TGF-β-activated Kinase 1 Expression Research Articles

TAK1 MEDIATES HMGB1-INDUCED MACROPHAGE ACTIVATION IN OSTEOARTHRITIS

Osteoarthritis, the most common degenerative joint disease, significantly impairs life quality and labor capability of patients. Synovial inflammation, initiated by HMGB1 (High mobility group box 1)-induced activation of macrophage, precedes other pathological changes. As an upstream regulator of NF-κB (nuclear factor-kappa B) and MAPK (mitogen-activated protein kinase) signaling pathway, TAK1 (TGF-β activated kinase 1) participates in macrophage activation, while its function in osteoarthritis remains unveiled. This study aims to investigate the role of TAK1 in the pathogenesis of osteoarthritis via both in vitro and in vivo approaches.We performed immunohistochemical staining for TAK1 in synovial tissue, both in osteoarthritis patients and healthy control. Besides, immunofluorescence staining for F4/80 as macrophage marker and TAK1 were conducted as well. TAK1 expression was examined in RAW264.7 macrophages stimulated by HMGB1 via qPCR (Quantitative polymerase chain reaction) and Western blotting, and the effect of TAK1 inhibitor (5z-7 oxozeaenol) on TNF-α production was evaluated by immunofluorescence staining. Further, we explored the influence of intra-articular shRNA (short hairpin RNA) targeting TAK1 on collagenase-induced osteoarthritis in mice.Immunohistochemical staining confirmed significant elevation of TAK1 in osteoarthritic synovium, and immunofluorescence staining suggested macrophages as predominant residence of TAK1. In HMGB1-stimulated RAW264.7 macrophages, TAK1 expression was up-regulated both in mRNA and protein level. Besides, TAK1 inhibitor significantly impairs the production of TNF-α by macrophages upon HMGB1 stimulation. Moreover, intra-articular injection of lentivirus loaded with shRNA targeting TAK1 (sh-TAK1) reduced peri-articular osteophyte formation in collagenase-induced osteoarthritis in mice.TAK1 exerts a potent role in the pathogenesis of osteoarthritis by mediating the activation of macrophages.

MicroRNA‑143‑3p levels are reduced in the peripheral blood of patients with gestational diabetes mellitus and influences pancreatic β‑cell function and viability.

Pancreatic β-cell dysfunction has been demonstrated to mediate key roles in the pathogenesis of gestational diabetes mellitus (GDM). Accumulating evidence has supported the functional involvement of microRNAs (miRNAs) in various types of diabetes, including GDM. However, the detailed biological effect of miRNAs in pancreatic β-cell dysfunction remains poorly understood. In the present study, microarray data of miRNAs in the blood plasma of patients with GDM were retrieved from the Gene Expression Omnibus dataset under the accession number GSE98043. Reverse transcription-quantitative PCR (RT-qPCR) was performed to measure the expression levels of miR-143-3p in the blood plasma isolated from 30 female patients with GDM women and 30 healthy female individuals. Subsequently, murine pancreatic β-cell line, MIN6 cells were treated with high glucose (HG) to construct in vitro cell models of GDM. miR-143-3p in HG-treated MIN6 cells was overexpressed or knocked down using miR-143-3p mimics and miR-143-3p inhibitor. Cell viability, insulin secretion and proinflammatory cytokine production were examined using CCK-8 and ELISA, respectively Cell apoptosis was measured by flow cytometry assay. The protein expression levels of proteins involved in the TAK1/NF-κB pathway were also assessed using western blot. The levels of miR-143-3p were found to be markedly lower in samples from patients with GDM, which were in turn negatively correlated with blood glucose levels. Overexpression of miR-143-3p in MIN6 cells significantly reversed HG-induced cell apoptosis and impairments in cell viability and insulin secretion. In addition, miR-143-3p overexpression attenuated HG-induced proinflammatory cytokine production by MIN6 cells. Subsequently, TGFβ-activated kinase 1 (TAK1), an upstream regulator of the NF-κB pathway, was found to be a direct target of miR-143-3p in pancreatic β cells through luciferase assays and western blot. Overexpression of TAK1 was revealed to abolish the curative effects of miR-143-3p on insulin secretion, cell viability and inflammatory response in HG-treated MIN6 cells. In addition, miR-143-3p could inactivate the NF-κB pathway by inhibiting TAK1 expression. Collectively, these results suggest that miR-143-3p levels are downregulated in the peripheral blood of patients with GDM. Therefore, miR-143-3p overexpression may serve as a method for preventing pancreatic β cell dysfunction by inhibiting the TAK1/NF-κB pathway.

TAK1 is essential for endothelial barrier maintenance and repair after lung vascular injury.

