Unspliced XBP1 Counteracts β-catenin to Inhibit Vascular Calcification

2021 ◽  
Author(s):  
Liu Yang ◽  
Rongbo Dai ◽  
Hao Wu ◽  
Zeyu Cai ◽  
Nan Xie ◽  
...  
Keyword(s):  
Vascular Calcification ◽  
Glycogen Synthase ◽  
Vascular Diseases ◽  
Proteasomal Degradation ◽  
Calcium Deposition ◽  
Alizarin Red ◽  
Protein Levels ◽  
Cardiovascular Morbidity And Mortality ◽  
Related Transcription Factor ◽  
Gsk 3Β

Background: Vascular calcification is a prevalent complication in chronic kidney disease and contributes to increased cardiovascular morbidity and mortality. XBP1 (X-box binding protein 1), existing as the unspliced (XBP1u) and spliced (XBP1s) forms, is a key component of the endoplasmic reticulum stress involved in vascular diseases. However, whether XBP1u participates in the development of vascular calcification remains unclear. Methods: We aim to investigate the role of XBP1u in vascular calcification.XBP1u protein levels were reduced in high phosphate (Pi)-induced calcified vascular smooth muscle cells (VSMCs), calcified aortas from mice with adenine diet-induced chronic renal failure (CRF) and calcified radial arteries from CRF patients. Results: Inhibition of XBP1u rather than XBP1s upregulated in the expression of the osteogenic markers runt-related transcription factor 2 (Runx2) and msh homeobox2 (Msx2), and exacerbated high Pi-induced VSMC calcification, as verified by calcium deposition and Alizarin red S staining. In contrast, XBP1u overexpression in high Pi-induced VSMCs significantly inhibited osteogenic differentiation and calcification. Consistently, SMC-specific XBP1 deficiency in mice markedly aggravated the adenine diet- and 5/6 nephrectomy-induced vascular calcification compared with that in the control littermates. Further interactome analysis revealed that XBP1u bound directly to β-catenin, a key regulator of vascular calcification, via aa 205-230 in its C-terminal degradation domain. XBP1u interacted with β-catenin to promote its ubiquitin-proteasomal degradation and thus inhibited β-catenin/T-cell factor (TCF)-mediated Runx2 and Msx2 transcription. Knockdown of β-catenin abolished the effect of XBP1u deficiency on VSMC calcification, suggesting a β-catenin-mediated mechanism. Moreover, the degradation of β-catenin promoted by XBP1u was independent of glycogen synthase kinase 3β (GSK-3β)-involved destruction complex. Conclusions: Our study identified XBP1u as a novel endogenous inhibitor of vascular calcification by counteracting β-catenin and promoting its ubiquitin-proteasomal degradation, which represents a new regulatory pathway of β-catenin and a promising target for vascular calcification treatment.

Targeting EZH2 Ameliorates the LPS-Inhibited PDLSC Osteogenesis via Wnt/β-Catenin Pathway

2021 ◽  
pp. 1-9
Author(s):  
Mosha Cheng ◽  
Qing Zhou
Keyword(s):  
Bone Morphogenetic Protein 2 ◽  
Periodontal Ligament Stem Cells ◽  
Alizarin Red ◽  
Protein Levels ◽  
Inflammatory Conditions ◽  
Morphogenetic Protein ◽  
Related Transcription Factor ◽  
Underlying Mechanisms ◽  
Factor Α ◽  
Osteoblast Maturation

