Prodomains regulate the synthesis, extracellular localisation and activity of TGF-β superfamily ligands

All transforming growth factor-β (TGF-β) ligands are synthesised as precursor molecules consisting of a signal peptide, an N-terminal prodomain and a C-terminal mature domain. During synthesis, prodomains interact non-covalently with mature domains, maintaining the molecules in a conformation competent for dimerisation. Dimeric precursors are cleaved by proprotein convertases, and TGF-β ligands are secreted from the cell non-covalently associated with their prodomains. Extracellularly, prodomains localise TGF-β ligands within the vicinity of their target cells via interactions with extracellular matrix proteins, including fibrillin and perlecan. For some family members (TGF-β1, TGF-β2, TGF-β3, myostatin, GDF-11 and BMP-10), prodomains bind with high enough affinity to suppress biological activity. The subsequent mechanism of activation of these latent TGF-β ligands varies according to cell type and context, but all activating mechanisms directly target prodomains. Thus, prodomains control many aspects of TGF-β superfamily biology, and alterations in prodomain function are often associated with disease.     

Prodomains regulate the synthesis,extracellular localisation and activity of TGF-β superfamily ligands

All transforming growth factor-β (TGF-β) ligands are synthesised as precursor molecules consisting of a signal peptide, an N-terminal prodomain and a C-terminal mature domain. During synthesis, prodomains interact non-covalently with mature domains, maintaining the molecules in a conformation competent for dimerisation. Dimeric precursors are cleaved by proprotein convertases, and TGF-β ligands are secreted from the cell non-covalently associated with their prodomains. Extracellularly, prodomains localise TGF-β ligands within the vicinity of their target cells via interactions with extracellular matrix proteins, including fibrillin and perlecan. For some family members (TGF-β1, TGF-β2, TGF-β3, myostatin, GDF-11 and BMP-10), prodomains bind with high enough affinity to suppress biological activity. The subsequent mechanism of activation of these latent TGF-β ligands varies according to cell type and context, but all activating mechanisms directly target prodomains. Thus, prodomains control many aspects of TGF-β superfamily biology, and alterations in prodomain function are often associated with disease.     

Differential regulation of TNFα, IL-1β, IL-6, IL-8, TNFβ, and IL-10 by pentoxifylline

Pentoxifylline (PTX) is a methylxanthine drug known to inhibit the production of tumor necrosis factor-alpha (TNFα), which plays a key role in inflammation. Recent studies also revealed that other cytokines may be inhibited by PTX. We investigated PTX effects on production and mRNA expression of TNFα, IL-1β, IL-6, IL-8, TNFβ and IL-10. Cytokine release was studied in 1/10 diluted whole blood culture (WB) and in peripheral blood mononuclear cell (PBMC) culture. Cytokine production was triggered in both culture systems by endotoxin (LPS) or by phorbol ester (PMA) plus phytohemagglutinin (PHA). Our results showed that expression and production of TNFα and TNFβ were inhibited by PTX in a dose-dependent manner. Moreover, we observed that depending on the way of activating cells, PTX induced an up- or a down-regulation (in PMA+PHA or LPS stimulated cells, respectively) for IL-1 and IL-6 release. We also noted that the effects of PTX on IL-6, IL-8 and IL-10 production were different in WB and in PBMC culture. In conclusion PTX acts on cytokine in a complex manner depending on cellular environment and on the method of activation.     

Roles of TNF-α, GSK-3β and RANKL in the occurrence and development of diabetic osteoporosis

