scholarly journals The mTORC2 Regulator Homer1 Modulates Protein Levels and Sub-Cellular Localization of the CaSR in Osteoblast-Lineage Cells

2021 ◽  
Vol 22 (12) ◽  
pp. 6509
Author(s):  
Mark S. Rybchyn ◽  
Tara Clare Brennan-Speranza ◽  
David Mor ◽  
Zhiqiang Cheng ◽  
Wenhan Chang ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Bone Cells ◽  
Cell Protein ◽  
Human Osteoblasts ◽  
Beneficial Effects ◽  
Protein Levels ◽  
Calcium Sensing ◽  
Mtorc1 Activation ◽  
Osteoblast Lineage

We recently found that, in human osteoblasts, Homer1 complexes to Calcium-sensing receptor (CaSR) and mediates AKT initiation via mechanistic target of rapamycin complex (mTOR) complex 2 (mTORC2) leading to beneficial effects in osteoblasts including β-catenin stabilization and mTOR complex 1 (mTORC1) activation. Herein we further investigated the relationship between Homer1 and CaSR and demonstrate a link between the protein levels of CaSR and Homer1 in human osteoblasts in primary culture. Thus, when siRNA was used to suppress the CaSR, we observed upregulated Homer1 levels, and when siRNA was used to suppress Homer1 we observed downregulated CaSR protein levels using immunofluorescence staining of cultured osteoblasts as well as Western blot analyses of cell protein extracts. This finding was confirmed in vivo as the bone cells from osteoblast specific CaSR−/− mice showed increased Homer1 expression compared to wild-type (wt). CaSR and Homer1 protein were both expressed in osteocytes embedded in the long bones of wt mice, and immunofluorescent studies of these cells revealed that Homer1 protein sub-cellular localization was markedly altered in the osteocytes of CaSR−/− mice compared to wt. The study identifies additional roles for Homer1 in the control of the protein level and subcellular localization of CaSR in cells of the osteoblast lineage, in addition to its established role of mTORC2 activation downstream of the receptor.

scholarly journals The mTORC2 Regulator homer1 Modulates Protein Levels and Sub-Cellular Localization of the CaSR in Human Osteoblasts.

2021 ◽  
Author(s):  
Mark S Rybchyn ◽  
Tara C Brennan-Speranza ◽  
David Mor ◽  
Zhiqiang Cheng ◽  
Wenhan Chang ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Bone Cells ◽  
Cell Protein ◽  
Osteosarcoma Cell ◽  
Wild Type ◽  
Human Osteoblasts ◽  
Beneficial Effects ◽  
Protein Levels ◽  
Mtorc1 Activation

We recently found that in human osteoblasts Homer1 complexes to CaSR and mediates AKT initiation via mTORC2 leading to beneficial effects in osteoblasts including -catenin stabilization and mTORC1 activation (doi: 10.1074/jbc.RA118.006587). Herein we further investigated the relationship between Homer1 and CaSR and demonstrate a link between the protein levels of CaSR and Homer1 in human osteoblasts in primary culture. Thus, when siRNA was used to suppress the CaSR, we observed upregulated Homer1 levels and when siRNA was used to suppress Homer1 we observed downregulated CaSR protein levels using immunofluorescence staining of cultured osteoblasts as well as western blot analyses of cell protein extracts. This finding was confirmed in vivo as the bone cells from osteoblast specific CaSR(-/-) mice showed increased Homer1 expression compared to wild-type. Furthermore, when the commonly used osteosarcoma cell lines MG63 and SAOS-2 were compared to primary osteoblasts, higher levels of Homer1 protein were associated with increased protein levels of the CaSR as well as mTOR and Rictor. CaSR and Homer1 protein were both expressed in osteocytes embedded in the long bones of wild-type mice, and immunofluorescent studies of these cells revealed that Homer1 protein sub-cellular localization was markedly altered in the osteocytes of CaSR(-/-) mice compared to wt. The study identifies additional roles for Homer1 in the control of the protein level and subcellular localization of CaSR in cells of the osteoblast lineage, in addition to its established role of mTORC2 activation downstream of the receptor.

