Myeloid-derived cullin 3 promotes STAT3 phosphorylation by inhibiting OGT expression and protects against intestinal inflammation

Signal transducer and activator of transcription 3 (STAT3) is a key mediator of intestinal inflammation and tumorigenesis. However, the molecular mechanism that modulates STAT3 phosphorylation and activation is not fully understood. Here, we demonstrate that modification of STAT3 with O-linked β-N-acetylglucosamine (O-GlcNAc) on threonine 717 (T717) negatively regulates its phosphorylation and targets gene expression in macrophages. We further found that cullin 3 (CUL3), a cullin family E3 ubiquitin ligase, down-regulates the expression of the O-GlcNAc transferase (OGT) and inhibits STAT3 O-GlcNAcylation. The inhibitory effect of CUL3 on OGT expression is dependent on nuclear factor E2–related factor-2 (Nrf2), which binds to the Ogt promoter region and increases gene transcription. Myeloid deletion of Cul3 led to defective STAT3 phosphorylation in colon macrophages, which was accompanied by exacerbated colonic inflammation and inflammation-driven tumorigenesis. Thus, this study identifies a new form of posttranslational modification of STAT3, modulating its phosphorylation, and suggests the importance of immunometabolism on colonic inflammation and tumorigenesis.

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Absolute Amounts and Status of the Nrf2-Keap1-Cul3 Complex within Cells

Molecular and Cellular Biology ◽

10.1128/mcb.00389-16 ◽

2016 ◽

Vol 36 (24) ◽

pp. 3100-3112 ◽

Cited By ~ 35

Author(s):

Tatsuro Iso ◽

Takafumi Suzuki ◽

Liam Baird ◽

Masayuki Yamamoto

Keyword(s):

Transcription Factor ◽

Recombinant Protein ◽

Ubiquitin Ligase ◽

Stress Responses ◽

Quantitative Information ◽

Related Factor ◽

Electrophilic Stress ◽

Cullin 3 ◽

Electrophilic Agent ◽

Nrf2 Protein

The transcription factor Nrf2 (NF-E2-related-factor 2) is essential for the oxidative and electrophilic stress responses. Keap1 (Kelch-like-ECH-associated-protein 1), an adaptor for a cullin-3 (Cul3)-based ubiquitin ligase, regulates Nrf2 activity through proteasomal degradation, and acts as a sensor for oxidative and electrophilic stresses. The Keap1-Cul3 complex is a critical regulator of the cellular Nrf2 level, and yet quantitative information regarding their endogenous intracellular concentrations in homeostatic conditions and during stress responses is unknown. We analyzed the absolute amounts of the Nrf2, Keap1, and Cul3 proteins in five murine cell lines by comparison with serial dilutions of purified recombinant protein standards in combination with quantitative immunoblot analyses. In the basal state, the amount of Nrf2 was maintained at lower levels than those of Keap1 and Cul3 proteins, whereas the electrophilic agent diethylmaleate dramatically increased Nrf2 to a level greater than that of Keap1 and Cul3, resulting in the accumulation of Nrf2 in the nucleus. In contrast, Keap1 and Cul3 did not display any changes in their abundance, subcellular localization, or interaction in response to electrophilic stimuli. Our results demonstrate that the regulation of the Nrf2 protein level during stress responses is mediated by the activity but not the composition of the Nrf2-Keap1-Cul3 complex.

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Selective activation of STAT3 in human monocytes stimulated by G-CSF: implication in inhibition of LPS-induced TNF-α production

AJP Cell Physiology ◽

10.1152/ajpcell.00387.2003 ◽

2004 ◽

Vol 286 (6) ◽

pp. C1302-C1311 ◽

Cited By ~ 42

Author(s):

Saori Nishiki ◽

Fumihiko Hato ◽

Noriko Kamata ◽

Erina Sakamoto ◽

Taro Hasegawa ◽

...

