胰岛素受体底物1通过PI3K/Akt通路下调大鼠成骨细胞NFκB和BAX表达促进成骨细胞增殖
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摘要
目的：
糖尿病影响骨代谢，诱发糖尿病性骨病。胰岛素受体底物1（insulin receptor substrate1, IRS1）是胰岛素信号转导通路中受体后水平的重要信号蛋白。研究发现IRS1不仅促进骨形成，而且影响骨吸收，IRS1主要作用于成骨细胞影响骨转换，但机制尚不完全清楚。抑制成骨细胞NFκB(nuclear factor kappa-B，NFκB)通路增强成骨细胞分化、矿化和骨形成，但直接调节成骨细胞NFκB的基因仍需确定。高糖环境导致凋亡蛋白BAX（Bcl2–associated X protein，BAX）表达增加。研究发现IRS1具有抗凋亡能力，但IRS1如何调控成骨细胞BAX尚不清楚。
糖尿病影响骨代谢，诱发糖尿病性骨病。1型糖尿病病人骨密度降低，2型糖尿病病人骨密度正常、升高或降低。试验证据清楚表明无论1型还是2型糖尿病病人骨折风险性均增加。糖尿病病人骨折风险性增加与成骨细胞骨形成和破骨细胞骨吸收不平衡相关。多种机制参与糖尿病性骨病的发生。胰岛素和IGF1信号通路在糖尿病发挥重要作用，且与骨代谢密切相关。研究报导糖尿病病人骨量和胰岛素剂量、尿C肽呈正相关，外源性和内源性胰岛素可能影响糖尿病病人骨代谢平衡。胰岛素和IGF1通过经典细胞内信号转导途径影响骨代谢，下游IRS1/2、Akt(Protein Kinase B，Akt)和MEK(MAPK/ERK kinase，MEK)作用机制尚不清楚。除经典途径外，胰岛素和IGF1可能通过成骨细胞Wnt (wingless-int)和BMP2(bone morphogenic protein2)通路促进骨骼发育。胰岛素直接影响成骨细胞代谢。体外研究发现生理剂量胰岛素促进成骨细胞增殖、胶原合成、碱性磷酸酶产生，目前仍不完全清楚胰岛素促进骨形成机制。胰岛素促进成骨细胞发育和骨钙素表达，骨钙素参与骨矿化和钙离子的动态平衡。IGF1通过自分泌和旁分泌方式促进成骨细胞样细胞增殖、分化和细胞外基质产生，促进骨形成。青春期时期，IGF1决定长骨生长、骨骼发育和骨量增加，成年阶段维持骨量。IGF1增加成骨细胞数目和活性以增加骨形成；抑制破骨细胞分化以减少骨吸收。
IRS1是胰岛素和IGF1信号通路重要靶分子，且对维持正常骨代谢有重要意义。IRS1基因敲除小鼠骨形成和骨吸收均减少。IRS1基因自发性突变影响成年后骨骼表型，表现为高胰岛素血症及低骨密度等。IRS1基因敲除小鼠骨折难愈合提示IRS1参与骨修复，而且其作用不能被IRS其他亚型和IGF通路替代。IRS1缺陷小鼠骨愈合受损，这种受损在IRS1重新表达后恢复。IRS1主要经成骨细胞调节骨转换，其中机制仍不完全清楚。大量体外研究已证明IGF1和IRS/PI3K/Akt通路促进成骨细胞骨分化。抑制IRS1减少成骨细胞寿命、增殖、矿化和分化。IRS1通过促进前成骨细胞增殖和分化增加骨形成，产生更多胶原增加骨基质；IRS1不仅促进骨形成和矿化，也经由MMPs(matrix metallopeptidases，MMPs)和RANKL/TNFRSF11B(tumor necrosis factor receptor superfamily, member11b)比率影响骨吸收，最终调节骨循环。此外，予以IRS1干扰RNA抑制RUNX2(runt-related transcription factor2, RUNX2)、 ALP(alkaline phosphatase, ALP)和BSP(bone sialoprotein, BSP)表达。
PI3K/Akt通路与骨代谢关系密切。越来越多证据表明许多信号分子在骨发挥其特异性效应通过选择性激活成骨细胞PI3K/Akt通路。此外PI3K/Akt可以与其他信号通路和基因调控网络共同调控骨细胞发育，也有证据表明PI3K/Akt信号通路失控可能与某些骨骼病变相关。激活PI3K/Akt通路促进成骨细胞增殖。高糖诱导的氧化应激激活PI3K/Akt通路抑制成骨分化。破骨细胞产生趋化因子SIP (Sphingosine-1-Phosphate，S1P)激活PI3K/Akt信号通路，导致成骨细胞分化标记物包括碱性磷酸酶表达上调，并促进成骨细胞迁移。破骨细胞分泌骨吸收介质通过PI3K/Akt信号通路促进成骨细胞前体细胞分化和钙化。HGF(Hepatocyte growth factor，HGF)激活PI3K/Akt信号通路，增加骨桥蛋白(Osteopontin, OPN)表达调控骨矿化。在成骨细胞和钙化血管平滑肌细胞，Xie H等发现omentin1激活PI3K/Akt信号通路激活OPG和抑制RANKL产生，减轻OPG/小鼠动脉钙化和骨质流失。Akt磷酸化抑制糖皮质激素诱导的成骨细胞凋亡。胰岛素依赖型糖尿病通过抑制Akt，减弱骨形成。激活Akt，增加破骨细胞形成和增强破骨细胞分化。Akt蛋白家族有3个亚型：Akt1,Akt2和Akt3。Akt1和Akt2均在成骨细胞和破骨细胞大量表达，除了Akt3。虽然Akt蛋白单亚型敲除小鼠表现出温和的表型，但是Akt1和Akt2都敲除小鼠出现严重骨骼发育受损和侏儒症。Akt1促进成骨细胞和破骨细胞分化和存活，Akt1缺陷抑制成骨细胞RANKL表达，引起破骨细胞自主功能障碍，损害骨吸收功能。Akt1和Akt2都敲除下调RANKL诱导的NFκB p50的DNA结合能力，抑制破骨细胞分化。
NFκB与骨代谢密切相关。由RANKL（nuclear factor kB ligand，RANKL）、肿瘤坏死因子（Tumor necrosis factor，TNF）或者白介素1（interleukin1，IL1）激活的NFκB信号通路能够诱导破骨分化基因表达，延长破骨细胞寿命，增加骨吸收。成骨细胞NFκB活性降低时，JNK活性增强，导致骨形成增强。抑制NFκB通路可以逆转TNFα抑制的成骨细胞分化。Julien M等发现在成熟成骨细胞NFκB通过一系列信号转导抑制成骨细胞矿化。体外抑制成骨细胞NFκB信号通路，增强了成骨细胞矿化，但是破骨细胞数量没有改变。NFκB激活导致成骨细胞释放IL6，成骨细胞释放IL6导致破骨细胞激活。但直接调节成骨细胞NFκB的基因仍需确定。
