IRS-2 branch of IGF-1 receptor signaling is essential for appropriate timing of myelination

Insulin-like growth factor (IGF)-1 increases proliferation, inhibits apoptosis and promotes differentiation of oligodendrocytes and their precursor cells, indicating an important function for IGF-1 receptor (IGF-1R) signaling in myelin development. The insulin receptor substrates (IRS), IRS-1 and -2 serve as intracellular IGF-1R adaptor proteins and are expressed in neurons, oligodendrocytes and their precursors. To address the role of IRS-2 in myelination, we analyzed myelination in IRS-2 deficient (IRS-2(-/-)) mice and age-matched controls during postnatal development. Interestingly, expression of the most abundant myelin proteins, myelin basic protein and proteolipid protein was reduced in IRS-2(-/-) brains at postnatal day 10 (P10) as compared to controls. myelin basic protein immunostaining in P10-IRS-2(-/-) mice revealed a reduced immunostaining, but an unchanged regional distribution pattern. In cerebral myelin isolates at P10 unaltered relative expression of different myelin proteins was found, indicating quantitatively reduced but not qualitatively altered myelination. Interestingly, up-regulation of IRS-1 expression and increased IGF-1R signaling were observed in IRS-2(-/-) mice at P10-14, indicating a compensatory mechanism to overcome IRS-2 deficiency. Adult IRS-2(-/-) mice showed unaltered myelination and motor function. Furthermore, in neuronal/brain-specific insulin receptor knockout mice myelination was unchanged. Thus, our experiments reveal that IGF-1R/IRS-2 mediated signals are critical for appropriate timing of myelination in vivo.     

TLR3 contributes to persistent autophagy and heart failure in mice after myocardial infarction

Toll‐like receptors (TLRs) are essential immunoreceptors involved in host defence against invading microbes. Recent studies indicate that certain TLRs activate immunological autophagy to eliminate microbes. It remains unknown whether TLRs regulate autophagy to play a role in the heart. This study examined this question. The activation of TLR3 in cultured cardiomyocytes was observed to increase protein levels of autophagic components, including LC3‐II, a specific marker for autophagy induction, and p62/SQSTM1, an autophagy receptor normally degraded in the final step of autophagy. The results of transfection with a tandem mRFP‐GFP‐LC3 adenovirus and use of an autophagic flux inhibitor chloroquine both suggested that TLR3 in cardiomyocytes promotes autophagy induction without affecting autophagic flux. Gene‐knockdown experiments showed that the TRIF‐dependent pathway mediated the autophagic effect of TLR3. In the mouse model of chronic myocardial infarction, persistent autophagy was observed, concomitant with up‐regulated TLR3 expression and increased TLR3‐Trif signalling. Germline knockout (KO) of TLR3 inhibited autophagy, reduced infarct size, attenuated heart failure and improved survival. These protective effects were abolished by in vivo administration of an autophagy inducer rapamycin. Similar to the results obtained in cultured cardiomyocytes, TLR3‐KO did not prevent autophagic flux in mouse heart. Additionally, this study failed to detect the involvement of inflammation in TLR3‐KO‐derived protection, as wild‐type and TLR3‐KO hearts were comparable in inflammatory activity. It is concluded that up‐regulated TLR3 expression and signalling contributes to persistent autophagy following MI, which promotes heart failure and lethality.     

