INAUGURAL ARTICLE by a Recently Elected Academy Member:Functional redundancy of type I and type II receptors in the regulation of skeletal muscle growth by myostatin and activin A

Myostatin (MSTN) is a transforming growth factor-β (TGF-β) family member that normally acts to limit muscle growth. The function of MSTN is partially redundant with that of another TGF-β family member, activin A. MSTN and activin A are capable of signaling through a complex of type II and type I receptors. Here, we investigated the roles of two type II receptors (ACVR2 and ACVR2B) and two type I receptors (ALK4 and ALK5) in the regulation of muscle mass by these ligands by genetically targeting these receptors either alone or in combination specifically in myofibers in mice. We show that targeting signaling in myofibers is sufficient to cause significant increases in muscle mass, showing that myofibers are the direct target for signaling by these ligands in the regulation of muscle growth. Moreover, we show that there is functional redundancy between the two type II receptors as well as between the two type I receptors and that all four type II/type I receptor combinations are utilized in vivo. Targeting signaling specifically in myofibers also led to reductions in overall body fat content and improved glucose metabolism in mice fed either regular chow or a high-fat diet, demonstrating that these metabolic effects are the result of enhanced muscling. We observed no effect, however, on either bone density or muscle regeneration in mice in which signaling was targeted in myofibers. The latter finding implies that MSTN likely signals to other cells, such as satellite cells, in addition to myofibers to regulate muscle homeostasis.

Myostatin (MSTN) is a secreted signaling molecule that normally acts to limit skeletal muscle growth (for review, see ref. 1). Mice lacking MSTN exhibit dramatic increases in muscle mass throughout the body, with individual muscles growing to about twice the normal size (2). MSTN appears to play two distinct roles in regulating muscle size, one to regulate the number of muscle fibers that are formed during development and a second to regulate the growth of those fibers postnatally. The sequence of MSTN has been highly conserved through evolution, with the mature MSTN peptide being identical in species as divergent as humans and turkeys (3). The function of MSTN has also been conserved, and targeted or naturally occurring mutations in MSTN have been shown to cause increased muscling in numerous species, including cattle (3–5), sheep (6), dogs (7), rabbits (8), rats (9), swine (10), goats (11), and humans (12). Numerous pharmaceutical and biotechnology companies have developed biologic agents capable of blocking MSTN activity, and these have been tested in clinical trials for a wide range of indications, including Duchenne and facioscapulohumeral muscular dystrophy, inclusion body myositis, muscle atrophy following falls and hip fracture surgery, age-related sarcopenia, Charcot–Marie–Tooth disease, and cachexia due to chronic obstructive pulmonary disease, end-stage kidney disease, and cancer.The finding that certain inhibitors of MSTN signaling can increase muscle mass even in Mstn^−/− mice revealed that the function of MSTN as a negative regulator of muscle mass is partially redundant with at least one other TGF-β family member (13, 14), and subsequent studies have identified activin A as one of these cooperating ligands (15, 16). MSTN and activin A share many key regulatory and signaling components. For example, the activities of both MSTN and activin A can be modulated extracellularly by naturally occurring inhibitory binding proteins, including follistatin (17, 18) and the follistatin-related protein, FSTL-3 or FLRG (19, 20). Moreover, MSTN and activin A also appear to share receptor components. Based on in vitro studies, MSTN is capable of binding initially to the activin type II receptors, ACVR2 and ACVR2B (also called ActRIIA and ActRIIB) (18) followed by engagement of the type I receptors, ALK4 and ALK5 (21). In previous studies, we presented genetic evidence supporting a role for both ACVR2 and ACVR2B in mediating MSTN signaling and regulating muscle mass in vivo. Specifically, we showed that mice expressing a truncated, dominant-negative form of ACVR2B in skeletal muscle (18) or carrying deletion mutations in Acvr2 and/or Acvr2b (13) have significantly increased muscle mass. One limitation of the latter study, however, was that we could not examine the consequence of complete loss of both receptors using the deletion alleles, as double homozygous mutants die early during embryogenesis (22). Moreover, the roles that the two type I receptors, ALK4 and ALK5, play in regulating MSTN and activin A signaling in muscle in vivo have not yet been documented using genetic approaches. Here, we present the results of studies in which we used floxed alleles for each of the type II and type I receptor genes in order to target these receptors alone and in combination in muscle fibers. We show that these receptors are functionally redundant and that signaling through each of these receptors contributes to the overall control of muscle mass.     

