低氧对成肌细胞体外分化的影响及其机制的研究
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摘要
氧在能量代谢和机体内环境稳定以及一系列生命活动中起着重要的调节作用。氧浓度的改变,作为一种重要的生理或病理性的调节因素,影响着从胚胎发生发育到机体正常功能的维持以及功能紊乱,疾病,衰老整个生命过程。大气中氧浓度为21%,而在组织水平,氧浓度显著地低于环境中大气氧浓度,动脉血中氧浓度为12%,成年组织中平均的氧浓度大约为3%,成熟骨骼肌细胞中的氧浓度大约在1~10%。哺乳动物骨骼肌中存在着少量的肌肉干细胞,肌肉干细胞可激活、增殖形成单核的成肌细胞,并融合分化为多核肌管。从骨骼肌中分离获得的肌卫星细胞在体外培养时被统称为成肌细胞。成肌细胞在出生后骨骼肌的损伤修复和维持中发挥着重要的作用。而生理性的低氧条件才是成肌发生过程中的最适条件,目前关于低氧对成肌细胞增殖和分化的影响的研究报道甚少。本研究选取C2C12细胞作为实验对象,观察生理性低氧(3% O2)对成肌细胞体外分化的影响,并探讨低氧调控成肌细胞分化过程中可能参与的信号机制。
我们首先进行了免疫荧光和免疫细胞化学实验,从表型的水平观察了C2C12细胞分别在常氧和低氧(3% O2)条件下的分化情况。结果表明,与常氧条件下相比,低氧条件下诱导分化的C2C12细胞几乎很少融合形成肌管并表达成肌终末分化的标志蛋白MHC。Western Blot分析也进一步证实了这一结果。利用RT-PCR和Western Blot分析检测了C2C12细胞中MRFs的表达变化,结果显示,低氧下调了MRFs在mRNA和蛋白水平的表达。以上结果提示,低氧(3% O2)抑制了成肌细胞的体外分化。
在明确了低氧抑制成肌细胞体外分化这一现象后,我们进一步探讨了低氧抑制成肌细胞体外分化过程中可能参与的信号机制。低氧条件下HIF-1α的稳定和激活是细胞对低氧诱导反应的一个主要的特点。为了研究HIF-1α是否参与了低氧对成肌细胞体外分化的抑制作用,我们利用RT-PCR和Western Blot分析检测了低氧条件下诱导分化的C2C12细胞中HIF-1α在蛋白水平和转录水平的表达,结果表明,C2C12细胞中的HIF-1α蛋白在常氧条件下稳定,没有发生降解,HIF-1α在蛋白水平和转录水平几乎不受低氧环境的影响。同时检测了HIF-1α在诱导分化的C2C12细胞中的定位,发现低氧没有影响HIF-1α蛋白在细胞中的分布。以上结果表明,低氧抑制成肌细胞的体外分化可能是不依赖于HIF-1信号通路的。
在研究过程中,我们发现在低氧抑制成肌细胞体外分化过程中Smad3蛋白的表达水平降低,然而,在C2C12细胞中过表达外源性的Smad3没有能够逆转低氧对成肌细胞分化的抑制作用。结果提示,Smad3蛋白在低氧抑制成肌细胞分化过程中不起关键的作用,Smad3蛋白水平的降低可能是低氧抑制成肌细胞体外分化过程中的一个伴随事件。随后我们研究低氧抑制成肌细胞体外分化过程中Smad3蛋白水平降低的原因时发现,Smad3的活性形式磷酸化Smad3蛋白的表达水平在低氧抑制成肌细胞体外分化的过程中下调,与Smad3蛋白水平的降低趋势相平行。有文献报道,磷酸化Smad3蛋白经泛素-蛋白酶体途径降解,于是我们使用了26S蛋白酶体的抑制剂MG-132,发现在C2C12细胞的分化培养液中加入MG-132后,引起了低氧条件下磷酸化Smad3蛋白的积累。以上结果提示,在低氧抑制成肌细胞体外分化的过程中,低氧加速了磷酸化Smad3蛋白的泛素-蛋白酶体途径的降解。
ERK1/2 MAPK信号通路在成肌分化过程中扮演着一定的角色。我们研究发现在诱导C2C12细胞分化过程中ERK1/2磷酸化激活,而在低氧抑制成肌细胞体外分化过程中ERK1/2的激活受到抑制。那么,是否ERK1/2 MAPK信号通路参与了低氧抑制成肌细胞体外分化的调控呢?我们瞬时转染激活型的MEK1(E)表达质粒,在C2C12细胞中过表达ERK1/2的上游激酶MEK1从而磷酸化激活ERK1/2,发现在低氧条件下,过表达激活型的MEK1(E)能够部分地逆转低氧对C2C12细胞分化的抑制作用。同时,利用免疫荧光实验检测了瞬时转染激活型MEK1(E)后,C2C12细胞在低氧条件下的分化情况。结果显示,在低氧(3% O2)条件下,表达MHC蛋白的阳性细胞的数目与转染空载体组相比有所增加。以上结果提示,ERK1/2 MAPK信号通路可能参与了低氧抑制成肌细胞体外分化的调控。
综合本研究的结果,结合本实验室前期的研究工作(3% O2促进成肌细胞的增殖)我们发现了一个新的低氧调控肌肉发育的效应:~3% O2浓度的生理性低氧环境促进成肌细胞的增殖,同时抑制成肌细胞的分化。在研究低氧抑制成肌细胞体外分化的机制时,发现了可能存在与通常低氧诱导的效应通路不同的机制,ERK1/2 MAPK信号通路可能参与了低氧抑制成肌细胞体外分化的过程。
Oxygen plays an important role in energy metabolization and homeostasis as well as a variety of life processes. Changes of oxygen concentration, as an important physiological and pathological regulator, might influence the whole life span, from embryogenesis and development to the maintenance of normal function, dysfunction, disease and aging. The concentration of oxygen in atmosphere is 21%, whereas the level of oxygen at the tissue in vivo is significantly less. Arterial blood is about 12% oxygen, and the mean tissue level of oxygen is about 3%, the