Effect of aerobic exercise and particulate matter exposure duration on the diversity of gut microbiota.

Inhalation of ambient particulate matter (PM) can disrupt the gut microbiome, while exercise independently influences the gut microbiome by promoting beneficial bacteria. In this study, we analyzed changes in gut microbial diversity and composition in response to combined interventions of PM exposure and aerobic exercise, extending up to 12 weeks. This investigation was conducted using mice, categorized into five groups: control group (Con), exercise group (EXE), exercise group followed by 3-day exposure to PM (EXE + 3-day PM), particulate matter exposure (PM), and PM exposure with concurrent treadmill exercise (PME). Notably, the PM group exhibited markedly lower alpha diversity and richness compared to the Con group and our analysis of beta diversity revealed significant variations among the intervention groups. Members of the Lachnospiraceae family showed significant enhancement in the exercise intervention groups (EXE and PME) compared to the Con and PM groups. The biomarker Lactobacillus, Coriobacteraceae, and Anaerofustis were enriched in the EXE group, while Desulfovibrionaceae, Mucispirillum schaedleri, Lactococcus and Anaeroplasma were highly enriched in the PM group. Differential abundance analysis revealed that Paraprevotella, Bacteroides, and Blautia were less abundant in the 12-week PM exposure group than in the 3-day PM exposure group. Moreover, both the 3-day and 12-week PM exposure groups exhibited a reduced relative abundance of Bacteroides uniformis, SMB53, and Staphylococcus compared to non-PM exposure groups. These findings will help delineate the possible roles and associations of altered microbiota resulting from the studied interventions, paving the way for future mechanistic research.

Glucocorticoids alleviate particulate matter-induced COX-2 expression and mitochondrial dysfunction through the Bcl-2/GR complex in A549 cells.

Exposure to particulate matter (PM) causes mitochondrial dysfunction and lung inflammation. The cyclooxygenase-2 (COX-2) pathway is important for inflammation and mitochondrial function. However, the mechanisms by which glucocorticoid receptors (GRs) suppress COX-2 expression during PM exposure have not been elucidated yet. Hence, we examined the mechanisms underlying the dexamethasone-mediated suppression of the PM-induced COX-2/prostaglandin E2 (PGE2) pathway in A549 cells. The PM-induced increase in COX-2 protein, mRNA, and promoter activity was suppressed by glucocorticoids; this effect of glucocorticoids was antagonized by the GR antagonist RU486. COX-2 induction was correlated with the ability of PM to increase reactive oxygen species (ROS) levels. Consistent with this, antioxidant treatment significantly abolished COX-2 induction, suggesting that ROS is involved in PM-mediated COX-2 induction. We also observed a low mitochondrial membrane potential in PM-treated A549 cells, which was reversed by dexamethasone. Moreover, glucocorticoids significantly enhanced Bcl-2/GR complex formation in PM-treated A549 cells. Glucocorticoids regulate the PM-exposed induction of COX-2 expression and mitochondrial dysfunction and increase the interaction between GR and Bcl-2. These findings suggest that the COX-2/PGE2 pathway and the interaction between GR and Bcl-2 are potential key therapeutic targets for the suppression of inflammation under PM exposure.

Regulation of nuclear DNA damage response by mitochondrial morphofunctional pathway.

Cells are constantly challenged by genotoxic stresses that can lead to genome instability. The integrity of the nuclear genome is preserved by the DNA damage response (DDR) and repair. Additionally, these stresses can induce mitochondria to transiently hyperfuse; however, it remains unclear whether canonical DDR is linked to these mitochondrial morphological changes. Here, we report that the abolition of mitochondrial fusion causes a substantial defect in the ATM-mediated DDR signaling. This deficiency is overcome by the restoration of mitochondria fusion. In cells with fragmented mitochondria, genotoxic stress-induced activation of JNK and its translocation to DNA lesion are lost. Importantly, the mitochondrial fusion machinery of MFN1/MFN2 associates with Sab (SH3BP5) and JNK, and these interactions are indispensable for the Sab-mediated activation of JNK and the ATM-mediated DDR signaling. Accordingly, the formation of BRCA1 and 53BP1 foci, as well as homology and end-joining repair are impaired in cells with fragmented mitochondria. Together, these data show that mitochondrial fusion-dependent JNK signaling is essential for the DDR, providing vital insight into the integration of nuclear and cytoplasmic stress signals.

