Chronic infection with Helicobacter pylori cagA-positive strains is the strongest risk factor for gastric cancer.The cagA gene product,CagA,is delivered into gastric epithelial cells via the bacterial type IV secretio...Chronic infection with Helicobacter pylori cagA-positive strains is the strongest risk factor for gastric cancer.The cagA gene product,CagA,is delivered into gastric epithelial cells via the bacterial type IV secretion system.Delivered CagA then undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala(EPIYA)motifs in its C-terminal region and acts as an oncogenic scaffold protein that physically interacts with multiple host signaling proteins in both tyrosine phosphorylation-dependent and-independent manners.Analysis of CagA using in vitro cultured gastric epithelial cells has indicated that the nonphysiological scaffolding actions of CagA cell-autonomously promote the malignant transformation of the cells by endowing the cells with multiple phenotypic cancer hallmarks:sustained proliferation,evasion of growth suppressors,invasiveness,resistance to cell death,and genomic instability.Transgenic expression of CagA in mice leads to in vivo oncogenic action of CagA without any overt inflammation.The in vivo oncogenic activity of CagA is further potentiated in the presence of chronic inflammation.Since Helicobacter pylori infection triggers a proinflammatory response in host cells,a feedforward stimulation loop that augments the oncogenic actions of CagA and inflammation is created in CagA-injected gastric mucosa.Given that Helicobacter pylori is no longer colonized in established gastric cancer lesions,the multistep nature of gastric cancer development should include a“hit-and-run”process of CagA action.Thus,acquisition of genetic and epigenetic alterations that compensate for CagA-directed cancer hallmarks may be required for completion of the“hit-and-run”process of gastric carcinogenesis.展开更多
Phytochromes （Phy） and phytochrome-interacting factor （PIF） transcription factors constitute a major signal- ing module that controls plant development in response to red and far-red light. A low red：far-red rati...Phytochromes （Phy） and phytochrome-interacting factor （PIF） transcription factors constitute a major signal- ing module that controls plant development in response to red and far-red light. A low red：far-red ratio is interpreted as shading by neighbor plants and induces cell elongation--a phenomenon called shade-avoidance syndrome （SAS）. PAR1 and its closest homolog PAR2 are negative regulators of SAS; they belong to the HLH transcription factor family that lacks a typical basic domain required for DNA binding, and they are believed to regulate gene expressions through DNA binding transcription factors that are yet to be identified. Here, we show that light signal stabilizes PAR1 protein and PAR1 interacts with PIF4 and inhibits PIF4-mediated gene activation. DNA pull-down and chromatin immunoprecipitation （CHIP） assays showed that PAR1 inhibits PIF4 DNA binding in vitro and in vivo. Transgenic plants overexpressing PAR1 （PARIOX） are insensitive to gibberellin （GA） or high temperature in hypocotyl elongation, similarly to the pifq mutant. In addition to PIF4, PAR1 also interacts with PRE1, a HLH transcription factor activated by brassinosteroid （BR） and GA. Overexpression of PRE1 largely suppressed the dwarf phenotype of PARIOX. These results indicate that PAR1-PRE1 and PAR1-PIF4 het- erodimers form a complex HLH/bHLH network regulating cell elongation and plant development in response to light and hormones.展开更多