Expression of insulin‐like growth factor‐1 receptor in metastatic uveal melanoma and implications for potential autocrine and paracrine tumor cell growth

We investigated the importance of the insulin‐like growth factor‐1 receptor (IGF‐1R) in hepatic metastases of uveal melanoma. The expression pattern of IGF‐1R in archival tissue samples of hepatic metastasis from 24 patients was analyzed by immunohistochemistry. All the samples of hepatic metastases stained positive for IGF‐1R. To investigate the biological role of IGF‐1R on the growth of metastatic uveal melanoma, a long‐term cell line obtained from a hepatic metastasis (TJU‐UM001) was evaluated. TJU‐UM001 expressed cell surface IGF‐1R (>90%) and proliferated in response to exogenous and endogenous insulin‐like growth factor‐1 (IGF‐1). Correlatively, anti‐IGF‐1R antibody completely blocked IGF‐1‐induced growth of TJU‐UM001 cells. IGF‐1 preferentially induced phosphorylation of Akt (S473) in quiescent TJU‐UM001 cells, and this was blocked by anti‐IGF‐1R antibody. This study suggests that autocrine and paracrine mechanisms underlie IGF‐1‐induced growth of metastatic uveal melanoma and underscore the potential benefit of IGF‐1 or IGF‐1R antagonism in treatment for metastatic uveal melanoma.     

Up‐regulated TLR4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

It remains unclear whether and how cardiomyocytes contribute to the inflammation in chronic heart failure (CHF). We recently reviewed the capacity of cardiomyocytes to initiate inflammation, by means of expressing certain immune receptors such as toll‐like receptors (TLRs) that respond to pathogen‐ and damage‐associated molecular patterns (PAMP and DAMP). Previous studies observed TLR4‐mediated inflammation within days of myocardial infarction (MI). This study examined TLR4 expression and function in cardiomyocytes of failing hearts after 4 weeks of MI in rats. The increases of TLR4 mRNA and proteins, as well as inflammatory cytokine production, were observed in both the infarct and remote myocardium. Enhanced immunostaining for TLR4 was observed in cardiomyocytes but not infiltrating leucocytes. The injection of lentivirus shRNA against TLR4 into the infarcted heart decreased inflammatory cytokine production and improved heart function in vivo. Accordingly, in cardiomyocytes isolated from CHF hearts, increases of TLR4 mRNA and proteins were detected. More robust binding of TLR4 with lipopolysaccharide (LPS), a PAMP ligand for TLR4, and heat shock protein 60 (HSP60), a DAMP ligand for TLR4, was observed in CHF cardiomyocytes under a confocal microscope. The maximum binding capacity (B_max) of TLR4 was increased for LPS and HSP60, whereas the binding affinity (Kd) was not significantly changed. Furthermore, both LPS and HSP60 induced more robust production of inflammatory cytokines in CHF cardiomyocytes, which was reduced by TLR4‐blocking antibodies. We conclude that the expression, ligand‐binding capacity and pro‐inflammatory function of cardiomyocyte TLR4 are up‐regulated after long‐term MI, which promote inflammation and exacerbate heart failure.     

绵羊皮肤中GHR、IGF-1和IGF-1R基因表达的发育性变化及品种特点

采用相对定量反转录多聚酶链式反应 (RT-PCR)方法, 以18S rRNA作内标, 研究了罗米丽(Romilly Hillys)×中国美利奴(新疆军垦型)杂交一代优质细毛羊和哈萨克粗毛羊皮肤中生长激素受体(GHR)、胰岛素样生长因子1(IGF-1)和胰岛素样生长因子1受体(IGF-1R) mRNA发育性变化并进行了品种间比较。分别于30、60、90、135、180和255日龄称重、采毛样, 并于30、90、135和255日龄采皮样。结果表明: 粗毛羊和细毛羊体重、羊毛生长的发育模式没有明显的差异, 30~135日龄体重迅速增加, 135~255日龄增重十分缓慢; 30~135日龄羊毛日增长逐渐增加, 135~180日龄羊毛生长十分缓慢, 而180~255日龄又上升到较高水平。粗毛羊皮肤中GHR mRNA在30~90日龄显著增加 (P<0.05), 90日龄达到高峰, 此后显著下降(P<0.05); 细毛羊在135日龄时GHR mRNA极显著地升高(P<0.01), 此后又极显著地下降。粗毛羊皮肤中IGF-1、IGF-1R mRNA 30~90日龄上升, 90日龄之后极显著下降(P<0.01); 细毛羊皮肤中IGF-1、IGF-1R mRNA出生时较高, 然后逐渐下降。品种之间比较, 细毛羊GHR mRNA出现高峰晚于粗毛羊, 135日龄高峰时显著地高于粗毛羊; 粗毛羊IGF-1、IGF-1R mRNA在90日龄出现高峰, 并极显著或显著地高于细毛羊; 粗毛羊90日龄前GHR、IGF-1和IGF-1R mRNA高于细毛羊, 之后低于细毛羊。结果提示: 绵羊皮肤中GHR、IGF-1和IGF-1R基因表达有特定的发育模式, 并存在品种差异。     

