Regulation Of MAPK Activation Research Articles

Calmodulin-dependent protein kinase kinase 2 as a novel therapeutic target against osteoarthritis

Purpose: Osteoarthritis (OA) is a disabling disease that affects more than 250 million people worldwide causing tremendous individual and socioeconomic burden. OA is a complex disease encompassing multiple molecular/biological mechanisms that result in progressive cartilage loss, subchondral bone remodeling, joint tissue inflammation, and osteophytes formation. OA is often diagnosed at a late stage and treatment is confined to management of symptoms due to a lack of clinically available therapies to stop or delay the disease progression. Thus, there is a critical need to identify novel therapeutic targets that could prevent and/or delay multiple facets of OA pathogenesis. Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) plays an important role in bone remodeling and macrophage-mediated inflammation. Hence, we hypothesized that genetic ablation or pharmacological inhibition of CaMKK2 will suppress inflammation and subchondral bone remodeling associated with OA. Methods: Male wild-type (WT) and/or Camkk2-/- mice (both C57BL6; n = 5 per group) underwent unilateral destabilization of medical meniscus (DMM) or sham surgery. WT mice undergoing sham and DMM surgeries were administered STO-609 (CaMKK2 inhibitor; 10 μM/kg body weight) or saline solution tri-weekly by i.p injection for 8 weeks. Damage to the joint tissues was evaluated using microcomputed tomography (microCT) and histology. The impact of CaMKK2 deficiency in cartilage pathophysiology was assessed in vitro using primary WT and Camkk2-/- murine articular chondrocytes (MACs) stimulated by interleukin-1β (IL-1β; 10 ng/ml). Quantitative real-time PCR was used to assess the expression of markers of cartilage matrix and turnover as well as inflammation. Results: Three dimensional microCT analysis of tibial subchondral bone showed a significant increase of tissue volume (TV; by 40%) and bone volume (BV/TV; 18%) in saline-treated DMM mice relative to sham-operated control mice. In addition, the thickness of subchondral bone plate (SPB) was 32% higher in DMM mice compared to sham operated cohorts. These results demonstrated an enhanced subchondral bone sclerosis after DMM surgery. Interestingly, such increases in TV and BV/TV were significantly diminished in CaMKK2 inhibitor-treated WT and Camkk2-/- mice, indicating protection from subchondral bone sclerosis induced by DMM. Moreover, SBP thickness was significantly lower in STO-609 treated WT and Camkk2-/- mice, compared to saline treated WT cohorts. Histological analyses revealed lower OARSI score and prevention of cartilage breakdown following DMM surgery in STO-609 treated WT and Camkk2-/- mice. Moreover, inhibition or loss of CaMKK2 also resulted in significantly reduced macrophage infiltration and inflammation of the synovium following DMM. Further, data obtained from isolated MACs reveal that the genetic ablation of CaMKK2 was able to counteract the effects of IL-1β by affecting the expression levels of inflammatory (iNOS, COX2, TNF-α, and IL6) cytokines and markers of cartilage catabolism (MMP2, MMP-13, and ADAMTS5). In addition, Camkk2-/- MACs expressed significantly higher levels of extracellular matrix proteins such as Col2a1, COMP and glycosaminoglycans (GAGs) in response to IL-1β, compared to WT. Whereas an increase in the activation of ERK was observed, the activity of p38 MAPK was significantly inhibited in theCamkk2-/- MACs. Inhibition of ERK with a specific inhibitor resulted in an increase of inflammatory and catabolism markers in Camkk2-/- MACs, indicating that the protective effects of CaMKK2 loss on OA pathogenesis is potentially through its regulation of MAPK activity. Conclusions: In conclusion, targeting CaMKK2 offers an overall protection of joint tissues in trauma-induced OA, owing to a suppression of inflammation and catabolism through a regulation of the ERK pathway as well as an increase in cartilage matrix production. Our study provides novel information on the role of CaMKK2 in OA pathogenesis and opens new avenues to design innovative therapeutic strategies against OA.

Transduction of Functionally Contrasting Signals by Two Mycobacterial PPE Proteins Downstream of TLR2 Receptors.

