippocampus is a temporal lobe structure critical for cognition, such as learning, memory, and attention, as well as emotional responses. Hippocampal dysfunction can lead to persistent anxiety and/or depression. However, how millions of neurons in the hippocampus are molecularly and structurally organized to engage their divergent functions remains unknown. Here, we genetically target a subset of neurons expressing the coagulation factor c homolog (COCH) gene. COCH-expressing neurons or COCH neurons are topographically segregated in the distal region of the ventral CA3 hippocampus and express Mtf1 and Cacna1h. MTF1 activation of Cacna1h transcription in COCH neurons encodes the ability of COCH neurons to burst action potentials and cause social-stress-induced anxiety-like behaviors by synapsing directly with a subset of GABAergic inhibitory neurons in the lateral septum. Together, this study provides a molecular and circuitry-based framework for understanding how COCH neurons in the hippocampus are assembled to engage social behavior.

Temporal transcriptomic alterations of cadmium exposed human iPSC-derived renal proximal tubule-like cells.

Cadmium is a well-studied environmental pollutant where the kidney and particularly the proximal tubule cells are especially sensitive as they are exposed to higher concentrations of cadmium than other tissues. Here we investigated the temporal transcriptomic alterations (TempO-Seq) of human induced pluripotent stem cell (iPSC)-derived renal proximal tubule-like (PTL) cells exposed to 5 µM cadmium chloride for 1, 2, 4, 8, 12, 16, 20, 24, 72 and 168 h. There was an early activation (within 4 h) of the metal and oxidative stress responses (metal-responsive transcription factor-1 (MTF1) and nuclear factor erythroid-2-related factor 2 (Nrf2) genes). The Nrf2 response returned to baseline within 24 h. The Activator Protein 1 (AP-1) regulated genes HSPA6 and FOSL-1 followed the Nrf2 time course. While the MTF1 genes also spiked at 4 h, they remained strongly elevated over the entire exposure period. The data and cell culture model utilised will be useful in further research aimed at the refinement of safe human exposure limits for cadmium, other metals and their mixtures.

[Transcription Factor MTF-1 Involved in the Cellular Response to Zinc].

Heavy metals, both toxic and essential, have long been an important research focus in life science. To investigate the intracellular actions of heavy metals at the molecular level, I have been exploring protein factors involved in induction of metallothionein (MT) genes by heavy metals that specifically bind to a metal responsive element (MRE) in the region upstream of the human MT-IIA gene. Purification of a zinc-dependent MRE-binding factor, and cloning of its cDNA identified a sequence identical to that of metal-responsive transcription factor-1 (MTF-1). MTF-1, which is characterized by six tandem repeats of the C2H2 type zinc finger motif, is indispensable for induction of MT gene expression by multiple types of heavy metal, but zinc is the only metal that can directly activate MTF-1 binding to the MRE, indicating that other heavy metal signals act through zinc as a second messenger. Functional analysis of various MTF-1 point mutants revealed several cysteine (Cys) residues critical for DNA binding and/or transactivation activity. Interestingly, six finger motifs seem to mediate several MTF-1 functions other than DNA binding. Immunohistochemical analyses of various mouse tissues revealed selective expression of MTF-1 in spermatocytes among the testicular cells, suggesting roles relevant to spermatogenesis. The zinc regulon, under the control of MTF-1, will likely provide good clues to aid in unraveling novel functions of intracellular zinc ions.

Gawky modulates MTF-1-mediated transcription activation and metal discrimination.

Metal-induced genes are usually transcribed at relatively low levels under normal conditions and are rapidly activated by heavy metal stress. Many of these genes respond preferentially to specific metal-stressed conditions. However, the mechanism by which the general transcription machinery discriminates metal stress from normal conditions and the regulation of MTF-1-meditated metal discrimination are poorly characterized. Using a focused RNAi screening in Drosophila Schneider 2 (S2) cells, we identified a novel activator, the Drosophila gawky, of metal-responsive genes. Depletion of gawky has almost no effect on the basal transcription of the metallothionein (MT) genes, but impairs the metal-induced transcription by inducing the dissociation of MTF-1 from the MT promoters and the deficient nuclear import of MTF-1 under metal-stressed conditions. This suggests that gawky serves as a 'checkpoint' for metal stress and metal-induced transcription. In fact, regular mRNAs are converted into gawky-controlled transcripts if expressed under the control of a metal-responsive promoter, suggesting that whether transcription undergoes gawky-mediated regulation is encrypted therein. Additionally, lack of gawky eliminates the DNA binding bias of MTF-1 and the transcription preference of metal-specific genes. This suggests a combinatorial control of metal discrimination by gawky, MTF-1, and MTF-1 binding sites.

Transcriptional responses of four slc30a/znt family members and their roles in Zn homeostatic modulation in yellow catfish Pelteobagrus fulvidraco.

