Ser727 Phosphorylation Research Articles

Endothelin-1 stimulates expression of cyclin D1 and S-phase kinase–associated protein 2 by activating the transcription factor STAT3 in cultured rat astrocytes

Brain injury-mediated induction of reactive astrocytes often leads to glial scar formation in damaged brain regions. Activation of signal transducer and activator of transcription 3 (STAT3), a member of the STAT family of transcription factors, plays a pivotal role in inducing reactive astrocytes and glial scar formation. Endothelin-1 (ET-1) is a vasoconstrictor peptide, and its levels increase in brain disorders and promote astrocytic proliferation through ETB receptors. To clarify the mechanisms underlying ET-1-mediated astrocytic proliferation, here we examined its effects on STAT3 in cultured rat astrocytes. ET-1 treatment stimulated Ser-727 phosphorylation of STAT3 in the astrocytes, but Tyr-705 phosphorylation was unaffected, and ET-induced STAT3 Ser-727 phosphorylation was reduced by the ETB antagonist BQ788. ET-1 stimulated STAT3 binding to its consensus DNA-binding motifs. Monitoring G1/S phase cell cycle transition through bromodeoxyuridine (BrdU) incorporation, we found that ET-1 increases BrdU incorporation into the astrocytic nucleus, indicating cell cycle progression. Of note, STAT3 chemical inhibition (with stattic or 5,15-diphenyl-porphine (5,15-DPP)) or siRNA-mediated STAT3 silencing reduced ET-induced BrdU incorporation. Moreover, ET-1 increased astrocytic expression levels of cyclin D1 and S-phase kinase-associated protein 2 (SKP2), which were reduced by stattic, 5,15-DPP, and STAT3 siRNA. ChIP-based PCR analysis revealed that ET-1 promotes the binding of SAT3 to the 5'-flanking regions of rat cyclin D1 and SKP2 genes. Our results suggest that STAT3-mediated regulation of cyclin D1 and SKP2 expression underlies ET-induced astrocytic proliferation.

Leptin attenuates cerebral ischemic injury in rats by modulating the mitochondrial electron transport chain via the mitochondrial STAT3 pathway.

BackgroundAccording to recent studies, leptin may exert a neuroprotective function by affecting the phosphorylation of signal transducer and activator of transcription 3 (STAT3). During stress, STAT3 regulates mitochondrial oxidative stress and reduces apoptosis.ObjectiveIn the present study, we hypothesized that leptin increases STAT3 phosphorylation in the mitochondria and protects against mitochondrial oxidative stress in rats subjected to permanent middle cerebral artery occlusion (MCAO).ResultsLeptin reduced reactive oxygen species (ROS) production, and we confirmed that the mechanism underlying this change involved the enzymatic activities of mitochondrial respiratory chain complexes I and II. In addition, leptin increased the level of STAT3 Ser727 phosphorylation in the mitochondria.ConclusionsBased on these results, leptin may regulate mitochondrial respiratory chain enzymatic activities via mitochondria‐targeted STAT3 to reduce ROS production and protect brain tissues from mitochondrial oxidative stress during cerebral ischemia.

Immune response in the relapsing-remitting experimental autoimmune encephalomyelitis in mice: The role of the NF-κB signaling pathway

Characteristics of the mouse model of relapsing-remitting experimental autoimmune encephalomyelitis (rEAE) closely resemble manifestations of multiple sclerosis in humans. In the present study, we investigated the mechanisms of inflammatory response, focusing on NF-κB pathway activation. Cytokine response in rEAE mice was multiphasic: the early phase was characterized by the increase in interferon-γ level in plasma. In the later stage, the level of interleukin-17, but not of interferon-γ, was increased. The early phase of rEAE was also accompanied by increased RelA/p65 phosphorylation at Ser276 in spleen cells, whereas the rEAE maintenance phase was characterized by RelA/p65 phosphorylation at Ser536 and IKK phosphorylation. The IKKα/β inhibitor reduced interleukin-17 and interferon-γ levels in plasma and alleviated rEAE symptoms. The IKKα/β inhibitor decreased IKK and p65(Ser536) phosphorylation, but doubled p65(Ser276) phosphorylation in rEAE mice. The increased RelA/p65(Ser276) phosphorylation coincided in time with the production of interferon-γ, Hsp72, and the early phase of IL-17 generation, whereas increased RelA/p65(Ser536) phosphorylation coincided with the activation of IKK, SAPK/JNK, and p53, as well as the late phase of IL-17 production, indicating the role of the RelA/p65 phosphorylation events in the induction and maintenance of rEAE.

