Sorafenib and edaravone protect against renal fibrosis induced by unilateral ureteral obstruction via inhibition of oxidative stress, inflammation, and RIPK-3/MLKL pathway.

Renal fibrosis is the common endpoint of nearly all chronic and progressive nephropathies. Cell death and sterile inflammation are the main characteristics of renal fibrosis, which can lead to end-stage renal failure. The inflammatory reaction triggered by tissue damage is strongly related to necroptosis, a type of caspase-independent, regulated cell death. Using an animal model of unilateral ureteral obstruction (UUO), the anti-fibrotic effects of sorafenib (SOF), a multi-kinase inhibitor, and edaravone (EDV), a potent antioxidant and free radical scavenger, were examined in rats with obstructive nephropathy. Experimentally, animals were divided randomly into five groups: sham; UUO; UUO + SOF (5 mg/kg/day, P.O.); UUO + EDV (20 mg/kg/day, P.O.); and UUO + SOF + EDV groups. The kidney function biomarkers, oxidant/antioxidant status, renal mRNA expressions of TNF-α, collagen-1α, protein expressions of RIPK-1, RIPK-3, MLKL, caspase-8, HYP, MPO, and TNF-α were all significantly modulated by UUO. Administration of either SOF or EDV significantly attenuated cellular and molecular changes induced by UUO. Also, histopathological changes were improved. Moreover, SOF in combination with EDV, significantly improved UUO-induced renal fibrosis compared with each drug alone. Collectively, administration of either SOF or EDV or both of them significantly attenuated the rats with obstructive nephropathy, possibly by blocking the RIPK-3/MLKL necroptotic pathway and suppressing renal oxidative stress and inflammation.

Molecular mechanisms of extracellular-ATP-mediated colorectal cancer progression: Implication of purinergic receptors-mediated nucleocytoplasmic shuttling of HuR.

One of the leading causes of cancer-related deaths worldwide is colorectal cancer (CRC). Extracellular ATP (e-ATP) and purinergic receptors (P2R) play a central role in CRC proliferation and progression. Human antigen R (HuR) is becoming more and more understood to be essential for the expression of genes linked to cancer. The current study demonstrates that ATP can mediate CRC (Caco-2 cells) progression via induction of HuR nucleocytoplasmic shuttling and subsequent expression of cancer-related genes, a consequence mostly mediated via the P2R receptor. It was also noted that suppression of HuR activity by using dihydrotanshinone I (DHTS) prevents cancer-related gene expression and subsequent CRC (Caco-2 cells) progression induced by ATP. The expression of cyclin A2/cyclin-dependent kinase 2 (CDK2), Bcl-2, ProT-α, hypoxia-inducible factor1-α (HIF1-α), vascular endothelial growth factor A (VEGF-A), transforming growth factor-ß (TGF-ß) and matrix metallopeptidase 9 (MMP-9) induced by ATP were highly reduced in the presence of either PPADS (non-selective P2R antagonist) or DHTS. In addition, e-ATP-induced Caco-2 cell proliferation as well as cell survival were highly reduced in the presence of either PPADS or DHTS or selective CDK-2 inhibitor (Roscovitine) or selective Bcl-2 inhibitor (ABT-263). Furthermore, it was found that MMP-9 is critical for Caco-2 cells migration induced by e-ATP as demonstrated by a clear reduction in cells migration in the presence of a selective MMP-9 inhibitor (Marimastat). Collectively, these data demonstrate that ATP through P2R activation can induce HuR nucleocytoplasmic shuttling that could be translated into an increase in cancer-related genes expression and subsequent, cell proliferation and progression.

P2 X 7 receptor is a critical regulator of extracellular ATP-induced profibrotic genes expression in rat kidney: implication of transforming growth factor-ß/Smad signaling pathway.

