1,25-Dihydroxyvitamin D₃ suppresses inflammation-induced expression of plasminogen activator inhibitor-1 by blocking nuclear factor-κB activation

Plasminogen activator inhibitor (PAI)-1 is a major fibrinolytic inhibitor. High PAI-1 is associated with increased renal and cardiovascular disease risk. Previous studies demonstrated PAI-1 down-regulation by 1,25-dihydroxyvitamin D? (1,25(OH)?D?), but the molecular mechanism remains unknown. Here we show that exposure of mouse embryonic fibroblasts to TNFα or LPS led to a marked induction of PAI-1, which was blunted by 1,25(OH)?D?, NF-κB inhibitor or p65 siRNA, suggesting the involvement of NF-κB in 1,25(OH)?D?-induced repression. In mouse Pai-1 promoter a putative cis-κB element was identified at -299. EMSA and ChIP assays showed that TNF-α increased p50/p65 binding to this κB site, which was disrupted by 1,25(OH)?D?. Luciferase reporter assays showed that PAI-1 promoter activity was induced by TNFα or LPS, and the induction was blocked by 1,25(OH)?D?. Mutation of the κB site blunted TNFα, LPS or 1,25(OH)?D? effects. 1,25(OH)?D? blocked IκBα degradation and arrested p50/p65 nuclear translocation. In mice LPS stimulated PAI-1 expression in the heart and macrophages, and the stimulation was blunted by pre-treatment with a vitamin D analog. Together these data demonstrate that 1,25(OH)?D? down-regulates PAI-1 by blocking NF-κB activation. Inhibition of PAI-1 production may contribute to the reno- and cardio-protective effects of vitamin D.     

1,25-dihydroxyvitamin D3 impairs NF-κB activation in human naïve B cells

1α,25-dihydroxyvitamin D₃ (calcitriol), the bioactive metabolite of vitamin D, modulates the activation and inhibits IgE production of anti-CD40 and IL-4 stimulated human peripheral B cells. Engagement of CD40 results in NF-κB p50 activation, which is essential for the class switch to IgE. Herein, we investigated by which mechanism calcitriol modulates NF-κB mediated activation of human naïve B cells. Naïve B cells were predominantly targeted by calcitriol in comparison with memory B cells as shown by pronounced induction of the VDR target gene cyp24a1. Vitamin D receptor activation resulted in a strongly reduced p105/p50 protein and mRNA expression in human naïve B cells. This effect is mediated by impaired nuclear translocation of p65 and consequently reduced binding of p65 to its binding site in the p105 promoter. Our data indicate that the vitamin D receptor reduces NF-κB activation by interference with NF-κB p65 and p105. Thus, the vitamin D receptor inhibits costimulatory signal transduction in naïve B cells, namely by reducing CD40 signaling.     

Forkhead transcription factor FoxO1 inhibits insulin- and transforming growth factor-β-stimulated plasminogen activator inhibitor-1 expression

Elevated levels of plasminogen activator inhibitor-1 (PAI-1) are considered a risk factor for chronic liver disease in patients with hyperinsulinemia. Insulin increases the expression of PAI-1, and inactivates the forkhead box-containing protein FoxO1. We were interested in whether the inactivation of FoxO1 is involved in the activation of PAI-1 expression under conditions of insulin stimulation. Here, we examined whether adenoviral-mediated expression of a constitutively active form of FoxO1 (Ad-CA-FoxO1) inhibited insulin-stimulated PAI-1 expression in human HepG2 hepatocellular liver carcinoma cells and mouse AML12 hepatocytes. Treatment of cells with insulin increased PAI-1 gene expression, and this effect was abolished by Ad-CA-FoxO1. Insulin also increased the transforming growth factor (TGF)-β-induced expression of PAI-1 mRNA, and Ad-CA-FoxO1 inhibited this effect. Transient transfection assays using a reporter gene under the control of the PAI-1 promoter revealed that CA-FoxO1 inhibits Smad3-stimulated PAI-1 promoter activity. Taken together, our results indicate that FoxO1 inhibits PAI-1 expression through the inhibition of TGF-β/Smad-mediated signaling pathways. Our data also suggest that in the hyperinsulinemic state, FoxO1 is inactivated by increased levels of insulin, and does not function as an inhibitor of TGF-β-induced PAI-1 expression.     