partial saturation of oxygen found in mature skeletal muscle is reported to be between 1 to 10%. A few muscle stem cells exist in adult mammalian skeletal muscle. Muscle stem cells can be activated to initiate proliferation and give rise to spindly myoblasts, which undergo differentiation and cell fusion into multinucleated myotubes. Muscle satellite cells isolated from skeletal muscle and cultured in vitro are called myoblasts. It is obvious that the tissue-specific myoblasts are required for maintenance and repair of postnatal skeletal muscle. So, physiological hypoxia is actually optimum condition for myogenesis. There have been few studies on effects of hypoxia on proliferation and differentiation of myoblasts up to now. In the present study we observed effects of 3% O2 on myogenic differentiation of C2C12 myoblasts and probed possible mechanisms involved in the inhibition of myogenic differentiation by hypoxia.
We firstly observed the differentiation of C2C12 myoblasts in normoxia or 3% O2 by using immunofluorescence and immunocytochemistry analysis. The results showed that C2C12 myoblasts cultured in DM under 3% O2 conditions hardly fused into myotubes and expressed MHC protein compared with cells cultured under normoxic conditions, which was also confirmed by Western Blot analysis. RT-PCR and Western Blot analysis detected the expression of MRFs in C2C12 myoblasts, and hypoxia down-regulated the expression of MRFs at both mRNA and protein levels.
The above data indicated that 3% O2 inhibited myogenic differentiation of myoblasts. After the phenomenon of the inhibition of myogenic differentiation by hypoxia was demonstrated, we further probed possible mechanisms involved in the inhibition of myogenic differentiation by hypoxia. HIF-1 is an important transcription factor expressed in response to hypoxic conditions. To investigate whether HIF-1|áwas involved in the inhibition of myogenic differentiation by hypoxia, RT-PCR and Western Blot analysis detected the expression of HIF-1|áat both mRNA and protein levels in C2C12 myoblasts cultured in DM under hypoxic conditions. The results showed that HIF-1|áprotein in C2C12 myoblasts was stable and not degraded under normoxic conditions, and the expression of HIF-1|áwas hardly influenced by oxygen conditions at both protein level and transcriptional level. Meanwhile, we detected the location of HIF-1|áprotein in differentiating C2C12 myoblasts and found that hypoxia did not influence the location of HIF-1|áprotein. These above results suggest that the inhibition of myogenic differentiation by hypoxia might be independent of HIF-1 signaling.