Human hepatocellular carcinoma tumor xenografts

Castrated or sham-operated male athymic mice were inoculated with cells from the human hepatocellular carcinoma cell line PLC/PRF/5. There were no significant differences between the two groups with respect to the number of animals developing tumors, the time to tumor development, or the subsequent rate of increase in either tumor base area or mouse serum alpha-fetoprotein concentration. Androgen receptors were assayed in nuclei obtained from three separate liver cancer cell lines and from normal adult human liver. Similar concentrations, ranging from 235 to 550 fmol/mg DNA, of nuclear androgen receptors were detected in all tissues. Low percentages of androgen receptors were retained on DNA-cellulose. Although the presence of receptors implies the potential for metabolic effects of androgens in normal and malignant liver, our in vivostudies suggest that castration does not alter significantly the growth of liver cancer xenografts in athymic mice.     

Basic fibroblast growth factor mRNA, bFGF peptide and FGF receptor in epiretinal membranes of intraocular proliferative disorders (PVR and PDR)

Basic fibroblast growth factor (bFGF) has been shown to be involved in epiretinal membrane formation in proliferative vitreoretinal disorders. However, up to now, little knowledge exists, as to the actual cellular source of this potent mitogen.We examined 20 epiretinal membranes from patients with proliferative diabetic retinopathy (PDR) (n = 12) and proliferative vitreoretinopathy (PVR) (n = 8) for the presence of bFGF peptide, fibroblast growth factor receptor-1 (FGFR-1) and bFGF messenger ribonucleic acid (mRNA).Using a specific antibody, we detected bFGF peptide in most (8/10) examined PDR membranes and in all (8/8) PVR membranes. Moreover, we found positive staining for the corresponding receptor.Local production of bFGF in epiretinal membranes was confirmed by nonisotopic in situ hybridisation for bFGF mRNA in some (4/7) examined PDR membranes and some (3/4) examined PVR membranes. All membranes which contained bFGF mRNA were also positive for bFGF peptide.In conclusion, bFGF is produced and stored in epiretinal membranes. Together with the corresponding receptor, bFGF may play a role in the auto- and paracrine control of the proliferative processes at the vitroretinal interface.Abbreviations aFGF acidic fibroblast growth factor - bFGF basic fibroblast growth factor - FGFR-1 fibroblast growth factor receptor-1 - mRNA messenger ribonucleic acid     

Effect of UVB 311 nm irradiation on normal human skin

Ultraviolet radiation B (UVB) on the skin induces erythema, inflammation and modifications of the immune system. These changes have been reported after excessive short-term or long-term exposure to broad spectrum UVB. In this study, we examined the effects of local repetitive UVB irradiation of 311 nm wavelength on the skin of seven young volunteers. Skin biopsies were taken before and after UVB irradiation, and we immunohistochemically analyzed the expression of CD1a and HLA-DR antigens of Langerhans cells (LC), the possible infiltration of dermis/epidermis by CD11b macrophages, the modifications or the induction of intercellular adhesion molecule-1 (ICAM-1), E-selectin and vascular cell adhesion molecule-1 (VCAM-1) involved in the binding of leukocytes to the endothelial surface and the development of perivascular infiltrates of LFA-1⁺ mononuclear cells. We also determined the expression of substance P receptors (SPR) using biotinylated substance P (SPB). Exposure of UVB 311 nm induced a drastic reduction of CD1a⁺ cells and a moderate increase of HLA-DR⁺ dendritic cells in the epidermis without infiltration by CD11b macrophages. An increase of the binding of SPB to upper layer epidermal cells was noted in five of seven biopsies. In the dermis, vessel-associated ICAM-1 expression increased and an induction of E-selectin occurred on nearly 20 to 40% of endothelial cells, but VCAM-1 expression remained undetectable. The percentage of LFA-1⁺ cells did not change significantly after irradiation. These observations may be compatible with a selective role of UVB 311 nm on the skin immune response.     

