TGF-β mediates suppression of adipogenesis by estradiol through connective tissue growth factor induction

In the bone marrow cavity, adipocyte numbers increase, whereas osteoblast progenitor numbers decrease with aging. Because adipocytes and osteoblasts share a common progenitor, it is possible that this shift is due to an increase in adipocyte-lineage cells at the expense of osteoblast-lineage commitment. Estrogens inhibit adipocyte differentiation, and in both men and women, circulating estrogens correlate with bone loss with aging. In bone cells, estrogens stimulate expression of TGF-β and suppress mesenchymal cell adipogenesis. Using a tripotential mesenchymal cell line, we have examined whether estradiol suppression of adipocyte differentiation is due to stimulation of TGF-β and the mechanism by which TGF-β suppresses adipogenesis. We observed that estradiol-mediated suppression of adipogenic gene expression required at least 48 h treatment. TGF-β expression increased within 24 h of estradiol treatment, and TGF-β inhibition reversed estradiol influences on adipogenesis and adipocyte gene expression. Connective tissue growth factor (CTGF) mediates TGF-β suppression of adipogenesis in mouse 3T3-L1 cells. CTGF expression was induced within 24 h of TGF-β treatment, whereas estradiol-mediated induction required 48 h treatment. Moreover, estradiol-mediated induction of CTGF was abrogated by TGF-β inhibition. These data support that estradiol effects on adipogenesis involves TGF-β induction, which then induces CTGF to suppress adipogenesis.     

Effect of atorvastatin on expression of TLR4 and NF-κB p65 in atherosclerotic rabbits

ObjectiveTo study the effect of atorvastatin on atherosclerotic rabbits.MethodsA total of 60 New Zealand male rabbits were randomly divided into the normal group, model group and atorvastatin group. The replication rabbit atherosclerotic model with immune injury combined with a high fat diet feeding was used. All rabbits were sacrificed after 3 months. TLR4 and NF-κB p65 were observed by HE staining, immunohistochemistry and western blotting.ResultsThe expression of TLR4, NF-κB p65 were significantly increased in the model group compared with the normal group. The expression of TLR4 and NF-κB p65 decreased significantly in the atorvastatin group, and there was no difference compared with the normal group.ConclusionsThe effect of atorvastatin on atherosclerosis may be achieved by the inhibition of the expression of TLR4 and NF-κB p65.     

Regulation of TNFα and IL1β in rheumatoid arthritis synovial fibroblasts by leukotriene B4

Arachidonic acid metabolites, such as leukotriene B4 (LTB4), are known to play an important role in pathogenesis of rheumatoid arthritis (RA). Apart from leukocytes, RA synovial fibroblasts (RASF) produce a broad array of inflammatory mediators to recruit, retain and activate immune cells and resident mesenchymal cells in the joints to promote ongoing inflammation and tissue destruction. To determine how LTB4 may contribute to this process, RASF was cultured from synovial tissues collected from RA patients undergoing total knee replacement. The level of LTB4 in culture medium was determined using ELISA, and expression of LTB4 receptors (BLT1 and BLT2) by RT-PCR. In the presence of exogenous LTB4, mRNA and protein levels of tumor necrosis factor-α (TNFα) and interleukin 1β (IL1β) were determined by real-time PCR and ELISA. Furthermore, we examined the effects of leukotrienes synthesis inhibitors, MK886 and bestatin, on mRNA and protein levels of TNFα and IL1β in RASF. We found that LTB4 was present at a low concentration in the culture medium of RASF, and the major LTB4 receptor expressed in RASF was BLT2. LTB4 synthesis was activated by treatment with LIT (LPS, ionomycin and thapsogargin), and suppressed by MK886 and bestatin. Exogenous LTB4 remarkably increased the expression of TNFα and IL1β at both the mRNA level and the protein level. In contrast, MK886 and bestatin significantly inhibited their expression. These data suggested that LTB4 contributed to RA by regulating the expression of TNFα and IL1β in RASF. BLT2 was probably the major receptor mediating such effects.     

