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Suberosin inhibits proliferation of human peripheral blood mononuclear cells through the modulation of the transcription factors NF-AT and NF-kappaB.

ABSTRACT: BACKGROUND AND PURPOSE: Extracts of Plumbago zeylanica containing suberosin exhibit anti-inflammatory activity. We purified suberosin from such extracts and studied its effects on a set of key regulatory events in the proliferation of human peripheral blood mononuclear cells (PBMC) stimulated by phytohemagglutinin (PHA). EXPERIMENTAL APPROACH: Proliferation of PBMC in culture was measured by uptake of 3H-thymidine; production of cytokines and cyclins by Western blotting and RT-PCR. Transcription factors NF-AT and NF-kappaB were assayed by immunocytochemistry and EMSA. KEY RESULTS: Suberosin suppressed PHA-induced PBMC proliferation and arrested cell cycle progression from the G1 transition to the S phase. Suberosin suppressed, in activated PBMC, transcripts of interleukin-2 (IL-2), interferon-gamma (IFN-gamma), and cyclins D3, E, A, and B. DNA binding activity and nuclear translocation of NF-AT and NF-kappaB induced by PHA were blocked by suberosin. Suberosin decreased the rise in intracellular Ca2+ concentration ([Ca2+]i) in PBMC stimulated with PHA. Suberosin did not affect phosphorylation of p38 and JNK but did reduce activation of ERK in PHA-treated PBMC. Pharmacological inhibitors of NF-kappaB, NF-AT, and ERK decreased expression of mRNA for the cyclins, IL-2, and IFN-gamma and cell proliferation in PBMC activated by PHA. CONCLUSIONS AND IMPLICATIONS: The inhibitory effects of suberosin on PHA-induced PBMC proliferation, were mediated, at least in part, through reduction of [Ca2+]i, ERK, NF-AT, and NF-kappaB activation, and early gene expression in PBMC including cyclins and cytokines, and arrest of cell cycle progression in the cells. Our observations provide an explanation for the anti-inflammatory activity of P. zeylanica.

SUBMITTER: Chen YC

PROVIDER: S-EPMC2013892 | biostudies-other | 2007 Feb

REPOSITORIES: biostudies-other

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Project description:In this work we present evidence that the p53 tumor suppressor protein and NF-kappaB transcription factors could be related through common descent from a family of ancestral transcription factors regulating cellular proliferation and apoptosis. P53 is a homotetrameric transcription factor known to interact with the ankyrin protein 53BP2 (a fragment of the ASPP2 protein). NF-kappaB is also regulated by ankyrin proteins, the prototype of which is the IkappaB family. The DNA binding sequences of the two transcription factors are similar, sharing 8 out of 10 nucleotides. Interactions between the two proteins, both direct and indirect, have been noted previously and the two proteins play central roles in the control of proliferation and apoptosis.Using previously published structure data, we noted a significant degree of structural alignment between p53 and NF-kappaB p65. We also determined that IkappaBalpha and p53 bind in vitro through a specific interaction in part involving the DNA binding region of p53, or a region proximal to it, and the amino terminus of IkappaBalpha independently or cooperatively with the ankyrin 3 domain of IkappaBalpha In cotransfection experiments, kappaBalpha could significantly inhibit the transcriptional activity of p53. Inhibition of p53-mediated transcription was increased by deletion of the ankyrin 2, 4, or 5 domains of IkappaBalpha Co-precipitation experiments using the stably transfected ankyrin 5 deletion mutant of kappaBalpha and endogenous wild-type p53 further support the hypothesis that p53 and IkappaBalpha can physically interact in vivo.The aggregate results obtained using bacterially produced IkappaBalpha and p53 as well as reticulocyte lysate produced proteins suggest a correlation between in vitro co-precipitation in at least one of the systems and in vivo p53 inhibitory activity. These observations argue for a mechanism involving direct binding of IkappaBalpha to p53 in the inhibition of p53 transcriptional activity, analogous to the inhibition of NF-kappaB by kappaBalpha and p53 by 53BP2/ASPP2. These data furthermore suggest a role for ankyrin proteins in the regulation of p53 activity. Taken together, the NFkappaB and p53 proteins share similarities in structure, DNA binding sites and binding and regulation by ankyrin proteins in support of our hypothesis that the two proteins share common descent from an ancestral transcriptional factor.

