Regulation Of NF-kappaB Activation Research Articles

Mineralocorticoid and glucocorticoid receptors differentially regulate NF-kappaB activity and pro-inflammatory cytokine production in murine BV-2 microglial cells

BackgroundMicroglia, the resident macrophage-like cells in the brain, regulate innate immune responses in the CNS to protect neurons. However, excessive activation of microglia contributes to neurodegenerative diseases. Corticosteroids are potent modulators of inflammation and mediate their effects by binding to mineralocorticoid receptors (MR) and glucocorticoid receptors (GR). Here, the coordinated activities of GR and MR on the modulation of the nuclear factor-κB (NF-κB) pathway in murine BV-2 microglial cells were studied.MethodsBV-2 cells were treated with different corticosteroids in the presence or absence of MR and GR antagonists. The impact of the glucocorticoid-activating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) was determined by incubating cells with 11-dehydrocorticosterone, with or without selective inhibitors. Expression of interleukin-6 (IL-6), tumor necrosis factor receptor 2 (TNFR2), and 11β-HSD1 mRNA was analyzed by RT-PCR and IL-6 protein expression by ELISA. NF-κB activation and translocation upon treatment with various corticosteroids were visualized by western blotting, immunofluorescence microscopy, and translocation assays.ResultsGR and MR differentially regulate NF-κB activation and neuroinflammatory parameters in BV-2 cells. By converting inactive 11-dehydrocorticosterone to active corticosterone, 11β-HSD1 essentially modulates the coordinated action of GR and MR. Biphasic effects were observed for 11-dehydrocorticosterone and corticosterone, with an MR-dependent potentiation of IL-6 and tumor necrosis factor-α (TNF-α) expression and NF-κB activation at low/moderate concentrations and a GR-dependent suppression at high concentrations. The respective effects were confirmed using the MR ligand aldosterone and the antagonist spironolactone as well as the GR ligand dexamethasone and the antagonist RU-486. NF-κB activation could be blocked by spironolactone and the inhibitor of NF-κB translocation Cay-10512. Moreover, an increased expression of TNFR2 was observed upon treatment with 11-dehydrocorticosterone and aldosterone, which was reversed by 11β-HSD1 inhibitors and/or spironolactone and Cay-10512.ConclusionsA tightly coordinated GR and MR activity regulates the NF-κB pathway and the control of inflammatory mediators in microglia cells. The balance of GR and MR activity is locally modulated by the action of 11β-HSD1, which is upregulated by pro-inflammatory mediators and may represent an important feedback mechanism involved in resolution of inflammation.

MicroRNA-146a inhibits G protein-coupled receptor-mediated activation of NF-κB by targeting CARD10 and COPS8 in gastric cancer

BackgroundGastric cancer is the second most common cause of cancer-related death in the world. Inflammatory signals originating from gastric cancer cells are important for recruiting inflammatory cells and regulation of metastasis of gastric cancer. Several microRNAs (miRNA) have been shown to be involved in development and progression of gastric cancer. miRNA-146a (miR-146a) is a modulator of inflammatory signals, but little is known about its importance in gastric cancer. We therefore wanted to identify targets of miR-146a in gastric cancer and examine its biological roles.ResultsThe expression of miR-146a was evaluated by quantitative PCR (qPCR) and found up-regulated in the gastrin knockout mice, a mouse model of gastric cancer, and in 73% of investigated human gastric adenocarcinomas. Expression of miR-146a by gastric cancer cells was confirmed by in situ hybridization. Global analysis of changes in mRNA levels after miR-146a transfection identified two transcripts, caspase recruitment domain-containing protein 10 (CARD10) and COP9 signalosome complex subunit 8 (COPS8), as new miR-146a targets. qPCR, Western blotting and luciferase assays confirmed these transcripts as direct miR-146a targets. CARD10 and COPS8 were shown to be part of the G protein-coupled receptor (GPCR) pathway of nuclear factor-kappaB (NF-kappaB) activation. Lysophosphatidic acid (LPA) induces NF-kappaB activation via this pathway and over-expression of miR-146a inhibited LPA-induced NF-kappaB activation, reduced LPA-induced expression of tumor-promoting cytokines and growth factors and inhibited monocyte attraction.ConclusionsmiR-146a expression is up-regulated in a majority of gastric cancers where it targets CARD10 and COPS8, inhibiting GPCR-mediated activation of NF-kappaB, thus reducing expression of NF-kappaB-regulated tumor-promoting cytokines and growth factors. By targeting components of several NF-kappaB-activating pathways, miR-146a is a key component in the regulation of NF-kappaB activity.

