GSK-3α Is a Novel Target of CREB and CREB-GSK-3α Signaling Participates in Cell Viability in Lung Cancer

Overexpression or activation of cyclic AMP-response element-binding protein (CREB) has been known to be involved in several human malignancies, including lung cancer. Genes regulated by CREB have been reported to suppress apoptosis, induce cell proliferation, inflammation, and tumor metastasis. However, the critical target genes of CREB in lung cancer have not been well understood. Here, we identified GSK-3α as one of the CREB target genes which is critical for the viability of lung cancer cells. The CREB knockdown significantly reduced the expression of GSK-3α and the direct binding of CREB on the promoter of GSK3A was identified. Kaplan-Meier analysis with a public database showed a prognostic significance of aberrant GSK-3α expression in lung cancer. Inhibition of GSK-3α suppressed cell viability, colony formation, and tumor growth. For the first time, we demonstrated that GSK-3α is regulated by CREB in lung cancer and is required for the cell viability. These findings implicate CREB-GSK-3α axis as a novel therapeutic target for lung cancer treatment.     

Reciprocal Regulation of HIF-1α and LincRNA-p21 Modulates the Warburg Effect

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Elongation Factor-1α Is a Novel Protein Associated with Host Cell Invasion and a Potential Protective Antigen of Cryptosporidium parvum

The phylum Apicomplexa comprises obligate intracellular parasites that infect vertebrates. All invasive forms of Apicomplexa possess an apical complex, a unique assembly of organelles localized to the anterior end of the cell and involved in host cell invasion. Previously, we generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina sporozoites. This antigen was highly conserved among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium. In the present study, we identified the apical cytoskeletal antigen of Cryptosporidium parvum (C. parvum) and further characterized this antigen in C. parvum to assess its potential as a target molecule against cryptosporidiosis. Indirect immunofluorescence demonstrated that the reactivity of 6D-12-G10 with C. parvum sporozoites was similar to those of anti-β- and anti-γ-tubulins antibodies. Immunoelectron microscopy with the 6D-12-G10 mAb detected the antigen both on the sporozoite surface and underneath the inner membrane at the apical region of zoites. The 6D-12-G10 mAb significantly inhibited in vitro host cell invasion by C. parvum. MALDI-TOF/MS and LC-MS/MS analysis of tryptic peptides revealed that the mAb 6D-12-G10 target antigen was elongation factor-1α (EF-1α). These results indicate that C. parvum EF-1α plays an essential role in mediating host cell entry by the parasite and, as such, could be a candidate vaccine antigen against cryptosporidiosis.     

Regulation of Basolateral Cl− Channels in Airway Epithelial Cells: The Role of Nitric Oxide

The presence of basolateral Cl⁻ channels in airway epithelium has been reported in several studies, but little is known about their role in the regulation of anion secretion. The purpose of this study was to characterize regulation of these channels by nitric oxide (NO) in Calu-3 cells. Transepithelial measurements revealed that NO donors activated a basolateral Cl⁻ conductance sensitive to 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and anthracene-9-carboxylic acid. Apical membrane permeabilization studies confirmed the basolateral localization of NO-activated Cl⁻ channels. Experiments using 8-bromo cyclic guanosine monophosphate (8Br-cGMP) and selective inhibitors of soluble guanylyl cyclase and inducible NO synthase (1H-[1, 2, 4] oxadiazolol-[4, 3-a] quinoxalin-1-one [ODQ] and 1400W [N-(3-Aminomethyl)benzyl)acetamidine], respectively) demonstrated that NO activated Cl⁻ channels via a cGMP-dependent pathway. Anion replacement and ³⁶Cl⁻ flux studies showed that NO affected both Cl⁻ and HCO ₃ ⁻ secretion. Two different types of Cl⁻ channels are known to be present in the basolateral membrane of epithelial cells: Zn²⁺-sensitive ClC-2 and DIDS-sensitive bestrophin channels. S-Nitrosoglutathione (GSNO) activated Cl⁻ conductance in the presence of Zn²⁺ ions, indicating that ClC-2 channel function was not affected by GSNO. In contrast, DIDS completely inhibited GSNO-activated Cl⁻ conductance. Bestrophin immunoprecipitation studies showed that under control conditions bestrophin channels were not phosphorylated but became phosphorylated after GSNO treatment. The presence of bestrophin in airway epithelia was confirmed using immunohistochemistry. We conclude that basolateral Cl⁻ channels play a major role in the NO-dependent regulation of anion secretion in Calu-3 cells.     

