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ObjectiveEstrogen-related receptor γ (ERRγ) is a metabolic regulator with no identified physiological ligands. This study investigates whether calcium is an ERRγ ligand that mediates the effects of glucagon and whether cadmium, which mimics the effects of calcium, disrupts metabolism through ERRγ.MethodHepG2, MCF-7, and HEK293T transfected with ERRγ were treated with glucagon, calcium, cadmium, ERRγ agonist, or ERRγ inhibitor. Cells were then collected for in vitro assays including real-time qPCR, Western blot, ChIP, immunofluorescence, mutational analysis, or gene set enrichment analysis. Molecular dynamics simulations were performed to study mutation sites.ResultsIn HepG2 cells, treatment with glucagon, calcium, or cadmium re-localized ERRγ to the cell nucleus, recruited ERRγ to estrogen-related response elements, induced the expression of ERRγ-regulated genes, and increased extracellular glucose that was blocked by an ERRγ antagonist. In MCF-7 cells and HEK293T cells transfected with ERRγ, similar treatments induced the expression of metabolic genes. Mutational analysis identified S303, T429, and E452 in the ligand-binding domain as potential interaction sites. Molecular dynamics simulations showed that calcium induced changes in ERRγ similar to ERRγ agonist.ConclusionThe results suggest that calcium is a potential ligand of ERRγ that mediates the effects of glucagon and cadmium disrupts metabolism through ERRγ.

ObjectiveRealgar possesses important medical properties. This article aims to evaluate realgar and emerging artificially optimized realgar to ensure safe clinical use.MethodsMultiple techniques were employed to test natural realgar and artificially optimized realgar. Soluble arsenic content in representative samples were measured. Natural realgar and artificially optimized realgar were administered to KM mice via gavage for 28 days, and the extent of liver and kidney tissue damage, arsenic accumulation and form of arsenic were measured.ResultsNatural realgar and artificially optimized realgar can be distinguished by their physical properties or spectral signatures. ICP-MS and EPMA identified different contents of elements between two groups. In simulated gastric and intestinal fluids, only As (III) and As (V) were detected. Toxicity experiments in vivo demonstrate that both groups caused minimal liver and kidney damage at a dose of 30 mg·kg−1. At a dose of 180 mg·kg−1, artificially optimized realgar caused significantly greater liver and kidney damage.ConclusionThe differences between natural realgar and artificially optimized realgar were successfully distinguished through several methods. In vitro experiments showed that As is the main component exerting their medicinal effects. In vivo toxicity tests demonstrated that at higher dose, artificially optimized realgar exhibited significantly higher toxicity, suggesting that natural and artificially optimized realgar have different toxic properties.

The estrogen receptor alpha (ERα) plays a central role in the etiology, progression, and treatment of breast cancers. Constitutively activating somatic mutations Y537S and D538G, in the ligand binding domain (LBD) of ESR1, are associated with acquired resistance to endocrine therapies. We have previously shown that the metalloestrogen calcium activates ERα through an interaction with the LBD of the receptor. This study shows that cadmium activates ERα through a mechanism similar to calcium and contributes to, and further increases, the constitutive activity of the ERα mutants Y537S and D538G. Mutational analysis identified C381, N532A, H516A/N519A/E523A, and E542/D545A on the solvent accessible surface of the LBD as possible calcium/metal interaction sites. In contrast to estradiol, which did not increase the activity of the Y537S and D538G mutants, cadmium increased the activity of the constitutive mutants. Mutation of the calcium/metal interaction sites in Y537S and D538G mutants resulted in a significant decrease in constitutive activity and cadmium induced activity. Mutation of calcium/metal interaction sites in wtERα diminished binding of the receptor to the enhancer of estrogen responsive genes and the binding of nuclear receptor coactivator 1 and RNA polymerase II. In contrast to wtERα, mutation of the calcium/metal interaction sites in the Y537S and D538G mutants did not diminish binding to DNA but prevented a stable interaction with the coactivator and polymerase. Growth assays further revealed that calcium channel blockers and chelators significantly decreased the growth of MCF7 cells expressing these constitutively active mutants. Taken together, the results suggest that exposure to cadmium plays a role in the etiology, progression, and response to treatment of breast cancer due, in part, to its ability to activate ERα.