Here,we used reverse transcription-PCR(RT-PCR) and western blot to detect protease-activated receptor(PAR) 1,PAR 2 and PAR 4 expression in cancer tissues and cell lines of esophageal squamous cell carcinoma,and invest...Here,we used reverse transcription-PCR(RT-PCR) and western blot to detect protease-activated receptor(PAR) 1,PAR 2 and PAR 4 expression in cancer tissues and cell lines of esophageal squamous cell carcinoma,and investigated the co-relationship between PAR expression and clinic-pathological data for esophageal cancer.The methylation of PAR4 gene promoter involved in esophageal carcinoma was also analyzed.By comparing the mRNA expressions of normal esophageal tissue and human esophageal epithelial cells(HEEpiC),we found that among the 28 cases of esophageal squamous cell carcinoma,PAR1(60%) and PAR2(71%) were elevated in 17 and 20 cases,respectively,and PAR4(68%) expression was lowered in 19 cases.Whereas,in human esophageal squamous cells(TE-1 and TE-10),PAR1 and PAR2 expression was increased but PAR4 was decreased.Combined with clinical data,the expression of PAR1 in poorly differentiated(P=0.016) and middle and lower parts of the esophagus(P=0.016) was higher; expression of PAR4 in poorly differentiated carcinoma was lower(P=0.049).Regarding TE-1 and TE-10 protein expression,we found that in randomized esophageal carcinoma,PAR1(P=0.027) and PAR2(P=0.039) expressions were increased,but lowered for PAR4(P=0.0001).In HEEpiC,TE-1,TE-10,esophageal and normal esophagus tissue samples(case No.7),the frequency of methylation at the 19 CpG loci of PAR4 was 35.4%,95.2%,83.8%,62.6% and 48.2%,respectively.Our results indicate that the expression of PAR1 and PAR2 in esophageal squamous cell carcinoma is increased but PAR4 is decreased.Hypermethylation of the promoter of the PAR4 gene may contribute to reduced expression of PAR4 in esophageal squamous cell carcinoma.展开更多
Astrocytes are important cellular centers of cholesterol synthesis and metabolism that help maintain normal physiological function at the organism level.Spinal cord injury results in aberrant cholesterol metabolism by...Astrocytes are important cellular centers of cholesterol synthesis and metabolism that help maintain normal physiological function at the organism level.Spinal cord injury results in aberrant cholesterol metabolism by astrocytes and excessive production of oxysterols,which have profound effects on neuropathology.25-Hydroxycholesterol(25-HC),the main product of the membrane-associated enzyme cholesterol-25-hydroxylase(CH25H),plays important roles in mediating neuroinflammation.However,whether the abnormal astrocyte cholesterol metabolism induced by spinal cord injury contributes to the production of 25-HC,as well as the resulting pathological effects,remain unclear.In the present study,spinal cord injury-induced activation of thrombin was found to increase astrocyte CH25H expression.A protease-activated receptor 1 inhibitor was able to attenuate this effect in vitro and in vivo.In cultured primary astrocytes,thrombin interacted with protease-activated receptor 1,mainly through activation of the mitogen-activated protein kinase/nuclear factor-kappa B signaling pathway.Conditioned culture medium from astrocytes in which ch25h expression had been knocked down by siRNA reduced macrophage migration.Finally,injection of the protease activated receptor 1 inhibitor SCH79797 into rat neural sheaths following spinal cord injury reduced migration of microglia/macrophages to the injured site and largely restored motor function.Our results demonstrate a novel regulatory mechanism for thrombin-regulated cholesterol metabolism in astrocytes that could be used to develop anti-inflammatory drugs to treat patients with spinal cord injury.展开更多
Whitmania pigra has been used as a traditional Chinese medicine(TCM) for promoting blood circulation, alleviating blood coagulation, activating meridians and relieving stasis for several hundred years. However, the th...Whitmania pigra has been used as a traditional Chinese medicine(TCM) for promoting blood circulation, alleviating blood coagulation, activating meridians and relieving stasis for several hundred years. However, the therapeutic components of this species, especially proteins and peptides were poorly exploited. Until now only a few of them were obtained by using chromatographic isolation and purification. In recent decade, transcriptome techniques were rapidly developed, and have been used to fully reveal the functional components of many animal venoms. In the present study, the c DNA of the salivary gland of Whitmania pigra was sequenced by illumina and the transcriptome was assembled by using Trinity. The proteome were analysed by LC-MS/MS. Based on the data of the transcriptome and the