力生长因子及其E肽对成骨细胞分化的影响

力生长因子(mechano growth factor,MGF)是新近发现的一种生长因子,由Igf-1基因剪接变异产生,拉伸刺激会促使成骨细胞表达力生长因子.比较分析了MGF及其羧基端E肽(MGF-Ct24E)对成骨细胞前体细胞MC3T3-E1分化的影响.结果显示:MGF和MGF-Ct24E具有显著激活胞外信号调节激酶1/2(Erk1/2)的作用,并降低了成骨细胞碱性磷酸酶、Ⅰ型胶原的表达,促进了骨桥蛋白(OPN)的表达,减少了核心绑定因子(corebinding factor1,Cbfα-1)的核转运量,对成骨细胞的分化具有延迟效应,这种效应通过抑制剂PD98059抑制Erk1/2的活化得到逆转;MGF还能显著激活磷脂酰肌醇3-激酶信号通路中的蛋白激酶B(Akt),该活化作用对成骨细胞分化是必需的,钙沉积分析显示长期培养下的细胞MGF促进了矿化节结的形成.这些结果说明,MGF-Ct24E对成骨细胞的分化具有抑制作用,这种作用与Erk1/2的活化有关,MGF因为包含E肽和IGF-1部分,能分别激活Erk1/2和Akt,因此对成骨细胞分化表现出双重效用,在成骨细胞分化早期,具有一定的延迟效应,而在分化晚期对成骨...     

A new pro-migratory activity on human myogenic precursor cells for a synthetic peptide within the E domain of the mechano growth factor

Duchenne muscular dystrophy (DMD) is an inherited disease that leads to progressive muscle wasting. Myogenic precursor cell transplantation is an approach that can introduce the normal dystrophin gene in the muscle fibers of the patients. Unfortunately, these myogenic precursor cells do not migrate well in the muscle and thus many injections have to be done to enable a good graft success. Recent reports have shown that there is extensive splicing of the IGF-1 gene in muscles. The MGF isoform contains a C-terminal 24 amino acids peptide in the E domain (MGF-Ct24E) that has intrinsic properties. It can promote the proliferation while delaying the differentiation of C(2)C(12) cells. Here, we demonstrated that this synthetic peptide is a motogenic factor for human precursor myogenic cells in vitro and in vivo. Indeed, MGF-Ct24E peptide can modulate members of the fibrinolytic and metalloproteinase systems, which are implicated in the migration of myogenic cells. MGF-Ct24E peptide enhances the expression of u-PA, u-PAR and MMP-7 while reducing PAI-1 activity. Moreover, it has no effect on the gelatinases MMP-2 and -9. Those combined effects can favour cell migration. Finally, we present some results suggesting that the MGF-Ct24E peptide induces these cell responses through a mechanism that does not involve the IGF-1 receptor. Thus, this MGF-Ct24E peptide has a new pro-migratory activity on human myogenic precursor cells that may be helpful in the treatment of DMD. Those results reinforce the possibility that the IGF-1Ec isoform may produce an E domain peptide that can act as a cytokine.     

Age-related loss of skeletal muscle function; impairment of gene expression

Mechano Growth Factor (MGF) is derived from the insulin-like growth factor (IGF-I) but its sequence differs from the systemic IGF-I produced by the liver. MGF is expressed by mechanically overloaded muscle and is involved in tissue repair and adaptation. It is expressed as a pulse following muscle damage and involved in the activation of muscle satellite (stem) cells. These donate nuclei to the muscle fibers that are required for repair and for the hypertrophy processes which may have similar regulatory mechanisms. Muscles in the elderly are unable to upregulate MGF in response to exercise. This is also true in certain diseases and this helps to explain muscle loss in those conditions. There is evidence that MGF is a local tissue repair factor as well as a growth factor and that it has an important role in damage limitation and inducing repair in other post-mitotic tissues. As there is no cell replacement in these tissues there has to be an effective local cellular repair mechanism. With advancing years this seems to become deficient and there is an increased chance that the damaged cells will undergo cell death leading to progressive loss of tissue function.     

