UHRF1 silences gelsolin to inhibit cell death in early stage cervical cancer.

Persistent infection with high-risk strains of human papillomavirus (HPV) is the primary cause of cervical cancer, the fourth most common cancer among women worldwide. Two oncoproteins encoded by the HPV genome, E6 and E7, are required for epigenetic modifications that promote cervical cancer development. We found that knockdown of HPV E6/E7 by siRNA reduced the levels of ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) but increased the levels of gelsolin (GSN) in early stage cervical cancer cells. In addition, we found that UHRF1 levels were increased and GSN levels were decreased in early stage cervical cancer compared with those in normal cervical tissues, as shown by Western blot analysis, immunohistochemistry, and analysis of the Oncomine database. Moreover, knockdown of UHRF1 resulted in increased cell death in cervical cancer cell lines. Treatment of E6/E7-transformed HaCaT (HEK001) cells and HeLa cells with the DNA-hypomethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor Trichostatin A increased GSN expression levels. UHRF1 knockdown in HEK001 cells by siRNA or the UHRF1 antagonist thymoquinone increased GSN levels, induced cell cycle arrest and apoptosis, and increased the levels of p27 and cleaved PARP. Those results indicate that upregulation of UHRF1 by HPV E6/E7 causes GSN silencing and a reduction of cell death in early stage cervical cancer, suggesting that GSN might be a useful therapeutic target in early stage cervical cancer.

Cross-talk between CD38 and TTP Is Essential for Resolution of Inflammation during Microbial Sepsis.

The resolution phase of acute inflammation is essential for tissue homeostasis, yet the underlying mechanisms remain unclear. We demonstrate that resolution of inflammation involves interactions between CD38 and tristetraprolin (TTP). During the onset of acute inflammation, CD38 levels are increased, leading to the production of Ca2+-signaling messengers, nicotinic acid adenine dinucleotide phosphate (NAADP), ADP ribose (ADPR), and cyclic ADPR (cADPR) from NAD(P)+. To initiate the onset of resolution, TTP expression is increased by the second messengers, NAADP and cADPR, which downregulate CD38 expression. The activation of TTP by Sirt1-dependent deacetylation, in response to increased NAD+ levels, suppresses the acute inflammatory response and decreases Rheb expression, inhibits mTORC1, and induces autophagolysosomes for bacterial clearance. TTP may represent a mechanistic target of anti-inflammatory agents, such as carbon monoxide. TTP mediates crosstalk between acute inflammation and autophagic clearance of bacteria from damaged tissue in the resolution of inflammation during sepsis.

Metformin inhibits cervical cancer cell proliferation via decreased AMPK O-GlcNAcylation.

Metformin is a widely used drug for the treatment of type 2 diabetes. Antidiabetic drugs are also known to influence cancer progression, as high glucose levels affect both cancer and diabetes. Metformin induces cell cycle arrest in cancer cells, but the underlying mechanism remains unclear in cervical cancer system. Here, we examined how metformin affects cell cycle arrest and apoptosis in cervical cancer cells. Western blot analysis showed that levels of O-linked N-acetylglucosamine (O-GlcNAc) and O-GlcNAc transferase (OGT) were increased in cervical cancer cells; these effects were reversed by metformin treatment. Immunoprecipitation analysis was used to examine the interplay between O-GlcNAcylation and phosphorylation in HeLa cells, revealing that metformin decreased O-GlcNAcylated AMP-activated protein kinase (AMPK) and increased levels of phospho-AMPK compared to untreated cells. These results were associated with decreased cell cycle arrest and apoptotic cell death in HeLa cells, as shown by flow cytometry. Moreover, 6-diazo-5-oxo-L-norleucine (a glutamine fructose-6-phosphate aminotransferase inhibitor) or thiamet G (an O-GlcNAcase inhibitor) decreased or increased levels of O-GlcNAcylated AMPK, and increased or decreased levels of phosphorylated AMPK, respectively, suggesting that O-GlcNAc modification affects AMPK activation. Of note, we found that metformin treatment of HeLa cells increased the levels of p21 and p27 (which are AMPK-dependent cell cycle inhibitors), leading to increased cell cycle arrest and apoptosis in HeLa cells compared to untreated cells. These findings suggest that metformin may serve as a useful antiproliferative drug in cervical cancer cells, with potential therapeutic benefit.

