HECT (Homologous to the E6-AP Carboxyl Terminus)-Type Ubiquitin E3 Ligase ITCH Attenuates Cardiac Hypertrophy by Suppressing the Wnt/β-Catenin Signaling Pathway

The HECT (homologous to the E6-AP carboxyl terminus)-type ubiquitin E3 ligase ITCH is an enzyme that plays an important role in ubiquitin-proteasomal protein degradation. Disheveled proteins (Dvl1 [disheveled protein 1], Dvl2, and Dvl3) are the main components of the Wnt/β-catenin signaling pathway, which is involved in cardiac hypertrophy. The aim of this study was to examine the role of ITCH during development of cardiac hypertrophy. Thoracic transverse aortic constriction (TAC) was performed in transgenic mice with cardiac-specific overexpression of ITCH (ITCH-Tg) and wild-type mice. Cardiac hypertrophy after TAC was attenuated in ITCH-Tg mice, and the survival rate was higher for ITCH-Tg mice than for wild-type mice. Protein interaction between ITCH and Dvls was confirmed with immunoprecipitation in vivo and in vitro. Expression of key molecules of the Wnt/β-catenin signaling pathway (Dvl1, Dvl2, GSK3β [glycogen synthase kinase 3β], and β-catenin) was inhibited in ITCH-Tg mice compared with wild-type mice. Notably, the ubiquitination level of Dvl proteins increased in ITCH-Tg mice. Protein and mRNA expression levels of ITCH increased in response to Wnt3a stimulation in neonatal rat cardiomyocytes. Knockdown of ITCH using small-interfering RNA increased cardiomyocyte size and augmented protein expression levels of Dvl proteins, phospho-GSK3β, and β-catenin after Wnt3a stimulation in cardiomyocytes. Conversely, overexpression of ITCH attenuated cardiomyocyte hypertrophy and decreased protein expression levels of Dvl proteins, phospho-GSK3β and β-catenin. In conclusion, ITCH targets Dvl proteins for ubiquitin-proteasome degradation in cardiomyocytes and attenuates cardiac hypertrophy by suppressing the Wnt/β-catenin signaling pathway.

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P1615HECT-Type Ubiquitin E3 Ligase ITCH attenuates cardiac hypertrophy by suppressing Wnt signaling pathway

European Heart Journal ◽

10.1093/eurheartj/ehz748.0374 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

J Goto ◽

Y Otaki ◽

T Watanabe ◽

T Aono ◽

K Watanabe ◽

...

Keyword(s):

Cardiac Hypertrophy ◽

Signaling Pathway ◽

Wnt Signaling Pathway ◽

E3 Ligase ◽

Pressure Overload ◽

Aortic Constriction ◽

Ubiquitin E3 Ligase ◽

Ubiquitin Proteasome ◽

Canonical Wnt ◽

Interfering Rna

Abstract Background The homologous to the E6-AP carboxyl terminus (HECT)–type ubiquitin E3 ligase ITCH is an enzyme that plays an important role in ubiquitin proteasomal protein degradation. Dishevelled proteins (Dvl1, Dvl2 and Dvl3), which are involved in canonical Wnt/β catenin signaling pathway, play a role in cardiac hypertrophy. Purpose The aim of this study was to examine whether ITCH interacts with Dvls and prevents cardiac hypertrophy induced by pressure overload. Methods and results We confirmed the protein interaction between ITCH and Dvls in cardiomyocytes. Overexpression of ITCH decreased protein expression levels of Dvls, phospho-GSK3β and β-catenin. Conversely, knockdown of ITCH using small interfering RNA augmented canonical Wnt/β catenin signaling pathway. Thoracic transverse aortic constriction (TAC) was performed in transgenic mice with cardiac-specific overexpression of ITCH (ITCH-Tg) and wild-type (WT) mice. The canonical Wnt/β catenin signaling pathway was inhibited and cardiac hypertrophy was attenuated in ITCH-Tg mice compared with WT mice after TAC. Overexpression of ITCH in cardiomyocytes Conclusion We demonstrated that ITCH targets Dvls for ubiquitin-proteasome degradation in cardiomyocytes and ameliorates cardiac hypertrophy by suppressing canonical Wnt/β catenin signaling pathway.