This study was designed to investigate the potential of extracellular adenosine 5'-triphosphate (ATP) via the P2 X 7 receptor to activate the renal fibrotic processes in rats. The present study demonstrates that administration of ATP rapidly activated transforming growth factor-ß (TGF-ß) to induce phosphorylation of Smad-2/3. Renal connective tissue growth factor (CTGF) and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA and protein expressions were also increased following ATP administration. A decrease in TGF-ß amount in serum as well as renal Smad-2/3 phosphorylation was noticed in animals pre-treated with the specific antagonist of P2 X 7 receptor, A 438,079. In addition, a significant reduction in mRNA and protein expression of CTGF and TIMP-1were also observed in the kidneys of those animals. Collectively, the current findings demonstrate that ATP has the ability to augment TGF-ß-mediated Smad-2/3 phosphorylation and enhance the expression of the pro-fibrotic genes, CTGF and TIMP-1, an effect that is largely mediated via P2 X 7 receptor.

Hepatoprotective effects of carvedilol and crocin against leflunomide-induced liver injury.

Leflunomide-induced liver injury has been an important problem since its approval. Although, severe cases of leflunomide-induced liver injury leading to hospitalization are rare, the risk is higher with concurrent liver disease or use of other hepatotoxic drugs. The current study was conducted to investigate the potential protective effects of carvedilol and crocin alone and in combination against leflunomide-induced hepatic injury and to clarify the possible mechanism(s) through which carvedilol and crocin may elicit their effects. Fifty male albino mice were allocated into five groups: normal control group, leflunomide group, carvedilol group, crocin group, and combination group. These groups were given vehicle, leflunomide, leflunomide plus carvedilol, leflunomide plus crocin, and leflunomide plus combination of carvedilol and crocin, respectively. The study was conducted for 8 weeks, and different parameters were assessed. The results demonstrated that leflunomide significantly increased the serum levels of AST, ALT, ALP, hepatic MDA, nitrite, mTOR gene, PI3K gene, TGF-ß, and the pathological changes alongside with the significant decrease of serum albumin, total protein, hepatic catalase, and GSH. While the coadministration of carvedilol, crocin and their combination with leflunomide significantly decreased the serum levels of AST, ALT, ALP, hepatic MDA, mTOR gene, PI3K gene, TGF-ß, and the pathological changes alongside with the significant elevation of serum albumin, total protein, hepatic catalase, and GSH. This study is suggesting several solutions for Leflunomide-induced hepatotoxicity demonstrated by the protective effect of the antihypertensive drug carvedilol, the natural product crocin, and their combination which was demonstrated to be superior to each drug alone.

PEG-SOD attenuates the mitogenic ERK1/2 signaling cascade induced by cyclosporin A in the liver and kidney of albino mice.

The calcineurin inhibitor, cyclosporin A (CsA) is one of the most common immunosuppressive agents used in organ transplantation. However, its clinical use is often limited by several unwanted effects including nephrotoxicity and hepatotoxicity. By using immunohistochemical and ELISA techniques, it was found that CsA administration causes a rapid activation of a disintegrin and metalloproteases-17 (ADAM-17), epidermal growth factor receptor (EGFR) and subsequent ERK1/2 phosphorylation in the liver and kidney of albino mice. Furthermore, this study presents mechanistic relevance of this signaling cascade involving reactive oxygen species (ROS)-mediated ADAM-17/EGFR/ERK1/2 activation as indicated by a clear reduction in ADAM-17 and EGFR activities as well as ERK1/2 phosphorylation when the animals pretreated with Polyethylene glycol-superoxide dismutase (PEG-SOD) before CsA administration. Collectively, our findings demonstrate that CsA has the ability to activate ADAM-17-mediated EGFR/ERK1/2 phosphorylation in the liver and kidney of albino mice in ROS-dependent manner. Finally, these data may support the concept of using antioxidant therapy as a valuable approach for the prevention of CsA-induced nephrotoxicity and hepatotoxicity.

Cyclosporin A activates human hepatocellular carcinoma (HepG2 cells) proliferation: implication of EGFR-mediated ERK1/2 signaling pathway.

