{"title":"反义寡核苷酸抑制表明ikappab - α在系膜细胞中不形成NF-kappaB的负自我调节环。","authors":"R B Auwardt, S J Mudge, C Chen, D A Power","doi":"10.1159/000020662","DOIUrl":null,"url":null,"abstract":"<p><p>The IkappaB proteins are important in the regulation of the NF-kappaB/Rel group of transcription factors which are pivotal in the inflammatory response. IkappaB-alpha is itself upregulated by activation of NF-kappaB and is postulated to be part of a negative feedback loop. This role of IkappaB-alpha has been challenged, however, by recent evidence that demonstrates (1) continued activation of NF-kappaB in mesangial and endothelial cells despite the resynthesis of IkappaB-alpha protein and (2) that inhibition of the transactivating activity of NF-kappaB by corticosteroids can be dissociated from a rise in IkappaB-alpha protein. We investigated the role of IkappaB-alpha in mesangial cells using a phosphorothioate antisense oligonucleotide directed against the translational start point of IkappaB-alpha. If IkappaB-alpha does function as a negative feedback inhibitor in these cells, then reducing IkappaB-alpha levels should lead to an increase in NF-kappaB activity. We first demonstrated that IkappaB-alpha protein resynthesis following stimulation could be specifically reduced. We then showed that NF-kappaB DNA binding was not increased with antisense treatment following stimulation. Finally, NF- kappaB-dependent gene signalling after stimulation (determined through an NF-kappaB luciferase reporter and upregulation of the mRNA of known NF-kappaB-responsive genes MCP-1 and IkappaB-alpha) was reduced rather than increased. These data suggest that IkappaB-alpha does not form a negative autoregulatory loop for NF-kappaB in mesangial cells and may actually reduce NF-kappaB activity. This may have relevance to therapies directed at inhibition of NF-kappaB activity in mesangial cell diseases.</p>","PeriodicalId":12179,"journal":{"name":"Experimental nephrology","volume":"8 3","pages":"144-51"},"PeriodicalIF":0.0000,"publicationDate":"2000-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000020662","citationCount":"15","resultStr":"{\"title\":\"Inhibition with antisense oligonucleotide suggests that IkappaB-alpha does not form a negative autoregulatory loop for NF-kappaB in mesangial cells.\",\"authors\":\"R B Auwardt, S J Mudge, C Chen, D A Power\",\"doi\":\"10.1159/000020662\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>The IkappaB proteins are important in the regulation of the NF-kappaB/Rel group of transcription factors which are pivotal in the inflammatory response. IkappaB-alpha is itself upregulated by activation of NF-kappaB and is postulated to be part of a negative feedback loop. This role of IkappaB-alpha has been challenged, however, by recent evidence that demonstrates (1) continued activation of NF-kappaB in mesangial and endothelial cells despite the resynthesis of IkappaB-alpha protein and (2) that inhibition of the transactivating activity of NF-kappaB by corticosteroids can be dissociated from a rise in IkappaB-alpha protein. We investigated the role of IkappaB-alpha in mesangial cells using a phosphorothioate antisense oligonucleotide directed against the translational start point of IkappaB-alpha. If IkappaB-alpha does function as a negative feedback inhibitor in these cells, then reducing IkappaB-alpha levels should lead to an increase in NF-kappaB activity. We first demonstrated that IkappaB-alpha protein resynthesis following stimulation could be specifically reduced. We then showed that NF-kappaB DNA binding was not increased with antisense treatment following stimulation. Finally, NF- kappaB-dependent gene signalling after stimulation (determined through an NF-kappaB luciferase reporter and upregulation of the mRNA of known NF-kappaB-responsive genes MCP-1 and IkappaB-alpha) was reduced rather than increased. These data suggest that IkappaB-alpha does not form a negative autoregulatory loop for NF-kappaB in mesangial cells and may actually reduce NF-kappaB activity. This may have relevance to therapies directed at inhibition of NF-kappaB activity in mesangial cell diseases.</p>\",\"PeriodicalId\":12179,\"journal\":{\"name\":\"Experimental nephrology\",\"volume\":\"8 3\",\"pages\":\"144-51\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2000-05-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.1159/000020662\",\"citationCount\":\"15\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Experimental nephrology\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1159/000020662\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Experimental nephrology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1159/000020662","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
Inhibition with antisense oligonucleotide suggests that IkappaB-alpha does not form a negative autoregulatory loop for NF-kappaB in mesangial cells.
The IkappaB proteins are important in the regulation of the NF-kappaB/Rel group of transcription factors which are pivotal in the inflammatory response. IkappaB-alpha is itself upregulated by activation of NF-kappaB and is postulated to be part of a negative feedback loop. This role of IkappaB-alpha has been challenged, however, by recent evidence that demonstrates (1) continued activation of NF-kappaB in mesangial and endothelial cells despite the resynthesis of IkappaB-alpha protein and (2) that inhibition of the transactivating activity of NF-kappaB by corticosteroids can be dissociated from a rise in IkappaB-alpha protein. We investigated the role of IkappaB-alpha in mesangial cells using a phosphorothioate antisense oligonucleotide directed against the translational start point of IkappaB-alpha. If IkappaB-alpha does function as a negative feedback inhibitor in these cells, then reducing IkappaB-alpha levels should lead to an increase in NF-kappaB activity. We first demonstrated that IkappaB-alpha protein resynthesis following stimulation could be specifically reduced. We then showed that NF-kappaB DNA binding was not increased with antisense treatment following stimulation. Finally, NF- kappaB-dependent gene signalling after stimulation (determined through an NF-kappaB luciferase reporter and upregulation of the mRNA of known NF-kappaB-responsive genes MCP-1 and IkappaB-alpha) was reduced rather than increased. These data suggest that IkappaB-alpha does not form a negative autoregulatory loop for NF-kappaB in mesangial cells and may actually reduce NF-kappaB activity. This may have relevance to therapies directed at inhibition of NF-kappaB activity in mesangial cell diseases.