363 MicroRNAs Implicated in the Regulation of NF-kappaB Activity in Glioma Tumours

363 MicroRNAs Implicated in the Regulation of NF-kappaB Activity in Glioma Tumours

Angiotensin II Type 2 Receptor: A Novel Modulator of Inflammation in Pancreatic Ductal Adenocarcinoma Through Regulation of NF-KappaB Activity

Angiotensin II Type 2 Receptor: A Novel Modulator of Inflammation in Pancreatic Ductal Adenocarcinoma Through Regulation of NF-KappaB Activity

Human Trim5α has additional activities that are uncoupled from retroviral capsid recognition

Trim5α is a host antiviral protein that recognizes incoming retroviral capsids in the cytoplasm and prevents productive infections. Although present in most mammals, the state of the Trim5 gene is dynamic in that primates have one copy with several splice variants, while rodents and cows have multiple copies. Mouse Trim30 (one of the mouse Trim5α homologs) has been shown to negatively regulate NF-kappaB activation by targeting upstream signaling intermediates TAB2 and TAB3 for degradation. We show that human Trim5α also affects levels of TAB2, resulting in abrogation of TAB2-dependent NF-kappaB activation. Surprisingly, unlike mouse Trim30, human and rhesus Trim5α are able to activate NF-kappaB-driven reporter gene expression in a dose-dependent manner. We show that Trim5α uses distinct domains for the distinct abilities of affecting TAB2 levels, regulating NF-kappaB, and recognizing retroviral capsids. Our results demonstrate functions of Trim5α that are not dependent on recognizing the retroviral capsid.

Preserved Immune Functions and Controlled Leukocyte Oxidative Stress in Naturally Long-lived Mice: Possible Role of Nuclear Factor Kappa B

In order to verify the survival biomarker role of several immune functions, and to determine the oxidation and inflammation mechanisms underlying variability in the aging process, we have investigated a variety of immune functions and oxidative stress parameters as well as activation of the nuclear factor kappa B (NF kappaB) in peritoneal leukocytes from four different age groups of mice, including natural extreme longevity. Immune cells from naturally long-lived animals showed preservation of immune function in response to stimuli and controlled oxidative stress as well as nuclear factor kappa B activation in resting conditions. Moreover, leukocytes from extreme long-lived animals showed increased catalase activity when compared with the adults. In contrast, the old and very old animal groups showed impaired immune function and increased oxidation as well as NF kappaB activation. Our results support preserved immune function as a biomarker of extended survival and point to controlled regulation of NF kappaB activity as a key mechanism restraining oxidative stress in immune cells and contributing to reach longevity.

Sustained Inflammation Due to Nuclear Factor-Kappa B Activation in Irradiated Human Arteries