TGF–β-activated kinase 1 (TAK1) plays crucial roles in innate and adaptive immune responses and is required for embryonic vascular development. However, TAK1’s role in regulating vascular barrier integrity is not well defined. Here we show that endothelial TAK1 kinase function is required to maintain and repair the injured lung endothelial barrier. We observed that inhibition of TAK1 with 5Z-7-oxozeaenol markedly reduced expression of β-catenin (β-cat) and VE-cadherin at endothelial adherens junctions and augmented protease-activated receptor-1 (PAR-1)- or toll-like receptor-4 (TLR-4)-induced increases in lung vascular permeability. In inducible endothelial cell (EC)-restricted TAK1 knockout (TAK1i∆EC) mice, we observed that the lung endothelial barrier was compromised and in addition, TAK1i∆EC mice exhibited heightened sensitivity to septic shock. Consistent with these findings, we observed dramatically reduced β-cat expression in lung ECs of TAK1i∆EC mice. Further, either inhibition or knockdown of TAK1 blocked PAR-1- or TLR-4-induced inactivation of glycogen synthase kinase 3β (GSK3β), which in turn increased phosphorylation, ubiquitylation, and degradation of β-cat in ECs to destabilize the endothelial barrier. Importantly, we showed that TAK1 inactivates GSK3β through AKT activation in ECs. Thus our findings in this study point to the potential of targeting the TAK1-AKT-GSK3β axis as a therapeutic approach to treat uncontrolled lung vascular leak during sepsis.

Intestinal ischemia-reperfusion induces the release of IL-17A to regulate cell inflammation, apoptosis and barrier damage.

Intestinal ischemia-reperfusion (I/R) injury promotes the release of IL-17A, and previous studies have indicated that TGF-β activated kinase 1 (TAK1) is an important signaling molecule in the regulatory function of IL-17A. The present study aimed to explore the potential effects of IL-17A release in intestinal I/R injury, and to investigate the underlying regulatory mechanisms. Initially, the expression levels of TAK1 and JNK in a hypoxia/reoxygenation model were determined, and the effects of TAK1-knockdown on JNK phosphorylation and the viability, inflammation, apoptosis and barrier function of Caco-2 cells were assessed using Cell Counting Kit-8, reverse transcription-quantitative PCR, TUNEL and transepithelial electrical resistance assays, respectively. Subsequently, an antibody targeting IL-17A was used, and the effects of the IL-17A antibody on the expression levels of TAK1 as well as cell viability, inflammation, apoptosis and barrier function were determined. The results of the present study demonstrated that TAK1-knockdown markedly reduced JNK phosphorylation and improved the levels of cell viability, inflammation, apoptosis and barrier function via the MAPK signaling pathway. In addition, treatment with the IL-17A antibody inhibited the expression of TAK1, and reversed the aforementioned effects of TAK1 on Caco-2 cells. In conclusion, intestinal I/R induces the release of IL-17A to regulate cell viability, inflammation, apoptosis and barrier damage via the TAK1/MAPK signaling pathway.

Microglial TLR4-induced TAK1 phosphorylation and NLRP3 activation mediates neuroinflammation and contributes to chronic morphine-induced antinociceptive tolerance

Background and purposeThe aim of this work was to investigate the role and signal transduction of toll-like receptor 4 (TLR4), TGF-β-activated kinase 1 (TAK1) and nod-like receptor protein 3 (NLRP3) in microglial in the development of morphine-induced antinociceptive tolerance. MethodsTLR4 and NLRP3 knockout mice and 5Z-7-oxozeaeno (a selective inhibitor against TAK1 activity) were used to observe their effect on the development of morphine tolerance. Intrathecal injections of morphine (0.75 mg/kg once daily for 7 days) were used to establish anti-nociceptive tolerance, which was measured by the tail-flick test. Spinal TLR4, TAK1, and NLRP3 expression levels and phosphorylation of TAK1 were evaluated by Western blotting and immunofluorescence. ResultsRepeated treatment with morphine increased total expression of spinal TLR4, TAK1, and NLRP3 and phosphorylation of TAK1 in wild-type mice. TLR4 knockout attenuated morphine-induced tolerance and inhibited the chronic morphine-induced increase in NLRP3 and phosphorylation of TAK1. Compared with controls, mice that received 5Z-7-oxozeaenol showed decreased development of morphine tolerance and inhibition on repeated morphine-induced increase of NLRP3 but not TLR4. NLRP3 knockout mice showed resistance to morphine-induced analgesic tolerance with no effect on chronic morphine-induced expression of TLR4 and TAK1. TLR4, TAK1, and NLRP3 were collectively co-localized together and with the microglia marker Iba1. ConclusionsMicroglial TLR4 regulates TAK1 expression and phosphorylation and results in NLRP3 activation contributes to the development of morphine tolerance through regulating neuroinflammation. Targeting TLR4-TAK1-NLRP3 signaling to regulate neuro-inflammation will be alternative therapeutics and strategies for chronic morphine-induced antinociceptive tolerance.

TAK1 Phosphorylates RASSF9 and Inhibits Esophageal Squamous Tumor Cell Proliferation by Targeting the RAS/MEK/ERK Axis.