68 - Mitochondrial STAT3 serine phosphorylation by zinc reduces ROS generation at reperfusion in rat hearts

STAT3 plays a role in the regulation of mitochondrial respiration. This study tested if STAT3 serine phosphorylation by zinc could alter mitochondrial ROS generation in the setting of ischemia/reperfusion in the heart. Under normoxic conditions, treatment of isolated rat hearts with ZnCl2 increased mitochondrial STAT3 phosphorylation at Ser727. In isolated rat hearts subjected to ischemia followed by reperfusion, ZnCl2 increased mitochondrial phospho-STAT3. Moreover, ZnCl2 prevented mitochondrial ROS generation and dissipation of mitochondrial membrane potential (ΔΨm) at reoxygenation through Ser727 phosphorylation. Finally, ZnCl2 suppression of succinate dehydrogenase (SDH) activity upon the onset of reperfusion was nullified by the Ser727 mutation. In conclusion, zinc inhibits mitochondrial ROS generation at reperfusion by increasing mitochondrial STAT3 phosphorylation at Ser727. The inhibition of SDH activity may account for the role of STAT3 in the inhibitory action of zinc on ROS generation at reperfusion.

MTOR/STAT-3 pathway mediates mesenchymal stem cell-secreted hepatocyte growth factor protective effects against lipopolysaccharide-induced vascular endothelial barrier dysfunction and apoptosis.

Mesenchymal stem cells (MSCs) protect the endothelial barrier complex and survival, implicated in the pathogenesis of acute lung injury (ALI) via paracrine hepatocyte growth factor (HGF). However, the mechanism of HGF in endothelial regulation remains unclear. Here, we introduced a coculture protocol of pulmonary microvascular endothelial cells (PMVECs) and overexpression of the HGF gene of MSCs (MSC-HGF). Immunofluorescence and endothelial permeability analysis revealed that MSC-HGF protected endothelial tight junction protein occludin expression and attenuated cellular permeability as well as endothelial apoptosis. To investigate the novel mechanism mammalian TOR (mTOR)/ signal transducer and activator of transcription 3 (STAT-3) signaling in HGF protective effects against endothelial barrier and apoptosis, we used recombinant mouse HGF in endothelial cells. In addition, we used mTOR inhibitor rapamycin to inhibit the mTOR pathway. Our study demonstrated that rapamycin decreased the protective effects of HGF on the endothelium by decreasing tight junction protein occludin expression and cell proliferation, and raising lipopolysaccharide (LPS)-induced endothelial permeability, endothelial cell injury factors ET-1 and vWF. Similarly, the protective effects of HGF on reducing endothelial barrier and apoptosis were weakened when PMVECs were treated with the STAT-3 inhibitor S3I-201. Moreover, mTOR/STAT-3 were activated by HGF demonstrated as raising mTOR (Ser2448) and STAT3 (Ser727) phosphorylation proteins, leading to endothelial barrier improvement and survival. Reversely, rapamycin or S3I-201 inhibited mTOR/STAT-3 activation. Taken together, our findings highlight that the activation of the mTOR/STAT-3 pathway provides novel mechanistic insights into MSC-secreted HGF protection against LPS-induced vascular endothelial permeability dysfunction and apoptosis, which contributes to decreasing microvascular loss and lung injury.

PO-294 BRAFV600E/PTEn-loss status is associated with interleukin (IL)−8 expression in preclinical models of colorectal cancer (CRC)

IntroductionMutational status in CRC is a strong predictor for overall survival; unfortunately, tumour microenvironment (TME) and tumor-stroma interactions (TSI) also increase cancer cells’ drug resistance, leading to an urgent need to better understand the molecular mechanisms of acquired tumor-resistance, which remains crucial to determine overall patient benefit.Material and methodsProduction of IL-8 and vascular endothelial growth factor (VEGF) was determined by ELISA under standardised culture conditions. Modulation of cytokine production after exposure to selective inhibitors of the MAPK and PI3K pathways was assessed, by both ELISA assay and real time-PCR. BRAF, MEK1, ERK1 and ERK2 expression were modulated using siRNAs specifically targeting these genes.Results and discussionsCRC cell lines harbouring both BRAFV600E and PTEN-loss expressed the highest levels of IL-8 and a ROC curve-based prediction algorithm based on these two mutations had 68% accuracy in predicting IL-8 production (p=0.002); on the other hand, VEGF levels inversely correlated with KRAS mutational status. IL-8 is tightly and transcriptionally controlled by activation of the MEK/ERK pathway, as the MEK inhibitor trametinib downregulated ERK Thr202/Tyr204 phosphorylation in the nuclear compartment and profoundly suppressed IL-8 production regardless of the genetic background; conversely, the selective BRAF inhibitor dabrafenib downregulated IL-8 only in BRAFV600E contexts, but upregulated its production in parallel with ERK Thr202/Tyr204 and STAT3 Ser727 phosphorylation in the nuclear compartment of BRAF-wt CRC cells. PI3K/mTOR inhibitors did not significantly modulate IL-8 production, confirming that the IL-8 release is downstream the BRAF/MEK/ERK pathway.In order to better investigate the role of MAPK pathway in IL-8 regulation, components of the MAPK-signalling pathways (BRAF, MEK, ERK1 and ERK2) were silenced by siRNA: IL-8 production was significantly downregulated after BRAF, MEK, or ERK2 silencing, regardless of the BRAF mutational status; conversely, no effects on IL-8 expression were observed after ERK1 silencing.ConclusionThese results showed that, in preclinical models of CRC, IL-8 expression is regulated by a BRAF/MEK/ERK2/STAT3 axis, and differential modulation of IL-8 after pharmacological treatments is dependent on BRAF mutational status. Such evidence allows better understanding of the molecular mechanisms of chemokine regulation, which can, in turn, contribute to the development of new targeted therapies in CRC.