Keyword(s):

Inhibitory Effect ◽

Janus Kinase ◽

Cytokine Signaling ◽

Human Monocytes ◽

Selective Activation ◽

Extracellular Signal ◽

Stat3 Phosphorylation ◽

Tnf Α ◽

Transcription 3

Lipopolysaccharide (LPS) induced tumor necrosis factor (TNF)-α production in human monocytes, which was dependent on activation of extracellular signal-regulated kinase (ERK), p38, c-Jun NH2-terminal kinase (JNK), and nuclear factor (NF)-κB. LPS-induced TNF-α production was inhibited by granulocyte colony-stimulating factor (G-CSF) and interleukin (IL)-10. G-CSF, like IL-10, exerted the inhibitory effect even when simultaneously added with LPS. Among the signaling pathways, signal transducer and activator of transcription 3 (STAT3) was selectively activated in monocytes stimulated by G-CSF or IL-10. G-CSF-mediated inhibition of LPS-induced TNF-α production as well as G-CSF-induced STAT3 phosphorylation and suppressor of cytokine signaling 3 mRNA expression were prevented by pretreatment of monocytes with AG-490, an inhibitor of Janus kinase 2. G-CSF did not affect LPS-induced activation of ERK, p38, JNK, and NF-κB, indicating that G-CSF affects the pathway downstream or independently of these signaling molecules. G-CSF-induced, but not IL-10-induced, STAT3 phosphorylation was attenuated in the presence of LPS. These findings suggest that G-CSF, like IL-10, inhibits LPS-induced TNF-α production in human monocytes through selective activation of STAT3, and the immunomodulation observed in vivo by G-CSF administration may be partly ascribed to the direct effect of G-CSF on monocyte functions.

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Angiopoietin-Like Growth Factor Involved in Leptin Signaling in the Hypothalamus

International Journal of Molecular Sciences ◽

10.3390/ijms22073443 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3443

Author(s):

Yunseon Jang ◽

Jun Young Heo ◽

Min Joung Lee ◽

Jiebo Zhu ◽

Changjun Seo ◽

...

Keyword(s):

Growth Factor ◽

Brain Regions ◽

Whole Body ◽

Signal Transducers ◽

Leptin Signaling ◽

Stat3 Phosphorylation ◽

Hypothalamic Regulation ◽

Induced Signal ◽

Body Energy ◽

Transcription 3

The hypothalamic regulation of appetite governs whole-body energy balance. Satiety is regulated by endocrine factors including leptin, and impaired leptin signaling is associated with obesity. Despite the anorectic effect of leptin through the regulation of the hypothalamic feeding circuit, a distinct downstream mediator of leptin signaling in neuron remains unclear. Angiopoietin-like growth factor (AGF) is a peripheral activator of energy expenditure and antagonizes obesity. However, the regulation of AGF expression in brain and localization to mediate anorectic signaling is unknown. Here, we demonstrated that AGF is expressed in proopiomelanocortin (POMC)-expressing neurons located in the arcuate nucleus (ARC) of the hypothalamus. Unlike other brain regions, hypothalamic AGF expression is stimulated by leptin-induced signal transducers and activators of transcription 3 (STAT3) phosphorylation. In addition, leptin treatment to hypothalamic N1 cells significantly enhanced the promoter activity of AGF. This induction was abolished by the pretreatment of ruxolitinib, a leptin signaling inhibitor. These results indicate that hypothalamic AGF expression is induced by leptin and colocalized to POMC neurons.

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Intranuclear Crosstalk between Extracellular Regulated Kinase1/2 and Signal Transducer and Activator of Transcription 3 Regulates JEG-3 Choriocarcinoma Cell Invasion and Proliferation

The Scientific World JOURNAL ◽

10.1155/2013/259845 ◽

2013 ◽

Vol 2013 ◽

pp. 1-10 ◽

Cited By ~ 10

Author(s):

Diana M. Morales-Prieto ◽

Stephanie Ospina-Prieto ◽

Wittaya Chaiwangyen ◽

Maja Weber ◽

Sebastian Hölters ◽

...

Keyword(s):