Intertwined Relationship of Mitochondrial Metabolism, Gut Microbiome and Exercise Potential.

The microbiome has emerged as a key player contributing significantly to the human physiology over the past decades. The potential microbial niche is largely unexplored in the context of exercise enhancing capacity and the related mitochondrial functions. Physical exercise can influence the gut microbiota composition and diversity, whereas a sedentary lifestyle in association with dysbiosis can lead to reduced well-being and diseases. Here, we have elucidated the importance of diverse microbiota, which is associated with an individual's fitness, and moreover, its connection with the organelle, the mitochondria, which is the hub of energy production, signaling, and cellular homeostasis. Microbial by-products, such as short-chain fatty acids, are produced during regular exercise that can enhance the mitochondrial capacity. Therefore, exercise can be employed as a therapeutic intervention to circumvent or subside various metabolic and mitochondria-related diseases. Alternatively, the microbiome-mitochondria axis can be targeted to enhance exercise performance. This review furthers our understanding about the influence of microbiome on the functional capacity of the mitochondria and exercise performance, and the interplay between them.

Effect of Aerobic Exercise on Oxidative Stress and Inflammatory Response During Particulate Matter Exposure in Mouse Lungs.

Regular exercise provides several health benefits that can improve the cardiovascular and musculoskeletal systems, but clear evidence on the effect of exercise-induced hyperventilation in particulate matter (PM) exposure is still lacking. This study aimed to investigate the effects of exercise in PM exposure on reactive oxygen species (ROS) generation, inflammatory response, and mitochondrial integrity in human lung epithelial cells (A549), as well as in mouse lung tissue. In in vitro experiments, PM treatment was shown to significantly increased ROS production, and reduced cell viability and mitochondrial function in A549 cells. The mice were divided into four groups for an in vivo exercise experiment: control (CON), PM inhalation (PI), PM inhalation during exercise (PIE), and exercise (EX) groups. The PI and PIE groups were exposed to 100 µg/m3 of PM for 1 h per day for a week. The PIE and EX groups performed treadmill exercises every day for 1 h at 20 m/min for a week. The levels of pro-inflammatory markers (IL-6 and TNF-α) were significantly higher in the PI group than in the CON group (P < 0.001 and P < 0.01, respectively). The carbonyl protein level was decreased in EX vs. PI (P < 0.001). Mitochondrial fission (Drp1) content was significantly decreased in the EX vs. CON group (P < 0.01), but anti-mitochondrial fission (P-Drp1 Ser637) was increased in the EX vs. PI group (P < 0.05). Mitochondrial autophagy (mitophagy), which is an assessment of mitochondrial integrity, was markedly increased in PI vs. CON (P < 0.001), but the level was reversed in PIE (P < 0.05). Lung fibrosis was increased in PI vs. CON group (P < 0.001), however, the cells were rescued in the PIE (P < 0.001). The number of apoptotic cells was remarkably increased in the PI vs. CON group (P < 0.001), whereas the level was decreased in the PIE (P < 0.001). Taken together, these results showed that short-term exposure to PM triggers oxidative stress, pro-inflammatory responses, and apoptosis in the lungs, but the PM-induced adverse effects on the lung tissue are not exacerbated by exercise-induced PM hyperventilation but rather has a protective effect.

Voluntary exercise training improves body weight of leptin-deficient ob/ob mice by altering hepatic stearoyl-CoA desaturase 1 and deleted in breast cancer 1 protein levels.

PURPOSE: Deleted in breast cancer 1 (DBC1) ablation causes obesity, and stearoyl-CoA desaturase 1 (SCD1) induces the biosynthesis of monounsaturated fatty acids. This study examined whether voluntary wheel running (VWR) alters SCD-1 and DBC1 protein levels in the liver of leptin-deficient ob/ob mice. METHODS: Twenty-five Ob/Ob mice were divided into two groups (ob/ob-Sed and ob/ob-Ex). The expression of DBC1 and SCD1 in the mouse liver was determined using western blotting. RESULTS: After 10 weeks, VWR significantly reduced body weight without affecting the fatty acid synthase and CD36 protein levels. The average daily running distance was 4.0±1.0 km/day. This improvement was associated with changes in the hepatic SCD1 and DBC1 levels. Hepatic SCD-1 protein levels increased significantly, and DBC1 protein levels decreased in ob/ob-Sed animals. On the other hand, VWR inhibited the obesity-induced increase in SCD1 expression and impaired the obesity-induced decrease in DBC1 expression in the liver of leptin-deficient ob/ob mice. CONCLUSION: This is the first study showing that VWR has strong effects on hepatic SCD1 and DBC1 in ob/ob mice, and provides key insights into the effects of exercise on obesity.