Serum markers of apoptosis in the early period of heart transplantation

Context and objective: To assess the relationship between levels of serum markers of apoptosis and rejection grades in heart transplant (HTx).

Materials and methods: A prospective study was conducted in 91 HTx. We correlated apoptosis markers and biopsy samples. The apoptosis markers were: TRAIL, TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, sFas, sTNF-R1 and sTNF-R2.

Results: The only significant correlation with rejection grade was sFas (r?=?0.329; p?=?0.005). Cyclosporine showed a proapoptotic effect (sTNF-R1 0.02 and sTNF-R2 0.02) and everolimus an antiapoptotic effect (sTNF-R1 r?=??0.523; p?=?0.0001 and sTNF-R2 r?=??0.405; p?=?0.0001).

Conclusions: The utility of specific apoptosis markers in peripheral blood for diagnosis of acute cellular rejection is low. Everolimus may have an anti-apoptotic effect.     

Crosstalk between the extracellular domain of the ErbB2 receptor and IGF-1 receptor signaling

Insulin-like growth factor 1 receptor (IGF-1R) plays an important role in cell growth and malignant transformation. To investigate IGF-1R-dependent signaling events and its effects on apoptosis induction and cellular proliferation, we generated a constitutively active, ligand-independent IGF-1R variant. We fused the cytoplasmic domain of the IGF-1R to the extracellular and transmembrane domains of the oncogenic ErbB2 receptor (ErbB2^V→E/IGF-1). A fusion protein in which the wild-type sequence of the ErbB2 receptor was used, served as a control (ErbB2^V/IGF-1R). ErbB2^V/IGF-1R, ErbB2^V→E/IGF-1R and IGF-1R were stably transfected into interleukin 3 (IL-3)-dependent BaF/3 cells. ErbB2^V→E/IGF-1R expressing cells exhibited ligand-independent, constitutive tyrosine phosphorylation of the receptor fusion protein. Constitutively, activated ErbB2^V→E/IGF-1R conferred IL-3 independence for growth and survival to the transfected BaF/3 cells. Constitutive activation of the IGF-1R results in cellular growth and protection against apoptosis upon IL-3 withdrawal in BaF/3 cells.     

高龄老年心衰患者外周血淋巴细胞GRK2的表达研究

目的:观察高龄老年心衰患者外周血淋巴细胞GRK2的表达及其与心脏射血分数(EF)的关系。方法:选取80岁以上心衰患者16例,按EF分为两组:EF45%组(n=7),EF≥45%组(n=9),80岁以上高龄健康老人作为对照组(n=8),分别抽取外周血2ml,分离淋巴细胞,提取RNA,检测GRK2mRNA的表达。结果:EF45%组的高龄心衰患者外周血淋巴细胞GRK2mRNA的表达高于EF≥45%组(P0.05),且两组明显高于对照组。随着EF的减低,外周血淋巴细胞GRK2mRNA的表达增加。结论:随着EF的减低,高龄心衰患者外周血淋巴细胞GRK2mRNA的表达增加,外周血淋巴细胞GRK2的检测有助于判断高龄老年心衰患者心功能状况及临床治疗心衰疗效的判定。     

mTORC2 promotes type I insulin-like growth factor receptor and insulin receptor activation through the tyrosine kinase activity of mTOR

