受体介导的基因转移技术

通过受体对DNA-配体复合物的特异识别和内吞, 可以将外源基因导入特定的细胞内进行表达,称为受体介导的基因转移技术. 对该项技术的基本方法、体内外应用研究进展以及发展方向等进行了介绍和综述.     

Insulin Receptor Sub-Types in a Human Lymphoid-Derived Cell Line (IM-9): Differential Regulation by Insulin,Dexamethasone and Monensin

Abstract

The cells of the human IM-9 lymphocyte-derived line contain a sub-population of insulin binding sites which differ from classical insulin binding sites in their higher binding affinity for insulin-like growth factor II (IGF-II) and insulin-like growth factor I (IGF-I). These atypical insulin binding sites are identified on IM-9 cells by [¹²⁵I]IGF-II binding.

To determine whether the atypical and classical insulin receptors of IM-9 cells were subject to different modes of in vivo regulation, we treated IM-9 cells with agents known to alter the surface expression of insulin receptors - insulin, dexamethasone and monensin. We then measured insulin and IGF-II binding to the surface of the washed cells.

Pretreatment of IM-9 cells with 1 μM insulin for 20 h at 37°C induced a 44–48% decrease in the number of high affinity insulin binding sites, but no change in the number of IGF-II binding sites. In contrast, the surface expression of both insulin and IGF-II binding sites (classical and atypical insulin receptors) increased 1.3 to 1.7-fold after treatment with dexamethasone (200 nM) and decreased 30 to 45% after monensin (1 μM). These results suggest that atypical and classical insulin receptors are differentially susceptible to down-regulation by insulin.     

LRP6 Enhances Glucose Metabolism by Promoting TCF7L2-Dependent Insulin Receptor Expression and IGF Receptor Stabilization in Humans

1. Download : Download high-res image (198KB)
2. Download : Download full-size image

Highlights? Nondiabetic LRP6 mutation carriers are hyperinsulinemic and insulin resistant ? IR expression is reduced in skeletal muscles of the LRP6 mutation carriers ? Wnt/LRP6 regulate the insulin receptor and IGFR expression ? The LRP6 mutation reduces TCF7L2-dependent IR expression and enhances mTOR activity     

胰岛素受体介导的信号传递

就胰岛素与其受体结合后, 信号传递的过程及参与信号传递的细胞内信号分子进行了综述.胰岛素作为一种重要激素,参与机体的新陈代谢, 调节细胞的生长分化.其发挥生理功能的第一步是与靶细胞膜上的受体相结合, 激活胰岛素受体的酪氨酸激酶活性, 随之磷酸化细胞内的信号分子, 从而使胰岛素的刺激信号转化为细胞反应.     

Signal Transduction Cross Talk Mediated by Jun N-Terminal Kinase-Interacting Protein and Insulin Receptor Substrate Scaffold Protein Complexes