Celecoxib exerts protective effects in the vascular endothelium via COX-2-independent activation of AMPK-CREB-Nrf2 signalling

Although concern remains about the athero-thrombotic risk posed by cyclo-oxygenase (COX)-2-selective inhibitors, recent data implicates rofecoxib, while celecoxib appears equivalent to NSAIDs naproxen and ibuprofen. We investigated the hypothesis that celecoxib activates AMP kinase (AMPK) signalling to enhance vascular endothelial protection. In human arterial and venous endothelial cells (EC), and in contrast to ibuprofen and naproxen, celecoxib induced the protective protein heme oxygenase-1 (HO-1). Celecoxib derivative 2,5-dimethyl-celecoxib (DMC) which lacks COX-2 inhibition also upregulated HO-1, implicating a COX-2-independent mechanism. Celecoxib activated AMPKα(Thr172) and CREB-1(Ser133) phosphorylation leading to Nrf2 nuclear translocation. Importantly, these responses were not reproduced by ibuprofen or naproxen, while AMPKα silencing abrogated celecoxib-mediated CREB and Nrf2 activation. Moreover, celecoxib induced H-ferritin via the same pathway, and increased HO-1 and H-ferritin in the aortic endothelium of mice fed celecoxib (1000 ppm) or control chow. Functionally, celecoxib inhibited TNF-α-induced NF-κB p65(Ser536) phosphorylation by activating AMPK. This attenuated VCAM-1 upregulation via induction of HO-1, a response reproduced by DMC but not ibuprofen or naproxen. Similarly, celecoxib prevented IL-1β-mediated induction of IL-6. Celecoxib enhances vascular protection via AMPK-CREB-Nrf2 signalling, a mechanism which may mitigate cardiovascular risk in patients prescribed celecoxib. Understanding NSAID heterogeneity and COX-2-independent signalling will ultimately lead to safer anti-inflammatory drugs.

Zinc improves mitochondrial respiratory function and prevents mitochondrial ROS generation at reperfusion by phosphorylating STAT3 at Ser727

Serine 727 (Ser727) phosphorylation of STAT3 plays a role in the regulation of mitochondrial respiration. This study aimed to test if zinc could regulate mitochondrial respiration through phosphorylation of STAT3 at Ser727 in the setting of ischemia/reperfusion in the heart. Under normoxic conditions, treatment of isolated rat hearts with ZnCl2 increased cytosolic STAT3 phosphorylation at Ser727 followed by phospho-STAT3 translocation to mitochondria. In isolated rat hearts subjected to 30 min regional ischemia followed by 20 min of reperfusion, ZnCl2 given 5 min before the onset of reperfusion also increased mitochondrial phospho-STAT3. ZnCl2 enhanced ERK phosphorylation and PD98059 reversed the effect of ZnCl2 on STAT3 phosphorylation. ZnCl2 improved the mitochondrial oxidative phosphorylation at reperfusion. This effect was abolished by STAT3S727A, a mutant in which Ser727 is replaced with alanine, in H9c2 cells subjected to hypoxia/reoxygenation. In addition, ZnCl2 increased the mRNA level of the complex I subunit ND6, which was also reversed by STAT3S727A. Moreover, ZnCl2 attenuated mitochondrial ROS generation and dissipation of mitochondrial membrane potential (ΔΨm) at reoxygenation through Ser727 phosphorylation. Finally, ZnCl2 suppression of succinate dehydrogenase (SDH) activity upon the onset of reperfusion was nullified by the Ser727 mutation. In conclusion, zinc improves cardiac oxidative phosphorylation and inhibits mitochondrial ROS generation at reperfusion by increasing mitochondrial STAT3 phosphorylation at Ser727 via ERK. The preservation of ND6 mtDNA and the inhibition of SDH activity may account for the role of STAT3 in the beneficial action of zinc on the mitochondrial oxidative phosphorylation and ROS generation at reperfusion.

