ATF3 negatively regulates adiponectin receptor 1 expression

Adiponectin is an adipocyte-derived hormone that has antidiabetic and antiatherogenic effects through two membrane receptors, adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2). Although it has been reported that the expression of AdipoR1 and AdipoR2 is regulated under physiological and pathophysiological states, their regulation is largely unknown. Previously, we demonstrated that endoplasmic reticulum (ER) stress or obesity-inducible ATF3 negatively regulates the expression of adiponectin and AdipoR2. Here, we investigated the regulation of another adiponectin receptor, AdipoR1 by ATF3, to determine if ATF3 may contribute to impairment of adiponectin signaling by repressing the expression of both adiponectin and adiponectin receptors. We found that treatment with thapsigargin, a stimulator of ATF3 expression as an inducer of ER stress, decreased AdipoR1 expression in insulin-sensitive cells (HepG2, C2C12) and insulin secreting cells (MIN6N8). Furthermore, overexpression of lentivirus carrying-ATF3 decreased AdipoR1 expression in those cells, demonstrating that ATF3 downregulates AdipoR1 expression. Next, we investigated the effects of ATF3 on human AdipoR1 promoter activity and identified an ATF3-responsive region in the promoter. Both thapsigargin treatment and ATF3 expression repressed AdipoR1 promoter activity. Transfection studies using mutant constructs containing 5′-deletions in the human AdipoR1 promoter revealed that putative ATF/CRE site is located between the −248 and −224, TGACGCGG. Chromatin immunoprecipitation assay demonstrated that ATF3 directly binds to human AdipoR1 promoter spanning from −248 to −224. Finally, deletion of the putative ATF/CRE site abrogated ATF3-mediated transrepression of the AdipoR1 promoter. Importantly, ATF3 expression was increased in hyperglycemia or TNF-α-treated C2C12 cells in which AdipoR1 expression was decreased, suggesting that ATF3 may contribute to downregulation of AdipoR1 by hyperglycemia and TNF-α. Collectively, these results demonstrate that ATF3 negatively regulates human AdipoR1 expression via binding to an ATF3-responsive region in the promoter, which plays an important role in attenuation of adiponectin signaling and induction of insulin resistance.     

GATA4 negatively regulates osteoblast differentiation by downregulation of Runx2

Osteoblasts are specialized mesenchymal cells that are responsible for bone formation. In this study, we examine the role of GATA4 in osteoblast differentiation. GATA4 was abundantly expressed in preosteoblast cells and gradually down-regulated during osteoblast differentiation. Overexpression of GATA4 in osteoblastic cells inhibited alkaline phosphatase activity and nodule formation in osteogenic conditioned cell culture system. In addition, overexpression of GATA4 attenuated expression of osteogenic marker genes, including Runx2, alkaline phosphatase, bone sialoprotein, and osteocalcin, all of which are important for osteoblast differentiation and function. Overexpression of GATA4 attenuated Runx2 promoter activity, whereas silencing of GATA4 increased Runx2 induction. We found that GATA4 interacted with Dlx5 and subsequently decreased Dlx5 binding activity to Runx2 promoter region. Our data suggest that GATA4 acts as a negative regulator in osteoblast differentiation by downregulation of Runx2. [BMB Reports 2014; 47(8): 463-468]     

Tolfenamic acid negatively regulates YAP and TAZ expression in human cancer cells

Several diseases are associated with improper regulation of the Hippo pathway, which plays an important role in cell proliferation and cancer metastasis. Overactivation of the YAP and TAZ proteins accelerates cell proliferation, invasion, and migration during tumorigenesis. Tolfenamic acid (TA) is a non-steroidal anti-inflammatory drug (NSAID) that exhibits activity against various types of cancer. In this study, we observed that TA decreased YAP and TAZ protein levels in cancer cells. TA increased the phosphorylation of YAP and TAZ, leading to the degradation of YAP and TAZ in the cytoplasm and nucleus. TA predominantly affected multiple phosphodegron sites in the YAP and TAZ and lowered 14-3-3β protein expression, causing YAP and TAZ to enter the ubiquitination pathway. Proteins that affect YAP and TAZ regulation, such as NAG-1 and several YAP/TAZ E3 ligases, were not involved in TA-mediated YAP/TAZ degradation. In summary, our results indicate that TA affects phosphodegron sites on YAP/TAZ, demonstrating a novel effect of TA in tumorigenesis.     