Role and Interrelationship of Gα Protein,Hydrogen Peroxide,and Nitric Oxide in Ultraviolet B-Induced Stomatal Closure in Arabidopsis Leaves

Heterotrimeric G proteins have been shown to transmit ultraviolet B (UV-B) signals in mammalian cells, but whether they also transmit UV-B signals in plant cells is not clear. In this paper, we report that 0.5 W m⁻² UV-B induces stomatal closure in Arabidopsis (Arabidopsis thaliana) by eliciting a cascade of intracellular signaling events including Gα protein, hydrogen peroxide (H₂O₂), and nitric oxide (NO). UV-B triggered a significant increase in H₂O₂ or NO levels associated with stomatal closure in the wild type, but these effects were abolished in the single and double mutants of AtrbohD and AtrbohF or in the Nia1 mutants, respectively. Furthermore, we found that UV-B-mediated H₂O₂ and NO generation are regulated by GPA1, the Gα-subunit of heterotrimeric G proteins. UV-B-dependent H₂O₂ and NO accumulation were nullified in gpa1 knockout mutants but enhanced by overexpression of a constitutively active form of GPA1 (cGα). In addition, exogenously applied H₂O₂ or NO rescued the defect in UV-B-mediated stomatal closure in gpa1 mutants, whereas cGα AtrbohD/AtrbohF and cGα nia1 constructs exhibited a similar response to AtrbohD/AtrbohF and Nia1, respectively. Finally, we demonstrated that Gα activation of NO production depends on H₂O₂. The mutants of AtrbohD and AtrbohF had impaired NO generation in response to UV-B, but UV-B-induced H₂O₂ accumulation was not impaired in Nia1. Moreover, exogenously applied NO rescued the defect in UV-B-mediated stomatal closure in the mutants of AtrbohD and AtrbohF. These findings establish a signaling pathway leading to UV-B-induced stomatal closure that involves GPA1-dependent activation of H₂O₂ production and subsequent Nia1-dependent NO accumulation.Heterotrimeric G proteins, composed of α-, β-, and γ-subunits, are a key intracellular signaling molecule in both mammalian and plant systems. Classically, upon signal reception by a receptor coupled to the heterotrimer, the Gα-subunit separates from the Gβγ dimer, and either Gα or the Gβγ dimer can act as a functional unit and induce downstream signaling (Oldham and Hamm, 2008). In contrast to mammalian cells, where multiple α, β, and γ genes exist, there is only one prototypical Gα (GPA1), one Gβ (AGB1), and two known Gγ (AGG1 and AGG2) genes in Arabidopsis (Arabidopsis thaliana; Temple and Jones, 2007). Despite the comparative simplicity of players, G proteins have been shown to participate in multiple signaling pathways in Arabidopsis, including developmental processes, phytohormone responses, and responses to biotic and abiotic environmental signals such as pathogens, ozone, drought, and light (Assmann, 2005; Temple and Jones, 2007; Warpeha et al., 2007; Okamoto et al., 2009; Nilson and Assmann, 2010).Depletion of the stratospheric ozone layer results in increased levels of the sun’s UV-B radiation (280–315 nm) at the Earth’s surface. Although this influx of shortwave photons with high energy implies serious effects for all living organisms (Frohnmeyer and Staiger, 2003), UV-B is also a key environmental signal that initiates diverse responses in a range of organisms (Jansen and Bornman, 2012). Thus, understanding the mechanism of UV-B signal transduction in cells is very important. In recent years, significant progress has been made in identifying the molecular players and understanding the early mechanisms and functions of the UV-B perception and signaling pathway in plants. The perception of UV-B by UV RESISTANCE LOCUS8 (UVR8) followed by the interaction among UVR8, CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), and ELONGATED HYPOCOTYL5 (HY5) has emerged as a primary mechanism of the UV-B response that is crucial for UV-B acclimation and tolerance (Rizzini et al., 2011; Christie et al., 2012; Heijde and Ulm, 2012; Jansen and Bornman, 2012). However, few of the molecular players involved in UV-B signal transduction are currently known. In mammalian cells, heterotrimeric G proteins have been shown to mediate various UV-B-induced cellular responses, such as secretion of heparin-binding epidermal growth factor (HB-EGF), activation of mitogen-activated protein kinases, cyclooxygenase2 expression, and apoptosis in human keratinocytes (Seo et al., 2004, 2007; Seo and Juhnn, 2010), suggesting that G proteins are important molecular players in UV-B signal transduction. However, at present, whether G proteins participate in the responses of plant cells to UV-B is not known.Stomata