Mammalian target of rapamycin (mTOR) is a core component of raptor-mTOR (mTORC1) and rictor-mTOR (mTORC2) complexes that control diverse cellular processes. Both mTORC1 and mTORC2 regulate several elements downstream of type I insulin-like growth factor receptor (IGF-IR) and insulin receptor (InsR). However, it is unknown whether and how mTOR regulates IGF-IR and InsR themselves. Here we show that mTOR possesses unexpected tyrosine kinase activity and activates IGF-IR/InsR. Rapamycin induces the tyrosine phosphorylation and activation of IGF-IR/InsR, which is largely dependent on rictor and mTOR. Moreover, mTORC2 promotes ligand-induced activation of IGF-IR/InsR. IGF- and insulin-induced IGF-IR/InsR phosphorylation is significantly compromised in rictor-null cells. Insulin receptor substrate (IRS) directly interacts with SIN1 thereby recruiting mTORC2 to IGF-IR/InsR and promoting rapamycin- or ligand-induced phosphorylation of IGF-IR/InsR. mTOR exhibits tyrosine kinase activity towards the general tyrosine kinase substrate poly(Glu-Tyr) and IGF-IR/InsR. Both recombinant mTOR and immunoprecipitated mTORC2 phosphorylate IGF-IR and InsR on Tyr1131/1136 and Tyr1146/1151, respectively. These effects are independent of the intrinsic kinase activity of IGF-IR/InsR, as determined by assays on kinase-dead IGF-IR/InsR mutants. While both rictor and mTOR immunoprecitates from rictor^+/+ MCF-10A cells exhibit tyrosine kinase activity towards IGF-IR and InsR, mTOR immunoprecipitates from rictor^−/− MCF-10A cells do not induce IGF-IR and InsR phosphorylation. Phosphorylation-deficient mutation of residue Tyr1131 in IGF-IR or Tyr1146 in InsR abrogates the activation of IGF-IR/InsR by mTOR. Finally, overexpression of rictor promotes IGF-induced cell proliferation. Our work identifies mTOR as a dual-specificity kinase and clarifies how mTORC2 promotes IGF-IR/InsR activation.     

Insulin‐like growth factor binding protein 4 inhibits proliferation of bone marrow mesenchymal stem cells and enhances growth of neurospheres derived from the stem cells

Insulin‐like growth factor binding protein 4 (IGFBP‐4) was reported to trigger cellular senescence and reduce cell growth of bone marrow mesenchymal stem cells (BMSCs), but its contribution to neurogenic differentiation of BMSCs remains unknown. In the present study, BMSCs were isolated from the femur and tibia of young rats to investigate effects of IGFBP‐4 on BMSC proliferation and growth of neurospheres derived from BMSCs. Bone marrow mesenchymal stem cell proliferation was assessed using CCK‐8 after treatment with IGFBP‐4 or blockers of IGF‐IR and β‐catenin. Phosphorylation levels of Akt, Erk, and p38 in BMSCs were analysed by Western blotting. Bone marrow mesenchymal stem cells were induced into neural lineages in NeuroCult medium; the number and the size of BMSC‐derived neurospheres were counted after treatment with IGFBP‐4 or the blockers. It was shown that addition of IGFBP‐4 inhibited BMSC proliferation and immunodepletion of IGFBP‐4 increased the proliferation. The blockade of IGF‐IR with AG1024 increased BMSC proliferation and reversed IGFBP‐4‐induced proliferation inhibition; however, blocking of β‐catenin with FH535 did not. p‐Erk was significantly decreased in IGFBP‐4‐treated BMSCs. IGFBP‐4 promoted the growth of neurospheres derived from BMSCs, as manifested by the increases in the number and the size of the derived neurospheres. Both AG1024 and FH535 inhibited the formation of NeuroCult‐induced neurospheres, but FH535 significantly inhibited the growth of neurospheres in NeuroCult medium with EGF, bFGF, and IGFBP‐4. The data suggested that IGFBP‐4 inhibits BMSC proliferation through IGF‐IR pathway and promotes growth of BMSC‐derived neurospheres via stabilizing β‐catenin.     