Transforming growth factor-β1 suppresses hepatitis B virus replication by the reduction of hepatocyte nuclear factor-4α expression

Several studies have demonstrated that cytokine-mediated noncytopathic suppression of hepatitis B virus (HBV) replication may provide an alternative therapeutic strategy for the treatment of chronic hepatitis B infection. In our previous study, we showed that transforming growth factor-beta1 (TGF-β1) could effectively suppress HBV replication at physiological concentrations. Here, we provide more evidence that TGF-β1 specifically diminishes HBV core promoter activity, which subsequently results in a reduction in the level of viral pregenomic RNA (pgRNA), core protein (HBc), nucleocapsid, and consequently suppresses HBV replication. The hepatocyte nuclear factor 4alpha (HNF-4α) binding element(s) within the HBV core promoter region was characterized to be responsive for the inhibitory effect of TGF-β1 on HBV regulation. Furthermore, we found that TGF-β1 treatment significantly repressed HNF-4α expression at both mRNA and protein levels. We demonstrated that RNAi-mediated depletion of HNF-4α was sufficient to reduce HBc synthesis as TGF-β1 did. Prevention of HNF-4α degradation by treating with proteasome inhibitor MG132 also prevented the inhibitory effect of TGF-β1. Finally, we confirmed that HBV replication could be rescued by ectopic expression of HNF-4α in TGF-β1-treated cells. Our data clarify the mechanism by which TGF-β1 suppresses HBV replication, primarily through modulating the expression of HNF-4α gene.     

Cepharanthine suppresses osteoclast formation by modulating the nuclear factor-κB and nuclear factor of activated T-cell signaling pathways

The increased activation of osteoclasts is the major manifestation of several lytic bone diseases, including osteoporosis, rheumatoid arthritis, aseptic loosening of orthopedic implants, Paget disease and malignant bone diseases. One important bone-protective therapy in these diseases focuses on the inhibition of osteoclast differentiation and resorptive function. Given that the deleterious side-effects of currently available drugs, it is beneficial to search for effective and safe medications from natural compounds. Cepharanthine (CEP) is a compound extracted from Stephania japonica and has been found to have antioxidant and anti-inflammatory effects. In this study, we found that CEP inhibited receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast formation and bone-resorbing activities using osteoclastogenesis and bone resorption assay. By polymerase chain reaction, we also found that CEP inhibited the expression of osteoclast-differentiation marker genes including Ctsk, Calcr, Atp6v0d2, Mmp9 and Nfatc1. Mechanistic analyses including Western blot and luciferase reporter assay revealed that CEP inhibited RANKL-induced activation of NF-κB and nuclear factor of activated T-cell, which are essential for the formation of osteoclast. Collectively, these data suggested that CEP can potentially be used as an alternative therapy for preventing or treating osteolytic diseases.     

Aristolochic acid downregulates monocytic matrix metalloproteinase-9 by inhibiting nuclear factor-κB activation