Bruton's tyrosine kinase regulates TLR7/8-induced TNF transcription via nuclear factor-κB recruitment

Tumour necrosis factor (TNF) is produced by primary human macrophages in response to stimulation by exogenous pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs) via Toll-like receptor (TLR) signalling. However, uncontrolled TNF production can be deleterious and hence it is tightly controlled at multiple stages. We have previously shown that Bruton's tyrosine kinase (Btk) regulates TLR4-induced TNF production via p38 MAP Kinase by stabilising TNF messenger RNA. Using both gene over-expression and siRNA-mediated knockdown we have examined the role of Btk in TLR7/8 mediated TNF production. Our data shows that Btk acts in the TLR7/8 pathway and mediates Ser-536 phosphorylation of p65 RelA and subsequent nuclear entry in primary human macrophages. These data show an important role for Btk in TLR7/8 mediated TNF production and reveal distinct differences for Btk in TLR4 versus TLR7/8 signalling.

Induction of cancer cell stemness by depletion of macrohistone H2A1 in hepatocellular carcinoma.

Hepatocellular carcinomas (HCC) contain a subpopulation of cancer stem cells (CSCs), which exhibit stem cell-like features and are responsible for tumor relapse, metastasis, and chemoresistance. The development of effective treatments for HCC will depend on a molecular-level understanding of the specific pathways driving CSC emergence and stemness. MacroH2A1 is a variant of the histone H2A and an epigenetic regulator of stem-cell function, where it promotes differentiation and, conversely, acts as a barrier to somatic-cell reprogramming. Here, we focused on the role played by the histone variant macroH2A1 as a potential epigenetic factor promoting CSC differentiation. In human HCC sections we uncovered a significant correlation between low frequencies of macroH2A1 staining and advanced, aggressive HCC subtypes with poorly differentiated tumor phenotypes. Using HCC cell lines, we found that short hairpin RNA-mediated macroH2A1 knockdown induces acquisition of CSC-like features, including the growth of significantly larger and less differentiated tumors when injected into nude mice. MacroH2A1-depleted HCC cells also exhibited reduced proliferation, resistance to chemotherapeutic agents, and stem-like metabolic changes consistent with enhanced hypoxic responses and increased glycolysis. The loss of macroH2A1 increased expression of a panel of stemness-associated genes and drove hyperactivation of the nuclear factor kappa B p65 pathway. Blocking phosphorylation of nuclear factor kappa B p65 on Ser536 inhibited the emergence of CSC-like features in HCC cells knocked down for macroH2A1. Conclusion: The absence of histone variant macroH2A1 confers a CSC-like phenotype to HCC cells in vitro and in vivo that depends on Ser536 phosphorylation of nuclear factor kappa B p65; this pathway may hold valuable targets for the development of CSC-focused treatments for HCC. (Hepatology 2018;67:636-650).

Under-expression of CK2β subunit in ccRCC represents a complementary biomarker of p-STAT3 Ser727 that correlates with patient survival.

Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive subtype of renal cancer. STAT3 pathway is altered in these tumors and p-STAT3 Ser727 is an independent prognostic factor for ccRCC. Protein kinase CK2 is altered in different types of tumors and overexpression of CK2α is considered predictive of bad prognosis and metastatic risk. CK2 subunits analyses in ccRCC samples showed increased CK2α/α’ nuclear content in all cases, but decreased cytosolic CK2β (CK2βcyt) levels in the more advanced tumors. Stable downregulation of CK2β in renal proximal tubular (HK-2) and clear cell adenocarcinoma (786-O) cells triggered changes in E-cadherin, vimentin and Snail1 protein levels indicative of epithelial-to-mesenchymal transition (EMT), and increased HIF-α. Moreover, CK2β was required in order to observe STAT3 Ser727 phosphorylation in HK-2 but not in 786-O cells.We also observed that CK2β improved the prognostic value of p-STAT3 Ser727, as CK2βcyt>41 (median value) discriminates patients free of disease for a period of 10 years upon surgery, from those with CK2βcyt<41, when p-STAT3 Ser727levels are low.We conclude that CK2β down-regulation might represent a mechanism to support EMT and angiogenesis and that CK2βcyt levels are instrumental to refine prognosis of ccRCC patients with low p-STAT3 Ser727 levels.

Susceptibility of Mycobacterium tuberculosis-infected host cells to phospho-MLKL driven necroptosis is dependent on cell type and presence of TNFα