The aim of this study was to investigate gene expression networks related to cardiovascular disease in radiated human arteries. Recent epidemiological studies have shown that radiotherapy is associated with cardiovascular disease years after treatment. However, the molecular mechanisms underlying late effects of radiation are poorly described. Arterial biopsies from radiated and nonradiated human conduit arteries, from the same patient, were simultaneously harvested during microvascular free tissue transfer for cancer-reconstruction in 13 patients, 4 to 500 weeks from radiation treatment. Radiated and nonradiated arteries were compared, with Affymetrix (Santa Clara, California) microarrays on a subset of the material to generate candidate genes. A Taqman (Applied Biosystems, Foster City, California) low-density array of 45 selected genes was designed for analysis of the whole material. Thirteen genes were synchronously expressed in all patients (p = 0.0015), including CCL8, CCL3, CXCL2, DUSP5, FGFR2, HMOX1, HOXA9, IL-6, MMP-1, PTX3, RDH10, SOD2, and TNFAIP3. A majority of differentially regulated genes related to the nuclear factor-kappa B (NF-kappaB) signaling pathway and were dysregulated even years after radiation. The NF-kappaB activation was confirmed by immunohistochemistry and immunofluorescence. In the present study, we found sustained inflammation due to NF-kappaB activation in human radiated arteries. The results are supported by previous in vitro findings suggesting that deoxyribonucleic acid injury, after radiation, activates NF-kappaB. We also suggest that HOXA9 might be involved in the regulation of NF-kappaB activation. The observed sustained inflammatory response can explain cardiovascular disease years after radiation.

Suppression of the NF-kB signalling pathway by ergolide, sesquiterpene lactone, in HeLa cells

We have previously reported that ergolide, a sesquiterpene lactone isolated from Inula britannica, suppresses inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression by inhibiting nuclear factor-kappaB (NF-kappaB) in RAW 264.7 macrophages. In this study, we show that ergolide suppresses the DNA binding activity of NF-kappaB and nuclear translocation of NF-kappaB p65 subunit, leading to the inhibition of NF-kappaB-dependent gene transcription in 12-O-tetradecanoylphorbol 13acetate (TPA)-stimulated HeLa cells. We also show that ergolide decreases the degradation and phosphorylation of IkappaB, an inhibitory protein of NF-kappaB, and this effect is accompanied by a simultaneous reduction of IkappaB kinase (IKK) activity. However, ergolide does not inhibit in-vitro IKK activity directly, suggesting the possible involvement of upstream IKK kinases in the regulation of NF-kappaB activation. Furthermore, ergolide-mediated protein kinase Calpha (PKCalpha) inhibition is involved in reduction of NF-kappaB inhibition, as demonstrated by the observation that dominant negative PKCalpha, but not p44/42 MAPK and p38 MAPK, inhibits TPA-stimulated reporter gene expression. Taken together, our results suggest that ergolide suppresses NF-kappaB activation through the inhibition of PKCalpha-IKK activity, providing insight for PKCalpha as a molecular target for anti-inflammatory drugs.

Inhibition of NF-kappaB-mediated gene transcription by the human A2B adenosine receptor in Chinese hamster ovary cells.

NF-kappaB is a transcription factor that plays a vital role in regulating inducible gene expression in immune and inflammatory responses. In view of the well documented effects of adenosine on immune and inflammatory responses, we have explored whether adenosine A1, A2B and A3 receptors regulate NF-kappaB activity in transfected Chinese hamster ovary (CHO) cells using a luciferase reporter gene construct. No increases in NF-kappaB activity were observed in CHO-A1, -A2B and -A3 cells stimulated with the non-selective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine. Elevating intracellular cyclic AMP (cAMP) levels using forskolin (direct activator of adenylyl cyclase) and rolipram (type IV, cAMP-specific phosphodiesterase inhibitor), inhibited NF-kappaB activity in CHO cells. Adenosine A2B receptor stimulation also inhibited NF-kappaB activity, whereas adenosine A1 and A3 receptor activation had no effect. These data reflect the known coupling of adenosine A2B receptors to increases in cAMP. In conclusion, adenosine A1, A2B and A3 receptors do not directly activate NF-kappaB in CHO cells. However, adenosine A2B receptor activation significantly inhibited NF-kappaB activity. Inhibition of NF-kappaB activity by the adenosine A2B receptor may contribute to the anti-inflammatory effects of adenosine.