As pathogen-associated molecular pattern sensors, the TLRs can detect diverse ligands to elicit either proinflammatory or anti-inflammatory responses, but the mechanism that dictates such contrasting immune responses is not well understood. In this work, we demonstrate that proline-proline-glutamic acid (PPE)17 protein of Mycobacterium tuberculosis induces TLR1/2 heterodimerization to elicit proinflammatory-type response, whereas PPE18-induced homodimerization of TLR2 triggers anti-inflammatory type responses. Ligation of TLR1/2 caused an increased recruitment of IL-1R-associated kinase (IRAK)1, MyD88, and protein kinase C (PKC)ε to the downstream TLR-signaling complex that translocated PKCε into the nucleus in an IRAK1-dependent manner. PKCε-mediated phosphorylation allowed the nuclear IRAK3 to be exported to the cytoplasm, leading to increased activation of ERK1/2, stabilization of MAPK phosphatase 1 (MKP-1), and induction of TNF-α with concomitant downregulation of p38MAPK. Silencing of TLR1 inhibited PPE17-triggered cytoplasmic export of IRAK3 as well as TNF-α induction, suggesting an important role of TLR1/2 heterodimer in regulating proinflammatory responses via the IRAK3-signaling pathway. In contrast, PPE18-mediated homodimerization of TLR2 caused poorer cytoplasmic export of nuclear IRAK3 and MKP-1 stabilization, resulting in increased p38MAPK activation. Our study hints to a novel mechanism that implicates PKCε-IRAK3-MKP-1 signaling in the regulation of MAPK activity and inflammatory cascades downstream of TLR2 in tuberculosis.

Endogenous secreted phospholipase A2 group X regulates cysteinyl leukotrienes synthesis by human eosinophils

Phospholipase A2s mediate the rate-limiting step in the formation of eicosanoids such as cysteinyl leukotrienes (CysLTs). Group IVA cytosolic PLA2α (cPLA2α) is thought to be the dominant PLA2 in eosinophils; however, eosinophils also have secreted PLA2 (sPLA2) activity that has not been fully defined. To examine the expression of sPLA2 group X (sPLA2-X) in eosinophils, the participation of sPLA2-X in the formation of CysLTs, and the mechanism by which sPLA2-X initiates the synthesis of CysLTs in eosinophils. Peripheral blood eosinophils were obtained from volunteers with asthma and/or allergy. Arabbit polyclonal anti-sPLA2-X antibody identified sPLA2-X by Western blot. Weused confocal microscopy to colocalize the sPLA2-X to intracellular structures. An inhibitor of sPLA2-X (ROC-0929) that does not inhibit other mammalian sPLA2s, as well as inhibitors of the mitogen-activated kinase cascade (MAPK) and cPLA2α, was used to examine the mechanism of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-mediated formation of CysLT. Eosinophils express the mammalian sPLA2-X gene (PLA2G10). The sPLA2-X protein is located in the endoplasmic reticulum, golgi, and granules of eosinophils and moves to the granules and lipid bodies during fMLP-mediated activation. Selective sPLA2-X inhibition attenuated the fMLP-mediated release of arachidonic acid and CysLT formation by eosinophils. Inhibitors of p38, extracellular-signal-regulated kinases 1/2 (p44/42 MAPK), c-Jun N-terminal kinase, and cPLA2α also attenuated the fMLP-mediated formation of CysLT. The sPLA2-X inhibitor reduced the phosphorylation of p38 and extracellular-signal-regulated kinases 1/2 (p44/42 MAPK) as well as cPLA2α during cellular activation, indicating that sPLA2-X is involved in activating the MAPK cascade leading to the formation of CysLT via cPLA2α. We further demonstrate that sPLA2-X is activated before secretion from the cell during activation. Short-term priming with IL-13 and TNF/IL-1β increased the expression of PLA2G10 by eosinophils. These results demonstrate that sPLA2-X plays a significant role in the formation of CysLTs by human eosinophils. The predominant role of the enzyme is the regulation of MAPK activation that leads to the phosphorylation of cPLA2α. The sPLA2-X protein is regulatedby proteolytic cleavage, suggesting that an inflammatory environment may promote the formation of CysLTs through this mechanism. These results have important implications forthe treatment of eosinophilic disorders such as asthma.

Regulation of Angiopoietin‐1/Tie‐2 Receptor Signaling in Endothelial Cells by Dual‐Specificity Phosphatases 1, 4, and 5

BackgroundAngiopoietin‐1 (Ang‐1) promotes survival and migration of endothelial cells, in part through the activation of mitogen‐activated protein kinase (MAPK) pathways downstream of Tie‐2 receptors. Dual‐specificity phosphatases (DUSPs) dephosphorylate phosphotyrosine and phosphoserine/phosphothreonine residues on target MAPKs. The mechanisms by which DUSPs modulate MAPK activation in Ang‐1/Tie‐2 receptor signaling are unknown in endothelial cells.Methods and ResultsExpression of various DUSPs in human umbilical vein endothelial cells exposed to Ang‐1 was measured. The functional roles of DUSPs in Ang‐1‐induced regulation of MAPK activation, endothelial cell survival, migration, differentiation, and permeability were measured using selective siRNA oligos. Ang‐1 differentially induces DUSP1, DUSP4, and DUSP5 in human umbilical vein endothelial cells through activation of the PI‐3 kinase, ERK1/2, p38, and SAPK/JNK pathways. Lack‐of‐function siRNA screening revealed that DUSP1 preferentially dephosphorylates p38 protein and is involved in Ang‐1‐induced cell migration and differentiation. DUSP4 preferentially dephosphorylates ERK1/2, p38, and SAPK/JNK proteins and, under conditions of serum deprivation, is involved in Ang‐1‐induced cell migration, several antiapoptotic effects, and differentiation. DUSP5 preferentially dephosphorylates ERK1/2 proteins and is involved in cell survival and inhibition of permeability.ConclusionsDUSP1, DUSP4, and DUSP5 differentially modulate MAPK signaling pathways downstream of Tie‐2 receptors, thus highlighting the importance of these phosphatases to Ang‐1‐induced angiogenesis.