embedded in the epidermis of terrestrial plants are important for CO₂ absorption and water transpiration and are possible points of entry for pathogens. Thus, the regulation of stomatal apertures is extremely important for the survival of plants. Phenotypic analyses of Arabidopsis mutants lacking the Gα- or Gβ-subunit show that these G proteins are involved in stomatal movement regulated by abscisic acid (ABA; Wang et al., 2001; Pandey and Assmann, 2004; Liu et al., 2007; Fan et al., 2008; Zhang et al., 2011), sphingosine-1-P (Coursol et al., 2003, 2005), phosphatidic acid (PA; Mishra et al., 2006), extracellular calmodulin (ExtCaM; Chen et al., 2004; Li et al., 2009), extracellular ATP (Hao et al., 2012), and the pathogen-associated molecular pattern flg22 (Zhang et al., 2008), suggesting that plant G proteins respond to various stimuli as key regulators of stomatal movement. On exposure to UV-B radiation, many plant species exhibit decreases in stomatal conductance and/or aperture under growth chamber, greenhouse, and field conditions (Musil and Wand, 1993; Nogués et al., 1999; Jansen and Noort, 2000). However, in some species, UV-B has been reported to induce either stomatal opening or stomatal closure, perhaps depending on the metabolic state of guard cells (Jansen and Noort, 2000). Furthermore, UV-B-inhibited photosynthesis is partially caused by stomatal limitation (He et al., 2004). Thus, understanding the mechanism of stomatal movement regulated by UV-B is extremely important for improving the resistance of plants to enhanced UV-B radiation, but, to date, it is poorly understood.Recently, compelling evidence emerged that hydrogen peroxide (H₂O₂) and nitric oxide (NO) function as signaling molecules in plants, mediating a range of responses to environmental stress including UV-B radiation (Neill et al., 2002; Qiao and Fan, 2008; Wilson et al., 2008). Increasing evidence also points to the role for H₂O₂ and NO as essential components in guard cell signaling. For example, both H₂O₂ and NO have been implicated in ABA-, salicylic acid (SA)-, ethylene-, ExtCaM-, and darkness-induced stomatal closure. Furthermore, several main cellular players in stomatal movement, such as mitogen-activated protein kinases, protein phosphatases, cytoskeleton, and ion channels, have already been identified as likely targets downstream of H₂O₂ or NO (Neill et al., 2008; Wang and Song, 2008; Huang et al., 2009; Li et al., 2009; Wilkins et al., 2011; Yemets et al., 2011). G protein signaling to the membrane-bound H₂O₂ synthetic enzyme, NADPH oxidase, has been implicated in the development of disease resistance and the apoptotic hypersensitive response in rice (Oryza sativa; Suharsono et al., 2002). Production of reactive oxygen species (ROS) in response to the air pollutant ozone is also impaired in a mutant lacking the Gα subunit (Joo et al., 2005). The heterotrimeric G proteins also participate in ROS metabolism in plant cells (Wei et al., 2008; Zhao et al., 2010). During stomatal movement, G proteins mediate H₂O₂ production induced by ABA (Zhang et al., 2011), ExtCaM (Chen et al., 2004; Li et al., 2009), and extracellular ATP (Hao et al., 2012) as well as NO production induced by ExtCaM in guard cells (Li et al., 2009). In addition, phospholipase Dα and its product PA, which interact with GPA1 during ABA inhibition of stomatal opening (Mishra et al., 2006), also promote ABA-induced ROS production (Zhang et al., 2009). These observations suggest that G proteins may be key regulators of H₂O₂ and NO production in plant cells, including guard cells. With regard to the stomatal movement regulated by UV-B radiation, our previous studies showed that H₂O₂ and NO generation are required for UV-B-induced stomatal closure (He et al., 2005, 2011a, 2011b). However, whether the UV-B-induced production of H₂O₂ and NO in guard cells is also regulated by G proteins remains unknown.In this study, we use Arabidopsis mutants (e.g. GPA1 null mutants gpa1-1 and gpa1-2; Nia1-2, Nia2-1, and Nia1-2/Nia2-5, which are defective in NO production; and AtrbohD, AtrbohF, and AtrbohD/AtrbohF, which are defective in producing H₂O₂) and pharmacological reagents to show that the G protein is involved in the regulation of UV-B-induced stomatal closure in Arabidopsis via sequential elucidation of H₂O₂ and NO, two key regulators of UV-B regulation of stomatal movements. Our results establish a linear signaling cascade in which the Gα protein transmits UV-B signals to elicit H₂O₂, which then elicits NO in guard cells to regulate UV-B-dependent stomatal closure.     