As a histone methyltransferase, enhancer of zeste homolog 2 (EZH2), suppresses osteoblast maturation and is involved in inflammation. However, the role of EZH2 in human periodontal ligament stem cells (PDLSCs) under inflammation still needs to be further investigated. This study aimed to identify the underlying mechanisms and explore the function of EZH2 in PDLSC osteogenesis under inflammation. PDLSCs were treated with sh-EZH2, DZNep or DKK1 under inflammation. The alkaline phosphatase (ALP) activity, alizarin red staining, and osteogenesis-related protein levels were analyzed. Lipopolysaccharide (LPS)-induced inflammation restrained osteogenic differentiation. Under inflammation, the upregulation of EZH2 suppressed the expression of osteogenic markers, including osteocalcin, runt-related transcription factor 2, and bone morphogenetic protein-2, the activity of ALP, and the accumulation of mineralization through the Wnt/β-catenin pathway. EZH2 knockdown inhibited the levels of proinflammatory cytokines such as interleukin-6 and tumor necrosis factor-α. These results suggested that LPS-induced overexpression of EZH2 suppressed PDLSC osteogenesis under inflammatory conditions through the Wnt/β-catenin pathway. These findings give new insights into the physiological differentiation and pathological inflammation of PDLSC osteogenesis, and provide an underlying therapeutic target for periodontitis.

scholarly journals RTEF-1 Inhibits Vascular Smooth Muscle Cell Calcification through Regulating Wnt/β-Catenin Signaling Pathway

2021 ◽  
Author(s):  
Jingjing Cong ◽  
Bei Cheng ◽  
Jinyu Liu ◽  
Ping He
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Glycogen Synthase ◽  
Critical Role ◽  
Transcriptional Enhancer ◽  
Alizarin Red ◽  
Gsk 3Β ◽  
Enhancer Factor

AbstractVascular calcification (VC) is highly prevailing in cardiovascular disease, diabetes mellitus, and chronic kidney disease and, when present, is associated with cardiovascular events and mortality. The osteogenic differentiation of vascular smooth muscle cells (VSMCs) is regarded as the foundation for mediating VC. Related transcriptional enhancer factor (RTEF-1), also named as transcriptional enhanced associate domain (TEAD) 4 or transcriptional enhancer factor-3 (TEF-3), is a nuclear transcriptional factor with a potent effect on cardiovascular diseases, apart from its oncogenic role in the canonical Hippo pathway. However, the role and mechanism of RTEF-1 in VC, particularly in calcification of VSMCs, are poorly understood. Our results showed that RTEF-1 was reduced in calcified VSMCs. RTEF-1 significantly ameliorated β-glycerophosphate (β-GP)-induced VSMCs calcification, as detected by alizarin red staining and calcium content assay. Also, RTEF-1 reduced alkaline phosphatase (ALP) activity and decreased expressions of osteoblast markers such as Osteocalcin and Runt-related transcription factor-2 (Runx2), but increased expression of contractile protein, including SM α-actin (α-SMA). Additionally, RTEF-1 inhibited β-GP-activated Wnt/β-catenin pathway which plays a critical role in calcification and osteogenic differentiation of VSMCs. Specifically, RTEF-1 reduced the levels of Wnt3a, p-β-catenin (Ser675), glycogen synthase kinase-3β (GSK-3β), and p-GSK-3β (Ser9), but increased the levels of p-β-catenin (Ser33/37). Also, RTEF-1 increased the ratio of p-β-catenin (Ser33/37) to β-catenin proteins and decreased the ratio of p-GSK-3β (Ser9) to GSK-3β protein. LiCl, a Wnt/β-catenin signaling activator, was observed to reverse the protective effect of RTEF-1 overexpression on VSMCs calcification induced by β-GP. Accordingly, Dickkopf-1 (Dkk1), a Wnt antagonist, attenuated the role of RTEF-1 deficiency in β-GP-induced VSMCs calcification. Taken together, we concluded that RTEF-1 ameliorated β-GP-induced calcification and osteoblastic differentiation of VSMCs by inhibiting Wnt/β-catenin signaling pathway.

scholarly journals Inhibition of Gastrin-Releasing Peptide Attenuates Phosphate-Induced Vascular Calcification