Pterostilbene 4'-ß-Glucoside Attenuates LPS-Induced Acute Lung Injury via Induction of Heme Oxygenase-1.

Heme oxygenase-1 (HO-1) can exert anti-inflammatory and antioxidant effects. Acute lung injury (ALI) is associated with increased inflammation and influx of proinflammatory cells and mediators in the airspaces and lung parenchyma. In this study, we demonstrate that pterostilbene 4'-ß-glucoside (4-PG), the glycosylated form of the antioxidant pterostilbene (PTER), can protect against lipopolysaccharide- (LPS-) or Pseudomonas aeruginosa- (P. aeruginosa-) induced ALI when applied as a pretreatment or therapeutic post-treatment, via the induction of HO-1. To determine whether HO-1 mediates the antioxidant and anti-inflammatory effects of 4-PG, we subjected mice genetically deficient in Hmox-1 to LPS-induced ALI and evaluated histological changes, HO-1 expression, and proinflammatory cytokine levels in bronchoalveolar lavage (BAL) fluid. 4-PG exhibited protective effects on LPS- or P. aeruginosa-induced ALI by ameliorating pathological changes in lung tissue and decreasing proinflammatory cytokines. In addition, HO-1 expression was significantly increased by 4-PG in cells and in mouse lung tissues. The glycosylated form of pterostilbene (4-PG) was more effective than PTER in inducing HO-1 expression. Genetic deletion of Hmox-1 abolished the protective effects of 4-PG against LPS-induced inflammatory responses. Furthermore, we found that 4-PG decreased both intracellular ROS levels and mitochondrial (mt) ROS production in a manner dependent on HO-1. Pharmacological application of the HO-1 reaction product carbon monoxide (CO), but not biliverdin or iron, conferred protection in Hmox-1-deficient macrophages. Taken together, these results demonstrate that 4-PG can increase HO-1 expression, which plays a critical role in ameliorating intracellular and mitochondrial ROS production, as well as in downregulating inflammatory responses induced by LPS. Therefore, these findings strongly suggest that HO-1 mediates the antioxidant and anti-inflammatory effects of 4-PG.

O-linked N-acetylglucosamine transferase enhances secretory clusterin expression via liver X receptors and sterol response element binding protein regulation in cervical cancer.

O-linked N-acetylglucosamine transferase (OGT) expression is increased in various cancer types, indicating the potential importance of O-GlcNAcylation in tumorigenesis. Secretory clusterin (sCLU) is involved in cancer cell proliferation and drug resistance, and recently, liver X receptors (LXRs) and sterol response element binding protein-1 (SREBP-1) were reported to regulate sCLU transcription. Here, we found that sCLU is significantly increased in cervical cancer cell lines, which have higher expression levels of O-GlcNAc and OGT than keratinocytes. OGT knockdown decreased expression of LXRs, SREBP-1 and sCLU through hypo-O-GlcNAcylation of LXRs. Additionally, treatment with Thiamet G, O-GlcNAcase OGA inhibitor, increased expression of O-GlcNAcylation and sCLU, and high glucose increased levels of LXRs, SREBP-1 and sCLU in HeLa cells. Moreover, OGT knockdown induced G0/G1 phase cell cycle arrest and late apoptosis in cisplatin-treated HeLa cells, and decreased viability compared to OGT intact HeLa cells. Taken together, these findings suggest that OGT, O-GlcNAcylated LXRs, and SREBP-1 increase sCLU expression in cervical cancer cells, which contributes to drug resistance.

Quercetin induces cell death in cervical cancer by reducing O-GlcNAcylation of adenosine monophosphate-activated protein kinase.

