mRNA and protein expression of transcription factor c—fos in burned rats and their effects on wound healing

Objective:To explore the expression of mRNA and its protein in burned rats and their effects of burn wound healing.Methods:A partial-thickness burn of 30% total body surface area was created on the back of 40 Wistar rats.In situ hybridization and immunohistochemical methods were used to exaluate the location and the amount of the c-fos mRNA and its protein in normal skin and the burned skin,respectively,at 3h,6h,1d,3d,7d and 14d after burn.Results:Under a light microscope,both the expression of c-for mRNA and its protein could be found in the normal skin,but their induction levels were much higher in the burned skin.The level of for protein expression reached peak at 3h after burn while that of c-for mRNA reached peak at 6h after burn.Conclusions:The expression of c-fos can be induced by burns.And the peak level expression of c-for mRNA comes later than that of c-fos protein.It indicates that the action of fos protein is induced by post-translational modification of pre-existing fos molecules.     

Role of the renin angiotensin system in TNF-α and Shiga-toxin-induced tissue factor expression

Current evidence implicates a prothrombotic state in the development of Shiga-toxin (Stx)-mediated hemolytic uremic syndrome (HUS). We recently reported that Stx modulates procoagulant activity by enhancing functional tissue factor (TF) activity on cytokine-activated human glomerular endothelial cells (HGECs). Since angiotensin II (Ang II), the key effector of the renin angiotensin system (RAS), has been shown to increase TF expression in vascular tissue, we examined the possible involvement of Ang II in TF expression in HGECs. HGECs were exposed to tumor necrosis factor (TNF)-α ± Stx-1 ± Ang II. Exogenous Ang II significantly increased TF activity and TF mRNA in TNF-α- ± Stx-1-activated HGECs. This increase was mediated via Ang II type I receptor (AT₁R), as losartan, an AT₁R inhibitor, attenuated Ang-II-induced TF activity. To study the effect of endogenous Ang II in TF expression by TNF-α ± Stx-1, HGECs were incubated with losartan or an AT₂R inhibitor (PD 123319) or an angiotensin-converting enzyme inhibitor (enalapril). Losartan but not PD 123319 decreased TF activity induced by TNF-α ± Stx-1 (P < 0.05). Enalapril, also, dose dependently, downregulated TF expression in HGECs exposed to TNF-α ± Stx-1 (P < 0.05). AT₁R mRNA was upregulated in TNF-α- ± Stx-1-activated HGECs (P < 0.05). These data indicate that TF expression in TNF-α- and Stx-1-activated HGECs is enhanced by exogenous Ang II and that endogenous Ang II production may be upregulated by TNF-α ± Stx-1. Hence, local RAS activation may be important in the development of the thrombotic microangiopathy observed in HUS. This paper was presented in part as an abstract at the American Society of Nephrology, 38 Annual Meeting and Scientific Exposition, Philadelphia, PA, USA, November 2005 (Journal of the American Society of Nephrology 2005; 16 Supplement November: PO327) and at the Pediatric Academic Societies’ Annual Meeting, San Francisco, CA, USA, May 2006 (E-PAS 2006:59: 2830.32). This work was supported by National Institutes of Health grants HL 33095, DK 71253 (EFG) and DK 58950 (JRI).     

TNF-α decreases expression of Thrombomudulin and endothelial protein C receptor in human endothelial cells

Introduction. Thrombomodulin (TM) and endothelial protein C receptor (EPCR) are intrinsic anticoagulant factors of normal endothelial cells. The objective of this study was to determine the effects of TNF-α on TM and EPCR gene expression in human endothelial cells as well as the effect of a spice and coloring food compound curcumin as a potential therapeutic agent. Methods. Human umbilical vein endothelial cells (HUVECs) were cultured without or with TNF-α (2.0 ng/ml) for different times. The expression of TM and EPCR was determined by real-time RT-PCR, Western blot, and ELISA. Dose-dependent study of TNF-α in 6 h culture was also performed. Blocking effect of anti-inflammatory agent curcumin (5, 10, and 25 μM) on TNF-α was determined. Results. HUVECs treated with TNF-α (2.0 ng/ml) reduced TM mRNA levels by 80, 97, 94, and 97% at 3, 6, 12, and 24 h, respectively (P < 0.01). Dose-dependent study showed that TM mRNA levels of HUVECs were decreased by 86, 89, 92, 91, and 94% after treatment of TNF-α (0, 0.25, 0.5, 1, 2, and 4 ng/ml) for 6 h, respectively (P < 0.01). TM protein levels were significantly reduced by 69% in TNF-α-treated cells as compared to controls (P < 0.01). Secreted protein and activity of TM in the supernatant of HUVEC cultures were also significantly reduced after TNF-α treatment. In addition, EPCR mRNA and protein levels were significantly reduced in TNF-α-treated group as compared to controls (P < 0.05). Furthermore, these effects were observed in other types of endothelial cells from human coronary arteries, lungs, and skins. Curcumin effectively blocked these effects of TNF-α on down regulation of TM and EPCR. Conclusions. These data demonstrate that TNF-α significantly decreases expression of TM and EPCR at both mRNA and protein levels in several human endothelial cells. Curcumin can effectively block TNF-α-induced endothelial dysfunction. This study suggests a new molecular mechanism of inflammation associated thrombosis and a new strategy to prevent this problem.     