Scaffold proteins have been established as important mediators of signal transduction specificity. The insulin receptor substrate (IRS) proteins represent a critical group of scaffold proteins that are required for signal transduction by the insulin receptor, including the activation of phosphatidylinositol 3 kinase. The c-Jun NH₂-terminal kinase (JNK)-interacting proteins (JIPs) represent a different group of scaffold molecules that are implicated in the regulation of the JNK. These two signaling pathways are functionally linked because JNK can phosphorylate IRS1 on the negative regulatory site Ser-307. Here we demonstrate the physical association of these signaling pathways using a proteomic approach that identified insulin-regulated complexes of JIPs together with IRS scaffold proteins. Studies using mice with JIP scaffold protein defects confirm that the JIP1 and JIP2 proteins are required for normal glucose homeostasis. Together, these observations demonstrate that JIP proteins can influence insulin-stimulated signal transduction mediated by IRS proteins.The c-Jun NH₂-terminal kinase (JNK)-interacting proteins (JIPs) are implicated in the regulation of the JNK signal transduction pathway (8, 28). The JIP1 and JIP2 proteins are structurally related with similar modular domains (SH3 and PTB) and binding sites for the mixed-lineage protein kinase (MLK) group of mitogen-activated protein kinase (MAPK) kinase kinases, the MAPK kinase MKK7, and JNK (19). These JIP proteins also interact with the microtubule motor protein kinesin, several guanine nucleotide exchange factors, the phosphatase MKP7, Src-related protein kinases, and AKT to form multifunctional protein complexes (19).One potential physiological role of JIP scaffold proteins is the response to metabolic stress, insulin resistance, and diabetes. Several lines of evidence support this hypothesis. First, JIP1 is required for metabolic stress-induced activation of JNK in white adipose tissue (12). Second, MLKs that interact with JIP proteins are implicated as essential components of a signaling pathway that mediates the effects of metabolic stress on JNK activation (13). Third, studies have demonstrated that the human Jip1 gene may contribute to the development of type 2 diabetes, because a Jip1 missense mutation was found to segregate with type 2 diabetes (26). Collectively, these data suggest that JIP proteins play a role in the cellular response to metabolic stress and the regulation of insulin resistance.It is established that the insulin receptor substrate (IRS) group of scaffold proteins plays a central role in insulin signaling (27). Treatment of cells with insulin causes tyrosine phosphorylation of the insulin receptor, the recruitment of IRS proteins to the insulin receptor, and the subsequent tyrosine phosphorylation of IRS proteins on multiple residues that act as docking sites for insulin-regulated signaling molecules, including phosphatidylinositol 3 kinase (27). Negative regulation of IRS proteins is implicated as a mechanism of insulin resistance and can be mediated by multiple pathways, including IRS protein phosphorylation and degradation. Thus, the mTOR/p70^S6K (21, 22, 24) and the SOCS-1/3 (20) signaling pathways can regulate IRS protein degradation. Multisite phosphorylation on Ser/Thr residues can also regulate IRS protein function, including JNK phosphorylation of IRS1 on the inhibitory site Ser-307 that prevents recruitment of IRS1 to the activated insulin receptor (2).The IRS and JIP groups of scaffold proteins may function independently to regulate JNK-dependent and insulin-dependent signal transduction. However, functional connections between these scaffold proteins have been identified. Thus, studies using Jip1^−/− mice demonstrate that JIP1 is required for high-fat-diet-induced JNK activation in white adipose tissue, IRS1 phosphorylation on the inhibitory site Ser-307, and insulin resistance (12). These data suggest that JIP scaffold proteins function cooperatively with IRS proteins to regulate signal transduction by the insulin receptor. The purpose of this study was to examine cross talk between the JIP and IRS scaffold complexes. We demonstrate that the JIP and IRS scaffold complexes physically interact in an insulin-dependent manner and confirm that JIP proteins influence normal glucose homeostasis.     

Dexamethasone regulation of cFos mRNA in osteoprogenitors

The glucocorticoid, dexamethasone (Dex), has a beneficial effect on osteogenesis by increasing the activation and differentiation of osteoblastic cells. We investigated the effect of Dex on osteoblasts and detected changes in pattern of expression of cFos mRNA. cFos is an early response gene that is expressed as unspliced and spliced mRNAs. We analyzed the regulation of cFos mRNA in correlation with the cell proliferation status and following the induction by Dex. Treatment of osteoblastic cells with Dex for 30 min resulted in elevated levels of spliced form of cFos mRNA, which was maintained with extended treatment of cultured cells for 24 h. In addition, we demonstrated interaction between glucocorticoid receptor (GR) and cFos mRNA. This study provides evidence for the action of Dex on osteoblasts through dynamic cell responses involving with cFos.     