One of the most common causes of cancer mortality worldwide is hepatocellular carcinoma (HCC). Extracellular signal-regulated kinase (ERK1/2) pathway has been shown to play an important role in the development and progression of HCC. Here, we demonstrate that the immunosuppressive agent cyclosporin A (CsA) has the ability to increase the cellular growth in HCC (HepG2 cells) via activation of ERK1/2 signaling cascade. It was found that ERK1/2 phosphorylation induced by CsA was highly reduced in the presence of the reactive oxygen species (ROS) scavenger polyethylene glycol-superoxide dismutase (PEG-SOD). Furthermore, it was observed that inhibition of metalloproteinase activity using TAPI-2 prevents ERK1/2 activation by CsA. Moreover, a disintegrin and metalloproteinase domain 17 (ADAM-17) activity was found to be critical for ERK phosphorylation by CsA. In addition, CsA-induced ERK phosphorylation was highly reduced in the presence of either neutralizing anti-heparin-binding-epidermal growth factor (HB-EGF) antibody or UO126 (MEK inhibitor). By using the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478, it was found that EGFR is critical for ERK phosphorylation induced by CsA. Furthermore, CsA-induced cell proliferation was strongly reduced in the presence of either PEG-SOD or TAPI-2 or neutralizing anti-ADAM17 antibody or neutralizing anti-HB-EGF antibody or AG1478 or UO126. Collectively, these data demonstrate that CsA has the ability to activate ERK1/2 signaling cascade that could be translated into an increase in HepG2 cell proliferation. Furthermore, these data support the role of ROS, ADAM-17, and EGFR in ERK1/2 signaling activation and subsequent cell proliferation induced by CsA in HepG2 cells.

Vitamin E inhibits cyclosporin A-induced CTGF and TIMP-1 expression by repressing ROS-mediated activation of TGF-ß/Smad signaling pathway in rat liver.

Cyclosporin A (CsA) is the most common immunosuppressive drug used in organ transplantation. However, the clinical use of CsA is often limited by several side effects including hepatotoxicity. In the present study, it was found that administration of CsA causes a rapid activation of TGF-ß/Smad signaling cascade and subsequent expression of the profibrotic genes connective tissue growth factor (CTGF) and tissue inhibitors of matrix metallproteinases-1 (TIMP-1) in rat liver. In addition, Smad phosphorylation and subsequent CTGF and TIMP-1 expression were markedly reduced in the presence of neutralizing monoclonal TGFß1-3 antibody. Furthermore, CsA administration significantly increased the serum levels of the liver enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) as well as lipid peroxidation in hepatic tissues. Moreover, significant reduction in the hepatic content of reduced glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) was observed in CsA-alone-treated animals. Histopathological changes were also observed in CsA-alone-treated rats. Pretreatment of animals with Vitamin E (Vit E) before CsA administration significantly reduced TGF-ß level as well as Smad phosphorylation and subsequent CTGF and TIMP-1 expression. Furthermore, administration of PEG-SOD clearly attenuated TGF-ß/Smad signaling induced by CsA. Moreover, concomitant administration of Vit E along with CsA significantly ameliorated the histopathological changes and improved liver function as well as the antioxidant capacity. Finally, this study shows that the immunosuppressive efficiency of CsA was not altered in the presence of Vit E. These data may support the concept of using antioxidant therapy as a valuable approach for the prevention of CsA-induced tissue fibrosis.

The potential antifibrotic impact of apocynin and alpha-lipoic acid in concanavalin A-induced liver fibrosis in rats: Role of NADPH oxidases 1 and 4.

Liver fibrosis results from chronic inflammation that precipitates excessive accumulation of extracellular matrix. Oxidative stress is involved in its pathogenesis. This study aimed to elucidate the potential antifibrotic effect of the NADPH oxidase (NOX) inhibitor, apocynin against concanavalin A (ConA)-induced immunological model of liver fibrosis, and to investigate the ability of the antioxidant, alpha-lipoic acid (α-LA) to potentiate this effect. Rats were treated with apocynin and/or α-LA for six weeks. Hepatotoxicity indices, oxidative stress, insulin, NOXs, inflammatory and liver fibrosis markers were assessed. Treatment of animals with apocynin and α-LA significantly ameliorated the changes in liver functions and histopathological architecture induced by ConA. Liver fibrosis induced by ConA was evident where alpha-smooth muscle actin and transforming growth factor- beta1 were elevated, which was further confirmed by Masson's trichrome stain and increased hydroxyproline. Co-treatment with apocynin and α-LA significantly reduced their expression. Besides, apocynin and α-LA significantly ameliorated oxidative stress injury evoked by ConA, as evidenced by enhancing reduced glutathione content, antioxidant enzymes activities and decreasing lipid peroxides. ConA induced a significant elevation in serum insulin level and inflammatory markers; tumor necrosis factor-alpha, interleukin-6 and nuclear factor kappa b. Furthermore, the mRNA tissue expression of NOXs 1 and 4 was found to be elevated in the ConA group. All these elevations were significantly reduced by apocynin and α-LA co-treatment. These findings indicate that using apocynin and α-LA in combination possess marked antifibrotic effects, and that NOX enzymes are partially involved in the pathogenesis of ConA-induced liver fibrosis.

