Insulin rescues ES cell-derived neural progenitor cells from apoptosis by differential regulation of Akt and ERK pathways

Transplantation of embryonic stem (ES) cell-derived neural progenitor cells (ES-NPCs) is one promising technology for the treatment of spinal cord injury. Promoting ES-NPC survival at the lesion site is critical for the successful treatment. We tested the role of insulin in promoting mouse ES-NPC survival. Cultured ES-NPCs survived when maintained in normoxia but underwent apoptosis when exposed to hypoxia. Insulin rescued ES-NPCs from hypoxia-induced cell death. This effect could be blocked by the phosphatidylinositol 3-kinase (PI3K)/Akt pathway inhibitor LY294002. In contrast, mitogen-activated protein kinase (MAP)/extracellular-signal-regulated kinase (ERK) pathway inhibitor U0126 potentiated insulin-mediated survival. Immunoblots revealed that insulin upregulated activation of Akt and inhibited ERK activation through the PI3K pathway. In addition, we showed that insulin reduced the activation of caspase-3, the key executor of apoptosis. In summary, our data suggest that insulin prevent apoptosis in ES-NPCs by activating Akt and inhibiting ERK through the PI3K pathway.     

Hormone‐sensitive lipase in skeletal muscle: regulatory mechanisms

Aim: The enzymatic regulation of intramuscular triacylglycerol (TG) breakdown has until recently not been well understood. Our aim was to elucidate the role of hormone‐sensitive lipase (HSL), which controls TG breakdown in adipose tissue. Methods: Isolated rat muscle as well as exercising humans were studied. Results: The presence of HSL was demonstrated in all muscle fibre types by Western blotting of muscle fibres isolated by collagenase treatment or after freeze‐drying. The content of HSL varies between fibre types, being higher in oxidative than in glycolytic fibres. Analysed under conditions optimal for HSL, neutral lipase activity in muscle can be stimulated by adrenaline as well as by contractions. These increases are abolished by presence of anti‐HSL antibody during analysis. Moreover, immunoprecipitation with affinity‐purified anti‐HSL antibody causes similar reductions in muscle HSL protein concentration and in measured neutral lipase responses to contractions. The immunoreactive HSL in muscle is stimulated by adrenaline via beta‐adrenergic activation of protein kinase A (PKA). From findings in adipocytes it is likely that PKA phosphorylates HSL at residues Ser⁵⁶³, Ser⁶⁵⁹ and Ser⁶⁶⁰. Contraction probably also enhances muscle‐HSL activity by phosphorylation, because the contraction‐induced increase in HSL activity is increased by the protein phosphatase inhibitor okadaic acid and reversed by alkaline phosphatase. A novel signalling pathway in muscle by which HSL activity may be stimulated by protein kinase C (PKC) via extracellular signal regulated kinase (ERK) has been demonstrated. In contrast to previous findings in adipocytes, in muscle activation of ERK is not necessary for stimulation of HSL by adrenaline. However, contraction‐induced HSL activation is mediated by PKC, at least partly via the ERK pathway. In fat cells ERK is known to phosphorylate HSL at Ser⁶⁰⁰. So, phosphorylation of different sites may explain that in muscle the effects of contractions and adrenaline on HSL activity are partially additive. In line with the view that the two stimuli act by different mechanisms, training increases the contraction‐mediated, but diminishes the adrenaline mediated HSL activation in muscle. Conclusion: The existence and regulation of HSL in skeletal muscle indicate a role of HSL in muscle TG metabolism.     

低氧预适应小鼠脑内ERK1/2磷酸化水平和蛋白表达量的改变

目的：初步探讨细胞外信号调节激酶（Extraeellular signal-regulated kinases，ERK1／2）在脑低氧预适应发生发展过程中的作用。方法：按已建小鼠整体低氧预适应模型，将BALB／C小鼠（18-22g，雌雄不限）随机分为正常对照（H0）、早期（H1-H4）和延迟性（U5-H6）低氧预适应等7组（每组至少6只动物）。应用SDS-PAGE和Western blot等生化技术，并结合Gel Doc凝胶成像系统，半定量检测小鼠脑组织内ERK1／2的磷酸化水平和蛋白表达量。结果：①早期低氧预适应形成过程中，随低氧暴露次数的增加（H1-H4），小鼠海马和皮层组织内ERK1／2磷酸化水平显著降低（P〈0．05，n=6），而ERK1／2蛋白表达量并无显著变化；②延迟性低氧预适应中（H5-H6），小鼠大脑皮层和海马组织内ERK1／2的蛋白表达量显著降低（P〈0．05，n=6）。结论：ERK1／2的活性降低（磷酸化水平降低），以及ERK1／2蛋白表达量下调可能分别参与了脑早期低氧预适应和晚期延迟性低氧预适应的发生发展过程。     