Objective: To investigate the roles of TNF-α, GSK-3β and RANKL in the occurrence and development of diabetic osteoporosis. Methods: Diabetic rat model was established; tissue section technology was used to observe the situation of osteoporosis in diabetic rats; rat serum levels of OC, RANKL, GSK-3β, P38mapk, TNF-α and INS were detected by Elisa assay; osteoblasts and osteoclasts were primarily cultured and identified by immunohistochemistry and tartrate-resistant acid phosphatase (TRAP) staining respectively. The effects of GSK-3β inhibitors, lithium chloride, TNF-α antagonists and RANKL antagonists on the proliferation of osteoblasts and osteoclasts were evaluated; quantitative PCR was used to assess the effects of GSK-3β inhibitors, lithium chloride, on TNF-α and RANKL gene expression in osteoblasts and osteoclasts, and the effects of TNF-α and RANKL antagonists on GSK-3β gene expression in osteoblasts and osteoclasts. Results: Diabetic rat model was successfully established; osteoblasts and osteoclasts were successfully isolated and cultured. Elisa experiments showed that in diabetic model group, the levels of RANKL, GSK-3β, P38mapk and TNF-α were significantly increased, while the levels of osteocalcin (OC) and insulin (INS) were significantly reduced; MTT results showed that osteoclast proliferation in GSK-3β inhibitor and lithium chloride groups were weaker than the untreated group, while osteoclast proliferation in TNF-α antagonist group and RANKL antagonist Group was very close to the untreated group. Osteoblast proliferation in GSK-3β inhibitor and lithium chloride groups were weaker than the untreated group, while osteoblast proliferation in TNF-α antagonist group and RANKL antagonist group was higher than the untreated group. In all of the corresponding groups, cell proliferation in the diabetic group was stronger than the untreated group. In GSK-3β inhibitor and lithium oxide groups, TNF-α and RANKL gene expression levels were elevated, but TNF-α and RANKL gene expression levels in the diabetic group were slightly lower than the control group. GSK-3β gene expression level in TNF-α antagonist group and RANKL antagonist group was reduced; GSK-3β gene expression level in diabetic group was lower than the control group. Conclusion: In diabetic rats, TNF-α, GSK-3β and RANKL levels were elevated; GSK-3β could promote the proliferation of osteoblasts and osteoclasts, and inhibit the expression of TNF-α and RANKL; TNF-α and RANKL can suppress the proliferation of osteoblasts while had little effect on osteoclast proliferation; they also can promote the GSK-3β gene expression; interactions between the three broke the balance between osteoblasts and osteoclasts, leading to osteoporosis.     

Coordinated age-dependent and pancreatic-specific expression of mouse Reg2Reg3α, and Reg3β genes

Reg family proteins such as Reg1 and islet neogenesis-associated protein (INGAP) have long been implicated in the growth and/or neogenesis of pancreatic islet cells. Recent reports further suggest similar roles to be played by new members such as Reg2, Reg3α, and Reg3β. We have studied their age-, isoform-, and tissue-specific expressions. RNA and protein were isolated from C57BL/6 mice aged 7, 30, and 90 days. Using real-time polymerase chain reaction, the levels of Reg gene expression in the pancreas were 20-600-fold higher than that in other tissues (?duodenum>stomach>liver); gene expression of Reg2, Reg3α, and Reg3β was age dependent as it was hardly detectable at day 7, increased drastically at day 30, and significantly decreased at day 90; the levels of pancreatic proteins displayed similar age-dependent variations. Using dual-labeled immunofluorescence, Reg2, Reg3α, and Reg3β were abundantly expressed in most acinar cells of the pancreas, in contrast to INGAP which exhibited stepwise increases from day 7 to day 90 and colocalized with the α-cells. These new Reg genes were mainly expressed in the pancreas, with clear age-dependent and isoform-specific patterns.     

Coordinated age-dependent and pancreatic-specific expression of mouse Reg2Reg3α, and Reg3β genes

Reg family proteins such as Reg1 and islet neogenesis-associated protein (INGAP) have long been implicated in the growth and/or neogenesis of pancreatic islet cells. Recent reports further suggest similar roles to be played by new members such as Reg2, Reg3α, and Reg3β. We have studied their age-, isoform-, and tissue-specific expressions. RNA and protein were isolated from C57BL/6 mice aged 7, 30, and 90 days. Using real-time polymerase chain reaction, the levels of Reg gene expression in the pancreas were 20–600-fold higher than that in other tissues (?duodenum>stomach>liver); gene expression of Reg2, Reg3α, and Reg3β was age dependent as it was hardly detectable at day 7, increased drastically at day 30, and significantly decreased at day 90; the levels of pancreatic proteins displayed similar age-dependent variations. Using dual-labeled immunofluorescence, Reg2, Reg3α, and Reg3β were abundantly expressed in most acinar cells of the pancreas, in contrast to INGAP which exhibited stepwise increases from day 7 to day 90 and colocalized with the α-cells. These new Reg genes were mainly expressed in the pancreas, with clear age-dependent and isoform-specific patterns.     

Investigation of immunohistochemical ERα, ERβ and ERβcx expressions in normal and neoplastic breast tissues

