A Brief Ischemic Postconditioning Protects Against Amyloid-ß Peptide Neurotoxicity by Downregulating MLK3-MKK3/6-P38MAPK Signal in Rat Hippocampus.

BACKGROUND: Oligomeric amyloid-ß peptide (Aß) is associated with dysfunctional neuronal networks and neuronal loss in the development of Alzheimer's disease (AD). Ischemic postconditioning protects against post-ischemic excitotoxicity, oxidative stress, and inflammatory process that have also been implicated in the pathogenesis of AD. Evaluating the roles of ischemic postconditioning in oligomeric Aß-induced neurotoxicity and underlying signal events may provide potential strategy for medical therapy in AD. OBJECTIVES: The aim of the present study was to explore whether and how a brief ischemic postconditioning protects against Aß neurotoxicity in rat hippocampus. METHODS: Oligomeric Aß25-35 (20 nmol/rat) or Aß1-42 (5 nmol/rat) was infused by intracerebroventricular injection in adult male Sprague-Dawley rats. Ischemic postconditioning, a brief episode of global brain ischemia (3 min), was conducted at 1, 3, or 7 days after Aß treatment, respectively. RESULTS: A brief ischemic postconditioning reduced neuronal loss and inhibited the activation of MLK3, MKK3/6, and P38MAPKs in rat hippocampal CA1 and CA3 subfields after Aß oligomer infusion. An N-methyl-D-aspartate (NMDA) receptor antagonist amantadine, but not non-NMDA receptor antagonist CNQX, reversed the MLK3-MKK3/6-P38MAPK signal events and beneficial effect of ischemic postconditioning on neuronal survival. Such reversion was also realized by NVP-AAM077, a GluN2A-subunit-selective NMDA receptor antagonist. Moreover, posttreatment with low doses of NMDA (5 nmol-40 nmol/rat) suppressed the Aß-induced P38MAPK signaling and imitated the neuroprotection of ischemic postconditioning against Aß neurotoxicity. CONCLUSIONS: Ischemic postconditioning provides neuroprotection against Aß neurotoxicity by moderate upregulation of NMDA receptor signaling, especially GluN2A-containing NMDA receptor pathway, and thereafter downregulation of MLK3-MKK3/6-P38MAPK signal events.

Autophagy-induced senescence is regulated by p38α signaling.

Apoptosis and senescence are two mutually exclusive cell fate programs that can be activated by stress. The factors that instruct cells to enter into senescence or apoptosis are not fully understood, but both programs can be regulated by the stress kinase p38α. Using an inducible system that specifically activates this pathway, we show that sustained p38α activation suffices to trigger massive autophagosome formation and to enhance the basal autophagic flux. This requires the concurrent effect of increased mitochondrial reactive oxygen species production and the phosphorylation of the ULK1 kinase on Ser-555 by p38α. Moreover, we demonstrate that macroautophagy induction by p38α signaling determines that cancer cells preferentially enter senescence instead of undergoing apoptosis. In agreement with these results, we present evidence that the induction of autophagy by p38α protects cancer cells from chemotherapy-induced apoptosis by promoting senescence. Our results identify a new mechanism of p38α-regulated basal autophagy that controls the fate of cancer cells in response to stress.

Gossypetin is a novel MKK3 and MKK6 inhibitor that suppresses esophageal cancer growth in vitro and in vivo.

Gossypetin as a hexahydroxylated flavonoid found in many flowers and Hibiscus. It exerts various pharmacological activities, including antioxidant, antibacterial and anticancer activities. However, the anticancer capacity of gossypetin has not been fully elucidated. In this study, gossypetin was found to inhibit anchorage-dependent and -independent growth of esophageal cancer cells. To identify the molecular target(s) of gossypetin, various signaling protein kinases were screened and results indicate that gossypetin strongly attenuates the MKK3/6-p38 signaling pathway by directly inhibiting MKK3 and MKK6 protein kinase activity in vitro. Mechanistic investigations showed that arginine-61 in MKK6 is critical for binding with gossypetin. Additionally, the inhibition of cell growth by gossypetin is dependent on the expression of MKK3 and MKK6. Gossypetin caused G2 phase cell cycle arrest and induced intrinsic apoptosis by activating caspases 3 and 7 and increasing the expression of BAX and cytochrome c. Notably, gossypetin suppressed patient-derived esophageal xenograft tumor growth in an in vivo mouse model. Our findings suggest that gossypetin is an MKK3 and MKK6 inhibitor that could be useful for preventing or treating esophageal cancer.