ABSTRACT An important feature of Mycobacterium tuberculosis pathogenesis is the ability to control cell death in infected host cells, including inhibition of apoptosis and stimulation of necrosis. Recently an alternative form of programmed cell death, necroptosis, has been described where necrotic cell death is induced by apoptotic stimuli under conditions where apoptotic execution is inhibited. We show for the first time that M. tuberculosis and TNFα synergise to induce necroptosis in murine fibroblasts via RIPK1-dependent mechanisms and characterized by phosphorylation of Ser345 of the MLKL necroptosis death effector. However, in murine macrophages M. tuberculosis and TNFα induce non-necroptotic cell death that is RIPK1-dependent but independent of MLKL phosphorylation. Instead, M. tuberculosis-infected macrophages undergo RIPK3-dependent cell death which occurs both in the presence and absence of TNFα and involves the production of mitochondrial ROS. Immunocytochemical staining for MLKL phosphorylation further demonstrated the occurrence of necroptosis in vivo in murine M. tuberculosis granulomas. Phosphorylated-MLKL immunoreactivity was observed associated with the cytoplasm and nucleus of fusiform cells in M. tuberculosis lesions but not in proximal macrophages. Thus whereas pMLKL-driven necroptosis does not appear to be a feature of M. tuberculosis-infected macrophage cell death, it may contribute to TNFα-induced cytotoxicity of the lung stroma and therefore contribute to necrotic cavitation and bacterial dissemination.

N-Palmitoylethanolamine-Oxazoline as a New Therapeutic Strategy to Control Neuroinflammation: Neuroprotective Effects in Experimental Models of Spinal Cord and Brain Injury.

Modulation of N-acylethanolamine-hydrolyzing acid amidase (NAAA) represents a potential alternative strategy in the treatment of neuroinflammation. Recent studies showed that pharmacological modulation of NAAA could be achieved with the oxazoline of palmitoylethanolamide (PEA; PEA-OXA). The aim of this study was to evaluate the neuroprotective effects of PEA-OXA in the secondary neuroinflammatory events induced by spinal and brain trauma in mice. Animals were subjected to spinal cord and brain injury models and PEA-OXA (10 mg/kg) was administered both intraperitoneally and orally 1 h and 6 h after trauma. PEA-OXA treatment markedly reduced the histological alterations induced by spinal cord injury (SCI) and traumatic brain injury (TBI) and ameliorated the motor function and behavioral deficits, as well. In addition, the expression of neurotrophic factors, such as glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor, and neurotrophin-3 were increased by PEA-OXA treatment. Moreover, PEA-OXA also significantly decreased glial fibrillary acidic protein hyperexpression, the nuclear translocation of nuclear factor (NF)-κB, phosphorylation of Ser536 on the NF-κB subunit p65, and degradation of IκB-α, as well as diminished the expression of pro-inflammatory mediators such as cyclooxygenase-2 (COX-2), inducible nitric oxide synthase, tumor necrosis factor (TNF)-α and interleukin (IL)-1β. The modulation of intracellular NAAA by PEA-OXA treatment could thus represent a novel therapy to control neuroinflammatory conditions associated with SCI and TBI.

RECQ5 Helicase Cooperates with MUS81 Endonuclease in Processing Stalled Replication Forks at Common Fragile Sites during Mitosis

The MUS81-EME1 endonuclease cleaves late replication intermediates at common fragile sites (CFSs) during early mitosis to trigger DNA-repair synthesis that ensures faithful chromosome segregation. Here, we show that these DNA transactions are promoted by RECQ5 DNA helicase in a manner dependent on its Ser727 phosphorylation by CDK1. Upon replication stress, RECQ5 associates with CFSs in early mitosis through its physical interaction with MUS81 and promotes MUS81-dependent mitotic DNA synthesis. RECQ5 depletion or mutational inactivation of its ATP-binding site, RAD51-interacting domain, or phosphorylation site causes excessive binding of RAD51 to CFS loci and impairs CFS expression. This leads to defective chromosome segregation and accumulation of CFS-associated DNA damage in G1 cells. Biochemically, RECQ5 alleviates the inhibitory effect of RAD51 on 3'-flap DNA cleavage by MUS81-EME1 through its RAD51 filament disruption activity. These data suggest that RECQ5 removes RAD51 filaments stabilizing stalled replication forks at CFSs and hence facilitates CFS cleavage by MUS81-EME1.

Mixed Lineage Kinase 3 Mediates the Induction of CXCL10 by a STAT1-Dependent Mechanism During Hepatocyte Lipotoxicity.