Is Vitamin D Binding Protein a Novel Predictor of Labour?

Vitamin D binding protein (VDBP) has previously been identified in the amniotic fluid and cervicovaginal fluid (CVF) of pregnant women. The biological functions of VDBP include acting as a carrier protein for vitamin D metabolites, the clearance of actin that is released during tissue injury and the augmentation of the pro-inflammatory response. This longitudinal observational study was conducted on 221 healthy pregnant women who spontaneously laboured and delivered either at term or preterm. Serial CVF samples were collected and VDBP was measured by ELISA. Binary logistic regression analysis was performed to assess the utility of VDBP as a predictor of labour. VDBP in the CVF did not change between 20 and 35 weeks'' gestation. VDBP measured in-labour was significantly increased 4.2 to 7.4-fold compared to 4–7, 8–14 and 15–28 days before labour (P<0.05). VDBP concentration was 4.3-fold significantly higher at 0–3 days compared to 15–28 days pre-labour (P<0.05). The efficacy of VDBP to predict spontaneous labour onset within 3 days provided a positive and negative predictive value of 82.8% and 95.3% respectively (area under receiver operator characteristic curve  = 0.974). This longitudinal study of pregnant women suggests that VDBP in the CVF may be a useful predictor of labour.     

Nitric Oxide and Protein Disulfide Isomerase Explain the Complexities of Unfolded Protein Response Following Intra-hippocampal Aβ Injection

Several pathways involved in regulation of intracellular protein integrity are known as the protein quality control (PQC) system. Molecular chaperones as the main players are engaged in various aspects of PQC system. According to the importance of these proteins in cell survival, in the present study, we traced endoplasmic reticulum-specific markers and chaperone-mediated autophagy (CMA)-associated factors as two main arms of PQC system in intra-hippocampal amyloid beta (Aβ)-injected rats during 10 days running. Data analysis from Western blot indicated that exposure to Aβ activates immunoglobulin heavy-chain-binding protein (Bip) which is the upstream regulator of unfolded protein responses (UPR). Activation of UPR system eventually led to induction of pro-apoptotic factors like CHOP, calpain, and caspase-12. Moreover, our data revealed that protein disulfide isomerase activity dramatically decreased after Aβ injection, which could be attributed to the increased levels of nitric oxide. Besides, Aβ injection induced levels of 2 members of heat shock proteins (Hsp) 70 and 90. Elevated levels of Hsps family members are accompanied by increased levels of lysosome-associated membrane protein type-2A (Lamp-2A) that are involved in CMA. Despite the reduction in CHOP, calpain, caspase-12, and Lamp-2A protein levels, the levels of molecular chaperones Bip, Hsps70, and 90 increased 10 days after Aβ injection in comparison to the control group. Based on our results, 10 days after Aβ injection, despite the activation of protective chaperones, markers associated with neurotoxicity were still elevated.     