Identification of Functional MKK3/6 and MEK1/2 Homologs from Echinococcus granulosus and Investigation of Protoscolecidal Activity of Mitogen-Activated Protein Kinase Signaling Pathway Inhibitors In Vitro and In Vivo.

Cystic echinococcosis is a zoonosis caused by the larval stage of Echinococcus granulosussensu lato There is an urgent need to develop new drugs for the treatment of this disease. In this study, we identified two new members of mitogen-activated protein kinase (MAPK) cascades, MKK3/6 and MEK1/2 homologs (termed EgMKK1 and EgMKK2, respectively), from E. granulosussensu stricto Both EgMKK1 and EgMKK2 were expressed at the larval stages. As shown by yeast two-hybrid and coimmunoprecipitation analyses, EgMKK1 interacted with the previously identified Egp38 protein but not with EgERK. EgMKK2, on the other hand, interacted with EgERK. In addition, EgMKK1 and EgMKK2 displayed kinase activity toward the substrate myelin basic protein. When sorafenib tosylate, PD184352, or U0126-ethanol (EtOH) was added to the medium for in vitro culture of E. granulosus protoscoleces (PSCs) or cysts, an inhibitory and cytolytic effect was observed via suppressed phosphorylation of EgMKKs and EgERK. Nonviability of PSCs treated with sorafenib tosylate or U0126-EtOH, and not with PD184352, was confirmed through bioassays, i.e., inoculation of treated and untreated protoscoleces into mice. In vivo treatment of E. granulosussensu stricto-infected mice with sorafenib tosylate or U0126-EtOH for 4 weeks demonstrated a reduction in parasite weight, but the results did not show a significant difference. In conclusion, the MAPK cascades were identified as new targets for drug development, and E. granulosus was efficiently inhibited by their inhibitors in vitro The translation of these findings into in vivo efficacy requires further adjustment of treatment regimens using sorafenib tosylate or, possibly, other kinase inhibitors.

Pharmacological targeting of p38 MAP-Kinase 6 (MAP2K6) inhibits the growth of esophageal adenocarcinoma.

Drug repurposing with a better understanding of the underlying mechanism has provided new avenues to find treatment for malignancies. Esophageal adenocarcinoma (EAC) is a rapidly increasing cancer with a dismal 5-year survival rate of <15%. Lack of efficient treatment options contributes to the high mortality rate of EAC. To find new therapy against EAC we performed unbiased drug screening of an FDA-approved drug library and identified that the cardiac glycosides including Ouabain, Digoxin and Digitoxin efficiently inhibit the proliferation of EAC cell lines (OE33 and OE19) both in vitro and in vivo. RNA-Sequencing analysis combined with RNAi screening revealed that Ouabain suppresses the proliferation of EAC cells through downregulation of p38 MAP-Kinase 6 (MAP2K6, also known as MKK6). Consistently, shRNA-mediated knockdown of MKK6 reduced the proliferation of EAC cells and tumor growth. Further analysis demonstrated that MKK6 inhibition leads to the reduced levels of the transcription factor SOX9. In line with this finding, deletion of SOX9 with CRISPR/Cas9 resulted in decreased proliferation of EACs in 3D organoid culture and reduced tumor growth. Together these findings establish a druggable axis that can be harnessed for therapeutic gain against EAC.

Discovery of 2-arylquinazoline derivatives as a new class of ASK1 inhibitors.

The development of a new series of apoptosis signal-regulating kinase 1 (ASK1) inhibitors is described. Starting from purine, pyrimidine and quinazoline scaffolds identified by high throughput screening, we used tools of structure-based drug design to develop a series of potent kinase inhibitors, including 2-arylquinazoline derivatives 12 and 23, with submicromolar inhibitory activities against ASK1. Kinetic analysis demonstrated that the 2-arylquinazoline scaffold ASK1 inhibitors described herein are ATP competitive.

Discovery of a Covalent Kinase Inhibitor from a DNA-Encoded Small-Molecule Library × Protein Library Selection.