研究发现凋亡可能是骨质疏松症第三大常见原因，估计60~80%成骨细胞正处于凋亡。成骨细胞凋亡增加松质骨生成。Bcl2（B-cell lymphoma2, Bcl2）家族长久以来被认为是凋亡主要调节机制，其中Bcl2抑制凋亡，BAX促进凋亡，二者独立调节凋亡。研究发现糖尿病大鼠凋亡显著增加。糖尿病时，高糖环境引起细胞损伤，BAX激活和引起其他促凋亡蛋白如细胞色素C释放。1型糖尿病小鼠骨中成骨细胞凋亡增加。一些研究表明IRS1具有抗凋亡能力，IRS1可通过调节Bcl2调控凋亡。
本研究组前期试验显示2型糖尿病合并骨质疏松症的发病可能与骨组织IGF1、IRS1、IRS2表达受抑制有关，同时可能与骨组织PI3K、Akt表达降低、NFκB表达升高有关。
本研究通过构建pEGFP-N1-IRS1表达载体，转染体外培养的大鼠成骨细胞，同时给予PI3K激酶特异性抑制剂LY294002，观察在有或无LY294002条件下成骨细胞NFκB、BAX表达变化及成骨细胞细胞周期变化，为阐明糖尿病骨病的发生机制提供一条新思路。
方法：
第一部分：提取Wister大鼠肝脏RNA，逆转录生成cDNA。根据NCBI提供的IRS1基因编码序列，委托DNA合成公司合成带适当酶切位点的引物，以cDNA作为模板合成IRS1，插入pEGFP-N1质粒，获得过表达IRS1的表达载体。体外转染人Hela细胞。
第二部分：酶消化联合组织块法体外培养Wister大鼠成骨细胞，倒置显微镜下观察成骨细胞形态，传代至第二代时茜素红染色鉴定成骨细胞。传代至第二代时pEGFP-N1-IRS1表达载体脂质体法LipofectamineTM2000瞬时转染成骨细胞，同时应用PI3K激酶特异性抑制剂LY294002阻断PI3K/Akt通路，免疫荧光和Western blotting法观察成骨细胞NFκB表达变化。
第三部分：酶消化联合组织块法体外培养Wister大鼠成骨细胞，传代至第二代时pEGFP-N1-IRS1脂质体法LipofectamineTM2000瞬时转染成骨细胞，同时应用LY294002阻断PI3K/Akt通路，观察成骨细胞IRS1/PI3K/Akt通路及BAX的表达变化。同时检测成骨细胞细胞周期。
结果：
第一部分：构建质粒经核酸内切酶酶切分析、PCR分析初步判断重组质粒中所插入的片段与预期长度一致。经过测序鉴定，合成的IRS1序列与登录在GenBank上的大鼠IRS1基因序列(GenBank登录号: NM_012969.1)高度同源,测序结果发现3个碱基与已发表的IRS1序列不同: NM_012969.1的第414、1824、2631位碱基分别是C、C和A，而合成的IRS1片段这3个碱基突变为T、T和G，但合成的IRS1序列翻译的蛋白质和NM_012969.1翻译的蛋白质一致。荧光显微镜下IRS1-GFP融合蛋白在Hela细胞成功表达。
第二部分：酶消化联合组织块法体外培养Wister大鼠成骨细胞，经HE染色和茜素红染色证明成骨细胞培养成功。荧光显微镜下IRS1-GFP融合蛋白在成骨细胞成功表达；RT-PCR结果表明IRS1表达载体转染组IRS1mRNA表达水平明显增高；Western blotting结果显示IRS1组pAktThr308蛋白水平相对对照组显著增加，LY294002阻断这种作用。免疫荧光和Western Blotting结果均表明IRS1组NFκB p65蛋白水平相对对照组降低，IRS1+LY294002组NFκB p65蛋白表达水平相对IRS1组增加。
第三部分：Western Blotting结果表明IRS1组BAX蛋白水平相对对照组显著降低，IRS1+LY294002组BAX蛋白表达水平相对IRS1组增加。细胞周期分析结果表明IRS1组S期增加，促进细胞增殖；IRS1+LY294002组S期降低。
结论：
1成功构建pEGFP-N1-IRS1表达载体，IRS1-GFP融合蛋白在体外成功表达。
2pEGFP-N1-IRS1成功激活成骨细胞PI3K/Akt通路，抑制NFκBp65蛋白表达，PI3K抑制剂LY294002逆转这种作用，推断IRS1通过PI3K/Akt通路抑制NFκB通路。
3pEGFP-N1-IRS1诱导的PI3K/Akt通路抑制BAX蛋白表达，PI3K抑制剂LY294002逆转这种作用，推断IRS1通过PI3K/Akt通路抑制BAX表达，促进细胞增殖。
Objective:
Diabetes affects bone metabolism, and leads to diabetic osteopathy. Insulin receptor substrate1is an important receptor protein of insulin signal transduction pathway. IRS1not only promotes bone formation but also plays an important role in bone resorption, and IRS1regulates bone turnover mainly via osteoblasts, but the underlying mechanism is not clear. Inhibition of NFκB in osteoblast, promotes osteoblast differentiation, mineralization and increases bone formation. However, the genes that directly regulate NFκB in osteoblast remain to be determined. High concentration of glucose could up-regulate the expression of BAX. Several reports have already indicated that IRS1possesses an antiapoptotic activity. However, IRS1how to regulate BAX in osteoblast remains to be determined.