Saturated fatty acids (SFA) and their toxic metabolites contribute to hepatocyte lipotoxicity in nonalcoholic steatohepatitis (NASH). We previously reported that hepatocytes, under lipotoxic stress, express the potent macrophage chemotactic ligand C-X-C motif chemokine 10 (CXCL10), and release CXCL10-enriched extracellular vesicles (EV) by a mixed lineage kinase (MLK) 3-dependent mechanism. In the current study, we sought to examine the signaling pathway responsible for CXCL10 induction during hepatocyte lipotoxicity. Here, we demonstrate a role for signal transducer and activator of transcription (STAT) 1 in regulating CXCL10 expression. Huh7 and HepG2 cells were treated with lysophosphatidylcholine (LPC), the toxic metabolite of the SFA palmitate. In LPC-treated hepatocytes, CXCL10 induction is mediated by a mitogen activated protein kinase (MAPK) signaling cascade consisting of a relay kinase module of MLK3, MKK3/6, and p38. P38 in turn induces STAT1 Ser727 phosphorylation and CXCL10 upregulation in hepatocytes, which is reduced by genetic or pharmacological inhibition of this MAPK signaling cascade. The binding and activity of STAT1 at the CXCL10 gene promoter were identified by chromatin immunoprecipitation and luciferase gene expression assays. Promoter activation was attenuated by MLK3/STAT1 inhibition or by deletion of the consensus STAT1 binding sites within the CXCL10 promoter. In lipotoxic hepatocytes, MLK3 activates a MAPK signaling cascade, resulting in the activating phosphorylation of STAT1, and CXCL10 transcriptional upregulation. Hence, this kinase relay module and/or STAT1 inhibition may serve as a therapeutic target to reduce CXCL10 release, thereby attenuating NASH pathogenesis. J. Cell. Biochem. 118: 3249-3259, 2017. © 2017 Wiley Periodicals, Inc.

Identification of microRNA-181A-5P and microRNA-4454 as mediators of facet cartilage degeneration through the activation of zinc finger protein 440 mediated NF-KB signaling pathway and potential circulating biomarkers for detecting the severity of facet joint osteoarthritis in blood

Purpose: Osteoarthritis (OA) of the spine (facet joints, FJ) is one of the major causes of severe low back pain and disability worldwide. However, specific mechanisms associated with facet cartilage degeneration during FJ OA are largely unknown. For the first time, in this study we aimed to investigate the role of microRNAs (miRNAs) in the pathophysiology of facet cartilage degeneration during FJ OA and the potential as biomarkers for detecting and determining the severity of FJ OA in human blood plasma. Methods: Based on MRI grading (by Weishaupt et al) and histopathology, we first established and validated a patient cohort (n = 203) (Group 1 patients [control group]: normal or mild facet cartilage degeneration and Group 2 patients [FJ OA group]: moderate to severe facet cartilage degeneration). Using this large and unique cohort, we screened 2,100 miRNAs using miRNA-array and differentially regulated the expressions of miRNAs in facet cartilage. miRNAs were further tested by qPCR analysis and their correlation with FJ OA severities were evaluated based on MRI grading score (grade 0; normal, grade 1; mild, grade 2; moderate and grade 3; severe). Human FJ OA chondrocytes were cultured and transfected with miRNA enhancers/inhibitors (or control enhancer/inhibitor) with/without interleukin (IL)-1β to determine the effect of miRNA enhancement/inhibition on the expression of catabolic (MMP13)/inflammatory (IL6, MCP1, TNFA)/anabolic (COL2A1)/cell death (PARP p85) markers by qPCR and western blot analysis. Target genes and signaling pathways modulated by miRNAs were also identified using mirDIP/pathDIP. Furthermore, we injected the miR-181a-5p in-vivo mimic (enhancer) into FJs of rats to see the effect of the overexpression in animal FJ cartilage. Finally, we tested the expression of two miRNAs in human blood plasma obtained from patients with various FJ OA severities (from grade 0 to grade 3) using qPCR analysis to identify any correlations between the circulating levels of two miRNAs in human blood plasma and FJ OA severity based on MRI grading. Results: Out of 2,100 miRNAs, we specifically identified 2 miRNAs (miR-181a-5p and miR-4454) whose expression were markedly up-regulated in FJ OA cartilage compared to control facet cartilage. Our data from patients with varying degrees of FJ OA disease severity showed that the expression levels of miR-181a-5p and miR-4454 in human facet cartilage were markedly elevated with increased degeneration and exhibited significant correlation with clinical disease severity based on MRI grading. We then treated FJ OA chondrocytes with miR-181a-5p or miR-4454 enhancer/inhibitor and showed that treatment with miR-181a-5p or miR-4454 enhancer significantly elevated the expression of inflammatory (IL6, MCP1, TNFA)/catabolic (MMP13)/cell death (PARP p85) markers and reduced expression of type II collagen (COL2A1). Inhibition of these two miRNAs was able to reverse these destructive effects compared to control enhancer/inhibitor in facet chondrocytes treated with IL-1β. Our study using mirDIP/pathDIP also identified that in facet cartilage both miR-181a-5p and miR-4454 signal zinc finger protein 440 (ZNF440) via modulation of the NF-κB pathway. Induction of NF-κB signaling using IL-1β treatment elevated miR-181a-5p and miR-4454 expression, which mediated a feedback loop to sustain NF-κB signaling through ZNF440 expression. FJ OA chondrocytes co-transfected with miR-181a-5p or miR-4454 mimic (or control mimic) and ZNF440 siRNA (or control siRNA) regulated phosphorylation of Ser536 NF-κB-p65 and reduction of IκB expression compared to control. Furthermore, by injecting miR-181a-5p in vivo-mimic (enhancer) in rat FJs, we observed a FJ OA phenotype in facet cartilage associated with enhanced catabolic activity and chondrocyte apoptosis in vivo. Finally, we determined that circulating forms of miR-181a-5p and miR-4454 in human blood plasma were sufficiently detectable and elevated in patient blood plasma with increased degree of FJ OA severity based on MRI grading. Conclusions: Using clinical, in vitro functional and in vivo studies, we have uniquely identified miR-181a-5p and miR-4454 as active players of facet cartilage degeneration during FJ OA through the activation of ZNF440-mediated NF-κB signaling pathway and potential circulating biomarkers in human blood plasma for detecting and determining the severity of FJ OA.