Propolis alleviates concanavalin A-induced hepatitis by modulating cytokine secretion and inhibition of reactive oxygen species.

Viral hepatitis-induced oxidative stress accompanied by increased levels of transforming growth factor-ß (TGF-ß) and hepatic fibrosis are hallmarks of hepatitis C virus infection. The present study was designed to investigate the potential protective effect of propolis against liver injury induced by concanavalin A (Con A), a T-cell-dependent model that causes an immune-mediated hepatitis in a similar pattern to the one induced by viral infections. In the present study, rats were randomly divided into four groups. The first group (control) was administered the vehicle of Con A (i.v.) for 24 h. The second group received Con A (12 mg/kg body weight i.v.) for 24 h. The third group received propolis (300 mg/kg by oral gavage) 5 days before and concurrently with Con A for 24 h. The last group received propolis alone. Following a single injection of Con A, histopathological changes as well as significant reduction in albumin level were observed. In addition, serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin were significantly increased. These increases correlated with an increase in lipid peroxidation and downregulation of reduced glutathione (GSH) as well as superoxide dismutase (SOD) and catalase activities in liver tissue. Furthermore, these changes were associated with an increase in serum levels of the inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) as well as the profibrotic cytokine TGF-ß. Moreover, TGF- ß activation was accompanied with an increase in Smad phosphorylation. Interestingly, concomitant administration of propolis along with Con A significantly attenuated all these negative effects and improved liver function indicating that propolis has the ability to protect rats from Con A-induced hepatitis.

All-trans retinoic acid potentiates cisplatin-induced kidney injury in rats: impact of retinoic acid signaling pathway.

Cisplatin (cis-diammine dichloroplatinum (II), CDDP) is a widely used drug for treatment of various types of cancers. However, CDDP-induced nephrotoxicity remains the main dose-limiting side effect. Retinoids are a group of vitamin A-related compounds that exert their effects through retinoid receptors activation. In this study, we investigated the effect of CDDP treatment on retinoic acid receptor-α (RAR-α) and retinoid X receptor-α (RXR-α) expression. In addition, we investigated the possible modulatory effects of RAR agonist, all-trans retinoic acid (ATRA), on CDDP-induced nephrotoxicity. Rats were treated with saline, DMSO, CDDP, ATRA, or CDDP/ATRA. Twenty-four hours after the last ATRA injection, rats were killed; blood samples were collected; kidneys were dissected; and biochemical, immunohistochemical, and histological examinations were performed. Our results revealed that CDDP treatment significantly increased serum levels of creatinine and urea, with concomitant decrease in serum albumin. Moreover, reduced glutathione (GSH) content as well as superoxide dismutase (SOD) and catalase (CAT) activities were significantly reduced with concurrent increase in kidney malondialdehyde (MDA) content following CDDP treatment. Furthermore, CDDP markedly upregulated tubular RAR-α, RXR-α, fibrin, and inducible nitric oxide synthase (iNOS) protein expression. Although administration of ATRA to control rats did not produce marked alterations in kidney function parameters, administration of ATRA to CDDP-treated rats significantly exacerbated CDDP-induced nephrotoxicity. In addition, CDDP/ATRA co-treatment significantly increased RAR-α, RXR-α, fibrin, and iNOS protein expression compared to CDDP alone. In conclusion, we report, for the first time, the crucial role of retinoid receptors in CDDP-induced nephrotoxicity. Moreover, our findings indicate that co-administration of ATRA with CDDP, although beneficial on the therapeutic effects, their deleterious effects on the kidney may limit their clinical use.

Molecular mechanisms of the protective role of wheat germ oil against cyclosporin A-induced hepatotoxicity in rats.