Dna Binding ◽

Binding Capacity ◽

Mapk Signaling ◽

Signaling Molecules ◽

Signal Transducer ◽

Stat3 Phosphorylation ◽

Functional Implications ◽

Choriocarcinoma Cells ◽

Cell Invasiveness ◽

Transcription 3

Invasiveness of trophoblast and choriocarcinoma cells is in part mediated via leukemia inhibitory factor- (LIF-) induced activation of signal transducer and activator of transcription 3 (STAT3). The regulation of STAT3 phosphorylation at its ser727 binding site, possible crosstalk with intracellular MAPK signaling, and their functional implications are the object of the present investigation. JEG-3 choriocarcinoma cells were cultured in presence/absence of LIF and the specific ERK1/2 inhibitor (U0126). Phosphorylation of signaling molecules (p-STAT3 (ser727 and tyr705) and p-ERK1/2 (thr 202/tyr 204)) was assessed per Western blot. Immunocytochemistry confirmed results, but also pinpointed the location of phosphorylated signaling molecules. STAT3 DNA-binding capacity was studied with a colorimetric ELISA-based assay. Cell viability and invasion capability were assessed by MTS and Matrigel assays. Our results demonstrate that LIF-induced phosphorylation of STAT3 (tyr705 and ser727) is significantly increased after blocking ERK1/2. STAT3 DNA-binding capacity and cell invasiveness are enhanced after LIF stimulation and ERK1/2 blockage. In contrast, proliferation is enhanced by LIF but reduced after ERK1/2 inhibition. The findings herein show that blocking ERK1/2 increases LIF-induced STAT3 phosphorylation and STAT3 DNA-binding capacity by an intranuclear crosstalk, which leads to enhanced invasiveness and reduced proliferation.

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Cycles of autoubiquitination and deubiquitination regulate the ERAD ubiquitin ligase Hrd1

eLife ◽

10.7554/elife.50903 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 8

Author(s):

Brian G Peterson ◽

Morgan L Glaser ◽

Tom A Rapoport ◽

Ryan D Baldridge

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Ubiquitin Ligase ◽

Ground State ◽

Inhibitory Effect ◽

The Other ◽

Misfolded Proteins ◽

Deubiquitinating Enzyme ◽

Transmembrane Segment

Misfolded proteins in the lumen of the endoplasmic reticulum (ER) are retrotranslocated into the cytosol and polyubiquitinated before being degraded by the proteasome. The multi-spanning ubiquitin ligase Hrd1 forms the retrotranslocation channel and associates with three other membrane proteins (Hrd3, Usa1, Der1) of poorly defined function. The Hrd1 channel is gated by autoubiquitination, but how Hrd1 escapes degradation by the proteasome and returns to its inactive ground state is unknown. Here, we show that autoubiquitination of Hrd1 is counteracted by Ubp1, a deubiquitinating enzyme that requires its N-terminal transmembrane segment for activity towards Hrd1. The Hrd1 partner Hrd3 serves as a brake for autoubiquitination, while Usa1 attenuates Ubp1’s deubiquitination activity through an inhibitory effect of its UBL domain. These results lead to a model in which the Hrd1 channel is regulated by cycles of autoubiquitination and deubiquitination, reactions that are modulated by the other components of the Hrd1 complex.

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Inhibition of O-GlcNAc Transferase Alters the Differentiation and Maturation Process of Human Monocyte Derived Dendritic Cells

Cells ◽

10.3390/cells10123312 ◽

2021 ◽

Vol 10 (12) ◽

pp. 3312

Author(s):

Matjaž Weiss ◽

Marko Anderluh ◽

Martina Gobec

Keyword(s):

Dendritic Cells ◽

Posttranslational Modification ◽

Human Monocyte ◽

T Cell Differentiation ◽

Maturation Process ◽

Hla Dr ◽

Glcnac Transferase ◽

And Function

The O-GlcNAcylation is a posttranslational modification of proteins regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase. These enzymes regulate the development, proliferation and function of cells, including the immune cells. Herein, we focused on the role of O-GlcNAcylation in human monocyte derived dendritic cells (moDCs). Our study suggests that inhibition of OGT modulates AKT and MEK/ERK pathways in moDCs. Changes were also observed in the expression levels of relevant surface markers, where reduced expression of CD80 and DC-SIGN, and increased expression of CD14, CD86 and HLA-DR occurred. We also noticed decreased IL-10 and increased IL-6 production, along with diminished endocytotic capacity of the cells, indicating that inhibition of O-GlcNAcylation hampers the transition of monocytes into immature DCs. Furthermore, the inhibition of OGT altered the maturation process of immature moDCs, since a CD14medDC-SIGNlowHLA-DRmedCD80lowCD86high profile was noticed when OGT inhibitor, OSMI-1, was present. To evaluate DCs ability to influence T cell differentiation and polarization, we co-cultured these cells. Surprisingly, the observed phenotypic changes of mature moDCs generated in the presence of OSMI-1 led to an increased proliferation of allogeneic T cells, while their polarization was not affected. Taken together, we confirm that shifting the O-GlcNAcylation status due to OGT inhibition alters the differentiation and function of moDCs in in vitro conditions.