机械生长因子E肽的功能及研究展望

机械生长因子(MGF)E肽是胰岛素样生长因子Ⅰ(IGF-Ⅰ)基因剪接后的一段长40个氨基酸残基的延伸肽,其编码基因由IGF-Ⅰ基因的外显子5、6及部分外显子4组成。近年来的实验证明,MGFE肽能独立发挥促进肌肉肥大、修复肌肉损伤、保护神经元、提高心脏功能等多种重要的生理作用,有望对肌肉萎缩、肌营养不良、神经退行性疾病及大脑局部缺血等相关病症的新型药物开发产生重大推动,引起了国内外学者的广泛关注。     

IGF-I synergizes with FGF-2 to stimulate oligodendrocyte progenitor entry into the cell cycle

Secreted peptide growth factors are critical extracellular signals that interact to promote the proliferation, differentiation, and survival of progenitor cells in developing tissues. IGF-I signaling through the IGF type I receptor provides a mitogenic signal for numerous cell types, including stem and progenitor cells. We have utilized the O-2A oligodendrocyte progenitor to study the mechanism of IGF-I mitogenic actions since these progenitors respond to IGF-I in vitro, and gene targeting studies in mice have demonstrated that IGF-I is essential for normal oligodendrocyte development in vivo. The goal of this study was to elucidate the mechanism by which IGF-I promotes the proliferation of oligodendrocyte progenitors in the context of other mitogens critical for their proliferation. Results presented here show that IGF-I significantly amplified the actions of FGF-2 and PDGF to promote DNA synthesis in O-2A progenitors. Investigation of cell cycle kinetics revealed that IGF-I had no significant effect on the rate of cell cycle progression. Instead, IGF-I promoted increased recruitment of O-2A progenitors into the S phase of the cell cycle. These studies support a role for IGF-I as a cell cycle progression factor for progenitor cells.     

The structure-function relationships of insulin-like growth factor 1 Ec in C2C12 cells

Insulin-like growth factor 1 (IGF1) is a crucial growth factor, that regulates skeletal muscles development during cell growth and repair. Recently, its alternative splicing variant, named IGF1Ec, also named mechano-growth factor (MGF), has gained attentions as a new damage repair factor. However, the structure-function relationships of IGF1Ec have not been fully clarified due to contradictory reports. In this study, we systematically investigated physiologic responses of C2C12 muscle cells to IGF1Ec, IGF1 and MGF E peptide. Our data indicate that while the N-terminal sequence of IGF1Ec, which is homolog in part with IGF1, promotes proliferation; the C-terminal sequence of IGF1Ec, which is identical to MGF E, promotes differentiation and migration of C2C12 cells. Our results suggest that MGF E cannot completely replace all the functions of IGF1Ec on muscle repair and regeneration, and elucidate the relationships between structure and function of IGF1Ec.     

Novel activities of pro-IGF-I E peptides: regulation of morphological differentiation and anchorage-independent growth in human neuroblastoma cells

Insulin-like growth factor-I (IGF-I) is translated as a pre-pro-peptide that is posttranslationally processed to its mature form by proteolytic removal of the signal peptide and the E-domain peptide. Contrary to the mature human (h) IGF-I, the recombinant rtEa4 -peptide significantly reduced the anchorage-independent cell growth in human neuroblastoma cells (SK-N-F1), shown by colony formation assay in vitro. Significant inhibition of colony formation is also observed in SK-N-F1 cells stably transfected with a bicistronic expression construct encoding a secretory form of the rtEa4 peptide. Furthermore, treatment with the recombinant rtEa4 peptide, but not the mature hIGF-I, resulted in morphological differentiation of SK-N-F1 cells characterized by long neurite outgrowth. Similar morphological differentiation is also observed in SK-N-F1 cell clones stably transfected with the rtEa4 peptide expression construct. A spectrum of biological activities similar to those of rtEa4 peptide is also observed in the synthetic hEb peptide, but not-the hEa peptide. Results of further characterization reveal that neurites induced by rtEa4 or hEb peptide contain neuronal-specific MAP-2, Tau, and neurofilament (NF-160), accompanied by an increased expression of the neuronal marker gene neuropeptide tyrosine (NPY). Furthermore, effects of signal transduction inhibitors are indicative of the involvement of MAP-kinase PI-3-kinase cascades. The activation of ERK-1/-2 is markedly increased in response to rtEa-4 or hEb peptide stimulation, further indicating the involvement of MAPK signaling cascade. These unique biological activities exhibited by the rtEa4 or hEb peptide suggest that E peptide of the pro-IGF-I may play distinct roles in regulating cell growth and differentiation in neuroblastoma cells.     

