Transforming Growth Factor (TGF)-β-activated Kinase 1 (TAK1) Activation Requires Phosphorylation of Serine 412 by Protein Kinase A Catalytic Subunit α (PKACα) and X-linked Protein Kinase (PRKX)

TGF-β-activated kinase 1 (TAK1) is a key kinase in mediating Toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R) signaling. Although TAK1 activation involves the phosphorylation of Thr-184 and Thr-187 residues at the activation loop, the molecular mechanism underlying the complete activation of TAK1 remains elusive. In this work, we show that the Thr-187 phosphorylation of TAK1 is regulated by its C-terminal coiled-coil domain-mediated dimerization in an autophosphorylation manner. Importantly, we find that TAK1 activation in mediating downstream signaling requires an additional phosphorylation at Ser-412, which is critical for TAK1 response to proinflammatory stimuli, such as TNF-α, LPS, and IL-1β. In vitro kinase and shRNA-based knockdown assays reveal that TAK1 Ser-412 phosphorylation is regulated by cAMP-dependent protein kinase catalytic subunit α (PKACα) and X-linked protein kinase (PRKX), which is essential for proper signaling and proinflammatory cytokine induction by TLR/IL-1R activation. Morpholino-based in vivo knockdown and rescue studies show that the corresponding site Ser-391 in zebrafish TAK1 plays a conserved role in NF-κB activation. Collectively, our data unravel a previously unknown mechanism involving TAK1 phosphorylation mediated by PKACα and PRKX that contributes to innate immune signaling.     

Bruton's Tyrosine Kinase and Protein Kinase C μ Are Required for TLR7/9-Induced IKKα and IRF-1 Activation and Interferon-β Production in Conventional Dendritic Cells

Stimulation of TLR7/9 by their respective ligands leads to the activation of IκB kinase α (IKKα) and Interferon Regulatory Factor 1 (IRF-1) and results in interferon (IFN)-β production in conventional dendritic cells (cDC). However, which other signaling molecules are involved in IKKα and IRF-1 activation during TLR7/9 signaling pathway are not known. We and others have shown that Bruton''s Tyrosine Kinase (BTK) played a part in TLR9-mediated cytokine production in B cells and macrophages. However, it is unclear if BTK participates in TLR7/9-induced IFN-β production in cDC. In this study, we show that BTK is required for IFN-β synthesis in cDC upon TLR7/9 stimulation and that stimulated BTK-deficient cDC are defective in the induction of IKKα/β phosphorylation and IRF-1 activation. In addition, we demonstrate that Protein Kinase C µ (PKCµ) is also required for TLR7/9-induced IRF-1 activation and IFN-β upregulation in cDC and acts downstream of BTK. Taken together, we have uncovered two new molecules, BTK and PKCµ, that are involved in TLR7/9-triggered IFN-β production in cDC.     

VRK2 activates TNFα/NF-κB signaling by phosphorylating IKKβ in pancreatic cancer

NF-κB signaling is active in more than 50% of patients with pancreatic cancer and plays an important role in promoting the progression of pancreatic cancer. Revealing the activation mechanism of NF-κB signaling is important for the treatment of pancreatic cancer. In this study, the regulation of TNFα/NF-κB signaling by VRK2 (vaccinia-related kinase 2) was investigated. The levels of VRK2 protein were examined by immunohistochemistry (IHC). The functions of VRK2 in the progression of pancreatic cancer were examined using CCK8 assay, anchorage-independent assay, EdU assay and tumorigenesis assay. The regulation of VRK2 on the NF-κB signaling was investigated by immunoprecipitation and invitro kinase assay. It was discovered in this study that the expression of VRK2 was upregulated in pancreatic cancer and that the VRK2 expression level was significantly correlated with the pathological characteristics and the survival time of patients. VRK2 promoted the growth, sphere formation and subcutaneous tumorigenesis of pancreatic carcinoma cells as well as the organoid growth derived from the pancreatic cancer mouse model. Investigation of the molecular mechanism indicated that VRK2 interacts with IKKβ, phosphorylating its Ser177 and Ser181 residues and thus activating the TNFα/NF-κB signaling pathway. An IKKβ inhibitors abolished the promotive effect of VRK2 on the growth of organoids. The findings of this study indicate that VRK2 promotes the progression of pancreatic cancer by activating the TNFα/NF-κB signaling pathway, suggesting that VRK2 is a potential therapeutic target for pancreatic cancer.     