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Increased Hypothalamic Signal Transducer and Activator of Transcription 3 Phosphorylation after Hindbrain Leptin Injection

Endocrinology ◽

10.1210/en.2009-0854 ◽

2010 ◽

Vol 151 (4) ◽

pp. 1509-1519 ◽

Cited By ~ 20

Author(s):

Marieke Ruiter ◽

Patricia Duffy ◽

Steven Simasko ◽

Robert C. Ritter

Keyword(s):

Body Weight ◽

Food Intake ◽

Fourth Ventricle ◽

Leptin Receptor ◽

Janus Kinase ◽

Signal Transducer ◽

Solitary Tract ◽

Stat3 Signaling ◽

Stat3 Phosphorylation ◽

Transcription 3

Reduction of food intake and body weight by leptin is attributed largely to its action in the hypothalamus. However, the signaling splice variant of the leptin receptor, LRb, also is expressed in the hindbrain, and leptin injections into the fourth cerebral ventricle or dorsal vagal complex are associated with reductions of feeding and body weight comparable to those induced by forebrain leptin administration. Although these observations suggest direct hindbrain action of leptin on feeding and body weight, the possibility that hindbrain leptin administration also activates the Janus kinase/signal transducer and activator of transcription 3 (STAT3) signaling in the hypothalamus has not been investigated. Confirming earlier work, we found that leptin produced comparable reductions of feeding and body weight when injected into the lateral ventricle or the fourth ventricle. We also found that lateral and fourth ventricle leptin injections produced comparable increases of STAT3 phosphorylation in both the hindbrain and the hypothalamus. Moreover, injection of 50 ng of leptin directly into the nucleus of the solitary tract also increased STAT3 phosphorylation in the hypothalamic arcuate and ventromedial nuclei. Increased hypothalamic STAT3 phosphorylation was not due to elevation of blood leptin concentrations and the pattern of STAT3 phosphorylation did not overlap distribution of the retrograde tracer, fluorogold, injected via the same cannula. Our observations indicate that even small leptin doses administered to the hindbrain can trigger leptin-related signaling in the forebrain, and raise the possibility that STAT3 phosphorylation in the hypothalamus may contribute to behavioral and metabolic changes observed after hindbrain leptin injections.

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Yu Ping Feng San Exert Anti-Angiogenesis Effects through the Inhibition of TSLP-STAT3 Signaling Pathways in Hepatocellular Carcinoma

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2019/1947156 ◽

2019 ◽

Vol 2019 ◽

pp. 1-16 ◽

Cited By ~ 2

Author(s):

Qin Yuan ◽

Fei Yao ◽

Liang Zhou ◽

Guoqiang Liang ◽

Xiudao Song ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

High Performance ◽

Inhibitory Effect ◽

Tube Formation ◽

Significant Inhibition ◽

Related Factor ◽

Related Factors ◽

Chemotherapy Drugs ◽

Stat3 Signaling ◽

Stat3 Signaling Pathway

Background. Clinically, Yu ping feng san (YPFS) has been extensively used as a medication for treating immune deficiency, and YPFS is combined with chemotherapy drugs to treat cancer, including hepatocellular carcinoma (HCC), lung cancer, and pancreatic cancer. Previous research has shown that YPFS has a therapeutic effect on HCC by improving the immunosuppressive state of the liver cancer microenvironment. The present study aimed to investigate the effect of YPFS on angiogenesis of HCC. Methods. High-performance liquid chromatography (HPLC) was used to certify the composition of YPFS. An orthotopic transplanted model of murine HCC was entrenched. Immunohistochemistry was used to observe the changes of the microvessel density (MVD). The MTT assay was used to detect the cell viability. ELISA was performed to analyze the expression of related factors. Western blot was used to analyze the protein expression. Tube formation assay was used to analyze the anti-angiogenic efficiency. Results. YPFS significantly reduced the tumor volume and weight, thus exerted the growth inhibitory effect. The level of MVD and VEGF was obviously decreased in YPFS-treated HCC-bearing mice, and the YPFS treatment also reduced the VEGF level in Hepa1-6 cells. Further study revealed that the expression of TSLP/TSLPR and p-STAT3/STAT3 was decreased by YPFS. The level of MVD and VEGF and the expression of TSLP/TSLPR and p-STAT3/STAT3 in tumor tissue and Hepa1-6 cells were suppressed by incubation with the anti-TSLP antibody, whereas treatment with the anti-TSLP antibody in YPFS-treated cells did not cause further significant inhibition compared with the cells treated only with YPFS. More importantly, YPFS inhibited proliferation, expression of p-STAT3/STAT3, and tube formation of HUVECs induced by TSLP. Conclusions. These results indicated that YPFS attenuated the activation of the TSLP-STAT3 signaling pathway by inhibiting the immune-related factor-TSLP, thereby inhibiting the formation of hepatic microvessels and exerting an anti-HCC effect.