We found that the expression level of Smad3 protein decreased during the inhibition of myogenic differentiation by hypoxia. However, the forced expression of exogenous Smad3 did not rescue the inhibition of myogenic differentiation by hypoxia. These data indicated that Smad3 protein did not play a vital role during the inhibition of myogenic differentiation by hypoxia. In addition, down-regulation of Smad3 protein might be a concomitant event during the inhibition of myogenic differentiation by hypoxia. Subsequently, we investigated the possible cause of Smad3 protein decrease and found that the expression level of phospho-Smad3 protein deceased during the inhibition of myogenic differentiation by hypoxia, which was paralleled by a decrease in Smad3 protein under hypoxic conditions. It was reported that the eventual fate of receptor-activated Smads was controlled by ubiquitin-mediated proteolysis. We added MG-132, the inhibitor of the 26S proteasome, into cultures and found that MG-132 induced accumulation of phospho-Smad3 in C2C12 myoblasts cultured in hypoxia. These results suggest that hypoxia-induced loss of phospho-Smad3 protein involve its degradation by the ubiquitin-proteasome pathway.
ERK1/2 MAPK pathway plays a role in myogenesis. Further investigation on the mechanism involved in the inhibition of myogenic differentiation by hypoxia showed that ERK1/2 were phosphorylated and activated in differentiating C2C12 myoblasts, however, ERK1/2 activation was suppressed during the inhibition of myogenic differentiation by hypoxia. To investigate whether ERK1/2 MAPK pathway was involved in the inhibition of myogenic differentiation by hypoxia, we transiently transfected a constitutively active MEK1(E) construct, which phosphorylated and activated ERK1/2 in C2C12 myoblasts. Western Blot analysis showed that the level of MHC expression in C2C12 myoblasts transfected with MEK1(E) was higher than that in cells transfected with empty vector control under hypoxic conditions, which was also confirmed by immunofluorescence analysis. These results indicated that increasing ERK1/2 activity by forced expression of MEK1(E) could partially reverse the inhibition of myogenic differentiation by hypoxia. These suggest that ERK1/2 MAPK pathway might be involved in the inhibition of myogenic differentiation by hypoxia.
Taken together, combining with our previous laboratory results that 3% O2 promoted proliferation of myoblasts, our findings indicated a new effect of hypoxia on skeletal muscle development that physiological hypoxia (3% O2) promoted proliferation of myoblasts and inhibited myogenic differentiation of myoblasts. During investigating possible mechanisms involved in the inhibition of myogenic differentiation by hypoxia, we found a possible mechanism that ERK1/2 MAPK pathway might be involved in hypoxia-mediated inhibition of myogenic differentiation.