proteome, a potential antiplatelet protein named pigrin was found. Pigrin was cloned and expressed using P. pastoris GS115. The antiplatelet and antithrombotic bioactivities of pigrin were tested by using aggregometer and the rat arterio-venous shunt thrombosis model, respectively. The bleeding time of pigrin was measured by a mice tail cutting method. The docking of pigrin and protease-activated receptor 1(PAR1) or collagen were conducted using the ZDOCK Server. Pigrin was able to selectively inhibit platelet aggregation stimulated by PAR1 agonist and collagen. Pigrin attenuated thrombotic formation in vivo in rat, while did not prolong bleeding time at its effective dosage. There are significant differences in the key residues participating in binding of Pigrin-Collagen complex from Pigrin-PAR1 complex. In conclusion, a novel PAR1 inhibitor pigrin was found from the leech Whitmania pigra. This study helped to elucidate the mechanism of the leech for the treatment of cardiovascular disorder.展开更多
目的探讨中期因子(midkine,MK)在人乳腺癌MDA-MB-231细胞体外血管生成中的作用及其机制。方法采用shRNA干扰技术降低MDA-MB-231细胞MK表达,应用Western blot技术检测肿瘤细胞中内皮蛋白C受体(endothelial protein C receptor,EPCR)的表...目的探讨中期因子(midkine,MK)在人乳腺癌MDA-MB-231细胞体外血管生成中的作用及其机制。方法采用shRNA干扰技术降低MDA-MB-231细胞MK表达,应用Western blot技术检测肿瘤细胞中内皮蛋白C受体(endothelial protein C receptor,EPCR)的表达;干扰MK和EPCR表达或通过抗体阻断活化蛋白酶激活受体1(protease-activated receptor 1,PAR1)作用后,制备肿瘤条件培养基作用于人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs),通过CCK-8试剂盒检测HUVECs增殖、Transwell小室检测迁移以及Matrigel表面培养检测脉管形成能力。结果干扰MK表达后,EPCR表达随之降低。干扰MK和EPCR低表达后,HUVECs增殖、迁移及脉管形成能力均低于对照组(P<0.05),EPCR干扰组低于MK干扰组(P<0.05)。应用抗PAR1作用后,HUVECs增殖、迁移及脉管形成能力低于对照组和EPCR干扰组(P<0.05)。结论 MK可通过EPCR/PAR1通路促进乳腺癌MDA-MB-231细胞体外血管生成。展开更多
基金Our studies presented in this review article were supported by Grants-in-Aid for Scientific Research“S”(#16H06373 to M.H.),“A”(#22240085 and#25250016 to M.H.)and“C”(#19K05945 to A.T.-K.)Grants-in-Aid for Innovative Areas(#3205,#22114001,and#22114002 to M.H.)+4 种基金Grants-in-Aid for Japan Society for the Promotion of Science(JSPS)Fellows(#07J03878 to A.T.-K.and#19J12668 to C.T.K.)Grants-in-Aid for Young Scientists“B”(#24700965 and#15K18399 to A.T.-K.)the Graduate Program for Leaders in Life Innovation(GPLLI,to C.T.K.)from the Ministry of Education,Culture,Sports,Science and Technology(MEXT)of Japan,by CREST(#120200000396 to M.H.)Japan Science and Technology Agency,by the Uehara Memorial Foundation(#137 in 2016 to A.T.-K.)by the Max-Planck Society,Germany。
文摘Chronic infection with Helicobacter pylori cagA-positive strains is the strongest risk factor for gastric cancer.The cagA gene product,CagA,is delivered into gastric epithelial cells via the bacterial type IV secretion system.Delivered CagA then undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala(EPIYA)motifs in its C-terminal region and acts as an oncogenic scaffold protein that physically interacts with multiple host signaling proteins in both tyrosine phosphorylation-dependent and-independent manners.Analysis of CagA using in vitro cultured gastric epithelial cells has indicated that the nonphysiological scaffolding actions of CagA cell-autonomously promote the malignant transformation of the cells by endowing the cells with multiple phenotypic cancer hallmarks:sustained proliferation,evasion of growth suppressors,invasiveness,resistance to cell death,and genomic instability.Transgenic expression of CagA in mice leads to in vivo oncogenic action of CagA without any overt inflammation.The in vivo oncogenic activity of CagA is further potentiated in the presence of chronic inflammation.Since Helicobacter pylori infection triggers a proinflammatory response in host cells,a feedforward stimulation loop that augments the oncogenic actions of CagA and inflammation is created in CagA-injected gastric mucosa.Given that Helicobacter pylori is no longer colonized in established gastric cancer lesions,the multistep nature of gastric cancer development should include a“hit-and-run”process of CagA action.Thus,acquisition of genetic and epigenetic alterations that compensate for CagA-directed cancer hallmarks may be required for completion of the“hit-and-run”process of gastric carcinogenesis.