Diabetes affects bone metabolism, and leads to diabetic osteopathy. Diabetes mellitus type1(DM1) patients have a lower bone mineral density than healthy individuals whereas diabetes mellitus type2(DM2) patients have a normal, lower or higher bone mass. Experimental evidence clearly demonstrated an increased fracture risk in T1DM and in T2DM. Patients with diabetes mellitus have an increased risk for fractures, which is related to an imbalance between osteoblastic bone formation and osteoclastic resorption. Several mechanisms are likely to be involved in the pathogenesis of diabetes osteopathy. Insulin and IGF1signaling pathway play an important role in diabetes, as well as in bone metabolism. In humans with diabetes, there is a positive correlation between bone mineral density and insulin dose or urinary C-peptide excretion, suggesting that endogenous and exogenous insulin may affect skeletal homeostasis in diabetes. Insulin and IGF1exert their cellular activities via conserved intracellular signaling proteins to affect bone metabolism. Genetic manipulation of these signaling proteins, such as IRS1/2、Akt and MEK, has uncovered a significant role for these signal transduction pathways in skeletal homeostasis. In addition to effect on skeletal physiology via canonical signaling pathways, insulin and IGF1may crosstalk with Wnt and BMP2signaling pathways in cells of the osteoblast lineage and thereby promote skeletal development. Insulin exerts direct anabolic actions in osteoblasts. Studies in vitro have shown that physiological dose of insulin promotes osteoblast proliferation, collagen synthesis, and alkaline phosphatase production; nevertheless, studies do not clarify what receptors or pathways insulin may use to promote osteogenesis. Insulin promotes osteoblast development and osteocalcin expression. Osteocalcin participates in mineralization and calcium ion homeostasis. IGF1acts in autocrine and paracrine ways, and stimulates proliferation, differentiation, and extracellular matrix production in osteoblastic like-cell lines and finally bone formation. IGF1plays important roles in stimulating longitudinal bone growth, skeletal maturation, and acquisition of bone mass in childhood, whereas in adults they are important in the maintenance of bone mass. IGF1can increase bone formation by stimulation of osteoblast number and activity, and reduce bone resorption by restriction of differentiation of osteoclast.
IRS1, which is a main target molecule of insulin/IGF1receptor signaling, have been shown to play important roles in maintaining normal bone turn-over. Both bone formation and resorption are decreased in Irs1gene knockout mice. A novel spontaneous mutation of Irs1in mice results in hyperinsulinemlia and low bone mass. Irs1knockout mice Irs1/fractures fail to heal indicating that IRS1has a role in bone repair that cannot be compensated by other IGF signaling pathways or IRS isoforms. Bone healing impaired in IRS1deficient mice can be corrected with re-expression of IRS1within the fracture site. The IRS1regulates bone turnover mainly via osteoblast, but the underlying mechanism is not clear. Numerous in vitro studies have documented the ability of IGF1and IRS/PI3K/Akt to promote osteoblast differentiation. The downregulation of IRS1expression reduces osteoblast survival, proliferation, mineralization and differentiation. IRS1can increase the bone formation by promoting the proliferation and differentiation of preosteoblasts and increase the bone matrix by producing more collagen. IRS1not only promotes bone formation and mineralization but also might play roles in bone resorption partly via the regulation of MMPs and RANKL/TNFRSF11B ratio, thus regulates the bone turnover. Moreover, small interfering RNA (siRNA) against IRS1suppresses mRNA expression of RUNX2, ALP and BSP.
PI3K/Akt pathway is closely related with bone metabolism. There is an increasing amount of evidence that the many signaling molecules exert some of their bone-specific effects in part via selectively activating the PI3K/Akt pathway in osteoblasts. There is further data demonstrating that PI3K/Akt has the capacity to specifically cross-talk with other signaling pathways and transcriptional networks controlling bone cells' development. There is also evidence that perturbations in the PI3K/Akt pathway may well be responsible for certain bone pathologies. PI3K/Akt activation induces osteoblasts proliferation. High glucose-induced oxidative stress activated PI3K/Akt pathway to inhibited osteogenic differentiation. Osteoclast-derived S1P activates the PI3K/Akt signaling pathway resulting in the upregulation of osteoblast differentiation markers including alkaline phosphatase, and promoting migration. The osteoclast bone resorption medium promotes the differentiation and calcification of MC3T3-E1cells through PI3K/Akt pathway activation. Hepatocyte growth factor has been demonstrated to enhance PI3K, Akt and stimulate Osteopontin (OPN) production. OPN is present during bone mineralization. Xie H et al. reported that omentin1activated the PI3K/Akt signalling pathway, which in turn stimulated OPG and decreased RANKL expression in calcifying vascular smooth muscle cells and osteoblasts, resulting in alleviation of arterial calcification and bone loss in the OPG/mice. Glucocorticoid-induced osteoblast apoptosis is mediated by suppression of Akt phosphorylation. Insulin-dependent diabetes mellitus decreases osteoblastogenesis through inhibition of Akt activation. There are three Akt family members—Akt1, Akt2, and Akt3. Akt1and Akt2, but not Akt3, are abundantly expressed in both bone cells: osteoblasts and osteoclasts. Although single KO mice of Akt isoform showed a mild phenotype, double KO mice of Akt1/Akt2showed severely impaired bone development and dwarfism. Akt1promotes differentiation and survival of osteoblast and osteoclast. Akt1deficiency caused impairment of bone resorption via cell autonomous dysfunction in osteoclasts because of reduced RANKL expression in osteoblasts. Knockdown of Akt1and Akt2inhibited osteoclast differentiation because of downregulation of RANKL-induced NFκB p50DNA binding activity.