IntroductionRealgar has a long history ofuse in traditional medicines. However, the mechanism through which Realgar or Realgar-Indigo naturalis formula (RIF) exert therapeutic effects is only partially understood.MethodsIn this study, 60 feces and 60 ileum samples from rats administered with realgar or RIF were collected to examine the gut microbiota.ResultsThe results showed that realgar and RIF influenced different microbiota in both feces and ileum. Compared with realgar, RIF at low dosage (0.1701 g/3 ml) significantly increased the microbiota diversity. LEfSe and random forest analyses showed that the bacterium Bacteroidales was significantly altered after RIF administration, and it was predicted that these microorganisms contribute to the inorganic arsenic metabolic process.DiscussionOur results suggest that realgar and RIF may exert their therapeutic effects through influencing microbiota. The low dose of RIF had greater effects on increasing the diversity of microbiota, and Bacteroidales in feces might participate in the inorganic arsenic metabolic process to exert therapeutic effects for realgar.

Agarwood, a highly valuable resin/wood combination with diverse pharmacological activities but scarce supply, has a long history of being used as a medicine in several medical systems. Grafted Kynam agarwood (GKA) has been cultivated successfully recently and has the qualities meeting the definition of premium Kynam agarwood. However, there are few comprehensive comparisons between GKA and normal agarwood in terms of traits, global composition, and activity, and some key issues for GKA to be adopted into the traditional Chinese medical (TCM) system have not been elaborated. The two types of agarwood samples were evaluated in terms of trait characteristics, physicochemical indicators, key component groups, and global compositional profile. Furthermore, a molecular docking was performed to investigate the active ingredients. In vitro activity assays were performed to evaluate the activation of adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK) by GKA and normal agarwood. The results revealed that, overall, the traits, microscopic characteristics, chemical composition types, and bioactivity between GKA and normal agarwood were similar. The main differences were the content of resin (ethanolic extract content), the content of key component groups, and the composition of the different parent structural groups of 2-(2-phenethyl) chromones (PECs). The contents of total PEC and ethanol extract content of GKA were significantly higher than those of normal agarwood. The MS-based high-throughput analysis revealed that GKA has higher concentrations of sesquiterpenes and flindersia-type 2-(2-phenylethyl) chromones (FTPECs) (m/z 250-312) than normal agarwood. Molecular docking revealed that parent structural groups of FTPECs activated multiple signaling pathways, including the AMPK pathway, suggesting that FTPECs are major active components in GKA. The aim of this paper is to describe the intrinsic reasons for GKA as a high-quality agarwood and a potential source for novel drug development. We combined high-throughput mass spectrometry and multivariate statistical analysis to infer the different components of the two types of agarwood. Then we combined virtual screening and in vitro activity to construct a component/pharmacodynamic relationship to explore the causes of the activity differences between agarwood with different levels of quality and to identify potentially valuable lead compounds. This strategy can also be used for the comprehensive study of other TCMs with different qualities.

Mutations of hepatocyte growth factor activator inhibitor (HAI)-2 in humans cause sodium loss in the gastrointestinal (GI) tract in patients with syndromic congenital sodium diarrhea (SCSD). Aberrant regulation of HAI-2 target protease(s) was proposed as the cause of the disease. Here functional linkage of HAI-2 with two membrane-associated serine proteases, matriptase and prostasin was analyzed in Caco-2 cells and the human GI tract. Immunodepletion-immunoblot analysis showed that significant proportion of HAI-2 is in complex with activated prostasin but not matriptase. Unexpectedly, prostasin is expressed predominantly in activated forms and was also detected in complex with HAI-1, a Kunitz inhibitor highly related to HAI-2. Immunohistochemistry showed a similar tissue distribution of prostasin and HAI-2 immunoreactivity with the most intense labeling near the brush borders of villus epithelial cells. In contrast, matriptase was detected primarily at the lateral plasma membrane, where HAI-1 was also detected. The tissue distribution profiles of immunoreactivity against these proteins, when paired with the species detected suggests that prostasin is under tight control by both HAI-1 and HAI-2 and matriptase by HAI-1 in human enterocytes. Furthermore, HAI-1 is a general inhibitor of prostasin in a variety of epithelial cells. In contrast, HAI-2 was not found to be a significant inhibitor for prostasin in mammary epithelial cells or keratinocytes. The high levels of constitutive prostasin zymogen activation and the selective prostasin inhibition by HAI-2 in enterocytes suggest that dysregulated prostasin proteolysis may be particularly important in the GI tract when HAI-2 function is lost and/or dysregulated.