Rosiglitazone ameliorates insulin resistance in brown adipocytes of Wistar rats by impairing TNF-α induction of p38 and p42/p44 mitogen-activated protein kinases

Aims/hypothesis TNF- caused insulin resistance on glucose uptake and on insulin signalling in fetal brown adipocytes. Since treatment with TNF- activates stress kinases, including c-jun NH2 terminal kinase (JNK), and p42/p44 and p38 mitogen-activated protein kinases (MAPK), we explored the contribution of these pathways to insulin resistance by TNF-. Rosiglitazone is used to treat Type 2 diabetes as it improves insulin sensitivity in vivo. However, its ability to ameliorate TNF--induced insulin resistance in brown adipocytes remains to be explored.Methods We used fetal rat primary brown adipocytes cultured with TNF-, with or without stress kinase inhibitors or rosiglitazone, and further stimulated with insulin. Then, we measured glucose uptake and GLUT4 translocation. To determine the insulin signalling cascade, we submitted cells to lysis, immunoprecipitation and immunoblotting.Results Exposure to TNF- for 24 h impairs insulin stimulation of the phosphatidylinositol (PI) 3-kinase activity associated with IRS-2 and Akt activity. Pretreatment with PD98059 or PD169316, which inhibit p42/p44MAPK and p38MAPK respectively, restored insulin signalling and insulin-induced glucose uptake in the presence of TNF-. However, in the presence of SP600125, an inhibitor of JNK, TNF- still produced insulin resistance. Rosiglitazone ameliorated insulin resistance by TNF- in brown adipocytes, restoring completely insulin-stimulated glucose uptake and insulin-induced GLUT4 translocation to plasma membrane in parallel to the insulin signalling cascade IRS-2/PI 3-kinase/Akt.Conclusions/interpretation Rosiglitazone treatment impaired TNF- activation of p38 and p42/p44MAPK, restoring insulin signalling and leading to normalisation of glucose uptake.     

Simultaneous inactivation of Par-4 and PTEN in vivo leads to synergistic NF-κB activation and invasive prostate carcinoma

Prostate cancer is one of the most common neoplasias in men. The tumor suppressor Par-4 is an important negative regulator of the canonical NF-κB pathway and is highly expressed in prostate. Here we show that Par-4 expression is lost in a high percentage of human prostate carcinomas, and this occurs in association with phosphatase and tensin homolog deleted from chromosome 10 (PTEN) loss. Par-4 null mice, similar to PTEN-heterozygous mice, only develop benign prostate lesions, but, importantly, concomitant Par-4 ablation and PTEN-heterozygosity lead to invasive prostate carcinoma in mice. This strong tumorigenic cooperation is anticipated in the preneoplastic prostate epithelium by an additive increase in Akt activation and a synergistic stimulation of NF-κB. These results establish the cooperation between Par-4 and PTEN as relevant for the development of prostate cancer and implicate the NF-κB pathway as a critical event in prostate tumorigenesis.     

Expression and significance of nuclear factor κB p65 in colon tissues of rats with TNBS-induced colitis

AIM: To investigate the role of NF-κB in the pathogenesis of TNBS-induced colitis in rats. METHODS: Thirty-two healthy adult Sprague-Dawley (SD) rats were randomly divided into four groups of eight each: normal, NS, model Ⅰ, model Ⅱ groups in our study. Rat colitis model was established through 2-,4-,6-trinitrobenzene sulfonic acid (TNBS) enema. At the end of four weeks, the macroscopical and histological changes of the colon were examined and mucosa myeloperoxidase (MPO)activities assayed. NF-~B p65 expression was determined by Western blot assessment in cytoplasmic and nuclear extracts of colon tissue, and the expressions of TNF-α (and ICAM-1 protein in colon tissue were examined by immunohistochemistry. The relativities between expression of NF-κB p65 and other parameters were analyzed. RESULTS: TNBS enema resulted in pronounced pathological changes of colonic mucosa in model Ⅱ group (macroscopic and histological injury indices 6.25±1.39 and 6.24±1.04, respectively), which were in accordance with the significantly elevated MPO activity (1.69+0.11). And the nuclear level of NF-κB and expression of TNF-α, ICAM-1 in rats of model Ⅱ group were higher than that of normal control(9.7±1.96 vs 1.7±0.15, 84.09±14.52 vs 16.03±6.21,77.69±8.09 vs 13.41±4.91 P<0.01), Linear correlation analysis revealed that there were strong correlations between the nuclear level of NF-κB and the tissue positive expression of TNF-α and ICAM-1, MPO activities, macroscopical and histological indices in TNBS-induced colitis, respectively (r = 0.8235, 0.8780, 0.8572, 0.9152,0.8247; P<0.05). CONCLUSION: NF-κB plays a pivotal role in the pathogenesis of ulcerative colitis, which might account for the up-regulation the expression of TNF-α and ICAM-1.     