CONTEXT: Cyclosporin A (CsA) is one of the most important immunosuppressive agents. However, its clinical use is strongly limited by several side effects including hepatotoxicity which remains a major clinical problem. Involvement of reactive oxygen species (ROS) in CsA-induced hepatotoxicity has been reported. OBJECTIVE: This study investigates the potential protective role of wheat germ oil (WGO) as an antioxidant against CsA-induced hepatotoxicity. MATERIALS AND METHODS: Twenty-four male Wistar albino rats (six animals in each group) received castor oil, the vehicle of CsA i.p. (control) or either CsA (25 mg/kg/d i.p.), WGO (900 mg/kg/d by oral gavage), or CsA in combination with WGO daily for 21 d. RESULTS: CsA administration significantly increased serum levels of the liver enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST). In addition, an increase in lipid peroxidation, inducible NO-synthase (iNOS), and NF-κB expression were observed in hepatic tissues of CsA-alone-treated rats. Furthermore, significant reduction in the hepatic content of reduced glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) was also observed in CsA-alone-treated animals. Moreover, histopathological changes occurred in CsA-alone-treated rats. Concomitant administration of WGO along with CsA improved all these parameters. Most interestingly, the immunosuppressive effect of CsA was not affected by WGO. CONCLUSION: The present study suggests that concomitant use of WGO might be useful in reducing liver toxicity induced by CsA via inhibition of ROS, iNOS, and NF-κB expression.

Molecular mechanisms of PDGF-AA expression induced by the dsRNA-mimetic poly (I:C) and IL-18.

Several animal studies suggest a role of platelet-derived growth factors (PDGFs) particularly A and B in atherosclerosis. Previously, it has been shown that viral infections have the ability to initiate and accelerate atherosclerosis in animal models. Recently, it has been reported that IL-18 has a pro-atherogenic character. Moreover, viral infections have been shown to be associated with induction of IL-18 bioactivity. By using human predendritic KG1 cells, we sought to assess PDGF-AA production under the influence of IL-18 and the byproduct of viral replication, dsRNA-mimetic poly (I:C). Here we demonstrate that poly (I:C) and IL-18 have the ability to induce PDGF-AA expression. In addition, costimulation of KG-1 cells with both IL-18 plus poly (I:C) shows an additive effect on PDGF-AA production. Furthermore, we demonstrate that neither p38 nor SAPK/JNK is required for PDGF-AA production by both PIC and IL-18. However, the expression of PDGF-AA has been found to be associated with increased activation of NF-κB and enhancement of DNA-binding capacity of NF-κB as shown by electrophoretic mobility shift assay (EMSA) and supershift analysis. Collectively, this study demonstrates that the byproduct of viral replication, dsRNA [poly (I:C)], and IL-18 have the ability to induce PDGF-AA in NF-κB-dependent manner. Furthermore, dsRNA act in an additive way with IL-18 to induce PDGF-AA which plays a major role in atherosclerosis. These data might help to understand the pro-atherogenic character of IL-18 and molecular mechanisms of viral infection-induced atherosclerosis.

Cyclosporin A and tacrolimus induce renal Erk1/2 pathway via ROS-induced and metalloproteinase-dependent EGF-receptor signaling.

We previously demonstrated that the widely used immunosuppressive drugs cyclosporin A (CsA) and tacrolimus (FK506), independent of immunophilin binding, can activate profibrogenic transforming growth factor ß (TGFß)/Smad signaling cascades in rat renal mesangial cells (MC). Here we report that both peptidyl-prolyl cis/trans isomerase (PPIase) inhibitors activate the extracellular-signaling regulated kinase (ERK) a member of the mitogen activated protein kinase (MAPK) and induce a rapid and transient increase in ERK phosphorylation. The MEK inhibitor U0126, the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC), a cell-permeant superoxide dismutase (SOD) and stigmatellin, an inhibitor of mitochondrial cytochrome bc1 complex strongly attenuated the increase in ERK1/2 phosphorylation triggered by PPIase inhibitors. Moreover, neutralizing antibodies against heparin binding-epidermal growth factor (HB-EGF), and inhibition of the EGF receptor by either small interfering (si)RNA or AG1478, demonstrate that ERK activation by both PPIase inhibitors is mediated via HB-EGF-induced EGF receptor (EGFR) tyrosine kinase activation. The strong inhibitory effects achieved by GM6001 and TAPI-2 furthermore implicate the involvement of a desintegrin and metalloproteinase 17 (ADAM17). Concomitantly, the PPIase inhibitor-induced ADAM17 secretase activity was significantly reduced by SOD and stigmatellin thus suggesting that mitochondrial ROS play a primary role in PPIase inhibitor-induced and ADAM17-mediated HB-EGF shedding. Functionally, both immunosuppressants caused a strong increase in MC proliferation which was similarly impeded when cells were treated in the presence of NAC, TAPI-2 or AG1478, respectively. Our data suggest that CsA and FK506, via ROS-dependent and ADAM17-catalyzed HB-EGF shedding induce the mitogenic ERK1/2 signaling cascade in renal MC.