Estrogen receptor alpha (ERα) is a well-established prognostic marker in breast cancer. The role of estrogen receptor beta (ERβ) in breast cancers is still under investigation. We aimed to investigate the clinicopathological significance and immunohistochemical expression patterns of ERα, total ERβ (ERβ) and its spliced variant ERβcx in normal breast, ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). Our study population comprised 10 normal breasts, 26 DCISs and 44 IDCs. Immunohistochemical expression of these markers was investigated in sections of formalin-fixed, paraffin-embedded blocks by 2 observers. In invasive ductal carcinomas, ERβ expression had a significant positive correlation with ERα expression (p=0.013), while ERβcx expression was significantly associated with the presence of lymphovascular invasion (p=0.046). There was a significant relationship between ERα expression and low histological grade (p<0.0001). Similarly, ERα+/ERβ+ tumors (p=0.004) and ERα+/ERβcx+ tumors (p=0.008) were significantly associated with low histological grade, too. ERα expression (p=0.009), ERβcx expression (p=0.048) and ERα+/ERβ+ coexpression (p=0.002) increased significantly in progression from normal breast to invasive ductal carcinoma. Expression of ERα correlates with less aggressive phenotypic features, and ERβ expression is positively correlated with ERα expression in breast cancer. ERβcx is associated with aggressive features and can take part in the progression of invasive carcinoma. Increase in ERα+/ERβ+ coexpression, ERα expression and ERβcx expression in breast cancer progression indicates an enhancement in ER expressions or an alteration in expression patterns of different ER variants during mammary carcinogenesis.     

Mechanism of crosstalk inhibition of IL-6 signaling in response to LPS and TNFα

Negative regulation of cytokine signaling is critical for the generation of the appropriate cellular outcome in response to signals, and can be modulated by other concomitant extracellular stimuli (“crosstalk”). Using both genetic and pharmacological manipulations we have investigated the mechanisms by which the pro-inflammatory stimuli, lipopolysaccharide (LPS) and Tumor necrosis factor α (TNFα), negatively regulate interleukin-6 (IL-6) signaling in primary mouse macrophages. Analysis of suppressor of cytokine signalling 3 (SOCS3)-deficient macrophages reveal that SOCS3 is necessary but surprisingly, not sufficient for the complete crosstalk inhibition of IL-6 signaling induced by LPS and TNFα. Analysis of macrophages from gp130 (Y757F) mutant mice suggest that SH2 domain-containing tyrosine phosphatase (SHP2) activity does not explain the residual inhibitory effect of these pro-inflammatory stimuli. In addition, p38 mitogen-activated protein kinase (p38) activation also negatively regulates IL-6 signaling independent of its parallel and necessary action to induce SOCS3 expression. Finally, we have identified an additional, novel mechanism of crosstalk inhibition: a reduction in total cellular levels of gp130 following stimulation with LPS and TNFα.     

Dysfunction of microvascular endothelial cells induced by TNFαand its molecular mechanism

Dysfunction of microvascular endothelial cells induced by TNFαand its molecular mechanism     

Umbelliprenin induced production of IFN-γ and TNF-α, and reduced IL-10, IL-4, Foxp3 and TGF-β in a mouse model of lung cancer

Umbelliprenin is a member of the 7-prenyloxycoumarins with potential therapeutic properties such as cytotoxic effects on various cancer cells. The present study investigates the effect of umbelliprenin on predominance of Th1 and Th2 responses in Lewis lung cancer (LLC) mouse model. The cytotoxic effect of umbelliprenin was explored on LLC cells and mouse splenocytes by MTT assay. Mice into which LLC had been transplanted were treated with umbelliprenin on alternate days, at 2.5?mg/200?µl intraperitoneally. Foxp3, TNF-α and TGF-β mRNA expressions were assessed in tumor and lung tissues of LLC mice. In addition, IL-10, IFN-γ and IL-4 levels were determined in sera and also in splenocyte culture supernatants at the presence of tumor cell lysate (10?µg/ml) and Con A (3?µg/ml) after 72?h. Results showed the cytotoxic effects of umbelliprenin on LLC cells (IC₅₀?=?51.6?±?5.4?µM) while no adverse effect was seen at this concentration on normal splenocytes. TNF-α mRNA expression in both lung and tumor tissues was increased. However, Foxp3 and TGF-β expressions were decreased in tumor tissues. Serum level of IFN-γ was elevated in the umbelliprenin treated cancerous mice compared to the control group while IL-10 and IL-4 secretions were reduced. Tumor size was also decreased in umbelliprenin treated group. In summary, umbelliprenin has shown a partially Th1 bias with a reduction of regulatory immune response. Although the mechanism behind this action is not known, it is speculated that upon changing the Th1/Th2 balance in favour of Th1, umbelliprenin induces its antitumor activity.     

