Herein, the mechanisms of transactivation of gene expression by mouse metal response element-binding transcription factor 1 (MTF-1) were investigated. Evidence obtained from coimmunoprecipitation assays revealed that exposure of the cells to zinc resulted in the rapid formation of a multiprotein complex containing MTF-1, the histone acetyltransferase p300/CBP, and the transcription factor Sp1. Down-regulation of endogenous p300 expression by small interfering RNA transfection significantly decreased zinc-dependent metallothionein I (MT-I) gene transcription without altering induction of zinc transporter 1 (ZnT1). MTF-1 independently facilitated the recruitment of Sp1 and p300 to the protein complex in response to zinc. Mutagenesis demonstrated that the acidic domain, one of three transactivation domains of MTF-1, is required for recruitment of p300 but not Sp1 as well as for zinc-dependent activation of MT-I gene transcription. Furthermore, mutation of leucine residues (L3A) within a nuclear exclusion signal in the MTF-1 acidic domain impaired recruitment of p300 and zinc-dependent activation of the MT-I gene. Nuclear magnetic resonance characterization of an isolated protein fragment corresponding to the MTF-1 acidic region demonstrated that this region is largely unstructured in the presence and absence of excess stoichiometric amounts of zinc. This suggests that the mechanism by which MTF-1 recruits p300 to this complex involves extrinsic-zinc-dependent steps. These studies reveal a novel zinc-responsive mechanism requiring an acidic region of MTF-1 that functions as a nuclear exclusion signal as well as participating in formation of a coactivator complex essential for transactivation of MT-I gene expression.Metal response element (MRE)-binding transcription factor 1 (MTF-1) activates metallothionein (MT) gene expression in response to several stresses, including exposure to metal ions (zinc and cadmium), hypoxia, oxidative stress, and elevated temperature (3,25). Central to the mechanism of action of vertebrate MTF-1 on MT genes is its ability to function as a zinc sensor, and an increase in available zinc may be a unifying theme during many stresses. However, mouse MTF-1 is also essential during embryonic development of the liver (41), and this transcription factor integrates a diverse set of environmental signals and modulates the expression of many genes (26). MTF-1 interacts with or cooperates with a diverse set of factors, including NF-B (8), hypoxia-inducible factor 1␣ (HIF-1␣) (28, 29), USF (4), Sp1 (30), heat shock transcription factor 1 (34, 38), and ribosomal protein S35 (1).Studies of the mechanism by which MTF-1 regulates transcription of vertebrate MT-I genes in response to zinc suggest that the reversible binding of zinc directly with the unique, highly conserved six-zinc-finger domain regulates the affinity of binding of the fingers to short, cis-acting DNA sequences, termed MREs, found in multiple copies in the MT-I (and other) gene promoters. Increased available zinc in the cell, resulting f...

Objective: Endothelial cells (EC) in obese adipose tissue (AT) are exposed to a chronic proinflammatory environment that may induce a mesenchymal-like phenotype and altered function. The objective of this study was to establish whether endothelial-to-mesenchymal transition (EndoMT) is present in human AT in obesity and to investigate the effect of such transition on endothelial function and the endothelial particulate secretome represented by extracellular vesicles (EV). Approach and Results: We identified EndoMT in obese human AT depots by immunohistochemical co-localization of CD31 or vWF and α-SMA (alpha-smooth muscle actin). We showed that AT EC exposed in vitro to TGF-β (tumor growth factor-β), TNF-α (tumor necrosis factor-α), and IFN-γ (interferon-γ) undergo EndoMT with progressive loss of endothelial markers. The phenotypic change results in failure to maintain a tight barrier in culture, increased migration, and reduced angiogenesis. EndoMT also reduced mitochondrial oxidative phosphorylation and glycolytic capacity of EC. EVs produced by EC that underwent EndoMT dramatically reduced angiogenic capacity of the recipient naïve ECs without affecting their migration or proliferation. Proteomic analysis of EV produced by EC in the proinflammatory conditions showed presence of several pro-inflammatory and immune proteins along with an enrichment in angiogenic receptors. Conclusions: We demonstrated the presence of EndoMT in human AT in obesity. EndoMT in vitro resulted in production of EV that transferred some of the functional and metabolic features to recipient naïve EC. This result suggests that functional and molecular features of EC that underwent EndoMT in vivo can be disseminated in a paracrine or endocrine fashion and may induce endothelial dysfunction in distant vascular beds.

The tweety family of genes encodes large-conductance chloride channels and has been implicated in a wide array of cellular processes including cell division, cell adhesion, regulation of calcium activity, and tumorigenesis, particularly in neuronal cells. However, their expression patterns during early development remain largely unknown. Here, we describe the spatial and temporal patterning of ttyh1, ttyh2, and ttyh3 in Xenopus laevis during early embryonic development. Ttyh1 and ttyh3 are initially expressed at the late neurula stage are and primarily localized to the developing nervous system; however ttyh1 and ttyh3 both show transient expression in the somites. By swimming tadpole stages, all three genes are expressed in the brain, spinal cord, eye, and cranial ganglia. While ttyh1 is restricted to proliferative, ventricular zones, ttyh3 is primarily localized to postmitotic regions of the developing nervous system. Ttyh2, however, is strongly expressed in cranial ganglia V, VII, IX and X. The differing temporal and spatial expression patterns of ttyh1, ttyh2, and ttyh3 suggest that they may play distinct roles throughout embryonic development.

Diabetic non-healing wounds are a major clinical problem. The mechanisms leading to poor wound healing in diabetes are multifactorial but unresolved inflammation may be a major contributing factor. The complement system (CS) is the most potent inflammatory cascade in humans and contributes to poor wound healing in animal models. Signal transducer and activator of transcription 4 (STAT4) is a transcription factor expressed in immune and adipose cells and contributes to upregulation of some inflammatory chemokines and cytokines. Persistent CS and STAT4 expression in diabetic wounds may thus contribute to chronic inflammation and delayed healing. The purpose of this study was to characterize CS and STAT4 in early diabetic wounds using db/db mice as a diabetic skin wound model. The CS was found to be activated early in the diabetic wounds as demonstrated by increased anaphylatoxin C5a in wound fluid and C3-fragment deposition by immunostaining. These changes were associated with a 76% increase in nucleated cells in the wounds of db/db mice vs. controls. The novel classical CS inhibitor, Peptide Inhibitor of Complement C1 (PIC1) reduced inflammation when added directly or saturated in an acellular skin scaffold, as reflected by reduced CS components and leukocyte infiltration. A significant increase in expression of STAT4 and the downstream macrophage chemokine CCL2 and its receptor CCR2 were also found in the early wounds of db/db mice compared to non-diabetic controls. These studies provide evidence for two new promising targets to reduce unresolved inflammation and to improve healing of diabetic skin wounds.