Genistein regulates the IL-1 beta induced activation of MAPKs in human periodontal ligament cells through G protein-coupled receptor 30

Periodontal ligament (PDL) cells are fibroblasts that play key roles in tissue integrity, periodontal inflammation and tissue regeneration in the periodontium. The periodontal tissue destruction in periodontitis is mediated by host tissue-produced inflammatory cytokines, including interleukin-1β (IL-1β). Here, we report the expression of G protein-coupled receptor 30 (GPR30, also known as G protein-coupled estrogen receptor 1 GPER) in human PDL cells and its regulation by IL-1β. IL-1β-induced GPR30 expression in human PDL cells leads to the activation of multiple signaling pathways, including MAPK, NF-κB and PI3K. In contrast, genistein, an estrogen receptor ligand, postpones the activation of MAPKs induced by IL-1β. Moreover, the inhibition of GPR30 by G15, a GPR30-specific antagonist, eliminates this delay. Thus, genistein plays a role in the regulation of MAPK activation via GPR30, and GPR30 represents a novel target regulated by steroid hormones in PDL cells.

Impaired dephosphorylation renders G6PD-knockdown HepG2 cells more susceptible to H 2O 2-induced apoptosis

Glucose-6-phosphate dehydrogenase (G6PD) plays a key role in the regeneration of NADPH and maintenance of cellular redox balance. In the present study, we investigate the effect of G6PD deficiency on H 2O 2-elicited signaling in HepG2 cells. H 2O 2 was found to inhibit cellular protein tyrosine phosphatase (PTP) activity, resulting in activation of MAPKs. MKP-1 expression increased in the late phase of H 2O 2 signaling. Using RNAi technology, we found that G6PD knockdown enhanced the inhibitory effect of H 2O 2 on PTPs and led to sustained MAPK activation. This was accompanied by delayed expression and inhibition of MKP-1. Using a pharmacological inhibitor and siRNA, we demonstrate that MKP-1 acts as a regulator of MAPK activation in H 2O 2 signaling. The prolonged MAPK activation in G6PD-knockdown cells was associated with an increased susceptibility to H 2O 2-induced apoptosis and growth retardation. Treatment with p38 and JNK inhibitors or N-acetylcysteine ameliorated such cellular effect, while triptolide and MKP-1-siRNA did the opposite. Glucose oxidase treatment had similar effects as addition of H 2O 2. Taken together, these findings suggest that G6PD knockdown enhances the magnitude and duration of H 2O 2-induced MAPK signaling through inhibition of cellular PTPs, and the resultant anomalous signaling may lead to cell demise.

Regulation of Mapk Activity in Response to Dietary Sodium in the Rat Adrenal Gland

Stimulation of aldosterone biosynthesis by angiotensin II (AII) is thought to be mediated via the PLC, IP3 and intracellular calcium signalling pathway. MAPK (p42/p44) is involved in cell proliferation, and is also activated by AII, but its role in the adrenal response to dietary sodium is unclear. To study the relationship between AII receptor (ATR), MAPK and PKC isoforms, PKCα and PKϵ, mature Wistar rats were maintained on low or high sodium diets for 1 week. In adrenals from animals on a sodium deplete diet, total ligand binding to both ATR subtypes decreased in the zona glomerulosa (ZG). Under these conditions, active MAPK in the ZG decreased paralleling a decrease in active PKCα. In the inner zones (IZ), largely reflecting medullary events, low sodium did not affect MAPK activity. However active PKCα decreased. In adrenals from sodium-loaded animals, type 2 ATR (AT2R) binding was reduced in the ZG, while type 1 ATR (AT1R) increased in the IZ. Active MAPK increased in ZG, as did active PKCα and PKϵ. In IZ, ERK, PKCα and PKϵ were unchanged. These results suggest that in the ZG and IZ, two different modes of MAPK regulation may exist, utilising different PKC isoforms.