1 alpha(OH)D3 (ETALPHA) treatment and receptor studies in 16 patients with chronic and myeloproliferative disorders

10 patients with CLL and 2 with CML were treated with gradually increasing doses of 1 alpha(OH)D3, up to 4 micrograms daily during 6 wk. 3 patients with preleukemia and 1 with myelofibrosis were treated with 2 micrograms daily of 1 alpha(OH)D3 for a prolonged period up to 17 wk. The treatment with 1 alpha (OH)D3 did not result in changes of disease parameters in any of the patients under study. Receptor studies for 1,25(OH)2D3 were performed in 8 CLL patients and revealed only 1 patient with increased specific receptor binding capacity. The maximum tolerable dose of 1 alpha(OH)D3 varied individually, but was in the range of 2-4 micrograms daily.     

肾上腺素能β1受体亚型特异性抗体的制备及鉴定

【目的】制备和鉴定 β1受体亚型特异性抗体。【方法】人工合成 β1受体细胞膜外第二环 197 2 2 2位氨基酸序列作为抗原。连接钥孔冒贝血蓝蛋白 (KLH)增加抗原性后免疫兔获得抗血清。通过凝胶双扩散实验和ELISA法鉴定其效价 ;通过免疫荧光法及ELISA法鉴定其特异性 ;通过离体蛙心灌流实验鉴定其药理活性。【结果】该抗血清效价高 (分别为 1∶6 4和 1∶10 6)、特异性强 ,能和心肌 β1受体发生特异性结合 (1∶10 4 ～ 1∶10 5) ,为异丙肾上腺素的非竞争性拮抗剂。 (pD2 ′ =1 6 2 )。【结论】成功制备的 β1受体亚型特异性抗体可能成为进一步研究 β1受体分布、功能和定量的有力工具。     

多柔比星肾病肾损伤早期核因子-κB和血管紧张素ⅠⅡ的表达及关系探讨

目的　探讨在多柔比星 (阿霉素 )肾病综合征 (NS)幼年大鼠肾损伤过程中核因子 (NF) κB和血管紧张素ATⅠ、ATⅡ的表达及其相关性。方法　 4周龄雄性Wistar大鼠单侧肾切除加腹腔注射阿霉素造成NS模型 ,分别以免疫组织化学和原位杂交检测ATⅠ、ATⅡ和NF κB。结果　肾病组随着病变时间的延长 ,NF κB和ATⅠ、ATⅡ表达的强度和部位均呈增强趋势 ,治疗组在相同时间点则两者都有不同程度下调 (P <0 .0 5 )。结论　在阿霉素肾病损伤过程中NF κB和ATⅠ、ATⅡ起着介导作用。     

~(99)Tc-MDP对类风湿性关节炎患者外周血单核细胞产生细胞因子的影响

目的 :探讨“云克”(99Tc MDP)治疗类风湿性关节炎 (RA)的免疫机制。方法 :采用ELISA双抗体夹心法 ,观察“云克”体外对RA患者外周血单核细胞 (PBMC)产生白介素 1(IL 1)和可溶性白介素 2受体 (sIL 2R)的影响。结果 :“云克”有抑制RA患者IL 1的分泌及细菌脂多糖 (LPS)的促分泌作用 ,并对可溶性白介素 2受体的自发分泌及植物血素 (PHA)诱导分泌均有抑制作用。结论 :“云克”对RA的治疗机制可能与其降低IL 1和sIL 2R的作用有关     

胃癌雌激素受体表达及新一代抗受体药物在胃癌治疗中的探讨

目的　探讨胃癌雌激素受体 (ER)实际表达率 ,为临床使用新一代抗ER药物 (toremifene ,TOR)治疗胃癌提供临床病理依据。方法　用免疫组化法对 349例胃癌标本进行ER的检测 ,其中 2 99例用ABC(avidinbiotinperoxidasecomplex)法 ,5 0例用葡聚糖聚合物技术二步法进行检测。在检测ER的同时也检测 p5 3及PCNA的表达。结果　两种检测方法的ER阳性率分别为 2 .3% (7/ 2 99)及 0 % (0 / 5 0 ) ,p5 3阳性率 37.1% (111/ 2 99)及 4 6 %(2 3/ 5 0 ) ,PCNA 94 .3% (2 82 / 2 99)及 96 % (48/ 5 0 )。结论　胃癌细胞可表达ER但实际表达率很低 ,而且存在质和量的变化。tamoxifen (TAM )及TOR的抗胃癌效应需要进一步研究