Lymphotoxin α, a novel target of posttranscriptional gene regulation by HuR in HepG2 cells.

The role of the RNA-binding protein human antigen R (HuR) in hepatocarcinogenesis is still elusive. By employing short hairpin (sh)RNA-dependent knockdown approach, we demonstrate that lymphotoxin α (LTα) is a target of posttranscriptional gene regulation by HuR in hepatocellular carcinoma (HepG2) cells. Consequently, the increased mRNA decay upon HuR depletion significantly affects lymphotoxin expression at both, the mRNA and protein level. Biotin-pulldown assay showed that HuR specifically interacts with the 3'-untranslated region (3'-UTR) of the LTα mRNA. Furthermore, electrophoretic mobility shift assay (EMSA) implicates that the RNA-binding critically depends on the RNA recognition motif 2 (RRM2) and the hinge region of HuR.

The cytoskeletal inhibitors latrunculin A and blebbistatin exert antitumorigenic properties in human hepatocellular carcinoma cells by interfering with intracellular HuR trafficking.

The impact of the RNA-binding protein HuR for the post-transcriptional deregulation of tumor-relevant genes is well established. Despite of elevations in HuR expression levels, an increase in cytoplasmic HuR abundance in many cases correlates with a high grade of malignancy. Here, we demonstrated that administration of the actin-depolymerizing macrolide latrunculin A, or blebbistatin, an inhibitor of myosin II ATPase activity, caused a dose- and time-dependent reduction in the high cytoplasmic HuR content of HepG2 and Huh7 hepatocellular carcinoma (HCC) cells. Subcellular fractionation revealed that in addition, both inhibitors strongly attenuated cytoskeletal and membrane-bound HuR abundance and conversely increased the HuR amount in nuclear cell fractions. Concomitant with changes in intracellular HuR localization, both cytoskeletal inhibitors markedly decreased the half-lives of cyclooxygenase-2 (COX-2), cyclin A and cyclin D1 encoding mRNAs resulting in a significant reduction in their expression levels in HepG2 cells. Importantly, a similar reduction in the expression of these HuR targets was achieved by a RNA interference (RNAi)-mediated knockdown of either HuR or nonmuscle myoin IIA. Using polysomal fractionation, we further demonstrate that the decrease in cytoplasmic HuR by latrunculin A or blebbistatin is accompanied by a marked change in the allocation of HuR and its mRNA cargo from polysomes to ribonucleoprotein (RNP) particles. Functionally, the basal migration and prostaglandin E2 synthesis are similarly impaired in inhibitor-treated and stable HuR-knockdown HepG2 cells. Our data demonstrate that interfering with the actomyosin-dependent HuR trafficking may comprise a valid therapeutic option for antagonizing pathologic posttranscriptional gene expression by HuR and furthermore emphasize the potential benefit of HuR inhibitory strategies for treatment of HCC.

Thrombin receptor protease-activated receptor 4 is a key regulator of exaggerated intimal thickening in diabetes mellitus.

BACKGROUND: Diabetes mellitus predisposes to thrombotic and proliferative vascular remodeling, to which thrombin contributes via activation of protease-activated receptor (PAR) 1. However, the use of PAR-1 inhibitors to suppress remodeling may be limited by severe bleeding. We recently reported upregulation of an additional thrombin receptor, PAR-4, in human vascular smooth muscle cells exposed to high glucose and have now examined PAR-4 as a novel mediator linking hyperglycemia, hypercoagulation, and vascular remodeling in diabetes mellitus. METHODS AND RESULTS: PAR-4 expression was increased in carotid atherectomies and saphenous vein specimens from diabetic versus nondiabetic patients and in aorta and carotid arteries from streptozotocin-diabetic versus nondiabetic C57BL/6 mice. Vascular PAR-1 mRNA was not increased in diabetic mice. Ligated carotid arteries from diabetic mice developed more extensive neointimal hyperplasia and showed greater proliferation than arteries from nondiabetic mice. The augmented remodeling response was absent in diabetic mice deficient in PAR-4. At the cellular level, PAR-4 expression was controlled via the mRNA stabilizing actions of human antigen R, which accounted for the stimulatory actions of high glucose, angiotensin II, and H2O2 on PAR-4 expression, whereas cicaprost via protein kinase A activation counteracted this effect. CONCLUSIONS: PAR-4 appears to play a hitherto unsuspected role in diabetic vasculopathy. The development of PAR-4 inhibitors might serve to limit mainly proliferative processes in restenosis-prone diabetic patients, particularly those patients in whom severe bleeding attributed to selective PAR-1 blockade or complete thrombin inhibition must be avoided or those who do not require anticoagulation.