Contradictory effects of short- and long-term hyperglycemias on ischemic injury of myocardium via intracellular signaling pathway

Although clinical diabetes mellitus is obviously a high risk factor for myocardial infarction, there is disagreement about the sensitivity of ischemic injury of an infarcted myocardium in experimental studies. The present study evaluated the influences of different durations of hyperglycemia on ischemic and reperfusion injuries of the myocardium, and focused on extracellular signal-regulated kinase 1/2 (ERK1/2), which plays an important role in the intracellular signaling pathway and is reported to be associated with myocardial protection against heart injury. Short- and long-term hyperglycemias were induced in rats by streptozotocin (STZ) injection and the rats were examined 4 (4WDM) and 20 weeks (20WDM) after the treatment. Ischemia and reperfusion were induced by occlusion and reperfusion (I/R) of the left coronary artery (LCA). I/R-induced infarct size was determined using triphenyltetrazolium chloride (TTC) staining. After 20 weeks of STZ treatment (20WDM+I/R), the infarct size in the rat heart increased by 65.2 +/- 4.3%, whereas after 4 weeks of STZ treatment (4WDM+I/R), the infarct size decreased compared with the time-matched I/R group (43.1 +/- 3.6% and 59.5 +/- 5.6%, respectively). The number of dead myocytes including necrotic and apoptotic cells was determined using horseradish peroxidase (HRP) and terminal deoxynucleotide nick-end labeling (TUNEL) methods. The number of dead myocytes decreased in the 4WDM+I/R group, while the number of dead myocytes increased markedly in the 20WDM+I/R group, compared with the time-matched I/R group. The increment of ERK1/2 phosphorylation in the 4WDM group and the slight enhancement of this phosphorylation by I/R treatment were observed by western blotting. However, in the 20WDM group, the level of ERK1/2 phosphorylation reduced by approximately 1/3 compared with the time-matched control group; moreover, I/R treatment did not enhance the phosphorylation level. This study demonstrated that short- and long-term hyperglycemias exert opposite influences on ischemic myocardial injury, and these contradictory influences may depend on an ERK1/2-mediated intracellular signaling pathway.     

Intracellular messengers involved in spontaneous pain,heat hyperalgesia,and mechanical allodynia induced by intrathecal dihydroxyphenylglycine

We investigated the role of two intracellular second messengers, extracellular signal-regulated protein kinase (ERK) and protein kinase C (PKC), in a model of persistent pain using intrathecal (i.t.) (R,S)-3,5-dihydroxyphenylglycine (DHPG). Spontaneous nociceptive behaviours (SNBs), mechanical allodynia (von Frey thresholds) and heat hyperalgesia (plantar test latencies) induced by DHPG were measured in animals pretreated i.t. with membrane permeable inhibitors of ERK (PD 98059) and PKC (GF 109203X). Spinal administration of PD 98059 dose-dependently reduced SNBs, and attenuated both mechanical allodynia and heat hyperalgesia induced by DHPG. GF 109203X treatment also reduced SNBs and heat hyperalgesia, but did not affect mechanical allodynia induced by DHPG. Neither PD 98059, nor GF 109203X, altered mechanical or thermal thresholds in saline-injected control rats. These results suggest that both ERK and PKC are involved in persistent pain associated with the i.t. administration of DHPG.     

Oxygen glucose deprivation inhibits the growth and ERK phosphorylation of neural progenitor cells in vitro

The neurogenic regions such as subventricular zone of the lateral ventricles could become ischemic in some clinical situations due to the blockage of blood vessels by blood clots. Hence the aim of this study is to investigate the effects of OGD on the growth of neural progenitor cells and the phosphorylation of ERK, which plays an important role in the growth of these cells. Oxygen glucose deprivation (OGD) for 4 h decreased the growth of neural progenitor cells in vitro and also decreased the phosphorylation of extracellular signal regulated kinase (ERK). Inhibition of the ERK pathway for 4 h using U0126 (10 μM) also decreased the growth of progenitor cells. These data suggest that a decline in the phospho-ERK content might decrease the growth of progenitor cells following OGD.     