我们首先进行了免疫荧光和免疫细胞化学实验,从表型的水平观察了C2C12细胞分别在常氧和低氧(3% O2)条件下的分化情况。结果表明,与常氧条件下相比,低氧条件下诱导分化的C2C12细胞几乎很少融合形成肌管并表达成肌终末分化的标志蛋白MHC。Western Blot分析也进一步证实了这一结果。利用RT-PCR和Western Blot分析检测了C2C12细胞中MRFs的表达变化,结果显示,低氧下调了MRFs在mRNA和蛋白水平的表达。以上结果提示,低氧(3% O2)抑制了成肌细胞的体外分化。
在明确了低氧抑制成肌细胞体外分化这一现象后,我们进一步探讨了低氧抑制成肌细胞体外分化过程中可能参与的信号机制。低氧条件下HIF-1α的稳定和激活是细胞对低氧诱导反应的一个主要的特点。为了研究HIF-1α是否参与了低氧对成肌细胞体外分化的抑制作用,我们利用RT-PCR和Western Blot分析检测了低氧条件下诱导分化的C2C12细胞中HIF-1α在蛋白水平和转录水平的表达,结果表明,C2C12细胞中的HIF-1α蛋白在常氧条件下稳定,没有发生降解,HIF-1α在蛋白水平和转录水平几乎不受低氧环境的影响。同时检测了HIF-1α在诱导分化的C2C12细胞中的定位,发现低氧没有影响HIF-1α蛋白在细胞中的分布。以上结果表明,低氧抑制成肌细胞的体外分化可能是不依赖于HIF-1信号通路的。
在研究过程中,我们发现在低氧抑制成肌细胞体外分化过程中Smad3蛋白的表达水平降低,然而,在C2C12细胞中过表达外源性的Smad3没有能够逆转低氧对成肌细胞分化的抑制作用。结果提示,Smad3蛋白在低氧抑制成肌细胞分化过程中不起关键的作用,Smad3蛋白水平的降低可能是低氧抑制成肌细胞体外分化过程中的一个伴随事件。随后我们研究低氧抑制成肌细胞体外分化过程中Smad3蛋白水平降低的原因时发现,Smad3的活性形式磷酸化Smad3蛋白的表达水平在低氧抑制成肌细胞体外分化的过程中下调,与Smad3蛋白水平的降低趋势相平行。有文献报道,磷酸化Smad3蛋白经泛素-蛋白酶体途径降解,于是我们使用了26S蛋白酶体的抑制剂MG-132,发现在C2C12细胞的分化培养液中加入MG-132后,引起了低氧条件下磷酸化Smad3蛋白的积累。以上结果提示,在低氧抑制成肌细胞体外分化的过程中,低氧加速了磷酸化Smad3蛋白的泛素-蛋白酶体途径的降解。
ERK1/2 MAPK信号通路在成肌分化过程中扮演着一定的角色。我们研究发现在诱导C2C12细胞分化过程中ERK1/2磷酸化激活,而在低氧抑制成肌细胞体外分化过程中ERK1/2的激活受到抑制。那么,是否ERK1/2 MAPK信号通路参与了低氧抑制成肌细胞体外分化的调控呢?我们瞬时转染激活型的MEK1(E)表达质粒,在C2C12细胞中过表达ERK1/2的上游激酶MEK1从而磷酸化激活ERK1/2,发现在低氧条件下,过表达激活型的MEK1(E)能够部分地逆转低氧对C2C12细胞分化的抑制作用。同时,利用免疫荧光实验检测了瞬时转染激活型MEK1(E)后,C2C12细胞在低氧条件下的分化情况。结果显示,在低氧(3% O2)条件下,表达MHC蛋白的阳性细胞的数目与转染空载体组相比有所增加。以上结果提示,ERK1/2 MAPK信号通路可能参与了低氧抑制成肌细胞体外分化的调控。
综合本研究的结果,结合本实验室前期的研究工作(3% O2促进成肌细胞的增殖)我们发现了一个新的低氧调控肌肉发育的效应:~3% O2浓度的生理性低氧环境促进成肌细胞的增殖,同时抑制成肌细胞的分化。在研究低氧抑制成肌细胞体外分化的机制时,发现了可能存在与通常低氧诱导的效应通路不同的机制,ERK1/2 MAPK信号通路可能参与了低氧抑制成肌细胞体外分化的过程。
Oxygen plays an important role in energy metabolization and homeostasis as well as a variety of life processes. Changes of oxygen concentration, as an important physiological and pathological regulator, might influence the whole life span, from embryogenesis and development to the maintenance of normal function, dysfunction, disease and aging. The concentration of oxygen in atmosphere is 21%, whereas the level of oxygen at the tissue in vivo is significantly less. Arterial blood is about 12% oxygen, and the mean tissue level of oxygen is about 3%, the partial saturation of oxygen found in mature skeletal muscle is reported to be between 1 to 10%. A few muscle stem cells exist in adult mammalian skeletal muscle. Muscle stem cells can be activated to initiate proliferation and give rise to spindly myoblasts, which undergo differentiation and cell fusion into multinucleated myotubes. Muscle satellite cells isolated from skeletal muscle and cultured in vitro are called myoblasts. It is obvious that the tissue-specific myoblasts are required for maintenance and repair of postnatal skeletal muscle. So, physiological hypoxia is actually optimum condition for myogenesis. There have been few studies on effects of hypoxia on proliferation and differentiation of myoblasts up to now. In the present study we observed effects of 3% O2 on myogenic differentiation of C2C12 myoblasts and probed possible mechanisms involved in the inhibition of myogenic differentiation by hypoxia.