Tim-3 negatively regulates IL-12 expression by monocytes in HCV infection

T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) is a newly identified negative immunomodulator that is up-regulated on dysfunctional T cells during viral infections. The expression and function of Tim-3 on human innate immune responses during HCV infection, however, remains poorly characterized. In this study, we report that Tim-3 is constitutively expressed on human resting CD14⁺ monocyte/macrophages (M/M_Ø) and functions as a cap to block IL-12, a key pro-inflammatory cytokine linking innate and adaptive immune responses. Tim-3 expression is significantly reduced and IL-12 expression increased upon stimulation with Toll-like receptor 4 (TLR4) ligand - lipopolysaccharide (LPS) and TLR7/8 ligand - R848. Notably, Tim-3 is over-expressed on un-stimulated as well as TLR-stimulated M/M_Ø, which is inversely associated with the diminished IL-12 expression in chronically HCV-infected individuals when compared to healthy subjects. Up-regulation of Tim-3 and inhibition of IL-12 are also observed in M/M_Ø incubated with HCV-expressing hepatocytes, as well as in primary M/M_Ø or monocytic THP-1 cells incubated with HCV core protein, an effect that mimics the function of complement C1q and is reversible by blocking the HCV core/gC1qR interaction. Importantly, blockade of Tim-3 signaling significantly rescues HCV-mediated inhibition of IL-12, which is primarily expressed by Tim-3 negative M/M_Ø. Tim-3 blockade reduces HCV core-mediated expression of the negative immunoregulators PD-1 and SOCS-1 and increases STAT-1 phosphorylation. Conversely, blocking PD-1 or silencing SOCS-1 gene expression also decreases Tim-3 expression and enhances IL-12 secretion and STAT-1 phosphorylation. These findings suggest that Tim-3 plays a crucial role in negative regulation of innate immune responses, through crosstalk with PD-1 and SOCS-1 and limiting STAT-1 phosphorylation, and may be a novel target for immunotherapy to HCV infection.     

Tunicamycin negatively regulates BMP2-induced osteoblast differentiation through CREBH expression in MC3T3E1 cells

Tunicamycin, an endoplasmic reticulum (ER) stress inducer, specifically inhibits N-glycosylation. The cyclic AMP (cAMP) response element-binding protein H (CREBH) was previously shown to be regulated by UPR-dependent proteolytic cleavage in the liver. On the other hand, the role of CREBH in other tissues is unknown. In the present study, tunicamycin increased the level of CREBH activation (cleavage) as well as mRNA expression in osteoblast cells. Adenoviral (Ad) overexpression of CREBH suppressed BMP2-induced expression of alkaline phosphatase (ALP) and osteocalcin (OC). Interestingly, the BMP2-induced OASIS (structurally similar to CREBH, a positive regulator of osteoblast differentiation) expression was also inhibited by CREBH overexpression. In addition, inhibition of CREBH expression using siRNA reversed the tunicamycin-suppressed ALP and OC expression. These results suggest that CREBH inhibited osteoblast differentiation via suppressing BMP2-induced ALP, OC and OASIS expression in mouse calvarial derived osteoblasts.     

PIKfyve negatively regulates exocytosis in neurosecretory cells

Regulated secretion depends upon a highly coordinated series of protein-protein and protein-lipid interactions. Two phosphoinositides, phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3-phosphate, are important for the ATP-dependent priming of the secretory apparatus prior to Ca(2+)-dependent exocytosis. Mechanisms that control phosphoinositide levels are likely to play an important role in priming fine tuning. Here we have investigated the involvement of PIKfyve, a phosphoinositide 5-kinase that can phosphorylate phosphatidylinositol 3-phosphate to produce phosphatidylinositol 3,5-bisphosphate on large dense core vesicle exocytosis from neuroendocrine cells. PIKfyve localizes to a subpopulation of secretory granules in chromaffin and PC12 cells. Nicotine stimulation promoted recruitment of PIKfyve-EGFP onto secretory vesicles in PC12 cells. YM-201636, a selective inhibitor of PIKfyve activity, and PIKfyve knockdown by small interfering RNA potentiated secretory granule exocytosis. Overexpression of PIKfyve or its yeast orthologue Fab1p inhibited regulated secretion in PC12 cells, whereas a catalytically inactive PIKfyve mutant had no effect. These results demonstrate a novel inhibitory role for PIKfyve catalytic activity in regulated secretion and provide further evidence for a fine tuning of exocytosis by 3-phosphorylated phosphoinositides.