文摘Phytochromes （Phy） and phytochrome-interacting factor （PIF） transcription factors constitute a major signal- ing module that controls plant development in response to red and far-red light. A low red：far-red ratio is interpreted as shading by neighbor plants and induces cell elongation--a phenomenon called shade-avoidance syndrome （SAS）. PAR1 and its closest homolog PAR2 are negative regulators of SAS; they belong to the HLH transcription factor family that lacks a typical basic domain required for DNA binding, and they are believed to regulate gene expressions through DNA binding transcription factors that are yet to be identified. Here, we show that light signal stabilizes PAR1 protein and PAR1 interacts with PIF4 and inhibits PIF4-mediated gene activation. DNA pull-down and chromatin immunoprecipitation （CHIP） assays showed that PAR1 inhibits PIF4 DNA binding in vitro and in vivo. Transgenic plants overexpressing PAR1 （PARIOX） are insensitive to gibberellin （GA） or high temperature in hypocotyl elongation, similarly to the pifq mutant. In addition to PIF4, PAR1 also interacts with PRE1, a HLH transcription factor activated by brassinosteroid （BR） and GA. Overexpression of PRE1 largely suppressed the dwarf phenotype of PARIOX. These results indicate that PAR1-PRE1 and PAR1-PIF4 het- erodimers form a complex HLH/bHLH network regulating cell elongation and plant development in response to light and hormones.
基金supported by the National Natural Foundation of China(81160302)the Major Research Project of Yunnan Province(2011FZ109)Research project of Yunnan Education Bureau(2014Y153)
文摘Here,we used reverse transcription-PCR(RT-PCR) and western blot to detect protease-activated receptor(PAR) 1,PAR 2 and PAR 4 expression in cancer tissues and cell lines of esophageal squamous cell carcinoma,and investigated the co-relationship between PAR expression and clinic-pathological data for esophageal cancer.The methylation of PAR4 gene promoter involved in esophageal carcinoma was also analyzed.By comparing the mRNA expressions of normal esophageal tissue and human esophageal epithelial cells(HEEpiC),we found that among the 28 cases of esophageal squamous cell carcinoma,PAR1(60%) and PAR2(71%) were elevated in 17 and 20 cases,respectively,and PAR4(68%) expression was lowered in 19 cases.Whereas,in human esophageal squamous cells(TE-1 and TE-10),PAR1 and PAR2 expression was increased but PAR4 was decreased.Combined with clinical data,the expression of PAR1 in poorly differentiated(P=0.016) and middle and lower parts of the esophagus(P=0.016) was higher; expression of PAR4 in poorly differentiated carcinoma was lower(P=0.049).Regarding TE-1 and TE-10 protein expression,we found that in randomized esophageal carcinoma,PAR1(P=0.027) and PAR2(P=0.039) expressions were increased,but lowered for PAR4(P=0.0001).In HEEpiC,TE-1,TE-10,esophageal and normal esophagus tissue samples(case No.7),the frequency of methylation at the 19 CpG loci of PAR4 was 35.4%,95.2%,83.8%,62.6% and 48.2%,respectively.Our results indicate that the expression of PAR1 and PAR2 in esophageal squamous cell carcinoma is increased but PAR4 is decreased.Hypermethylation of the promoter of the PAR4 gene may contribute to reduced expression of PAR4 in esophageal squamous cell carcinoma.