scholarly journals Honokiol Alleviates Methionine-Choline Deficient Diet-Induced Hepatic Steatosis and Oxidative Stress in C57BL/6 Mice by Regulating CFLAR-JNK Pathway

2020 ◽  
Vol 2020 ◽  
pp. 1-14
Author(s):  
Ting Zhai ◽  
Wei Xu ◽  
Yayun Liu ◽  
Kun Qian ◽  
Yanling Xiong ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Insulin Resistance ◽  
Regulatory Gene ◽  
Deficient Diet ◽  
Jnk Pathway ◽  
Beneficial Effects ◽  
Protein Levels ◽  
And Oxidative Stress

Background. Honokiol (HNK) has been reported to possess various beneficial effects in the context of metabolic disorders, including fatty liver, insulin resistance, and oxidative stress which are closely related to nonalcoholic steatohepatitis (NASH), however with no particular reference to CFLAR or JNK. Methods. C57BL/6 mice were fed methionine-choline-deficient (MCD) diet and administered simultaneously with HNK (10 and 20 mg/kg once a day, ig) for 6 weeks, and NCTC1469 cells were pretreated, respectively, by oleic acid (OA, 0.5 mmol/L) plus palmitic acid (PA, 0.25 mmol/L) for 24 h, and adenovirus-down Cflar for 24 h, then exposed to HNK (10 and 20 μmol/L) for 24 h. Commercial kits, H&E, MT, ORO staining, RT-qPCR, and Western blotting were used to detect the biomarkers, hepatic histological changes, and the expression of key genes involved in NASH. Results. The in vivo results showed that HNK suppressed the phosphorylation of JNK (pJNK) by activating CFLAR; enhanced the mRNA expression of lipid metabolism-related genes Acox, Cpt1α, Fabp5, Gpat, Mttp, Pparα, and Scd-1; and decreased the levels of hepatic TG, TC, and MDA, as well as the levels of serum ALT and AST. Additionally, HNK enhanced the protein expression of oxidative stress-related key regulatory gene NRF2 and the activities of antioxidases HO-1, CAT, and GSH-Px and decreased the protein levels of prooxidases CYP4A and CYP2E1. The in vivo effects of HNK on the expression of CLFAR, pJNK, and NRF2 were proved by the in vitro experiments. Moreover, HNK promoted the phosphorylation of IRS1 (pIRS1) in both tested cells and increased the uptake of fluorescent glucose 2-NBDG in OA- and PA-pretreated cells. Conclusions. HNK ameliorated NASH mainly by activating the CFLAR-JNK pathway, which not only alleviated fat deposition by promoting the efflux and β-oxidation of fatty acids in the liver but also attenuated hepatic oxidative damage and insulin resistance by upregulating the expression of NRF2 and pIRS1.

scholarly journals CHIP promotes Runx2 degradation and negatively regulates osteoblast differentiation

2008 ◽  
Vol 181 (6) ◽  
pp. 959-972 ◽  
Author(s):  
Xueni Li ◽  
Mei Huang ◽  
Huiling Zheng ◽  
Yinyin Wang ◽  
Fangli Ren ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Osteoblast Differentiation ◽  
Degradation Mechanism ◽  
Interacting Protein ◽  
E3 Ligases ◽  
Protein Levels ◽  
C Terminus ◽  
Osteoblast Lineage

Runx2, an essential transactivator for osteoblast differentiation, is tightly regulated at both the transcriptional and posttranslational levels. In this paper, we report that CHIP (C terminus of Hsc70-interacting protein)/STUB1 regulates Runx2 protein stability via a ubiquitination-degradation mechanism. CHIP interacts with Runx2 in vitro and in vivo. In the presence of increased Runx2 protein levels, CHIP expression decreases, whereas the expression of other E3 ligases involved in Runx2 degradation, such as Smurf1 or WWP1, remains constant or increases during osteoblast differentiation. Depletion of CHIP results in the stabilization of Runx2, enhances Runx2-mediated transcriptional activation, and promotes osteoblast differentiation in primary calvarial cells. In contrast, CHIP overexpression in preosteoblasts causes Runx2 degradation, inhibits osteoblast differentiation, and instead enhances adipogenesis. Our data suggest that negative regulation of the Runx2 protein by CHIP is critical in the commitment of precursor cells to differentiate into the osteoblast lineage.