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Effects of WNT1 c.110 T>C and c.505G>T mutations on osteoblast differentiation via the WNT1/β-catenin signaling pathway

Journal of Orthopaedic Surgery and Research ◽

10.1186/s13018-021-02495-2 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Bashan Zhang ◽

Rong Li ◽

Wenfeng Wang ◽

Xueming Zhou ◽

Beijing Luo ◽

...

Keyword(s):

Protein Expression ◽

Signaling Pathway ◽

Osteoblast Differentiation ◽

Missense Mutations ◽

Wild Type ◽

Expression Levels ◽

Type Group ◽

In The Wild ◽

Mrna And Protein Expression ◽

Gsk 3Β

Abstract Background WNT1 c.110 T>C and c.505G>T missense mutations have been identified in patients with osteogenesis imperfecta (OI). Whether these mutations affect osteoblast differentiation remains to be determined. This study aimed to investigate the effects of WNT1 c.110 T>C and c.505G>T mutations on osteoblast function, gene expression, and pathways involved in OI. Methods Empty vector (negative control), wild-type WNT1, WNT1 c.110 T>C, WNT1 c.505G>T, and WNT1 c.884C>A (positive control) mutant plasmids were constructed and transfected into preosteoblast (MC3T3-E1) cells to investigate their effect on osteoblast differentiation. The expressions of osteoblast markers, including BMP2, RANKL, osteocalcin, and alkaline phosphatase (ALP), were determined using quantitative real-time polymerase chain reaction (RT-qPCR), western blotting (WB), enzyme-linked immunosorbent assay, and ALP staining assay, respectively. The mRNA and protein expression levels of WNT1 or the expression levels of the relevant proteins involved in the WNT1/β-catenin signaling pathway were also determined using RT-qPCR, WB, and immunofluorescence (IF) assays after the different plasmids were transfected into MC3T3-E1 cells. Results Compared with those in the wild-type group, in the mutation groups, the mRNA and protein expression levels of BMP2 were suppressed, the expressions of osteocalcin and ALP were inhibited, and the mRNA and protein expression levels of RANKL were enhanced in MC3T3-E1 cells. WB and IF assays revealed that the protein expression levels of WNT1 in MC3T3-E1 cells were downregulated in the mutation groups compared with those in the wild-type WNT1 group. Furthermore, the expression levels of nonphosphorylated β-catenin (non-p-β-catenin) and phosphorylated GSK-3β (p-GSK-3β) were downregulated in the mutation groups compared with those in the wild-type group. However, no significant changes in the expression level of non-p-β-catenin or p-GSK-3β were observed in the mutation groups. Conclusions WNT1 c.110 T>C and c.505G>T mutations may alter the proliferation and osteogenic phenotype of MC3T3-E1 linked to the progression of OI via the inhibition of the WNT1/β-catenin signaling pathway. This is the first study to confirm the effect of WNT1 c.110 T>C and c.505G>T missense mutations on osteoblast differentiation and propose a new molecular mechanism for OI development.

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Bakuchiol protects against pathological cardiac hypertrophy by blocking NF-κB signaling pathway

Bioscience Reports ◽

10.1042/bsr20181043 ◽

2018 ◽

Vol 38 (5) ◽

Cited By ~ 2

Author(s):

Zheng Wang ◽

Lu Gao ◽

Lili Xiao ◽

Lingyao Kong ◽

Huiting Shi ◽

...