Insulin Receptor Signaling in Cones

In humans, age-related macular degeneration and diabetic retinopathy are the most common disorders affecting cones. In retinitis pigmentosa (RP), cone cell death precedes rod cell death. Systemic administration of insulin delays the death of cones in RP mouse models lacking rods. To date there are no studies on the insulin receptor signaling in cones; however, mRNA levels of IR signaling proteins are significantly higher in cone-dominant neural retina leucine zipper (Nrl) knock-out mouse retinas compared with wild type rod-dominant retinas. We previously reported that conditional deletion of the p85α subunit of phosphoinositide 3-kinase (PI3K) in cones resulted in age-related cone degeneration, and the phenotype was not rescued by healthy rods, raising the question of why cones are not protected by the rod-derived cone survival factors. Interestingly, systemic administration of insulin has been shown to delay the death of cones in mouse models of RP lacking rods. These observations led to the hypothesis that cones may have their own endogenous neuroprotective pathway, or rod-derived cone survival factors may be signaled through cone PI3K. To test this hypothesis we generated p85α^−/−/Nrl^−/− double knock-out mice and also rhodopsin mutant mice lacking p85α and examined the effect of the p85α subunit of PI3K on cone survival. We found that the rate of cone degeneration is significantly faster in both of these models compared with respective mice with competent p85α. These studies suggest that cones may have their own endogenous PI3K-mediated neuroprotective pathway in addition to the cone viability survival signals derived from rods.     

Caspase-mediated Cleavage of Insulin Receptor Substrate

Apoptosis is an important mechanism for maintaining tissue homeostasis. The efficient induction and execution of apoptosis are essential for cell clearance in specific developmental situations. Insulin-like growth factor (IGF)-I is a survival factor for epithelial cells in the mammary gland, and its withdrawal or inhibition leads to apoptosis. In this paper we describe a novel mechanism that may lead to suppression of an IGF-I-mediated signaling pathway through cleavage of insulin receptor substrate (IRS). During the process of forced weaning, when mammary epithelial cells rapidly enter apoptosis in vivo, IRS-1 and IRS-2 disappear. We have used cultured mammary epithelial cells to demonstrate that IRS removal can be mediated through the action of caspase 10. Caspase 10 activation and IRS-1 cleavage are regulated by a MKK1-signaling pathway but not by a phosphatidylinositol-3 kinase pathway nor by the extracellular proapoptotic ligands FasL, tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL), or transforming growth factor-beta3. In addition we show that the loss of IRS-1 after MKK1 inhibition prevents IGF-mediated phosphorylation of FKHRL1.     

异二聚体介导的mRNA核输出机制

真核生物mRNA在细胞核内被转录,而到达细胞质内翻译成为蛋白质,因此跨越核孔的核输出过程是必须的。现在已确定异二聚体TAP/NXT(在酵母中为Mex67p/Mtr2p)在此过程中是最基本的元件,此外其他相关因子如Aly、UAP56等也参与了这个复杂的过程,包括结合、输出、解离和载体输入。本文简要介绍了mRNA输出的基本机制。     

ASGP受体介导的体内基因转移

我们针对肝细胞特异的ＡＳＧＰ（去唾液酸糖蛋白）受体,构建了一种具有肝细胞特异导向性的基因转移载体系统,该载体包括两种共价结合的功能成分：一种为ＡＳＧＰ,作为配体,与肝细胞表面特异的ＡＳＧＰ受体结合;另一种为多聚左旋赖氨酸（ｐｏｌｙ－Ｌ－ｌｙｓｉｎｅ）,与ＤＮＡ以强静电作用相结合。小鼠尾静脉注射３２Ｐ－ＤＮＡ－ｐｏｌｙ－Ｌ－ｌｙｓｉｎｅ－ＡＳＯＲ（去唾液酸ａｌ酸性糖蛋白）或等量的３２Ｐ－ＤＮＡ、ＡＳＯＲ、ｐｏｌｙ－ＤＬ－ｌｙｓｉｎｅ单体混合物,２０小时后,小鼠肝、脾、肾各组织的放射性计数结果表明该载体系统具有较强的肝组织特异性。     