We previously reported interaction determination using unpurified proteins (IDUP), a method to selectively amplify DNA sequences encoding ligand:target pairs from a mixture of DNA-linked small molecules and unpurified protein targets in cell lysates. In this study, we applied IDUP to libraries of DNA-encoded bioactive compounds and DNA-tagged human kinases to identify ligand:protein binding partners out of 32 096 possible combinations in a single solution-phase library × library experiment. The results recapitulated known small molecule:protein interactions and also revealed that ethacrynic acid is a novel ligand and inhibitor of MAP2K6 kinase. Ethacrynic acid inhibits MAP2K6 in part through alkylation of a nonconserved cysteine residue. This work validates the ability of IDUP to discover ligands for proteins of biomedical relevance.

Anti-Inflammatory Effects of Chloranthalactone B in LPS-Stimulated RAW264.7 Cells.

Chloranthalactone B (CTB), a lindenane-type sesquiterpenoid, was obtained from the Chinese medicinal herb Sarcandra glabra, which is frequently used as a remedy for inflammatory diseases. However, the anti-inflammatory mechanisms of CTB have not been fully elucidated. In this study, we investigated the molecular mechanisms underlying these effects in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. CTB strongly inhibited the production of nitric oxide and pro-inflammatory mediators such as prostaglandin E2, tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß), and IL-6 in RAW264.7 cells stimulated with LPS. A reverse-transcription polymerase chain reaction assay and Western blot further confirmed that CTB inhibited the expression of inducible nitric oxide synthase, cyclooxygenase-2, TNF-α, and IL-1ß at the transcriptional level, and decreased the luciferase activities of activator protein (AP)-1 reporter promoters. These data suggest that inhibition occurred at the transcriptional level. In addition, CTB blocked the activation of p38 mitogen-activated protein kinase (MAPK) but not c-Jun N-terminal kinase or extracellular signal-regulated kinase 1/2. Furthermore, CTB suppressed the phosphorylation of MKK3/6 by targeting the binding sites via formation of hydrogen bonds. Our findings clearly show that CTB inhibits the production of inflammatory mediators by inhibiting the AP-1 and p38 MAPK pathways. Therefore, CTB could potentially be used as an anti-inflammatory agent.

Fragment-based drug discovery of potent and selective MKK3/6 inhibitors.

The MAPK signaling cascade, comprised of several linear and intersecting pathways, propagates signaling into the nucleus resulting in cytokine and chemokine release. The Map Kinase Kinase isoforms 3 and 6 (MKK3 and MKK6) are responsible for the phosphorylation and activation of p38, and are hypothesized to play a key role in regulating this pathway without the redundancy seen in downstream effectors. Using FBDD, we have discovered efficient and selective inhibitors of MKK3 and MKK6 that can serve as tool molecules to help further understand the role of these kinases in MAPK signaling, and the potential impact of inhibiting kinases upstream of p38.

Overexpression of prohibitin-1 inhibits RANKL-induced activation of p38-Elk-1-SRE signaling axis blocking MKK6 activity.

Prohibitin-1 (PHB) regulates diverse cellular processes by controlling several signaling pathways. In this study, we investigated the functional involvement of PHB in osteoclast differentiation. PHB expression was time-dependently increased by RANKL in BMMs. However, the retroviral over-expression of PHB strongly inhibited the expression of c-Fos and NFATc1, and activation of p38-Elk-1-SRE signaling pathway. Anti-osteoclastogenic action of PHB was significantly inhibited by constitutively active forms of MKK6, but not Elk-1. Collectively, PHB negatively regulates the formation of mature osteoclasts via inhibition of MKK6 activity that affects the activation of the p38-Elk-1 signaling axis required for the expression of c-Fos and NFATc1.

ß-Amyloid-evoked apoptotic cell death is mediated through MKK6-p66shc pathway.