基金supported by the National Natural Science Foundation of ChinaNo.81971826 (to AG)+5 种基金the China Postdoctoral Science FoundationNo.2020M681 689 (to YH)the Scientific Research Project of The Health Commission of Jiangsu ProvinceNo.ZDB2020003 (to AG)the Basic Scientific Research Projects of NantongNo.JC2020041 (to YH)
文摘Astrocytes are important cellular centers of cholesterol synthesis and metabolism that help maintain normal physiological function at the organism level.Spinal cord injury results in aberrant cholesterol metabolism by astrocytes and excessive production of oxysterols,which have profound effects on neuropathology.25-Hydroxycholesterol(25-HC),the main product of the membrane-associated enzyme cholesterol-25-hydroxylase(CH25H),plays important roles in mediating neuroinflammation.However,whether the abnormal astrocyte cholesterol metabolism induced by spinal cord injury contributes to the production of 25-HC,as well as the resulting pathological effects,remain unclear.In the present study,spinal cord injury-induced activation of thrombin was found to increase astrocyte CH25H expression.A protease-activated receptor 1 inhibitor was able to attenuate this effect in vitro and in vivo.In cultured primary astrocytes,thrombin interacted with protease-activated receptor 1,mainly through activation of the mitogen-activated protein kinase/nuclear factor-kappa B signaling pathway.Conditioned culture medium from astrocytes in which ch25h expression had been knocked down by siRNA reduced macrophage migration.Finally,injection of the protease activated receptor 1 inhibitor SCH79797 into rat neural sheaths following spinal cord injury reduced migration of microglia/macrophages to the injured site and largely restored motor function.Our results demonstrate a novel regulatory mechanism for thrombin-regulated cholesterol metabolism in astrocytes that could be used to develop anti-inflammatory drugs to treat patients with spinal cord injury.
基金supported by China Pharmaceutical University initiative program(No.CPU2018GY15)the National Natural Science Foundation for Young Scientists of China(No.81603647)
文摘Whitmania pigra has been used as a traditional Chinese medicine(TCM) for promoting blood circulation, alleviating blood coagulation, activating meridians and relieving stasis for several hundred years. However, the therapeutic components of this species, especially proteins and peptides were poorly exploited. Until now only a few of them were obtained by using chromatographic isolation and purification. In recent decade, transcriptome techniques were rapidly developed, and have been used to fully reveal the functional components of many animal venoms. In the present study, the c DNA of the salivary gland of Whitmania pigra was sequenced by illumina and the transcriptome was assembled by using Trinity. The proteome were analysed by LC-MS/MS. Based on the data of the transcriptome and the proteome, a potential antiplatelet protein named pigrin was found. Pigrin was cloned and expressed using P. pastoris GS115. The antiplatelet and antithrombotic bioactivities of pigrin were tested by using aggregometer and the rat arterio-venous shunt thrombosis model, respectively. The bleeding time of pigrin was measured by a mice tail cutting method. The docking of pigrin and protease-activated receptor 1(PAR1) or collagen were conducted using the ZDOCK Server. Pigrin was able to selectively inhibit platelet aggregation stimulated by PAR1 agonist and collagen. Pigrin attenuated thrombotic formation in vivo in rat, while did not prolong bleeding time at its effective dosage. There are significant differences in the key residues participating in binding of Pigrin-Collagen complex from Pigrin-PAR1 complex. In conclusion, a novel PAR1 inhibitor pigrin was found from the leech Whitmania pigra. This study helped to elucidate the mechanism of the leech for the treatment of cardiovascular disorder.