Peroxiredoxin IV Regulates Pro-Inflammatory Responses in Large Yellow Croaker (Pseudosciaena crocea) and Protects against Bacterial Challenge

In this study, we applied a comparative proteomic approach to the analysis of differentially expressed proteins in the spleens of large yellow croaker following treatment with an inactivated trivalent bacterial vaccine. Twenty-four altered proteins were identified by MALDI-TOF or MALDI-TOF-TOF, including immune-related proteins, antioxidant proteins, signal transducers, protein biosynthesis and catabolism modulators, and carbonic anhydrases. Three Prx family members, namely, Prx I, Prx II, and Prx IV, were upregulated after treatment with the vaccine, indicating potentially important roles for these antioxidant proteins in the antibacterial immune response. Large yellow croaker Prx IV (LycPrxIV), which has thiol-dependent peroxidase activity, was constitutively expressed in all tissues examined. Immunoelectron microscopy showed that LycPrxIV was primarily localized to the rER or peroxisome in spleen cells of healthy fish, and its synthesis on the rER increased following treatment with bacterial vaccine. Suppression of LycPrxIV by siRNA resulted in an increase in NF-kappaB activity in spleen tissues, while in vivo administration of recombinant LycPrxIV (rLycPrxIV) caused a decrease in NF-kappaB activity, indicating that LycPrxIV negatively regulates NF-kappaB activation. Likewise, siRNA-mediated knockdown of LycPrxIV increased the expression of TNF-alpha and CC chemokine, and downregulated the expression of IL-10. However, injection of fish with rLycPrxIV induced the opposite expression pattern of these cytokines, suggesting a role for LycPrxIV in regulating pro-inflammatory responses. Bacterial challenge experiments showed that suppression of LycPrxIV expression by siRNA significantly increased fish mortality as compared to controls, whereas rLycPrxIV provided a protective effect. Together, our data suggest that LycPrxIV may regulate pro-inflammatory responses to protect large yellow croaker from bacterial challenge, revealing a novel antibacterial mechanism in fish.

Differential Regulation of Mitogen- and Stress-activated Protein Kinase-1 and -2 (MSK1 and MSK2) by CK2 following UV Radiation

Mitogen- and stress-activated protein kinases, MSK1 and the closely related isoform MSK2, are nuclear kinases that are activated following mitogen stimulation or cellular stress, including UV radiation, by the ERK1/2 and p38 MAPK signaling cascades, respectively. However, factors that differentially regulate MSK1 and MSK2 have not been well characterized. Here we report that the CK2 protein kinase, which contributes to NF-kappaB activation following UV radiation in a p38-dependent manner, physically interacts with MSK2 but not MSK1 and that CK2 inhibition specifically impairs UV-induced MSK2 kinase activation. A putative site of CK2 phosphorylation was mapped to MSK2 residue Ser(324) and when substituted to alanine (S324A) also compromised MSK2 activity. RNA interference-mediated depletion of MSK2 in human MDA-MB-231 cells, but not MSK1 depletion, resulted in impaired UV-induced phosphorylation of NF-kappaB p65 at Ser(276) in vivo, which was restored by the ectopic expression of MSK2 but not by MSK2-S324A. Furthermore, UV radiation led to the activation of NF-kappaB-responsive gene expression in MDA-MB-231 cells and induced p65 transactivation capacity that was dependent on MSK2, MSK2 residue Ser(324), and p65-Ser(276). These results suggest that MSK1 and MSK2 are differentially regulated by CK2 during the UV response and that MSK2 is the major protein kinase responsible for the UV-induced phosphorylation of p65 at Ser(276) that positively regulates NF-kappaB activity in MDA-MB-231 cells.

The Regulatory Logic of the NF- B Signaling System

NF-kappaB refers to multiple dimers of Rel homology domain (RHD) containing polypeptides, which are controlled by a stimulus-responsive signaling system that mediates the physiological responses to inflammatory intercellular cytokines, pathogen exposure, and developmental signals. The NF-kappaB signaling system operates on transient or short timescales, relevant to inflammation and immune responses, and on longer-term timescales relevant to cell differentiation and organ formation. Here, we summarize our current understanding of the kinetic mechanisms that allow for NF-kappaB regulation at these different timescales. We distinguish between the regulation of NF-kappaB dimer formation and the regulation of NF-kappaB activity. Given the number of regulators and reactions involved, the NF-kappaB signaling system is capable of integrating a multitude of signals to tune NF-kappaB activity, signal dose responsiveness, and dynamic control. We discuss the prevailing mechanisms that mediate signaling cross talk.