Reduced trabecular bone mass and strength in mice overexpressing Gα11 protein in cells of the osteoblast lineage

G protein-coupled receptors (GPCRs) require G proteins for intracellular signaling to regulate a variety of growth and maintenance processes, including osteogenesis and bone turnover. Bone maintenance events may be altered by changes in the activity or level of G proteins, which then modify signaling in bone cells such as osteoblasts. We have previously reported increased levels of Gα₁₁ protein and signaling to phospholipase C/protein kinase C pathways in response to dexamethasone in osteoblastic UMR 106-01 cells. Here we generated pOBCol3.6-GNA11 transgenic mice that overexpress Gα₁₁ protein in cells of the osteoblast lineage (G₁₁-Tg mice). G₁₁-Tg mice exhibit an osteopenic phenotype characterized by significant reductions in trabecular bone mineral density, thickness, number and strength. The numbers of osteoblasts and osteocytes were unchanged in G₁₁-Tg bone, but early markers of osteoblast differentiation, Alp and Bsp, were increased while the late stage differentiation marker Ocn was not changed suggesting reduced osteoblast maturation in G₁₁-Tg trabecular bone which was accompanied by a decreased bone formation rate. Furthermore, in vitro cultures of G₁₁-Tg primary osteoblasts show delayed osteoblast differentiation and mineralization. Histological analyses also revealed increased osteoclast parameters, accompanied by elevated mRNA expression of Trap and Ctsk. mRNA levels of Rankl and M-csf were elevated in vitro in bone marrow stromal cells undergoing osteogenesis and in trabecular bone in vivo. Together, these findings demonstrate that increasing Gα₁₁ protein expression in osteoblasts can alter gene expression and result in a dual mechanism of trabecular bone loss.     

The association between retraction of the torn rotator cuff and increasing expression of hypoxia inducible factor 1α and vascular endothelial growth factor expression: an immunohistological study

Background  

Differing levels of tendon retraction are found in full-thickness rotator cuff tears. The pathophysiology of tendon degeneration and retraction is unclear. Neoangiogenesis in tendon parenchyma indicates degeneration. Hypoxia inducible factor 1α (HIF) and vascular endothelial growth factor (VEGF) are important inducers of neoangiogenesis. Rotator cuff tendons rupture leads to fatty muscle infiltration (FI) and muscle atrophy (MA). The aim of this study is to clarify the relationship between HIF and VEGF expression, neoangiogenesis, FI, and MA in tendon retraction found in full-thickness rotator cuff tears.     

Relations of transcription expression of IL—2 with nuclear factor of activated T cells as well as changes of C—Fos and C—Jun after trauma

Objective:To observe the relations among expression of interleukin-2(IL-2)in spleen lymphocytes,DNA binding activity of nuclear factor of activated T cells(NFAT)and expression of the partly family members C-Fos,C-Jun after trauma.Methods:A murine closed trauma model was used,animals were sacrificed6,12hours and 1,4,7,10,14days,respectively after injury,Spleen lymplocytes were isolated from injured mice and stimulated with concanavalin-A,The culture supernatants were harvested and assayed for IL-2activity,Total RNA was extracted from spleen lymphocytes and assayed for IL-2mRNA.Nuclear protein was extracted,and the DNA binding activity of NFAT was measured using an electrophoretic mobility shift assay(EMSA),the expressions of C-Fos,C-Jun protein determined by Western blot analysis.Results:The expressions of IL-2 activity and IL-2mRNA in spleen lymphocytes were decreased in injured mice compared with those in control mice,and the most obvious decrease appeared on the 4th day after injury,The DNA binding activity of NFAT decreased gradually and reached the minimum that was only41%of the control on the 4th day after injury,which was cloely associated with the decline of IL-2activity and IL-2mRNA.An decrease in the expression ofC-Fos on the lst and 4th day after injury,trauma had no significant effect on the C-Jun expression.Conclusions:These results suggest that the inhibition of IL-2 expression is partly due to the impairment in the activation of NFAT in injured mice;and the decline in the DNA binding activity of NFAT is partly due to trauma block in the C-Fos expression.     