NFκB is closely related to bone metabolism. NFκB signaling pathway activated by RANKL, TNF or IL1induces osteoclast differentiation gene expression and prolongs the life of osteoclasts, and increases bone resorption. A reduction in NFκB activity in osteoblasts results in an increase in bone formation via an increase in JNK activity. Inhibition of NFκB pathways reverses the TNFα suppression of osteoblast differentiation. Julien et al have found that NFκB represses osteoblast mineralization through a series of transcriptional and signaling events in mature osteoblasts. Inhibition of NFκB in osteoblasts in vitro leads to an increase in osteoblast mineralization. Interestingly, there is no change in the number of osteoclasts. NFκB translocates to the nucleus of the osteoblast, which leads to release of interleukin6(IL6). Release of IL6from osteoblasts results in the activation of the osteoclasts. However, the genes that directly regulate NFκB in osteoblasts remain to be identified.
Several studies have showed that apoptosis maybe the third most common cause of osteoporosis, and60–80%of osteoblasts are estimated to originally assembled at the resorption pit die by apoptosis. Dysapoptosis of osteoblasts increases cancellous bone formation. Members of the Bcl2family, including Bcl2and Bax, are the main regulators of apoptosis which promote (BAX) or inhibit (Bcl2) apoptosis. Each of them regulates apoptosis independently. Apoptosis is dramatically increased in diabetic rats. In diabetes, where the hyperglycemic environment causes cellular damage, BAX can become activated and form pores as a passage for other proapoptotic proteins, such as cytochrome c, to be released. Dying osteoblasts increases in bones of T1-diabetic mice. Several reports have already indicated that IRS1possesses an antiapoptotic activity, and IRS1modulates its antiapoptotic function by Bcl2.
My group pre-study has showed that the incidence of type2diabetes with osteoporosis maybe associated with inhibition of IGF1, IRS1and IRS2, reduction of PI3K, Akt and increase of NFκB in bone tissue. In this study, we constructed pEGFP-N1-IRS1expression vector, transfected rat osteoblast in vitro, and observed the expression of NFκB p65, BAX and cell cycle, in absence or presence of PI3K inhibitor (LY294002), to clarify a new mechanism under diabetic osteopathy.
Methods:
Part I: To extract liver RNA of Wister rat, reverse transcriptase to generate cDNA. To synthesize IRS1using cDNA as template and primers with appropriate restriction sites, insert IRS1into pEGFP-N1resulting in overexpression IRS1vector. To transfect human Hela cell in vitro.
Part II: We used enzymatic digestion-tissue explant in vitro to culture Wister rat osteoblast, observed osteoblast under microscope. Passaging to the second generation of osteoblast, alizarin red stained osteoblasts. We transfected the second generation osteoblast using LipofectamineTM2000, and observed the expression of IRS1/PI3K/Akt and NFκB p65, in absence or presence of PI3K inhibitor (LY294002).
Part III: We used enzymatic digestion-tissue explant in vitro to culture Wister rat osteoblast, transfected the second generation of osteoblast with pEGFP-N1-IRS1, and observed the expression of BAX and cell cycle, in absence or presence of LY294002.
Results:
Part I: The fragment inserted into plasmid is consistent with the expected length, via endonuclease restriction analysis and PCR analysis. Constructed IRS1sequence via sequencing is highly homologous with rat IRS1gene sequence in GenBank (GenBank accession number: NM_012969.1). Three bases of IRS1sequences are different from the published IRS1sequence: the414,1824,2631nucleotide are C, C and A, yet in constructed IRS1the three bases are mutated to T, T and G, but the protein translation of synthetic IRS1fragments is consistent with the protein translation of published IRS1sequence. IRS1-GFP is successfully expressed in Hela cells under fluorescence microscope.
Part II: To culture osteoblast from Wister rat in vitro by enzymatic digestion-tissue explant, osteoblasts are proved successfully cultured by HE staining and alizarin red staining. IRS1-GFP protein is successfully expressed in group (IRS1) under fluorescence microscope; RT-PCR results suggest that IRS1mRNA level is significantly increased in group (IRS1); western blotting results show that the level of pAktThr308protein, target receptor of IRS1signaling, was increased in group (IRS1), which was abolished by incubation of LY294002. Both immunofluorescence staining and western blotting results show that NFκBp65protein level is decreased in group (IRS1), compared to control; while NFκBp65protein level is increased in group (IRS1+LY294002), compared to group (IRS1).
Part III: BAX protein level is decreased in group (IRS1) compared to control; the effect induced by pEGFP-N1-IRS1was abolished by incubation of LY294002. Cell cycle analysis result shows that IRS1on cell proliferation was observed as an increase in cells in the S phases.
Conclusions:
1Recombinant pEGFP-N1-IRS1is successfully constructed, and IRS1-GFP is successfully expressed in vitro.
2pEGFP-N1-IRS1successfully activates PI3K/Akt pathway in osteoblasts, thus inhibites NFκBp65expression in osteoblasts, which was abolished by incubation of LY294002. IRS1-induced PI3K/Akt pathway inhibites NFκB pathway.
3IRS1-induced PI3K/Akt pathway promotes osteoblasts proliferation and inhibites BAX expression in osteoblasts, which is abolished by incubation of LY294002. IRS1-induced PI3K/Akt pathway inhibites BAX expression, led to promoting osteoblasts proliferation.