TGF‐β‐activated kinase 1 (TAK1), a serine/threonine kinase, is a key intermediate in several signaling pathways. However, its role in tumorigenesis is still not understood well. In this study, it is found that TAK1 expression decreases in esophageal tumor tissues and cell lines. In vitro experiments demonstrate that proliferation of esophageal tumor cells is enhanced by knockdown of TAK1 expression and attenuated by elevated expression of TAK1. Using a subcutaneous tumor model, these observations are confirmed in vivo. Based on the results from co‐immunoprecipitation coupled with mass spectrometry, Ras association domain family 9 (RASSF9) is identified as a downstream target of TAK1. TAK1 phosphorylates RASSF9 at S284, which leads to reduced RAS dimerization, thereby blocking RAF/MEK/ERK signal transduction. Clinical survey reveals that TAK1 expression is inversely correlated with survival in esophageal cancer patients. Taken together, the data reveal that TAK1‐mediated phosphorylation of RASSF9 at Ser284 negatively regulates esophageal tumor cell proliferation via inhibition of the RAS/MEK/ERK axis.

IL-1β dysregulates cGMP signaling in the newborn lung.

Cyclic guanosine monophosphate (cGMP) signaling is an important regulator of newborn lung function and development. Although cGMP signaling is decreased in many models of newborn lung injury, the mechanisms are poorly understood. We determined how IL-1β regulates the expression of the α1-subunit of soluble guanylate cyclase (sGCα1), a prime effector of pulmonary cGMP signaling. Physiologic levels of IL-1β were discovered to rapidly decrease sGCα1 mRNA expression in a human fetal lung fibroblast cell line (IMR-90 cells) and protein levels in primary mouse pup lung fibroblasts. This sGCα1 expression inhibition appeared to be at a transcriptional level; IL-1β treatment did not alter sGCα1 mRNA stability, although it reduced sGCα1 promoter activity. Transforming growth factor-β (TGFβ)-activated kinase-1 (TAK1) was determined to be required for IL-1β's regulation of sGCα1 expression; TAK1 knockdown protected sGCα1 mRNA expression in IL-1β-treated IMR-90 cells. Moreover, heterologously expressed TAK1 was sufficient to decrease sGCα1 mRNA levels in those cells. Nuclear factor-κB (NF-κB) signaling played a critical role in the IL-1β-TAK1-sGCα1 regulatory pathway; chromatin immunoprecipitation studies demonstrated enhanced activated NF-κB subunit (RelA) binding to the sGCα1 promoter after IL-1β treatment unless treated with an IκB kinase-2 inhibitor. Also, this NF-κB signaling inhibition protected sGCα1 expression in IL-1β-treated fibroblasts. Lastly, using transgenic mice in which active IL-1β was conditionally expressed in lung epithelial cells, we established that IL-1β expression is sufficient to stimulate TAK1 and decrease sGCα1 protein expression in the newborn lung. Together these results detail the role and mechanisms by which IL-1β inhibits cGMP signaling in the newborn lung.

IL‐1β inhibits cGMP signaling enzyme expression in perinatal fibroblasts via NF‐κB signaling

Lung injury causes important morbidity and mortality in newborns by disrupting pulmonary vascular function and alveolar development. Recent evidence suggests that IL‐1β signaling plays a primary role in the pathogenesis of newborn lung disease. But the mechanisms are incompletely understood. Previously we showed that nitric oxide (NO) and cGMP signaling regulate pulmonary blood flow and development in newborns. Furthermore, we determined that disruption of cGMP signaling during newborn lung injury accentuates pulmonary disease. Accordingly, here we determined whether IL‐1β inhibits cGMP signaling in the newborn lung.We used a human fetal lung fibroblast cell line (IMR‐90 cells) and primary mouse pup lung fibroblasts to test whether IL‐1β down‐regulates the expression of soluble guanylate cyclase α1 subunit (sGCα1). This is because fibroblasts have an important role in regulating newborn lung function and alveolar development and sGCα1 is a prime initiator of cGMP signaling upon NO stimulation. We determined that physiologic levels of recombinant IL‐1β rapidly decreased sGCα1 mRNA expression in the IMR‐90 cells and protein levels in the primary lung fibroblasts. This inhibition of sGCα1 expression appeared to be at a transcriptional level. This is because IL‐1β treatment did not further decrease sGCα1 mRNA levels in the fetal lung fibroblasts treated with actinomycin D. TGFβ‐activated kinase 1 (TAK1) was determined to mediate IL‐1β’s regulation of sGCα1 expression; TAK1 knock‐down protected sGCα1 mRNA expression in IL‐1β‐treated IMR‐90 cells. Moreover, heterologous expression of TAK1 in these cells revealed that this kinase is sufficient to decrease sGCα1 mRNA levels. Nuclear factor‐kappaB (NF‐κB) signaling played a critical role in the IL‐1β‐TAK1‐sGCα1 regulatory pathway; NF‐κB signaling inhibition by an IKK2 inhibitor PHA‐408 was determined to protect sGCα1 mRNA and protein expression in IL‐1β‐treated perinatal lung fibroblasts. Lastly, using tissue samples and soluble proteins obtained from transgenic mice in which active IL‐1β was conditionally expressed in lung epithelial cells, we established that IL‐1β expression is sufficient to stimulate TAK1 activation and decrease sGCα1 protein expression in the newborn lung. Together these results indicate that IL‐1β has a direct role in decreasing cGMP signaling in the newborn lung. We speculate that targeting IL‐1β and NF‐κB signaling might protect cGMP signaling in injured newborn lungs.Support or Funding InformationNIH HL096779 and HL147863 and the Swedish Government’s Grants for Medical Research supported this work.

