Anti-hypertensive effects of PPARgamma ligands through the inhibition of Rho/Rho kinase pathway

Although PPARgamma ligands have an antihypertensive effect in vivo, the precise mechanism has not been fully elucidated. We examined their effects on Rho/Rho kinase pathway, a key regulator of vascular tone. In cultured rat aortic smooth muscle cells, Rho kinase activated by angiotensin II was suppressed by the pretreatment with pioglitazone and troglitazone. The roles of Vav, a GTP/GDP exchange factor upregulating Rho kinase activity, and SHP-2, protein tyrosine phosphatase that dephosphorylated Vav and subsequently inactivated Rho kinase were examined. Both pioglitazone and troglitazone upregulated SHP-2, particularly in the cytosolic fraction, and the SHP-2-bound Vav, and reduced the phosphorylation of Vav. These mechanisms may contribute to the hemodynamic, in addition to metabolic, action by PPARgamma ligands in hypertensive, metabolic syndrome.     

The Rho kinase signaling pathway participates in tubular mitochondrial oxidative injury and apoptosis in uric acid nephropathy

IntroductionOxidative stress is a pathologic feature of hyperuricemia that is highly prevalent and that contributes to kidney tubular interstitial fibrosis. Rho-kinase is closely related to mitochondrial-induced oxidative stress. Herein, we designed a study to explore the expression and role of Rho-kinase in hyperuricemia nephropathy. The secondary objective was to investigate whether the Rho-kinase signaling pathway regulates hyperuricemic tubular oxidative injury and apoptosis via the mitochondrial pathway in addition to the mechanisms that are involved.Materials and methodsHK-2 cells were divided into the following five groups: normal; uric acid (UA); UA+Fasudil; UA+ROCK1 si-RNA; and UA+sc-siRNA. Rho-kinase activity, mitochondrial oxidative injury, and apoptosis-related protein levels were measured in each group. A t-test was used to analyze the difference between groups.ResultsMyosin phosphatase target subunit 1 (MYPT1) overexpression was shown in HK-2 cells, which was caused by UA. High concentrations of UA also up-regulated Rho-kinase expression and mitochondrial and apoptosis-related protein expression, while treatment with fasudil and ROCK1 si-RNA significantly attenuated these responses.ConclusionThe Rho-kinase signaling pathway participates in tubular mitochondrial oxidative injury and apoptosis via regulating mitochondrial dyneins/biogenic genes in UA nephropathy, which suggests that the mitochondrial pathway might be a potential therapeutic target for hyperuricemia nephropathy.     

Tumour necrosis factor-alpha increases extravasation of virus particles into tumour tissue by activating the Rho A/Rho kinase pathway

Tumour Necrosis Factor alpha (TNF) is a pleiotropic pro-inflammatory cytokine with known vascular permeabilising activity. It is employed during isolated limb perfusion to enhance delivery of chemotherapeutic drugs into tumour tissue. The use of conditionally-replicating lytic viruses, so called 'oncolytic virotherapy', provides a new approach to cancer treatment that is currently limited by the low efficiency of extravasation of viral particles into tumours. We report here evidence that TNF significantly enhances the delivery of virus particles through the endothelial layer to allow access to tumour cells both in vitro and in vivo. Intravenous administration of TNF resulted in a 3- to 6-fold increase in EL4 tumour uptake of Evans Blue/Albumin, adenovirus and long-circulating polymer coated adenovirus. Interestingly, endothelial permeabilisation could be suppressed in vitro and in vivo by Y-27632, a Rho kinase inhibitor, without inhibiting viral infection. These data indicate that TNF can enhance the delivery of virus particles into tumours through a Rho A/Rho kinase dependent mechanism and may be a valuable strategy for increasing the delivery of oncolytic viruses and other therapeutic agents.     