scholarly journals A systematic dissection of human primary osteoblasts in vivo at single-cell resolution

2020 ◽  
Author(s):  
Yun Gong ◽  
Junxiao Yang ◽  
Xiaohua Li ◽  
Cui Zhou ◽  
Yu Chen ◽  
...  
Keyword(s):  
Bone Formation ◽  
Single Cell ◽  
Bone Cells ◽  
Expression Patterns ◽  
Cellular Heterogeneity ◽  
Human Osteoblasts ◽  
Primary Osteoblasts ◽  
Human Primary Osteoblasts

AbstractOsteoblasts are multifunctional bone cells, which play essential roles in bone formation, angiogenesis regulation, as well as maintenance of hematopoiesis. Although both in vivo and in vitro studies on mice have identified several potential osteoblast subtypes based on their different transition stages or biological responses to external stimuli, the categorization of primary osteoblast subtypes in vivo in humans has not yet been achieved. Here, we used single-cell RNA sequencing (scRNA-seq) to perform a systematic cellular taxonomy dissection of freshly isolated human osteoblasts. Based on the gene expression patterns and cell lineage reconstruction, we identified three distinct cell clusters including preosteoblasts, mature osteoblasts, and an undetermined rare osteoblast subpopulation. This novel subtype was mainly characterized by the nuclear receptor subfamily 4 group A member 1 and 2 (NR4A1 and NR4A2), and its existence was confirmed by immunofluorescence staining. Trajectory inference analysis suggested that the undetermined cluster, together with the preosteoblasts, are involved in the regulation of osteoblastogenesis and also give rise to mature osteoblasts. Investigation of the biological processes and signaling pathways enriched in each subpopulation revealed that in addition to bone formation, preosteoblasts and undetermined osteoblasts may also regulate both angiogenesis and hemopoiesis. Finally, we demonstrated that there are systematic differences between the transcriptional profiles of human osteoblasts in vivo and mouse osteoblasts both in vivo and in vitro, highlighting the necessity for studying bone physiological processes in humans rather than solely relying on mouse models. Our findings provide novel insights into the cellular heterogeneity and potential biological functions of human primary osteoblasts at the single-cell level, which is an important and necessary step to further dissect the biological roles of osteoblasts in bone metabolism under various (patho-) physiological conditions.

scholarly journals Beneficial Effects of Vitamins K and D3 on Redox Balance of Human Osteoblasts Cultured with Hydroxyapatite-Based Biomaterials

Cells ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 325 ◽  
Author(s):  
Ewa Ambrożewicz ◽  
Marta Muszyńska ◽  
Grażyna Tokajuk ◽  
Grzegorz Grynkiewicz ◽  
Neven Žarković ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Bone Cells ◽  
Antioxidant Properties ◽  
Redox Balance ◽  
The Body ◽  
Human Osteoblasts ◽  
Redox Imbalance ◽  
Beneficial Effects ◽  
Dna Biosynthesis ◽  
Bone Damage