Project description:BACKGROUND:Psoriasis is a T cell-mediated autoimmune disease in patients with elevated levels of proinflammatory cytokines belonging mainly to the Th1 pathway. We investigated whether treatment of psoriasis patients with methotrexate (MTX), along with micronutrients, modulated mRNA expression of Th1 and Th2 components and whether expression of these components correlated with psoriasis severity. METHODS:Thirty plaque-type psoriasis patients with Psoriasis Area and Severity Index (PASI) scores greater than 10 were recruited; these were 15 non-micronutrients taking- (NMT) patients treated with MTX daily (0.2-0.3 mg/kg/week), and 15 micronutrients taking- (MT) patients treated with MTX plus a micronutrient supplement daily, for 12 weeks. Blood samples were collected immediately before treatment (baseline) and after 12 weeks of treatment. Taqman quantitative real-time polymerase chain reaction (qPCR) was applied to analyze the expression of the Th1 components T-bet, interleukin-12 (IL-12), and interferon-gamma (IFN-?), and the Th2 components GATA-3 and interleukin-4 (IL-4). Disease severity was measured using the PASI scoring system. RESULTS:Significant clinical improvement in the MT group coincided with significant down-regulation of Th1 and up-regulation of Th2 markers (P<0.05). With respect to the PASI-75, (defined as a 75% or greater reduction in the PASI score) cut-off point, expression of IFN-? in the MT group with PASI scores above 75 was significantly less than that of patients in the NMT group (P=0.05). Also, GATA3 and IL-4 mRNA expression in the MT group with PASI scores greater than above 75 was significantly greater than that of patients in the NMT group (P=0.05 and 0.04, respectively). CONCLUSION:Based on significant attenuation of the PASI score, which correlated with upregulation of Th2 pathway markers in the MT group, we recommend administration of micronutrients combined with MTX for psoriasis patients. Our results contribute to a better understanding of methotrexate immunepathogenesis mechanisms and their correlations to clinical responses in psoriasis.

Project description:Neph3 (filtrin) is expressed in the glomerular podocytes where it localizes at the specialized cell adhesion structures of the foot processes called slit diaphragms which form the outermost layer of the glomerular filtration barrier. Neph3 protein shows homology and structural similarity to Neph1, Neph2 and nephrin, which all are crucial for maintaining the normal glomerular ultrafiltration function. The exact function of Neph3 in the kidney is not known but we have previously shown that the level of Neph3 mRNA is decreased in proteinuric diseases. This suggests that Neph3 may play a role in the pathogenesis of kidney damage, and emphasizes the need to analyze the regulatory mechanisms of Neph3 gene. In this study we investigated the transcriptional regulation of Neph3 gene by identifying transcription factors that control Neph3 expression.We cloned and characterized approximately 5 kb fragment upstream of the Neph3 gene. Neph3 proximal promoter near the transcription start site was found to be devoid of TATA and CAAT boxes, but to contain a highly GC-rich area. Using promoter reporter gene constructs, we localized the main activating regulatory region of Neph3 gene in its proximal promoter region from -105 to -57. Within this region, putative transcription factor binding sites for NF-kappaB and Sp1 were found by computational analysis. Mutational screening indicated that NF-kappaB and Sp1 response elements are essential for the basal transcriptional activity of the Neph3 promoter. Co-transfection studies further showed that NF-kappaB and Sp1 regulate Neph3 promoter activity. In addition, overexpression of NF-kappaB increased endogenous Neph3 gene expression. Chromatin immunoprecipitation assay using cultured human podocytes demonstrated that both NF-kappaB and Sp1 interact with the Neph3 promoter.Our results show that NF-kappaB and Sp1 are key regulators of Neph3 expression at the basal level in podocytes, therefore providing new insight into the molecular mechanisms that contribute to the expression of Neph3 gene.

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Suberosin inhibits proliferation of human peripheral blood mononuclear cells through the modulation of the transcription factors NF-AT and NF-kappaB.

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