Modulating TAK1 Expression Inhibits YAP and TAZ Oncogenic Functions in Pancreatic Cancer.

YAP and TAZ are central determinants of malignancy; however, their functions remain still undruggable. We identified TGFβ-activated kinase 1 (TAK1) as a central hub integrating the most relevant signals sustaining pancreatic cancer aggressiveness and chemoresistance. Glycogen synthase kinase (GSK)3 is known to stabilize TAK1, and its inhibition causes a reduction in TAK1 levels. Here, we hypothesized that TAK1 could sustain YAP/TAZ program, and thus, modulation of TAK1 expression through the inhibition of GSK3 could impair YAP/TAZ functions in pancreatic cancer.Differentially expressed transcripts between pancreatic cancer cells expressing scramble or TAK1-specific shRNA were annotated for functional interrelatedness by ingenuity pathway analysis. TAK1 expression was modulated by using different GSK3 inhibitors, including LY2090314. In vivo activity of LY2090314 alone or in combination with nab-paclitaxel was evaluated in an orthotopic nude mouse model.Differential gene expression profiling revealed significant association of TAK1 expression with HIPPO and ubiquitination pathways. We measured a significant downregulation of YAP/TAZ and their regulated genes in shTAK1 cells. TAK1 prevented YAP/TAZ proteasomal degradation in a kinase independent manner, through a complex with TRAF6, thereby fostering their K63-ubiquitination versus K48-ubiquitination. Pharmacologic modulation of TAK1 by using GSK3 inhibitors significantly decreased YAP/TAZ levels and suppressed their target genes and oncogenic functions. In vivo, LY2090314 plus nab-paclitaxel significantly prolonged mice survival duration.Our study demonstrates a unique role for TAK1 in controlling YAP/TAZ in pancreatic cancer. LY2090314 is a novel agent that warrants further clinical development in combination with nab-paclitaxel for the treatment of pancreatic cancer.

Abstract 3339: Modulating TAK1 expression through the inhibition of GSK3 impairs YAP/TAZ oncogenic functions in pancreatic cancer

Abstract Background: Resistance to chemotherapeutic drugs poses one of the greatest challenges in pancreatic cancer (PC) treatment. TGFβ-activated kinase 1 (TAK1) has been demonstrated to drive chemoresistance in PC through the phosphorylation-dependent activation of the NF-κB transcription factor. The transcriptional regulators Yes Associated Protein (YAP) and transcriptional coactivator with a PDZ binding domain (TAZ) are central determinants of malignancy and have been identified as a critical oncogenic effectors of KRAS in PC. We hypothesized that TAK1 could drive PC aggressiveness by sustaining YAP and TAZ oncogenic activities. Methods: TAK1 expression was silenced by shRNA in AsPC1, Panc1, and MDA-Panc28 cell lines. GSK3 was targeted by using the small molecule inhibitor LY2090314. The expression of TAK1, YAP/TAZ and their target genes in PC cell lines was studied by both Western blot and qPCR. Cell migration was assessed by transwell assays, and stemness by both spheroid formation and percentage of cancer stem cells (CSC). SRB assays were used to assess the in vitro chemopotentiation of nab-paclitaxel, gemcitabine, oxaliplatin, and SN-38. In vivo activity of LY2090314 alone or in combination with nab-paclitaxel was evaluated in an orthotopic nude mouse model with luciferase-expressing AsPC1 tumors. Expression of YAP/TAZ target genes in tumor samples was measured by both qPCR and IHC. Results: knockdown of TAK1 resulted in inhibition of K48-linked ubiquitination and proteasomal degradation of YAP and TAZ, which was independent on TAK1 kinase activity. Significant reduction of proliferation, migration and chemoresistance of shTAK1 PC cell lines was also observed. Treatment with LY2090314 induced silencing of TAK1, as well as a remarkable reduction of the expression of both YAP and TAZ and their targets. In combination, the GSK3 inhibitor strongly potentiated the cytotoxic activities of chemotherapeutic agents in all three PC cell lines. In nude mice, i.p. admnistration of LY2090314 plus nab-paclitaxel significantly reduced tumor burden. Conclusions: Our study identified GSK3 and TAK1 as major modulators of YAP and TAZ stability and oncogenic activities, thus indicating that inhibition of TAK1 expression by modulating GSK3 activity could represent a valid approach to revert in vivo the intrinsic chemoresistance of PC. Citation Format: Raffaela Santoro, Marco Zanotto, Geny Piro, Carmine Carbone, Giampaolo Tortora, Davide Melisi. Modulating TAK1 expression through the inhibition of GSK3 impairs YAP/TAZ oncogenic functions in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3339.

