The role of Raf kinase inhibitor protein (RKIP) in health and disease

Raf kinase inhibitor protein (RKIP) is a member of the phosphatidylethanolamine-binding protein (PEBP) family. RKIP plays a pivotal modulatory role in several protein kinase signaling cascades. RKIP binds inhibits Raf-1-mediated phosphorylation of MEK through binding to Raf-1. Protein kinase C (PKC) phosphorylates RKIP, resulting in release of Raf-1 and activation of MEK and ERK. The phosphorylated RKIP binds to and inhibits G-protein-coupled receptor kinase, resulting in sustained G-protein signaling. The regulatory role that RKIP has in cell signaling is reflected in its role in physiology and pathophysiology. RKIP is involved in neural development, cardiac function and spermatogenesis and appears to have serine protease activity. In addition to its roles in physiology, dysregulated RKIP expression has the potential to contribute to pathophysiological processes including Alzheimer's disease and diabetic nephropathy. RKIP has been shown to fit the criteria of being a metastasis suppressor gene, including having decreased expression in prostate cancer metastases and restoring RKIP expression in a prostate cancer cell line diminishes metastasis in a murine model. Clearly, RKIP has multiple molecular and cellular functions. In this review, RKIP's molecular roles in intracellular signaling, its physiological functions and its role in disease are described.     

In vitro differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs), derived from Wharton's jelly, into choline acetyltransferase (ChAT)-positive cells

We isolated and expanded fibroblast-like cells from the Wharton's jelly of human umbilical cord successfully. Immunocytochemistry showed that they were positive for several markers of mesenchymal stem cells (CD73, CD90, and CD105) and integrin markers (CD29 and CD44), but negative for a hematopoietic cell maker (CD45) and an endothelial cell marker (CD31). Their differentiation into osteocytes and adipocytes under specific conditions indicated that they had multi-lineage differentiation potential. Therefore these results proved that the cells we obtained from Wharton's jelly were human umbilical cord mensenchymal stem cells (hUCMSCs). Using immunocytochemistry and Western blotting analysis, we found that after treatment with neuronal induction medium [NIM; consisting of brain-derived neurotrophic factor (BDNF) and low-serum media] for 14 days, hUCMSCs expressed a neuronal specific marker, microtubule associated protein 2 (MAP2), and extended neurite-like processes. After treatment with NIM, supplemented with hippocampal cholinergic neurostimulating peptide (HCNP) or rat denervated hippocampal extract [rDHE; derived from rat fimbria fornix (FF) transected hippocampus], hUCMSCs expressed choline acetytransferase (ChAT) and this action could be enhanced when cells were cultured with NIM, supplemented with HCNP and rDHE in combination. ELISA showed that these ChAT-positive cells could secrete acetylcholine (ACh). These findings indicate that hUCMSCs possess the potential of differentiation into functional ChAT-positive cells in vitro and provide a new candidate of cells for the cell transplantation to treat Alzheimer's disease (AD).     

Bone marrow-derived mesenchymal stem cells prevent the apoptosis of neuron-like PC12 cells via erythropoietin expression

Hippocampal cholinergic neurostimulating peptide (HCNP) induces the synthesis of acetylcholine in medial septal nucleus in vitro and in vivo. HCNP precursor protein (HCNP-pp) is a multifunctional protein that participates in a number of signaling pathways, including MAPK/extracellular signal and G-protein-coupled receptor kinase 2. We recently demonstrated that the amount of collapsin response mediator protein-2 (CRMP-2) is increased in hippocampus of HCNP-pp transgenic mice. To clarify the interaction between HCNP/HCNP-pp and CRMP-2 and its role in synaptic function, we investigated whether HCNP-pp is localized to the synapse and if it affects protein expression. Here, we demonstrate that HCNP-pp co-localizes with CRMP-2 at presynaptic terminals. Furthermore, HCNP-pp overexpression increases synaptophysin levels. These findings suggest that HCNP-pp, in association with CRMP-2, plays an important role in presynaptic function in the hippocampus.     