Expression and significance of nuclear factor κB p65 in colon tissues of rats with TNBS-induced colitis

AIM: To investigate the role of NF-κB in the pathogenesis of TNBS-induced colitis in rats. METHODS: Thirty-two healthy adult Sprague-Dawley (SD) rats were randomly divided into four groups of eight each: normal, NS, model Ⅰ, model Ⅱ groups in our study. Rat colitis model was established through 2-,4-,6-trinitrobenzene sulfonic acid (TNBS) enema. At the end of four weeks, the macroscopical and histological changes of the colon were examined and mucosa myeloperoxidase (MPO) activities assayed. NF-κB p65 expression was determined by Western blot assessment in cytoplasmic and nuclear extracts of colon tissue, and the expressions of TNF-α and ICAM-1 protein in colon tissue were examined by immunohistochemistry. The relativities between expression of NF-κB p65 and other parameters were analyzed. RESULTS: TNBS enema resulted in pronounced pathological changes of colonic mucosa in model Ⅱ group (macroscopic and histological injury indices 6.25±1.39 and 6.24±1.04, respectively), which were in accordance with the significantly elevated MPO activity (1.69±0.11). And the nuclear level of NF-κB and expression of TNF-α, ICAM-1 in rats of model II group were higher than that of normal control (9.7±1.96 vs1.7±0.15, 84.09±14.52 vs 16.03±6.21, 77.69±8.09 vs 13.41±4.91 P<0.01), Linear correlation analysis revealed that there were strong correlations between the nuclear level of NF-κB and the tissue positive expression of TNF-α and ICAM-1, MPO activities, macroscopical and histological indices in TNBS-induced colitis, respectively (r= 0.8235, 0.8780, 0.8572, 0.9152, 0.8247; P<0.05). CONCLUSION: NF-κB plays a pivotal role in the pathogenesis of ulcerative colitis, which might account for the up-regulation the expression of TNF-α and ICAM-1.     

Paeonol ameliorates CFA-induced inflammatory pain by inhibiting HMGB1/TLR4/NF-κB p65 pathway

Metabolic Brain Disease - The enhanced release of inflammatory cytokines mediated by high mobility group box1 (HMGB1) leads to pain sensation, and has been implicated in the etiology of...     

NF-κB-dependent synergistic regulation of CXCL10 gene expression by IL-1β and IFN-γ in human intestinal epithelial cell lines

Background and aims Little is known about the intestinal epithelial expression and secretion of CXCL10 (IP-10), a chemokine involved in recruiting T cells and monocytes. We aimed to study CXCL10 gene expression and regulation by the pro-inflammatory cytokines interleukin (IL)-1β, interferon (IFN)-γ and tumour necrosis factor (TNF)-α in intestinal epithelial cell lines. Materials and methods CXCL10 expression and secretion kinetics were assessed in Caco-2, HT-29 and DLD1 human colon epithelial cells, treated with IL-1β, TNF-α, IFN-γ alone or in combination with each other by real-time polymerase chain reaction (PCR), Northern blotting and enzyme-linked immunoabsorbent assay (ELISA). Transient transfections with TGL-IP10 (CXCL10 promoter) and TGL-IP10-κB2 mutant promoter and gelshifts and supershifts for nuclear factor (NF)-κB were also performed. Results Real-time PCRs and ELISA experiments revealed that IL-1β was the strongest and earliest inducer of CXCL10 messenger ribonucleic acid (mRNA) expression and protein secretion in Caco-2 cell line, whereas INF-γ had a delayed kinetics. There was a strong synergistic effect of either TNF-α or IL-1β with IFN-γ both on CXCL10 mRNA expression and protein secretion in all three cell lines. Real-time PCR and ELISA experiments using a specific NF-κB inhibitor and transfection experiments with a NF-κB-binding defective CXCL10 promoter construct revealed that the induction of CXCL10 by IL-1β and its synergism with IFN-γ is NF-κB dependent. Conclusion These data demonstrate that in colonic epithelial cells, depending on the cellular context and utilizing the NF-κB pathway, IL-1β alone and/or in synergism with IFN-γ may play a major role in the induction of CXCL10.     