Domain-specific phosphomimetic mutation allows dissection of different protein kinase C (PKC) isotype-triggered activities of the RNA binding protein HuR.

The ubiquitous mRNA binding protein human antigen R (HuR) participates in the post-transcriptional regulation of many AU-rich element (ARE)-bearing mRNAs. Previously, by using in vitro kinase assay, we have identified serines (Ser) 158, 221 and 318 as targets of protein kinase C (PKC)-triggered phosphorylation. In this study, we tested whether GFP- or GST-tagged HuR constructs bearing a phosphomimetic Ser (S)-to-Asp (D) substitution at the different PKC target sites, would affect different HuR functions including HuR nucleo-cytoplasmic redistribution and binding to different types of ARE-containing mRNAs. The phosphomimetic GFP-tagged HuR protein bearing a phosphomimetic substitution in the hinge region of HuR (HuR-S221D) showed an increased cytoplasmic abundance when compared to wild-type HuR. Conversely, data from in vitro kinase assay and electrophoretic mobility shift assay (EMSA), implicates that phosphorylation at Ser 221 is not relevant for mRNA binding of HuR. Quantification of in vitro binding affinities of GST-tagged wild-type HuR and corresponding HuR proteins bearing a phosphomimetic substitution in either RRM2 (HuR-S158D) or in RRM3 (HuR-S318D) by microscale thermophoresis (MST) indicates a specific binding of wild-type HuR to type I, II or type III-ARE-oligonucleotides in the high nanomolar range. Interestingly, phosphomimetic mutation at position 158 or 318 had a negative influence on HuR binding to type I- and type II-ARE-mRNAs whereas it significantly enhanced HuR affinity to a type III-ARE substrate. Our data suggest that differential phosphorylation of HuR by PKCs at different HuR domains coordinates subcellular HuR distribution and leads to a preferential binding to U-rich bearing target mRNA.

RNA-dependent association with myosin IIA promotes F-actin-guided trafficking of the ELAV-like protein HuR to polysomes.

The role of the mRNA-binding protein human antigen R (HuR) in stabilization and translation of AU-rich elements (ARE) containing mRNAs is well established. However, the trafficking of HuR and bound mRNA cargo, which comprises a fundamental requirement for the aforementioned HuR functions is only poorly understood. By administering different cytoskeletal inhibitors, we found that the protein kinase Cδ (PKCδ)-triggered accumulation of cytoplasmic HuR by Angiotensin II (AngII) is an actin-myosin driven process functionally relevant for stabilization of ARE-bearing mRNAs. Furthermore, we show that the AngII-induced recruitment of HuR and its bound mRNA from ribonucleoprotein particles to free and cytoskeleton bound polysomes strongly depended on an intact actomyosin cytoskeleton. In addition, HuR allocation to free and cytoskeletal bound polysomes is highly sensitive toward RNase and PPtase and structurally depends on serine 318 (S318) located within the C-terminal RNA recognition motif (RRM3). Conversely, the trafficking of the phosphomimetic HuRS318D, mimicking HuR phosphorylation at S318 by the PKCδ remained PPtase resistant. Co-immunoprecipitation experiments with truncated HuR proteins revealed that the stimulus-induced association of HuR with myosin IIA is strictly RNA dependent and mediated via the RRM3. Our data implicate a microfilament dependent transport of HuR, which is relevant for stimulus-induced targeting of ARE-bearing mRNAs from translational inactive ribonucleoprotein particles to polysomes.

Regulation of the mRNA-binding protein HuR by posttranslational modification: spotlight on phosphorylation.