Hyper-O-GlcNAcylation is a general feature of cancer which contributes to various cancer phenotypes, including cell proliferation and cell growth. Quercetin, a naturally occurring dietary flavonoid, has been reported to reduce the proliferation and growth of cancer. Several reports of the anticancer effect of quercetin have been published, but there is no study regarding its effect on O-GlcNAcylation. The aim of this study was to investigate the anticancer effect of quercetin on HeLa cells and compare this with its effect on HaCaT cells. Cell viability and cell death were determined by MTT and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling assays. O-GlcNAcylation of AMP-activated protein kinase (AMPK) was examined by succinylated wheat germ agglutinin pulldown and immunoprecipitation. Immunofluorescence staining was used to detect the immunoreactivitiy of O-linked N-acetylglucosamine transferase (OGT) and sterol regulatory element binding protein 1 (SREBP-1). Quercetin decreased cell proliferation and induced cell death, but its effect on HaCaT cells was lower than that on HeLa cells. O-GlcNAcylation level was higher in HeLa cells than in HaCaT cells. Quercetin decreased the expression of global O-GlcNAcylation and increased AMPK activation by reducing the O-GlcNAcylation of AMPK. AMPK activation due to reduced O-GlcNAcylation of AMPK was confirmed by treatment with 6-diazo-5-oxo-L-norleucine. Our results also demonstrated that quercetin regulated SREBP-1 and its transcriptional targets. Furthermore, immunofluorescence staining showed that quercetin treatment decreased the immunoreactivities of OGT and SREBP-1 in HeLa cells. Our findings demonstrate that quercetin exhibited its anticancer effect by decreasing the O-GlcNAcylation of AMPK. Further studies are needed to explore how quercetin regulates O-GlcNAcylation in cancer.

O-GlcNAcylation of NF-κB Promotes Lung Metastasis of Cervical Cancer Cells via Upregulation of CXCR4 Expression.

C-X-C chemokine receptor 4 (CXCR4) stimulates cancer metastasis. NF-κB regulates CXCR4 expression in cancer cells, and O-GlcNAc modification of NF-κB promotes its transcriptional activity. Here, we determined whether CXCR4 expression is affected by O-GlcNAcylation of NF-κB in lung metastasis of cervical cancer. We found elevated levels of O-linked-N-actylglucosamine transferase (OGT) and O-GlcNAcylation in cervical cancer cells compared to those in non-malignant epithelial cells and detected increased expression of NF-κB p65 (p65) and CXCR4 in cervical cancer cells. Knockdown of OGT inhibited the O-GlcNAcylation of p65 and decreased CXCR4 expression levels in HeLa cells. Thiamet G treatment increased O-GlcNAcylated p65, which subsequently enhanced CXCR4 expression levels. Inhibition of O-GlcNAcylation by 6-Diazo-5-oxo-L-norleucine (DON) treatment decreased p65 activation, eventually inhibiting CXCR4 expression in HeLa cells. Lung tissues from mice engrafted with OGT-knockdown HeLa cells (shOGT) exhibited lower expression of Ki-67 and HPV E6 and E7 oncogenes compared to lung tissues from mice engrafted with control HeLa cells (shCTL). In addition, lung tissues from mice engrafted with shOGT cells exhibited lower p65 and CXCR4 immunoreactivity compared to tissues from mice engrafted with shCTL cells. Taken together, our data suggest that p65 O-GlcNAcylation promotes lung metastasis of cervical cancer cells by activating CXCR4 expression.

Carbon monoxide decreases interleukin-1ß levels in the lung through the induction of pyrin.