We have previously shown the involvement of p66shc in mediating apoptosis. Here, we demonstrate the novel mechanism of ß-Amyloid-induced toxicity in the mammalian cells. ß-Amyloid leads to the phosphorylation of p66shc at the serine 36 residue and activates MKK6, by mediating the phosphorylation at serine 207 residue. Treatment of cells with antioxidants blocks ß-Amyloid-induced serine phosphorylation of MKK6, reactive oxygen species (ROS) generation, and hence protected cells against ß-Amyloid-induced cell death. Our results indicate that serine phosphorylation of p66shc is carried out by active MKK6. MKK6 knock-down resulted in decreased serine 36 phosphorylation of p66shc. Co-immunoprecipitation results demonstrate a direct physical association between p66shc and WT MKK6, but not with its mutants. Increase in ß-Amyloid-induced ROS production was observed in the presence of MKK6 and p66shc, when compared to triple mutant of MKK6 (inactive) and S36 mutant of p66shc. ROS scavengers and knock-down against p66shc, and MKK6 significantly decreased the endogenous level of active p66shc, ROS production, and cell death. Finally, we show that the MKK6-p66shc complex mediates ß-Amyloid-evoked apoptotic cell death.

Crystal structure of non-phosphorylated MAP2K6 in a putative auto-inhibition state.

Mitogen-activated protein kinase kinase 6 (MAP2K6) plays a crucial role in the p38 MAP kinase signal cascade that regulates various stress-induced responses and is associated with pathological conditions. The crystal structure of human non-phosphorylated MAP2K6 (npMAP2K6) complexed with an ATP analogue was determined at 2.6 Å resolution and represents an auto-inhibition state of MAP2K6. Three characteristics of short α-helices configured in the activation loop region, termed activation helices (AH1, AH2 and AH3), are important in controlling the auto-inhibition mechanism. AH1 displaces the αC-helix, a component essential for forming the active configuration, away from the active site. AH1 and AH2 were found to enclose the γ-phosphate, the leaving group of ATP. A comparison with the related enzymes, MAP2K1 and MAP2K4 reveals that MAP2K6 has the unique auto-inhibition mechanism mediated by the three activation helices.

Oxidation-induced intramolecular disulfide bond inactivates mitogen-activated protein kinase kinase 6 by inhibiting ATP binding.

Mitogen-activated protein kinase kinase 6 (MKK6) is a member of the mitogen-activated protein kinase (MAPK) kinase (MAP2K) subfamily that specifically phosphorylates and activates the p38 MAPKs. Based on both biochemical and cellular assays, we found that MKK6 was extremely sensitive to oxidation: It was inactivated by oxidation and its kinase activity was fully restored upon treatment with a reducing agent. Detailed mechanistic studies showed that cysteines 109 and 196, two of the six cysteines in MKK6, formed an intramolecular disulfide bond upon oxidation that inactivated MKK6 by inhibiting its ATP binding. This mechanism is distinct from that seen in other redox-sensitive kinases. The two cysteines involved in intramolecular disulfide formation are conserved in all seven members of the MAP2K family. Consistently, we confirmed that other MAP2Ks were also sensitive to oxidation. Our work reveals that MKK6 and other MAP2Ks are a distinct class of cellular redox sensors.

The structure of the MAP2K MEK6 reveals an autoinhibitory dimer.

MAP2Ks are dual-specificity protein kinases functioning at the center of three-tiered MAP kinase modules. The structure of the kinase domain of the MAP2K MEK6 with phosphorylation site mimetic aspartic acid mutations (MEK6/DeltaN/DD) has been solved at 2.3 angstroms resolution. The structure reveals an autoinhibited elongated ellipsoidal dimer. The enzyme adopts an inactive conformation, based upon structural queues, despite the phosphomimetic mutations. Gel filtration and small-angle X-ray scattering analysis confirm that the crystallographically observed ellipsoidal dimer is a feature of MEK6/DeltaN/DD and full-length unphosphorylated wild-type MEK6 in solution. The interface includes the phosphate binding ribbon of each subunit, part of the activation loop, and a rare "arginine stack" between symmetry-related arginine residues in the N-terminal lobe. The autoinhibited structure likely confers specificity on active MAP2Ks. The dimer may also serve the function in unphosphorylated MEK6 of preventing activation loop phosphorylation by inappropriate kinases.

Vitamin E succinate induces NAG-1 expression in a p38 kinase-dependent mechanism.