Distribution of hippocampal cholinergic neurostimulating peptide (HCNP) immunoreactivity in the central nervous system of the rat

Hippocampal cholinergic neurostimulating peptide (HCNP), an undecapeptide isolated from the hippocampal tissue of young rats, enhances the cholinergic development in explant cultures of medial septal nuclei. This report concerns the distribution of HCNP immunoreactivity in the central nervous system (CNS) of 11- and 28-day-old Wistar rats; two affinity-purified anti HCNP antibodies were used. Immunoblot analyses of extracts of different regions of the brain revealed a single 23 kDa band that corresponded to the presumed HCNP precursor protein. Immunostaining of the various CNS structures of the 28-day-old rats was more intense than in those of 11-day-old animals. HCNP immunoreactivity was detected in neurons as well as in glia cells, particularly oligodendroglia. The perikarya of neurons in the cerebral cortex, hippocampus, limbic cortex, caudate, putamen, arcuate nucleus of hypothalamus, trigeminal subnuclei, rostroventrolateral reticular nucleus and dorsal horn of the spinal cord were positively stained. In addition, nerve fibers and terminals in the hypothalamic subnuclei, zona incerta, thalamic subnucleus, caudate, putamen, locus coeruleus, trigeminal subnuclei, dorsal motor nucleus of the vagus, dorsal horn of the spinal cord and intermediolateral column also displayed HCNP immunoreactivity. These observations would suggest that HCNP and its related molecules may have multifunctional roles in the CNS.     

海马胆碱能刺激肽促进神经干细胞向神经元分化

目的探讨海马胆碱能刺激肽(HCNP)在神经干细胞向神经元分化过程中所起的作用。方法切割SD大鼠右侧穹窿海马伞,14天后取切割穹窿海马伞侧海马制成海马提取液;将神经干细胞接种于24孔板中,分为HCNP与海马提取液联合培养组、HCNP组以及空白对照组,每组八孔;细胞分化至14天时行Tuj-1、MAP-2免疫荧光检测;计数Tuj-1、MAP-2阳性神经元数及细胞周长,并观察胞体大小和突起长度。结果联合培养组Tuj-1、MAP-2阳性神经元最多,胞体大,突起长;HCNP组神经元数量较前组少,胞体较小,突起数量和长度均小于前组;对照组则仅有少量胞体小、突起短的神经元。HCNP与切割穹窿海马伞侧海马提取液联合培养组神经元成熟度优于其它两组。结论 HCNP对神经干细胞向神经元的分化具有明显促进作用。     

利拉鲁肽对糖尿病前期大鼠胰岛海马胆碱能神经刺激肽前体蛋白的影响

目的明确利拉鲁肽对糖尿病前期OLETF大鼠的阻抑作用,观察对胰腺海马胆碱能神经刺激肽前体蛋白(hippocampal cholinergicneurostimulating peptide precursor protein,HCNP-pp)的影响。方法检测空腹及餐后2小时血糖,将处于糖耐量减低阶段12～14周龄OLETF大鼠随机分为3组,安慰剂组(PBO组)、100μg/kg利拉鲁肽处理组(L-100组)、200μg/kg利拉鲁肽处理组(L-200组),LETO大鼠为阴性对照组(LETO组)。12周后检测大鼠体重、空腹血糖、餐后2 h血糖和胰岛素,以免疫组织化学染色分析胰岛细胞形态学变化,应用蛋白印迹技术检测HCNP-pp及胰高血糖素样多肽-1受体(GLP-1R)蛋白含量;应用实时定量PCR检测HCNP-pp和LGP-1R、胆碱乙酰转移酶(ChAT)、3型毒蕈碱样受体(M3R)基因表达水平。结果PBO组OLETF大鼠空腹、餐后2 h血糖符合糖尿病诊断标准,而L-100组、L-200组及LETO组大鼠均未进展为糖尿病状态。PBO组OLETF大鼠体重及空腹胰岛素均明显高于L-100组、L-200组和LETO组(P<0.01)。与PBO组相比,L-200组、LETO组大鼠胰腺胰岛素阳性细胞表达密度明显增多(P<0.01),而与L-100组差别无统计学意义(P>0.05);L-100组、L-200组及LEITO组大鼠胰腺HCNP-pp蛋白相对含量明显减少(P<0.05或P<0.01);L-100组、L-200组及LEITO组大鼠胰腺GLP-1R蛋白相对含量明显增多(P<0.01);L-100组、L-200组和LETO组大鼠胰腺HCNP-pp mRNA水平明显下降(P<0.01);L-200组和LETO组大鼠胰GLP-1R,ChAT、M3R mRNA水平均明显增多(P<0.01);L-100组大鼠GLP-1R和ChAT mRNA水平均明显增多(P<0.01),而M3R mRNA水平则无变化(P>0.05)。结论利拉鲁肽可阻抑糖尿病前期进展,可能与胰腺HCNP-pp表达下降、GLP-1R表达增高及胰岛β细胞密度增加相关。利拉鲁肽改善胰岛素分泌状态,可能与胰腺HCNP增多、ChAT和M3R表达增高相关。     