Aristolochic acid (AA)-associated nephropathy was described as being characterized by a rapid progressive enhancement of interstitial renal fibrosis. Renal tissue fibrosis occurs because of an imbalance of extracellular matrix (ECM) accumulation and matrix metalloproteinase (MMP) activation. Much evidence indicates that inflammatory renal disease including monocyte and mesangial interactions is linked to the development and progression of renal remodeling. In this study, we found that AA showed concentration-dependent inhibition of tumor necrosis factor (TNF)-α-induced MMP-9 activation with an IC₅₀ value of 6.4 ± 0.5 μM in human monocytic THP-1 cells. A similar effect was also noted with different ratios of AAs (types I and II). However, AA had no inhibitory effect on the intact enzymatic activity of MMP-9 at a concentration of 20 μM. On the other hand, the level of tissue inhibitor of metalloproteinase (TIMP)-1 was not induced by AA, but it suppressed TNF-α-induced MMP-9 protein and messenger RNA expressions. AA also significantly inhibited TNF-α-induced IκBα degradation. Furthermore, an electrophoretic mobility shift assay and a reported gene study, respectively, revealed that AA inhibited TNF-α-induced NF-κB translocation and activation. In addition, compared to other NF-κB inhibitors, AA exerted significant inhibition of MMP-9 activation and monocyte chemotactic protein-1-directed invasion. From these results, we concluded that AA, a natural compound, inhibits TNF-α-induced MMP-9 in human monocytic cells possibly through the NF-κB signal pathway. These results also imply that AA may be involved in alteration of matrix homeostasis during renal fibrosis in vivo.     

1α,25-Dihydroxyvitamin D3; its role for homeostasis of keratinocytes

Epidermal homeostasis is influenced by a number of hormones and regulative growth factors that are maintained by a tightly regulated balance between cell proliferation, cell differentiation, and cell death. Vitamin D is one of those regulatory factors. It has recently been demonstrated that 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃) takes part in the regulation of the cell cycle by multiple and complex functions. This review discusses 1α,25(OH)₂D₃ and its analogues in connection with the renewal of epidermal keratinocytes as well as the molecular mechanisms underlying terminal differentiation or rather programmed cell death. Furthermore, interest is focused on the possible clinical application of vitamin D₃ analogues.     

1α,25-Dihydroxyvitamin D3 induces differentiation of human myeloid leukemia cells

A human myeloid leukemia cell line [HL-60] could be induced to differentiate into mature myeloid cells by 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃], the active form of vitamin D₃. At 10^?10–10^?8 M, 1α,25(OH)₂D₃ suppressed cell growth in a dose-dependent manner and markedly induced phagocytosis and C3 rosette formation. The potency of 1α,25(OH)₂D₃ in inducing differentiation was nearly equivalent to that of known synthetic inducers such as dimethyl sulfoxide, actinomycin D or a phorbol ester (12-o-tetra-decanoyl-phorbol-13-acetate). These results clearly indicate that 1α,25(OH)₂D₃, besides its well known biological effect in enhancing intestinal calcium transport and bone mineral mobilization activities, is involved in the cell grwoth and differentiation of HL-60 cells.     

Palmitate-induced PTP1B expression is mediated by ceramide-JNK and nuclear factor κB (NF-κB) activation

Palmitate induces PTP1B expression in skeletal muscle cells. The purpose of this study was to investigate the mechanisms responsible for palmitate-induced PTP1B expression in mouse skeletal muscle cells. Three truncated fragments of PTP1B promoter were cloned into PGL3-basic vector and the promoter activity of PTP1B was assessed in C2C12 cells exposed to palmitate either in the presence or in the absence of several inhibitors to study the biochemical pathways involved. EMSA was performed to examine binding of NF-κB to NF-κB consensus sequence and PTP1B oligonucelotides in the cells treated with palmitate. Lentiviral PTP1B-shRNA was used to knockdown PTP1B in myotubes. The phosphorylation and protein levels of IRS-1 and Akt were detected by western blot. 0.5mM palmitate induced PTP1B promoter activity in fragment -1715/+59 by 50% (p<0.01). Palmitate increased NF-κB binding to both NF-κB consensus sequence and one NF-κB sequence (-920 to -935) in PTP1B promoter. Incubation of C2C12 cells with different concentrations of C2-ceramide enhanced PTP1B promoter activity dose-dependently. Inhibitors of de novo ceramide synthesis prevented palmitate-induced PTP1B promoter activity in myotubes. In addition, inhibitor of JNK pathway prevented ceramide-induced PTP1B promoter activity in myotubes. Knockdown of PTP1B also prevented ceramide-reduced IRS-1 and Akt phosphorylations in the myotubes. Exposure of the cells to PMA and calphostin C, an inhibitor of PKC, did not affect the activity of PTP1B promoter. Our data provide the evidence that the mechanism by which palmitate increased the expression of PTP1B seems to be through a mechanism involving the activation of ceramide-JNK and NF-κB pathways.     