Human Keratinocytes Respond to Extracellular UTP by Induction of Hyaluronan Synthase 2 Expression and Increased Hyaluronan Synthesis

The release of nucleotides into extracellular space is triggered by insults like wounding and ultraviolet radiation, resulting in stimulatory or inhibitory signals via plasma membrane nucleotide receptors. As similar insults are known to activate hyaluronan synthesis we explored the possibility that extracellular UTP or its breakdown products UDP and UMP act as mediators for hyaluronan synthase (HAS) activation in human epidermal keratinocytes. UTP increased hyaluronan both in the pericellular matrix and in the culture medium of HaCaT cells. 10-100 μm UTP strongly up-regulated HAS2 expression, although the other hyaluronan synthases (HAS1, HAS3) and hyaluronidases (HYAL1, HYAL2) were not affected. The HAS2 response was rapid and transient, with the maximum stimulation at 1.5 h. UDP exerted a similar effect, but higher concentrations were required for the response, and UMP showed no stimulation at all. Specific siRNAs against the UTP receptor P2Y2, and inhibitors of UDP receptors P2Y6 and P2Y14, indicated that the response to UTP was mediated mainly through P2Y2 and to a lesser extent via UDP receptors. UTP increased the phosphorylation of p38, ERK, CREB, and Ser-727 of STAT3 and induced nuclear translocation of pCaMKII. Inhibitors of PKC, p38, ERK, CaMKII, STAT3, and CREB partially blocked the activation of HAS2 expression, confirming the involvement of these pathways in the UTP-induced HAS2 response. The present data reveal a selective up-regulation of HAS2 expression by extracellular UTP, which is likely to contribute to the previously reported rapid activation of hyaluronan metabolism in response to tissue trauma or ultraviolet radiation.

Specific tumor-derived CCL2 mediated by pyruvate kinase M2 in colorectal cancer cells contributes to macrophage recruitment in tumor microenvironment.

Development of colorectal cancer has been considered as a result of imbalance of pro- and anti-inflammatory intestinal microenvironment accompanied by macrophage recruitment. Despite macrophages are implicated in remodeling tumor microenvironment, the mechanism of macrophage recruitment is not fully elucidated yet. In this study, we reported clinical association of highly expressed pyruvate kinase M2 in colorectal cancer with macrophage attraction. The conditioned medium from Caco-2 and HT-29 cells with depleted pyruvate kinase M2 dramatically reduced macrophage recruitment, which is reversed by addition of, a critical chemotaxis factor to macrophage migration, rCCL2. Silencing of endogenous pyruvate kinase M2 markedly decreased CCL2 expression and secretion by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Endogenous pyruvate kinase M2 interacted with p65 and mediated nuclear factor-κB signaling pathway and mainly regulated phosphorylation of Ser276 on p65 nuclear factor-κB. In addition, inhibition of macrophage recruitment caused by pyruvate kinase M2 silencing was rescued by ectopic expression of p65. Interestingly, pyruvate kinase M2 highly expressed in colorectal cancer tissue, which is correction with macrophage distribution. Taken together, we revealed a novel mechanism of pyruvate kinase M2 in promoting colorectal cancer progression by recruitment of macrophages through p65 nuclear factor-κB-mediated expression of CCL2.

Signal Transducer and Activator of Transcription 1 Plays a Pivotal Role in RET/PTC3 Oncogene-induced Expression of Indoleamine 2,3-Dioxygenase 1