NF-κB as a Prognostic Marker and Therapeutic Target in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia is the most common adult leukemia and is currently incurable with conventional chemotherapeutic agents. Over the last few years, significant discoveries have been made regarding the biology that underpins this disease. These new insights have allowed us to develop more rational prognostic tools and identify promising novel therapeutic targets. In this review, we highlight the importance of both constitutive and inducible DNA binding of the transcription factor NF-kappaB in chronic lymphocytic leukemia. We describe the current knowledge regarding the activity and function of specific NF-kappaB subunits in this disease, and discuss the complex mechanisms that regulate NF-kappaB activation in vivo. In addition, we provide compelling evidence for the utility of the NF-kappaB subunit, Rel A, as a prognostic marker and as a therapeutic target in this disease, and we also describe how this protein may contribute to the drug resistance commonly encountered with this condition.

Induction of RIP-2 kinase by proinflammatory cytokines is mediated via NF-κB signaling pathways and involves a novel feed-forward regulatory mechanism

The transcription factor NF-kappaB (nuclear factor kappaB) is a central mediator of inflammatory and apoptotic signaling in the cell. The protein kinase RIP-2 is a member of the CARD protein family (caspase activation and recruitment domain, also known as CARD3, Ripk2, CARDIAK, RICK, and CCK), and has been shown to be an activator of NF-kappaB. In this study, it was demonstrated by transcriptional profiling and protein expression analysis that the inflammatory cytokines TNF-alpha, IL-1 beta, and IFN-gamma induced RIP-2 transcription and translation in endothelial cells. Two mechanistically distinct inhibitors of NF-kappaB signaling, sulfasalazine (NF-kappaB inhibitor) and WY-14643 [PPAR alpha (peroxisome proliferator-activated receptor alpha) agonist] that interfere with the transcription factor RELA (p65), suppressed TNF-alpha induced RIP-2 gene expression, which indicated that NF-kappaB signaling was involved in the cytokine-induced transcriptional activation of RIP-2 gene expression. Consistent with these observations, multiple NF-kappaB response elements were found in the upstream regions of the human and mouse RIP-2 genes. NF-kappaB-mediated regulation of RIP-2 gene and protein expression suggests an additional step in the regulation of NF-kappaB function as RIP-2 has been shown to positively modulate NF-kappaB by binding IKK gamma (I kappaB kinase gamma), a component of the IKK complex. These findings support a positive feed-forward mechanism of NF-kappaB regulation that involves NF-kappaB-dependent induction of RIP-2 transcription and a subsequent increase in RIP-2 protein levels in response to inflammatory cytokines. Elevated RIP-2 protein levels are then available to promote NF-kappaB function via interaction with IKK gamma. RIP-2 is the first reported NF-kappaB-dependent protein kinase that positively regulates NF-kappaB activity.

Angiotensin II Activates IκB Kinase Phosphorylation of RelA at Ser536 to Promote Myofibroblast Survival and Liver Fibrosis

