PKCδ-IRAK1 axis regulates oxidized LDL-induced IL-1β production in monocytes

This study examined the role of interleukin (IL)-1 receptor-associated kinase (IRAK) and protein kinase C (PKC) in oxidized LDL (Ox-LDL)-induced monocyte IL-1β production. In THP1 cells, Ox-LDL induced time-dependent secretory IL-1β and IRAK1 activity; IRAK4, IRAK3, and CD36 protein expression; PKCδ-JNK1 phosphorylation; and AP-1 activation. IRAK1/4 siRNA and inhibitor (INH)-attenuated Ox-LDL induced secreted IL-1β and pro-IL-1β mRNA and pro-IL-1β and mature IL-1β protein expression, respectively. Diphenyleneiodonium chloride (NADPH oxidase INH) and N-acetylcysteine (free radical scavenger) attenuated Ox-LDL-induced reactive oxygen species generation, caspase-1 activity, and pro-IL-1β and mature IL-1β expression. Ox-LDL-induced secretory IL-1β production was abrogated in the presence of JNK INH II, Tanshinone IIa, Ro-31-8220, Go6976, Rottlerin, and PKCδ siRNA. PKCδ siRNA attenuated the Ox-LDL-induced increase in IRAK1 kinase activity, JNK1 phosphorylation, and AP-1 activation. In THP1 macrophages, CD36, toll-like receptor (TLR)2, TLR4, TLR6, and PKCδ siRNA prevented Ox-LDL-induced PKCδ and IRAK1 activation and IL-1β production. Enhanced Ox-LDL and IL-1β in systemic inflammatory response syndrome (SIRS) patient plasma demonstrated positive correlation with each other and with disease severity scores. Ox-LDL-containing plasma induced PKCδ and IRAK1 phosphorylation and IL-1β production in a CD36-, TLR2-, TLR4-, and TLR6-dependent manner in primary human monocytes. Results suggest involvement of CD36, TLR2, TLR4, TLR6, and the PKCδ-IRAK1-JNK1-AP-1 axis in Ox-LDL-induced IL-1β production.     

α-MSH signalling via melanocortin 5 receptor promotes lipolysis and impairs re-esterification in adipocytes

The melanocortin system has a clear effect on the mobilisation of stored lipids in adipocytes. The aim of the current study was to investigate the role of melanocortin 5 receptor (MC5R) on α-melanocyte-stimulating hormone (α-MSH)-induced lipolysis in 3T3-L1 adipocytes. To this end, MC5R expression was decreased by small interfering RNA (siRNA), which significantly impaired the α-MSH stimulation of lipolysis, as determined by glycerol and nonesterified fatty-acid (NEFA) quantification. The functional role of α-MSH/MC5R on triglyceride (TG) hydrolysis was mediated by hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), perilipin 1 (PLIN1) and acetyl-CoA carboxylase (ACC). Immunofluorescence microscopy revealed that phosphorylated HSL clearly surrounded lipid droplets in α-MSH-stimulated adipocytes, whereas PLIN1 left the immediate periphery of lipids. These observations were lost when the expression of MC5R was suppressed.     

PKCα promotes insulin secretion via TRPC1 phosphorylation in INS-1E cells

ABSTRACT

Protein kinase C (PKC) is a class of phospholipid-dependent serine/threonine kinases that contribute to cell survival, migration, and invasion. Previous studies demonstrated that PKC participates in insulin secretion. However, the role of PKC in glucose-stimulated insulin secretion (GSIS) remains unclear. Herein, we demonstrated that PKC is an important mediator of insulin secretion and revealed a close relationship between PKC activation and insulin secretion in INS-1E cells. Meanwhile, the presence of PKCα was found to induce TRPC1 phosphorylation in INS-1E cells. TRPC1 phosphorylation levels increased by activating PKCα activity. Inhibition of PKCα activity reduced TRPC1 phosphorylation. Finally, we showed that TRPC1 could reverse the decrease in intracellular Ca²⁺ levels and reduced insulin secretion induced by treatment with PKCα inhibitor under high glucose conditions. In conclusion, our findings indicated that TRPC1 and PKCα are involved in promoting insulin secretion and that PKCα promotes insulin secretion via TRPC1 phosphorylation in INS-1E cells.     