The membrane-associated serine proteases matriptase and prostasin are believed to function in close partnership. Their zymogen activation has been reported to be tightly coupled, either as a matriptase-initiated proteolytic cascade or through a mutually dependent mechanism involving the formation of a reciprocal zymogen activation complex. Here we show that this putative relationship may not apply in the context of human matriptase and prostasin. First, the tightly coupled proteolytic cascade between matriptase and prostasin might not occur when modest matriptase activation is induced by sphingosine 1-phospahte in human mammary epithelial cells. Second, prostasin is not required and/or involved in matriptase autoactivation because matriptase can undergo zymogen activation in cells that do not endogenously express prostasin. Third, matriptase is not required for and/or involved in prostasin activation, since activated prostasin can be detected in cells expressing no endogenous matriptase. Finally, matriptase and prostasin both undergo zymogen activation through an apparently un-coupled mechanism in cells endogenously expressing both proteases, such as in Caco-2 cells. In these human enterocytes, matriptase is detected primarily in the zymogen form and prostasin predominantly as the activated form, either in complexes with protease inhibitors or as the free active form. The negligible levels of prostasin zymogen with high levels of matriptase zymogen suggests that the reciprocal zymogen activation complex is likely not the mechanism for matriptase zymogen activation. Furthermore, high level prostasin activation still occurs in Caco-2 variants with reduced or absent matriptase expression, indicating that matriptase is not required and/or involved in prostasin zymogen activation. Collectively, these data suggest that any functional relationship between natural endogenous human matriptase and prostasin does not occur at the level of zymogen activation.

The gene product of SPINT 2, that encodes a transmembrane, Kunitz-type serine protease inhibitor independently designated as HAI-2 or placenta bikunin (PB), is involved in regulation of sodium absorption in human gastrointestinal track. Here, we show that SPINT 2 is expressed as two species of different size (30-40- versus 25-kDa) due to different N-glycans on Asn-57. The N-glycan on 25-kDa HAI-2 appears to be of the oligomannose type and that on 30-40-kDa HAI-2 to be of complex type with extensive terminal N-acetylglucosamine branching. The two different types of N-glycan differentially mask two epitopes on HAI-2 polypeptide, recognized by two different HAI-2 mAbs. The 30-40-kDa form may be mature HAI-2, and is primarily localized in vesicles/granules. The 25-kDa form is likely immature HAI-2, that remains in the endoplasmic reticulum (ER) in the perinuclear regions of mammary epithelial cells. The two different N-glycans could, therefore, represent different maturation stages of N-glycosylation with the 25-kDa likely a precursor of the 30-40-kDa HAI-2, with the ratio of their levels roughly similar among a variety of cells. In breast cancer cells, a significant amount of the 30-40-kDa HAI-2 can translocate to and inhibit matriptase on the cell surface, followed by shedding of the matriptase-HAI-2 complex. The 25-kDa HAI-2 appears to have also exited the ER/Golgi, being localized at the cytoplasmic face of the plasma membrane of breast cancer cells. While the 25-kDa HAI-2 was also detected at the extracellular face of plasma membrane at very low levels it appears to have no role in matriptase inhibition probably due to its paucity on the cell surface. Our study reveals that N-glycan branching regulates HAI-2 through different subcellular distribution and subsequently access to different target proteases.

The estrogen receptor alpha (ERa) plays a central role in the etiology, progression, and treatment of breast cancers. Constitutively activating somatic mutations Y537S and D538G, in the ligand binding domain (LBD) of ESR1, are associated with acquired resistance to endocrine therapies. We have previously shown that the metalloestrogen calcium activates ERa through an interaction with the LBD of the receptor. This study shows that cadmium activates ERa through a mechanism similar to calcium and contributes to, and further increases, the constitutive activity of the ERa mutants Y537S and D538G. Mutational analysis identified C381, N532A, H516A/N519A/E523A, and E542/ D545A on the solvent accessible surface of the LBD as possible calcium/metal interaction sites. In contrast to estradiol, which did not increase the activity of the Y537S and D538G mutants, cadmium increased the activity of the constitutive mutants. Mutation of the calcium/metal interaction sites in Y537S and D538G mutants resulted in a significant decrease in constitutive activity and cadmium induced activity. Mutation of calcium/metal interaction sites in wtERa diminished binding of the receptor to the enhancer of estrogen responsive genes and the binding of nuclear receptor coactivator 1 and RNA polymerase II. In contrast to wtERa, mutation of the calcium/metal interaction sites in the Y537S and D538G mutants did not diminish binding to DNA but prevented a stable interaction with the coactivator and polymerase. Growth assays further revealed that calcium channel blockers and chelators significantly decreased the growth of MCF7 cells expressing these constitutively active mutants. Taken together, the results suggest that exposure to cadmium plays a role in the etiology, progression, and response to treatment of breast cancer due, in part, to its ability to activate ERa. [ABSTRACT FROM AUTHOR]