MicroRNA 143-5p regulates alpaca melanocyte migration, proliferation and melanogenesis.

microRNAs (miRNAs) have been shown to be closely involved in the control of melanogenesis and hair colour in mammals. Previous data also indicate that miR-143 regulates cell growth in melanoma. Here, we aimed to investigate the role of miR-143-5p in alpaca melanocytes. We found that miR-143-5p was highly expressed in the cytoplasm of alpaca melanocytes as demonstrated by an in situ hybridization assay. Prediction analysis revealed that miR-143-5p could regulate TGF-β-activated kinase 1 (TAK1) expression, which we confirmed by luciferase reporter assay, indicating that miR-143-5p controls TAK1 expression by directly targeting its 3' untranslated region (UTR). miR-143-5p overexpression decreased TAK1 expression, which led to increased melanocyte migration and proliferation, and downregulation of microphthalmia-associated transcription factor (MITF), which regulates melanin production. These results support a functional role for miR-143-5p in regulating alpaca melanocyte migration, proliferation and melanogenesis through direct targeting of TAK1.

Inhibition of BMP and of TGFβ receptors downregulates expression of XIAP and TAK1 leading to lung cancer cell death.

BackgroundBone morphogenetic proteins (BMP) are embryonic proteins that are part of the transforming growth factor (TGFβ) superfamily, which are aberrantly expressed in many carcinomas. Inhibition of BMP receptors with small molecule inhibitors decreases growth and induces death of lung cancer cells, which involves the downregulation of Id1 and Id3 by a Smad dependent mechanism. Developmentally, BMP and TGFβ signaling utilizes Smad-1/5 independent mechanisms to stabilize the expression of X-linked inhibitor of apoptosis protein (XIAP) and activate TGFβ activated kinase 1 (TAK1), which are known to be potent inhibitors of apoptosis. The role of BMP signaling in regulating XIAP and TAK1 in cancer cells is poorly understood. Furthermore, the interaction between the BMP and TGFβ signaling cascades in regulating the activation of TAK1 in cancer cells has not been elucidated.MethodsFeedback regulation between the BMP and TGFβ signaling pathways and their regulation of XIAP, TAK1, and Id1 were examined in lung cancer cells utilizing siRNA and inhibitors targeting BMP type I receptors, inhibitors of BMP and TGFβ type I receptors, and an inhibitor of BMP and TGFβ type I and type II receptors.ResultsWe show that upon inhibition of BMP signaling in lung cancer cells, the TGFβ signaling cascade is activated. Both the BMP and TGFβ pathways activate TAK1, which then increases the expression of Id1. Inhibition of TGFβ signaling increased Id1 expression except when BMP signaling is suppressed, which then causes a dose-related decrease in the expression of Id1. Inhibition of both BMP and TGFβ signaling enhances the downregulation of TAK1. Our data also suggests that the blockade of the BMP type II receptor enhances the downregulation XIAP, which is important in decreasing the activity of TAK1. Knockdown studies demonstrate that both XIAP and TAK1 regulate the survival of lung cancer cells.ConclusionsThis paper highlights that targeting the BMP and TGFβ type I and type II receptors causes a downregulation of XIAP, TAK1, and Id1 leading to cell death of lung cancer cells. Small molecule inhibitors targeting the BMP and TGFβ receptors represents a potential novel means to treat cancer patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0511-9) contains supplementary material, which is available to authorized users.

Blockade of TGF-β-activated kinase 1 prevents advanced glycation end products-induced inflammatory response in macrophages

Advanced glycation end products (AGEs), inflammatory-activated macrophages are essential in the initiation and progression of diabetic nephropathy (DN). TGF-β-activated kinase 1 (TAK1) plays a vital role in innate immune responses and inflammation. However, little information has been available about the effects of AGEs on the regulation of TAK1 expression and underlying mechanisms in AGEs-stimulated macrophage activation. We hypothesized TAK1 signal pathway in AGEs conditions could be a vital factor contributing to macrophage activation and inflammation. Thus, in the present study, we used bone marrow-derived macrophages (BMMs) to explore the functional role and potential mechanisms of TAK1 pathway under AGEs conditions. Results indicated that TAK1 played important roles in AGEs-induced mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B protein (NF-κB) activation, which regulated the production of monocyte chemo-attractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-α) in AGEs-stimulated macrophages. The results also suggested that TAK1 inhibitor (5Z-7-oxozeaenol) could inhibit AGEs-induced macrophage activation to down-regulate inflammatory cytokine production via MAPKs and NF-κB pathways, indicating that 5Z-7-oxozeaenol might be an immunoregulatory agent against AGEs-stimulated inflammatory response in DN.

