El-Sayed Akool

Nitric Oxide Increases the Decay of Matrix Metalloproteinase 9 mRNA by Inhibiting the Expression of mRNA-Stabilizing Factor HuR

ABSTRACT Dysregulation of extracellular matrix turnover is an important feature of many inflammatory processes. Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin-1 beta. We demonstrate that NO does strongly destabilize MMP-9 mRNA, since different luciferase reporter gene constructs containing the MMP-9 3′ untranslated region (UTR) displayed significant reduced luciferase activity in response to the presence of NO. Moreover, by use of an in vitro degradation assay we found that the cytoplasmic fractions of NO-treated cells contained a higher capacity to degrade MMP-9 transcripts than those obtained from control cells. An RNA electrophoretic mobility shift assay demonstrated that three of four putative AU-rich elements present in the 3′ UTR of MMP-9 were constitutively occupied by the mRNA-stabilizing factor HuR and that the RNA binding was strongly attenuated by the presence of NO. The addition of recombinant glutathione transferase-HuR prevented the rapid decay of MMP-9 mRNA, whereas the addition of a neutralizing anti-HuR antibody caused an acceleration of MMP-9 mRNA degradation. Furthermore, the expression of HuR mRNA and protein was significantly reduced by exogenously and endogenously produced NO. These inhibitory effects were mimicked by the cGMP analog 8-bromo-cGMP and reversed by LY-83583, an inhibitor of soluble guanylyl cyclase. These results demonstrate that NO acts in a cGMP-dependent mechanism to inhibit the expression level of HuR, thereby reducing the stability of MMP-9 mRNA.

Posttranslational Modification of the AU-Rich Element Binding Protein HuR by Protein Kinase Cδ Elicits Angiotensin II-Induced Stabilization and Nuclear Export of Cyclooxygenase 2 mRNA

ABSTRACT 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 Cδ (PKCδ), which physically interacts with nuclear HuR, thereby promoting its phosphorylation. Mapping of phosphorylation sites identified serines 221 and 318 as critical target sites for PKCδ-triggered HuR phosphorylation and AngII-induced HuR export to the cytoplasm. Posttranslational modification of HuR by PKCδ represents an important novel mode of HuR activation implied in renal COX-2 regulation.

ATP Potentiates Interleukin-1β-induced MMP-9 Expression in Mesangial Cells via Recruitment of the ELAV Protein HuR

Renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin (IL)-1β. We demonstrate here that the stable ATP analog adenosine 5′-O-(thiotriphosphate) (ATPγS) potently amplifies the cytokine-induced gelatinolytic content of mesangial cells mainly by an increase in the MMP-9 steady-state mRNA level. A Luciferase reporter gene containing 1.3 kb of the MMP-9 5′-promoter region showed weak responses to ATPγS but confered a strong ATP-dependent increase in Luciferase activity when under the additional control of the 3′-untranslated region of MMP-9. By in vitro degradation assay and actinomycin D experiments we found that ATPγS potently delayed the decay of MMP-9 mRNA. Gel-shift and supershift assays demonstrated that three AU-rich elements (AREs) present in the 3′-untranslated region of MMP-9 are constitutively bound by complexes containing the mRNA stabilizing factor HuR. The RNA binding of these complexes was markedly increased by ATPγS. Mutation of each ARE element strongly impaired the RNA binding of the HuR containing complexes. Reporter gene assays revealed that mutation of one ARE did not affect the stimulatory effects by ATPγS, but mutation of all three ARE motifs caused a loss of ATP-dependent increase in luciferase activity without affecting IL-1β-inducibility. By confocal microscopy we demonstrate that ATPγS increased the nucleo cytoplasmic shuttling of HuR and caused an increase in the cytosolic HuR level as shown by cell fractionation experiments. Together, our results indicate that the amplification of MMP-9 expression by extracellular ATP is triggered through mechanisms that likely involve a HuR-dependent rise in MMP-9 mRNA stability.