血清sST2在心力衰竭诊断、预后中的应用价值

心力衰竭(心衰)的发病率正随着人口老龄化的加速而显著上升,目前仍然是一个重大的公共健康问题。尽管近年来在心衰治疗方面取得了显著成效,但患者的生存率依旧很低,预后差,确诊心衰后5年内死亡率高达50%。如果能够对心衰进行快速有效的诊断并按危险程度进行合理分层,将为临床医生制定治疗方案提供重要的参考依据。生物标志物在心衰的诊断、疗效评估及预后判断方面都具有重要的意义。心力衰竭是一种复杂的疾病,涉及多种生理病理过程。心力衰竭时,神经内分泌系统被激活,同时伴随着血容量和心室壁压力增加,心室肌细胞分泌NT-proBNP/BNP,因此,其可作为心衰诊断和预后生物标志物。然而血浆中NT-proBNP/BNP易受到年龄、性别、体型、左室肥大、心动过速、右心室过载、低氧血症、肾脏功能等诸多因素影响。sST2作为一种新型心力衰竭标志物,近年来备受关注,它不仅能够反映心肌纤维化程度并预测是否发生心室重构,且不受年龄、性别、肾功能等因素的影响,同时具有更低的参考变化值和个体指数,更适合用于连续监测和指导治疗,是评价心力衰竭的理想指标之一。文中对近年来sST2在心衰诊断和预后方面的研究进展进行总结归纳,并对其发展...     

Adrenergic and muscarinic receptor regulation and therapeutic implications in heart failure

In end-stage heart failure the expression of different myocardial regulatory proteins involved in the -adrenergic cAMP signalling pathway is altered. The downregulation of -adrenoceptors and their uncoupling from the effector as well as an increased expression of the inhibitory GTP-binding protein seem to be the most important alterations. Since catecholamine levels are elevated in these patients and since some alterations can be restored after treatment with -adrenoceptor antagonists it was hypothesized that excessive -adrenergic stimulation could be involved in these alterations.In this article the changes of -adrenergic receptors, GTP-binding proteins, sarcoplasmic reticulum Ca²⁺-ATPase and of phospholamban found in heart failure are addressed with its possible therapeutic implications.     

Nordihydroguaiaretic acid inhibits insulin-like growth factor signaling, growth, and survival in human neuroblastoma cells

Neuroblastoma is a common pediatric malignancy that metastasizes to the liver, bone, and other organs. Children with metastatic disease have a less than 50% chance of survival with current treatments. Insulin-like growth factors (IGFs) stimulate neuroblastoma growth, survival, and motility, and are expressed by neuroblastoma cells and the tissues they invade. Thus, therapies that disrupt the effects of IGFs on neuroblastoma tumorigenesis may slow disease progression. We show that NVP-AEW541, a specific inhibitor of the IGF-I receptor (IGF-IR), potently inhibits neuroblastoma growth in vitro. Nordihydroguaiaretic acid (NDGA), a phenolic compound isolated from the creosote bush (Larrea divaricata), has anti-tumor properties against a number of malignancies, has been shown to inhibit the phosphorylation and activation of the IGF-IR in breast cancer cells, and is currently in Phase I trials for prostate cancer. In the present study in neuroblastoma, NDGA inhibits IGF-I-mediated activation of the IGF-IR and disrupts activation of ERK and Akt signaling pathways induced by IGF-I. NDGA inhibits growth of neuroblastoma cells and induces apoptosis at higher doses, causing IGF-I-resistant activation of caspase-3 and a large increase in the fraction of sub-G0 cells. In addition, NDGA inhibits the growth of xenografted human neuroblastoma tumors in nude mice. These results indicate that NDGA may be useful in the treatment of neuroblastoma and may function in part via disruption of IGF-IR signaling.     

