TGF-β Receptor Expression in Human Odontoblasts and Pulpal Cells

Transforming growth factor- (TGF-) isoform expression by odontoblasts leads to their sequestration within the dentine matrix, from where they may be released during caries and participate in the reparative processes. Two receptor types for TGF- have been implicated in TGF- induced signalling. The aim of this study was to characterise immunohistochemically the expression of these receptors in sound and carious human teeth to facilitate our understanding of the ability of these cells to respond to TGF- stimulation. Sound and carious human teeth were routinely processed and paraffin sections stained for TGF- receptors I and II, using the StrAviGen immunoperoxidase method. Strong specific staining for both receptor types was observed in the odontoblasts of healthy teeth with the greatest intensity seen with receptor I. Staining of weaker intensity was also observed for both receptors in the underlying cell rich area and pulp core. Similar patterns of staining were observed within carious tissues. We conclude that odontoblasts and other cells of the pulp of mature human molar teeth show the presence of both TGF- receptors I and II in health and disease with odontoblasts showing the strongest expression. Such findings may be important in the response of these cells to tissue injury.     

Characterization and Identification of Subpopulations of Mononuclear Preosteoclasts Induced by TNF-α in Combination with TGF-β in Rats

Osteoclasts are unique multinucleated cells formed by fusion of preosteoclasts derived from cells of the monocyte/macrophage lineage, which are induced by RANKL. However, characteristics and subpopulations of osteoclast precursor cells are poorly understood. We show here that a combination of TNF-α, TGF-β, and M-CSF efficiently generates mononuclear preosteoclasts but not multinucleated osteoclasts (MNCs) in rat bone marrow cultures depleted of stromal cells. Using a rat osteoclast-specific mAb, Kat1, we found that TNF-α and TGF-β specifically increased Kat1⁺c-fms⁺ and Kat1⁺c-fms⁻ cells but not Kat1⁻c-fms⁺ cells. Kat1⁻c-fms⁺ cells appeared in early stages of culture, but Kat1⁺c-fms⁺ and Kat1⁺c-fms⁻ cells increased later. Preosteoclasts induced by TNF-α, TGF-β, and M-CSF rapidly differentiated into osteoclasts in the presence of RANKL and hydroxyurea, an inhibitor of DNA synthesis, suggesting that preosteoclasts are terminally differentiated cells. We further analyzed the expression levels of genes encoding surface proteins in bone marrow macrophages (BMM), preosteoclasts, and MNCs. Preosteoclasts expressed itgam (CD11b) and chemokine receptors CCR1 and CCR2; however, in preosteoclasts the expression of chemokine receptors CCR1 and CCR2 was not up-regulated compared to their expression in BMM. However, addition of RANKL to preosteoclasts markedly increased the expression of CCR1. In contrast, expression of macrophage antigen emr-1 (F4/80) and chemokine receptor CCR5 was down-regulated in preosteoclasts. The combination of TNF-α, TGF-β, and M-CSF induced Kat1⁺CD11b⁺ cells, but these cells were also induced by TNF-α alone. In addition, MIP-1α and MCP-1, which are ligands for CCR1 and CCR2, were chemotactic for preosteoclasts, and promoted multinucleation of preosteoclasts. Finally, we found that Kat1⁺c-fms⁺ cells were present in bone tissues of rats with adjuvant arthritis. These data demonstrate that TNF-α in combination with TGF-β efficiently generates preosteoclasts in vitro. We delineated characteristics that are useful for identifying and isolating rat preosteoclasts, and found that CCR1 expression was regulated in the fusion step in osteoclastogenesis.     

Association of Six Well-Characterized Polymorphisms in TNF-α and TNF-β Genes with Sarcoidosis: A Meta-Analysis

Backgrounds

In this study, we aimed to investigate the association of six well-characterized polymorphisms in tumor necrosis factor alpha and beta (TNF-α and TNF-β) genes with the risk for sarcoidosis via a comprehensive meta-analysis.

Methods And Findings

The electronic MEDLINE (Ovid) and PubMed databases covering the period from the earliest possible year to June 2013 were searched. Total 13 qualified articles including 1584 patients with sarcoidosis and 2636 controls were recruited. The data were analyzed by RevMan software, and risk estimates were expressed as odds ratios (ORs) and 95% confidence intervals (95% CIs). Analyses of the full data set failed to identify any significant association of TNF-α gene -307A (OR=1.25; 95% CI: 0.98-1.59), -1031C (OR=0.88; 95% CI: 0.71-1.1), -863A (OR=0.89; 95% CI: 0.72-1.11), -238A (OR=0.97; 95% CI: 0.71-1.32), and -857T (OR=1.14; 95% CI: 0.74-1.77) alleles, but a significant association for TNF-β 252A allele (OR=1.65; 95%CI = 1.33-2.04; P<0.00001). Under a random-effects allelic model, there was marginally significant increased risk of sarcoidosis for -307A allele among Caucasians (OR=1.25; 95% CI: 0.96-1.62; P=0.09) but not among Asians (OR=2.12; 95% CI: 0.31-14.27; P=0.44). There was a low probability of publication bias as reflected by the fail-safe number.