Carbon monoxide (CO) can act as an anti-inflammatory effector in mouse models of lung injury and disease, through the downregulation of pro-inflammatory cytokines production, though the underlying mechanisms remain unclear. The nucleotide-binding oligomerization domain-, leucine-rich region-, and pyrin domain-containing-3 (NLRP3) inflammasome is a protein complex that regulates the maturation and secretion of pro-inflammatory cytokines, including interleukin-1ß (IL-1ß). In this report, we show that the CO-releasing molecule (CORM-2) can stimulate the expression of pyrin, a negative regulator of the NLRP3 inflammasome. CORM-2 increased the transcription of pyrin in the human leukemic cell line (THP-1) in the absence and presence of lipopolysaccharide (LPS). In THP-1 cells, CORM-2 treatment dose-dependently reduced the activation of caspase-1 and the secretion of IL-1ß, and increased the levels of IL-10, in response to LPS and adenosine 5'-triphosphate (ATP), an NLRP3 inflammasome activation model. Genetic interference of IL-10 by small interfering RNA (siRNA) reduced the effectiveness of CORM-2 in inhibiting IL-1ß production and in inducing pyrin expression. Genetic interference of pyrin by siRNA increased IL-1ß production in response to LPS and ATP, and reversed CORM-2-dependent inhibition of caspase-1 activation. CO inhalation (250 ppm) in vivo increased the expression of pyrin and IL-10 in lung and spleen, and decreased the levels of IL-1ß induced by LPS. Consistent with the induction of pyrin and IL-10, and the downregulation of lung IL-1ß production, CO provided protection in a model of acute lung injury induced by intranasal LPS administration. These results provide a novel mechanism underlying the anti-inflammatory effects of CO, involving the IL-10-dependent upregulation of pyrin expression.

O-linked-N-acetylglucosamine transferase is associated with metastatic spread of human papillomavirus E6 and E7 oncoproteins to the lungs of mice.

High-risk human papilloma virus (HPV) 16/18 infections are often found in lung cancer. The cellular mechanisms involved in the metastatic spread of HPV-infected cervical cancer cells remain largely elusive. High O-linked-N-acetylglucosamine (O-GlcNAc) modification has also been observed in lung cancer. In the present study, we assessed the relationship between O-GlcNAc transferase (OGT) and HPV 16/18 E6/E7, or C-X-C chemokine receptor type 4 (CXCR4), in HeLa cells and in lungs of xenografted mice. Depleting OGT with an OGT-specific shRNA significantly decreased levels of E6 and E7 oncoproteins in HeLa cells and xenograft tumors, and reduced tumor formation in vivo. Western blotting and immunofluorescence analysis showed significantly decreased expression levels of E6, E7, and HCF-1 in the lungs of xenografted mice treated with an OGT-specific shRNA compared to those treated with non-targeting shRNA. Additionally, levels of E7 or OGT co-localized with Ki-67 were significantly decreased in the lungs of xenografted mice treated with OGT-specific shRNA compared to those treated with non-targeting shRNA. Moreover, levels of CXCR4 were significantly decreased in HeLa cells and in the lungs of xenografted mice treated with OGT-specific shRNA compared to those treated with non-targeting shRNA; this may be related to reduced adhesion or invasion of circulating HPV-positive tumor cells. These findings provide novel evidence that OGT functions in metastatic spread of HPV E6/E7-positive tumor cells to the lungs through E6/E7, HCF-1 and CXCR4, suggesting OGT might be a therapeutic target for HPV-positive lung cancer.

O-linked N-acetylglucosamine transferase promotes cervical cancer tumorigenesis through human papillomaviruses E6 and E7 oncogenes.

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) increases O-GlcNAc modification (O-GlcNAcylation), and transcriptional co-regulator host cell factor 1 (HCF-1) is one of OGT targets. High-risk Human Papillomaviruses (HPVs) encode E6 and E7 oncoproteins, which promote cervical cancer. Here, we tested whether O-GlcNAc modification of HCF-1 affects HPV E6 and E7 expressions and tumorigenesis of cervical cancer. We found that depleting OGT with OGT-specific shRNA significantly decreased levels of E6 and E7 oncoproteins, and cervical cancer tumorigenesis, while OGT overexpression greatly increased levels of E6 and E7 oncoproteins. Notably, OGT overexpression caused dose-dependent increases in the transcriptional activity of E6 and E7, and this activity was decreased when HCF-1 was depleted with HCF-1-specific siRNA. Moreover, OGT depletion reduced proliferation, invasion, and metastasis in cervical cancer cells. Further, high glucose enhanced the interaction between OGT and HCF-1, paralleling increased levels of E6 and E7 in cervical cancer cells. Most importantly, we found that reducing OGT in HeLa cells caused decreased tumor growth in vivo. These findings identify OGT as a novel cellular factor involved in E6 and E7 expressions and cervical cancer tumorigenesis, suggesting that targeting OGT in cervical cancer may have potential therapeutic benefit.