Insulin-like growth factor 1 (IGF-I) improves hepatic differentiation of human bone marrow-derived mesenchymal stem cells

The ability of MSCs (mesenchymal stem cells) to differentiate between other cell types makes these cells an attractive therapeutic tool for cell transplantation. This project was designed to improve transdifferentiation of human MSCs into liver cells using IGF-I (insulin-like growth factor 1) which, despite its important role in liver development, has not been used for in vitro hepatic differentiation. In the present study, the MSCs derived from healthy human bone marrow samples were cultured and characterized by immunophenotyping and differentiation potential into osteoblast and adipocytes. Transdifferentiation into hepatocyte-like cells was performed in the presence/absence of IGF-I in combination with predefined hepatic differentiation cocktail. To evaluate transdifferentiation, morphological features, immuno-cytochemical staining of specific biological markers and hepatic functions were assessed. Morphological assessment and evaluation of glycogen content, albumin and AFP (α-feto protein) expression as well as albumin and urea secretion revealed statistically significant difference between experimental groups compared with the control. Morphology and function (albumin secretion) of IGF-I-treated cells were significantly better than IGF-I-free experimental group. To the best of our knowledge, our study is the first to demonstrate that the combination of IGF-I with the predefined hepatic differentiation cocktail will significantly improve the morphological features of the differentiated cells and albumin secretion.     

Milk growth factor (MGF) induces transformation into ATDC5 cells, prechondrocytes, and cooperates with retinoic acid to transform the cells into new forms

The effects of milk growth factor (MGF) showed the transformation of ATDC5 prechondrocytes and differed from that of retinoic acid (RA) as follows. MGF (200 ng/ml) did not suppress the proliferation of ATDC5 cells, though RA (1 x 10(-7) M) suppressed the cell proliferation. However, MGF showed the result as RA, which was verified to suppress the production of proteoglycan. The synthesis of vimentin in ATDC5 cells was slightly induced by RA, but its withdrawal induced the large-scale induction and the fibril formation of vimentin, which may indicate that the cells became fibroblastic cells, namely dedifferentiation. MGF, which hardly induced the vimentin synthesis in ATDC5 cells, induced its synthesis under control by the withdrawal. MGF suppressed the synthesis of alpha-smooth muscle actin (alpha-SM-actin), which was apt to reverse in its withdrawal. However, RA did not affect this synthesis of ATDC5 cells. The combination of MGF and RA enlarged the cells and enhanced the synthesis of vimentin due to RA under control, however, almost terminated alpha-SM-actin-synthesis in the cells. And its effect is almost irreversible. Furthermore, the combination of MGF and RA prevented the induction of fibroblasts due to RA in the cells. And the withdrawal of the mixture transformed prechondrocytes into hypertrophic cells. Then, MGF contributes to bone metabolism in prechondrocyte.     

Insulin-like growth factor I (IGF-I) receptors and IGF-I action in oligodendrocytes from rat brains.

Oligodendrocyte progenitor cells were prepared by mechanical dissociation of 1-day-old rat brain cultures. These cells undergo proliferation and differentiation into oligodendrocytes as demonstrated by the expression of proliferation and differentiation-related specific antigens. We have used this unique culture system to characterize insulin-like growth factor I (IGF-I) receptors and their action in the central nervous system (CNS). 125I-IGF-I specifically binds to these cultures with high affinity. Competition-inhibition data suggest that IGF-I is most potent in competing for 125I-IGF-I binding, followed by IGF-II and insulin. Scatchard analyses of the binding data indicate a curvilinear plot with a Kd for high affinity of 0.2 nM, and a Bmax of 247 fmol/mg, and a Kd for low affinity of 3.2 nM and Bmax of 1213 fmol/mg protein. Covalent cross-linking followed by SDS-PAGE analysis demonstrated a radioactive band of Mr 135,000 which corresponds to the alpha subunit of the IGF-I receptor. Solution hybridization/RNase protection assay produced a single protected band corresponding to IGF-I receptor messenger RNA, further confirming the presence of these receptors. Incubation of progenitor cells with IGF-I resulted in a time- and concentration-dependent increase in [3H]thymidine incorporation and cell numbers. This effect appears to be mediated by IGF-I receptors since IGF-II and insulin were proportionately less potent. In addition to its effect on proliferation, IGF-I also increased the number of 4E7- and GC-antigen positive cells. These observations indicate that oligodendrocytes in primary culture express specific IGF-I receptors and that the interaction of IGF-I with these receptors results in the proliferation as well as differentiation of oligodendrocytes.     