NAG-1 (nonsteroidal anti-inflammatory drug-activated gene), a member of the transforming growth factor-beta superfamily, is involved in many cellular processes, such as inflammation, apoptosis/survival, and tumorigenesis. Vitamin E succinate (VES) is the succinate derivative of alpha-tocopherol and has antitumorigenic activity in a variety of cell culture and animal models. In the current study, the regulation and role of NAG-1 expression in PC-3 human prostate carcinoma cells by VES was examined. VES treatment induced growth arrest and apoptosis as well as an increase in NAG-1 protein and mRNA levels in a time- and concentration-dependent manner. VES treatment induced nuclear translocation and activation of p38 kinase. Pretreatment with p38 kinase inhibitor blocked the VES-induced increase in NAG-1 protein and mRNA levels, whereas an inhibition of protein kinase C, Akt, c-Jun NH(2)-terminal kinase, or MEK activity had no effect on VES-induced NAG-1 levels. Forced expression of constitutively active MKK6, an upstream kinase for p38, induced an increase in NAG-1 promoter activity, whereas p38 kinase inhibitor blocked MKK6-induced increase in NAG-1 promoter activity. VES treatment resulted in >3-fold increase in the half-life of NAG-1 mRNA in a p38 kinase-dependent manner and transient transfection experiment showed that VES stabilizes NAG-1 mRNA through AU-rich elements in 3'-untranslated region of NAG-1 mRNA. The inhibition of NAG-1 expression by small interfering RNA significantly blocked VES-induced poly(ADP-ribose) polymerase cleavage, suggesting that NAG-1 may play an important role in VES-induced apoptosis. These results indicate that VES-induced expression of NAG-1 mRNA/protein is regulated by transcriptional/post-transcriptional mechanism in a p38 kinase-dependent manner and NAG-1 can be chemopreventive/therapeutic target in prostate cancer.

A dominant function of p38 mitogen-activated protein kinase signaling in receptor activator of nuclear factor-kappaB ligand expression and osteoclastogenesis induction by Aggregatibacter actinomycetemcomitans and Escherichia coli lipopolysaccharide.

BACKGROUND AND OBJECTIVE: Lipopolysaccharide from gram-negative bacteria is one of the microbial-associated molecular patterns that initiate the immune/inflammatory response, leading to the tissue destruction observed in periodontitis. The aim of this study was to evaluate the role of the p38 mitogen-activated protein kinase (MAPK) signaling pathway in lipopolysaccharide-induced receptor activator of nuclear factor-kappaB ligand (RANKL) expression by murine periodontal ligament cells. MATERIAL AND METHODS: Expression of RANKL and osteoprotegerin mRNA was studied by reverse transcription-polymerase chain reaction upon stimulation with lipopolysaccharide from Escherichia coli and Aggregatibacter actinomycetemcomitans. The biochemical inhibitor SB203580 was used to evaluate the contribution of the p38 MAPK signaling pathway to lipopolysaccharide-induced RANKL and osteoprotegerin expression. Stable cell lines expressing dominant-negative forms of MAPK kinase (MKK)-3 and MKK6 were generated to confirm the role of the p38 MAPK pathway. An osteoclastogenesis assay using a coculture model of the murine monocytic cell line RAW 264.7 was used to determine if osteoclast differentiation induced by lipopolysaccharide-stimulated periodontal ligament was correlated with RANKL expression. RESULTS: Inhibiting p38 MAPK prior to lipopolysaccharide stimulation resulted in a significant decrease of RANKL mRNA expression. Osteoprotegerin mRNA expression was not affected by lipopolysaccharide or p38 MAPK. Lipopolysaccharide-stimulated periodontal ligament cells increased osteoclast differentiation, an effect that was completely blocked by osteoprotegerin and significantly decreased by inhibition of MKK3 and MKK6, upstream activators of p38 MAPK. Conditioned medium from murine periodontal ligament cultures did not increase osteoclast differentiation, indicating that periodontal ligament cells produced membrane-bound RANKL. CONCLUSION: Lipopolysaccharide resulted in a significant increase of RANKL in periodontal ligament cells. The p38 MAPK pathway is required for lipopolysaccharide-induced membrane-bound RANKL expression in these cells.

MKK3/6-p38 MAPK signaling is required for IL-1beta and TNF-alpha-induced RANKL expression in bone marrow stromal cells.