Synergistic Effects of Cilostazol and Probucol on ER Stress-Induced Hepatic Steatosis via Heme Oxygenase-1-Dependent Activation of Mitochondrial Biogenesis.

The selective type-3 phosphodiesterase inhibitor cilostazol and the antihyperlipidemic agent probucol have antioxidative, anti-inflammatory, and antiatherogenic properties. Moreover, cilostazol and probucol can regulate mitochondrial biogenesis. However, the combinatorial effect of cilostazol and probucol on mitochondrial biogenesis remains unknown. Endoplasmic reticulum (ER) stress is a well-known causative factor of nonalcoholic fatty liver disease (NAFLD) which can impair mitochondrial function in hepatocytes. Here, we investigated the synergistic effects of cilostazol and probucol on mitochondrial biogenesis and ER stress-induced hepatic steatosis. A synergistic effect of cilostazol and probucol on HO-1 and mitochondrial biogenesis gene expression was found in human hepatocellular carcinoma cells (HepG2) and murine primary hepatocytes. Furthermore, in an animal model of ER stress involving tunicamycin, combinatorial treatment with cilostazol and probucol significantly increased the expression of HO-1 and mitochondrial biogenesis-related genes and proteins, whereas it downregulated serum ALT, eIF2 phosphorylation, and CHOP expression, as well as the lipogenesis-related genes SREBP-1c and FAS. Based on these results, we conclude that cilostazol and probucol exhibit a synergistic effect on the activation of mitochondrial biogenesis via upregulation of HO-1, which confers protection against ER stress-induced hepatic steatosis.

Resveratrol Induces Glioma Cell Apoptosis through Activation of Tristetraprolin.

Tristetraprolin (TTP) is an AU-rich elements (AREs)-binding protein, which regulates the decay of AREs-containing mRNAs such as proto-oncogenes, anti-apoptotic genes and immune regulatory genes. Despite the low expression of TTP in various human cancers, the mechanism involving suppressed expression of TTP is not fully understood. Here, we demonstrate that Resveratrol (3,5,4'-trihydroxystilbene, Res), a naturally occurring compound, induces glioma cell apoptosis through activation of tristetraprolin (TTP). Res increased TTP expression in U87MG human glioma cells. Res-induced TTP destabilized the urokinase plasminogen activator and urokinase plasminogen activator receptor mRNAs by binding to the ARE regions containing the 3' untranslated regions of their mRNAs. Furthermore, TTP induced by Res suppressed cell growth and induced apoptosis in the human glioma cells. Because of its regulation of TTP expression, these findings suggest that the bioactive dietary compound Res can be used as a novel anti-cancer agent for the treatment of human malignant gliomas.

Cilostazol attenuates murine hepatic ischemia and reperfusion injury via heme oxygenase-dependent activation of mitochondrial biogenesis.