MGF E peptide improves anterior cruciate ligament repair by inhibiting hypoxia-induced cell apoptosis and accelerating angiogenesis

Severe hypoxic microenvironment endangers cell survival of anterior cruciate ligament (ACL) fibroblasts and is harmful to ACL repair and regeneration. In the current study, we explored the effects of mechanogrowth factor (MGF) E peptide on the hypoxia-induced apoptosis of ACL fibroblasts and relevant mechanisms. It demonstrated that severe hypoxia promoted hypoxia-inducible factor-1α (HIF-1α) expression and caused cell apoptosis of ACL fibroblasts through increasing caspase 3/7/9 messenger RNA (mRNA), cleaved caspase 3 and proapoptotic proteins expression levels but decreasing antiapoptotic proteins expression levels. Fortunately, MGF E peptide effectively protected ACL fibroblasts against hypoxia-induced apoptosis through regulating caspase 3/7/9 mRNA, cleaved caspase 3 and apoptosis-relevant proteins expression levels. Simultaneously, mitochondrial, @@@MEK-ERK1/2 (extracellular-signal-regulated kinase 1/2), and phosphoinositide-3-kinase-protein kinase B (PI3K-Akt) pathways were involved in MGF E peptide regulating hypoxia-induced apoptosis of ACL fibroblasts. In rabbit ACL rupture model, MGF E peptide also decreased HIF-1α expression levels, cell apoptosis, and facilitated cell proliferation. In addition, MGF could accelerate angiogenesis after ACL injury probably owing to its recruitment of proangiogenesis cells by stromal cell-derived factor 1α/CXCR4 axis and stimulation of vascular endothelial growth factor α expression level. In conclusion, our findings suggested that MGF E peptide could be utilized for ACL repair and regeneration and supplied experimental support for its application in clinical ACL treatment as a potential strategy.     

Insulin-Like Growth Factor I Receptor Expression and Function in Nerve Growth Factor-Differentiated PC12 Cells

Abstract: Receptors for insulin-like growth factor I (IGF-I) were studied on PC12EY cells, a subclone of PC12. Differentiation of PC12EY cells with nerve growth factor (NGF) did not alter either the number of IGF-I receptors nor their affinity for IGF-I. IGF-I receptors remained fully functional during differentiation, promoting increases in thymidine incorporation, glucose uptake, amino acid uptake, and the phosphorylation of the S6 protein of the ribosomes. IGF-I also increased the proportion of differentiated cells found in S-phase. But although the addition of IGF-I to naive cells caused an increase in cell number, there was no comparable increase when IGF-I was added to differentiated cells. Thus, although the receptor for IGF-I continues to be present and functional, IGF-I fails to induce cell proliferation in differentiated PC12 cells.     

力生长因子表达调节及其功能机制

力生长因子(mechano growth factor,MGF)是胰岛素样生长因子1（insulin-like growth factor-1,IGF-1)的选择性剪接变异体,具有力敏感性.本文综述了MGF在组织细胞中的表达、调节及其功能机制的最新研究.首先阐述了MGF在多种细胞和组织中的表达和功能,其次说明MGF表达受应力、损伤、激素、温度等多种因素的调节.综述各项研究显示,MGF不依靠IGF-1R发挥作用,而是直接激活骨骼肌卫星细胞,促肌细胞增殖,进而促骨骼肌肥大,修复受损肌肉. 通过激活Erk磷酸化促成肌细胞增殖、保护心肌细胞,上调血红素加氧酶-1（heme oxygenase-1,HO-1）,激活蛋白激酶Cε(protein kinase Cε,PKCε）和NF-E2-相关因子2（NF-E2-related factor2,Nrf2 )发挥保护神经的作用.另外,MGF发挥作用还可能与Wnt/β-catenin信号有关.对MGF作用及其作用机制深入研究有助于未来MGF在临床的运用.     