We firstly observed the differentiation of C2C12 myoblasts in normoxia or 3% O2 by using immunofluorescence and immunocytochemistry analysis. The results showed that C2C12 myoblasts cultured in DM under 3% O2 conditions hardly fused into myotubes and expressed MHC protein compared with cells cultured under normoxic conditions, which was also confirmed by Western Blot analysis. RT-PCR and Western Blot analysis detected the expression of MRFs in C2C12 myoblasts, and hypoxia down-regulated the expression of MRFs at both mRNA and protein levels.
The above data indicated that 3% O2 inhibited myogenic differentiation of myoblasts. After the phenomenon of the inhibition of myogenic differentiation by hypoxia was demonstrated, we further probed possible mechanisms involved in the inhibition of myogenic differentiation by hypoxia. HIF-1 is an important transcription factor expressed in response to hypoxic conditions. To investigate whether HIF-1|áwas involved in the inhibition of myogenic differentiation by hypoxia, RT-PCR and Western Blot analysis detected the expression of HIF-1|áat both mRNA and protein levels in C2C12 myoblasts cultured in DM under hypoxic conditions. The results showed that HIF-1|áprotein in C2C12 myoblasts was stable and not degraded under normoxic conditions, and the expression of HIF-1|áwas hardly influenced by oxygen conditions at both protein level and transcriptional level. Meanwhile, we detected the location of HIF-1|áprotein in differentiating C2C12 myoblasts and found that hypoxia did not influence the location of HIF-1|áprotein. These above results suggest that the inhibition of myogenic differentiation by hypoxia might be independent of HIF-1 signaling.
We found that the expression level of Smad3 protein decreased during the inhibition of myogenic differentiation by hypoxia. However, the forced expression of exogenous Smad3 did not rescue the inhibition of myogenic differentiation by hypoxia. These data indicated that Smad3 protein did not play a vital role during the inhibition of myogenic differentiation by hypoxia. In addition, down-regulation of Smad3 protein might be a concomitant event during the inhibition of myogenic differentiation by hypoxia. Subsequently, we investigated the possible cause of Smad3 protein decrease and found that the expression level of phospho-Smad3 protein deceased during the inhibition of myogenic differentiation by hypoxia, which was paralleled by a decrease in Smad3 protein under hypoxic conditions. It was reported that the eventual fate of receptor-activated Smads was controlled by ubiquitin-mediated proteolysis. We added MG-132, the inhibitor of the 26S proteasome, into cultures and found that MG-132 induced accumulation of phospho-Smad3 in C2C12 myoblasts cultured in hypoxia. These results suggest that hypoxia-induced loss of phospho-Smad3 protein involve its degradation by the ubiquitin-proteasome pathway.
ERK1/2 MAPK pathway plays a role in myogenesis. Further investigation on the mechanism involved in the inhibition of myogenic differentiation by hypoxia showed that ERK1/2 were phosphorylated and activated in differentiating C2C12 myoblasts, however, ERK1/2 activation was suppressed during the inhibition of myogenic differentiation by hypoxia. To investigate whether ERK1/2 MAPK pathway was involved in the inhibition of myogenic differentiation by hypoxia, we transiently transfected a constitutively active MEK1(E) construct, which phosphorylated and activated ERK1/2 in C2C12 myoblasts. Western Blot analysis showed that the level of MHC expression in C2C12 myoblasts transfected with MEK1(E) was higher than that in cells transfected with empty vector control under hypoxic conditions, which was also confirmed by immunofluorescence analysis. These results indicated that increasing ERK1/2 activity by forced expression of MEK1(E) could partially reverse the inhibition of myogenic differentiation by hypoxia. These suggest that ERK1/2 MAPK pathway might be involved in the inhibition of myogenic differentiation by hypoxia.