Differential modulation of the cytokine-induced MMP-9/TIMP-1 protease-antiprotease system by the mTOR inhibitor rapamycin.

The mTOR-inhibitor rapamycin is a potent drug used in many immunosuppressive and antiinflammatory therapeutic regimes. In renal transplantation despite its beneficial roles rapamycin in some cases can promote renal fibrosis in the kidney but the underlying mechanisms are unknown. In this study, we tested for possible modulatory effects of rapamycin on the cytokine-triggered matrix metalloproteinase 9 (MMP-9)/tissue inhibitor of metalloproteinase (TIMP)-1 protease-antiprotease system which is critically involved in renal inflammation and fibrosis. Treatment of rat mesangial cells (MC) with rapamycin dose-dependently reduced the interleukin 1ß (IL-1ß)-triggered increase in gelatinolytic levels as demonstrated by zymography. The reduction in the extracellular MMP-9 content by rapamycin coincided with an attenuation in cytokine-induced steady-state MMP-9 mRNA levels. Conversely, rapamycin caused a dose-dependent increase in cytokine-evoked TIMP-1 expression in a Smad binding element (SBE)-dependent manner. Surprisingly, the attenuation of MMP-9 mRNA levels by rapamycin is accompanied by a potentiation of IL-1ß-induced MMP-9 promoter activity in which the stimulatory effects by rapamycin are mainly attributed to a proximal AP-1 binding site. Furthermore, the rapamycin-dependent potentiation of MMP-9 expression is accompanied by an amplification of cytokine-triggered activities of nuclear factor κB (NF-κB) and activator protein 1 (AP-1) transcription factors. Importantly, rapamycin-triggered increase in MMP-9 promoter activity is fully impaired when we used a MMP-9 reporter construct which is under the additional control of the 3' untranslated region (3'-UTR) of MMP-9. Collectively, these data imply that rapamycin inhibits the cytokine-induced MMP-9 mainly through posttranscriptional events and thereby exerts profibrotic activities.

The dsRNA-mimetic poly (I:C) and IL-18 synergize for IFNgamma and TNFalpha expression.

Interleukin (IL)-18 bioactivity and dsRNA sensing by receptors of innate immunity are key components of anti-viral host defense. Despite extensive data on signal transduction activated by both pathways knowledge on cross-communication is incomplete. By using human PBMC and predendritic KG1 cells, as prototypic IL-18-responsive cellular models, we sought to assess cytokine production under the influence of IL-18 and the dsRNA-mimetic poly (I:C). Here, we report on potent synergy between both mediators concerning pro-inflammatory IFNgamma and TNFalpha production. KG1 data revealed that synergistic induction likely relied on TLR3 and was associated with prolonged/increased activation of NF-kappaB, as detected by IkappaB analysis and luciferase reporter assays, respectively. Moreover, extended activation of JNK was mediated by IL-18/poly (I:C). Although vital for innate immunity, overwhelming induction of inflammatory cytokines during viral infections poses the threat of serious collateral tissue damage. The stunning synergism inherent to IL-18/dsRNA-induced TNFalpha/IFNgamma detected herein may contribute to this pathological phenomenon.

Rapamycin induces the TGFbeta1/Smad signaling cascade in renal mesangial cells upstream of mTOR.