Grape powder extract attenuates tumor necrosis factor α-mediated inflammation and insulin resistance in primary cultures of human adipocytes

Grapes are rich in phenolic phytochemicals that possess anti-oxidant and anti-inflammatory properties. However, the ability of grape powder extract (GPE) to prevent inflammation and insulin resistance in human adipocytes caused by tumor necrosis factor α (TNFα), a cytokine elevated in plasma and white adipose tissue (WAT) of obese, diabetic individuals, is unknown. Therefore, we examined the effects of GPE on markers of inflammation and insulin resistance in primary cultures of newly differentiated human adipocytes treated with TNFα. We found that GPE attenuated TNFα-induced expression of inflammatory genes including interleukin (IL)-6, IL-1β, IL-8, monocyte chemoattractant protein (MCP)-1, cyclooxygenase (COX)-2 and Toll-like receptor (TLR)-2. GPE attenuated TNFα-mediated activation of extracellular signal-related kinase (ERK) and c-Jun NH₂-terminal kinase (JNK) and activator protein-1 (AP-1, i.e., c-Jun). GPE also attenuated TNFα-mediated IκBα degradation and nuclear factor-kappa B (NF-κB) activity. Finally, GPE prevented TNFα-induced expression of protein tyrosine phosphatase (PTP)-1B and phosphorylation of serine residue 307 of insulin receptor substrate-1 (IRS-1), which are negative regulators of insulin sensitivity, and suppression of insulin-stimulated glucose uptake. Taken together, these data demonstrate that GPE attenuates TNFα-mediated inflammation and insulin resistance in human adipocytes, possibly by suppressing the activation of ERK, JNK, c-Jun and NF-κB.     

Asprosin attenuates insulin signaling pathway through PKCδ-activated ER stress and inflammation in skeletal muscle

It has been reported that asprosin is a novel adipokine which is augmented in mice and humans with type 2 diabetes (T2DM). Asprosin stimulates hepatic gluconeogenesis under fasting conditions. However, the roles of asprosin in inflammation, endoplasmic reticulum (ER) stress, and insulin resistance in skeletal muscle has not been studied. In the currents study, elevated levels of asprosin expression were observed in adipocytes under hyperlipidemic conditions. Treatment of C2C12 myocytes with asprosin-induced ER stress markers (phosphorylated inositol-requiring enzyme 1 and eukaryotic initiation factor 2, and CHOP expression) as well as inflammation markers (interleukin-6 expression, phosphorylated IκB, and nuclear translocated nuclear factor-κβ). Finally, asprosin treatment promoted exacerbation of insulin sensitivity as determined by levels of insulin receptor substrate 1 and Akt phosphorylation as well as glucose uptake. Moreover, treatment of asprosin augmented protein kinase C-δ (PKCδ) phosphorylation and nuclear translocation, but suppressed messenger RNA expression of sarcoplasmic reticulum Ca²⁺ ATPase 2b in both C2C12 myocytes and in mouse soleus skeletal muscle. These asprosin-induced effects were markedly decreased in small interfering (si) RNA-mediated PKCδ-knockdown in C2C12 myocytes. These results suggest that asprosin results in impairment of insulin sensitivity in skeletal muscle through PKCδ-associated ER stress/inflammation pathways and may be a valuable strategy for management of insulin resistance and T2DM.     