Avenanthramide C suppresses hypoxia-induced cyclooxygenase-2 expression through sirtuin1 activation in non-small-cell lung cancer cells.

Avenanthramide C (AVC), found mainly in oats, mediates anti-inflammatory activities by reducing the anti-inflammatory cytokine levels. This study investigated the effects of AVC on hypoxia-induced cyclooxygenase-2 (COX-2) expression in A549 cells. AVC suppressed the hypoxia-induced increase in COX-2 protein levels and promoter activity. We also observed that the effects of AVC were reversed by a SIRT1 inhibitor, indicating that the inhibitory effects of AVC on hypoxia-induced COX-2 expression are mediated by SIRT1. Therefore, AVC inhibits the hypoxic induction of COX-2 expression via SIRT1 activation. Our results suggest that AVC could be beneficial for preventing lung inflammation under hypoxia.

Dual targeting of RIG-I and MAVS by MARCH5 mitochondria ubiquitin ligase in innate immunity.

The mitochondrial antiviral signaling (MAVS) protein on the mitochondrial outer membrane acts as a central signaling molecule in the RIG-I-like receptor (RLR) signaling pathway by linking upstream viral RNA recognition to downstream signal activation. We previously reported that mitochondrial E3 ubiquitin ligase, MARCH5, degrades the MAVS protein aggregate and prevents persistent downstream signaling. Since the activated RIG-I oligomer interacts and nucleates the MAVS aggregate, MARCH5 might also target this oligomer. Here, we report that MARCH5 targets and degrades RIG-I, but not its inactive phosphomimetic form (RIG-IS8E). The MARCH5-mediated reduction of RIG-I is restored in the presence of MG132, a proteasome inhibitor. Upon poly(I:C) stimulation, RIG-I forms an oligomer and co-expression of MARCH5 reduces the expression of this oligomer. The RING domain of MARCH5 is necessary for binding to the CARD domain of RIG-I. In an in vivo ubiquitination assay, MARCH5 transfers the Lys 48-linked polyubiquitin to Lys 193 and 203 residues of RIG-I. Thus, dual targeting of active RIG-I and MAVS protein oligomers by MARCH5 is an efficient way to switch-off RLR signaling. We propose that modulation of MARCH5 activity might be beneficial for the treatment of chronic immune diseases.

Data on the mode of binding between avenanthramides and IKKß domains in a docking model.

The data presented in this article are related to the research paper entitled "Anti-inflammatory effect of avenanthramides via NF-κB pathways in C2C12 skeletal muscle cells." (Kang et al., in press) [1] This article includes experimental procedures used to analyze the mode of binding between and IkB kinase (IKKß) and avenanthramides which are a group of phenolic alkaloids found in oats. The protein-ligand docking and the computer simulation method of molecular dynamics (MD) for studying the physical interactions of molecules were performed.

Anti-inflammatory effect of avenanthramides via NF-κB pathways in C2C12 skeletal muscle cells.

Avenanthramides (Avns), the polyphenol compounds found only in oats, have been shown to exhibit anti-inflammatory effects mainly by inhibiting nuclear factor (NF)-κB activation in select cell lines. However, the molecular mechanism by which Avns regulate the NF-κB pathway is still unclear. The purpose of this study was to investigate (1) the molecular mechanism by which three main fractions of Avns (AvnA, AvnB and AvnC) interact with IκB Kinase ß (IKKß); and (2) whether this interaction results in reduced inflammatory responses in skeletal muscle cells. The protein-ligand docking and molecular dynamics simulation studies suggest that Avns acted as an allosteric inhibitor for modulating IKKß's affinity for the NF-κB complex. Thus, Avns reduced IKKß kinase activity in response to tert-butyl hydroperoxide (tBHP) stimulation and attenuated tBHP-induced TNFα and IL-1ß mRNA expression. Furthermore, the three-fold increases in cyclooxygenase-2 (COX-2) protein and luciferase activity with tBHP treatment were reduced by 50% with Avns (P < .01), along with decreased prostaglandin E2 levels (P < .01). These data indicate that Avns are potent inhibitors of NFκB-mediated inflammatory response due to the downregulation of IKKß activity in C2C12 cells.