Hydroxyapatite-based biomaterials are commonly used in surgery to repair bone damage. However, the introduction of biomaterials into the body can cause metabolic alterations, including redox imbalance. Because vitamins D3 and K (K1, MK-4, MK-7) have pronounced osteoinductive, anti-inflammatory, and antioxidant properties, it is suggested that they may reduce the adverse effects of biomaterials. The aim of this study was to investigate the effects of vitamins D3 and K, used alone and in combination, on the redox metabolism of human osteoblasts (hFOB 1.19 cell line) cultured in the presence of hydroxyapatite-based biomaterials (Maxgraft, Cerabone, Apatos, and Gen-Os). Culturing of the osteoblasts in the presence of hydroxyapatite-based biomaterials resulted in oxidative stress manifested by increased production of reactive oxygen species and decrease of glutathione level and glutathione peroxidase activity. Such redox imbalance leads to lipid peroxidation manifested by an increase of 4-hydroxynonenal level, which is known to influence the growth of bone cells. Vitamins D3 and K were shown to help maintain redox balance and prevent lipid peroxidation in osteoblasts cultured with hydroxyapatite-based biomaterials. The strongest effect was observed for the combination of vitamin D3 and MK-7. Moreover, vitamins promoted growth of the osteoblasts, manifested by increased DNA biosynthesis. Therefore, it is suggested that the use of vitamins D3 and K may protect redox balance and support the growth of osteoblasts affected by hydroxyapatite-based biomaterials.

scholarly journals Investigation of the curative effects of palm vitamin E tocotrienols on autoimmune arthritis disease in vivo

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Zaida Zainal ◽  
Afiqah Abdul Rahim ◽  
Ammu Kutty Radhakrishnan ◽  
Sui Kiat Chang ◽  
Huzwah Khaza’ai
Keyword(s):  
Vitamin E ◽  
Rat Model ◽  
Bone Erosion ◽  
Cartilage Destruction ◽  
Intradermal Injection ◽  
Dark Agouti ◽  
Therapeutic Effects ◽  
Beneficial Effects ◽  
Protein Levels

AbstractThe tocotrienol-rich fraction (TRF) from palm oil contains vitamin E, which possesses potent antioxidant and anti-inflammatory activities. Rheumatoid arthritis (RA) is a chronic joint inflammatory disease characterised by severe joint pain, cartilage destruction, and bone erosion owing to the effects of various pro-inflammatory mediators and cytokines. Here, we investigated the therapeutic effects of TRF in a rat model of collagen-induced arthritis (CIA). Arthritis was induced by a single intradermal injection of collagen type II in Dark Agouti (DA) rats. Rats were then treated with or without TRF by oral gavage from day 28 after the first collagen injection. Arthritic rats supplemented with TRF showed decreased articular index scores, ankle circumferences, paw volumes, and radiographic scores when compared with untreated rats. The untreated arthritic rats showed higher plasma C-reactive protein levels (p < 0.05) and production of pro-inflammatory cytokines than arthritic rats fed TRF. Moreover, there was a marked reduction in the severity of histopathological changes observed in arthritic rats treated with TRF compared with that in untreated arthritic rats. Overall, the results show that TRF had beneficial effects in this rat model of RA.

scholarly journals (–)-Epicatechin Suppresses Angiotensin II-induced Cardiac Hypertrophy via the Activation of the SP1/SIRT1 Signaling Pathway

2017 ◽  
Vol 41 (5) ◽  
pp. 2004-2015 ◽  
Author(s):  
Zeng-xiang Dong ◽  
Lin Wan ◽  
Ren-jun Wang ◽  
Yuan-qi Shi ◽  
Guang-zhong Liu ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Cardiac Hypertrophy ◽  
Signaling Pathway ◽  
Cardiomyocyte Hypertrophy ◽  
Ang Ii ◽  
Beneficial Effects ◽  
Protein Levels ◽  
Qrt Pcr