Akt-dependent Activation of mTORC1 Complex Involves Phosphorylation of mTOR (Mammalian Target of Rapamycin) by IκB Kinase α (IKKα)

The serine/threonine protein kinase Akt promotes cell survival, growth, and proliferation through phosphorylation of different downstream substrates. A key effector of Akt is the mammalian target of rapamycin (mTOR). Akt is known to stimulate mTORC1 activity through phosphorylation of tuberous sclerosis complex 2 (TSC2) and PRAS40, both negative regulators of mTOR activity. We previously reported that IκB kinase α (IKKα), a component of the kinase complex that leads to NF-κB activation, plays an important role in promoting mTORC1 activity downstream of activated Akt. Here, we demonstrate IKKα-dependent regulation of mTORC1 using multiple PTEN null cancer cell lines and an animal model with deletion of IKKα. Importantly, IKKα is shown to phosphorylate mTOR at serine 1415 in a manner dependent on Akt to promote mTORC1 activity. These results demonstrate that IKKα is an effector of Akt in promoting mTORC1 activity.     

Negative Regulation of TLR Inflammatory Signaling by the SUMO-deconjugating Enzyme SENP6

The signaling of Toll-like receptors (TLRs) induces host defense against microbial invasion. Protein posttranslational modifications dynamically shape the strength and duration of the signaling pathways. It is intriguing to explore whether de-SUMOylation could modulate the TLR signaling. Here we identified SUMO-specific protease 6 (SENP6) as an intrinsic attenuator of the TLR-triggered inflammation. Depletion of SENP6 significantly potentiated the NF-κB-mediated induction of the proinflammatory genes. Consistently, SENP6-knockdown mice were more susceptible to endotoxin-induced sepsis. Mechanistically, the small ubiquitin-like modifier 2/3 (SUMO-2/3) is conjugated onto the Lysine residue 277 of NF-κB essential modifier (NEMO/IKKγ), and this impairs the deubiquitinase CYLD to bind NEMO, thus strengthening the inhibitor of κB kinase (IKK) activation. SENP6 reverses this process by catalyzing the de-SUMOylation of NEMO. Our study highlights the essential function of the SENP family in dampening TLR signaling and inflammation.     

The Induction of Lipocalin-2 Protein Expression in Vivo and in Vitro

Lipocalin-2 (LCN2) is secreted from adipocytes, and its expression is up-regulated in obese and diabetic mice and humans. LCN2 expression and secretion have been shown to be induced by two proinflammatory cytokines, IFNγ and TNFα, in cultured murine and human adipocytes. In these studies, we demonstrated that IFNγ and TNFα induced LCN2 expression and secretion in vivo. Although we observed a strong induction of LCN2 expression and secretion from white adipose tissue (WAT) depots, the induction of LCN2 varied among different insulin-sensitive tissues. Knockdown experiments also demonstrated that STAT1 is required for IFNγ-induced lipocalin-2 expression in murine adipocytes. Similarly, knockdown of p65 in adipocytes demonstrated the necessity of the NF-κB signaling pathway for TNFα-mediated effects on LCN2. Activation of ERKs by IFNγ and TNFα also affected STAT1 and NF-κB signaling through modulation of serine phosphorylation. ERK activation-induced serine phosphorylation of both STAT1 and p65 mediated the additive effects of IFNγ and TNFα on LCN2 expression. Our results suggest that these same mechanisms occur in humans as we observed STAT1 and NF-κB binding to the human LCN2 promoter in chromatin immunoprecipitation assays performed in human fat cells. These studies substantially increase our knowledge regarding the requirements and mechanisms used by proinflammatory cytokines to induce LCN2 expression.     

