The aging brain, a key target for the future: The protein kinase C involvement

The brain represents the primary centre for the regulation and control of all our body activities, receiving and interpreting sensory impulses and transmitting information to the periphery. Most importantly, it is also the seat of consciousness, thought, emotion and especially memory, being in fact able to encode, store and recall any information. Memory is really what makes possible so many of our complex cognitive functions, including communication and learning, and surely without memory, life would lose all of its glamour and purpose. Age-associated mental impairment can range in severity from forgetfulness at the border with pathology to dementia, such as in Alzheimer's disease. In recent years, one of the most relevant observations of research on brain aging relates to data indicating that age-related cognitive decline is not only due to neuronal loss, as previously thought; instead, scientists now believe that age-associated functional changes have more to do with the dysfunctions occurring over time. Within this context a prominent role is certainly played by signal transduction cascades which guarantee neuronal cell to elaborate coordinated responses to the multiple signals coming from the outside and to adapt itself to the environmental changes and requests. This review will focus the attention on protein kinase C pathway, with a particular interest on its activation process, and on the role of protein-lipid and protein-protein interactions to selectively localize the cellular responses. Furthermore, information is emerging and will be discussed on the possibility of mRNA stabilization through PKC activation. This review will also approach the issue on how alterations of these molecular cascades may have implications in physiological and pathological brain aging, such as Alzheimer's disease.     

柴胡疏肝散、四君子汤对肝郁、脾虚大鼠Th细胞蛋白激酶C表达的影响

目的研究柴胡疏肝散、四君子汤对肝郁、脾虚大鼠Th细胞蛋白激酶C(PKC)表达的影响及其意义。方法应用RT—PCR法研究柴胡疏肝散、四君子汤对Th细胞PKCmRNA表达的影响。结果脾虚时PKCmRNA表达水平明显低于正常对照组 (P <0 .0 5 ) ,四君子汤治疗后恢复正常 (P <0 .0 5 ) ,柴胡疏肝散治疗后仍明显低于正常对照组 ;肝郁时PKCmRNA表达水平明显高于正常对照组 (P <0 .0 5 ) ,柴胡疏肝散治疗后恢复正常 (P <0 .0 5 ) ,但四君子汤治疗效应不明显。结论肝脾两脏在T细胞尤其是Th0的活化中具有相关性 ;PKC参与了肝脾这种相关的发生。     

卵巢癌中蛋白激酶C的表达及其临床意义

目的　探讨上皮性卵巢癌组织蛋白激酶C (proteinkinaseC ,PKC)的表达和化疗耐药的关系 ,以及与P -糖蛋白 (P -gp)的相关性。方法　用免疫组化S -P法检测 35例卵巢上皮性肿瘤组织、 2 0例卵巢良性肿瘤组织和 2 0例正常卵巢组织中PKC和P -gp的表达 ,并进行相关临床因素分析。结果　①PKC、P -gp在卵巢恶性肿瘤中的表达明显高于在良性及正常组织中的表达 ;并且PKC和P -gp的表达有相关性 (P <0 0 5 ) ;②卵巢癌PKC的表达与临床病理因素无直接关系 ;③恶性肿瘤中 ,初治和复发的PKC表达阳性率分别为 34 8%和 75 % ;④化疗对PKC表达阳性和阴性卵巢癌患者的有效率分别为 2 3 5 %、 6 6 7% (P <0 0 5 ) ;⑤PKC表达阴性患者的预后优于阳性者 (P =0 0 39)。结论　PKC表达与卵巢癌组织化疗耐药明显相关 ,可能在P -gp介导的卵巢癌多药耐药中起重要作用。     