Noonan syndrome-associated SHP2/PTPN11 mutants cause EGF-dependent prolonged GAB1 binding and sustained ERK2/MAPK1 activation

Noonan syndrome is a developmental disorder with dysmorphic facies, short stature, cardiac defects, and skeletal anomalies, which can be caused by missense PTPN11 mutations. PTPN11 encodes Src homology 2 domain-containing tyrosine phosphatase 2 (SHP2 or SHP-2), a protein tyrosine phosphatase that acts in signal transduction downstream to growth factor, hormone, and cytokine receptors. We compared the functional effects of three Noonan syndrome-causative PTPN11 mutations on SHP2's phosphatase activity, interaction with a binding partner, and signal transduction. All SHP2 mutants had significantly increased basal phosphatase activity compared to wild type, but that activity varied significantly between mutants and was further increased after epidermal growth factor stimulation. Cells expressing SHP2 mutants had prolonged extracellular signal-regulated kinase 2 activation, which was ligand-dependent. Binding of SHP2 mutants to Grb2-associated binder-1 was increased and sustained, and tyrosine phosphorylation of both proteins was prolonged. Coexpression of Grb2-associated binder-1-FF, which lacks SHP2 binding motifs, blocked the epidermal growth factor-mediated increase in SHP2's phosphatase activity and resulted in a dramatic reduction of extracellular signal-regulated kinase 2 activation. Taken together, these results document that Noonan syndrome-associated PTPN11 mutations increase SHP2's basal phosphatase activity, with greater activation when residues directly involved in binding at the interface between the N-terminal Src homology 2 and protein tyrosine phosphatase domains are altered. The SHP2 mutants prolonged signal flux through the RAS/mitogen-activated protein kinase (ERK2/MAPK1) pathway in a ligand-dependent manner that required docking through Grb2-associated binder-1 (GAB1), leading to increased cell proliferation.     

乳腺浸润性导管癌组织学分级与BAD、p-BAD及p-ERK蛋白的表达

目的:探讨乳腺浸润性导管癌不同组织学分级间BAD及其磷酸化形式p-BAD和上游作用因子ERK的活性形式p-ERK蛋白表达的变化。方法:应用免疫组化SP法检测83例乳腺浸润性导管癌中BAD、p-BAD及p-ERK蛋白表达及应用TUNEL法原位观察不同组织学分级间凋亡指数的差异。结果:组织学分级越高,凋亡指数及BAD、p-BAD及p-ERK蛋白表达均越高,且在高分化与低分化组间差异均具有统计学意义。BAD与凋亡指数存在正相关性(r=1.000,P=0.018),BAD与p-BAD存在正相关性(r=0.999,P=0.031),p-BAD与p-ERK存在正相关性(r=0.997,P=0.040)。结论:BAD、p-BAD及p-ERK在乳腺浸润性导管癌蛋白表达水平呈现出肿瘤分化级别越低而表达越强的特征,这与肿瘤恶性进程中细胞发生凋亡与增殖的变化有关。     

不同乳腺疾病中ERK、PI3-K mRNA的表达

目的:检测正常乳腺组织、良性增生、非典型增生和乳腺癌中ERK、PI3-KmRNA的表达,探讨其在乳腺癌发生、发展中的作用及关系。方法:应用RT-PCR方法检测ERK、PI3-KmRNA在正常乳腺组织及不同乳腺疾病中的表达。结果:ERK2和PI3-KmRNA在乳腺癌组织中表达明显高于其他乳腺组织,分别为100%(37/37)和94.59%(35/37)。ERK2mRNA在乳腺癌、非典型增生、良性增生和正常组织的表达强度依次减弱;PI3-KmRNA表达在乳腺癌中明显增高,而在非典型增生和良性增生和正常组织中的表达无明显差别。结论:MAPK/ERK途径参与乳腺癌发生的信号转导,而PI3-K途径参与乳腺癌后期进展阶段的信号转导。