Indoleamine 2,3-dioxygenase 1 (IDO1) is a single chain oxidoreductase that catalyzes tryptophan degradation to kynurenine. In cancer, it exerts an immunosuppressive function as part of an acquired mechanism of immune escape. Recently, we demonstrated that IDO1 expression is significantly higher in all thyroid cancer histotypes compared with normal thyroid and that its expression levels correlate with T regulatory (Treg) lymphocyte densities in the tumor microenvironment. BRAFV600E- and RET/PTC3-expressing PcCL3 cells were used as cellular models for the evaluation of IDO1 expression in thyroid carcinoma cells and for the study of involved signal transduction pathways. BRAFV600E-expressing PcCL3 cells did not show IDO1 expression. Conversely, RET/PTC3-expressing cells were characterized by a high IDO1 expression. Moreover, we found that, the STAT1-IRF1 pathway was instrumental for IDO1 expression in RET/PTC3 expressing cells. In detail, RET/PTC3 induced STAT1 overexpression and phosphorylation at Ser-727 and Tyr-701. STAT1 transcriptional regulation appeared to require activation of the canonical NF-κB pathway. Conversely, activation of the MAPK and PI3K-AKT pathways primarily regulated Ser-727 phosphorylation, whereas a physical interaction between RET/PTC3 and STAT1, followed by a direct tyrosine phosphorylation event, was necessary for STAT1 Tyr-701 phosphorylation. These data provide the first evidence of a direct link between IDO1 expression and the oncogenic activation of RET in thyroid carcinoma and describe the involved signal transduction pathways. Moreover, they suggest possible novel molecular targets for the abrogation of tumor microenvironment immunosuppression. The detection of those targets is becoming increasingly important to yield the full function of novel immune checkpoint inhibitors.

DYRK1A Is a Therapeutic Target in B-ALL in Children with Down Syndrome

Children with Down syndrome have a spectrum of associated disorders including a 20-fold increased incidence of B-cell acute lymphoblastic leukemia (DS-ALL). Although a number of genetic alterations have been found in this ALL subtype, such as activating mutations in JAK2 and overexpression of CRLF2, the mechanisms by which trisomy 21 promotes the leukemia are largely unknown. Previous studies have implicated chromosome 21 genes HMGN1 and DYRK1A in both malignant and normal lymphopoiesis.DYRK1A is a member of the dual-specificity tyrosine phosphorylation-regulated kinase family that has been well studied in non-hematopoietic tissues. Its targets include proteins that regulate multiple pathways including cell signaling, cell cycle, and brain development. We have previously shown that DYRK1A is a megakaryoblastic leukemia-promoting gene through its negative regulation of NFAT transcription factors. Furthermore, in studies with a conditional Dyrk1a knock-out mouse, we found that the kinase is required for lymphoid, but not myeloid cell development. In developing lymphocytes, Dyrk1a regulates the cell cycle by destabilizing cyclin D3. Consequently, loss of Dyrk1a resulted in the failure of these cells to switch from a proliferative to quiescent phase for subsequent maturation (Thompson et al. J. Exp. Med. 2015 212:953-70). Despite this deficiency in exiting the cell cycle, Dyrk1a-deficient lymphocytes also exhibit impaired proliferation before undergoing apoptosis. These data reveal a critical role for DYRK1A in lymphopoiesis and suggest that it may be a target for therapeutic intervention.We assayed the activity of the highly selective and potent DYRK1 inhibitor, EHT 1610, in multiple ALL cell lines. EHT 1610 inhibited the growth of Jurkat and MHH-CALL-4 cells with EC50s of 0.83mM and 0.49mM, respectively. Next, we treated primary human ALL blasts with EHT 1610 and the less selective DYRK1A inhibitor harmine. Growth of 16 out of 30 specimens, which included DS-ALL, pre-B ALL, and T-ALL, was sensitive to DYRK1A inhibition at doses between 0.5 and 10mM. Of note, growth of 9 of the 11 of the DS-ALL samples was inhibited by EHT 1610. This result indicates that the increased dosage of DYRK1A in DS samples sensitizes the cells to DYRK1A inhibition.To further study the contributions of DYRK1A to normal and malignant lymphopoiesis, we performed phosphoproteomic analysis on primary murine pre-B cells treated with EHT 1610. After 2 hours of EHT 1610 treatment, the cells were collected and analyzed for changes in the phosphoproteome. Phosphorylation of 36 proteins was significantly altered. Bioinformatics analysis led to the identification of a number of notable pathways that appear to be regulated by DYRK1A including cell cycle, cell division and mitosis, RNA metabolism, and JAK-STAT signaling. Differentially phosphorylated proteins included geminin, which is important in cell division and whose loss enhances megakaryopoiesis, and POLR2M, which is intriguing because DYRK1A phosphorylates the CTD of RNA Pol II and binds chromatin at specific sites in glioblastoma cells. Another interesting target is STAT3, which is phosphorylated by DYRK1A on Ser727, a residue whose phosphorylation is required for maximal STAT3 activation. Treatment of murine pre-B cells with EHT 1610 significantly reduced the level of phosphorylation of Ser727 and Tyr705, suggesting that DYRK1A may provide a priming event for STAT3 activation similar to its priming effect on GSK3b phosphorylation. Consistent with a role for JAK/STAT signaling and STAT3 activity, B-ALL cells were highly sensitive to ruxolitinib therapy. Taken together, our study suggests that DYRK1A is a therapeutic target in DS-ALL and likely functions in part by enhancing JAK/STAT signaling. DisclosuresNo relevant conflicts of interest to declare.