RhoA/Rho kinase pathway contributes to the pathogenesis of thermal hyperalgesia in diabetic mice

Diabetic neuropathy is one of the most common complications of diabetes and causes various problems in daily life. Several investigations have noted that many factors in the spinal cord are involved in the symptoms of painful diabetic neuropathy, and there are very few effective therapeutic regimens. In the present study, we sought to elucidate the role of the RhoA/Rho kinase (ROCK) pathway in thermal hyperalgesia in diabetic mice. The intracellular localization of RhoA and the expression of eNOS were measured by western blotting. Thermal hyperalgesia was assessed by the tail-flick test and mechanical allodynia was assessed by automated von Frey filament test in streptozotocin(STZ)-induced diabetic mice. The spinal cord of STZ-treated diabetic mice showed increased membrane-bound RhoA compared to non-diabetic control. Treatment with the RhoA inhibitor exoenzyme C3, Clostridium botulinum, and the ROCK inhibitor Y27632 attenuated thermal hyperalgesia and mechanical allodynia in diabetic mice. Moreover, daily treatment with simvastatin attenuated all of those changes in diabetic mice. The expression of eNOS and NO metabolite contents in the spinal cord was decreased in diabetic mice, and these changes were normalized by treatment with simvastatin. The present results show that HMG-CoA reductase inhibitors have an inhibitory effect on thermal hyperalgesia in diabetic mice, which is mediated by an increase in NO production through the inhibition of RhoA/ROCK pathways. These results suggest that ROCK inhibitors and HMG-CoA inhibitors may be attractive compounds to relieve the symptoms of painful diabetic neuropathies.     

Hyperosmolar glucose induces vasoconstriction through Rho/Rho‐kinase pathway in the rat aorta

Rho/Rho‐kinase signalling pathway plays a substantial role in vascular contractions. In this study, we investigated any roles of Rho/Rho‐kinase pathway in the vasoconstriction of the rat conductance and capacitance vessels by hyperosmolar glucose solution. Isolated aortic, mesenteric and renal rings were suspended and exposed to hyperosmolar glucose, sucrose and NaCl in the organ chambers filled with Krebs solution gassed with 95% O₂ and 5% CO₂ and maintained at 37 °C. The effect of a Rho‐kinase inhibitor, (+)‐(R)‐trans‐4‐(1‐aminoethyl)‐N‐(4‐pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y‐27632, 10^?5 m ), was tested on the contraction induced by hypertonic solutions. Endothelial integrity was also assessed after hyperosmolar glucose exposure. Moreover, the activity and expression of Rho‐kinase (ROCK‐2) as well as RhoA translocation were detected by Western blotting and enzyme‐linked immunosorbent assay‐based RhoA activity detection method detection kit. The vessels produced substantial contractions in response to hyperosmolar solutions. Y‐27632 significantly reduced hyperosmolarity‐induced vasoconstrictions (P < 0.05). Phosphorylation of myosin‐phosphatase target 1 increased after hyperosmolar glucose exposure, and this phosphorylation was significantly decreased by Y‐27632 (P < 0.05) in the aorta. Furthermore, RhoA translocation but not ROCK‐2 expression markedly increased by hyperosmolar glucose solution. These results may indicate that hyperosmolarity could induce vasoconstriction through Rho/Rho‐kinase signalling.     

IRBIT governs epithelial secretion in mice by antagonizing the WNK/SPAK kinase pathway

Fluid and HCO(3)(-) secretion are fundamental functions of epithelia and determine bodily fluid volume and ionic composition, among other things. Secretion of ductal fluid and HCO(3)(-) in secretory glands is fueled by Na(+)/HCO(3)(-) cotransport mediated by basolateral solute carrier family 4 member 4 (NBCe1-B) and by Cl(-)/HCO(3)(-) exchange mediated by luminal solute carrier family 26, member 6 (Slc26a6) and CFTR. However, the mechanisms governing ductal secretion are not known. Here, we have shown that pancreatic ductal secretion in mice is suppressed by silencing of the NBCe1-B/CFTR activator inositol-1,4,5-trisphosphate (IP(3)) receptor-binding protein released with IP(3) (IRBIT) and by inhibition of protein phosphatase 1 (PP1). In contrast, silencing the with-no-lysine (WNK) kinases and Ste20-related proline/alanine-rich kinase (SPAK) increased secretion. Molecular analysis revealed that the WNK kinases acted as scaffolds to recruit SPAK, which phosphorylated CFTR and NBCe1-B, reducing their cell surface expression. IRBIT opposed the effects of WNKs and SPAK by recruiting PP1 to the complex to dephosphorylate CFTR and NBCe1-B, restoring their cell surface expression, in addition to stimulating their activities. Silencing of SPAK and IRBIT in the same ducts rescued ductal secretion due to silencing of IRBIT alone. These findings stress the pivotal role of IRBIT in epithelial fluid and HCO(3)(-) secretion and provide a molecular mechanism by which IRBIT coordinates these processes. They also have implications for WNK/SPAK kinase-regulated processes involved in systemic fluid homeostasis, hypertension, and cystic fibrosis.     