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 737 ◽  
Author(s):  
Hyun-Joo Park ◽  
Yeon Kim ◽  
Mi-Kyoung Kim ◽  
Jae Joon Hwang ◽  
Hyung Joon Kim ◽  
...  
Keyword(s):  
Vascular Calcification ◽  
Vascular System ◽  
Ex Vivo ◽  
Calcium Deposition ◽  
Gastrin Releasing Peptide ◽  
Cardiovascular Morbidity And Mortality ◽  
Potential Strategy ◽  
Grp Receptor

Vascular calcification is the pathological deposition of calcium/phosphate in the vascular system and is closely associated with cardiovascular morbidity and mortality. Here, we investigated the role of gastrin-releasing peptide (GRP) in phosphate-induced vascular calcification and its potential regulatory mechanism. We found that the silencing of GRP gene and treatment with the GRP receptor antagonist, RC-3095, attenuated the inorganic phosphate-induced calcification of vascular smooth muscle cells (VSMCs). This attenuation was caused by inhibiting phenotype change, apoptosis and matrix vesicle release in VSMCs. Moreover, the treatment with RC-3095 effectively ameliorated phosphate-induced calcium deposition in rat aortas ex vivo and aortas of chronic kidney disease in mice in vivo. Therefore, the regulation of the GRP-GRP receptor axis may be a potential strategy for treatment of diseases associated with excessive vascular calcification.

scholarly journals Altered Wnt signalling in the teenage suicide brain: focus on glycogen synthase kinase-3β and β-catenin

2013 ◽  
Vol 16 (5) ◽  
pp. 945-955 ◽  
Author(s):  
Xinguo Ren ◽  
Hooriyah S. Rizavi ◽  
Mansoor A. Khan ◽  
Yogesh Dwivedi ◽  
Ghanshyam N. Pandey
Keyword(s):  
Protein Expression ◽  
Glycogen Synthase ◽  
Wnt Signalling ◽  
Signalling Pathway ◽  
Glycogen Synthase Kinase ◽  
Mrna Levels ◽  
Protein Levels ◽  
Wnt Signalling Pathway ◽  
Gsk 3Β ◽  
Teenage Suicide

Abstract Glycogen synthase kinase (GSK)-3β and β-catenin are important components of the Wnt signalling pathway, which is involved in numerous physiological functions such as cognition, brain development and cell survival. Their abnormalities have been implicated in mood disorders and schizophrenia. Teenage suicide is a major public health concern; however, very little is known about its neurobiology. In order to examine if abnormalities of GSK-3β and β-catenin are associated with teenage suicide, we determined the gene and protein expression of GSK-3β and β-catenin in the prefrontal cortex (PFC) and hippocampus obtained from 24 teenage suicide victims and 24 normal control subjects. Protein expression was determined using Western blot with specific antibodies and gene expression (mRNA levels) was determined using the real-time polymerase chain reaction method. No significant change was observed in the GSK-3β protein levels either in the PFC or hippocampus of suicide victims compared to controls. However, protein levels of pGSK-3β-ser9 were significantly decreased in the PFC and hippocampus of suicide victims compared to normal controls. We also found that GSK-3β mRNA levels were significantly decreased in the PFC but not in the hippocampus of teenage suicide victims compared to controls. Mean protein and mRNA levels of β-catenin were significantly decreased in both the PFC and hippocampus of teenage suicide group compared to controls. The observation that there is a decrease in β-catenin and pGSK-3β-ser9 in the PFC and hippocampus of teenage suicide victims does indicate a disturbance in the Wnt signalling pathway in teenage suicide.

scholarly journals Melatonin attenuates vascular calcification by activating mitochondrial fusion and mitophagy via an AMPK/OPA1 signaling pathway

2019 ◽  
Author(s):  
chenweiren chen ◽  
jia qi yang ◽  
fang liu ◽  
xue qin shen ◽  
yuan sha
Keyword(s):  
Vascular Calcification ◽  
Mitochondrial Fusion ◽  
Calcium Deposition ◽  
Protective Effects ◽  
Melatonin Treatment ◽  
Western Blots ◽  
Alizarin Red ◽  
Mitofusin 2 ◽  
Alp Activity ◽  
Compound C