The ubiquitous mRNA-binding protein human antigen R (HuR) and its neuronal relatives (HuB, HuC, HuD) participate in the post-transcriptional regulation of many AU-rich element-bearing mRNAs. In addition to its originally described role in controlling mRNA decay, the binding of HuR to target mRNAs can affect many aspects of mRNA processing including splicing, polyadenylation, intracellular trafficking, translation and modulation of mRNA repression by miRNAs. In accordance to the growing list of signalling events which are involved in regulating these different HuR functions, recent data implicate that posttranslational modification, namely protein kinase-triggered phosphorylation of HuR plays a crucial role in connecting extracellular signal inputs to a specific post-transcriptional program by HuR. Notably, in addition to directly targeting HuR functions, posttranslational modifications of HuR have a major impact on the sequestration and binding to various HuR ligand proteins as has been demonstrated e.g. for the 14-3-3 chaperones. However, the detailed mechanisms of how a specific modification of HuR coordinates different aspects in HuR regulation are currently poorly understood. Due to the fact that most of the described HuR activities are closely related to its subcellular localization and the binding to cargo mRNA, this review will focus on these aspects of HuR functions and their control by posttranslational modification, particularly by HuR phosphorylations by different protein kinases.

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.

Ahnak1 is a tuneable modulator of cardiac Ca(v)1.2 calcium channel activity.

Ahnak1 has been implicated in the beta-adrenergic regulation of the cardiac L-type Ca(2+) channel current (I (CaL)) by its binding to the regulatory Cavß(2) subunit. In this study, we addressed the question whether ahnak1/Cavß(2) interactions are essential or redundant for beta-adrenergic stimulation of I (CaL). Three naturally occurring ahnak1 variants (V5075 M, G5242R, and T5796 M) identified by genetic screening of cardiomyopathy patients did essentially not influence the in vitro Cavß(2) interaction as assessed by recombinant proteins. But, we observed a robust increase in Cavß(2) binding by mutating Ala at position 4984 to Pro which creates a PxxP consensus motif in the ahnak1 protein fragment. Surface plasmon resonance measurements revealed that this mutation introduced an additional Cavß(2) binding site. The functionality of A4984P was supported by the specific action of the Pro-containing ahnak1-derived peptide (P4984) in beta-adrenergic regulation of I (CaL). Patch clamp recordings on cardiomyocytes showed that intracellular perfusion of P4984 markedly reduced I (CaL) response to the beta-adrenergic agonist, isoprenaline, while the Ala-containing counterpart failed to affect I (CaL). Interestingly, I (CaL) of ahnak1-deficient cardiomyocytes was not affected by peptide application. Moreover, I (CaL) of ahnak1-deficient cardiomyocytes showed intact beta-adrenergic responsiveness. Similarly isolated ahnak1-deficient mouse hearts responded normally to adrenergic challenge. Our results indicate that ahnak1 is not essential for beta-adrenergic up-regulation of I (CaL) and cardiac contractility in mice. But, tuning ahnak1/Cavß(2) interaction provides a tool for modulating the beta-adrenergic response of I (CaL).

Redox regulation of human protease-activated receptor-2 by activated factor X.

Activated factor X (FXa) exerts coagulation-independent actions such as proliferation of vascular smooth muscle cells (SMCs) through the protease-activated receptors PAR-1 and PAR-2. Both receptors are upregulated upon vascular injury but the underlying mechanisms have not been defined. We examined if FXa regulates PAR-1 and PAR-2 in human vascular SMCs. FXa increased PAR-2 mRNA, protein, and cell-surface expression and augmented PAR-2-mediated mitogenesis. PAR-1 was not influenced. The regulatory action of FXa on PAR-2 was concentration-dependent and mimicked by a PAR-2-selective activating peptide. PAR-2 regulation was not influenced by the thrombin inhibitor argatroban or PAR-1 siRNA. FXa increased dichlorofluorescein diacetate fluorescence and 8-isoprostane formation and induced expression of the NADPH oxidase subunit NOX-1. NOX-1 siRNA prevented FXa-stimulated PAR-2 regulation, as did ebselen and cell-permeative and impermeative forms of catalase. Exogenous H(2)O(2) increased PAR-2 expression and mitogenic activity. FXa promoted nuclear translocation and PAR-2/DNA binding of nuclear factor κB (NF-κB); NF-κB inhibition prevented PAR-2 regulation by FXa. FXa also promoted PAR-2 mRNA stabilization through increased human antigen R (HuR)/PAR-2 mRNA binding and cytoplasmic shuttling. HuR siRNA abolished FXa-stimulated PAR-2 expression. Thus FXa induces functional expression of PAR-2 but not of PAR-1 in human SMCs, independent of thrombin formation, via a mechanism involving NOX-1-containing NADPH oxidase, H(2)O(2), NF-κB, and HuR.

Regulation of human vascular protease-activated receptor-3 through mRNA stabilization and the transcription factor nuclear factor of activated T cells (NFAT).