Keyword(s):

Cardiac Hypertrophy ◽

Signaling Pathway ◽

Pressure Overload ◽

Neonatal Rat ◽

Oral Gavage ◽

Aortic Banding ◽

Rat Cardiomyocytes ◽

Pathological Cardiac Hypertrophy ◽

First Time

Bakuchiol (Bak), a monoterpene phenol isolated from the seeds of Psoralea corylifolia, has been widely used to treat a large variety of diseases in both Indian and Chinese folkloric medicine. However, the effects of Bak on cardiac hypertrophy remain unclear. Therefore, the present study was designed to determine whether Bak could alleviate cardiac hypertrophy. Mice were subjected to aortic banding (AB) to induce cardiac hypertrophy model. Bak of 1 ml/100 g body weight was given by oral gavage once a day from 1 to 8 weeks after surgery. Our data demonstrated for the first time that Bak could attenuate pressure overload-induced cardiac hypertrophy and could attenuate fibrosis and the inflammatory response induced by AB. The results further revealed that the effect of Bak on cardiac hypertrophy was mediated by blocking the activation of the NF-κB signaling pathway. In vitro studies performed in neonatal rat cardiomyocytes further proved that the protective effect of Bak on cardiac hypertrophy is largely dependent on the NF-κB pathway. Based on our results, Bak shows profound potential for its application in the treatment of pathological cardiac hypertrophy, and we believe that Bak may be a promising therapeutic candidate to treat cardiac hypertrophy and heart failure.

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Xihuang Pill inhibits the development of DMBA combined estrogen and progesterone induced breast precancerous lesions rats by PI3K/AKT/mTOR signaling pathway

10.21203/rs.3.rs-151758/v1 ◽

2021 ◽

Author(s):

Huanfang Fan ◽

Dehui Li ◽

Na Guo ◽

Chunxia Sun ◽

Jingfei Dong ◽

...

Keyword(s):

Protein Expression ◽

Signaling Pathway ◽

Breast Tissue ◽

Precancerous Lesions ◽

Precancerous Lesion ◽

Mtor Signaling ◽

Mtor Signaling Pathway ◽

Vegf Mrna ◽

Expression Levels ◽

Pten Mrna

Abstract Objective. To study the inhibitory effect of Xihuang Pill on the development of DMBA combined estrogen and progesterone induced breast precancerous lesions rats by PI3K/AKT/mTOR signaling pathway, and to explore the effect of Xihuang Pill in preventing and treating breast cancer. Method. Establishment of a rat model of breast precancerous lesion with DMBA combined estrogen and progesterone sequential induction for 10 weeks. Xihuang Pill was administered by gavage continuously for 4 weeks. Take rat breast tissue and stain with hematoxylin- eosin (HE). The pathomorphological changes were observed with light microscope; TUNEL staining to detect cell apoptosis in breast tissue; Western blot was used to detect the protein expression of P-PI3K, P-AKT (S473), P-AKT (T308), PTEN, P-Tuberin/TSC2, P-Tuberin (p-S939), p-mTOR, P-4E-BP1 in breast tissues. The qRT-PCR was used to detect the gene expression of PTEN mRNA and VEGF mRNA. Immunohistochemistry was used to detect the protein expression of P-S6, p-p70s6k and VEGF. Result. Compared with the disease model group, the low, middle and high dose Xihuang Pill groups could significantly reduce the degree of breast pathology, and the number of apoptosis of breast precancerous lesions cells increased with the increase of Xihuang Pill dose; The expression levels of P-PI3K, P-AKT (S473), P-AKT (T308), p-mTOR, P-4E-BP1, p-S6, p-p70S6K, VEGF protein and VEGF mRNA dropped with the increase of Xihuang Pill dose. The expression levels of PTEN, P-Tuberin/TSC2, P-Tuberin (p-S939) protein and PTEN mRNA elevated with the increase of Xihuang Pill dose. Conclusion. Xihuang Pill can promote the apoptosis of breast precancerous lesion cells and reduce the proliferation of vascular endothelial cells, and then inhibit the progression of breast precancerous lesions. Its mechanism probably associated with the regulation of PI3K/AKT/mTOR pathway related gene protein expression.

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LCZ696, an Angiotensin Receptor-Neprilysin Inhibitor, Improves Cardiac Hypertrophy and Fibrosis and Cardiac Lymphatic Remodeling in Transverse Aortic Constriction Model Mice

BioMed Research International ◽

10.1155/2020/7256862 ◽

2020 ◽

Vol 2020 ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

Qing Ge ◽

Li Zhao ◽

Chen Liu ◽

Xiaoming Ren ◽

Yi-hui Yu ◽

...