TRIF Mediates Toll-like Receptor 5-induced Signaling in Intestinal Epithelial Cells

Toll-like receptors (TLRs) associate with adaptor molecules (MyD88, Mal/TIRAP, TRAM, and TRIF) to mediate signaling of host-microbial interaction. For instance, TLR4 utilizes the combination of both Mal/TIRAP-MyD88 (MyD88-dependent pathway) and TRAM-TRIF (MyD88-independent pathway). However, TLR5, the specific receptor for flagellin, is known to utilize only MyD88 to elicit inflammatory responses, and an involvement of other adaptor molecules has not been suggested in TLR5-dependent signaling. Here, we found that TRIF is involved in mediating TLR5-induced nuclear factor κB (NFκB) and mitogen-activated protein kinases (MAPKs), specifically JNK1/2 and ERK1/2, activation in intestinal epithelial cells. TLR5 activation by flagellin permits the physical interaction between TLR5 and TRIF in human colonic epithelial cells (NCM460), whereas TLR5 does not interact with TRAM upon flagellin stimulation. Both primary intestinal epithelial cells from TRIF-KO mice and TRIF-silenced NCM460 cells significantly reduced flagellin-induced NFκB (p105 and p65), JNK1/2, and ERK1/2 activation compared with control cells. However, p38 activation by flagellin was preserved in these TRIF-deficient cells. TRIF-KO intestinal epithelial cells exhibited substantially reduced inflammatory cytokine (keratinocyte-derived cytokine, macrophage inflammatory protein 3α, and IL-6) expression upon flagellin, whereas control cells from TRIF-WT mice showed robust cytokine expression by flagellin. Compare with TRIF-WT mice, TRIF-KO mice were resistant to in vivo intestinal inflammatory responses: flagellin-mediated exacerbation of colonic inflammation and dextran sulfate sodium-induced experimental colitis. We conclude that in addition to MyD88, TRIF mediates TLR5-dependent responses and, thereby regulates inflammatory responses elicited by flagellin/TLR5 engagement. Our findings suggest an important role of TRIF in regulating host-microbial communication via TLR5 in the gut epithelium.     

A Novel MEK-ERK-AMPK Signaling Axis Controls Chemokine Receptor CCR7-dependent Survival in Human Mature Dendritic Cells

Chemokine receptor CCR7 directs mature dendritic cells (mDCs) to secondary lymph nodes where these cells regulate the activation of T cells. CCR7 also promotes survival in mDCs, which is believed to take place largely through Akt-dependent signaling mechanisms. We have analyzed the involvement of the AMP-dependent kinase (AMPK) in the control of CCR7-dependent survival. A pro-apoptotic role for AMPK is suggested by the finding that pharmacological activators induce apoptosis, whereas knocking down of AMPK with siRNA extends mDC survival. Pharmacological activation of AMPK also induces apoptosis of mDCs in the lymph nodes. Stimulation of CCR7 leads to inhibition of AMPK, through phosphorylation of Ser-485, which was mediated by G_i/Gβγ, but not by Akt or S6K, two kinases that control the phosphorylation of AMPK on Ser-485 in other settings. Using selective pharmacological inhibitors, we show that CCR7-induced phosphorylation of AMPK on Ser-485 is mediated by MEK and ERK. Coimmunoprecipitation analysis and proximity ligation assays indicate that AMPK associates with ERK, but not with MEK. These results suggest that in addition to Akt-dependent signaling mechanisms, CCR7 can also promote survival of mDCs through a novel MEK1/2-ERK1/2-AMPK signaling axis. The data also suggest that AMPK may be a potential target to modulate mDC lifespan and the immune response.     