The study characterized their regulatory functions of four znt members (znt1, znt2, znt6 and znt8) in Zn homeostasis in vertebrates. We found that the -1281/-1296 bp locus on the znt1 promoter, the -1/-16 bp locus on the znt2 promoter, the -825/-839 bp locus on the znt6 promoter, the -165/-180 bp locus and the -274/-292 bp STAT3 locus on the znt8 promoter were functional MTF-1 binding sites and had metal responsive element (MRE). Zn incubation increased activities of four znt promoters, which was mediated by MRE sites on znt1, znt2, znt6 and znt8 promoters and by STAT3 binding site on znt8 promoter. Moreover, Zn activated the transcription of these znts genes through MTF-1-MRE-dependent pathway. Zn incubation up-regulated the mRNA and total protein expression of ZnT1, ZnT2 and ZnT8 at both 24 h and 48 h. Overall, for the first time, this study offered novel insights for regulatory mechanism of Zn homeostasis in vertebrates.

The hypoxia sensitive metal transcription factor MTF-1 activates NCX1 brain promoter and participates in remote postconditioning neuroprotection in stroke.

Remote limb ischemic postconditioning (RLIP) is an experimental strategy in which short femoral artery ischemia reduces brain damage induced by a previous harmful ischemic insult. Ionic homeostasis maintenance in the CNS seems to play a relevant role in mediating RLIP neuroprotection and among the effectors, the sodium-calcium exchanger 1 (NCX1) may give an important contribution, being expressed in all CNS cells involved in brain ischemic pathophysiology. The aim of this work was to investigate whether the metal responsive transcription factor 1 (MTF-1), an important hypoxia sensitive transcription factor, may (i) interact and regulate NCX1, and (ii) play a role in the neuroprotective effect mediated by RLIP through NCX1 activation. Here we demonstrated that in brain ischemia induced by transient middle cerebral occlusion (tMCAO), MTF-1 is triggered by a subsequent temporary femoral artery occlusion (FAO) and represents a mediator of endogenous neuroprotection. More importantly, we showed that MTF-1 translocates to the nucleus where it binds the metal responsive element (MRE) located at -23/-17 bp of Ncx1 brain promoter thus activating its transcription and inducing an upregulation of NCX1 that has been demonstrated to be neuroprotective. Furthermore, RLIP restored MTF-1 and NCX1 protein levels in the ischemic rat brain cortex and the silencing of MTF-1 prevented the increase of NCX1 observed in RLIP protected rats, thus demonstrating a direct regulation of NCX1 by MTF-1 in the ischemic cortex of rat exposed to tMCAO followed by FAO. Moreover, silencing of MTF-1 significantly reduced the neuroprotective effect elicited by RLIP as demonstrated by the enlargement of brain infarct volume observed in rats subjected to RLIP and treated with MTF-1 silencing. Overall, MTF-dependent activation of NCX1 and their upregulation elicited by RLIP, besides unraveling a new molecular pathway of neuroprotection during brain ischemia, might represent an additional mechanism to intervene in stroke pathophysiology.

Ferroportin-Dependent Iron Homeostasis Protects against Oxidative Stress-Induced Nucleus Pulposus Cell Ferroptosis and Ameliorates Intervertebral Disc Degeneration In Vivo.

Ferroptosis is a specialized form of regulated cell death that is charactered by iron-dependent lethal lipid peroxidation, a process associated with multiple diseases. However, its role in the pathogenesis of intervertebral disc degeneration (IVDD) is rarely investigated. This study is aimed at investigating the role of ferroptosis in oxidative stress- (OS-) induced nucleus pulposus cell (NPC) decline and the pathogenesis of IVDD and determine the underlying regulatory mechanisms. We used tert-butyl hydroperoxide (TBHP) to simulate OS conditions around human NPCs. Flow cytometry and transmission electron microscopy were used to identify ferroptosis, while iron assay kit, Perl's staining, and western blotting were performed to assay the intracellular iron levels. A ferroportin- (FPN-) lentivirus and FPN-siRNA were constructed and used to explore the relationship between FPN, intracellular iron homeostasis, and ferroptosis. Furthermore, hinokitiol, a bioactive compound known to specifically resist OS and restore FPN function, was evaluated for its therapeutic role in IVDD both in vitro and in vivo. The results indicated that intercellular iron overload plays an essential role in TBHP-induced ferroptosis of human NPCs. Mechanistically, FPN dysregulation is responsible for intercellular iron overload under OS. The increase in nuclear translocation of metal-regulatory transcription factor 1 (MTF1) restored the function of FPN, abolished the intercellular iron overload, and protected cells against ferroptosis. Additionally, hinokitiol enhanced the nuclear translocation of MTF1 by suppressing the JNK pathway and ameliorated the progression of IVDD in vivo. Taken together, our results demonstrate that ferroptosis and FPN dysfunction are involved in the NPC depletion and the pathogenesis of IVDD under OS. To the best of our knowledge, this is the first study to demonstrate the protective role of FPN in ferroptosis of NPCs, suggesting its potential used as a novel therapeutic target against IVDD.