Osthol inhibits fatty acid synthesis and release via PPARα/γ-mediated pathways in 3T3-L1 adipocytes

Osthol is an active constituent isolated from the fruit of Cnidium monnieri (L.) Cusson (Apiaceae), and has obvious therapeutic effect on fatty liver, but its mechanisms are not yet understood completely. One potential link between adipose tissue and fatty liver may be circulating fatty acids. In the present study, the effect of osthol on fatty acid synthesis and release in cultured 3T3-L1 adipocytes was observed. Following treatment of adipocytes with osthol, the intracellular levels of free fatty acids (FFA) and triacylglycerols as well as cultured supernatant level of FFA were decreased, and some lipogenic gene and protein expressions were also decreased, while the peroxisome proliferator-activated receptor (PPAR) α/γ mRNA expressions were increased. Osthol-reduced lipogenic gene expressions were decreased or abrogated after pretreatment with specific inhibitor(s) of PPARα and/or PPARγ.     

High dose of glucose promotes chondrogenesis via PKCα and MAPK signaling pathways in chick mesenchymal cells

We investigated the molecular mechanism of the glucose effect on the regulation of chondrogenesis. Exposure of chick wing bud mesenchymal cells to high concentrations of glucose stimulated chondrogenesis 2–fold to 2.5-fold without affecting cell proliferation. Glucose increased protein levels and the membrane translocation of protein kinase C alpha (PKC), leading to a reduction of extracellular signal-regulated kinase (ERK) phosphorylation. Phosphorylation of p38 was also increased in a PKC-independent manner by glucose treatment. Glucose also increased cell adhesion molecules such as fibronectin, integrin 1, and N-cadherin at early stages and then decreased these adhesion molecules at later stages of chondrogenesis. These alterations in protein level of adhesion molecules and in the phosphorylation of mitogen-activated protein kinases by glucose were blocked by inhibition of PKC or p38 but were synergistically increased by the inhibition of ERK. Therefore, high doses of glucose induce the down-regulation of ERK activity via PKC and the up-regulation of p38 and result in the stimulation of chondrogenesis of chick mesenchymal cells through modulating the expression of adhesion molecules.This work was supported by the Korea Research Foundation (KRF-2000-DP0352)     

Fibroblast growth factor-2 promotes catabolism via FGFR1-Ras-Raf-MEK1/2-ERK1/2 axis that coordinates with the PKCδ pathway in human articular chondrocytes

Fibroblast growth factor 2 (FGF-2) has been found to play an anti-anabolic and/or a catabolic role in adult human articular cartilage via regulation of multiple signaling pathways. Upon FGF-2 stimulation, a molecular crosstalk between the mitogen activated protein kinase (MAPK) and protein kinase C δ (PKCδ) pathways are initiated, where PKCδ positively regulates downstream MAPK signaling. In this study, we explored the relationship between fibroblast growth factor receptor 1 (FGFR1), Ras, and PKCδ in FGF-2 signaling in human articular chondrocytes. Pathway-specific inhibition using both chemical inhibitors and siRNA targeting FGFR1 demonstrated that, upon FGF-2 stimulation, FGFR1 controlled both Ras and PKCδ activation, which converged on the Raf-MEK1/2-ERK1/2 axis. No crosstalk was observed between Ras and PKCδ. Quantitative PCR analyses revealed that both Ras and PKCδ contributed to FGF-2-mediated upregulation of MMP-13, ADAMTS5, and repression of aggrecan gene. Correspondingly, FGF-2-mediated proteoglycan loss was effectively reversed by individual pathway-specific inhibitor of Ras, PKCδ, and ERK1/2 in both 3-dimensional alginate bead culture and cartilage organ culture systems. Our findings suggest that FGFR1 interacts with FGF-2 and then activates Ras and PKCδ, which concertedly drive MAPK signaling to mediate biological effects of FGF-2. Such an integration of dual inputs constitutes a novel mechanism of FGF-2 signaling cascade in human articular chondrocytes.     