Reduction of tumor necrosis factor α and interleukin-1β levels in human synovial tissue by interleukin-4 and glucocorticoid

Summary The effects of recombinant human interleukin-4 (IL-4) and the glucocorticoid, dexamethasone, on tumor necrosis factor (TNF) and interleukin-1 (IL-1) levels in cultures of rheumatoid and osteoarthritic synovial tissue were studied. Low concentrations of IL-4 and dexamethasone suppressed the levels of both cytokines in the supernatants of both types of tissue after stimulation with lipopolysaccharide (LPS); the IL-1 and TNF levels were measured by ELISA. It is suggested that it is the monocyte/macrophage in the synovial tissues that is responsive to the inhibitors. It is proposed that glucocorticoids may act on synovial tissue in this manner in vivo and IL-4 may do so if administered intraarticularly.     

Correlation between MMP-2 and NF-κ B expression of intracranial aneurysm

ObjectiveTo investigate the correlation between expressions of MMP-2 and NF-κ B in the intracranial aneurysm wall, and explore their role in the mechanism of the occurrence, growth and rupture of intracranial aneurysms.MethodsRT-PCR was used to detect the expression of MMP-2 and NF-κ B mRNA of 30 cases of intracranial aneurysm tissue and 10 cases of normal intracranial arterial tissue; Immunohistochemical method was used to detect the expression of MMP-2 and NF-κ B protein.ResultsThe semi-quantitative analysis of MMP-2 and NF-κ B in aneurysms tissues and normal tissues were statistically significant different from each other (P<0 05). Immunohistochemical staining results showed NF-κ B was expressed in different layers. The expression of them were positive in intimal and medial, and the expression sites were located in the nucleus. MMP-2 were expressed in different layers of the aneurysm wall, and the expressions were positive in media and extima. The MMP-2 and NF- κ B-positive expression of aneurysm wall were significantly higher than in normal cerebral arteries (P <0.05). MMP-2 and NF-κ B mRNA expression showed positive correlation in the aneurysm wall tissue (r = 0.689, P = 0.005).ConclusionsThe expressions of MMP-2 and NF-κ B in the intracranial aneurysm wall tissue were significantly higher than in the normal intracranial arterial tissues. They have a synergistic effect on the formation of intracranial aneurysms.     

TGF-β IL-6 axis mediates selective and adaptive mechanisms of resistance to molecular targeted therapy in lung cancer

The epidermal growth-factor receptor (EGFR) tyrosine kinase inhibitor erlotinib has been proven to be highly effective in the treatment of nonsmall cell lung cancer (NSCLC) harboring oncogenic EGFR mutations. The majority of patients, however, will eventually develop resistance and succumb to the disease. Recent studies have identified secondary mutations in the EGFR (EGFR T790M) and amplification of the N-Methyl-N′-nitro-N-nitroso-guanidine (MNNG) HOS transforming gene (MET) oncogene as two principal mechanisms of acquired resistance. Although they can account for approximately 50% of acquired resistance cases together, in the remaining 50%, the mechanism remains unknown. In NSCLC-derived cell lines and early-stage tumors before erlotinib treatment, we have uncovered the existence of a subpopulation of cells that are intrinsically resistant to erlotinib and display features suggestive of epithelial-to-mesenchymal transition (EMT). We showed that activation of TGF-β–mediated signaling was sufficient to induce these phenotypes. In particular, we determined that an increased TGF-β–dependent IL-6 secretion unleashed previously addicted lung tumor cells from their EGFR dependency. Because IL-6 and TGF-β are prominently produced during inflammatory response, we used a mouse model system to determine whether inflammation might impair erlotinib sensitivity. Indeed, induction of inflammation not only stimulated IL-6 secretion but was sufficient to decrease the tumor response to erlotinib. Our data, thus, argue that both tumor cell-autonomous mechanisms and/or activation of the tumor microenvironment could contribute to primary and acquired erlotinib resistance, and as such, treatments based on EGFR inhibition may not be sufficient for the effective treatment of lung-cancer patients harboring mutant EGFR.