The mTOR kinase inhibitor rapamycin (sirolimus) is a drug with potent immunosuppressive and antiproliferative properties. We found that rapamycin induces the TGFbeta/Smad signaling cascade in rat mesangial cells (MC) as depicted by the nuclear translocation of phospho-Smads 2, -3 and Smad-4, respectively. Concomitantly, rapamycin increases the nuclear DNA binding of receptor (R)- and co-Smad proteins to a cognate Smad-binding element (SBE) which in turn causes an increase in profibrotic gene expression as exemplified by the connective tissue growth factor (CTGF) and plasminogen activator inhibitor 1 (PAI-1). Using small interfering (si)RNA we demonstrate that Smad 2/3 activation by rapamycin depends on its endogenous receptor FK binding protein 12 (FKBP12). Mechanistically, Smad induction by rapamycin is initiated by an increase in active TGFbeta(1) as shown by ELISA and by the inhibitory effects of a neutralizing TGFbeta antibody. Using an activin receptor-like kinase (ALK)-5 inhibitor and by siRNA against the TGFbeta type II receptor (TGFbeta-RII) we furthermore demonstrate a functional involvement of both types of TGFbeta receptors. However, rapamycin did not compete with TGFbeta for TGFbeta-receptor binding as found in radioligand-binding assay. Besides SB203580, a specific inhibitor of the p38 MAPK, the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC) and a cell-permeable superoxide dismutase (SOD) mimetic strongly abrogated the stimulatory effects of rapamycin on Smad 2 and 3 phosphorylation. Furthermore, the rapid increase in dichlorofluorescein (DCF) formation implies that rapamycin mainly acts through ROS. In conclusion, activation of the profibrotic TGFbeta/Smad signaling cascade accompanies the immunosuppressive and antiproliferative actions of rapamycin.

Molecular mechanisms of nitric oxide-dependent inhibition of TPA-induced matrix metalloproteinase-9 (MMP-9) in MCF-7 cells.

Matrix metalloproteinase-9 (MMP-9) is implicated in the invasion and metastasis of breast cancer cells. We investigated the modulatory effects of nitric oxide (NO) on the 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced MMP-9 expression in MCF-7 cells. Different chemical NO donors inhibited the extracellular content of TPA-induced MMP-9 protein and MMP-9 activity as assessed by gelatin-zymography and ELISA, respectively. Concomitant with the reduction in the extracellular MMP-9 content NO strongly decreased the steady-state levels of MMP-9 mRNA which in turn leads to a lower recruitment of MMP-9 transcripts to polysomes and to a diminished MMP-9 translation. Reporter gene assays revealed that the inhibition in MMP-9 expression by NO is mainly attributed to a 0.67 kb fragment of the 5'-promoter region of the MMP-9 gene but independent of the 3'untranslated region thus indicating that MMP-9 suppression by NO mainly results from transcriptional events. Electrophoretic mobility shift assays (EMSA), showed that NO specifically interferes with the TPA-induced DNA binding affinity of c-Jun and c-Fos without affecting the TPA-induced increase in the levels of the transcription factors. Using pharmacological inhibitors and small interfering (si)RNA we found that PKCdelta is indispensably involved in the TPA-triggered MMP-9 expression. Concomitantly, the TPA-evoked increase in total PKC activity was strongly attenuated in the lysates from NO-treated MCF-7 cells, thus suggesting that NO attenuates TPA-triggered MMP-9 mainly through a direct inhibition of PKCdelta. Modulation of MMP-9 by NO highlights the complex roles of NO in the regulation of MMP-9 in breast cancer cells.

Molecular mechanisms of TGF beta receptor-triggered signaling cascades rapidly induced by the calcineurin inhibitors cyclosporin A and FK506.