Pkd1 regulates immortalized proliferation of renal tubular epithelial cells through p53 induction and JNK activation

Autosomal dominant polycystic kidney disease (ADPKD) is the most common human monogenic genetic disorder and is characterized by progressive bilateral renal cysts and the development of renal insufficiency. The cystogenesis of ADPKD is believed to be a monoclonal proliferation of PKD-deficient (PKD(-/-)) renal tubular epithelial cells. To define the function of Pkd1, we generated chimeric mice by aggregation of Pkd1(-/-) ES cells and Pkd1(+/+) morulae from ROSA26 mice. As occurs in humans with ADPKD, these mice developed cysts in the kidney, liver, and pancreas. Surprisingly, the cyst epithelia of the kidney were composed of both Pkd1(-/-) and Pkd1(+/+) renal tubular epithelial cells in the early stages of cystogenesis. Pkd1(-/-) cyst epithelial cells changed in shape from cuboidal to flat and replaced Pkd1(+/+) cyst epithelial cells lost by JNK-mediated apoptosis in intermediate stages. In late-stage cysts, Pkd1(-/-) cells continued immortalized proliferation with downregulation of p53. These results provide a novel understanding of the cystogenesis of ADPKD patients. Furthermore, immortalized proliferation without induction of p53 was frequently observed in 3T3-type culture of mouse embryonic fibroblasts from Pkd1(-/-) mice. Thus, Pkd1 plays a role in preventing immortalized proliferation of renal tubular epithelial cells through the induction of p53 and activation of JNK.     

S6K1 regulates hematopoietic stem cell self-renewal and leukemia maintenance

Hyperactivation of the mTOR pathway impairs hematopoietic stem cell (HSC) functions and promotes leukemogenesis. mTORC1 and mTORC2 differentially control normal and leukemic stem cell functions. mTORC1 regulates p70 ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E–binding (eIF4E-binding) protein 1 (4E-BP1), and mTORC2 modulates AKT activation. Given the extensive crosstalk that occurs between mTORC1 and mTORC2 signaling pathways, we assessed the role of the mTORC1 substrate S6K1 in the regulation of both normal HSC functions and in leukemogenesis driven by the mixed lineage leukemia (MLL) fusion oncogene MLL-AF9. We demonstrated that S6K1 deficiency impairs self-renewal of murine HSCs by reducing p21 expression. Loss of S6K1 also improved survival in mice transplanted with MLL-AF9–positive leukemic stem cells by modulating AKT and 4E-BP1 phosphorylation. Taken together, these results suggest that S6K1 acts through multiple targets of the mTOR pathway to promote self-renewal and leukemia progression. Given the recent interest in S6K1 as a potential therapeutic target in cancer, our results further support targeting this molecule as a potential strategy for treatment of myeloid malignancies.     

原钙黏附蛋白7在脂多糖诱导的肾小管上皮细胞焦亡中的作用

目的 探讨原钙黏附蛋白7（PCDH7）是否参与脂多糖（LPS）刺激引起的肾小管上皮细胞（HK-2）焦亡过程。方法 构建LPS刺激HK-2损伤模型,使用RT-PCR和蛋白免疫印迹法检测细胞中PCDH7、NLR家族Pyrin域蛋白3（NLRP3）、caspase-1、和IL-1β蛋白的表达变化。MTT法测定细胞活力,TUNEL法确定细胞焦亡率,ELISA法检测细胞分泌TNF-α、IL-6水平,全自动生化分析仪检测培养液中乳酸脱氢酶（LDH）含量。结果 与Control组比较,LPS组细胞生存率下降,细胞凋亡率升高,炎症因子TNF-α和IL-6水平升高,细胞损伤指标LDH含量明显升高,PCDH7的蛋白和mRNA表达明显下调,细胞焦亡指标NLRP3、caspase-1、IL-1β蛋白和mRNA表达明显上调（P均< 0.05）。结论 LPS通过下调PCDH7的表达,促进肾小管上皮细胞发生明显细胞焦亡损伤。     