Decoy receptor 3 suppresses TLR2-mediated B cell activation by targeting NF-κB

Decoy receptor 3 (DcR3) is a soluble protein in the TNFR superfamily. Its known ligands include Fas ligand, homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes, TNF-like molecule 1A, and heparan sulfate proteoglycans. DcR3 has been reported to modulate the functions of T cells, dendritic cells, and macrophages; however, its role in regulating B cell activation is largely unknown. In this study, we found that the DcR3.Fc fusion protein bound to human and mouse B cells and suppressed the activation of B cells. DcR3.Fc attenuated Staphylococcus aureus, IgM-, Pam(3)CSK(4)-, and LPS-mediated B cell proliferation but did not affect cytokine-induced B cell growth. In the presence of these mitogens, DcR3.Fc did not induce B cell apoptosis, suggesting that DcR3 may inhibit the signal(s) important for B cell activation. Because the combination of Fas.Fc, LT-βR.Fc (homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes receptor), and DR3.Fc (TNF-like molecule 1A receptor) did not suppress B cell proliferation and because the biological effect of DcR3.Fc on B cells was not blocked by heparin, we hypothesize that a novel ligand(s) of DcR3 mediates its inhibitory activity on B cells. Moreover, we found that TLR2-stimulated NF-κB p65 activation and NF-κB-driven luciferase activity were attenuated by DcR3.Fc. The TLR2-induced cytokine production by B cells was consistently reduced by DcR3. These results imply that DcR3 may regulate B cell activation by suppressing the activation of NF-κB.     

Effect of quercetin on Vibrio cholerae induced nuclear factor-κB activation and interleukin-8 expression in intestinal epithelial cells

Vibrio cholerae, the etiological agent of cholera, colonizes the small intestine, produces an enterotoxin and causes acute inflammatory response at intestinal epithelial cell surface. Pretreatment of intestinal epithelial cells with quercetin reduces the level of V. cholerae induced IL-8 in dose and time dependent manner as determined by ELISA and RT-PCR. Immunofluorescence studies showed that quercetin suppresses the translocation of p50 subunit of NF-κB. In vivo, quercetin administration produced a significant reduction of neutrophil infiltration in the intestinal epithelial layer of suckling mouse. Taken together, quercetin could inhibit the V. cholerae induced inflammation and may therefore find use in management of V. cholerae induced pathogenesis.     

1α,25-Dihydroxyvitamin D3 affects hormone production and expression of steroidogenic enzymes in human adrenocortical NCI-H295R cells

The current study presents data indicating that 1α,25-dihydroxyvitamin D₃ affects the production of hormones and expression of crucial steroidogenic enzymes in the human adrenocortical cell line NCI-H295R. This cell line is widely used as a model for adrenal steroidogenesis. Treatment of the cells with 1α,25-dihydroxyvitamin D₃ suppressed the levels of corticosterone, aldosterone, DHEA, DHEA-sulfate and androstenedione in the culture medium. In order to study the mechanisms behind this suppression of hormone production, we investigated the effects of 1α,25-dihydroxyvitamin D₃ on important genes and enzymes controlling the biosynthesis of adrenal hormones. The mRNA levels were decreased for CYP21A2 while they were increased for CYP11A1 and CYP17A1. No significant changes were observed in mRNA for CYP11B1, CYP11B2 or 3β-hydroxysteroid dehydrogenase (3βHSD). In similarity with the effects on mRNA levels, also the endogenous enzyme activity of CYP21A2 decreased after treatment with 1α,25-dihydroxyvitamin D₃. Interestingly, the two CYP17A1-mediated activities were influenced reciprocally — the 17α-hydroxylase activity increased whereas the 17,20-lyase activity decreased. The current data indicate that the 1α,25-dihydroxyvitamin D₃-mediated decrease in corticosterone and androgen production is due to suppression of the 21-hydroxylase activity by CYP21A2 and the 17,20-lyase activity by CYP17A1, respectively. In conclusion, the current study reports novel findings on 1α,25-dihydroxyvitamin D₃-mediated effects on hormone production and regulation of genes and enzymes involved in steroidogenesis in the adrenocortical NCI-H295R cell line, a model for human adrenal cortex.     