Abstract Background: Mitochondrial fusion/mitophagy play a role in cardiovascular calcification. Melatonin has been shown to protect against cardiovascular disease. This study sought to explore whether melatonin attenuates vascular calcification by regulating mitochondrial fusion/mitophagy via an AMP activated protein kinase/ Optic atrophy 1 (AMPK/OPA1) signaling pathway.Methods: The effects of melatonin on vascular calcification were investigated in vascular smooth muscle cells (VSMCs). Calcium deposits were visualised by Alizarin red staining. Calcium content and alkaline phosphatase (ALP) activity were used to evaluate osteogenic differentiation. Western blots were used to measure expression of runt-related transcription factor 2 (Runx2), mitofusin 2 (Mfn2), mito-light chain 3 II (LC3II) and cleaved caspase3. Results: Melatonin markedly reduced calcium deposition and ALP activity. Runx2 and cleaved caspase3 were found to be down-regulated and Mfn2 or mito-LC3II was found to be enhanced in response to melatonin, together with a decrease in mitochondrial superoxide levels. Melatonin also maintained mitochondrial function and promoted mitochondrial fusion/mitophagy via OPA1 pathway. But OPA1 deletion abolished the protective effects of melatonin on VSMC calcification. Melatonin treatment significantly increased the p-AMPK and OPA1 protein expression. Treatment with compound C ablated the benefit observed with melatonin treatment. Conclusions: Melatonin protects VSMC against calcification by promoting mitochondrial fusion/mitophagy via AMPK/OPA1 pathway.

MO441CALPROTECTIN IS A NOVEL CONTRIBUTING FACTOR IN VASCULAR CALCIFICATION AND A PREDICTOR OF CARDIOVASCULAR OUTCOME IN CKD PATIENTS*

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Ana Amaya Garrido ◽  
José M Valdivielso ◽  
Stanislas Faguer ◽  
Arnaud Del Bello ◽  
Benedicte Buffin-Meyer ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Calcification ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Calcium Deposition ◽  
Alizarin Red ◽  
Serum Proteome ◽  
Ckd Stage

Abstract Background and Aims Vascular calcification, leading to aortic stiffening and heart failure, is decisive risk factor for cardiovascular (CV) mortality in patients with chronic kidney disease (CKD). Promoted by bone mineral disorder and systemic inflammation in CKD patients, vascular calcification is a complex mechanism involving osteochondrogenic differentiation of vascular smooth muscle cells (VSMCs) and abnormal deposition of minerals in the vascular wall. Despite intensive research efforts in recent years, available treatments have limited effect and none of them prevent or reverse vascular calcification. The aim of this study was to analyse the serum proteome of CKD stage 3-4 patients in order to unravel new molecular changes associated to CV morbid-mortality and to decipher the role of novel candidates on vascular calcification to provide potential new therapeutic agents. Method In this study we used serum samples from two independent cohorts: 112 CKD stage 3-4 patients with a 4 years follow-up for CV events and 222 CKD stage 5 patients exhibiting a broad range of calcification degree determined by histological quantification in the epigastric and/or iliac artery. Serum proteome analysis was performed using tandem mass-spectrometry in a subcohort of 66 CKD3-4 patients and validation of protein candidates was performed using ELISA in the two full cohorts. Human primary vascular smooth muscle cells and mouse aortic rings were used for calcification assays. Calcium content was quantified using QuantiChrom calcium assay kit and calcium deposition was visualized by Alizarin Red and Von Kossa staining. Results Among 443 proteins detected in the serum of CKD3-4 patients, 134 displayed significant modified abundance in patients with CV events (n=32) compared to patients without (n=34). One of the most prominent changes was increased level of calprotectin (up to 8.6 fold, P<.0001). Using ELISA, we validated that higher serum calprotectin levels were strongly associated with higher probability of developing CV complications and increased mortality in CKD stage 3-4 patients (Figure A). Moreover, we showed that higher serum calprotectin was associated with increased vascular calcification levels in CKD stage 5 patients (Figure B). In vitro, calprotectin promoted calcification of human VSMCs (p<0.0001) (Figures C-D) and in mouse aortic rings (p<0.0001) (Figure E-F). Interestingly, these effects were significantly attenuated by paquinimod, a calprotectin inhibitor (Figures C-F). Conclusion Circulating calprotectin is a novel predictor of CV outcome and mortality in CKD patients. Calprotectin also shows calcification-inducing properties and its blockade by paquinimod alleviates its effects. Future experiments will consist in deciphering the signalling pathways involved in the regulation of calcification by calprotectin and evaluating in vivo the therapeutic potential of paquinimod on the development of medial vascular calcification lesions associated with CKD.

