Rosiglitazone suppresses RANKL-induced NFATc1 autoamplification by disrupting the physical interaction between NFATc1 and PPARγ.

Receptor activator of nuclear factor-κB ligand (RANKL) is required for initiation of osteoclastogenesis, with the signaling pathway including the NF-kB, c-Fos, and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) transcription factors. Because NFATc1 expression is autoamplified, we investigated the molecular mechanism by which peroxisome proliferator-activated receptor gamma (PPARγ) activation by the thiazolidinedione drug rosiglitazone decreases NFATc1 expression during RANKL stimulation. Western blotting demonstrated that rosiglitazone attenuated the increase in NFATc1 protein level induced by RANKL without affecting that of PPARγ. Immunofluorescence data indicated that rosiglitazone tended to suppress RANKL-induced NFATc1 nuclear translocation, partly by reducing calcineurin activity, as reflected by the observed decrease in nuclear NFATc1 abundance. On coimmunoprecipitation, the intensity of the physical interaction between NFATc1 and PPARγ was unexpectedly higher in the RANKL-stimulated group than in the control, but rosiglitazone reduced this to basal levels. Furthermore, RANKL failed to elevate mRNA expression of NFATc1 after PPARγ knockdown. ChIP assay indicated that rosiglitazone significantly reduced the binding of NFATc1 to its own promoter despite RANKL stimulation. These findings suggest that PPARγ activation by rosiglitazone blocks NFATc1 from binding to its own promoter, thereby reducing RANKL-induced NFATc1 autoamplification.

Regulation of Airway Inflammation by G-protein Regulatory Motif Peptides of AGS3 protein.

Respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), and lung infections have critical consequences on mortality and morbidity in humans. The aims of the present study were to examine the mechanisms by which CXCL12 affects MUC1 transcription and airway inflammation, which depend on activator of G-protein signaling (AGS) 3 and to identify specific molecules that suppress CXCL12-induced airway inflammation by acting on G-protein-coupled receptors. Herein, AGS3 suppresses CXCL12-mediated upregulation of MUC1 and TNFα by regulating Gαi. We found that the G-protein regulatory (GPR) motif peptide in AGS3 binds to Gαi and downregulates MUC1 expression; in contrast, this motif upregulates TNFα expression. Mutated GPR Q34A peptide increased the expression of MUC1 and TGFß but decreased the expression of TNFα and IL-6. Moreover, CXCR4-induced dendritic extensions in 2D and 3D matrix cultures were inhibited by the GPR Q34A peptide compared with a wild-type GPR peptide. The GPR Q34A peptide also inhibited CXCL12-induced morphological changes and inflammatory cell infiltration in the mouse lung, and production of inflammatory cytokines in bronchoalveolar lavage (BAL) fluid and the lungs. Our data indicate that the GPR motif of AGS3 is critical for regulating MUC1/Muc1 expression and cytokine production in the inflammatory microenvironment.

Silencing of MUC8 by siRNA increases P2Y2-induced airway inflammation.

Mucin hypersecretion and overproduction are frequent manifestations of respiratory disease. Determining the physiological function of airway mucin is presently considered more important than identifying the relevant signaling pathways. The lack of a full-length human mucin 8 (MUC8) cDNA sequence has hindered the generation of a Muc8 knockout mouse line. Thus, the precise physiological functions of MUC8 are unclear. Herein, we investigated the function of MUC8 using a small-interfering RNA (siRNA)-mediated genetic silencing approach in human airway epithelial cells. Herein, intracellular IL-1α production was stimulated by an ATP/P2Y2 complex. While ATP/P2Y2 increased IL-1α secretion in a time-dependent manner, treatment with P2Y2-specific siRNA significantly decreased IL-1α secretion. Moreover, ATP increased P2Y2-mediated upregulation of MUC8 expression; however, IL-1α significantly decreased the extent to which ATP/P2Y2 upregulated MUC8 expression. Interestingly, treatment with MUC8-specific siRNA decreased the production of anti-inflammatory cytokines (TGF-ß and IL-1 receptor antagonist) and increased the production of inflammatory cytokines (IL-1α and IL-6) in our system. In addition, siRNA-mediated knockdown of MUC8 expression dramatically increased the secretion of inflammatory chemokines and resulted in an approximately threefold decrease in cell chemotaxis. We propose that MUC8 may function as an anti-inflammatory mucin that participates in inflammatory response by attracting immune cells/cytokines to the site of inflammation. Our results provide new insight into the physiological function of MUC8 and enhance our understanding of mucin overproduction during airway inflammation.

Differential activation of diverse glutathione transferases of Clonorchis sinensis in response to the host bile and oxidative stressors.

