Pub Date : 2015-01-01DOI: 10.21767/2471-8153.100004
C. Malemud, A. Lewis, Meredith A Wylie, Evan C. Meszaros, Yelenna Skomorovska-Prokvolit, S. Mesiano
BACKGROUND Activation of the SAPK/MAPK signaling pathway by pro-inflammatory cytokines is known to induce apoptosis in cultured articular chondrocytes. C-28/I2, an immortalized human juvenile chondrocyte cell line was employed to determine the extent to which recombinant human (rh) forms of the pro-inflammatory cytokines, tumor necrosis factor-α (rhTNF-α,), interleukin-6 (rhIL-6) and oncostatin M (rhOSM) induced apoptosis. METHODS The induction of apoptosis in the presence or absence of these cytokines was measured by the DAPI/TUNEL assay, by whether or not pro-caspase-3 was activated and by the extent to which poly-ADP-ribose polymerase (PARP) was degraded. FINDINGS Only rhTNF-α, and rhIL-6 significantly increased apoptosis in C-28/I2 chondrocytes, although rhOSM exhibited a strong trend (p=0.067) towards increasing the frequency of apoptotic chondrocytes. The number of apoptotic C28/I2 chondrocytes was significantly increased (p=1.3 × 10-5) by the combination of rhTNF-α and U0126 (10 μM) compared to rhTNF-α alone. However apoptosis was not further increased by combining rhIL-6 with U0126. The LI-COR® western blot system showed that U0126 (10 μM) inhibited the phosphorylation of extracellular signal-regulated kinase-2 (p-ERK2) by phorbol myristate acetate-treated immortalized myometrial cells, U0126 (10 μM) did not alter total U-ERK2. Western blot analysis also revealed that the increased frequency of apoptotic C-28/I2 chondrocytes induced by rhTNF-α and rhOSM, but not rhIL-6, was associated with PARP degradation. However, none of the cytokines resulted in pro-caspase-3 activation. CONCLUSION These results showed that rhTNF-α and rhIL-6 were strong inducers of apoptosis in the immortalized C-28/I2 human chondrocyte cell line. They also suggested that inhibiting ERK2 phosphorylation via U0126-mediated inhibition of MEK1/2 activity, increased rhTNF-α-induced C-28/I2 chondrocyte apoptosis.
{"title":"U0126, an Inhibitor of MEK1/2, Increases Tumor Necrosis Factor-α-Induced Apoptosis, but not Interleukin-6 Induced Apoptosis in C-28/I2 Human Chondrocytes.","authors":"C. Malemud, A. Lewis, Meredith A Wylie, Evan C. Meszaros, Yelenna Skomorovska-Prokvolit, S. Mesiano","doi":"10.21767/2471-8153.100004","DOIUrl":"https://doi.org/10.21767/2471-8153.100004","url":null,"abstract":"BACKGROUND Activation of the SAPK/MAPK signaling pathway by pro-inflammatory cytokines is known to induce apoptosis in cultured articular chondrocytes. C-28/I2, an immortalized human juvenile chondrocyte cell line was employed to determine the extent to which recombinant human (rh) forms of the pro-inflammatory cytokines, tumor necrosis factor-α (rhTNF-α,), interleukin-6 (rhIL-6) and oncostatin M (rhOSM) induced apoptosis. METHODS The induction of apoptosis in the presence or absence of these cytokines was measured by the DAPI/TUNEL assay, by whether or not pro-caspase-3 was activated and by the extent to which poly-ADP-ribose polymerase (PARP) was degraded. FINDINGS Only rhTNF-α, and rhIL-6 significantly increased apoptosis in C-28/I2 chondrocytes, although rhOSM exhibited a strong trend (p=0.067) towards increasing the frequency of apoptotic chondrocytes. The number of apoptotic C28/I2 chondrocytes was significantly increased (p=1.3 × 10-5) by the combination of rhTNF-α and U0126 (10 μM) compared to rhTNF-α alone. However apoptosis was not further increased by combining rhIL-6 with U0126. The LI-COR® western blot system showed that U0126 (10 μM) inhibited the phosphorylation of extracellular signal-regulated kinase-2 (p-ERK2) by phorbol myristate acetate-treated immortalized myometrial cells, U0126 (10 μM) did not alter total U-ERK2. Western blot analysis also revealed that the increased frequency of apoptotic C-28/I2 chondrocytes induced by rhTNF-α and rhOSM, but