scholarly journals Glycogen synthase kinase-3-mediated phosphorylation of serine 73 targets sterol response element binding protein-1c (SREBP-1c) for proteasomal degradation

2016 ◽  
Vol 36 (1) ◽  
Author(s):  
Qingming Dong ◽  
Francesco Giorgianni ◽  
Sarka Beranova-Giorgianni ◽  
Xiong Deng ◽  
Robert N. O'Meally ◽  
...  
Keyword(s):  
Functional Role ◽  
Glycogen Synthase ◽  
Proteasomal Degradation ◽  
Glycogen Synthase Kinase ◽  
Glycogen Synthase Kinase 3 ◽  
Response Element ◽  
Element Binding Protein ◽  
Gsk 3Β

We have identified Serine 73 as a novel GSK-3β site on SREBP-1c that alters its affinity for SCAP, and proteasomal degradation. Phosphorylation of Serine 73 by GSK-3β during starvation (insulin-depleted stat) may lead to lower levels of SREBP-1c; conversely, de-phosphorylation of this site may be involved in stabilizing SREBP-1c by insulin (by blocking GSK-3β action). A functional role of this site needs to be corroborated in vivo.

scholarly journals Artesunate attenuates traumatic brain injury-induced impairments in rats

2020 ◽  
Vol 11 (1) ◽  
pp. 309-318
Author(s):  
Zhike Zhou ◽  
Jun Hou ◽  
Qinghua Li
Keyword(s):  
Rat Model ◽  
Brain Injuries ◽  
Glycogen Synthase ◽  
Combined Treatment ◽  
Potential Candidate ◽  
Behavioral Experiments ◽  
Protective Function ◽  
Protein Levels ◽  
Gsk 3Β ◽  
The Impact

AbstractBackgroundBlood–brain barrier (BBB) dysfunction and neuroinflammation induced by traumatic brain injuries (TBIs) cause a succession of secondary brain damage events and finally lead to a massive and progressive cerebral neuronal destruction. Artesunate, a semisynthetic artemisinin derivative, is a potential candidate for the management of cerebral damage induced by TBI due to its protective function to BBB and cerebral neurons.MethodsTo demonstrate the effect of artesunate to TBI-induced BBB dysfunction and neural damage, TBI rat model was constructed by cortical impact injury. Behavioral experiments were used to estimate the impact of the combined treatment on rats. Western blotting was performed to demonstrate the protein levels in the brain tissues of rats. Quantitative real-time PCRs were utilized to investigate the alteration in the expression of various RNA levels. The chemokine levels were estimated by ELISA.ResultsArtesunate treatment attenuated the impact caused by TBI on rat brain and improved the long-term neurological recover. Artesunate treatment protected the integrity of BBB and inhibited neuroinflammation. Artesunate treatment promoted the phosphorylation of Akt and inhibited the phosphorylation of glycogen synthase kinase (GSK)-3β in TBI rat model.ConclusionArtesunate protected rats from TBI-induced impairments of BBB and improved longer-term neurological outcomes.

scholarly journals SCFFBXO17 E3 ligase modulates inflammation by regulating proteasomal degradation of glycogen synthase kinase-3β in lung epithelia