Coupled bone turnover is directed by the expression of receptor-activated NF-kappaB ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG). Proinflammatory cytokines, such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) induce RANKL expression in bone marrow stromal cells. Here, we report that IL-1beta and TNF-alpha-induced RANKL requires p38 mitogen-activating protein kinase (MAPK) pathway activation for maximal expression. Real-time PCR was used to assess the p38 contribution toward IL-1beta and TNF-alpha-induced RANKL mRNA expression. Steady-state RANKL RNA levels were increased approximately 17-fold by IL-1beta treatment and subsequently reduced approximately 70%-90% when p38 MAPK was inhibited with SB203580. RANKL mRNA stability data indicated that p38 MAPK did not alter the rate of mRNA decay in IL-1beta-induced cells. Using a RANKL-luciferase cell line receptor containing a 120-kB segment of the 5' flanking region of the RANKL gene, reporter expression was stimulated 4-5-fold by IL-1beta or TNF-alpha treatment. IL-1beta-induced RANKL reporter expression was completely blocked with specific p38 inhibitors as well as dominant negative mutant constructs of MAPK kinase-3 and -6. In addition, blocking p38 signaling in bone marrow stromal cells partially inhibited IL-1beta and TNF-alpha-induced osteoclastogenesis in vitro. Results from these studies indicate that p38 MAPK is a major signaling pathway involved in IL-1beta and TNF-alpha-induced RANKL expression in bone marrow stromal cells.

Alteration of an essential NK cell signaling pathway by low doses of tributyltin in human natural killer cells.

Tributyltin (TBT), a toxic and widespread environmental contaminant, has been shown to inhibit natural killer (NK) cell cytotoxic function significantly. Inhibition of NK cell cytotoxic function has the potential to increase viral infections and tumor growth. Upon NK cell binding to lysis-sensitive tumor cells, an intracellular pathway is activated, which generally begins with activation of non-receptor protein tyrosine kinases (PTKs) and ends with mitogen-activated protein kinase (MAPK)-mediated release of lytic granules toward the contacted target cell. In the current studies, we used a cytotoxicity assay to examine how low doses (200nM or lower) of TBT affect cytotoxic function. Additionally, we investigated how low doses of TBT modulate the signaling pathway that dictates lytic granule exocytosis. A 1h exposure to 200, 100, 50 and 25nM TBT significantly decreased cytotoxic function 6d later. We also saw significant activation of p38 and p44/42 by as low as 50nM TBT within ten minutes of exposure. The observed activation of MAPKs, p38 and p44/42, implicated their upstream activators MAPK kinases (MAP2Ks). On examining MAP2Ks, MKK3/6 and MEK1/2, activation was seen within ten minutes. However, when the most upstream signaling molecules in this pathway, non-receptor protein tyrosine kinases (PTKs) such as Syk, ZAP-70, Pyk2 and Src were examined, no significant activation was seen. These data imply that upstream activators of MAP2Ks, MAP2K kinases (MAP3Ks), are activated by TBT exposures and/or that MAP2K phosphatases are being inhibited by TBT. Taken together, these data suggest that TBT-induced activation of MAPKs, p38 and p44/42, is caused by their upstream activators MAP2Ks, MKK3/6 and MEK1/2, respectively.

Osteoclast differentiation requires TAK1 and MKK6 for NFATc1 induction and NF-kappaB transactivation by RANKL.

Osteoclast (Oc) differentiation is fundamentally controlled by receptor activator of nuclear factor kappaB ligand (RANKL). RANKL signalling targets include mitogen-activated protein kinases (MAPKs), nuclear factor kappaB (NF-kappaB), and nuclear factor of activated T cells (NFAT)c1. In this study, we found that p38 MAPK upstream components transforming growth factor-beta-activated kinase 1 (TAK1), MKK3, and MKK6 increased by RANKL in an early stage of osteoclastogenesis from primary bone marrow cells, which led to enhanced p38 activation. Retroviral transduction of dominant-negative (DN) forms of TAK1 and MKK6, but not that of MKK3, reduced Oc differentiation. Transduction of TAK1-DN and MKK6-DN and treatment with the p38 inhibitor SB203580 attenuated NFATc1 induction by RANKL. TAK1-DN, MKK6-DN, and SB203580, but not MKK3-DN, also suppressed RANKL stimulation of NF-kappaB transcription activity in a manner dependent on p65 phosphorylation on Ser-536. These results indicate that TAK1 and MKK6 constitute the p38 signalling pathway to participate to Oc differentiation by RANKL through p65 phosphorylation and NFATc1 induction, and that MKK6 and MKK3 have differential roles in osteoclastogenesis from bone marrow precursors.