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TFEB activates Nrf2 by repressing its E3 ubiquitin ligase DCAF11 and promoting phosphorylation of p62

Scientific Reports ◽

10.1038/s41598-019-50877-8 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 4

Author(s):

Jee-Yun Park ◽

Sunhyo Kim ◽

Hee Young Sohn ◽

Young Ho Koh ◽

Chulman Jo

Keyword(s):

Oxidative Stress ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Biological Response ◽

Related Factor ◽

Mrna Levels ◽

Nuclear Factor ◽

Glutathione S Transferase ◽

First Time ◽

Cellular Stressors

Abstract Transcriptional factor EB (TFEB) and nuclear factor E2-related factor 2 (Nrf2) play crucial roles in the biological response against cellular stressors; however, their relationship has not yet been investigated. Here, we constructed human neuroglioma cell lines stably expressing TFEB. The expression of Nrf2-response genes, including heme oxygenase (HO)-1, glutathione-s-transferase-mu1 (GSTM1), and p62, was induced in the cell line, independent of oxidative stress. Of note, the protein level of Nrf2 was significantly increased, and its ubiquitinated fraction was reduced in stable cells compared to that in the control cells. Among E3 ubiquitin ligases known to be involved in the ubiquitination of Nrf2, DDB1 and Cullin4 associated factor 11 (DCAF11) was down-regulated at both protein and mRNA levels in stable cells, indicating that the repression of DCAF11 by TFEB may be mainly involved in the stabilization of Nrf2. In addition, the level of phosphorylated p62 at S349 was highly increased in stable cells compared to that in control cells, which could allow it to interfere with the association of Keap1 and Nrf2, thus stabilizing Nrf2. We suggest for the first time that TFEB could activate Nrf2 by increasing its stability under conditions devoid of oxidative stress.

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STAT3 regulates NF-κB recruitment to the IL-12p40 promoter in dendritic cells

Blood ◽

10.1182/blood-2004-04-1309 ◽

2005 ◽

Vol 105 (2) ◽

pp. 689-696 ◽

Cited By ~ 108

Author(s):

Frank Hoentjen ◽

R. Balfour Sartor ◽

Michitaka Ozaki ◽

Christian Jobin

Keyword(s):

Gene Expression ◽

Dendritic Cells ◽

Molecular Mechanisms ◽

Intestinal Inflammation ◽

Nuclear Translocation ◽

Green Fluorescence Protein ◽

Interleukin 10 ◽

Mrna Accumulation ◽

Stat3 Phosphorylation ◽

Regulatory Effects

Abstract Interleukin-10-deficient (IL-10-/-) mice develop an IL-12-mediated intestinal inflammation in the absence of endogenous IL-10. The molecular mechanisms of the dysregulated IL-12 responses in IL-10-/- mice are poorly understood. In this study, we investigated the role of nuclear factor-κ B (NF-κB) and signal transducers and activators of transcription 3 (STAT3) in lipopolysaccharide (LPS)-induced IL-12p40 gene expression in bone marrow derived-dendritic cells (BMDCs) isolated from wild-type (WT) and IL-10-/- mice. We report higher IL-12p40 mRNA accumulation and protein secretion in LPS-stimulated BMDCs isolated from IL-10-/- compared with WT mice. LPS-induced NF-κB signaling is similar in IL-10-/- and WT BMDCs as measured by IκBα phosphorylation and degradation, RelA phosphorylation and nuclear translocation, and NF-κB transcriptional activity, with no down-regulatory effects of exogenous IL-10. Chromatin immunoprecipitation demonstrated enhanced NF-κB (cRel, RelA) binding to the IL-12p40 promoter in IL-10-/- but not WT BMDCs. Interestingly, LPS induced STAT3 phosphorylation in WT but not IL-10-/- BMDCs, a process blocked by IL-10 receptor blocking antibody. Adenoviral gene delivery of a constitutively active STAT3 but not control green fluorescence protein (GFP) virus blocked LPS-induced IL-12p40 gene expression and cRel recruitment to the IL-12p40 promoter. In conclusion, dysregulated LPS-induced IL-12p40 gene expression in IL-10-/- mice is due to enhanced NF-κB recruitment to the IL-12p40 promoter in the absence of activated STAT3.

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