The protein kinase A inhibitor H89 acts on cell morphology by inhibiting Rho kinase

The small GTPase RhoA can retract cell extensions by acting on two Rho kinases (ROCKs). Activated protein kinase A (PKA) inhibits RhoA and induces extensions. The isoquinoline H89 inhibits PKA and thus should prevent the inactivation of RhoA. In kinase assays, H89 has been recently found to inactivate a ROCK-II also. Because H89 may be able to exert opposite effects on cell extensions, we have studied the effects of H89 on neurite formation in the neuroblastoma-glioma line NG 108-15, which expresses ROCK-I and ROCK-II. We found that H89 can indeed inhibit ROCKs and PKA. Because ROCKs act downstream of RhoA, the inhibitory effect of H89 on ROCKs is most prominent. The data indicate that H89 may not be used as an antagonist of PKA in systems in which ROCKs play a role.     

MAPK/ERK信号通路调节K562细胞中mdr1基因的诱导性表达

目的 观察多柔比星(doxorubicin,DOX)诱导K562细胞mdr1基因表达过程中丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)/细胞外信号调节激酶(ERK)信号通路的作用,探讨mdr1基因的转录调控机制.方法 多柔比星初始浓度为0.01 μg/ml,诱导K562细胞24 h后撤药继续培养至细胞状态恢复,加入多柔比星继续诱导24 h,浓度增加为0.02μg/ml.依上述方法 多柔比星浓度逐渐增加,直至0.05μg/ml.收集DOX浓度为0.01、0.03和0.05μg/ml时的细胞.RT-PCR检测mdr1基因表达,流式细胞仪检测mdr1基因编码的P糖蛋白(P-gP)的表达,Western blot 检测ERK活化情况.MAPK的抑制剂PD98059预处理K562细胞1 h后与多柔比星共同作用,逆转录(RT)-PCR和流式细胞仪分别检测mdr1基因和P-gP的表达情况.结果 经多柔比星作用后K562细胞中ERK磷酸化增强,同时mdr1基因转录上调,及其蛋白产物P-gP的表达也增加,当多柔比星浓度为0.05μg/ml时两者的表达均增加了5倍之多.而用PD98059预处理细胞后能明显抑制多柔比星诱导mdr1基因转录和蛋白表达,多柔比星浓度为0.03μg/ml时PD98059对mdr1基因表达的抑制率为(74.1±0.11)%,多柔比星浓度为0.05 μg/ml时抑制率为(70.2±0.14)%.结论 多柔比星能够诱导K562细胞mdr1基因表达,同时激活MAPK/ERK信号通路,阻断ERK的活化能抑制mdr1基因的诱导性表达.     

PP2A regulates upstream members of the c-jun N-terminal kinase mitogen-activated protein kinase signaling pathway

We have previously demonstrated that inhibition of the serine-threonine phosphatase PP2A resulted in increased c-jun N-terminal kinase (JNK) activity, and that the regulatory subunit, A/alpha of PP2A, was physically associated with the JNK. Because there exists additional examples of phosphatases serving as negative regulators of multiple members of mitogen-activated protein kinase (MAPK) pathways in Drosophila and yeast, we hypothesized that PP2A may serve a homologous function in mammalian cells affording the regulation of additional upstream kinases in the JNK pathway. In human monocytes, activation of JNK by LPS proceeds through the MAPK kinase kinase MEKK-1 and, subsequently, the MAPK kinases MKK4 and/or MKK7. Using the human monocyte cell line THP-1, we show that pharmacological manipulation of the activity of PP2A seemed to regulate not only JNK but also the upstream kinases MKK4 and MEKK-1. Using coimmunoprecipitation, overexpression of tagged recombinant JNK, and bacterial two-hybrid strategies, evidence for physical interactions between the structural subunit, PP2A-A/alpha and MEKK-1, MKK4, and JNK was observed. These studies suggest that the target of regulation by PP2A includes upstream kinases in the JNK MAPK pathway. Furthermore, PP2A-A/alpha seems to serve as a structural protein to foster protein-protein interactions affording specificity of the regulation among members of this MAP kinase pathway.     