糖尿病影响骨代谢，诱发糖尿病性骨病。胰岛素受体底物1（insulin receptor substrate1, IRS1）是胰岛素信号转导通路中受体后水平的重要信号蛋白。研究发现IRS1不仅促进骨形成，而且影响骨吸收，IRS1主要作用于成骨细胞影响骨转换，但机制尚不完全清楚。抑制成骨细胞NFκB(nuclear factor kappa-B，NFκB)通路增强成骨细胞分化、矿化和骨形成，但直接调节成骨细胞NFκB的基因仍需确定。高糖环境导致凋亡蛋白BAX（Bcl2–associated X protein，BAX）表达增加。研究发现IRS1具有抗凋亡能力，但IRS1如何调控成骨细胞BAX尚不清楚。
糖尿病影响骨代谢，诱发糖尿病性骨病。1型糖尿病病人骨密度降低，2型糖尿病病人骨密度正常、升高或降低。试验证据清楚表明无论1型还是2型糖尿病病人骨折风险性均增加。糖尿病病人骨折风险性增加与成骨细胞骨形成和破骨细胞骨吸收不平衡相关。多种机制参与糖尿病性骨病的发生。胰岛素和IGF1信号通路在糖尿病发挥重要作用，且与骨代谢密切相关。研究报导糖尿病病人骨量和胰岛素剂量、尿C肽呈正相关，外源性和内源性胰岛素可能影响糖尿病病人骨代谢平衡。胰岛素和IGF1通过经典细胞内信号转导途径影响骨代谢，下游IRS1/2、Akt(Protein Kinase B，Akt)和MEK(MAPK/ERK kinase，MEK)作用机制尚不清楚。除经典途径外，胰岛素和IGF1可能通过成骨细胞Wnt (wingless-int)和BMP2(bone morphogenic protein2)通路促进骨骼发育。胰岛素直接影响成骨细胞代谢。体外研究发现生理剂量胰岛素促进成骨细胞增殖、胶原合成、碱性磷酸酶产生，目前仍不完全清楚胰岛素促进骨形成机制。胰岛素促进成骨细胞发育和骨钙素表达，骨钙素参与骨矿化和钙离子的动态平衡。IGF1通过自分泌和旁分泌方式促进成骨细胞样细胞增殖、分化和细胞外基质产生，促进骨形成。青春期时期，IGF1决定长骨生长、骨骼发育和骨量增加，成年阶段维持骨量。IGF1增加成骨细胞数目和活性以增加骨形成；抑制破骨细胞分化以减少骨吸收。
IRS1是胰岛素和IGF1信号通路重要靶分子，且对维持正常骨代谢有重要意义。IRS1基因敲除小鼠骨形成和骨吸收均减少。IRS1基因自发性突变影响成年后骨骼表型，表现为高胰岛素血症及低骨密度等。IRS1基因敲除小鼠骨折难愈合提示IRS1参与骨修复，而且其作用不能被IRS其他亚型和IGF通路替代。IRS1缺陷小鼠骨愈合受损，这种受损在IRS1重新表达后恢复。IRS1主要经成骨细胞调节骨转换，其中机制仍不完全清楚。大量体外研究已证明IGF1和IRS/PI3K/Akt通路促进成骨细胞骨分化。抑制IRS1减少成骨细胞寿命、增殖、矿化和分化。IRS1通过促进前成骨细胞增殖和分化增加骨形成，产生更多胶原增加骨基质；IRS1不仅促进骨形成和矿化，也经由MMPs(matrix metallopeptidases，MMPs)和RANKL/TNFRSF11B(tumor necrosis factor receptor superfamily, member11b)比率影响骨吸收，最终调节骨循环。此外，予以IRS1干扰RNA抑制RUNX2(runt-related transcription factor2, RUNX2)、 ALP(alkaline phosphatase, ALP)和BSP(bone sialoprotein, BSP)表达。
PI3K/Akt通路与骨代谢关系密切。越来越多证据表明许多信号分子在骨发挥其特异性效应通过选择性激活成骨细胞PI3K/Akt通路。此外PI3K/Akt可以与其他信号通路和基因调控网络共同调控骨细胞发育，也有证据表明PI3K/Akt信号通路失控可能与某些骨骼病变相关。激活PI3K/Akt通路促进成骨细胞增殖。高糖诱导的氧化应激激活PI3K/Akt通路抑制成骨分化。破骨细胞产生趋化因子SIP (Sphingosine-1-Phosphate，S1P)激活PI3K/Akt信号通路，导致成骨细胞分化标记物包括碱性磷酸酶表达上调，并促进成骨细胞迁移。破骨细胞分泌骨吸收介质通过PI3K/Akt信号通路促进成骨细胞前体细胞分化和钙化。HGF(Hepatocyte growth factor，HGF)激活PI3K/Akt信号通路，增加骨桥蛋白(Osteopontin, OPN)表达调控骨矿化。在成骨细胞和钙化血管平滑肌细胞，Xie H等发现omentin1激活PI3K/Akt信号通路激活OPG和抑制RANKL产生，减轻OPG/小鼠动脉钙化和骨质流失。Akt磷酸化抑制糖皮质激素诱导的成骨细胞凋亡。胰岛素依赖型糖尿病通过抑制Akt，减弱骨形成。激活Akt，增加破骨细胞形成和增强破骨细胞分化。Akt蛋白家族有3个亚型：Akt1,Akt2和Akt3。Akt1和Akt2均在成骨细胞和破骨细胞大量表达，除了Akt3。虽然Akt蛋白单亚型敲除小鼠表现出温和的表型，但是Akt1和Akt2都敲除小鼠出现严重骨骼发育受损和侏儒症。Akt1促进成骨细胞和破骨细胞分化和存活，Akt1缺陷抑制成骨细胞RANKL表达，引起破骨细胞自主功能障碍，损害骨吸收功能。Akt1和Akt2都敲除下调RANKL诱导的NFκB p50的DNA结合能力，抑制破骨细胞分化。
NFκB与骨代谢密切相关。由RANKL（nuclear factor kB ligand，RANKL）、肿瘤坏死因子（Tumor necrosis factor，TNF）或者白介素1（interleukin1，IL1）激活的NFκB信号通路能够诱导破骨分化基因表达，延长破骨细胞寿命，增加骨吸收。成骨细胞NFκB活性降低时，JNK活性增强，导致骨形成增强。抑制NFκB通路可以逆转TNFα抑制的成骨细胞分化。Julien M等发现在成熟成骨细胞NFκB通过一系列信号转导抑制成骨细胞矿化。体外抑制成骨细胞NFκB信号通路，增强了成骨细胞矿化，但是破骨细胞数量没有改变。NFκB激活导致成骨细胞释放IL6，成骨细胞释放IL6导致破骨细胞激活。但直接调节成骨细胞NFκB的基因仍需确定。