Keyword(s):

Cardiac Hypertrophy ◽

Protein Expression ◽

Lymphatic System ◽

Lymphatic Vessels ◽

Mouse Heart ◽

Transverse Aortic Constriction ◽

Aortic Constriction ◽

Expression Levels ◽

Factor C ◽

Proinflammatory Factors

Cardiac hypertrophy and ventricular remodeling following heart failure are important causes of high mortality in heart disease patients. The cardiac lymphatic system has been associated with limited research, but it plays an important role in the improvement of myocardial edema and the promotion of fluid balance. LCZ696 is a novel combination of angiotensin and neprilysin inhibitors. Here, we studied the role played by LCZ696 during transverse aortic constriction (TAC) induced cardiac hypertrophy and changes in the lymphatic system. Mice undergoing aortic coarctation were constructed to represent a cardiac hypertrophy model and then divided into random groups that either received treatment with LCZ696 (60 mg/kg/d) or no treatment. Cardiac ultrasonography was used to detect cardiac function, and hematoxylin and eosin (H&E) and Masson staining were used to detect myocardial hypertrophy and fibrosis. The proinflammatory factors interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α) were detected in the blood and heart tissues of mice. The protein expression levels of lymphatic-specific markers, such as vascular endothelial growth factor C (VEGF-C), VEGF receptor 3 (VEGFR3), and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) were detected in mouse heart tissues. We also examined the colocalization of lymphatic vessels and macrophages by immunofluorescence. The results showed that LCZ696 significantly improved heart dysfunction, cardiac hypertrophy, and fibrosis and inhibited the expression of proinflammatory factors IL-6, IL-1β, and TNF-α in the circulating blood and heart tissues of mice. LCZ696 also decreased the protein expression levels of VEGF-C, VEGFR3, and LYVE-1 in mouse heart tissues, ameliorated the transport load of lymphatic vessels to macrophages, and improved the remodeling of the lymphatic system in the hypertrophic cardiomyopathy model induced by TAC.

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Role of myofibrillogenesis regulator-1 in myocardial hypertrophy

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00247.2005 ◽

2006 ◽

Vol 290 (1) ◽

pp. H279-H285 ◽

Cited By ~ 14

Author(s):

Xiuhua Liu ◽

Tianbo Li ◽

Sheng Sun ◽

Feifei Xu ◽

Yiguang Wang

Keyword(s):

Rna Interference ◽

Cardiac Hypertrophy ◽

Protein Expression ◽

Western Blotting ◽

Left Ventricular ◽

Neonatal Rat ◽

Heart Weight ◽

Homologous Gene ◽

Ang Ii ◽

Rat Cardiomyocytes

Myofibrillogenesis regulator-1 (MR-1) is a novel homologous gene, identified from a human skeletal muscle cDNA library, that interacts with contractile proteins and exists in human myocardial myofibrils. The present study investigated MR-1 protein expression in hypertrophied myocardium and MR-1 involvement in cardiac hypertrophy. Cardiac hypertrophy was induced by abdominal aortic stenosis (AAS) in Sprague-Dawley rats. Left ventricular (LV) hypertrophy was assessed by the ratio of LV wet weight to whole heart weight (LV/HW) or LV weight to body weight (LV/BW). Rat MR-1 (rMR-1) expression in the myocardium was detected by immunohistochemical and Western blotting analysis. Hypertrophy was induced by ANG II incubation in cultured neonatal rat cardiomyocytes. The effect of rMR-1 RNA interference on ANG II-induced hypertrophy was studied by transfection of cardiomyocytes with an RNA interference plasmid, pSi-1, which targets rMR-1. Hypertrophy in cardiomyocytes was assessed by [3H]Leu incorporation and myocyte size. rMR-1 protein expression in cardiomyocytes was detected by Western blotting. We found that AAS resulted in a significant increase in LV/HW and LV/BW: 89% and 86%, respectively ( P < 0.01). Immunohistochemistry and Western blot analysis demonstrated upregulated rMR-1 protein expression in hypertrophic myocardium. ANG II induced a 24% increase in [3H]Leu incorporation and a 65.8% increase in cell size compared with control cardiomyocytes ( P < 0.01), which was prevented by treatment with losartan, an angiotensin (AT1) receptor inhibitor, or transfection with pSi-1. rMR-1 expression increased in ANG II-induced hypertrophied cardiomyocytes, and pSi-1 transfection abolished the upregulation. These findings suggest that MR-1 is associated with cardiac hypertrophy in rats in vivo and in vitro.