Autocrine stimulation by insulin-like growth factor I is involved in the growth, tumorigenicity and chemoresistance of human esophageal carcinoma cells

Insulin-like growth factor I (IGF-I) receptor (IGF-IR)-mediated signals are known to be involved in cell growth and transformation and prevention of apoptosis. In this study, we demonstrated the coexpression of IGF-I and IGF-IR in human esophageal carcinoma tissues. We also demonstrated the IGF-I autocrine system in esophageal carcinoma cell lines. Both the CE48T/VGH and CE81T/VGH cell lines showed proliferative responses to IGF-I stimulation. Autokinase activity of IGF-IR in these cells can be triggered by the exogenous addition of IGF-I. In addition, an IGF-I peptide antagonist, JB1, specifically inhibited ligand-induced receptor autophosphorylation in a dose-dependent manner. Under serum-free conditions, JB1 also reduced the degree of IGF-IR phosphorylation and cell numbers. Furthermore, the addition of JB1 decreased the number of CE81T/VGH colonies formed in methyl cellulose agar and the size and the incidence of tumors which grew in mice with severe combined immunodeficiency. These results imply that an IGF-I autocrine system in human esophageal carcinoma cells could stimulate tumor growth. Finally, we found that IGF-I prevented the apoptosis of CE81T/VGH cells induced by chemotherapeutic drugs, such as cisplatin, 5-fluorouracil and camptothecin. Thus, interruption of IGF-IR function may provide a way to retard tumor growth and increase the sensitivity of esophageal carcinoma to chemotherapy.     

Expression of insulin-like growth factor I, insulin-like growth factor binding proteins, and collagen mRNA in mechanically loaded plantaris tendon.

Insulin-like growth factor I (IGF-I) is known to exert an anabolic effect on tendon fibroblast production of collagen. IGF-I's regulation is complex and involves six different IGF binding proteins (IGFBPs). Of these, IGFBP-4 and -5 could potentially influence the effect of IGF-I in the tendon because they both are produced in fibroblast; however, the response of IGFBP-4 and -5 to mechanical loading and their role in IGF-I regulation in tendinous tissue are unknown. A splice variant of IGF-I, mechano-growth factor (MGF) is upregulated and known to be important for adaptation in loaded muscle. However, it is not known whether MGF is expressed and upregulated in mechanically loaded tendon. This study examined the effect of mechanical load on tendon collagen mRNA in relation to changes in the IGF-I systems mRNA expression. Data were collected at 2, 4, 8 and 16 days after surgical removal of synergistic muscle to the plantaris muscle of the rat, thus increasing the load to plantaris muscle and tendon. Nearly a doubling of the tendon mass was observed after 16 days of loading. A rapid rise in tendon procollagen III mRNA was seen after 2 days whereas the increase in procollagen I mRNA was significant from day 8. MGF was expressed and upregulated in loaded tendon tissue with a faster response than IGF-I, which was increased from day 8. Finally, IGFBP-4 mRNA was increased with a time pattern similar to procollagen III, whereas IGFBP-5 decreased at day 8. In conclusion, loading of tendon tissue results in an upregulation of IGF-I, IGFBP-4, and procollagen and is associated with an increase in tendon mass. Also, MGF is expressed with an early upregulation in loaded tendon tissue. We suggest that the IGF-I system could be involved in collagen synthesis in tendon in response to mechanical loading.     

中脑神经前体细胞的体外分离、培养和特性

为了解中脑神经前体细胞的体外培养特性和建立中脑神经前体细胞的体外分化调控机制提供细胞模型。本实验采用含有丝分裂源表皮生长因子(EGF)的无血清培养基培养来源于大鼠胚胎E14.5天的中脑神经前体细胞,应用免疫细胞化学方法了解其前体细胞特性。结果发现中脑神经前体细胞呈神经前体细胞特征性标记Nestin免疫染色阳性,无分化细胞标记;细胞克隆实验证实中脑神经前体细 胞有自我更新能力;在EGF刺激下增殖迅速;当撤去EGF后置于含胎牛血清的培养基和被覆多聚赖氨酸(PLL)的培养皿内,中脑神经前体细胞可分化成神经元和星形胶质细胞。本试验证明我们培养的中脑神经前体细胞具有增殖、自我更新能力和多向分化潜能特性。     