Taken together, combining with our previous laboratory results that 3% O2 promoted proliferation of myoblasts, our findings indicated a new effect of hypoxia on skeletal muscle development that physiological hypoxia (3% O2) promoted proliferation of myoblasts and inhibited myogenic differentiation of myoblasts. During investigating possible mechanisms involved in the inhibition of myogenic differentiation by hypoxia, we found a possible mechanism that ERK1/2 MAPK pathway might be involved in hypoxia-mediated inhibition of myogenic differentiation.
引文
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[45] Beraud C, Henzel WJ, Baeuerle PA. Involvement of regulatory and catalytic subunits of phosphoinositide 3-kinase in NF-|êB activation. Proc Natl Acad Sci USA. 1999, 96: 429-434.
[46] Choy L, Skillington J, Derynck R. Roles of autocrine TGF-|?receptor and Smad signaling in adipocyte differentiation. J Cell Biol. 2000, 149: 667-682.
[47] Centrella M, Horowitz MC, Wozney JM, et al. Transforming growth factor-|?gene family members and bone. Endocr Rev. 1994, 15: 27-39.
[48] Olson EN. Interplay between proliferation and differentiation within the myogenic lineage. Dev Biol. 1992, 154: 261-272.
[49] Olson EN, Sternberg E, Hu JS, et al. Regulation of myogenic differentiation by type|? transforming growth factor. J Cell Biol. 1986, 103: 1799-1805.
[50] Massagu¨| J, Cheifetz S, Endo T, et al. Type|? transforming growth factor is an inhibitor of myogenic differentiation. Proc Natl Acad Sci. 1986, 83: 8206-8210.
[51] Liu D, Black BL, Derynck R. TGF-|? inhibits muscle differentiation through functional repression of myogenic transcription factors by Smad3. Genes Dev. 2001, 15: 2950-2966.
[52] Liu D, Kang JS, Derynck R. TGF-|?-activated Smad3 represses MEF2-dependent transcription in myogenic differentiation. EMBO J. 2004, 23: 1557-1566.
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[54] Akman HO, Zhang H, Siddiqui MAQ, et al. Response to hypoxia involves transforming growth factor-|?2 and Smad proteins in human endothelial cells. Blood. 2001, 98(12): 3324-3331.
[55] Zhang H, Akman HO, Smith ELP, et al. Cellular response to hypoxia involves signaling via Smad proteins. Blood. 2003, 101(6): 2253-2260.
[56] Sa??nchez-Elsner T, Botella LM, Velasco B, et al. Synergistic cooperation between hypoxia and transforming growth factor-|? pathways on human vascular endothelial growth factor gene expression. J Biol Chem. 2001, 276(42):38527-38535.
[57] Nakagawa T, Lan HY, Zhu HJ, et al. Differential regulation of VEGF by TGF-|? and hypoxia in rat proximal tubular cells. Am J Physiol Renal Physiol. 2004, 287: 658-664.
[58] Sa??nchez-Elsner T, Ram???rez JR, Rodriguez-Sanz F,et al. A cross-talk between hypoxia and TGF-|? orchestrates erythropoietin gene regulation through SP1 and Smads. J Mol Biol. 2004, 336: 9-24.
[59] Zhou S, Lechpammer S, Greenberger JS, et al. Hypoxia inhibition of adipocytogenesis in human bone marrow stromal cells requires transforming growth factor-|?/Smad3 signaling. J Biol Chem. 2005, 280(24): 22688-22696.
[60] Gredinger E, Gerber AN, Tamir Y, et al. Mitogen-activated Protein Kinase Pathway Is Involved in the Differentiation of Muscle Cells. J Biol Chem. 1998, 273(17): 10436-10444.
[61] Li J, Johnson SE. ERK2 is required for efficient terminal differentiation of skeletal myoblasts. Biochem Biophys Res Commun. 2006, 345(4): 1425-1433.
[62] Wu Z, Woodring PJ, Bhakta KS, et al. p38 and extracellular signal-regulated kinases regulate the myogenic program at multiple steps. Mol Cell Biol. 2000, 20(11): 3951-3964.
[63] Bennett AM, Tonks NK. Regulation of distinct stages of skeletal muscle differentiation by mitogen-activated protein kinases. Science. 1997, 278: 1288-1291.
