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The estrogen metabolite 2-methoxyestradiol regulates eukaryotic initiation factor 4E(eIF4E)and inhibits protein synthesis in MG63 osteosarcoma cells

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Genes & Diseases 2016年第2期3卷 153-158页

作者： Avudaiappan Maran Kristen L.Shogren michael j.yaszemski Department of Orthopedics Mayo ClinicRochesterMN 55905USA

Osteosarcoma is a primary bone tumor that affects children and young adults.The estrogen metabolite 2-methoxyestradiol(2-ME)induces cell death in osteosarcoma cells.To determine whether 2-ME actions involve the control of protein synthesis,we studied the effect of 2-ME on eukaryotic initiation factor 4E(eIF4E)and eIF4E-binding protein 1(4E-BP1)in MG63 osteosarcoma cells.Our results show that 2-ME treatment increases the association of eIF4E with 4E-BP1 in osteosarcoma cells.Also,2-ME decreases the binding of eIF4E protein to 7-methyl-guanosine cap structure,indicating that 2-ME treatment results in the inhibition of translational initiation.These findings are further supported by the inhibition of protein synthesis in 2-ME-treated osteosarcoma cells.Taken together,our studies show that 2-ME-mediated antitumor effects in osteosarcoma cells involve the regulation of protein synthesis,and translational machinery could serve as a target in the treatment of osteosarcoma.

关键词： 2-Methoxyestradiol 4E-BP Estrogen metabolite eIF4E Osteosarcoma

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Key changes in denervated muscles and their impact on regeneration and reinnervation

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Neural Regeneration Research 2014年第20期9卷 1796-1809页

作者： Peng Wu Aditya Chawla Robert j.Spinner Cong Yu michael j.yaszemski Anthony j.Windebank Huan Wang Department of Neurologic Surgery Mayo ClinicRochesterMNUSA Department of Hand Surgery Huashan HospitalFudan University Department of Orthopedic Surgery the Second Affiliated HospitalZhejiang University School of Medicine Department of Orthopedic Surgery Wake Forest School of MedicineWinston-SalemNCUSA Departments of Orthopedic Surgery and Biomedical Engineering Mayo ClinicRochesterMNUSA Department of Neurology Mayo ClinicRochesterMNUSA Shanghai Key Laboratory of Peripheral Nerve and Microsurgery

The neuromuscular junction becomes progressively less receptive to regenerating axons if nerve repair is delayed for a long period of time. It is difficult to ascertain the denervated muscle＇s residual receptivity by time alone. Other sensitive markers that closely correlate with the extent of denervation should be found. After a denervated muscle develops a fibrillation potential, muscle fiber conduction velocity, muscle fiber diameter, muscle wet weight, and maximal isometric force all decrease; remodeling increases neuromuscular junction fragmentation and plantar area, and expression of myogenesis-related genes is initially up-regulated and then down-regulated. All these changes correlate with both the time course and degree of denervation. The nature and time course of these denervation changes in muscle are reviewed from the literature to explore their roles in assessing both the degree of detrimental changes and the potential success of a nerve repair. Fibrillation potential amplitude, muscle fiber conduction velocity, muscle fiber diameter, mRNA expression levels of myogenic regulatory factors and nicotinic acetylcholine receptor could all reflect the severity and length of denervation and the receptiveness of denervated muscle to regenerating axons, which could possibly offer an important clue for surgical choices and predict the outcomes of delayed nerve repair.

关键词： nerve regeneration denervation reinnervation fibrillation potential muscle fiberconduction velocity muscle fiber diameter maximal isometric force neuromuscular junction geneexpression neural regeneration

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A novel triple immunoenzyme staining enables simultaneous identification of all muscle fiber types on a single skeletal muscle cryosection from normal, denervated or reinnervated rats

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Neural Regeneration Research 2017年第8期12卷 1357-1364页

作者： Peng Wu Shuya Zhang Robert j.Spinner michael Torres Lizardi Yudong Gu Cong Yu michael j.yaszemski Anthony j.Windebank Huan Wang Department of Neurological Surgery Mayo Clinic Rochester MN USA Department of Hand Surgery Huashan Hospital Fudan University Shanghai China Shanghai Tenth People's Hospital Tongji University Shanghai China Department of Neurology Mayo Clinic Rochesterl MN USA Department of Medicine New York Presbyterian Weill Cornell Medical Center New York NY USA Shanghai Key Laboratory of Peripheral Nerve and Microsurgery Shanghai China Departments of Orthopedic Surgery and Biomedical Engineering Mayo Clinic Rochester MN USA