BACKGROUND: Clonorchis sinensis causes chronic cumulative infections in the human hepatobiliary tract and is intimately associated with cholangiocarcinoma. Approximately 35 million people are infected and 600 million people are at risk of infections worldwide. C. sinensis excretory-secretory products (ESP) constitute the first-line effector system affecting the host-parasite interrelationship by interacting with bile fluids and ductal epithelium. However, the secretory behavior of C. sinensis in an environment close to natural host conditions is unclear. C. sinensis differs from Fasciola hepatica in migration to, and maturation in, the hepatic bile duct, implying that protein profile of the ESP of these two trematodes might be different from each other. METHODOLOGY/PRINCIPAL FINDINGS: We conducted systemic approaches to analyze the C. sinensis ESP proteome and the biological reactivity of C. sinensis glutathione transferases (GSTs), such as global expression patterns and induction profiles under oxidative stress and host bile. When we observed ex host excretion behavior of C. sinensis in the presence of 10% host bile, the global proteome pattern was not significantly altered, but the amount of secretory proteins was increased by approximately 3.5-fold. Bioactive molecules secreted by C. sinensis revealed universal/unique features in relation to its intraluminal hydrophobic residing niche. A total of 38 protein spots identified abundantly included enzymes involved in glucose metabolism (11 spots, 28.9%) and diverse-classes of glutathione transferases (GSTs; 10 spots, 26.3%). Cathepsin L/F (four spots, 10.5%) and transporter molecules (three spots, 7.9%) were also recognized. The universal secretory proteins found in other parasites, such as several enzymes involved in glucose metabolism and oxygen transporters, were commonly detected. C. sinensis secreted less cysteine proteases and fatty acid binding proteins compared to other tissue-invading or intravascular trematodes. Interestingly, secretion of a 28 kDa σ-class GST (Cs28σGST3) was significantly affected by the host bile, involving reduced secretion of the 28 kDa species and augmented secretion of Cs28σGST3-related high-molecular-weight 85 kDa protein. Oxidative stressors induced upregulated secretion of 28 kDa Cs28σGST3, but not an 85 kDa species. A secretory 26 kDa µ-class GST (Cs26µGST2) was increased upon treatment with oxidative stressors and bile juice, while another 28 kDa σ-class GST (Cs28σGST1) showed negligible responses. CONCLUSIONS/SIGNIFICANCE: Our results represent the first analysis of the genuine nature of the C. sinensis ESP proteome in the presence of host bile mimicking the natural host environments. The behavioral patterns of migration and maturation of C. sinensis in the bile ducts might contribute to the secretion of copious amounts of diverse GSTs, but a smaller quantity and fewer kinds of cysteine proteases. The Cs28σGST1 and its paralog(s) detoxify endogenous oxidative molecules, while Cs28σGST3 and Cs26µGST2 conjugate xenobiotics/hydrophobic substances in the extracellular environments, which imply that diverse C. sinensis GSTs might have evolved for each of the multiple specialized functions.

Peptidoglycan from Staphylococcus aureus increases MUC5AC gene expression via RSK1-CREB pathway in human airway epithelial cells.

Respiratory tract exposure to viruses, air pollutants, or bacterial pathogens can lead to pulmonary diseases. The molecular mechanism of mucous overproduction increased by these pathogens provides the knowledge for developing new therapeutic strategies. There is established in vitro data demonstrating that the overexpression of MUC5AC is induced by peptidoglycan (PGN) derived from Staphylococcus aureus. However, the mechanisms by which PGN activates MUC5AC gene expression in the airway remain unclear. The aim of this study was to identify the mechanism of PGN-induced MUC5AC gene expression. We found that PGN could induce MUC5AC gene expressions in a time- and dose-dependent manner. Moreover, activations of ERK1/2 and JNK increased after treatment of cells with PGN, whereas phosporylation of p38 was undetected. Of these MAPKs, pharmacologic inhibition of ERK1/2 decreased PGN-induced MUC5AC gene expression. In addition, we checked the activation of p90 ribosomal S6 kinase 1 (RSK1) as a downstream signaling target of ERK1/2 in PGN signaling. The activation of RSK1 was prevented by pretreatment with PD98059. We also found that RSK1 mediated the PGN-induced phosphorylation of cAMP response element-binding protein (CREB) and the transcription of MUC5AC. Furthermore, the cAMP-response element (CRE) in the MUC5AC promoter appears to be important for PGN-induced MUC5AC gene expression in NCI-H292 cells.

Inhibition of renal Na+/H+ exchange in cadmium-intoxicated rats.

Chronic exposure to cadmium (Cd) results in bicarbonaturia, leading to metabolic acidosis. To elucidate the mechanism(s) by which renal bicarbonate reabsorption is inhibited, we investigated changes in renal transporters and enzymes associated with bicarbonate reabsorption in Cd-intoxicated rats. Cd intoxication was induced by subcutaneous injections of CdCl(2) (2 mg Cd/kg per day) for 3 weeks. Cd intoxication resulted in a significant reduction in V(max) of Na(+)/H(+) antiport with no changes in K(Na) in the renal cortical brush-border membrane vesicles (BBMV). Western blotting of BBM proteins and indirect immunohistochemistry in renal tissue sections, using an antibody against Na(+)/H(+) exchange-3 (NHE3), showed a diminished expression of NHE3 protein in the BBM. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that NHE3 mRNA expression was reduced in the renal cortex. The activity of carbonic anhydrase IV (CA IV) in BBM was not changed. The protein abundance of Na(+)-HCO(3)(-) cotransporter-1 (NBC1) in whole kidney membrane fractions was slightly attenuated, whereas that of the Na(+)-K(+)-ATPase alpha-subunit was markedly elevated in Cd-intoxicated animals. These results indicate that Cd intoxication impairs NHE3 expression in the proximal tubule, thereby reducing the capacity for bicarbonate reabsorption, leading to bicarbonaturia in an intact animal.