not rhIL-6, was associated with PARP degradation. However, none of the cytokines resulted in pro-caspase-3 activation. CONCLUSION These results showed that rhTNF-α and rhIL-6 were strong inducers of apoptosis in the immortalized C-28/I2 human chondrocyte cell line. They also suggested that inhibiting ERK2 phosphorylation via U0126-mediated inhibition of MEK1/2 activity, increased rhTNF-α-induced C-28/I2 chondrocyte apoptosis.","PeriodicalId":91305,"journal":{"name":"Journal of autoimmune disorders","volume":"15 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2015-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75878090","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2015-01-01DOI: 10.21767/2471-8153.100006
M. Guerau-de-Arellano, Yue Liu, W. H. Meisen, D. Pitt, M. Racke, A. Lovett-racke
Genetic studies suggest that the immune system is the greatest genetic contributor to multiple sclerosis (MS) susceptibility. Yet, these immune-related genes do not explain why inflammation is limited to the CNS in MS. We hypothesize that there is an underlying dysregulation in the CNS of MS patients that makes them more vulnerable to CNS inflammation. The sparsity of CNS-related genes associated with MS suggests that epigenetic changes in the CNS may play a role. Thus, a miRNA profiling study was performed in NAWM of MS patients and control subjects to determine if specific CNS pathways can be identified that may be altered due to miRNA-mediated post-transcriptional dysregulation. There were 15 differentially expressed miRNAs found in the MS patients' NAWM. Pathway analysis indicated that the MAPK pathway and pathways associated with the blood-brain barrier were predicted to be significantly affected by these miRNAs. Using target predication and mRNA analysis, an inverse relationship was found between miR-191 and BDNF, SOX4, FZD5 and WSB1. The pathway and target analysis of the MS-associated miRNAs suggests that MS patients' CNS is more prone to inflammation and less capable of repair, yet enriched in neuroprotective mechanisms.
{"title":"Analysis of miRNA in Normal Appearing White Matter to Identify Altered CNS Pathways in Multiple Sclerosis.","authors":"M. Guerau-de-Arellano, Yue Liu, W. H. Meisen, D. Pitt, M. Racke, A. Lovett-racke","doi":"10.21767/2471-8153.100006","DOIUrl":"https://doi.org/10.21767/2471-8153.100006","url":null,"abstract":"Genetic studies suggest that the immune system is the greatest genetic contributor to multiple sclerosis (MS) susceptibility. Yet, these immune-related genes do not explain why inflammation is limited to the CNS in MS. We hypothesize that there is an underlying dysregulation in the CNS of MS patients that makes them more vulnerable to CNS inflammation. The sparsity of CNS-related genes associated with MS suggests that epigenetic changes in the CNS may play a role. Thus, a miRNA profiling study was performed in NAWM of MS patients and control subjects to determine if specific CNS pathways can be identified that may be altered due to miRNA-mediated post-transcriptional dysregulation. There were 15 differentially expressed miRNAs found in the MS patients' NAWM. Pathway analysis indicated that the MAPK pathway and pathways associated with the blood-brain barrier were predicted to be significantly affected by these miRNAs. Using target predication and mRNA analysis, an inverse relationship was found between miR-191 and BDNF, SOX4, FZD5 and WSB1. The pathway and target analysis of the MS-associated miRNAs suggests that MS patients' CNS is more prone to inflammation and less capable of repair, yet enriched in neuroprotective mechanisms.","PeriodicalId":91305,"journal":{"name":"Journal of autoimmune disorders","volume":"9 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2015-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83416334","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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