Regulation of intracellular calcium release and PP1α in a mechanism for 4-hydroxytamoxifen-induced cytotoxicity

Treatment with tamoxifen, or its metabolite 4-hydroxytamoxifen (4OHT), has cytostatic and cytotoxic effects on breast cancer cells in vivo and in culture. Although the effectiveness of 4OHT as an anti-breast cancer agent is due to its action as an estrogen receptor-alpha (ERα) antagonist, evidences show that 4OHT is also cytotoxic for ERα-negative breast cancer cells and can be effective therapy against tumors that lack estrogen receptors. These findings underscore 4OHT signaling complexities and belie the most basic understandings of 4OHT action and resistance. Here, we have investigated the effects of 4OHT on Ca²⁺ homeostasis and cell death in breast cancer cells in culture. Measurement of Ca²⁺ signaling in breast cancer cells showed that 4OHT treatment altered Ca²⁺ homeostasis and was cytotoxic for both an ERα+ and an ERα- cell line, MCF-7 and MDA-MB-231, respectively. Further investigation lead us to the novel discovery that 4OHT-induced increase of ATP-dependent Ca²⁺ release from the endoplasmic reticulum correlated with 4OHT-induced upregulation of protein phosphatase 1α (PP1α) and the inositol 1,4,5-trisphosphate receptor (IP3R). Blocking 4OHT-induced PP1α upregulation by siRNA strategy reduced the effects of 4OHT on both Ca²⁺ signaling and cytotoxicity. Results from these investigations strongly suggest a role for PP1α upregulation in a mechanism for 4OHT-induced changes to Ca²⁺ signaling that ultimately contribute to the cytotoxic effects of 4OHT. Aliccia Bollig, Liping Xu- contributed equally to this work     

Chenodeoxycholic Acid Reduces Hypoxia Inducible Factor-1α Protein and Its Target Genes

This study evaluated HIF-1α inhibitors under different hypoxic conditions, physiological hypoxia (5% O₂) and severe hypoxia (0.1% O₂). We found that chenodeoxy cholic acid (CDCA) reduced the amount of HIF-1α protein only under physiological hypoxia but not under severe hypoxia without decreasing its mRNA level. By using a proteasome inhibitor MG132 and a translation inhibitor cyclohexamide, we showed that CDCA reduced HIF-1α protein by decreasing its translation but not by enhancing its degradation. The following findings indicated that farnesoid X receptor (FXR), a CDCA receptor and its target gene, Small heterodimer partner (SHP) are not involved in this effect of CDCA. Distinctly from CDCA, MG132 prevented SHP and an exogenous FXR agonist, GW4064 from reducing HIF-1α protein. Furthermore a FXR antagonist, guggulsterone failed to prevent CDCA from decreasing HIF-1α protein. Furthermore, guggulsterone by itself reduced HIF-1α protein even in the presence of MG132. These findings suggested that CDCA and guggulsterone reduced the translation of HIF-1α in a mechanism which FXR and SHP are not involved. This study reveals novel therapeutic functions of traditional nontoxic drugs, CDCA and guggulsterone, as inhibitors of HIF-1α protein.     