Increased sensitivity of rheumatoid synoviocytes to Schnurri-3 expression in TNF-α and IL-17A induced osteoblastic differentiation

ObjectiveTo compare the effects of TNF-α and IL-17A on osteogenic differentiation of isolated fibroblast-like synoviocytes (FLS) from healthy donors, osteoarthritis (OA) and rheumatoid arthritis (RA) patients.MethodsFLS were cultured in osteogenic medium, with and without TNF-α and/or IL-17A. Extracellular matrix mineralization was evaluated by alizarin red staining and alkaline phosphatase activity (ALP) measurement. mRNA expression was analyzed by qRT-PCR for Wnt5a, BMP2 and Runx2, genes associated with osteogenesis, for DKK1 and RANKL, genes associated with osteogenesis inhibition and Schnurri-3, a new critical gene in the cross talk with osteoclasts. IL-6 and IL-8 production was measured by ELISA.ResultsIn osteogenic medium, matrix mineralization and increased ALP activity indicated that FLS can undergo osteogenic differentiation, which was increased with TNF-α and IL-17A. The expression of osteogenesis activators (BMP2 and Wnt5a) was increased with cytokines and that of the osteogenesis inhibitor DKK1 was decreased. There was no difference between all three cell types. In contrast, RA FLS were particularly sensitive to the synergistic increase of Shn3 with TNF-α and IL-17A. Levels of IL-6 and IL-8 were also higher for RA-FLS, compared to healthy and OA FLS.ConclusionIL-17A and/or TNF-α treatment favor an osteogenesis induction in isolated FLS, independent of their origin. RA-FLS were more sensitive to the synergistic increase of Schnurri-3 expression. Combined with the higher levels of inflammation, this may in turn activate osteoclastogenesis, leading to increased bone destruction seen in destructive arthritis.     

Observed correlation between the expression levels of catalytic subunit,Cβ2, of cyclic adenosine monophosphate–dependent protein kinase and prostate cancer aggressiveness

Background

Today overtreatment of indolent prostate cancers and undertreatment of aggressive prostate cancer are a major concern for patients, their families, and the health care system. New biomarkers distinguishing indolent and aggressive prostate cancer are needed to improve precision medicine. In prostate cancer, protein kinase A (PKA) is known to activate the androgen receptor and published data indicate that PKA subunits can act as predictive markers for response to radiation and chemotherapy. We have previously shown that the catalytic subunit, Cβ2, of PKA is up-regulated in prostate cancer and we would in this study investigate the potential of Cβ2 to become a prognostic biomarker in prostate cancer.

Caffeic acid 3,4-dihydroxy-phenethyl ester suppresses receptor activator of NF-κB ligand–induced osteoclastogenesis and prevents ovariectomy-induced bone loss through inhibition of mitogen-activated protein kinase/activator protein 1 and Ca2+–nuclear factor of activated T-cells cytoplasmic 1 signaling pathways

Receptor activator of NF-κB ligand (RANKL) stimulation leads to the activation of mitogen-activated protein kinase (MAPK)/AP-1 and Ca2+–nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) signaling pathways in osteoclastogenesis. Targeting these pathways has been an encouraging strategy for bone-related diseases, such as postmenopausal osteoporosis. In this study, we examined the effects of caffeic acid 3,4-dihydroxy-phenethyl ester (CADPE) on osteoclastogenesis. In mouse bone marrow monocytes (BMMs) and RAW264.7 cells, CADPE suppressed RANKL-induced osteoclast differentiation and actin-ring formation in a dose-dependent manner within non–growth inhibitory concentrations at the early stage, while CADPE had no effect on macrophage colony-stimulating factor (M-CSF)-induced proliferation and differentiation. At the molecular level, CADPE inhibited RANKL-induced phosphorylation of MAPKs, including extracellular signal-regulated kinases 1/2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK), without significantly affecting the NF-κB signaling pathway. CADPE abrogated RANKL-induced activator protein 1 (AP-1)/FBJ murine osteosarcoma viral oncogene homolog (c-Fos) nuclear translocation and activation. Overexpression of c-Fos prevented the inhibition by CADPE of osteoclast differentiation. Furthermore, CADPE suppressed RANKL-induced the tumor necrosis factor receptor associated factor 6 (TRAF6) interaction with c-src tyrosine kinase (c-Src), blocked RANKL-induced the phosphorylation of protein kinase B (AKT), and inhibited RANKL-induced Ca2+ oscillation. As a result, CADPE decreased osteoclastogenesis-related marker gene expression, including NFATc1, TRAP, cathepsin K, and c-Src. To test the effects of CADPE on osteoclast activity in vivo, we showed that CADPE prevented ovariectomy-induced bone loss by inhibiting osteoclast activity. Together, our data demonstrate that CADPE suppresses osteoclastogenesis and bone loss through inhibiting RANKL-induced MAPKs and Ca2+-NFATc1 signaling pathways. CADPE is a novel agent in the treatment of osteoclast-related diseases, such as osteoporosis.