The transcription factor nuclear factor-kappaB (NF)-kappaB promotes survival of hepatic myofibroblasts and fibrogenesis through poorly defined mechanisms. We investigated the activities of angiotensin II and I kappaB kinase (IKK) in regulation of NF-kappaB activity and the role of these proteins in liver fibrosis in rodents and humans. Phosphorylation of the NF-kappaB subunit RelA at serine 536 (P-Ser(536)-RelA) was detected by immunoblot and immunohistochemical analyses. P-Ser(536)-RelA function was assessed using vectors that expressed mutant forms of RelA, cell-permeable blocking peptides, and assays for RelA nuclear transport and apoptosis. Levels of P-Ser(536)-RelA were compared with degree of fibrosis in liver sections from chronically injured rats and patients with hepatitis C virus-mediated fibrosis who had been treated with the AT1 antagonist losartan. Constitutive P-Ser(536)-RelA is a feature of human hepatic myofibroblasts, both in vitro and in situ in diseased livers. Autocrine angiotensin II stimulated IKK-mediated phosphorylation of RelA at Ser(536), which was required for nuclear transport and transcriptional activity of NF-kappaB. Inhibition of angiotensin II, the angiotensin II receptor type 1 (AT1), or IKK blocked Ser(536) phosphorylation and stimulated myofibroblast apoptosis. Treatment of fibrotic rodent liver with the angiotensin converting enzyme (ACE) inhibitor captopril or the IKK inhibitor sulphasalazine resulted in loss of P-Ser(536)-RelA-positive myofibroblasts and fibrosis regression. In human liver samples, increased numbers of P-Ser(536)-RelA-positive cells were associated with fibrosis that regressed following exposure to losartan. An autocrine pathway that includes angiotensin II, IKK, and P-Ser(536)-RelA regulates myofibroblast survival and can be targeted to stimulate therapeutic regression of liver fibrosis.

Identification of polyubiquitin binding proteins involved in NF-κB signaling using protein arrays

Attachment of ubiquitin to proteins represents a central mechanism for the regulation of protein metabolism and function. In the NF-kappaB pathway, binding of NEMO to polyubiquitinated substrates initiates the pathway in response to cellular stimuli. Other polyubiquitin binding proteins can antagonize this pathway by competing with NEMO for polyubiquitin. We have used protein arrays to identify polyubiquitin binding proteins that regulate NF-kappaB activity. Using polyubiquitin as bait, protein arrays were screened and polyubiquitin binders identified. Novel polyubiquitin binders AWP1, CALCOCO2, N4BP1, RIO3, TEX27, TTC3, UBFD1 and ZNF313 were identified using this approach, while known NF-kappaB regulators including NEMO, A20, ABIN-1, ABIN-2, optineurin and p62 were also identified. Overexpressed AWP1 and RIO3 repressed NF-kappaB activity in a manner similar to optineurin, while siRNAs directed against AWP1 and RIO3 also reduced NF-κB activity. TNFalpha-dependent degradation of IkappaBalpha was also suppressed by overexpression of AWP1 and RIO3, possibly due to the polyubiquitin binding activity of these proteins. Protein array screening using polyubiquitin enabled rapid identification of many known and novel polyubiquitin binding proteins and the identification of novel NF-kappaB regulators.

Increase in transglutaminase 2 expression is associated with NF-kappaB activation in breast cancer tissues

Activation of NF-kappaB is reported in breast cancers. NF-kappaB inhibition in breast cancer cell lines results in an increase in apoptosis. However, the reason for continuous activation of this transcription factor in breast cancer is currently unclear. Interestingly, elevated transglutaminase 2 (TGase 2) expression is additionally observed in breast cancer. Recent studies showed that doxorubicin-resistant cells contain a higher level of TGase 2, compared to doxorubicin-sensitive cells. Moreover, increasing the level of TGase 2 in breast cancer cells by transient transfection leads to decreased expression of the inhibitory subunit ?of NF-kappaB (I-kappaBA), and up regulation of NF-kappaB activity. Our data show that TGase 2 expression is inversely correlated with the level of I-kappaBa in breast tumors, implying that the enzyme is responsible for the constitutive activation of NF-kappaB in breast tumors.

NF-kappaB signalling in chronic kidney disease

The mammalian NF-kappaB signalling pathway is an important intracellular transcription factor system that is induced in response to diverse extracellular stimuli. The hallmark of NF-kappaB activation is the nuclear translocation of dimeric Rel protein transcription factors, which regulate hundreds of kappaB-dependent genes that are involved in inflammation, immunity, apoptosis, cell proliferation and differentiation. In addition, cell-surface receptors (TNFR, Toll-like and angiotensin II, type 1 receptors), inhibitory kappaB kinases (IKK proteins), I kappaB proteins and factors regulating the post-translational modification of the Rel proteins (acetylation, phosphorylation), are other intracellular components that regulate NF-kappaB activation. Over the last decade, in vitro studies, animal models and human studies have provided evidence that upregulation of the canonical (RelA/p50) NF- kappaB isoform (in tubular epithelial cells, podocytes, mesangial cells, macrophages) has a pathogenic role in mediating chronic inflammation in chronic kidney disease (CKD). This review will examine current evidence regarding NF- kappaB isoforms and their potential role in the treatment of kidney failure due to CKD.