Role of brown adipose tissue in metabolic syndrome, aging, and cancer cachexia.

Brown adipose tissue (BAT) plays a fundamental role in maintaining body temperature by producing heat. BAT that had been know to exist only in mammals and the human neonate has received great attention for the treatment of obesity and diabetes due to its important function in energy metabolism, ever since it is recently reported that human adults have functional BAT. In addition, beige adipocytes, brown adipocytes in white adipose tissue (WAT), have also been shown to take part in whole body metabolism. Multiple lines of evidence demonstrated that transplantation or activation of BAT or/and beige adipocytes reversed obesity and improved insulin sensitivity. Furthermore, many genes involved in BATactivation and/or the recruitment of beige cells have been found, thereby providing new promising strategies for future clinical application of BAT activation to treat obesity and metabolic diseases. This review focuses on recent advances of BAT function in the metabolic aspect and the relationship between BAT and cancer cachexia, a pathological process accompanied with decreased body weight and increased energy expenditure in cancer patients. The underlying possible mechanisms to reduce BAT mass and its activity in the elderly are also discussed.

Brown adipose tissue activation by rutin ameliorates polycystic ovary syndrome in rat.

Polycystic ovary syndrome (PCOS) is a complex endocrinopathy that is characterized by anovulation, hyperandrogenism and polycystic ovary. However, there is a lack of effective treatment for PCOS at present because the pathologic cause of PCOS has not been elucidated. Although it has been known that brown adipose tissue transplantation ameliorates PCOS by activating endogenous BAT, BAT transplantation is not applicable in clinic. Therefore, BAT activation with natural compound could be an effective treatment strategy for PCOS patients. Here, we found that 3 weeks of rutin (a novel compound for BAT activation) treatment increased BAT activation, thereby it improved thermogenesis and systemic insulin sensitivity in dehydroepiandrosterone (DHEA)-induced PCOS rat. In addition, the expression levels of ovarian steroidogenic enzymes such as P450C17, aromatase, 3ß-HSD, 17ß-HSD and STAR were up-regulated in rutin-treated PCOS rat. Furthermore, acyclicity and the serum level of luteinizing hormone were normalized, and a large number of mature ovulated follicle with a reduction of cystic formation were observed in PCOS rat after rutin treatment. Finally, rutin treatment surprisingly improved fertility and birth defect in PCOS rat. Collectively, our results indicate that rutin treatment significantly improves systemic insulin resistance and ovarian malfunction in PCOS, and our findings in this study provide a novel therapeutic option for the treatment of PCOS by activating BAT with rutin.

Data on mitochondrial function in skeletal muscle of old mice in response to different exercise intensity.

Endurance exercise is securely linked to muscle metabolic adaptations including enhanced mitochondrial function ("Effects of exercise on mitochondrial oxygen uptake and respiratory enzyme activity in skeletal muscle" [1], "Effects of exercise on mitochondrial content and function in aging human skeletal muscle" [2]). However, the link between exercise intensity and mitochondrial function in aging muscle has not been fully investigated. In order to understand how strenuous exercise affects mitochondrial function in aged mice, male C57BL/6 mice at age 24 months were randomly assigned to 3 groups: non-exercise (NE), low-intensity (LE) and high-intensity treadmill exercise group (HE). Mitochondrial complex activity and respiration were measured to evaluate mitochondrial function in mouse skeletal muscle. The data described here are related to the research article entitled "Strenuous exercise induces mitochondrial damage in skeletal muscle of old mice" [3].

Red ginseng represses hypoxia-induced cyclooxygenase-2 through sirtuin1 activation.