Regulation of skin inflammation and angiogenesis by EC-SOD via HIF-1α and NF-κB pathways

Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that breaks down superoxide anion into oxygen and hydrogen peroxide in extracellular spaces and plays key roles in controlling pulmonary and vascular diseases in response to oxidative stresses. We aimed to investigate the role of EC-SOD in angiogenesis and inflammation in chronic inflammatory skin disorders such as psoriasis. Overexpressed EC-SOD reduced expression of angiogenic factors and proinflammatory mediators in hypoxia-induced keratinocytes and in ultraviolet B-irradiated mice, whereas the expression of the antiangiogenic factor tissue inhibitor of metalloproteinase-1 and anti-inflammatory cytokine interleukin-10 were increased. EC-SOD decreased new vessel formation, epidermal edema, and inflammatory cell infiltration in UVB-irradiated transgenic mice. Moreover, cells treated with recombinant human EC-SOD showed inhibited endothelial tube formation and cell proliferation. Overall, the antiangiogenic and anti-inflammatory effects of EC-SOD might be due to suppression of hypoxia-inducible factor-1α, protein kinase C, and nuclear factor-κB expression. Furthermore, EC-SOD expression in tissue from psoriasis patients was markedly decreased in psoriatic lesional and nonlesional skins from psoriasis patients in comparison to normal skin from healthy volunteers. Together, these results suggest that EC-SOD may provide a novel therapeutic approach to treating angiogenic and inflammatory skin diseases such as psoriasis.     

Differential roles of Sirt1 in HIF-1α and HIF-2α mediated hypoxic responses

Hypoxia-inducible factors 1α and 2α (HIF-1α and HIF-2α) determine cancer cell fate under hypoxia. Despite the similarities of their structures, HIF-1α and HIF-2α have distinct roles in cancer growth under hypoxia, that is, HIF-1α induces growth arrest whereas HIF-2α promotes cell growth. Recently, sirtuin 1 (Sirt1) was reported to fine-tune cellular responses to hypoxia by deacetylating HIF-1α and HIF-2α. Yet, the roles of Sirt1 in HIF-1α and HIF-2α functions have been controversial. We here investigated the precise roles of Sirt1 in HIF-1α and HIF-2α regulations. Immunological analyses revealed that HIF-1α K674 and HIF-2α K741 are acetylated by PCAF and CBP, respectively, but are deacetylated commonly by Sirt1. In the Gal4 reporter systems, Sirt1 was found to repress HIF-1α activity constantly in ten cancer cell-lines but to regulate HIF-2α activity cell type-dependently. Moreover, Sirt1 determined cell growth under hypoxia depending on HIF-1α and HIF-2α. Under hypoxia, Sirt1 promoted cell proliferation of HepG2, in which Sirt1 differentially regulates HIF-1α and HIF-2α. In contrast, such an effect of Sirt1 was not shown in HCT116, in which Sirt1 inactivates both HIF-1α and HIF-2α because conflicting actions of HIF-1α and HIF-2α on cell growth may be offset. Our results provide a better understanding of the roles of Sirt1 in HIF-mediated hypoxic responses and also a basic concept for developing anticancer strategy targeting Sirt1.     

HIF-1α Promotes Epithelial-Mesenchymal Transition and Metastasis through Direct Regulation of ZEB1 in Colorectal Cancer

It is well recognized that hypoxia-inducible factor 1 alpha (HIF-1α) is involved in cancer metastasis, chemotherapy and poor prognosis. We previously found that deferoxamine, a hypoxia-mimetic agent, induces epithelial-mesenchymal transition (EMT) in colorectal cancer. Therefore, here we explored a new molecular mechanism for HIF-1α contributing to EMT and cancer metastasis through binding to ZEB1. In this study, we showed that overexpression of HIF-1α with adenovirus infection promoted EMT, cell invasion and migration in vitro and in vivo. On a molecular level, HIF-1α directly binding to the proximal promoter of ZEB1 via hypoxia response element (HRE) sites thus increasing the transactivity and expression of ZEB1. In addition, inhibition of ZEB1 was able to abrogate the HIF-1α-induced EMT and cell invasion. HIF-1α expression was highly correlated with the expression of ZEB1 in normal colorectal epithelium, primary and metastatic CRC tissues. Interestingly, both HIF-1α and ZEB1 were positively associated with Vimentin, an important mesenchymal marker of EMT, whereas negatively associated with E-cadherin expression. These findings suggest that HIF-1α enhances EMT and cancer metastasis by binding to ZEB1 promoter in CRC. HIF-1α and ZEB1 are both widely considered as tumor-initiating factors, but our results demonstrate that ZEB1 is a direct downstream of HIF-1α, suggesting a novel molecular mechanism for HIF-1α-inducing EMT and cancer metastasis.     

