Insulin Receptor Substrate1Down-regulates NFκb And BAX To Promote Osteoblast Proliferation Via PI3K/Akt Signaling

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: NM₀12969.1）. 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.