Hepatic ischemia-reperfusion (I/R) can cause hepatocellular injury associated with the inflammatory response and mitochondrial dysfunction. We studied the protective effects of the phosphodiesterase inhibitor cilostazol in hepatic I/R and the roles of mitochondria and the Nrf2/heme oxygenase-1 (HO-1) system. Wild-type, Hmox1(-/-), or Nrf2(-/-) mice were subjected to hepatic I/R in the absence or presence of cilostazol followed by measurements of liver injury. Primary hepatocytes were subjected to cilostazol with the HO-1 inhibitor ZnPP, or Nrf2-specific siRNA, followed by assessment of mitochondrial biogenesis. Preconditioning with cilostazol prior to hepatic I/R protected against hepatocellular injury and mitochondrial dysfunction. Cilostazol reduced the serum levels of alanine aminotransferase, TNF-α, and liver myeloperoxidase content relative to control I/R-treated mice. In primary hepatocytes, cilostazol increased the expression of HO-1, and markers of mitochondrial biogenesis, PGC-1α, NRF-1, and TFAM, induced the mitochondrial proteins COX III and COX IV and increased mtDNA and mitochondria content. Pretreatment of primary hepatocytes with ZnPP inhibited cilostazol-induced PGC-1α, NRF-1, and TFAM mRNA expression and reduced mtDNA and mitochondria content. Genetic silencing of Nrf2 prevented the induction of HO-1 and mitochondrial biogenesis by cilostazol in HepG2 cells. Cilostazol induced hepatic HO-1 production and mitochondrial biogenesis in wild-type mice, but not in Hmox1(-/-) or Nrf2(-/-) mice, and failed to protect against liver injury in Nrf2(-/-) mice. These results suggest that I/R injury can impair hepatic mitochondrial function, which can be reversed by cilostazol treatment. These results also suggest that cilostazol-induced mitochondrial biogenesis was mediated by an Nrf-2- and HO-1-dependent pathway.

Carbon monoxide protects against hepatic ischemia/reperfusion injury by modulating the miR-34a/SIRT1 pathway.

Hepatic ischemia/reperfusion (I/R) injury can arise as a complication of liver surgery and transplantation. Sirtuin 1 (SIRT1), an NAD+-dependent deacetylase, modulates inflammation and apoptosis in response to oxidative stress. SIRT1, which is regulated by p53 and microRNA-34a (miR-34a), can modulate non-alcoholic fatty liver disease, fibrosis and cirrhosis. Since carbon monoxide (CO) inhalation can protect against hepatic I/R, we hypothesized that CO could ameliorate hepatic I/R injury by regulating the miR-34a/SIRT1 pathway. Livers from mice pretreated with CO, or PFT, a p53 inhibitor, displayed reduced production of pro-inflammatory mediators, including TNF-α, iNOS, interleukin (IL)-6, and IL-1ß after hepatic I/R injury. SIRT1 expression was increased by CO or PFT in the liver after I/R, whereas acetylated p65, p53 levels, and miR-34a expression were decreased. CO increased SIRT1 expression by inhibiting miR-34a. Both CO and PFT diminished pro-inflammatory cytokines production in vitro. Knockdown of SIRT1 in LPS-stimulated macrophages increased NF-κB acetylation, and increased pro-inflammatory cytokines. CO treatment reduced miR-34a expression and increased SIRT1 expression in oxidant-challenged hepatocytes; and rescued SIRT1 expression in p53-expressing or miR-34a transfected cells. In response to CO, enhanced SIRT1 expression mediated by miR-34a inhibition protects against liver damage through p65/p53 deacetylation, which may mediate inflammatory responses and hepatocellular apoptosis. The miR-34a/SIRT1 pathway may represent a therapeutic target for hepatic injury.

Tristetraprolin inhibits the growth of human glioma cells through downregulation of urokinase plasminogen activator/urokinase plasminogen activator receptor mRNAs.

Urokinase plasminogen activator (uPA) and urokinase plasminogen activator receptor (uPAR) play a major role in the infiltrative growth of glioblastoma. Downregulatoion of the uPA and uPAR has been reported to inhibit the growth glioblastoma. Here, we demonstrate that tristetraprolin (TTP) inhibits the growth of U87MG human glioma cells through downregulation of uPA and uPAR. Our results show that expression level of TTP is inversely correlated with those of uPA and uPAR in human glioma cells and tissues. TTP binds to the AU-rich elements within the 3' untranslated regions of uPA and uPAR and overexpression of TTP decreased the expression of uPA and uPAR through enhancing the degradation of their mRNAs. In addition, overexpression of TTP inhibited the growth and invasion of U87MG cells. Our findings implicate that TTP can be used as a promising therapeutic target to treat human glioma.