研究发现凋亡可能是骨质疏松症第三大常见原因，估计60~80%成骨细胞正处于凋亡。成骨细胞凋亡增加松质骨生成。Bcl2（B-cell lymphoma2, Bcl2）家族长久以来被认为是凋亡主要调节机制，其中Bcl2抑制凋亡，BAX促进凋亡，二者独立调节凋亡。研究发现糖尿病大鼠凋亡显著增加。糖尿病时，高糖环境引起细胞损伤，BAX激活和引起其他促凋亡蛋白如细胞色素C释放。1型糖尿病小鼠骨中成骨细胞凋亡增加。一些研究表明IRS1具有抗凋亡能力，IRS1可通过调节Bcl2调控凋亡。
本研究组前期试验显示2型糖尿病合并骨质疏松症的发病可能与骨组织IGF1、IRS1、IRS2表达受抑制有关，同时可能与骨组织PI3K、Akt表达降低、NFκB表达升高有关。
本研究通过构建pEGFP-N1-IRS1表达载体，转染体外培养的大鼠成骨细胞，同时给予PI3K激酶特异性抑制剂LY294002，观察在有或无LY294002条件下成骨细胞NFκB、BAX表达变化及成骨细胞细胞周期变化，为阐明糖尿病骨病的发生机制提供一条新思路。
方法：
第一部分：提取Wister大鼠肝脏RNA，逆转录生成cDNA。根据NCBI提供的IRS1基因编码序列，委托DNA合成公司合成带适当酶切位点的引物，以cDNA作为模板合成IRS1，插入pEGFP-N1质粒，获得过表达IRS1的表达载体。体外转染人Hela细胞。
第二部分：酶消化联合组织块法体外培养Wister大鼠成骨细胞，倒置显微镜下观察成骨细胞形态，传代至第二代时茜素红染色鉴定成骨细胞。传代至第二代时pEGFP-N1-IRS1表达载体脂质体法LipofectamineTM2000瞬时转染成骨细胞，同时应用PI3K激酶特异性抑制剂LY294002阻断PI3K/Akt通路，免疫荧光和Western blotting法观察成骨细胞NFκB表达变化。
第三部分：酶消化联合组织块法体外培养Wister大鼠成骨细胞，传代至第二代时pEGFP-N1-IRS1脂质体法LipofectamineTM2000瞬时转染成骨细胞，同时应用LY294002阻断PI3K/Akt通路，观察成骨细胞IRS1/PI3K/Akt通路及BAX的表达变化。同时检测成骨细胞细胞周期。
结果：
第一部分：构建质粒经核酸内切酶酶切分析、PCR分析初步判断重组质粒中所插入的片段与预期长度一致。经过测序鉴定，合成的IRS1序列与登录在GenBank上的大鼠IRS1基因序列(GenBank登录号: NM_012969.1)高度同源,测序结果发现3个碱基与已发表的IRS1序列不同: NM_012969.1的第414、1824、2631位碱基分别是C、C和A，而合成的IRS1片段这3个碱基突变为T、T和G，但合成的IRS1序列翻译的蛋白质和NM_012969.1翻译的蛋白质一致。荧光显微镜下IRS1-GFP融合蛋白在Hela细胞成功表达。
第二部分：酶消化联合组织块法体外培养Wister大鼠成骨细胞，经HE染色和茜素红染色证明成骨细胞培养成功。荧光显微镜下IRS1-GFP融合蛋白在成骨细胞成功表达；RT-PCR结果表明IRS1表达载体转染组IRS1mRNA表达水平明显增高；Western blotting结果显示IRS1组pAktThr308蛋白水平相对对照组显著增加，LY294002阻断这种作用。免疫荧光和Western Blotting结果均表明IRS1组NFκB p65蛋白水平相对对照组降低，IRS1+LY294002组NFκB p65蛋白表达水平相对IRS1组增加。
第三部分：Western Blotting结果表明IRS1组BAX蛋白水平相对对照组显著降低，IRS1+LY294002组BAX蛋白表达水平相对IRS1组增加。细胞周期分析结果表明IRS1组S期增加，促进细胞增殖；IRS1+LY294002组S期降低。
结论：
1成功构建pEGFP-N1-IRS1表达载体，IRS1-GFP融合蛋白在体外成功表达。
2pEGFP-N1-IRS1成功激活成骨细胞PI3K/Akt通路，抑制NFκBp65蛋白表达，PI3K抑制剂LY294002逆转这种作用，推断IRS1通过PI3K/Akt通路抑制NFκB通路。
3pEGFP-N1-IRS1诱导的PI3K/Akt通路抑制BAX蛋白表达，PI3K抑制剂LY294002逆转这种作用，推断IRS1通过PI3K/Akt通路抑制BAX表达，促进细胞增殖。
Objective:
Diabetes affects bone metabolism, and leads to diabetic osteopathy. Insulin receptor substrate1is an important receptor protein of insulin signal transduction pathway. IRS1not only promotes bone formation but also plays an important role in bone resorption, and IRS1regulates bone turnover mainly via osteoblasts, but the underlying mechanism is not clear. Inhibition of NFκB in osteoblast, promotes osteoblast differentiation, mineralization and increases bone formation. However, the genes that directly regulate NFκB in osteoblast remain to be determined. High concentration of glucose could up-regulate the expression of BAX. Several reports have already indicated that IRS1possesses an antiapoptotic activity. However, IRS1how to regulate BAX in osteoblast remains to be determined.