Differential Regulation of IL-8 by IL-1β and TNFα in Hyaline Membrane Disease

Mechanisms that regulate cytokine-mediated inflammation in the lungs of preterm infants, including factors which regulate production of the chemokine IL-8, remain poorly defined. Sequential bronchoalveolar lavage samples were obtained from preterm newborns with hyaline membrane disease over a 28-day period. Bronchoalveolar lavage cell cytokine relationships were evaluated and the differential regulation of IL-8 by IL-1 and TNF was studied in a short-term culture system. In vivo, IL-8 and IL-l protein levels correlated closely with each other and with macrophage counts. In cell culture, exogenous anti-IL-1 antibody led to a 40% maximum inhibition (approximately) of IL-8 production by lipopolysaccharide stimulated lung inflammatory cells. Comparable amounts of exogenous anti-TNF antibodies achieved a 15% maximum inhibition (approximately) of IL-8 production. Anti-IL-1 and anti-TNF antibodies in combination did not inhibit IL-8 production beyond that achieved by anti-IL-l antibody alone. These results, in preterm newborns, support the concept of lung inflammation mediated in part by a macrophage, IL-1, and IL-8 cell cytokine pathway. The results also suggest that factors other than IL-1 and TNF regulate IL-8 expression in the lungs of preterm infants.     

Immunohistochemical localization and biological activity of 3β-hydroxysteroid dehydrogenase and 5α-reductase in the brain of the frog,Rana esculenta,during development

The occurrence of several enzymes responsible for the biosynthesis of neurosteroids in the brain of adult frogs is now firmly established but the expression of these enzymes during ontogenesis has not yet been investigated. In the present report, we describe the immunohistochemical distribution and biological activity of 3β-hydroxysteroid dehydrogenase (3β-HSD) and 5α-reductase (5α-R) in the brain of the European green frog, Rana esculenta, during larval development. The spatio-temporal distribution of 3β-HSD and 5α-R immunoreactivities in the tadpole brain was generally different, although these two enzymes were occasionally detected in the same areas such as the olfactory bulbs and cerebellum. Identification of neurons based on their morphological aspect as well as labeling of astrocytes with an antiserum against glial fibrillary acidic protein (GFAP) revealed that, in the tadpole brain, 3β-HSD- and 5α-R-immunoreactive materials were contained in both neurons and glial cells. Incubation of tadpole brain explants with [³H]-pregnenolone resulted in the formation of several tritiated steroids including progesterone, 17-hydroxyprogesterone, androstenedione, 5α-dihydroprogesterone and 5α-dihydrotestosterone. The present study provides the first immunocytochemical mapping of two key steroidogenic enzymes in the developing frog brain. The data also indicate that neurosteroid biosynthesis occurs in the brain of tadpoles, as previously shown for adult amphibians, birds and mammals. The transient expression of steroidogenic enzymes in several regions of the tadpole brain suggests that, in amphibians, neurosteroids may be implicated in neurotrophic activities during larval development.     

TNFα, IL-1α and bFGF are Implicated in the Complex Disease of GM-CSF Transgenic Mice

Abstract

Transgenic mice aberrantly expressing the granulocyte-macrophage colony stimulating factor (GM-CSF) gene develop an unusual syndrome of blindness, tissue damage and wasting which is associated with accumulations of hemopoietic cells. In order to further characterize this disease state, we have used messenger RNA detection techniques to show that the genes for rumor necrosis factor (TNFα), interleukin-1α (IL-1α) and basic fibroblast growth factor (bFGF) are expressed at abnormally high levels in both macrophages and granulocytes in transgenic mice. Furthermore, since these cell types also express the GM-CSF transgene, it is likely that they are autocrine stimulated by GM-CSF. These observations raise the possibilities that, first, the expression of tumor necrosis factor α, interleukin-1α and basic fibroblast growth factor in hemopoietic cells is a direct consequence of their autostimulation by GM-CSF, and second, that these cytokines may be responsible for some aspects of the transgenic mouse disease.     

Is physical activity a part of who I am? A review and meta-analysis of identity,schema and physical activity

Two parallel literatures on the physical activity (PA) identity and schema constructs have the potential to supplement traditional social cognitive approaches used for PA promotion. The purpose of this paper was to review schema/identity research and appraise its relationship with PA via meta-analysis followed by thematic analyses of its correlates, as well as its proposed mechanisms on PA. Eligible studies were from English, peer-reviewed published articles that examined identity and/or schema in the context of PA. Searches were completed in June 2015 in five databases. Sixty-two independent data-sets (32 available for meta-analysis), primarily of modest quality, were identified. Results of the random effects meta-analysis showed that the point-estimate between identity/schema and behaviour was r?=?.44 (CI?=?.39?.48), and invariant to selected study characteristics. Thematic review showed that identity/schema was associated with commitment, ability, affective judgments, identified/integrated regulation and social comparison and predicted intention, self-regulatory efficacy, and self-regulation strategy use. It had reliable evidence as a moderator of the intention–behavior relationship, was associated with increases in the speed of processing of relevant information and created negative affect under hypothetical identity–behavior discrepant situations. While this initial research is promising, more rigorous research designs, including interventions to increase identity/schema, are warranted.