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Effect of CXCR4 on Apoptosis in Osteosarcoma Cells via the PI3K/Akt/NF-κβ Signaling Pathway

Cellular Physiology and Biochemistry ◽

10.1159/000489593 ◽

2018 ◽

Vol 46 (6) ◽

pp. 2250-2260 ◽

Cited By ~ 15

Author(s):

Chunming Jiang ◽

Shenglin Ma ◽

Runlei Hu ◽

Xuepeng Wang ◽

Maoqiang Li ◽

...

Keyword(s):

Protein Expression ◽

Signaling Pathway ◽

Cell Apoptosis ◽

Luciferase Reporter ◽

Osteosarcoma Cells ◽

Down Regulation ◽

Expression Levels ◽

Apoptotic Protein ◽

Effective Manner ◽

Human Osteosarcoma Cells

Background/Aims: Osteosarcoma, the most common primary bone malignancy, arises from primitive transformed cells of mesenchymal origin with the worldwide increasing morbidity and mortality. Previous studies found apoptosis of osteosarcoma cells was essential for an effective manner to improve the progress of osteosarcoma, and CXCR4 has been demonstrated to be relevant with various tumor progress and metastasis. Methods: The proliferation of cells transfected with CXCR4 shRNA and control shRNA were measured by BrdU assay. Apoptosis was detected by flow cytometry. Apoptotic protein expression levels were detected by Western blot. Caspase activity was detected by Colorimetric Assay Kits using microplate reader. Activation of NF-κβ signaling after CXCR4 down-regulation in osteosarcoma cells was examined by constructing NF-κβ promoter luciferase reporter plasmid. The expression and activation of NF-κβ Signaling relevant protein were analyzed to investigate the relationship between Akt and NF-κβ signaling after the down-regulation of CXCR4 in osteosarcoma cells. Results: Down-regulation of CXCR4 significantly reduced the cell proliferation, while remarkably increased the cell apoptosis and apoptotic protein expression levels in osteosarcoma cells. Furthermore, down-regulation of CXCR4 induced cell apoptosis was caspase dependent in osteosarcoma cells. This study also showed CXCR4 down-regulation induced apoptosis through inhibiting PI3K/Akt/NF-κβ signaling pathway. In addition, endoplasmic reticulum stress (ERS) activation was involved in cell apoptosis induced down-regulation of CXCR4. Knockdown of partial ERS relevant proteins followed down-regulation of CXCR4 significantly inhibited cell apoptosis and the apoptotic protein expression levels. Conclusions: Taken together, the results demonstrated that down-regulation of CXCR4 could induce apoptosis of human osteosarcoma cells through inhibiting PI3K/Akt/NF-κβ signaling pathway, indicating that CXCR4 could be vital for the clinical therapy of osteosarcoma.

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The Effect and Mechanism of KLF7 in the TLR4/NF-κB/IL-6 Inflammatory Signal Pathway of Adipocytes

Mediators of Inflammation ◽

10.1155/2018/1756494 ◽

2018 ◽

Vol 2018 ◽

pp. 1-12 ◽

Cited By ~ 1

Author(s):

Meixiu Zhang ◽

Cuizhe Wang ◽

Jinxiu Wu ◽

Xiaodan Ha ◽

Yuchun Deng ◽

...