3个六肽的胰岛素受体结合和体内生物活性

为了研究胰岛素受体结合部位的结构和功能,设计并用固相方法合成了３个六肽．在浓度大于１×１０３ｎｍｏｌ／Ｌ时,ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ－Ｖａｌ－Ｌｅｕ－Ｔｙｒ－Ｇｌｙ）具有明显的胰岛素受体结合活力;Ｈ－Ｐｈｅ－Ｐｈｅ－Ｖａｌ－Ｌｅｕ－Ｔｙｒ－Ｇｌｙ－ＯＨ的这一活力则不明显;而Ｈ－Ｇｌｙ－Ｇｌｕ－Ａｒｇ－Ｇｌｙ－Ｐｈｅ－Ｐｈｅ－ＯＨ则增强胰岛素和其受体的亲和性．然而,它们都没有体内生物活性．这表明：环六肽部分模拟了胰岛素受体结合部位的空间构象;胰岛素受体结合部位的疏水性和其中的Ｂ２３Ｇｌｙ－Ｂ２４Ｐｈｅ－Ｂ２５Ｐｈｅ对胰岛素和其受体的结合起重要作用．     

胰岛素对βTC-3 细胞胰岛素受体表达的作用

目的:观察外源性胰岛素对小鼠胰岛β细胞瘤细胞株βTC-3细胞胰岛素受体表达的影响。方法:采用免疫荧光细胞化学技术结合激光扫描共聚焦显微镜观察高浓度胰岛素(100 IU/ml)刺激不同时间(0 min、30 min、60 min、120 min、240 min),培养的βTC-3细胞胰岛素受体的表达,用Image pro plus软件对胰岛素受体的荧光强度进行了半定量分析。结果:与0 min比较,胰岛素孵育30 min、60 min、120 min、240 min时胰岛素受体荧光强度均明显下降(P<0.05)。结论:高浓度胰岛素孵育βTC3细胞后,可明显下调胰岛素受体的表达,这可能是高胰岛素血症导致胰岛素抵抗产生的机制之一。     

胰岛素对βTC-3细胞胰岛素受体表达的作用

目的：观察外源性胰岛素对小鼠胰岛β细胞瘤细胞株βTC-3细胞胰岛素受体表达的影响。方法：采用免疫荧光细胞化学技术结合激光扫描共聚焦显微镜观察高浓度胰岛素（100 IU/ml）刺激不同时间（0 min、30 min、60 min、120 min、240 min）,培养的βTC-3细胞胰岛素受体的表达,用Image pro plus软件对胰岛素受体的荧光强度进行了半定量分析。结果：与0 min比较,胰岛素孵育30 min、60 min、120 min、240 min时胰岛素受体荧光强度均明显下降（P〈0.05）。结论：高浓度胰岛素孵育βTC3细胞后,可明显下调胰岛素受体的表达,这可能是高胰岛素血症导致胰岛素抵抗产生的机制之一。     

胰岛素受体变异性剪接与肿瘤和2型糖尿病

    胰岛素受体基因第11号外显子因为变异性剪接而形成两种胰岛素受体,两者与配体胰岛素、胰岛素样生长因子的结合力以及分别诱导的信号传导通路、发挥的生物学效应存在显著差异。这种差异不仅可能是导致胰岛素抵抗、2型糖尿病的重要原因,也会影响肿瘤细胞的生长、增殖、抗凋亡。虽然具体的调节机制尚不明确,但高胰岛素血症及高血糖等代谢因素是影响胰岛素受体变异性剪接的重要原因,同时基因序列敲除试验证实,胰岛素受体基因水平的改变会影响胰岛素受体的变异性剪接。           

Effect of Dexamethasone on Insulin Secretion: Examination of Underlying Mechanisms

ObjectiveTo study the mechanism of increased insulin secretion in response to short-term administration of dexamethasone.MethodsMale Wistar rats were injected intraperitoneally with dexamethasone (dexamethasone; 200 mcg/kg body weight per day) or saline for 3 consecutive days. Insulin secretion in response to glucose, ionomycin, and KCl was quantified in islets isolated from the animals, and the amount of glucokinase was measured by Western blot.ResultsDexamethasone-treated animals had 1.18-fold higher fasting blood glucose concentration and 6.5-fold increase in fasting serum insulin concentration compared with findings from animals injected with saline. Compared with islets isolated from control rats, islets from dexamethasone-treated rats secreted more insulin at 60 minutes in response to 5.5 mM glucose (416.4 vs 115.6 fmoles/10 islets, P = .011) and in response to 16.6 mM glucose (985.5 vs 520.6 fmoles/10 islets, P = .014); no change in insulin secretion was observed at 10 minutes. Insulin secretion from islets of dexamethasone-treated rats and control rats was not differentially augmented in response to either ionomycin or potassium chloride. Glucokinase expression was not altered by treatment with dexamethasone.ConclusionsAugmentation of insulin secretion in response to glucose in the pancreatic islets from dexamethasone-treated rats is preserved in islets studied in vitro. The increase in glucose-stimulated insulin secretion appears to be mediated by steps upstream to β -cell membrane depolarization and the attended increase in intracellular calcium in the signaling pathway of insulin secretion. (Endocr Pract. 2010;16:763-769)     