BACKGROUND: Korean red ginseng (KRG) is a traditional herbal medicine made by steaming and drying the fresh ginseng, leading to chemical transformation of some components by heat. It ameliorates various inflammatory diseases and strengthens the endocrine, immune, and central nervous systems. The cyclooxygenase-2 (COX-2)/prostaglandin E2 pathway in hypoxic cancer cells has important implications for stimulation of inflammation and tumorigenesis. PURPOSE: In this study we examined the effects and the mechanism underlying Korean red ginseng water extract (KRG-WE) inhibition of hypoxia-induced COX-2 in human distal lung epithelial A549 cells. STUDY DESIGN: The effect of the KRG on suppression of hypoxia-induced COX-2 in A549 cells were determined by Western blot and/or qRT-PCR. The anti-invasive effect of KRG-WE was evaluated on A549 cells using matrigel invasion assay. The activation of glucocorticoid receptor (GR) and sirtuin1 (Sirt1) was examined by using specific inhibitors. RESULTS: We first observed that hypoxia induced COX-2 protein and mRNA levels and promoter activity were suppressed by KRG-WE. Second, we observed that hypoxia-induced cell migration is dramatically reduced by KRG-WE. Third, we found that the effect of KRG-WE was not antagonized by the GR antagonist RU486 implying that the effect is mediated other than GR pathway. Finally, we demonstrated that inhibition of Sirt1 abolished the effect of KRG-WE on hypoxia-induced COX-2 suppression and cell-invasion indicating that the suppression is mediated by Sirt1. CONCLUSION: Taken together, KRG-WE inhibits the hypoxic induction of COX-2 expression and cell invasion through Sirt1 activation. Our results imply that KRG-WE could be effective for suppression of inflammation under hypoxia.

Subglutinol A, an immunosuppressive α-pyrone diterpenoid from Fusarium subglutinans, acts as an estrogen receptor antagonist.

Subglutinol A is an immunosuppressive α-pyrone diterpenoid isolated from Fusarium subglutinans that exhibits osteogenic activity. Several non-steroid mycotoxins isolated from various strains of Fusarium fungi exhibit female steroid hormone activities. In this study, we characterized the estrogenic activity of subglutinol A (1). Subglutinol A blocked the 17ß-estradiol-induced activation of reporter plasmids and endogenous estrogen-responsive target genes in a dose-dependent manner and efficiently destabilized ER proteins as shown using the estrogen receptor antagonist ICI 182,780. Subglutinol A also displaced the specific binding of [(3)H]17ß-estradiol from ER in MCF-7 whole-cell ligand binding assays. These data demonstrate the potential of subglutinol A as an ER antagonist though its competition with 17ß-estradiol for direct ER association.

Strenuous exercise induces mitochondrial damage in skeletal muscle of old mice.

Strenuous exercise is known to cause excessive ROS generation and inflammation. However, the mechanisms responsible for the regulation of mitochondrial integrity in the senescent muscle during high-intensity exercise (HE) are not well studied. Here, we show that HE suppresses up-regulation of mitochondrial function despite increase in mitochondrial copy number, following excessive ROS production, proinflammatory cytokines and NFκB activation. Moreover, HE in the old group resulted in the decreasing of both fusion (Mfn2) and fission (Drp1) proteins that may contribute to alteration of mitochondrial morphology. This study suggests that strenuous exercise does not reverse age-related mitochondrial damage and dysfunction by the increased ROS and inflammation.

Prognostic significance of catalase expression and its regulatory effects on hepatitis B virus X protein (HBx) in HBV-related advanced hepatocellular carcinomas.

Hepatitis B virus X protein (HBx) plays a role in liver cancer development. We previously showed that ROS increased HBx levels and here, we investigated the role of antioxidants in the regulation of HBx expression and their clinical relevance. We found that overexpression of catalase induced a significant loss in HBx levels. The cysteine null mutant of HBx (Cys-) showed a dramatic reduction in its protein stability. In clonogenic proliferation assays, Huh7-X cells produced a significant number of colonies whereas Huh7-Cys- cells failed to generate them. The Cys at position 69 of HBx was crucial to maintain its protein stability and transactivation function in response to ROS. Among 50 HBV-related hepatocellular carcinoma (HCC) specimens, 72% of HCCs showed lower catalase levels than those of surrounding non-tumor tissues. In advanced stage IV, catalase levels in non-tumor tissues were increased whereas those in tumors were further reduced. Accordingly, patients with a high T/N ratio for catalase showed significantly longer survival than those with a low T/N ratio. Together, catalase expression in HCC patients can be clinically useful for prediction of patient survival, and restoration of catalase expression in HCCs could be an important strategy for intervention in HBV-induced liver diseases.

Glucocorticoids suppress hypoxia-induced COX-2 and hypoxia inducible factor-1α expression through the induction of glucocorticoid-induced leucine zipper.