Involvement of neuronal TGF‐β activated kinase 1 in the development of tolerance to morphine‐induced antinociception in rat spinal cord

Tolerance induced by morphine and other opiates remains a major unresolved problem in the clinical management of pain. There is now good evidence for the importance of MAPKs in morphine-induced antinociceptive tolerance. A member of the MAPK kinase kinase family, TGF-β activated kinase 1 (TAK1) is the common upstream kinase of MAPKs. Here, we have assessed the involvement of TAK1 in the development of tolerance to morphine-induced analgesia. The effects of an antagonist of TAK1 on morphine tolerance were investigated in vivo using the Randall-Selitto test, and the mechanism was investigated using Western blot and immunohistochemistry. The expression of TAK1 after chronic morphine exposure was also evaluated in vitro by immunohistochemistry. Chronic intrathecal morphine exposure up-regulated protein levels and phosphorylation of spinal TAK1. TAK1 immunoreactivity was co-localized with the neuronal marker NeuN. Intrathecal administration of 5Z-7-oxozeaenol (OZ), a selective TAK1 inhibitor, attenuated the loss of morphine analgesic potency and morphine-induced TAK1 up-regulation. Furthermore, OZ decreased the up-regulated expression of spinal p38 and JNK after repeated morphine exposure. In vitro studies demonstrated that sustained morphine treatment induced TAK1 up-regulation, which was reversed by co-administration of OZ. A bolus injection of OZ showed some reversal of established morphine antinociceptive tolerance. TAK1 played a pivotal role in the development of morphine-induced antinociceptive tolerance. Modulation of TAK1 activation by the selective inhibitor OZ in the lumbar spinal cord may prove to be an attractive adjuvant therapy to attenuate such tolerance.

MUC1-C activates the TAK1 inflammatory pathway in colon cancer

The mucin 1 (MUC1) oncoprotein has been linked to the inflammatory response by promoting cytokine-mediated activation of the NF-κB pathway. The TGF-β-activated kinase 1 (TAK1) is an essential effector of proinflammatory NF-κB signaling that also regulates cancer cell survival. The present studies demonstrate that the MUC1-C transmembrane subunit induces TAK1 expression in colon cancer cells. MUC1 also induces TAK1 in a MUC1+/−/IL-10−/− mouse model of colitis and colon tumorigenesis. We show that MUC1-C promotes NF-κB-mediated activation of TAK1 transcription and, in a positive regulatory loop, MUC1-C contributes to TAK1-induced NF-κB signaling. In this way, MUC1-C binds directly to TAK1 and confers the association of TAK1 with TRAF6, which is necessary for TAK1-mediated activation of NF-κB. Targeting MUC1-C thus suppresses the TAK1→NF-κB pathway, downregulates BCL-XL, and in turn sensitizes colon cancer cells to MEK inhibition. Analysis of colon cancer databases further indicates that MUC1, TAK1 and TRAF6 are upregulated in tumors associated with decreased survival and that MUC1-C-induced gene expression patterns predict poor outcomes in patients. These results support a model in which MUC1-C-induced TAK1→NF-κB signaling contributes to intestinal inflammation and colon cancer progression.

TAK1 inhibition accelerates cellular senescence of retinal pigment epithelial cells.

Oxidative stress and cellular senescence are known to contribute to the development of AMD; however, the mechanism is not fully understood. This study investigated the role of TGF-β-activated kinase 1 (TAK1) in the senescence of RPE cells as a model for the development of dry AMD. Cultured human RPE cells were treated with the TAK1 inhibitor 5Z-7-oxozeaenol for 1 hour, and then treated with 200 μM hydrogen peroxide for 1 hour. Human RPE cells that were not pretreated with TAK1 inhibitor for 1 hour served as controls. Senescence-associated β-galactosidase (SA-β-gal) activity was detected by histochemistry, and p53 expression by immunoblotting. Cell-cycle and apoptosis rate in RPE cells were determined by flow cytometry. The TAK1 expression in human RPE cells was high and was altered on oxidative stress. Transforming growth factor-β-activated kinase 1 inhibition led to reduction in cell proliferation, cell-cycle arrest at G0/G1, and increased SA-β-gal expression, all known to be features of cell senescence. Exposure of cells to oxidative stress combined with inhibition of TAK1 activity decreased the expression of apoptotic proteins, such as p53, and promoted cellular senescence. Aberrant TAK1 activity in RPE cells triggered their secretion of factors that induced hypertrophy and fibrotic changes in neighboring cells. The in vitro evidence indicated a role for TAK1 in the onset of senescence in RPE cells. The data shown hereby demonstrated that TAK1 activity is essential for maintaining normal function of RPE cells. Elucidation of its role in mechanisms underlying RPE cellular senescence induction may potentiate development of powerful tools for halting the development of dry AMD.