Toll-Like Receptor 9 Mediated Responses in Cardiac Fibroblasts

Altered cardiac Toll-like receptor 9 (TLR9) signaling is important in several experimental cardiovascular disorders. These studies have predominantly focused on cardiac myocytes or the heart as a whole. Cardiac fibroblasts have recently been attributed increasing significance in mediating inflammatory signaling. However, putative TLR9-signaling through cardiac fibroblasts remains non-investigated. Thus, our aim was to explore TLR9-signaling in cardiac fibroblasts and investigate the consequence of such receptor activity on classical cardiac fibroblast cellular functions. Cultivated murine cardiac fibroblasts were stimulated with different TLR9 agonists (CpG A, B and C) and assayed for the secretion of inflammatory cytokines (tumor necrosis factor α [TNFα], CXCL2 and interferon α/β). Expression of functional cardiac fibroblast TLR9 was proven as stimulation with CpG B and –C caused significant CXCL2 and TNFα-release. These responses were TLR9-specific as complete inhibition of receptor-stimulated responses was achieved by co-treatment with a TLR9-antagonist (ODN 2088) or chloroquine diphosphate. TLR9-stimulated responses were also found more potent in cardiac fibroblasts when compared with classical innate immune cells. Stimulation of cardiac fibroblasts TLR9 was also found to attenuate migration and proliferation, but did not influence myofibroblast differentiation in vitro. Finally, results from in vivo TLR9-stimulation with subsequent fractionation of specific cardiac cell-types (cardiac myocytes, CD45+ cells, CD31+ cells and cardiac fibroblast-enriched cell-fractions) corroborated our in vitro data and provided evidence of differentiated cell-specific cardiac responses. Thus, we conclude that cardiac fibroblast may constitute a significant TLR9 responder cell within the myocardium and, further, that such receptor activity may impact important cardiac fibroblast cellular functions.     

Agonism and Antagonism at the Insulin Receptor

Insulin can trigger metabolic as well as mitogenic effects, the latter being pharmaceutically undesirable. An understanding of the structure/function relationships between insulin receptor (IR) binding and mitogenic/metabolic signalling would greatly facilitate the preclinical development of new insulin analogues. The occurrence of ligand agonism and antagonism is well described for G protein-coupled receptors (GPCRs) and other receptors but in general, with the exception of antibodies, not for receptor tyrosine kinases (RTKs). In the case of the IR, no natural ligand or insulin analogue has been shown to exhibit antagonistic properties, with the exception of a crosslinked insulin dimer (B29-B’29). However, synthetic monomeric or dimeric peptides targeting sites 1 or 2 of the IR were shown to be either agonists or antagonists. We found here that the S961 peptide, previously described to be an IR antagonist, exhibited partial agonistic effects in the 1–10 nM range, showing altogether a bell-shaped dose-response curve. Intriguingly, the agonistic effects of S961 were seen only on mitogenic endpoints (³H-thymidine incorporation), and not on metabolic endpoints (¹⁴C-glucose incorporation in adipocytes and muscle cells). The agonistic effects of S961 were observed in 3 independent cell lines, with complete concordance between mitogenicity (³H-thymidine incorporation) and phosphorylation of the IR and Akt. Together with the B29-B’29 crosslinked dimer, S961 is a rare example of a mixed agonist/antagonist for the human IR. A plausible mechanistic explanation based on the bivalent crosslinking model of IR activation is proposed.