Keyword(s):

Protein Expression ◽

Signaling Pathway ◽

Transcriptional Activation ◽

Inflammatory Cytokine ◽

Luciferase Reporter ◽

Signal Pathways ◽

High Concentration ◽

Inflammatory Signaling ◽

Expression Levels ◽

Mrna And Protein Expression

Objective. To investigate the role and possible molecular mechanism of Krüppel-like factor 7 (KLF7) in the TLR4/NF-κB/IL-6 inflammatory signaling pathway activated by free fatty acids (FFA). Methods. The mRNA and protein expression levels of KLF7 and the factors of TLR4/NF-κB/IL-6 inflammatory signal pathways were detected by qRT-PCR and Western blotting after cell culture with different concentrations of palmitic acid (PA). The expression of KLF7 or TLR4 in adipocytes was upregulated or downregulated; after that, the mRNA and protein expression levels of these key factors were detected. KLF7 expression was downregulated while PA stimulated adipocytes, and then the mRNA and protein expressions of KLF7/p65 and downstream inflammatory cytokine IL-6 were detected. The luciferase reporter assay was used to determine whether KLF7 had a transcriptional activation effect on IL-6. Results. (1) High concentration of PA can promote the expression of TLR4, KLF7, and IL-6 in adipocytes. (2) TLR4 positively regulates KLF7 expression in adipocytes. (3) KLF7 positively regulates IL-6 expression in adipocytes. (4) PA promotes IL-6 expression via KLF7 in adipocytes. (5) KLF7 has a transcriptional activation on IL-6. Conclusion. PA promotes the expression of the inflammatory cytokine IL-6 by activating the TLR4/KLF7/NF-κB inflammatory signaling pathway. In addition, KLF7 may directly bind to the IL-6 promoter region and thus activate IL-6.

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Proteasome Inhibitors Restore to Normal the Decreased Levels of Protein Expression and Nucleolar Localization of Various Mutant Ribosomal S19 Proteins Identified in DBA Patients.

Blood ◽

10.1182/blood.v106.11.185.185 ◽

2005 ◽

Vol 106 (11) ◽

pp. 185-185 ◽

Cited By ~ 3

Author(s):

Aurore Cretien ◽

Alexis Proust ◽

Hanna Gazda ◽

Jorg Meerpohl ◽

Charlotte Marie Niemeyer ◽

...

Keyword(s):

Subcellular Localization ◽

Protein Expression ◽

Proteasome Inhibitors ◽

Nucleolar Localization ◽

Wild Type ◽

Mutant Proteins ◽

Expression Levels ◽

Type Protein ◽

Wild Type Protein ◽

Proteosomal Degradation

Abstract Mutations in ribosomal protein S19 (RPS19) gene have been found in 25% of patients affected with Diamond-Blackfan anemia (DBA), a congenital erythroblastopenia. We have previously shown that several RPS19 mutant proteins (V15F, InsAG36, W33stop, Y48stop, R56stop, M75stop, R94stop, 274del31, InsG238, G127Q and L131P) exhibit decreased levels of protein expression and do not localize to the nucleolus like the wild type protein in transfected Cos-7 cells. In contrast, other mutants (W52C, T55M, R56Q, R62W, 24Del18, G120S) exhibit normal levels of protein expression and normal nucleolar localization. We hypothesized that decreased levels of expression of mutant proteins such as V15F, G127Q, and L131P may be due to proteosomal degradation. In order to validate our hypothesis, we analyzed the effects of two proteasome inhibitors (MG132 and lactacystin) on mutant RPS19 protein expression levels and their subcellular localization. Following treatment with proteosome inhibitors, the mutant proteins with missense mutations (V15F, G127Q and L131P) were expressed at levels similar to that of wild type protein and localized in the nucleolus. Similarly, proteasome inhibitors also restored the expression levels and normal subcellular localization to RPS19 with non-sense mutations (InsG238, R94stop, and 274del31) that resulted in the translation of RPS19 protein with at least 80 aminoacids. In marked contrast, proteosome inhibitors failed to restore the expression levels of RPS19 with the non-sense mutants that led to synthesis of shortened proteins (InsAG36, W33stop, Y48stop, R56stop, M75stop). Even in the presence of proteosome inhibitors we noted a dramatic decrease in the levels of expression of these mutant proteins and proteins expressed were localized in the cytoplasm. Our findings imply an important role for proteosomal degradation pathway in regulating the expression levels of RPS19. They further suggest that proteasome inhibitors could be considered as a potential treatment for some steroid resistant DBA affected individuals with specific RPS19 mutations.