NLRC5 negatively regulates LTA-induced inflammation via TLR2/NF-κB and participates in TLR2-mediated allergic airway inflammation

NLRC5, the largest member of the Nod-like receptor (NLR) family, has been reported to play a pivotal role in regulating inflammatory responses. Recent evidence suggests that NLRC5 participates in Toll-like receptor (TLR) signaling pathways and negatively modulates nuclear factor-κB (NF-κB) activation. In this study, we investigated the interaction between NLRC5 and TLR2 in the NF-κB inflammatory signaling pathway and the involvement of NLRC5 in TLR2-mediated allergic airway inflammation. We knocked down TLR2 and NLRC5, respectively in the RAW264.7 macrophage cell line by small interfering RNA (siRNA) and then stimulated the knockdown cells with lipoteichoic acid (LTA). In comparison with the negative siRNA group, the level of NLRC5 expression was lower in the TLR2 siRNA group, with a reduction in the NF-κB-related inflammatory response. Conversely, in the NLRC5 knockdown cells, after LTA-treated the level of TLR2 expression did not change but the expression levels of both NF-κB pp65 and NLRP3 increased remarkably. Thus, we hypothesize that NLRC5 participates in the LTA-induced inflammatory signaling pathway and regulates the inflammation via TLR2/NF-κB. Similarly, in subsequent in vivo experiments, we demonstrated that the expression level of NLRC5 was significantly increased in the ovalbumin-induced allergic airway inflammation. However, this effect disappeared in TLR2-deficient (TLR2 ^−/−) mice and was accompanied by reduced levels of NF-κB expression and airway inflammation. In conclusion, NLRC5 negatively regulates LTA-induced inflammatory response via a TLR2/NF-κB pathway in macrophages and also participates in TLR2-mediated allergic airway inflammation.     

Heregulin-β promotes matrix metalloproteinase-7 expression via HER2-mediated AP-1 activation in MCF-7 cells

It has been reported that HER2 level is strongly correlated with the expression of MMP-7 in some carcinomas. HER2 is a preferred heterodimerization partner of EGFR, HER3, and HER4. HER2 overexpression is believed to enhance the signaling from these receptors in response to binding of their specific ligands. In this study, we show that heregulin-beta (HRG-beta) stimulation remarkably induced MMP-7 promoter activity and significantly enhanced the expression and activity of MMP-7 in MCF-7 cells overexpressing HER2. The expression of c-Jun and c-Fos and the level of the phosphorylated c-Jun were markedly increased after HRG-beta treatment in MCF-7/HER2 cells. Increased MMP-7 promoter activity was observed in MCF-7/c-Jun cells. The activity of the MMP-7 promoter induced by HRG-beta in MCF-7/HER2 cells could be inhibited by a dominant negative c-Jun mutant TAM67 and by the mutagenesis of the AP-1 site. c-Jun binding to MMP-7 promoter was confirmed by ChIP assays. The data indicate a close link among HRG-beta stimulation, HER signaling, and AP-1 activation. Our data suggest that HRG-beta-induced MMP-7 expression was regulated by HER2-mediated AP-1 activation in MCF-7 cells.     

β₂ long-acting and anticholinergic drugs control TGF-β1-mediated neutrophilic inflammation in COPD