Fibroblast Growth Factor Receptor 3 Interacts with and Activates TGFβ-Activated Kinase 1 Tyrosine Phosphorylation and NFκB Signaling in Multiple Myeloma and Bladder Cancer

Cancer is a major public health problem worldwide. In the United States alone, 1 in 4 deaths is due to cancer and for 2013 a total of 1,660,290 new cancer cases and 580,350 cancer-related deaths are projected. Comprehensive profiling of multiple cancer genomes has revealed a highly complex genetic landscape in which a large number of altered genes, varying from tumor to tumor, impact core biological pathways and processes. This has implications for therapeutic targeting of signaling networks in the development of treatments for specific cancers. The NFκB transcription factor is constitutively active in a number of hematologic and solid tumors, and many signaling pathways implicated in cancer are likely connected to NFκB activation. A critical mediator of NFκB activity is TGFβ-activated kinase 1 (TAK1). Here, we identify TAK1 as a novel interacting protein and target of fibroblast growth factor receptor 3 (FGFR3) tyrosine kinase activity. We further demonstrate that activating mutations in FGFR3 associated with both multiple myeloma and bladder cancer can modulate expression of genes that regulate NFκB signaling, and promote both NFκB transcriptional activity and cell adhesion in a manner dependent on TAK1 expression in both cancer cell types. Our findings suggest TAK1 as a potential therapeutic target for FGFR3-associated cancers, and other malignancies in which TAK1 contributes to constitutive NFκB activation.

TGF-β-Activated Kinase 1 (TAK1) Is an In Vivo Druggable Target for Reverting Pancreatic Cancer Chemoresistance

Context TAK1, a mitogen-activated protein kinase kinase kinase, functions in the activation of nuclear factor kappaB (NF-kappaB) and activator protein-1, which can suppress proapoptotic signaling pathways and thus promote resistance to chemotherapeutic drugs. However, it is not known if inhibition of TAK1 is effective in reducing chemoresistance to therapeutic drugs against pancreatic cancer. Methods : NF-kappaB activity was measured by luciferase reporter assay in human pancreatic cancer cell lines AsPc-1, PANC-1, and MDAPanc-28, in which TAK1 expression was silenced by small hairpin RNA. TAK1 kinase activity was targeted in AsPc-1, PANC-1, MDAPanc-28, and Colo357FG cells with exposure to increasing doses of a selective small-molecule inhibitor, LYTAK1, for 24 hours. To test the effect of LYTAK1 in combination with chemotherapeutic agents, AsPc-1, PANC-1, MDAPanc-28 cells, and control cells were treated with increasing doses of oxaliplatin, SN-38, or gemcitabine in combination with LYTAK1. In vivo activity of oral LYTAK1 was evaluated in an orthotopic nude mouse model (n=40, 5 per group) with luciferase-expressing AsPc-1 pancreatic cancer cells. Results AsPc-1 and MDAPanc-28 TAK1 knockdown cells had a statistically significantly lower NF-kappaB activity than did their respective control cell lines. TAK1 inhibitor LYTAK1 had potent in vitro cytotoxic activity in AsPc-1, PANC-1, MDAPanc-28, and Colo357FG cells, with IC 50 between 5 and 40 nM. LYTAK1 also potentiated the cytotoxicity of chemotherapeutic agents oxaliplatin, SN-38, and gemcitabine in AsPc-1, PANC-1, and MDAPanc-28 cells compared with control cells. In nude mice, oral administration of LYTAK1 plus gemcitabine statistically significantly reduced tumor burden and prolonged survival duration. Conclusions The results of this study suggest that genetic silencing or inhibition of TAK1 kinase activity in vivo is a potential therapeutic approach to reversal of the intrinsic chemoresistance of pancreatic cancer.

TGF-β-activated Kinase-1: A Potential Prognostic Marker for Clear Cell Renal Cell Carcinoma

TGF-β-activated kinase-1 (TAK1) has been found to be over-expressed in a variety of solid malignancies and related to tumor growth. The aim of this study was to evaluate the expression level of TAK1 in clear cell renal cell carcinoma (ccRCC) and assess its value as a novel prognostic marker. TAK1 mRNA was assessed in 51 paired ccRCC tissues and adjacent normal tissues (ADTs) by real-time PCR. Tissue TAK1 protein was also assessed in 91 ADTs and 177 samples of ccRCC immunohistochemically for evaluation of relationships with clinical characteristics. RT-PCR showed that TAK1 RNA level was significantly higher in ccRCC tissues than in the paired ADTs and immunohistochemistry confirmed higher expression of TAK1 protein in ccRCC samples compared with ADTs. TAK1 protein expression in 177 ccRCC samples was significantly correlated with T stage, N classification, metastasis, recurrence and Fuhrman grade, but not age and gender. Patients with low TAK1 levels had a better survival outcome. TAK1 expression and N stage were independent prognosis factors for the overall survival of ccRCC patients. Overexpression of TAK1 predicts a poor prognosis in patients with ccRCC, so that TAK1 may serve as a novel prognostic marker.