[64] Penn BH, Berkes CA, Bergstrom DA, et al. How to MEK muscle. Molecular Cell. 2001, 8(2): 245-246.
[65] Coolican SA, Samuel DS, Ewton DZ, et al. The mitogenic and myogenic actions of insulin-like growth factors utilize distinct signaling pathways. J Biol Chem. 1997, 272(10): 6653¨C6662.
[66] Khurana A, Dey CS. Subtype specific roles of mitogen activated protein kinases in L6E9 skeletal muscle cell differentiation. Mol Cell Biochem. 2002, 238: 27-39.
[67] Rodesch F, Simon P, Donner C, et al. Oxygen measurements in endometrial and trophoblastic tissues during early pregnancy. Obstet Gynecol. 1992, 80(2): 283-285.
[68] Li X, Zhu LL, Chen XP, et al. Effects of hypoxia on proliferation and differentiation of myoblasts. Med Hypotheses. 2007, 69(3): 629-636.
[69] Sambrook J, Frisch EF, Maniatis T. Melecular cloning: a laboratory manual. ColdSpring Harbor Laboratory Press, 1989, 2: 870-896.
[70] Olson EN, Klein WH. bHLH factors in muscle development: dead lines and commitments, what to leave in and what to leave out. Genes Dev. 1994, 8(1): 1-8.
[71] Lassar AB, Skapek SX, Novitch B. Regulatory mechanisms that coordinate skeletal muscle differentiation and cell cycle withdrawal. Curr Opin Cell Biol. 1994, 6(6): 788-794.
[72] Bruick RK. Oxygen sensing in the hypoxic response pathway: regulation of the hypoxia-inducible transcription factor. Genes Dev. 2003, 17: 2614- 2623.
[73] Safran M, Kaelin WG Jr. HIF hydroxylation and the mammalian oxygen-sensing pathway. J Clin Invest. 2003, 111(6): 779-783.
[74] Greijer AE, van der Wall E. The role of hypoxia inducible factor 1 (HIF-1) in hypoxia induced apoptosis. J Clin Pathol. 2004, 57(10): 1009-1014.
[75] Maxwell PH. Hypoxia-inducible factor as a physiological regulator. Exp Physiol. 2005, 90(6): 791-797.
[76] Giaccia AJ, Simon MC, Johnson R. The biology of hypoxia: the role of oxygen sensing in development, normal function, and disease. Genes Dev. 2004, 18: 2183-2194.
[77] Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer. 2003, 3: 721-732.
[78] Lo RS, Massague J. Ubiquitin-dependent degradation of TGF-|?-activated Smad2. Nature Cell Biol. 1999, 1: 472-478.
[79] Wrana JL, Attisano L. The Smad pathway. Cytokine Growth Factor Rev. 2000, 11(1-2): 5-13.
[80] Haddad JJ. Hypoxia and the regulation of mitogen-activated protein kinases: gene transcription and the assessment of potential pharmacologic therapeutic interventions. Int Immunopharmacol. 2004, 4(10-11): 1249-1285.
[81] Ross KR, Corey DA, Dunn JM, et al. SMAD3 expression is regulated by mitogen-activated protein kinase kinase-1 in epithelial and smooth muscle cells. Cell Signal. 2007, 19(5): 923-931.
[82] Zhu LL, Wu LY, Yew DT, et al. Effects of hypoxia on the proliferation and differentiation of NSCs. Mol Neurobiol. 2005, 31(1-3): 231-242.
[83] Poncelet AC, Schnaper HW, Tan R, et al. Cell Phenotype-specific Down-regulation of Smad3 Involves Decreased Gene Activation as Well as Protein Degradation. J Biol Chem. 2007, 282(21): 15534-15540.
[84] Dhillon AS, Hagan S, Rath O et al. MAP kinase signaling pathways in cancer.Oncogene. 2007, 26: 3279-3290.
[85] Meloche S, Pouyssegur J. The ERK1/2 mitogen-activated protein kinase pathway as a master regulator of the G1-to S-phase transition. Oncogene. 2007, 26: 3227-3239.