We quantified TGF-β1 and acetylcholine (ACh) concentrations in induced sputum supernatants (ISSs) from 18 healthy controls (HC), 22 healthy smokers (HS) and 21 COPDs. ISSs from HC, HS and COPD as well as rhTGF-β1 were also tested in neutrophil adhesion and in mAChR2, mAChR3 and ChAT expression experiments in human bronchial epithelial cells (16-HBE). Finally, we evaluated the effects of Olodaterol (a novel inhaled β(2)-adrenoceptor agonist) and Tiotropium Spiriva?, alone or in combination, on neutrophil adhesion and mAChRs and ChAT expression in stimulated 16-HBE. The results showed that 1) TGF-β1 and ACh concentrations are increased in ISSs from COPD in comparison to HC and HS, and TGF-β1 in HS is higher than in HC; 2) ISSs from COPD and HS caused increased neutrophil adhesion to 16-HBE when compared to ISSs from HC. The effect of ISSs from COPD was significantly reduced by TGF-β1 depletion or by the pretreatment with Olodaterol or Tiotropium alone or in combination, while the effect of ISSs from HS was significantly reduced by the pretreatment with Olodaterol alone; 3) mAChR2, mAChR3 and ChAT expression was increased in 16-HBE stimulated with ISSs from COPD and TGF-β1 depletion significantly reduced this effect on mAChR3 and ChAT expression; 4) rhTGF-β1 increased mAChR2, mAChR3 and ChAT expression in 16-HBE; 5) Olodaterol did not affect the expression of mAChRs and ChAT in 16-HBE. Our findings support the use of β? long-acting and anticholinergic drugs to control the bronchoconstriction and TGF-β1-mediated neutrophilic inflammation in COPD.     

20-Hydroxyecdysone inhibits inflammation via SIRT6-mediated NF-κB signaling in endothelial cells

20-Hydroxyecdysone (20E) is known to have numerous pharmacological activities and can be used to treat diabetes and cardiovascular diseases. However, the protective effects of 20E against endothelial dysfunction and its targets remain unclear. In the present study, we revealed that 20E treatment could modulate the release of the endothelium-derived vasomotor factors NO, PGI₂ and ET-1 and suppress the expression of ACE in TNF-α-induced 3D-cultured HUVECs. In addition, 20E suppressed the expression of CD40 and promoted the expression of SIRT6 in TNF-α-induced 3D-cultured HUVECs. The cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) and molecular docking results demonstrated that 20E binding increased SIRT6 stability, indicating that 20E directly bound to SIRT6 in HUVECs. Further investigation of the underlying mechanism showed that 20E could upregulate SIRT6 levels and that SIRT6 knockdown abolished the regulatory effect of 20E on CD40 in TNF-α-induced HUVECs, while SIRT6 overexpression further improved the effect of 20E. Moreover, we found that 20E could reduce the acetylation of NF-κB p65 (K310) through SIRT6, but the catalytic inactive mutant SIRT6 (H133Y) did not promote the deacetylation of NF-κB p65, suggesting that the inhibitory effect of 20E on NF-κB p65 was dependent on SIRT6 deacetylase activity. Additionally, our results indicated that 20E inhibited NF-κB via SIRT6, and the expression of CD40 was increased in HUVECs treated with SIRT6 siRNA and NF-κB inhibitor. In conclusion, the present study demonstrates that 20E exerts its effect through SIRT6-mediated deacetylation of NF-κB p65 (K310) to inhibit CD40 expression in ECs, and 20E may have therapeutic potential for the treatment of cardiovascular diseases.     

Nei-like 1 inhibition results in motor dysfunction and promotes inflammation in Parkinson’s disease mice model

Parkinson’s disease (PD) is well known as a neurodegenerative disorder with progressive loss of dopaminergic (DA) neurons. Nei-like 1 (NEIL1) is one of four mammalian DNA glycosylases involved in the progression of various diseases, including neuroinflammation. However, it is still unknown if the expression changes of NEIL1 could contribute to PD progression. In the present study, we established mouse model with PD using 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to explore the effects of NEIL1 on PD development. Here, we found that NEIL1 deletion significantly promoted the motor dysfunction in the wild type mice treated with 6-OHDA. Furthermore, DA neuronal loss was further accelerated by NEIL1 deletion in 6-OHDA-injected mice, as evidenced by the significantly reduced expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT). Furthermore, in PD mice induced by MPTP, remarkably reduced expression of NEIL1 was observed in nigra and striatum of mice. A strong positive correlation was detected in the expression of NEIL1 and the survival rate of DA neurons. Also, NEIL1 ablation further elevated the DA neuronal loss in MPTP-treated mice, accompanied with higher glial activation, as evidenced by the obvious up-regulation of glial fibrillary acidic protein (GFAP) and Ionized calcium-Binding Adapter molecule 1 (Iba1). Moreover, MPTP-triggered inflammation was highly aggravated by the loss of NEIL1 through inducing the expression of pro-inflammatory cytokines and chemokines. In contrast, promoting NEIL1 expression effectively reversedPD progression induced by MPTP in mice. Together, these results demonstrated that NEIL1 insufficiency might be a contributing factor for the progression of PD, which therefore could be considered as a novel candidate to develop effective treatments against PD progression.     