Diabetes affects bone metabolism, and leads to diabetic osteopathy. Diabetes mellitus type1(DM1) patients have a lower bone mineral density than healthy individuals whereas diabetes mellitus type2(DM2) patients have a normal, lower or higher bone mass. Experimental evidence clearly demonstrated an increased fracture risk in T1DM and in T2DM. Patients with diabetes mellitus have an increased risk for fractures, which is related to an imbalance between osteoblastic bone formation and osteoclastic resorption. Several mechanisms are likely to be involved in the pathogenesis of diabetes osteopathy. Insulin and IGF1signaling pathway play an important role in diabetes, as well as in bone metabolism. In humans with diabetes, there is a positive correlation between bone mineral density and insulin dose or urinary C-peptide excretion, suggesting that endogenous and exogenous insulin may affect skeletal homeostasis in diabetes. Insulin and IGF1exert their cellular activities via conserved intracellular signaling proteins to affect bone metabolism. Genetic manipulation of these signaling proteins, such as IRS1/2、Akt and MEK, has uncovered a significant role for these signal transduction pathways in skeletal homeostasis. In addition to effect on skeletal physiology via canonical signaling pathways, insulin and IGF1may crosstalk with Wnt and BMP2signaling pathways in cells of the osteoblast lineage and thereby promote skeletal development. Insulin exerts direct anabolic actions in osteoblasts. Studies in vitro have shown that physiological dose of insulin promotes osteoblast proliferation, collagen synthesis, and alkaline phosphatase production; nevertheless, studies do not clarify what receptors or pathways insulin may use to promote osteogenesis. Insulin promotes osteoblast development and osteocalcin expression. Osteocalcin participates in mineralization and calcium ion homeostasis. IGF1acts in autocrine and paracrine ways, and stimulates proliferation, differentiation, and extracellular matrix production in osteoblastic like-cell lines and finally bone formation. IGF1plays important roles in stimulating longitudinal bone growth, skeletal maturation, and acquisition of bone mass in childhood, whereas in adults they are important in the maintenance of bone mass. IGF1can increase bone formation by stimulation of osteoblast number and activity, and reduce bone resorption by restriction of differentiation of osteoclast.
IRS1, which is a main target molecule of insulin/IGF1receptor signaling, have been shown to play important roles in maintaining normal bone turn-over. Both bone formation and resorption are decreased in Irs1gene knockout mice. A novel spontaneous mutation of Irs1in mice results in hyperinsulinemlia and low bone mass. Irs1knockout mice Irs1/fractures fail to heal indicating that IRS1has a role in bone repair that cannot be compensated by other IGF signaling pathways or IRS isoforms. Bone healing impaired in IRS1deficient mice can be corrected with re-expression of IRS1within the fracture site. The IRS1regulates bone turnover mainly via osteoblast, but the underlying mechanism is not clear. Numerous in vitro studies have documented the ability of IGF1and IRS/PI3K/Akt to promote osteoblast differentiation. The downregulation of IRS1expression reduces osteoblast survival, proliferation, mineralization and differentiation. IRS1can increase the bone formation by promoting the proliferation and differentiation of preosteoblasts and increase the bone matrix by producing more collagen. IRS1not only promotes bone formation and mineralization but also might play roles in bone resorption partly via the regulation of MMPs and RANKL/TNFRSF11B ratio, thus regulates the bone turnover. Moreover, small interfering RNA (siRNA) against IRS1suppresses mRNA expression of RUNX2, ALP and BSP.
PI3K/Akt pathway is closely related with bone metabolism. There is an increasing amount of evidence that the many signaling molecules exert some of their bone-specific effects in part via selectively activating the PI3K/Akt pathway in osteoblasts. There is further data demonstrating that PI3K/Akt has the capacity to specifically cross-talk with other signaling pathways and transcriptional networks controlling bone cells' development. There is also evidence that perturbations in the PI3K/Akt pathway may well be responsible for certain bone pathologies. PI3K/Akt activation induces osteoblasts proliferation. High glucose-induced oxidative stress activated PI3K/Akt pathway to inhibited osteogenic differentiation. Osteoclast-derived S1P activates the PI3K/Akt signaling pathway resulting in the upregulation of osteoblast differentiation markers including alkaline phosphatase, and promoting migration. The osteoclast bone resorption medium promotes the differentiation and calcification of MC3T3-E1cells through PI3K/Akt pathway activation. Hepatocyte growth factor has been demonstrated to enhance PI3K, Akt and stimulate Osteopontin (OPN) production. OPN is present during bone mineralization. Xie H et al. reported that omentin1activated the PI3K/Akt signalling pathway, which in turn stimulated OPG and decreased RANKL expression in calcifying vascular smooth muscle cells and osteoblasts, resulting in alleviation of arterial calcification and bone loss in the OPG/mice. Glucocorticoid-induced osteoblast apoptosis is mediated by suppression of Akt phosphorylation. Insulin-dependent diabetes mellitus decreases osteoblastogenesis through inhibition of Akt activation. There are three Akt family members—Akt1, Akt2, and Akt3. Akt1and Akt2, but not Akt3, are abundantly expressed in both bone cells: osteoblasts and osteoclasts. Although single KO mice of Akt isoform showed a mild phenotype, double KO mice of Akt1/Akt2showed severely impaired bone development and dwarfism. Akt1promotes differentiation and survival of osteoblast and osteoclast. Akt1deficiency caused impairment of bone resorption via cell autonomous dysfunction in osteoclasts because of reduced RANKL expression in osteoblasts. Knockdown of Akt1and Akt2inhibited osteoclast differentiation because of downregulation of RANKL-induced NFκB p50DNA binding activity.