FK506 reduces calpain-regulated calcineurin activity in both the cytoplasm and the nucleus.

Excessive immune responses induced by ischemia-reperfusion injury (IRI) are known to lead to necrotic and apoptotic cell death, and calcineurin plays a major role in this process. Calcineurin dephosphorylates the nuclear factor of activated T-cells (NFAT), permitting its translocation into the nucleus. As a result, calcineurin promotes the release of pro-inflammatory cytokines, such as tumor necrosis factor-α. The overproduction of pro-inflammatory cytokines causes renal cell death. Calcineurin activity is regulated by calpain, a cysteine protease present in the nucleus. Calpain-mediated proteolysis increases the phosphatase activity of calcineurin, resulting in NFAT dephosphorylation. This process has been studied in cardiomyocytes but its role in renal IRI is unknown. Thus, we examined whether calpain regulates calcineurin in renal tubule nuclei. We established an in vivo renal IRI model in mice and identified the protective role of a calcineurin inhibitor, FK506, in this process. Calcineurin is expressed in the nucleus, where it is present in its calpain-cleaved form. FK506 reduced nuclear expression of calcineurin and prevented calcineurin-mediated NFAT activation. Our study shows clearly that FK506 reduces calpain-mediated calcineurin activity. Consequently, calcineurin could not maintain NFAT activation. FK506 reduced renal cell death by suppressing the transcription of pro-inflammatory cytokine genes. This study provides evidence that FK506 protects against inflammation in a renal IRI mouse model. We also provided a mechanism of calcineurin action in the nucleus. Therefore, FK506 could improve renal function by decreasing calcineurin activity in both the cytoplasm and the nucleus of renal tubule cells.

Hypoxia inducible factor-1α directly regulates nuclear clusterin transcription by interacting with hypoxia response elements in the clusterin promoter.

Differential transcription of the clusterin (CLU) gene yields two CLU isoforms, a nuclear form (nCLU) and a secretory form (sCLU), which play crucial roles in prostate tumorigenesis. Pro-apoptotic nCLU and anti-apoptotic sCLU have opposite effects and are differentially expressed in normal and cancer cells; however, their regulatory mechanisms at the transcriptional level are not yet known. Here, we examined the transcriptional regulation of nCLU in response to hypoxia. We identified three putative hypoxia response elements (HREs) in the human CLU promoter between positions -806 and +51 bp. Using a luciferase reporter, electrophoretic gel mobility shift, and chromatin immunoprecipitation assays, we further showed that hypoxia-inducible factor-1α (HIF-1α) bound directly to these sites and activated transcription. Exposure to the hypoxiamimetic compound CoCl2, incubation under 1% O2 conditions, or overexpression of HIF-1α enhanced nCLU expression and induced apoptosis in human prostate cancer PC3M cells. However, LNCaP prostate cancer cells were resistant to hypoxia-induced cell death. Methylation-specific PCR analysis revealed that the CLU promoter in PC3M cells was not methylated; in contrast, the CLU promoter in LNCap cells was methylated. Co-treatment of LNCaP cells with CoCl2 and a demethylating agent promoted apoptotic cell death through the induction of nCLU. We conclude that nCLU expression is regulated by direct binding of HIF-1α to HRE sites and is epigenetically controlled by methylation of its promoter region.

Tristetraprolin mediates anti-inflammatory effect of carbon monoxide against DSS-induced colitis.

Endogenous carbon monoxide (CO) exerts anti-inflammatory effects. Tristetraprolin (TTP) is known to destabilize pro-inflammatory transcripts. Here we found that exogenous CO enhanced the decay of TNF-α mRNA and suppressed TNF-α expression in LPS-activated macrophages from wild-type (WT) mice. However, TTP deficiency abrogated the effects of exogenous CO. While CO treatment prior to DSS administration in WT mice significantly reduced inflammatory cytokine levels and colitis, it failed to reduce the pro-inflammatory cytokine levels and colitis in TTP knockout (KO) mice. Our results demonstrate that TTP is a key factor mediating the anti-inflammatory action of CO in DSS-induced colitis.