Casuarinin suppresses TNF-α-induced ICAM-1 expression via blockade of NF-κB activation in HaCaT cells

Hippophae rhamnoides has been extensively used in oriental traditional medicines for treatment of asthma, skin diseases, gastric ulcers, and lung disorders. In this study, we isolated casuarinin from the leaves of H.rhamnoides and examined the effect of casuarinin on the TNF-α-induced ICAM-1 expression in a human keratinocytes cell line HaCaT. Pretreatment with casuarinin inhibited TNF-α-induced protein and mRNA expression of ICAM-1 and subsequent monocyte adhesiveness in HaCaT cells. Casuarinin significantly inhibited TNF-α-induced NF-κB activation. In addition, casuarinin inhibited activation of ERK and p38 MAPK in a dose-dependent manner. Furthermore, pretreatment with casuarinin decreased TNF-α-induced pro-inflammatory mediators, such as IL-1β, IL-6, IL-8, and MCP-1. These results demonstrated that casuarinin exerts its anti-inflammatory activity by suppressing TNF-α-induced expression of ICAM-1 and pro-inflammatory cytokines/chemokines via blockage of activation of NF-κB and ERK/p38 MAPK and can be used as a therapeutic agent against inflammatory skin diseases.     

Casuarinin suppresses TNF-α-induced ICAM-1 expression via blockade of NF-κB activation in HaCaT cells

The GRB2 associated binder 1 (GAB1) is an essential docking/adaptor protein for transmitting intracellular signals of the MET tyrosine kinase receptor activated by hepatocyte growth factor/scatter factor (HGF/SF). We found that in response to hours of HGF/SF treatment, the GAB1 protein level is degraded by a mechanism involving MET activity and the proteasomal machinery. We also showed that GAB1 is both multi- and poly-ubiquitinated in a CBL-dependent manner. A long term exposure to HGF/SF caused a more sustained down-regulation of GAB1 than of MET, associated with a loss of reactivation of the ERK MAP kinases to subsequent acute ligand treatment. These data demonstrate that GAB1 is ubiquitinated by CBL and degraded by the proteasome, and plays a role in negative-feedback regulation of HGF/SF–MET signaling.     

Synthesis and bioassay of 3-deoxy-1α-hydroxyvitamin D3, an active analog of 1α,25-dihydroxyvitamin D3

Two synthetic routes to 3-deoxy-1α-hydroxyvitamin D₃, an analog of 1α,25-dihydroxyvitamin D₃, are described. One involved the six-step conversion of 1α,2α-epoxy-6,6-ethylenedioxy-5α-cholestan-3- one to 1α-acetoxycholest-5-ene, whereas, in the second, the same intermediate was prepared from 1α-hydroxycholesterol. Conversion of the Δ⁵-sterol to the required 5,7-diene was accomplished most efficiently via 7-keto and 7-tosylhydrazone intermediates. Bioassay of 3-deoxy-1α-hydroxyvitamin D₃ in the rat establishes that the analog can fulfill all common vitamin D functions including stimulation of intestinal calcium transport, mobilization of calcium and phosphate from bone, stimulation of growth, and calcification of bone. Direct comparison indicates the compound to have $\text{1}\text{20}$ to $\text{1}\text{50}$ of the activity of 1α-hydroxyvitamin D₃, but it acts with a time course indistinguishable from the latter.