Porphyromonas gingivalis promotes malignancy and chemo-resistance via GSK3β-mediated mitochondrial oxidative phosphorylation in human esophageal squamous cell carcinoma

Our prior studies have confirmed that long-term colonization of Porphyromonas gingivalis (Pg) and overexpression of the inflammatory factor glycogen synthase kinase 3β (GSK3β) promote the malignant evolution of esophageal squamous cell carcinoma (ESCC). We aimed to investigate the functional mechanism by which Pg could promote ESCC malignancy and chemo-resistance through GSK3β-mediated mitochondrial oxidative phosphorylation (mtOXPHOS), and the clinical implications. The effects of Pg and GSK3β on mtOXPHOS, malignant behaviors and response to paclitaxel and cisplatin treatment of ESCC cells were evaluated by in vitro and in vivo studies. The results showed that Pg induced high expression of the GSK3β protein in ESCC cells and promoted the progression and chemo-resistance via GSK3β-mediated mtOXPHOS in human ESCC. Then, Pg infection and the expression of GSK3β, SIRT1 and MRPS5 in ESCC tissues were detected, and the correlations between each index and postoperative survival of ESCC patients were analysed. The results showed that Pg-positive ESCC patients with high-expression of GSK3β, SIRT1 and MRPS5 have significant short postoperative survival. In conclusion, we demonstrated that the effective removal of Pg and inhibition of its promotion of GSK3β-mediated mtOXPHOS may provide a new strategy for ESCC treatment and new insights into the aetiology of ESCC.     

Conditional overexpression of TGFβ1 promotes pulmonary inflammation,apoptosis and mortality via TGFβR2 in the developing mouse lung

Background

Earlier studies have reported that transforming growth factor beta 1(TGFβ1) is a critical mediator of hyperoxia-induced acute lung injury (HALI) in developing lungs, leading to impaired alveolarization and a pulmonary phenotype of bronchopulmonary dysplasia (BPD). However, the mechanisms responsible for the TGFβ1-induced inflammatory signals that lead to cell death and abnormal alveolarization are poorly understood. We hypothesized that TGFβ1 signaling via TGFβR2 is necessary for the pathogenesis of the BPD pulmonary phenotype resulting from HALI.

Methods

We utilized lung epithelial cell-specific TGFβ1 overexpressing transgenic and TGFβR2 null mutant mice to evaluate the effects on neonatal mortality as well as pulmonary inflammation and apoptosis in developing lungs. Lung morphometry was performed to determine the impaired alveolarization and multicolor flow cytometry studies were performed to detect inflammatory macrophages and monocytes in lungs. Apoptotic cell death was measured with TUNEL assay, immunohistochemistry and western blotting and protein expression of angiogenic mediators were also analyzed.

Results

Our data reveals that increased TGFβ1 expression in newborn mice lungs leads to increased mortality, macrophage and immature monocyte infiltration, apoptotic cell death specifically in Type II alveolar epithelial cells (AECs), impaired alveolarization, and dysregulated angiogenic molecular markers.

Conclusions

Our study has demonstrated the potential role of inhibition of TGFβ1 signaling via TGFβR2 for improved survival, reduced inflammation and apoptosis that may provide insights for the development of potential therapeutic strategies targeted against HALI and BPD.