Conclusions

This meta-analysis extended previous findings on the association between the TNF-α and TNF-β genetic polymorphisms and sarcoidosis, by showing that the TNF-β gene A252G polymorphism might be a potential risk factor for the development of sarcoidosis.     

Retinoic Acid Mediates Regulation of Network Formation by COUP-TFII and VE-Cadherin Expression by TGFβ Receptor Kinase in Breast Cancer Cells

Tumor development, growth, and metastasis depend on the provision of an adequate vascular supply. This can be due to regulated angiogenesis, recruitment of circulating endothelial progenitors, and/or vascular transdifferentiation. Our previous studies showed that retinoic acid (RA) treatment converts a subset of breast cancer cells into cells with significant endothelial genotypic and phenotypic elements including marked induction of VE-cadherin, which was responsible for some but not all morphological changes. The present study demonstrates that of the endothelial-related genes induced by RA treatment, only a few were affected by knockdown of VE-cadherin, ruling it out as a regulator of the RA-induced endothelial genotypic switch. In contrast, knockdown of the RA-induced gene COUP-TFII prevented the formation of networks in Matrigel but had no effect on VE-cadherin induction or cell fusion. Two pan-kinase inhibitors markedly blocked RA-induced VE-cadherin expression and cell fusion. However, RA treatment resulted in a marked and broad reduction in tyrosine kinase activity. Several genes in the TGFβ signaling pathway were induced by RA, and specific inhibition of the TGFβ type I receptor blocked both RA-induced VE-cadherin expression and cell fusion. Together these data indicate a role for the TGFβ pathway and COUP-TFII in mediating the endothelial transdifferentiating properties of RA.     

GSK-3α Is a Novel Target of CREB and CREB-GSK-3α Signaling Participates in Cell Viability in Lung Cancer

Overexpression or activation of cyclic AMP-response element-binding protein (CREB) has been known to be involved in several human malignancies, including lung cancer. Genes regulated by CREB have been reported to suppress apoptosis, induce cell proliferation, inflammation, and tumor metastasis. However, the critical target genes of CREB in lung cancer have not been well understood. Here, we identified GSK-3α as one of the CREB target genes which is critical for the viability of lung cancer cells. The CREB knockdown significantly reduced the expression of GSK-3α and the direct binding of CREB on the promoter of GSK3A was identified. Kaplan-Meier analysis with a public database showed a prognostic significance of aberrant GSK-3α expression in lung cancer. Inhibition of GSK-3α suppressed cell viability, colony formation, and tumor growth. For the first time, we demonstrated that GSK-3α is regulated by CREB in lung cancer and is required for the cell viability. These findings implicate CREB-GSK-3α axis as a novel therapeutic target for lung cancer treatment.     

Curcumin and Emodin Down-Regulate TGF-β Signaling Pathway in Human Cervical Cancer Cells

Cervical cancer is the major cause of cancer related deaths in women, especially in developing countries and Human Papilloma Virus infection in conjunction with multiple deregulated signaling pathways leads to cervical carcinogenesis. TGF-β signaling in later stages of cancer is known to induce epithelial to mesenchymal transition promoting tumor growth. Phytochemicals, curcumin and emodin, are effective as chemopreventive and chemotherapeutic compounds against several cancers including cervical cancer. The main objective of this work was to study the effect of curcumin and emodin on TGF-β signaling pathway and its functional relevance to growth, migration and invasion in two cervical cancer cell lines, SiHa and HeLa. Since TGF-β and Wnt/β-catenin signaling pathways are known to cross talk having common downstream targets, we analyzed the effect of TGF-β on β-catenin (an important player in Wnt/β-catenin signaling) and also studied whether curcumin and emodin modulate them. We observed that curcumin and emodin effectively down regulate TGF-β signaling pathway by decreasing the expression of TGF-β Receptor II, P-Smad3 and Smad4, and also counterbalance the tumorigenic effects of TGF-β by inhibiting the TGF-β-induced migration and invasion. Expression of downstream effectors of TGF-β signaling pathway, cyclinD1, p21 and Pin1, was inhibited along with the down regulation of key mesenchymal markers (Snail and Slug) upon curcumin and emodin treatment. Curcumin and emodin were also found to synergistically inhibit cell population and migration in SiHa and HeLa cells. Moreover, we found that TGF-β activates Wnt/β-catenin signaling pathway in HeLa cells, and curcumin and emodin down regulate the pathway by inhibiting β-catenin. Taken together our data provide a mechanistic basis for the use of curcumin and emodin in the treatment of cervical cancer.     