2017 ◽  
Vol 292 (18) ◽  
pp. 7452-7461 ◽  
Author(s):  
Tomeka Suber ◽  
Jianxin Wei ◽  
Anastasia M. Jacko ◽  
Ina Nikolli ◽  
Yutong Zhao ◽  
...  
Keyword(s):  
Glycogen Synthase ◽  
Proteasomal Degradation ◽  
Lung Epithelial Cells ◽  
Glycogen Synthase Kinase ◽  
Acceptor Site ◽  
Glycogen Synthase Kinase 3Β ◽  
Protein Subunit ◽  
Protein Levels ◽  
Ubiquitin Ligase Complex ◽  
F Box Protein

Glycogen synthase kinase-3β (GSK3β) has diverse biological roles including effects on cellular differentiation, migration, and inflammation. GSK3β phosphorylates proteins to generate phosphodegrons necessary for recognition by Skp1/Cullin-1/F-box (SCF) E3 ubiquitin ligases leading to subsequent proteasomal degradation of these substrates. However, little is known regarding how GSK3β protein stability itself is regulated and how its stability may influence inflammation. Here we show that GSK3β is degraded by the ubiquitin-proteasome pathway in murine lung epithelial cells through lysine 183 as an acceptor site for K48 polyubiquitination. We have identified FBXO17 as an F-box protein subunit that recognizes and mediates GSK3β polyubiquitination. Both endogenous and ectopically expressed FBXO17 associate with GSK3β, and its overexpression leads to decreased protein levels of GSK3β. Silencing FBXO17 gene expression increased the half-life of GSK3β in cells. Furthermore, overexpression of FBXO17 inhibits agonist-induced release of keratinocyte-derived cytokine (KC) and interleukin-6 (IL-6) production by cells. Thus, the SCFFBXO17 E3 ubiquitin ligase complex negatively regulates inflammation by targeting GSK3β in lung epithelia.

scholarly journals Glycogen Synthase Kinase 3β Phosphorylates p21WAF1/CIP1 for Proteasomal Degradation after UV Irradiation

2007 ◽  
Vol 27 (8) ◽  
pp. 3187-3198 ◽  
Author(s):  
Ji Young Lee ◽  
Su Jin Yu ◽  
Yun Gyu Park ◽  
Joon Kim ◽  
Jeongwon Sohn
Keyword(s):  
Signaling Pathway ◽  
Uv Irradiation ◽  
Glycogen Synthase ◽  
Proteasome Inhibitors ◽  
Proteasomal Degradation ◽  
Glycogen Synthase Kinase ◽  
Target Sequence ◽  
Glycogen Synthase Kinase 3Β ◽  
P21 Protein ◽  
Gsk 3Β

ABSTRACT UV irradiation has been reported to induce p21WAF1/CIP1 protein degradation through a ubiquitin-proteasome pathway, but the underlying biochemical mechanism remains to be elucidated. Here, we show that ser-114 phosphorylation of p21 protein by glycogen synthase kinase 3β (GSK-3β) is required for its degradation in response to UV irradiation and that GSK-3β activation is a downstream event in the ATR signaling pathway triggered by UV. UV transiently increased GSK-3β activity, and this increase could be blocked by caffeine or by ATR small interfering RNA, indicating ATR-dependent activation of GSK-3β. ser-114, located within the putative GSK-3β target sequence, was phosphorylated by GSK-3β upon UV exposure. The nonphosphorylatable S114A mutant of p21 was protected from UV-induced destabilization. Degradation of p21 protein by UV irradiation was independent of p53 status and prevented by proteasome inhibitors. In contrast to the previous report, the proteasomal degradation of p21 appeared to be ubiquitination independent. These data show that GSK-3β is activated by UV irradiation through the ATR signaling pathway and phosphorylates p21 at ser-114 for its degradation by the proteasome. To our knowledge, this is the first demonstration of GSK-3β as the missing link between UV-induced ATR activation and p21 degradation.

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