雌激素替代治疗大鼠海马结构PKC阳性细胞的变化

目的 :观察雌性大鼠行去势后以及雌激素替代治疗海马结构蛋白激酶C ( proteinkinaseC ,PKC)阳性细胞的变化 ,通过该模型研究绝经期后女性情绪烦躁、记忆下降等神经精神症状的分子机制。方法 :将大鼠分为对照组、去势 3个月组和去势后雌激素替代治疗 3个月组 ,用免疫组织化学方法检测海马结构PKC阳性细胞的变化。结果 :PKC阳性细胞主要分布于下托和齿状回的颗粒层 ,其中齿状回内的阳性细胞较下托的多。去势 3个月组与对照组相比 ,齿状回内的PKC阳性细胞没有显著性减少 (P >0 .0 5 ) ,下托PKC阳性细胞显著减少 (P <0 .0 5 ) ;雌激素治疗组与对照组相比 ,下托细胞数量有所减少 ,但无显著性差异 (P >0 .0 5 )。结论 :海马结构下托PKC阳性细胞的减少可能在绝经后女性情绪烦躁、记忆下降等症状中起重要的作用。     

Insulin inhibits phagocytosis in normal human neutrophils via PKCα/β-dependent priming of F-actin assembly

Objective: This study investigated the effects of insulin on the phagocytosis of C3bi – and IgG-opsonized yeast particles in normal human neutrophils. Methods: Neutrophils were incubated in different insulin concentrations for 30 minutes and stimulated by C3bi – or IgG-opsonized yeast particles. Phagocytosis was quantified by both light microscopy and FACscan flow cytometry. Laser confocal microscopy was used for quantification of F-actin levels. Results: Elevated insulin concentrations decreased neutrophil phagocytosis of both types of targets. This defect was shown to be in part due to a delayed phagocytosis in the presence of insulin. Following a 30 minute incubation, insulin was found to increase the accumulation of cortical F-actin, without affecting the total cellular F-actin content. The specific PKCα/β inhibitor, Go6976, abolished the insulin-mediated increase in cortical F-actin content and both Go6976 and the PKCα/β/δ/ε-specific inhibitor GF109203X reversed the inhibitory effects of insulin on phagocytosis. Conclusion: Hyperinsulinemia in vitro can inhibit phagocytosis of opsonized targets in normal human neutrophils. This effect of insulin is dependent on activation of PKCα and/or PKCβ, and these insulin signals may interfere with the dynamic assembly/disassembly and/or distribution of F-actin, which is required for the phagocytosis process. Received 8 July 2005; accepted 13 October 2005 without revision I. Ahnfelt-R?nne     

Autocrine motility factor (neuroleukin, phosphohexose isomerase) induces cell movement through 12-lipoxygenase-dependent tyrosine phosphorylation and serine dephosphorylation events

Autocrine motility factor (AMF) is one of the motility cytokines regulating tumor cell migration, therefore identification of the signaling pathway coupled with it has critical importance. Previous studies revealed several elements of this pathway predominated by lipoxygenase-PKC activations but the role for tyrosine kinases remained questionable. Motility cytokines frequently have mitogenic effect as well, producing activation of overlapping signaling pathways therefore we have used B16a melanoma cells as models where AMF has exclusive motility effect. Our studies revealed that in B16a cells AMF initiated rapid (1–5 min) activation of the protein tyrosine kinase (PTK) cascade inducing phosphorylation of 179, 125, 95 and 40/37 kD proteins which was mediated by upstream cyclo- and lipoxygenases. The phosphorylated proteins were localized to the cortical actin-stress fiber attachment zones in situ by confocal microscopy. On the other hand, AMF receptor activation induced significant decrease in overall serine-phosphorylation level of cellular proteins accompanied by serine phosphorylation of 200, 90, 78 and 65 kd proteins. The decrease in serine phosphorylation was independent of PTKs, PKC as well as cyclo- and lipoxygenases. However, AMF induced robust translocation of PKCα to the stress fibers and cortical actin suggesting a critical role for this kinase in the generation of the motility signal. Based on the significant decrease in serine phosphorylation after AMF stimulus in B16a cells we postulated the involvement of putative serine/threonine phosphatase(s) upstream lipoxygenase and activation of the protein tyrosine kinase cascade downstream cyclo- and lipoxygenase(s) in the previously identified autocrine motility signal. This revised version was published online in July 2006 with corrections to the Cover Date.     