Background/Aims: Flavonol (–)-epicatechin (EPI) is present in high amounts in cocoa and tea products, and has been shown to exert beneficial effects on the cardiovascular system. However, the precise mechanism of EPI on cardiomyocyte hypertrophy has not yet been determined. In this study, we examined whether EPI could inhibit cardiac hypertrophy. Methods: We utilised cultured neonatal mouse cardiomyocytes and mice for immunofluorescence, immunochemistry, qRT-PCR, and western blot analyses. Results: 1µM EPI significantly inhibited 1µM angiotensin II (Ang II)-induced increase of cardiomyocyte size, as well as the mRNA and protein levels of ANP, BNP and β-MHC in vitro. The effects of EPI were accompanied with an up-regulation of SP1 and SIRT1, and were abolished by SP1 inhibition. Up-regulation of SP1 could block Ang II-induced increase in cardiomyocyte size, as well as the mRNA and protein levels of ANP, BNP and β-MHC, and increase the protein levels of SIRT1 in vitro. Moreover, 1 mg/kg body weight/day EPI significantly inhibited mouse cardiac hypertrophy induced by Ang II, which could be eliminated by SP1 inhibition in vivo. Conclusion: Our data indicated that EPI inhibited AngII-induced cardiac hypertrophy by activating the SP1/SIRT1 signaling pathway.

scholarly journals Sodium valproate increases activity of the sirtuin pathway resulting in beneficial effects for spinocerebellar ataxia-3 in vivo

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Maxinne Watchon ◽  
Luan Luu ◽  
Katherine J. Robinson ◽  
Kristy C. Yuan ◽  
Alana De Luca ◽  
...  
Keyword(s):  
Spinocerebellar Ataxia ◽  
Sodium Valproate ◽  
Hek293 Cells ◽  
Spinocerebellar Ataxia Type ◽  
Transgenic Zebrafish ◽  
Zebrafish Model ◽  
Spinocerebellar Ataxia Type 3 ◽  
Beneficial Effects ◽  
Protein Levels

Abstract Machado-Joseph disease (MJD, also known as spinocerebellar ataxia type 3) is a fatal neurodegenerative disease that impairs control and coordination of movement. Here we tested whether treatment with the histone deacetylase inhibitor sodium valproate (valproate) prevented a movement phenotype that develops in larvae of a transgenic zebrafish model of the disease. We found that treatment with valproate improved the swimming of the MJD zebrafish, affected levels of acetylated histones 3 and 4, but also increased expression of polyglutamine expanded human ataxin-3. Proteomic analysis of protein lysates generated from the treated and untreated MJD zebrafish also predicted that valproate treatment had activated the sirtuin longevity signaling pathway and this was confirmed by findings of increased SIRT1 protein levels and sirtuin activity in valproate treated MJD zebrafish and HEK293 cells expressing ataxin-3 84Q, respectively. Treatment with resveratrol (another compound known to activate the sirtuin pathway), also improved swimming in the MJD zebrafish. Co-treatment with valproate alongside EX527, a SIRT1 activity inhibitor, prevented induction of autophagy by valproate and the beneficial effects of valproate on the movement in the MJD zebrafish, supporting that they were both dependent on sirtuin activity. These findings provide the first evidence of sodium valproate inducing activation of the sirtuin pathway. Further, they indicate that drugs that target the sirtuin pathway, including sodium valproate and resveratrol, warrant further investigation for the treatment of MJD and related neurodegenerative diseases. Graphical abstract

scholarly journals Suppression of Hepatic Lipogenesis and Enhancement of β-oxidation in Skeletal Muscle by Betulinic Acid Lead to the Reduction of Body Adiposity and Dyslipidemia in Mice (P21-043-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Hyun Kyung Kim ◽  
Gwang-woong Go
Keyword(s):  
Skeletal Muscle ◽  
Betulinic Acid ◽  
Fruits And Vegetables ◽  
Metabolic Diseases ◽  
Final Body Weight ◽  
Beneficial Effects ◽  
Protein Levels ◽  
Funding Sources ◽  
Body Adiposity