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Ubiquitin E3 Ligase, Tripartite Motif Protein 68 (TRIM68) Inhibits TCP-1 b Function by Proteasome-Mediated Degradation and May Overcome Imatinib-Resistance.

Blood ◽

10.1182/blood.v114.22.3789.3789 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3789-3789

Author(s):

Yasuhito Terui ◽

Ryoko Kuniyoshi ◽

Yuji Mishima ◽

Yuko Mishima ◽

Kiyohiko Hatake

Keyword(s):

Differential Display ◽

Ring Finger ◽

E3 Ligase ◽

K562 Cells ◽

Wild Type ◽

Mcf7 Cells ◽

Spry Domain ◽

Ubiquitin E3 Ligase ◽

Imatinib Resistant

Abstract Abstract 3789 Poster Board III-725 [Background] Imatinib mesylate is effective therapy against Philadelphia chromosome-positive leukemia, but the resistance develops in all phases of the disease. The identification of new proteins induced by imatinib may lead to find the novel potent molecular targets in imatinib-resistant CML. [Methods] K562 cells were treated with or without 1 mM imatinib for 24 hours, and then differential display between them was performed. TRIM68 expression was examined by RT-PCR, and in vivo ubiquitination or sumoylation assay was performed by transfection experiment and Western blot analysis. The substrates for TRIM68 were analyzed by mass spectorometry. [Results] As the results of RNA differential display, we found that the expression of TRIM68 mRNA was increased when the K562 cells were treated with 1 mM imatinib for 24 hours. TRIM68 protein possesses a RING finger domain at its N-terminal site. Since many RING-finger proteins have been identified as E3 ligases for ubiquitination or sumoylation (Meroni G, Diez-Roux G. TRIM/RBCC, a novel class of esingle protein RING finger' E3 ubiquitin ligases. Bioessays 2005; 27: 1147-57.), we examined whether TRIM68 functions as an E3 ligase for ubiquitination or sumoylation. To examine the function of TRIM68 as an E3 ligase, wild type TRIM68 and a RING domain deletion mutant of TRIM68 (TRIM68/¢R) genes were constructed into a mammalian expression vector and they were transfected into MCF7 cells. TRIM68 had auto-ubiquitination activity but not auto-sumoylation activity on the in vivo assays, suggesting that TRIM68 can be an ubiquitin E3 ligase but not sumo ligase. Moreover, wild type TRIM68 promoted the whole ubiqutination in the cells, whereas TRIM68/¢R prevented the ubiquitination inside of the cells. To identify the TRIM68-interacting proteins, we transfected FLAG-tagged wild type TRIM68 gene or B30.2/SPRY domain of TRIM68 gene into MCF7 cells, and immunoprecipitation with FLAG-M2 agarose was performed and mass spectrometric analysis was performed. As the results, we revealed that the members of molecular chaperone T-complex polypeptide 1 (TCP-1) complex, TCP-1 b and heat shock protein 70 (HSP70) interacted with TRIM68 at the B30.2/SPRY domain. Then, we examined whether TCP-1 b is one of the substrates for TRIM68-related ubiqutination. TCP-1 b was ubiquitinated by wild type TRIM68, but not by TRIM68/¢R. Furthermore, the ubiquitination of TCP-1 b was accumulated by the treatment with a proteasome inhibitor MG132. These suggested that TCP-1 b is one of the substrates for TRIM68. [Conclusions] We found that TRIM68 is induced by the treatment with imatinib and functions as an ubiquitin E3 ligase. Furthermore, we identified that TCP-1 b is a substrate of TRIM68. TRIM68 may inhibit the function of TCP-1 b as a chaperone by ubiquitination and proteasome-mediated degradation. TRIM68 is possible for a new target in the imatinib-resistant CML. Disclosures: No relevant conflicts of interest to declare.

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