Loss of von Hippel-Lindau Protein (VHL) Increases Systemic Cholesterol Levels through Targeting Hypoxia-Inducible Factor 2α and Regulation of Bile Acid Homeostasis

Cholesterol synthesis is a highly oxygen-dependent process. Paradoxically, hypoxia is correlated with an increase in cellular and systemic cholesterol levels and risk of cardiovascular diseases. The mechanism for the increase in cholesterol during hypoxia is unclear. Hypoxia signaling is mediated through hypoxia-inducible factor 1α (HIF-1α) and HIF-2α. The present study demonstrates that activation of HIF signaling in the liver increases hepatic and systemic cholesterol levels due to a decrease in the expression of cholesterol hydroxylase CYP7A1 and other enzymes involved in bile acid synthesis. Specifically, activation of hepatic HIF-2α (but not HIF-1α) led to hypercholesterolemia. HIF-2α repressed the circadian expression of Rev-erbα, resulting in increased expression of E4BP4, a negative regulator of Cyp7a1. To understand if HIF-mediated decrease in bile acid synthesis is a physiologically relevant pathway by which hypoxia maintains or increases systemic cholesterol levels, two hypoxic mouse models were assessed, an acute lung injury model and mice exposed to 10% O₂ for 3 weeks. In both models, cholesterol levels increased with a concomitant decrease in expression of genes involved in bile acid synthesis. The present study demonstrates that hypoxic activation of hepatic HIF-2α leads to an adaptive increase in cholesterol levels through inhibition of bile acid synthesis.     

The Collagen-binding Integrin α2β1 Is a Novel Interaction Partner of the Trimeresurus flavoviridis Venom Protein Flavocetin-A

Many snake venoms are known for their antithrombotic activity. They contain components that specifically target different platelet-activating receptors such as the collagen-binding integrin α2β1 and the von Willebrand factor receptor GPIb. In a search for an α2β1 integrin-blocking component from the venom of the habu snake (Trimeresurus flavoviridis), we employed two independent purification protocols. First, we used the integrin α2A domain, a major collagen-binding domain, as bait for affinity purification of an α2β1 integrin-binding toxin from the crude venom. Second, in parallel, we used classical protein separation protocols and tested for α2β1 integrin-inhibiting capabilities by ELISA. Using both approaches, we identified flavocetin-A as an inhibitor of α2β1 integrin. Hitherto, flavocetin-A has been reported as a GPIb inhibitor. However, flavocetin-A inhibited collagen-induced platelet aggregation even after GPIb was blocked with other inhibitors. Moreover, flavocetin-A antagonized α2β1 integrin-mediated adhesion and migration of HT1080 human fibrosarcoma cells, which lack any GPIb, on collagen. Protein chemical analyses proved that flavocetin-A binds to α2β1 integrin and its α2A domain with high affinity and in a cooperative manner, which most likely is due to its quaternary structure. Kinetic measurements confirmed the formation of a strong complex between integrin and flavocetin-A, which dissociates very slowly. This study proves that flavocetin-A, which has long been known as a GPIb inhibitor, efficiently targets α2β1 integrin and thus blocks collagen-induced platelet activation. Moreover, our findings suggest that the separation of GPIb- and α2β1 integrin-blocking members within the C-type lectin-related protein family is less strict than previously assumed.