Vitamin D Receptor Inhibits Nuclear Factor κB Activation by Interacting with IκB Kinase β Protein

1,25-Dihydroxyvitamin D (1,25(OH)₂D₃) is known to suppress NF-κB activity, but the underlying mechanism remains poorly understood. Here we show that the vitamin D receptor (VDR) physically interacts with IκB kinase β (IKKβ) to block NF-κB activation. 1,25(OH)₂D₃ rapidly attenuates TNFα-induced p65 nuclear translocation and NF-κB activity in a VDR-dependent manner. VDR overexpression inhibits IKKβ-induced NF-κB activity. GST pull-down assays and coimmunoprecipitation experiments demonstrated that VDR physically interacts with IKKβ and that this interaction is enhanced by 1,25(OH)₂D₃. Protein mapping reveals that VDR-IKKβ interaction occurs between the C-terminal portions of the VDR and IKKβ proteins. Reconstitution of VDR^−/− cells with the VDR C terminus restores the ability to block TNFα-induced NF-κB activation and IL-6 up-regulation. VDR-IKKβ interaction disrupts the formation of the IKK complex and, thus, abrogates IKKβ phosphorylation at Ser-177 and abolishes IKK activity to phosphorylate IκBα. Consequently, stabilization of IκBα arrests p65/p50 nuclear translocation. Together, these data define a novel mechanism whereby 1,25(OH)₂D₃-VDR inhibits NF-κB activation.     

Liver Kinase B1 Suppresses Lipopolysaccharide-induced Nuclear Factor κB (NF-κB) Activation in Macrophages

Liver kinase B1 (LKB1), a serine/threonine kinase, is a tumor suppressor and metabolic regulator. Recent data suggest that LKB1 is essential in regulating homeostasis of hematopoietic cells and immune responses. However, its role in macrophages and innate immune system remains unclear. Here we report that macrophage LKB1 inhibits pro-inflammatory signaling in response to LPS. LPS-induced pro-inflammatory cytokines and pro-inflammatory enzymes were monitored in bone marrow-derived macrophages isolated from myeloid cell-specific LKB1 knock out mice and their wild type littermate control mice. LPS induced higher levels of pro-inflammatory cytokines and pro-inflammatory enzymes in bone marrow-derived macrophages from LKB1 KO than those from wild type mice. Consistently, LPS induced higher levels of NF-κB activation in LKB1-deficient macrophages than those in wild type. Further, LPS stimulation significantly increased LKB1 phosphorylation at serine 428, which promoted its binding to IκB kinaseβ (IKKβ), resulting in the inhibition of NF-κB. Finally, LPS injection caused higher levels of cytokine release and more severe tissue injury in the lung tissues of LKB1 KO mice than in those of control mice. We conclude that LKB1 inhibits LPS-induced NF-κB activation in macrophages.     

AS-703026 Inhibits LPS-Induced TNFα Production through MEK/ERK Dependent and Independent Mechanisms

Chronic obstructive pulmonary disease (COPD) is characterized by intense lung infiltrations of immune cells (macrophages and monocytes). Lipopolysaccharide (LPS) activates macrophages/monocytes, leading to production of tumor necrosis factor α (TNFα) and other cytokines, which cause subsequent lung damages. In the current study, our results demonstrated that AS-703026, a novel MEK/ERK inhibitor, suppressed LPS-induced TNFα mRNA expression and protein secretion in RAW 264.7 murine macrophages, and in murine bone marrow-derived macrophages (BMDMs). Meanwhile, TNFα production in LPS-stimulated COPD patents’ peripheral blood mononuclear cells (PBMCs) was also repressed by AS-703026. At the molecular level, we showed that AS-703026 blocked LPS-induced MEK/ERK activation in above macrophages/monocytes. However, restoring ERK activation in AS-703026-treated RAW 264.7 cells by introducing a constitutive-actively (CA)-ERK1 only partially reinstated LPS-mediated TNFα production. Meanwhile, AS-703026 could still inhibit TNFα response in ERK1/2-depleted (by shRNA) RAW 264.7 cells. Significantly, we found that AS-703026 inhibited LPS-induced nuclear factor κB (NFκB) activation in above macrophages and COPD patients’ PBMCs. In vivo, oral administration of AS-703026 inhibited LPS-induced TNFα production and endotoxin shock in BALB/c mice. Together, we show that AS-703026 in vitro inhibits LPS-induced TNFα production in macrophages/monocytes, and in vivo protects mice from LPS-induced endotoxin shock. Thus, it could be further studied as a useful anti-inflammatory therapy for COPD patients.