Tonicity-responsive enhancer binding protein regulates the expression of aldose reductase and protein kinase C δ in a mouse model of diabetic retinopathy.

Recent studies revealed that Tonicity-responsive enhancer binding protein (TonEBP) directly regulates the transcription of aldose reductase (AR), which catalyzes the first step of the polyol pathway of glucose metabolism. Activation of protein kinase C δ (PKCδ) is dependent on AR and it has been linked to diabetic complications. However, whether TonEBP affects expressions of AR and PKCδ in diabetic retinopathy was not clearly shown. In this study, we used TonEBP heterozygote mice to study the role of TonEBP in streptozotocin (STZ)-induced diabetic retinopathy. We performed immunofluorescence staining and found that retinal expressions of AR and PKCδ were significantly reduced in the heterozygotes compared to wild type littermates, particularly in ganglion cell layer. To examine further the effect of TonEBP reduction in retinal tissues, we performed intravitreal injection of TonEBP siRNA and confirmed the decrease in AR and PKCδ levels. In addition, we found that a proapoptotic factor, Bax level was reduced and a survival factor, Bcl2 level was increased after injection of TonEBP siRNA, indicating that TonEBP mediates apoptotic cell death. In parallel, TonEBP siRNA was applied to the in vitro human retinal pigment epithelial (ARPE-19) cells cultured in high glucose media. We have consistently found the decrease in AR and PKCδ levels and changes in apoptotic factors for survival. Together, these results clearly demonstrated that hyperglycemia-induced TonEBP plays a crucial role in increasing AR and PKCδ levels and leading to apoptotic death. Our findings suggest that TonEBP reduction is an effective therapeutic strategy for diabetic retinopathy.

Decreased interaction between FoxO3a and Akt correlates with seizure-induced neuronal death.

Status epilepticus (SE) leads to neurodegeneration which likely contributes to the development of chronic temporal lobe epilepsy (TLE). Therefore, neuroprotection following SE is considered as a promising strategy for preventing chronic TLE, but molecular changes that occur following SE still remain unclear. The Forkhead homeobox type O (FoxO) family of Forkhead transcription factors mediates cell death in several pathological conditions, but the role of FoxO in the excitotoxic effects of kainic acid (KA) remains largely unknown. The present study examined how FoxO3a and its interaction with other proteins changed in response to excitotoxic stimuli in the mouse hippocampus after SE. Mice were given intraperitoneal injection of kainate and seizure behavior was monitored for 2h to ensure SE. Western blot analyses, co-immunoprecipitation experiments, sub-cellular fractionation and double immunofluorescence analyses were used to determine changes in levels of FoxO3a, Akt, Bim, cleaved caspase-3 and phospho-FoxO3a or phospho-Akt, and their interactions at 6 or 24h after KA treatment. We found that SE activated FoxO3a and increased levels of Bim or cleaved caspase-3, and decreased levels of phospho-FoxO3a or phospho-Akt in the hippocampus. In addition, we noted extensive hippocampal cell death at 24h after KA treatment, evidenced by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL), fluoro-jade B or anti-active caspase-3 staining. Furthermore, co-immunoprecipitation experiments revealed that phospho-Akt interaction with FoxO3a was significantly lowered in the hippocampus at 24h after KA treatment, paralleling enhanced Bim levels and Bim interaction with Bcl-xL. Moreover, double immunofluorescence analyses showed increased co-localization of FoxO3a or Bim and TUNEL in the hippocampi at 24h after KA treatment. Identifying molecular mechanism underlying SE-induced neuronal death can provide a novel strategy to protect against seizure-induced neuronal injury. We found that Akt-FoxO3a signaling relates to seizure-induced neuronal death, providing insight into neuroprotection following SE.