Expression of WNT5A in Idiopathic Pulmonary Fibrosis and Its Control by TGF-β and WNT7B in Human Lung Fibroblasts

The wingless (Wnt) family of signaling ligands contributes significantly to lung development and is highly expressed in patients with usual interstitial pneumonia (UIP). We sought to define the cellular distribution of Wnt5A in the lung tissue of patients with idiopathic pulmonary fibrosis (IPF) and the signaling ligands that control its expression in human lung fibroblasts and IPF myofibroblasts. Tissue sections from 40 patients diagnosed with IPF or UIP were probed for the immunolocalization of Wnt5A. Further, isolated lung fibroblasts from normal or IPF human lungs, adenovirally transduced for the overexpression or silencing of Wnt7B or treated with TGF-β1 or its inhibitor, were analyzed for Wnt5A protein expression. Wnt5A was expressed in IPF lungs by airway and alveolar epithelium, smooth muscle cells, endothelium, and myofibroblasts of fibroblastic foci and throughout the interstitium. Forced overexpression of Wnt7B with or without TGF-β1 treatment significantly increased Wnt5A protein expression in normal human smooth muscle cells and fibroblasts but not in IPF myofibroblasts where Wnt5A was already highly expressed. The results demonstrate a wide distribution of Wnt5A expression in cells of the IPF lung and reveal that it is significantly increased by Wnt7B and TGF-β1, which, in combination, could represent key signaling pathways that modulate the pathogenesis of IPF.     

Expression of TNF-α and IL-1β in splenic dendritic cells and their serum levels in mouse sparganosis

Sparganosis is a tissue invading helminthiasis infecting intermediate hosts, including humans. Strong immune responses are expected to occur in early phases of infection. Thus, we investigated cytokine expressions in splenic dendritic cells and in sera after experimental infection of mice. In splenic dendritic cells, TNF-α and IL-1β expression peaked at week 1 and week 3 post-infection (PI), respectively, and also early phase (week 2 PI) depressed cytokine expression was noticed. Serum IL-1β concentration increased significantly at week 2 PI and peaked at week 6 PI, and that of TNF-α peaked at week 6 PI. These results showed that pro-inflammatory cytokines, TNF-α and IL-1β, are chronologically regulated in mouse sparganosis.     

Transforming Growth Factor β1 (TGF-β1) Enhances Expression of Profibrotic Genes through a Novel Signaling Cascade and MicroRNAs in Renal Mesangial Cells

Increased expression of transforming growth factor-β1 (TGF-β1) in glomerular mesangial cells (MC) augments extracellular matrix accumulation and hypertrophy during the progression of diabetic nephropathy (DN), a debilitating renal complication of diabetes. MicroRNAs (miRNAs) play key roles in the pathogenesis of DN by modulating the actions of TGF-β1 to enhance the expression of profibrotic genes like collagen. In this study, we found a significant decrease in the expression of miR-130b in mouse MC treated with TGF-β1. In parallel, there was a down-regulation in miR-130b host gene 2610318N02RIK (RIK), suggesting host gene-dependent expression of this miRNA. TGF-β receptor 1 (TGF-βR1) was identified as a target of miR-130b. Interestingly, the RIK promoter contains three NF-Y binding sites and was regulated by NF-YC. Furthermore, NF-YC expression was inhibited by TGF-β1, suggesting that a signaling cascade, involving TGF-β1-induced decreases in NF-YC, RIK, and miR-130b, may up-regulate TGF-βR1 to augment expression of TGF-β1 target fibrotic genes. miR-130b was down-regulated, whereas TGF-βR1, as well as the profibrotic genes collagen type IV α 1 (Col4a1), Col12a1, CTGF, and PAI-1 were up-regulated not only in mouse MC treated with TGF-β1 but also in the glomeruli of streptozotocin-injected diabetic mice, supporting in vivo relevance. Together, these results demonstrate a novel miRNA- and host gene-mediated amplifying cascade initiated by TGF-β1 that results in the up-regulation of profibrotic factors, such as TGF-βR1 and collagens associated with the progression of DN.     