Identification of the TAK1-NF-κB Axis As Critical Regulator of AML Stem and Progenitor Cell Survival.

AbstractAbstract 2982A small population of leukemic stem cells is resistant to chemotherapy and is responsible for the leukemic out-growth and relapse in acute myeloid leukemia (AML) patients. Evasion of apoptosis might be one of the essential mechanisms involved in this process. In order to gain more insight into the differences in the apoptotic programming between normal and leukemic (stem) cells, we recently performed gene array analysis by comparing CD34+ AML cells versus CD34+ normal bone marrow (NBM) cells. Gene ontology (GO) analysis of the differentially expressed genes between AML and NBM cells revealed differences in GO terms metabolic processes and apoptosis. In order to characterize differences in apoptotic programming in more detail 429 apoptotic related genes were selected and cluster analysis showed that CD34+ AML and CD34+ NBM cells could be separated into two distinct groups. In particular TGF-β activated kinase 1 (TAK1)/MAP3K7 was one of the apoptosis-related genes that was significantly higher expressed in CD34+ AML cells compared to CD34+ NBM cells (p = 1.8e−7). This increased expression of TAK1 could be confirmed by Q-PCR, showing an increase of on average 5.8 fold in TAK1 expression in the studied CD34+ AML cells. In mice it has been demonstrated that TAK1 is required for the survival of hematopoietic cells which is largely dependent on TNFR1 and TNFR2. In accordance with these data, we showed that TAK1 is also necessary in human hematopoiesis. Colony-forming cell (CFC) assays showed that inhibition of TAK1 in human cord blood CD34+cells, either by shRNAs targeting TAK1 or the TAK1 inhibitor 5z–7-oxozeaenol, resulted in a 2 fold reduction in CFU-GM and BFU-E frequencies compared to control cells. The efficacy was strongly further enhanced by the addition of TNFα, which resulted in a 9.4 fold decrease in CFC colonies upon TAK1 inhibition.Subsequently, we questioned whether TAK1 inhibition would affect CD34+ AML cell survival. Treatment of the AML cell lines MOLM13, OCI-M3 and HL60 with the TAK1 inhibitor 5z–7-oxozeaenol alone only induced modest effects, but in combination with TNFα for 24 hrs a strong induction of apoptosis was observed (IC50 respectively = 23nM, 215nM and 60 nM). Comparable results were observed in HL60 cells transduced with shRNAs targeting TAK1 whereby a downmodulation of TAK1 resulted in a 5.4 fold increase in Annexin V+ cells upon TNFα addition. In accordance with previous data, Western blot analysis showed that TAK1 inhibition reduced the levels of p-IκBα, p-p38, p-ERK and p-C-JUN. To test which of these pathways would be important for cell survival, AML cell lines were treated with either the p38 inhibitor SB203580, MEK/ERK inhibitor U0126, JNK inhibitor SP600125 and the NF-κB inhibitor BMS-345541, alone or in combination with TNFα. Addition of the NF-κB inhibitor BMS-345541 induced apoptosis in OCI-M3 and MOLM13 which was significantly increased in combination with TNFα (2.4 fold, p = 0.02). In contrast, inhibition of p38, MEK/ERK and JNK, either alone or in combination with TNFα, did not induce cell death in the AML cell lines. These data suggest that cell death induced by TAK1 inhibition is mainly due to inhibition of the NF-κB pathway. To determine the effects of TAK1 inhibition on primary AML cells, long-term expansion of the leukemic stem cell enriched CD34+ AML cell fraction was evaluated in MS5 stromal co-cultures in the absence or presence of TAK1 inhibitor and/or TNFα. Combined treatment for a period of 2 weeks completely abrogated the out-growth of CD34+ AML cells, indicating that both leukemic progenitors as well as leukemic stem cells were targeted. In contrast, addition of the single agents did not efficiently reduce cell growth. Similarly, downmodulation of TAK1 using shRNAs strongly sensitized primary CD34+ AML cells for TNFα-induced apoptosis, showing a 6 fold increase in Annexin V+ cells compared to control cells. Results on the in vivo efficacy of TAK1 inhibition on primary AML cells are in progress.In conclusion, our results show that TAK1 is frequently overexpressed in CD34+ AML cells, and that inhibition of TAK1 in combination with TNFα is highly efficient in inducing apoptosis of leukemic stem/progenitor cells in a NF-κB-dependent manner. Disclosures:No relevant conflicts of interest to declare.