TGF-β1诱导人肾小管上皮细胞血小板反应蛋白1的表达

目的探讨在TGF-β1诱导下人肾小管上皮细胞(HKCs)血小板反应蛋白1(thrombospondin1,TSP-1)的表达变化。方法实验分二组:对照组(C组)用无血清培养基(FSM)培养人肾小管上皮细胞;TGF-β1组(T组):用含TGF-β120ng/ml的FSM培养HKCs。各组观察24、72和120h3个时相点,分别为C1、C2、C3和T1、T2、T3亚组。用免疫细胞化学及逆转录酶链反应(RT-PCR)方法检测TSP-1及其mRNA的表达的变化。结果T1组比C1组TSP-1表达无显著差异(P>0.05),T2、T3组与C组相应时相点比较TSP-1表达显著增高(P<0.01),T组组内比较随时间延长,TSP-1表达逐渐增高。结论TGF-β1能诱导HKCs表达TSP-1,呈时间依赖性。     

安体舒通和缬沙坦对肾小管上皮细胞凋亡的影响

目的探讨醛固酮受体拮抗剂和血管紧张素П-1型受体拮抗剂（AT1Ra）对大鼠单侧输尿管梗阻（UU0）模型肾间质纤维化的影响及其可能机制。方法Wistar大鼠行左侧输尿管结扎术，分为UU0模型组（n=11），安体舒通治疗组（12=12），缬沙坦治疗组（n=11）和[安体舒通＋缬沙坦治疗组（n=10）]，同时设假手术对照组（n=7）。术后第14天处死各组大鼠，进行HE和Massod染色，观察肾脏病理变化；比色法测定肾组织丙二醛（MDA）、超氧化物歧化酶（SOD）含量；原位末端标记法（TUNEL）与DNA电泳观察肾小管上皮细胞凋亡情况；Western blotting检测半胱氨酸天门冬氨酸蛋白酶-3（caspase-3）的蛋白表达。结果UU0组与其它组大鼠比较，肾组织MDA含量明显升高，SOD含量显著下降，肾脏病理改变加重，TUNEL染色及DNA琼脂糖凝胶电泳可见大量的肾小管上皮细胞凋亡，肾组织caspase-3的表达明显增加（P〈0．05）。各用药组与UU0组大鼠比较，肾组织MDA含量明显降低，SOD含量升高，肾小管上皮细胞凋亡和肾间质纤维化显著减轻，caspase-3的表达显著减少（P〈0．05）。结论安体舒通和缬沙坦均可显著改善UU0所致的肾间质纤维化，减轻肾小管上皮细胞的凋亡，部分下调肾组织caspase-3的表达，且联合用药效果更为显著。上述作用可能与该药物抑制氧化应激有关。     

Retracted Article: Aclarubicin regulates glioma cell growth and DNA damage through the SIRT1/PI3K/AKT signaling pathway

Aclarubicin (ACR), an anthracycline anti-tumor agent, is known to play important roles in cancer. Evidence has suggested that ACR has therapeutic effects on rats intracranially implanted with C6 glioma cells. However, the function and mechanism of ACR in glioma cells remain elusive. In this study, we examined the effects of ACR on glioma cell growth, apoptosis, and DNA damage. Our results showed that treatment with different concentrations of ACR (1, 2, and 5 μM) markedly impeded glioma cell survival, significantly decreased cell proliferation, and increased cell apoptosis and caspase-3 activity. Furthermore, ACR treatment promoted DNA damage through phosphorylation of ATM and CHK1 in U87 and U251 cells. Treatment with ACR also increased sirtuin 1 (SIRT1) expression and inhibited phosphatidylinositol 3′-kinase (PI3K)/AKT pathway activation. Interestingly, we found that AKT overexpression reversed the effects of ACR on glioma cell survival, proliferation, apoptosis, and DNA damage. Thus, our data suggest that ACR induces apoptosis and DNA damage in U87 and U251 cells through the SIRT1/PI3K/AKT signaling pathway.

Aclarubicin (ACR), an anthracycline anti-tumor agent, is known to play important roles in cancer.