Triple immunofluorescence staining has recently been developed to simultaneously identify all muscle fibers on a single cryosection which is helpful for clinical and basic research, but it has disadvantages such as fast photobleaching and unclear outlines of muscle fibers. Triple immunoenzyme staining（TIE） is likely to avoid these disadvantages. In this study, we aimed to establish a sensitive and specific TIE technique to identify fiber types in normal, denervated, and reinnervated rat muscles, and to develop a systematic sampling method for muscle fiber quantification. Tibialis anterior and soleus from normal, denervated, and reinnervated Lewis rat hind limbs were used. Five consecutive cryosections were cut from each muscle, including one for TIE and four for single immunoenzyme staining（SIE）. The TIE was performed using the polymerized reporter enzyme staining system for the first two antigens（A4.74 for My HC-IIA, BA-F8 for My HC-I） and alkaline phosphatase staining system for the third antigen（BF-F3 for My HC-IIB）, followed by corresponding detective systems and respective chromogens. The type of muscle fibers was quantified by systematic sampling at 12.5%, 25%, 33% and 50% of all muscle fibers, and was compared with that acquired from counting all the fibers（100%）. All muscle fiber phenotypes, including pure and hybrid, could be simultaneously identified on a single TIE cryosection with clear outlines. The fiber types on TIE slides matched well with their respective counterpart on the consecutive SIE slides with a 95% match rate. Systematic sampling of 12.5% fibers could represent the true fiber type distribution of the entire muscle section. Our results suggest that novel TIE can effectively visualize fiber types in normal, denervated or reinnervated rat muscles.

关键词： nerve regeneration muscle fiber phenotyping immunohistochemistry triple immunoenzymestaining myosin heavy chain rats neural regeneration

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STIM1 expression is associated with osteosarcoma cell survival

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Chinese journal of Cancer Research 2019年第1期31卷 203-211页

作者： jie Zang Dongqing Zuo Kristen L.Shogren Carl T.Gustafson Zifei Zhou michael A Thompson Ruiwei Guo Y.S.Prakash Lichun Lu Wei Guo Avudaiappan Maran michael j.yaszemski Department of Orthopedic Surgery Mayo ClinicRochesterMN 55905USA Musculoskeletal Tumor Center Peking University People's HospitalBeijing 100044China Department of Orthopaedics Shanghai General HospitalShanghai Jiao Tong University School of MedicineShanghai 200080China Department of Anesthesiology Mayo ClinicRochesterMN 55905USA Department of Cardiology Kunming General Hospital of Chengdu Military Command of PLA Department of Physiology and Biomedical Engineering Mayo ClinicRochesterMN 55905USA

Objective: To examine the role of store-operated calcium entry(SOCE) and stromal interaction molecule 1(STIM1) in survival and migration of osteosarcoma cells and investigate what blockade of store-operated Ca^(2+)contributes to the regulation of osteosarcoma cells.Methods: First, we examined the expression levels of STIM1 in osteosarcoma cell lines by Western analysis and in tissue specimens by immunohistochemistry. Second, we investigated the effect of SOCE and STIM1 on osteosarcoma cell viability using MTS assays and on cell proliferation using colony formation. Third, we investigated the role of SOCE and STIM1 in cell migration using wound healing assays and Boyden chamber assays. Finally, we studied the effect of SOCE on the nuclear factor of activated T-cells cytoplasmic 1(NFATc1)activity by luciferase assays.Results: STIM1 was overexpressed in osteosarcoma cell lines and tissue specimens and was associated with poor survival of osteosarcoma patients. Also, inhibition of SOCE and STIM1 decreased the cell viability and migration of osteosarcoma cells. Furthermore, our results showed that blockade of store-operated Ca^(2+) channels involved down-regulation of NFATc1 in osteosarcoma cells.Conclusions: STIM1 is essential for osteosarcoma cell functions, and STIM1 and Ca^(2+) entry pathway could be further explored as molecular targets in the treatment of osteosarcoma.

关键词： Calcium NFATc1 osteosarcoma SOCE STIM1

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