The calcineurin inhibitor (CNI)-induced renal fibrosis is attributed to an exaggerated deposition of extracellular matrix, which is mainly due to an increased expression of TGFbeta. Herein we demonstrate that the CNI cyclosporin A and tacrolimus (FK506), independent of TGFbeta synthesis, rapidly activate TGFbeta/Smad signaling in cultured mesangial cells and in whole kidney samples from CNI-treated rats. By EMSA, we demonstrate increased DNA binding of Smad-2, -3, and -4 to a cognate Smad-binding promoter element (SBE) accompanied by CNI-triggered activation of Smad-dependent expression of tissue inhibitor of metalloprotease-1 (TIMP-1) and connective tissue growth factor. Using an activin receptor-like kinase-5 (ALK-5) inhibitor and by small interfering RNA we depict a critical involvement of both types of TGFbeta receptors in CNI-triggered Smad signaling and fibrogenic gene expression, respectively. Mechanistically, CNI cause a rapid activation of latent TGFbeta, which is prevented in the presence of the antioxidant N-acetyl cysteine. A convergent activation of p38 MAPK is indicated by the partial blockade of CNI-induced Smad-2 activation by SB203580; conversely, both TGFbeta-RII and TGFbeta are critically involved in p38 MAPK activation by CNI. Activation of both signaling pathways is similarly triggered by reactive oxygen species. Finally, we show that neutralization of TGFbeta markedly reduced the CNI-dependent Smad activation in vitro and in vivo. Collectively, this study demonstrates that CNI via reactive oxygen species generation activate latent TGFbeta and thereby initiate the canonical Smad pathway by simultaneously activating p38 MAPK, which both synergistically induce Smad-driven gene expression.

Posttranslational modification of the AU-rich element binding protein HuR by protein kinase Cdelta elicits angiotensin II-induced stabilization and nuclear export of cyclooxygenase 2 mRNA.

The mRNA stabilizing factor HuR is involved in the posttranscriptional regulation of many genes, including that coding for cyclooxygenase 2 (COX-2). Employing RNA interference technology and actinomycin D experiments, we demonstrate that in human mesangial cells (hMC) the amplification of cytokine-induced COX-2 by angiotensin II (AngII) occurs via a HuR-mediated increase of mRNA stability. Using COX-2 promoter constructs with different portions of the 3' untranslated region of COX-2, we found that the increase in COX-2 mRNA stability is attributable to a distal class III type of AU-rich element (ARE). Likewise, the RNA immunoprecipitation assay showed AngII-induced binding of HuR to this ARE. Using the RNA pulldown assay, we demonstrate that the AngII-caused HuR assembly with COX-2 mRNA is found in free and cytoskeleton-bound polysomes indicative of an active RNP complex. Mechanistically, the increased HuR binding to COX-2-ARE by AngII is accompanied by increased nucleocytoplasmic HuR shuttling and depends on protein kinase Cdelta (PKCdelta), which physically interacts with nuclear HuR, thereby promoting its phosphorylation. Mapping of phosphorylation sites identified serines 221 and 318 as critical target sites for PKCdelta-triggered HuR phosphorylation and AngII-induced HuR export to the cytoplasm. Posttranslational modification of HuR by PKCdelta represents an important novel mode of HuR activation implied in renal COX-2 regulation.

Modulation of mRNA stability as a novel therapeutic approach.

During the last decade evidence has accumulated that modulation of mRNA stability plays a central role in cellular homeostasis, including cell differentiation, proliferation and adaptation to external stimuli. The functional relevance of posttranscriptional gene regulation is highlighted by many pathologies, wherein occurrence tightly correlates with a dysregulation in mRNA stability, including chronic inflammation, cardiovascular diseases and cancer. Most commonly, the cis-regulatory elements of mRNA decay are represented by the adenylate- and uridylate (AU)-rich elements (ARE) which are specifically bound by trans-acting RNA binding proteins, which finally determine whether mRNA decay is delayed or facilitated. Regulation of mRNA decay by RNA stabilizing and RNA destabilizing factors is furthermore controlled by different intrinsic and environmental stimuli. The modulation of mRNA binding proteins, therefore, illuminates a promising approach for the pharmacotherapy of those key pathologies mentioned above and characterized by a posttranscriptional dysregulation. Most promisingly, intracellular trafficking of many of the mRNA stability regulating factors is, in turn, regulated by some major signaling pathways, including the mitogen-activated protein kinase (MAPK) cascade, the AMP-activated kinase (AMPK) and the protein kinase (PK) C (PKC) family. In this review, we present timely examples of genes regulated by mRNA stability with a special focus on signaling pathways involved in the ARE-dependent mRNA decay. A better understanding of these processes may form the basis for the development of novel therapeutics to treat major human diseases.