Inhibition of Cytokine-induced Matrix Metalloproteinase 9 Expression by Peroxisome Proliferator-activated Receptor α Agonists Is Indirect and Due to a NO-mediated Reduction of mRNA Stability

Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin 1β (IL-1β). We tested whether ligands of the peroxisome proliferator-activated receptor (PPARα) could influence the cytokine-induced expression of MMP-9. Different PPARα agonists dose-dependently inhibited the IL-1β-triggered increase in gelatinolytic activity mainly by decreasing the MMP-9 steady-state mRNA levels. PPARα agonists on their own had no effects on MMP-9 mRNA levels and gelatinolytic activity. Surprisingly, the reduction of MMP-9 mRNA levels by PPARα activators contrasted with an amplification of cytokine-mediated MMP-9 gene promoter activity and mRNA expression. The potentiation of MMP-9 promoter activity functionally depends on an upstream peroxisome proliferator-responsive element-like binding site, which displayed an increased DNA binding of a PPARα immunopositive complex. In contrast, the IL-1β-induced DNA-binding of nuclear factor κB was significantly impaired by PPARα agonists. Most interestingly, in the presence of an inducible nitric-oxide synthase (iNOS) inhibitor, the PPARα-mediated suppression switched to a strong amplification of IL-1β-triggered MMP-9 mRNA expression. Concomitantly, activators of PPARα potentiated the cytokine-induced iNOS expression. Using actinomycin D, we found that NO, but not PPARα activators, strongly reduced the stability of MMP-9 mRNA. In contrast, the stability of MMP-9 protein was not affected by PPARα activators. In summary, our data suggest that the inhibitory effects of PPARα agonists on cytokine-induced MMP-9 expression are indirect and primarily due to a superinduction of iNOS with high levels of NO reducing the half-life of MMP-9 mRNA.

Molecular Mechanisms of TGFβ 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 TGFβ. Herein we demonstrate that the CNI cyclosporin A and tacrolimus (FK506), independent of TGFβ synthesis, rapidly activate TGFβ/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 TGFβ receptors in CNI-triggered Smad signaling and fibrogenic gene expression, respectively. Mechanistically, CNI cause a rapid activation of latent TGFβ, 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 TGFβ-RII and TGFβ 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 TGFβ 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 TGFβ and thereby initiate the canonical Smad pathway by simultaneously activating p38 MAPK, which both synergistically induce Smad-driven gene expression.

Rapamycin induces the TGFβ1/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 PKCδ 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 PKCδ. Modulation of MMP‐9 by NO highlights the complex roles of NO in the regulation of MMP‐9 in breast cancer cells. J. Cell. Physiol. 219: 276–287, 2009.

Molecular Mechanisms of Cyclosporin A Inhibition of the Cytokine-Induced Matrix Metalloproteinase-9 in Glomerular Mesangial Cells

The effects of the immunosuppressants cyclosporin A (CsA) and tacrolimus (FK506) on the IL-1beta-induced matrix metalloproteinase-9 (MMP-9) were investigated. Impairment of the protease-antiprotease balance contributes to renal fibrosis, which is observed collectively under long-term treatment with either immunosuppressant. It is demonstrated that CsA, in contrast to FK506, reduced the IL-1beta-induced MMP-9 content in conditioned media of mesangial cells, which coincides with a reduction in the cytokine-induced MMP-9 mRNA level. Similar to FK506, the VIVIT peptide, a specific inhibitor of the nuclear factor of activated T cells, did not affect the cytokine-induced MMP-9 level. Moreover, CsA caused a dose-dependent inhibition on the IL-1beta-induced luciferase activity of a 1.3-kb MMP-9 promoter fragment. Concomitant, electrophoretic mobility shift assay revealed that CsA selectively inhibits the cytokine-induced DNA binding of activator protein-1 and NF-kappaB. The effects on NF-kappaB binding were accompanied by a marked reduction in the nuclear content of the p65 subunit of NF-kappaB. Accordingly, CsA specifically impaired the IL-1beta-triggered degradation of inhibitory NF-kappaB. The suppressive effects by CsA on MMP-9 expression were accompanied by a reduction in the cytokine-induced phosphorylation of p42/p44 and c-Jun N-terminal Kinase (JNK). It is interesting that only the JNK inhibitor SP600125 impaired the cytokine-triggered MMP-9 level, suggesting that CsA, via inhibition of the JNK pathway, negatively interferes with the NF-kappaB-dependent transcriptional control of MMP-9. Interference with MMP-9 transcription may account for the accumulation of extracellular matrix underlying the high fibrotic potential of CsA during anti-inflammatory therapies with calcineurin inhibitors.

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 β (TGFβ)/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.

The dsRNA-mimetic poly (I:C) and IL-18 synergize for IFNγ and TNFα 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.