免疫调节剂对致痫大鼠脑内神经元及小胶质细胞内谷氨酸和蛋白激酶C免疫反应的影响

为了探讨免疫调节作用对癫痫发病的影响与相关机制,应用免疫细胞化学技术研究免疫调节剂干预戊四氮(pentyle-netetrazol,PTZ)致痫时,大鼠脑内及培养的小胶质细胞内谷氨酸(Glu)和蛋白激酶C(PKC)的表达变化。结果显示:在体实验中大鼠大脑皮质及海马,Glu和PKC在生理盐水对照组(NS组)、戊四氮组(PTZ组)、左旋咪唑+戊四氮组(LMS+PTZ组)、地塞米松+戊四氮组(DEX+PTZ组)均有不同的表达,且二种物质变化趋势基本一致。其中PTZ组较NS组表达增强,LMS+PTZ组进一步增强,DEX+PTZ组较PTZ组和LMS+PTZ组明显减弱。离体实验中,PTZ作用小胶质细胞2h即可引起Glu和PKC表达增多,6h达峰值,12h表达有所减弱,且PTZ组Glu和PKC表达较NS组明显增多,免疫增强剂(levamisole,LMS)可进一步增强Glu和PKC的表达;免疫抑制剂(dexamethasone,DEX)则下调其表达。以上结果提示免疫调节剂可能通过作用于神经元和小胶质细胞内Glu和PKC的表达而共同影响癫痫的发病机制和发病状态。     

Potent induction of human colon cancer cell uptake of chemotherapeutic drugs by N-myristoylated protein kinase C-α (PKC-α) pseudosubstrate peptides through a P-glycoprotein-independent mechanism

Phorbol ester protein kinase C (PKC) activators and PKC isozyme over-expression have been shown to significantly reduce intracellular accumulation of chemotherapeutic drugs, in association with the induction of multidrug resistance (MDR) in drug-sensitive cancer cells and enhancement of drug resistance in MDR cancer cells. These observations constitute solid evidence that PKC plays a significant role in the MDR phenotype of cancer cells. PKC-catalyzed phosphorylation of the drug-efflux pump P-glycoprotein was recently ruled out as a contributing factor in MDR. At present, the sole drug transport-related event that has been identified as a component of the role of PKC in MDR is PKC-induced expression of the P-glycoprotein-encoding gene mdr1. The objective of this study was to test the hypothesis that PKC can modulate the uptake of chemotherapeutic drugs in cancer cells independently of P-glycoprotein. We analyzed the effects of selective PKC activators/inhibitors on the uptake of radiolabelled cytotoxic drugs by cultured human colon cancer cells that lacked P-glycoprotein activity and did not express the drug efflux pump at the level of message (mdr1) or protein. We found that the selective PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) significantly reduced uptake of [¹⁴C] Adriamycin and [³H] vincristine in human colon cancer cells devoid of P-glycoprotein activity, and that PKC-inhibitory N-myristoylated PKC- pseudosubstrate synthetic peptides potently and selectively induced uptake of the cytotoxic drugs in the phorbol ester-treated and non-treated colon cancer cells. TPA treatment of the cells did not induce expression of either P-glycoprotein or its message mdr1. In contrast with [¹⁴C]Adriamycin and [³H] vincristine uptake, [³H] 5-fluorouracil uptake by the cells was unaffected by TPA and reduced by the PKC-inhibitory peptides. These results indicate that PKC activation can significantly reduce the uptake of multiple cytotoxic drugs by cancer cells independently of P-glycoprotein, and that N-myristoylated PKC- pseudosubstrate peptides potently and selectively induce uptake of multiple cytotoxic drugs in cultured human colon cancer cells by a novel mechanism that does not involve P-glycoprotein and may involve PKC isozyme inhibition. Thus, N-myristoylated PKC- pseudosubstrate peptides may offer a basis for the development of agents that reverse intrinsic drug resistance in human colon cancer.