Insulin-like growth factor-1 increases endothelin receptor A levels and action in cultured rat aortic smooth muscle cells

Insulin is known to cause an increase in endothelin-1 (ET-1) receptors in vascular smooth muscle cells (SMCs), but the effect of insulin-like growth factor 1 (IGF-1) on ET-1 receptor expression is not known. We therefore carried out the present study to determine the effect of IGF-1 on the binding of ET-1 to, and ET type A receptor (ETAR) expression and ET-1-induced 3H-thymidine incorporation in, vascular SMCs. In serum-free medium, IGF-1 treatment increased the binding of 125I-ET-1 to SMC cell surface ET receptors from a specific binding of 20.1%+/-3.1% per mg of protein in control cells to 45.1%+/-8.6% per mg of protein in cells treated with IGF-1 (10 nM). The effect of IGF-1 was dose-related, with a significant effect (1.4-fold) being seen at 1 nM. The minimal time for IGF-1 treatment to be effective was 30 min and the maximal effect was reached at 6 h. Immunoblotting analysis showed that ETAR expression in IGF-1-treated cells was increased by 1.7-fold compared to controls. Levels of ETAR mRNA measured by the RT-PCR method and Northern blotting were also increased by 2-fold in IGF-1-treated SMCs. These effects of IGF-1 were abolished by cycloheximide or genistein. Finally, ET-1-stimulated thymidine uptake and cell proliferation were enhanced by IGF-1 treatment, with a maximal increase of 3.2-fold compared to controls. In conclusion, in vascular SMCs, IGF-1 increases the expression of the ET-1 receptor in a dose- and time-related manner. This effect is associated with increased thymidine uptake and involves tyrosine kinase activation and new protein synthesis. These findings support the role of IGF-1 in the development of atherosclerotic, hypertensive, and diabetic vascular complications.     

microRNA-454-mediated NEDD4-2/TrkA/cAMP axis in heart failure: Mechanisms and cardioprotective implications

The current study aimed to investigate the mechanism by which miR-454 influences the progression of heart failure (HF) in relation to the neural precursor cell expressed, developmentally downregulated 4-2 (NEDD4-2)/tropomyosin receptor kinase A (TrkA)/cyclic adenosine 3',5'-monophosphate (cAMP) axis. Sprague-Dawley rats were used to establish a HF animal model via ligation of the left anterior descending branch of the coronary artery. The cardiomyocyte H9c2 cells were treated with H₂O₂ to stimulate oxidative stress injury in vitro. RT-qPCR and Western blot assay were subsequently performed to determine the expression patterns of miR-454, NEDD4-2, TrkA, apoptosis-related proteins and cAMP pathway markers. Dual-luciferase reporter gene assay coupled with co-immunoprecipitation was performed to elucidate the relationship between miR-454, NEDD4-2 and TrkA. Gain- or loss-of-function experiments as well as rescue experiments were conducted via transient transfection (in vitro) and adenovirus infection (in vivo) to examine their respective functions on H9c2 cell apoptosis and myocardial damage. Our results suggested that miR-454 was aberrantly downregulated in the context of HF, while evidence was obtained suggesting that it targeted NEDD4-2 to downregulate NEDD4-2 in cardiomyocytes. miR-454 exerted anti-apoptotic and protective effects on cardiomyocytes through inhibition of NEDD4-2, while NEDD4-2 stimulated ubiquitination and degradation of TrkA protein. Furthermore, miR-454 activated the cAMP pathway via the NEDD4-2/TrkA axis, which ultimately suppressed cardiomyocyte apoptosis and attenuated myocardial damage. Taken together, the key findings of the current study highlight the cardioprotective role of miR-454, which is achieved through activation of the cAMP pathway by impairing NEDD4-2-induced TrkA ubiquitination.