BACKGROUND AND PURPOSE: The COX-2/PGE2 pathway in hypoxic cancer cells has important implications for stimulation of inflammation and tumourigenesis. However, the mechanism by which glucocorticoid receptors (GRs) inhibit COX-2 during hypoxia has not been elucidated. Hence, we explored the mechanisms underlying glucocorticoid-mediated inhibition of hypoxia-induced COX-2 in human distal lung epithelial A549 cells. EXPERIMENTAL APPROACH: The expressions of COX-2 and glucocorticoid-induced leucine zipper (GILZ) in A549 cells were determined by Western blot and/or quantitative real time-PCR respectively. The anti-invasive effect of GILZ on A549 cells was evaluated using the matrigel invasion assay. KEY RESULTS: The hypoxia-induced increase in COX-2 protein and mRNA levels and promoter activity were suppressed by dexamethasone, and this effect of dexamethasone was antagonized by the GR antagonist RU486. Overexpression of GILZ in A549 cells also inhibited hypoxia-induced COX-2 expression levels and knockdown of GILZ reduced the glucocorticoid-mediated inhibition of hypoxia-induced COX-2 expression, indicating that the inhibitory effects of dexamethasone on hypoxia-induced COX-2 are mediated by GILZ. GILZ suppressed the expression of hypoxia inducible factor (HIF)-1α at the protein level and affected its signalling pathway. Hypoxia-induced cell invasion was also dramatically reduced by GILZ expression. CONCLUSION AND IMPLICATIONS: Dexamethasone-induced upregulation of GILZ not only inhibits the hypoxic-evoked induction of COX-2 expression and cell invasion but further blocks the HIF-1 pathway by destabilizing HIF-1α expression. Taken together, these findings suggest that the suppression of hypoxia-induced COX-2 by glucocorticoids is mediated by GILZ. Hence, GILZ is a potential key therapeutic target for suppression of inflammation under hypoxia.

Estrogen receptor beta inhibits transcriptional activity of hypoxia inducible factor-1 through the downregulation of arylhydrocarbon receptor nuclear translocator.

INTRODUCTION: Estrogen receptor (ER) ß is predicted to play an important role in prevention of breast cancer development and metastasis. We have shown previously that ERß inhibits hypoxia inducible factor (HIF)-1α mediated transcription, but the mechanism by which ERß works to exert this effect is not understood. METHODS: Vascular endothelial growth factor (VEGF) was measured in conditioned medium by enzyme-linked immunosorbent assays. Reverse transcription polymerase chain reaction (RT-PCR), Western blotting, immunoprecipitation, luciferase assays and chromatin immunoprecipitation (ChIP) assays were used to ascertain the implication of ERß on HIF-1 function. RESULTS: In this study, we found that the inhibition of HIF-1 activity by ERß expression was correlated with ERß's ability to degrade aryl hydrocarbon receptor nuclear translocator (ARNT) via ubiquitination processes leading to the reduction of active HIF-1α/ARNT complexes. HIF-1 repression by ERß was rescued by overexpression of ARNT as examined by hypoxia-responsive element (HRE)-driven luciferase assays. We show further that ERß attenuated the hypoxic induction of VEGF mRNA by directly decreasing HIF-1α binding to the VEGF gene promoter. CONCLUSIONS: These results show that ERß suppresses HIF-1α-mediated transcription via ARNT down-regulation, which may account for the tumour suppressive function of ERß.

Mammalian MST2 kinase and human Salvador activate and reduce estrogen receptor alpha in the absence of ligand.

Mammalian MST2 kinase plays an important role in cell proliferation, survival, and apoptosis. In search of interacting proteins of MST2, we found that estrogen receptor α (ERα) co-immunoprecipitates with MST2 and its adaptor protein human Salvador (hSAV). Using reporter assays, we observed that overexpression of MST2 and hSAV leads to ligand-independent activation of ERα in human breast cancer MCF-7 cells, which was attenuated by the knockdown of hSAV. Furthermore, using truncated mutants of hSAV, we observed that the C terminus of hSAV is necessary and sufficient for the induction of ERα transactivation. The expression of hSAV and MST2 results in the phosphorylation of ERα at serine residues 118 and 167 and represses ERα expression. We then investigated the incidence of MST2 and ERα expression with other tumor biomarkers using commercially available tissue microarrays. Among 40 breast cancer samples analyzed, 60% (24 out of 40) expressed MST2. Nineteen among the 40 cases were MST2-positive and ERα-negative, implying a correlation between expressions of MST2 with loss of ERα in breast tumor samples. This study suggests that MST and hSAV act as novel co-regulators of ERα and may play an important role in breast cancer pathogenesis.