NFκB is closely related to bone metabolism. NFκB signaling pathway activated by RANKL, TNF or IL1induces osteoclast differentiation gene expression and prolongs the life of osteoclasts, and increases bone resorption. A reduction in NFκB activity in osteoblasts results in an increase in bone formation via an increase in JNK activity. Inhibition of NFκB pathways reverses the TNFα suppression of osteoblast differentiation. Julien et al have found that NFκB represses osteoblast mineralization through a series of transcriptional and signaling events in mature osteoblasts. Inhibition of NFκB in osteoblasts in vitro leads to an increase in osteoblast mineralization. Interestingly, there is no change in the number of osteoclasts. NFκB translocates to the nucleus of the osteoblast, which leads to release of interleukin6(IL6). Release of IL6from osteoblasts results in the activation of the osteoclasts. However, the genes that directly regulate NFκB in osteoblasts remain to be identified.
Several studies have showed that apoptosis maybe the third most common cause of osteoporosis, and60–80%of osteoblasts are estimated to originally assembled at the resorption pit die by apoptosis. Dysapoptosis of osteoblasts increases cancellous bone formation. Members of the Bcl2family, including Bcl2and Bax, are the main regulators of apoptosis which promote (BAX) or inhibit (Bcl2) apoptosis. Each of them regulates apoptosis independently. Apoptosis is dramatically increased in diabetic rats. In diabetes, where the hyperglycemic environment causes cellular damage, BAX can become activated and form pores as a passage for other proapoptotic proteins, such as cytochrome c, to be released. Dying osteoblasts increases in bones of T1-diabetic mice. Several reports have already indicated that IRS1possesses an antiapoptotic activity, and IRS1modulates its antiapoptotic function by Bcl2.
My group pre-study has showed that the incidence of type2diabetes with osteoporosis maybe associated with inhibition of IGF1, IRS1and IRS2, reduction of PI3K, Akt and increase of NFκB in bone tissue. In this study, we constructed pEGFP-N1-IRS1expression vector, transfected rat osteoblast in vitro, and observed the expression of NFκB p65, BAX and cell cycle, in absence or presence of PI3K inhibitor (LY294002), to clarify a new mechanism under diabetic osteopathy.
Methods:
Part I: To extract liver RNA of Wister rat, reverse transcriptase to generate cDNA. To synthesize IRS1using cDNA as template and primers with appropriate restriction sites, insert IRS1into pEGFP-N1resulting in overexpression IRS1vector. To transfect human Hela cell in vitro.
Part II: We used enzymatic digestion-tissue explant in vitro to culture Wister rat osteoblast, observed osteoblast under microscope. Passaging to the second generation of osteoblast, alizarin red stained osteoblasts. We transfected the second generation osteoblast using LipofectamineTM2000, and observed the expression of IRS1/PI3K/Akt and NFκB p65, in absence or presence of PI3K inhibitor (LY294002).
Part III: We used enzymatic digestion-tissue explant in vitro to culture Wister rat osteoblast, transfected the second generation of osteoblast with pEGFP-N1-IRS1, and observed the expression of BAX and cell cycle, in absence or presence of LY294002.
Results:
Part I: The fragment inserted into plasmid is consistent with the expected length, via endonuclease restriction analysis and PCR analysis. Constructed IRS1sequence via sequencing is highly homologous with rat IRS1gene sequence in GenBank (GenBank accession number: NM_012969.1). Three bases of IRS1sequences are different from the published IRS1sequence: the414,1824,2631nucleotide are C, C and A, yet in constructed IRS1the three bases are mutated to T, T and G, but the protein translation of synthetic IRS1fragments is consistent with the protein translation of published IRS1sequence. IRS1-GFP is successfully expressed in Hela cells under fluorescence microscope.
Part II: To culture osteoblast from Wister rat in vitro by enzymatic digestion-tissue explant, osteoblasts are proved successfully cultured by HE staining and alizarin red staining. IRS1-GFP protein is successfully expressed in group (IRS1) under fluorescence microscope; RT-PCR results suggest that IRS1mRNA level is significantly increased in group (IRS1); western blotting results show that the level of pAktThr308protein, target receptor of IRS1signaling, was increased in group (IRS1), which was abolished by incubation of LY294002. Both immunofluorescence staining and western blotting results show that NFκBp65protein level is decreased in group (IRS1), compared to control; while NFκBp65protein level is increased in group (IRS1+LY294002), compared to group (IRS1).
Part III: BAX protein level is decreased in group (IRS1) compared to control; the effect induced by pEGFP-N1-IRS1was abolished by incubation of LY294002. Cell cycle analysis result shows that IRS1on cell proliferation was observed as an increase in cells in the S phases.
Conclusions:
1Recombinant pEGFP-N1-IRS1is successfully constructed, and IRS1-GFP is successfully expressed in vitro.
2pEGFP-N1-IRS1successfully activates PI3K/Akt pathway in osteoblasts, thus inhibites NFκBp65expression in osteoblasts, which was abolished by incubation of LY294002. IRS1-induced PI3K/Akt pathway inhibites NFκB pathway.
3IRS1-induced PI3K/Akt pathway promotes osteoblasts proliferation and inhibites BAX expression in osteoblasts, which is abolished by incubation of LY294002. IRS1-induced PI3K/Akt pathway inhibites BAX expression, led to promoting osteoblasts proliferation.
引文
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