HCNP与海马提取液对神经干细胞向胆碱能神经元分化的协同作用

目的:探讨海马胆碱能神经元刺激肽(hippocampal cholinergic neurostimulating peptide,HCNP)与海马提取液对海马神经干细胞向神经元及胆碱能神经元分化的协同作用。方法:取孕17 d SD大鼠海马组织进行神经干细胞的培养、增殖、传代,并将其接种于2块24孔板中,培养基中均含B27、微量脑源性神经营养因子(BD-NF),将培养孔分为6组:H+T组,加入HCNP和切割穹窿海马伞侧海马提取液;H+N组,加入HCNP和正常侧海马提取液;H组,加入HCNP;T组,加入切割穹窿海马伞侧海马提取液;N组,加入正常侧海马提取液;对照组,不加HCNP和海马提取液。分化14 d后行MAP-2/ChAT免疫荧光双标检测。结果:与其它各组相比,H+T组分化的MAP-2阳性神经元最多,ChAT/MAP-2双标阳性神经元占MAP-2阳性神经元的比例最高(P<0.05);与N组相比,H+N组MAP-2阳性神经元较多,ChAT/MAP-2双标阳性神经元占MAP-2阳性神经元的比例也较高(P<0.05);与对照组相比,H组T组均可见一定数量的MAP-2阳性神经元及ChAT/MAP-2双标阳性神经元(P<0.05);N组以及对照组中MAP-2阳性神经元的数量和ChAT/MAP-2双标阳性神经元占MAP-2阳性神经元的比例则最低。结论:在微量BDNF存在的情况下,HCNP可促进海马神经干细胞向神经元和胆碱能神经元分化,切割侧海马提取液与HCNP可发挥协同作用。     

Accumulation of hippocampal cholinergic neurostimulating peptide (HCNP)-related components in Hirano bodies

We have previously isolated from the hippocampus of young rats a novel peptide termed 'hippocampal cholinergic neurostimulating peptide' (HCNP) which specifically enhances the cholinergic activity of the septohippocampal system in vitro . Cloning and base sequence analysis of HCNP-specific cDNA from rat and human cDNA libraries revealed that the 1.1 kDa peptide aligns at the N-terminal region of its 21 kDa precursor protein. An affinity-purified rabbit antibody to rat HCNP prepared by us recognizes the C-terminal domain of the peptide. while an antibody against human HCNP binds to a large portion of the peptide. In this report we show that both antibodies react with HCNP-related components present in the soluble cytosol fraction of human brain tissue.
Immunohistochemical examination of human nervous system tissues from elderly individuals revealed that Hirano bodies in Sommer's sector of the hippocampus were specifically stained by anti-HCNP antibodies. The number of HCNP-positive Hirano bodies was greater in patients with Alzheimer's disease than in normal, age-matched individuals. The immunohistochemical results were substantiated by immunoelectron microscopy. The present findings indicate that HCNP-related components accumulate in Hirano bodies, suggesting that patients who bear these inclusions may have a disturbance of the septohippocampal cholinergic system, considered to be of importance for learning and memory formation.