Siva-1 Promotes K-48 Polyubiquitination of TRAF2 and Inhibits TCR-Mediated Activation of NF-kappaB

The proapoptotic protein Siva-1 plays an important role in some of the extrinsic and intrinsic apoptosis signaling pathways in cancer cells. Previously, we showed that Siva-1 inhibited the activity of the prosurvival transcription factor NF-kappaB. In the present study, upon TCR cross-linking of Jurkat T leukemia cells, we demonstrated that the inhibitory target of Siva-1 is upstream of the IKK complex in the NF-kappaB signaling pathway. Additionally, Siva-1 also suppressed the activity of another crucial transcription factor AP-1, and a common mediator of both these pathways is the adaptor protein TRAF2. Further, we observed that Siva-1 indeed interacted with TRAF2 and negatively regulated its activity by promoting K48-hnked polyubiquitination. Siva-1 specifically interacted with the ring finger domain of TRAF2, which is essential for its E3 hgase activity and its ability to subsequently activate NF-kappaB. TCR cross-linking of Jurkat T cells that lacked Siva-1 revealed significantly lowered K48- but elevated K63-ubiquitinated TRAF2 levels upon TCR cross-linking, suggesting that the differential pattern of ubiquitination in these cells essentially contributed to a robust and sustained activation of NF-kappaB. The above results demonstrated an important role for endogenous Siva-1 in negatively regulating NF-kappaB activation by targeting TRAF2.

Protein kinase D1 mediates NF-kappaB activation induced by cholecystokinin and cholinergic signaling in pancreatic acinar cells.

The transcription factor NF-kappaB plays a critical role in inflammatory and cell death responses during acute pancreatitis. Previous studies in our laboratory demonstrated that protein kinase C (PKC) isoforms PKCdelta and epsilon are key regulators of NF-kappaB activation induced by cholecystokinin-8 (CCK-8), tumor necrosis factor-alpha, and ethanol. However, the downstream participants in regulating NF-kappaB activation in exocrine pancreas remain poorly understood. Here, we demonstrate that protein kinase D1 (PKD1) is a key downstream target of PKCdelta and PKCepsilon in pancreatic acinar cells stimulated by two major secretagogues, CCK-8 and the cholinergic agonist carbachol (CCh), and that PKD1 is necessary for NF-kappaB activation induced by CCK-8 and CCh. Both CCK-8 and CCh dose dependently induced a rapid and striking activation of PKD1 in rat pancreatic acinar cells, as measured by in vitro kinase assay and by phosphorylation at PKD1 activation loop (Ser744/748) or autophosphorylation site (Ser916). The phosphorylation and activation of PKD1 correlated with NF-kappaB activity stimulated by CCK-8 or CCh, as measured by NF-kappaB DNA binding. Either inhibition of PKCdelta or epsilon by isoform-specific inhibitory peptides, genetic deletion of PKCdelta and epsilon in pancreatic acinar cells, or knockdown of PKD1 by using small interfering RNAs in AR42J cells resulted in a marked decrease in PKD1 and NF-kappaB activation stimulated by CCK-8 or CCh. Conversely, overexpression of PKD1 resulted in augmentation of CCK-8- and CCh-stimulated NF-kappaB activation. Finally, the kinetics of PKD1 and NF-kappaB activation during cerulein-induced rat pancreatitis showed that both PKD1 and NF-kappaB activation were early events during acute pancreatitis and that their time courses of response were similar. Our results identify PKD1 as a novel early convergent point for PKCdelta and epsilon in the signaling pathways mediating NF-kappaB activation in pancreatitis.