IGF-I and procollagen alpha1(I) are coexpressed in a subset of mesenchymal cells in active Crohn's disease

This study tested the hypothesis that insulin-like growth factor I (IGF-I) expression is increased at sites of fibrosis in diseased intestine of patients with Crohn's disease (CD). IGF-I mRNA was quantified by RNase protection assay in uninvolved and involved intestine of 13 CD patients (10 ileum, 3 colon) and 7 ulcerative colitis (UC) patients (colon). In situ hybridization histochemistry compared the localization of IGF-I and procollagen alpha1(I) mRNAs. Masson's trichrome staining and immunohistochemistry for IGF-I precursor, alpha-smooth muscle actin (A), vimentin (V), desmin (D), and c-kit were used to examine the mesenchymal cell subtypes that express IGF-I and collagen in uninvolved and involved ileum and colon of CD patients and "normal" ileum and colon from noninflammatory controls. IGF-I mRNA was elevated in involved ileum and colon of patients with CD but not in involved colon of patients with UC. IGF-I and procollagen alpha1(I) mRNA showed overlapping distribution within fibrotic submucosa and muscularis propria of involved CD ileum and colon. In involved CD intestine, increased IGF-I precursor expression localized to mesenchymal cells in regions of tissue disorganization and fibrosis in muscularis mucosa, submucosa, and muscularis propria. In these regions, there were increased numbers of V(+) cells relative to normal or uninvolved intestine. Increased IGF-I expression was localized to cells with a phenotype typical of fibroblasts (V(+)/A(-)/D(-)), myofibroblasts (V(+)/A(+)/D(+)), and, to a lesser extent, cells with normal enteric smooth muscle phenotype (V(-)/A(+)/D(+)). We conclude that increased IGF-I expression in multiple mesenchymal cell subtypes and increased numbers of cells with fibroblast/myofibroblast phenotype are involved in fibrosis associated with CD.     

Expression of muscle anabolic and metabolic factors in mechanically loaded MLO-Y4 osteocytes

Lack of physical activity results in muscle atrophy and bone loss, which can be counteracted by mechanical loading. Similar molecular signaling pathways are involved in the adaptation of muscle and bone mass to mechanical loading. Whether anabolic and metabolic factors regulating muscle mass, i.e., insulin-like growth factor-I isoforms (IGF-I Ea), mechano growth factor (MGF), myostatin, vascular endothelial growth factor (VEGF), or hepatocyte growth factor (HGF), are also produced by osteocytes in bone in response to mechanical loading is largely unknown. Therefore, we investigated whether mechanical loading by pulsating fluid flow (PFF) modulates the mRNA and/or protein levels of muscle anabolic and metabolic factors in MLO-Y4 osteocytes. Unloaded MLO-Y4 osteocytes expressed mRNA of VEGF, HGF, IGF-I Ea, and MGF, but not myostatin. PFF increased mRNA levels of IGF-I Ea (2.1-fold) and MGF (2.0-fold) at a peak shear stress rate of 44Pa/s, but not at 22Pa/s. PFF at 22 Pa/s increased VEGF mRNA levels (1.8- to 2.5-fold) and VEGF protein release (2.0- to 2.9-fold). Inhibition of nitric oxide production decreased (2.0-fold) PFF-induced VEGF protein release. PFF at 22 Pa/s decreased HGF mRNA levels (1.5-fold) but increased HGF protein release (2.3-fold). PFF-induced HGF protein release was nitric oxide dependent. Our data show that mechanically loaded MLO-Y4 osteocytes differentially express anabolic and metabolic factors involved in the adaptive response of muscle to mechanical loading (i.e., IGF-I Ea, MGF, VEGF, and HGF). Similarly to muscle fibers, mechanical loading enhanced expression levels of these growth factors in MLO-Y4 osteocytes. Although in MLO-Y4 osteocytes expression levels of IGF-I Ea and MGF of myostatin were very low or absent, it is known that the activity of osteoblasts and osteoclasts is strongly affected by them. The abundant expression levels of these factors in muscle cells, in combination with low expression in MLO-Y4 osteocytes, provide a possibility that growth factors expressed in muscle could affect signaling in bone cells.