Expression of HIF-1α and Its Target Genes in the Nanorana parkeri Heart:Implications for High Altitude Adaptation

Hypoxia-inducible factor 1 alpha(HIF-1α) and its target genes vascular endothelial growth factor(VEGF) and transferrins(TF) play an important role in native endothermic animals' adaptation to the high altitude environments. For ectothermic animals – especially frogs – it remains undetermined whether HIF-1α and its target genes(VEGF and TF) play an important role in high altitude adaptation, too. In this study, we compared the gene sequences and expression of HIF-1α and its target genes(VEGF and TF) between three Nanorana parkeri populations from different altitudes(3008 m a.s.l., 3440 m a.s.l. and 4312 m a.s.l.). We observed that the c DNA sequences of HIF-1A exhibited high sequence similarity(99.38%) among the three altitudinally separated populations; but with increasing altitude, the expression of HIF-1A and its target genes(VEGF and TF) increased significantly. These results indicate that HIF-1α plays an important role in N. parkeri adaptation to the high altitude, similar to its role in endothermic animals.     

PPAR-α Expression Inversely Correlates with Inflammatory Cytokines IL-1β and TNF-α in Aging Rats

Dehydroepiandrosterone (DHEAS) was given the name “fountain of youth” in reference to its beneficial properties in memory, cognition and aging. Cultured cell studies showed that DHEAS may mediate its action by counteracting aging-associated inflammation via PPAR-α activation. In the present study, we demonstrated an age-dependent increase in IL-1β and TNF-α expression in the brain and the spleen of aging rats, while PPAR-α expression was decreased in the spleen of 18 month-old rats. Oral treatment with DHEAS increased PPAR-α mRNA in 3 month-old rats and decreased PPAR-α protein expression in 18 month-old rats in the spleen. In contrast, DHEAS did not alter cytokine expression in spleen and brain of the three age groups. These findings underline a differential role for DHEAS in PPAR-α expression that is age-dependent, and also, that beneficial effects of DHEAS on cognitive function are unlikely mediated by a decrease in cytokine expression.     

Mitotic Arrest and Apoptosis in Breast Cancer Cells Induced by Origanum majorana Extract: Upregulation of TNF-α and Downregulation of Survivin and Mutant p53

Background

In the present study, we investigated the effect of Origanum majorana ethanolic extract on the survival of the highly proliferative and invasive triple-negative p53 mutant breast cancer cell line MDA-MB-231.

Results

We found that O. majorana extract (OME) was able to inhibit the viability of the MDA-MB-231 cells in a time- and concentration-dependent manner. The effect of OME on cellular viability was further confirmed by the inhibition of colony growth. We showed, depending on the concentration used, that OME elicited different effects on the MDA-MB 231 cells. Concentrations of 150 and 300 µg/mL induced an accumulation of apoptotic–resistant population of cells arrested in mitotis and overexpressing the cyclin-dependent kinase inhibitor, p21 and the inhibitor of apoptosis, survivin. On the other hand, higher concentrations of OME (450 and 600 µg/mL) triggered a massive apoptosis through the extrinsic pathway, including the activation of tumor necrosis factor-α (TNF-α), caspase 8, caspase 3, and cleavage of PARP, downregulation of survivin as well as depletion of the mutant p53 in MDA-MB-231 cells. Furthermore, OME induced an upregulation of γ-H2AX, a marker of double strand DNA breaks and an overall histone H3 and H4 hyperacetylation.

Conclusion

Our findings provide strong evidence that O. majorana may be a promising chemopreventive and therapeutic candidate against cancer especially for highly invasive triple negative p53 mutant breast cancer; thus validating its complementary and alternative medicinal use.     

Leptin Increases TNF-α Expression and Production through Phospholipase D1 in Raw 264.7 Cells

Epidemiological evidence suggests that obesity is associated with inflammation of the respiratory tract and the pathogenesis of asthma. The purpose of this study was to examine the role of phospholipase D1 (PLD1) in leptin-induced expression of the proinflammatory cytokine, tumor necrosis factor (TNF)-α, and to suggest a molecular link between obesity and respiratory tract inflammation. We investigated whether leptin, a typical adipocytokine, plays a role in the expression of TNF-α through increased PLD1 activity in Raw 264.7. Leptin enhanced the activity of PLD1 through activation of PLCγ and Src, while PLD1 siRNA decreased the leptin-induced expression and production of TNF-α. Leptin-induced PLD activation was also inhibited by a PLCγ inhibitor (PAO) and Src kinase inhibitor (PP2), indicating that PLCγ and Src kinase are upstream activators of PLD1. Down-regulation of PLD1 also completely blocked activation of p70S6K, an activator of JNK. Leptin-induced expression of TNF-α was also prevented by inhibition of p70S6K and JNK. Taken together, these results indicate that PLD1 acts as an important regulator of leptin-induced expression of TNF-α by participating in the PLCγ/Src/PLD1/PA/p70S6K/JNK pathway.