Abstract Objectives Betulinic acid (BA) exists as a lipophilic conformation in nature, rich in fruits and vegetables, especially the bark of birch wood. Beneficial effects of BA on cancer, oxidative stress, and inflammation were previously reported. Studies of BA for anti-obesity and dyslipidemia have been recently emerged, however the outcomes are still not conclusive. We tested if BA improves obesity and/or dyslipidemia by incorporated mechanism of liver and skeletal muscle in vivo. Methods Five-week-old male C57BL/6 mice were fed Western type diet (45% kcal from fat with 1.25% cholesterol) ad libitum for 12 weeks. Mice were allocated into five groups: (−) control, BA 5 mg/kg, BA 15 mg/kg, BA 25 mg/kg, and fenofibrate as a (+) control. BA was orally administered every day. For proved phenotype alteration, we analyzed mRNA and protein expressions linked to lipogenesis and β-oxidation. Results Growth performance including final body weight, weight gain, and feed intake were not altered by BA. Body fat mass was decreased by all BA treated groups (P < 0.01) without changes of lean mass. Energy expenditure contributed by β-oxidation was increased by BA 25 (P < 0.001). Serum lipid profiles including triglyceride, free fatty acid, total cholesterol, and LDL cholesterol were significantly improved by all BA treated groups (P < 0.001). Lipid accumulation in the liver was reduced by BA 15 and 25 (all P < 0.001). The mRNA expressions of Acc1and Scd1were dose-dependently suppressed by betulinic acid in liver (P < 0.05). SREBP1 was diminished by BA treated groups (all P < 0.05). FAS showed reduced tendency by BA (P = 0.06). In protein levels, ACC1 and SCD1 were inhibited by BA treated groups (P < 0.001 and P < 0.05). FAS and SREBP1 tended to be reduced by BA (P = 0.40 and P = 0.70). LPL and CPT1, β-oxidation markers in skeletal muscle, were activated by BA 25 (P < 0.001 and P < 0.01). The activation of CPT1 was influenced by stimulating of AMPK-pT172 and ACC1-pS79 (all P < 0.05). Taken together, BA inhibitedde novolipogenesis in the liver and upregulated key proteins related to β-oxidation in skeletal muscle, contributing to the prevention of obesity and dyslipidemia. Conclusions We suggest that BA is a potent nutraceutical for prevention and treatment of chronic metabolic diseases including obesity and dyslipidemia. Funding Sources National Research Foundation of Korea.

scholarly journals Sodium valproate increases activity of the sirtuin pathway resulting in beneficial effects for spinocerebellar ataxia-3 in vivo

2021 ◽  
Author(s):  
Maxinne Watchon ◽  
Luan Luu ◽  
Katherine J. Robinson ◽  
Kristy C. Yuan ◽  
Alana De Luca ◽  
...  
Keyword(s):  
Spinocerebellar Ataxia ◽  
Sodium Valproate ◽  
Hek293 Cells ◽  
Transgenic Zebrafish ◽  
Zebrafish Model ◽  
Beneficial Effects ◽  
Protein Levels ◽  
Deacetylase Inhibitor ◽  
Spinocerebellar Ataxia 3

AbstractMachado-Joseph disease (MJD, also known as spinocerebellar ataxia-3) is a fatal neurodegenerative disease that impairs control and coordination of movement. Here we tested whether treatment with the histone deacetylase inhibitor sodium valproate (SV) prevented a movement phenotype that develops in larvae of a transgenic zebrafish model of the disease. We found that treatment with SV improved the swimming of the MJD zebrafish, increased levels of acetylated histones 3 and 4, but also increased expression of polyglutamine expanded human ataxin-3. Proteomic analysis of protein lysates generated from the treated and untreated MJD zebrafish also predicted that SV treatment had activated the sirtuin longevity signaling pathway and this was confirmed by findings of increased SIRT1 protein levels and sirtuin activity in SV treated MJD zebrafish and HEK293 cells expressing ataxin-3-84Q, respectively. Treatment with resveratrol (another compound known to activate the sirtuin pathway), also improved swimming in the MJD zebrafish. Co-treatment with SV alongside EX527, a SIRT1 activity inhibitor, prevented induction of autophagy by SV and the beneficial effects of SV on the movement in the MJD zebrafish, indicating that they were both dependent on sirtuin activity. These findings provide the first evidence of sodium valproate inducing activation of the sirtuin pathway. Further, they indicate that drugs that target the sirtuin pathway, including sodium valproate and resveratrol, warrant further investigation for the treatment of MJD and related neurodegenerative diseases.

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