Thrombin promotes vascular smooth muscle cell (SMC) proliferation and inflammation via protease-activated receptor (PAR)-1. A further thrombin receptor, PAR-3, acts as a PAR-1 cofactor in some cell-types. Unlike PAR-1, PAR-3 is dynamically regulated at the mRNA level in thrombin-stimulated SMC. This study investigated the mechanisms controlling PAR-3 expression. In human vascular SMC, PAR-3 siRNA attenuated thrombin-stimulated interleukin-6 expression and extracellular signal-regulated kinases 1/2 phosphorylation, indicating PAR-3 contributes to net thrombin responses in these cells. Thrombin slowed the decay of PAR-3 but not PAR-1 mRNA in the presence of actinomycin D and induced cytosolic shuttling and PAR-3 mRNA binding of the mRNA-stabilizing protein human antigen R (HuR). HuR siRNA prevented thrombin-induced PAR-3 expression. By contrast, forskolin inhibited HuR shuttling and destabilized PAR-3 mRNA, thus reducing PAR-3 mRNA and protein expression. Other cAMP-elevating agents, including the prostacyclin-mimetic iloprost, also down-regulated PAR-3, accompanied by decreased HuR/PAR-3 mRNA binding. Iloprost-induced suppression of PAR-3 was reversed with a myristoylated inhibitor of protein kinase A and mimicked by phorbol ester, an inducer of cyclooxygenase-2. In separate studies, iloprost attenuated PAR-3 promoter activity and prevented binding of nuclear factor of activated T cells (NFAT2) to the human PAR-3 promoter in a chromatin immunoprecipitation assay. Accordingly, PAR-3 expression was suppressed by the NFAT inhibitor cyclosporine A or NFAT2 siRNA. Thus human PAR-3, unlike PAR-1, is regulated post-transcriptionally via the mRNA-stabilizing factor HuR, whereas transcriptional control involves NFAT2. Through modulation of PAR-3 expression, prostacyclin and NFAT inhibitors may limit proliferative and inflammatory responses to thrombin after vessel injury.

High-constitutive HuR phosphorylation at Ser 318 by PKC{delta} propagates tumor relevant functions in colon carcinoma cells.

Overexpression of the messenger RNA (mRNA)-binding protein HuR is an important feature of many tumors and in most cases correlates with a high-grade malignancy. Since phosphorylation of HuR by protein kinase C δ (PKCδ) at serine (Ser) 318 implies an important mode in HuR regulation, we studied its functional role in dysregulated HuR and related functions in colon carcinoma cells. Coimmunoprecipitation experiments revealed a high-constitutive association of nuclear PKCδ with HuR. Using a phospho-Ser 318-specific HuR antibody, we found a strong increase in nuclear HuR phosphorylation in DLD-1 cells when compared with nontransformed CCD 841 colon epithelial cells. Importantly, a strong increase in HuR phosphorylation at Ser 318 was also found in tissue specimen from human colon carcinomas. Employing ribonucleoprotein-immunoprecipitation, we show that DLD-1 cells displayed a strong and constitutive RNA binding of HuR to cyclooxygenase-2 (COX-2) and cyclin A encoding mRNAs that was strongly impaired by rottlerin, an inhibitor of novel PKCs. Accordingly, rottlerin accelerated the decay of COX-2 and cyclin A encoding mRNAs concomitant with a reduced expression of both genes. Functionally, migration and invasion is similarly impaired in PKCδ- or HuR-small interfering RNA-depleted cells and in tumor cells transfected with a nonphosphorylatable serine-to-alanine 318 HuR construct. Conversely, expression of a phosphomimetic Ser 318 aspartic acid (D) HuR caused a significant increase in migration and proliferation of CCD 841 cells. Our data suggest that the increased HuR phosphorylation at Ser 318 by PKCδ reflects an important regulatory paradigm for aberrant HuR functions and emphasize the antitumorigenic potential of PKCδ inhibitory strategies.

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.

Tandem phosphorylation of serines 221 and 318 by protein kinase Cdelta coordinates mRNA binding and nucleocytoplasmic shuttling of HuR.

Stabilization of mRNA by the ubiquitous RNA binding protein human antigen R (HuR), a member of the embryonic lethal abnormal vision (ELAV) protein family, requires canonical binding to AU-rich element (ARE)-bearing target mRNA and export of nuclear HuR-mRNA complexes to the cytoplasm. In human mesangial cells (HMC) both processes are induced by angiotensin II (AngII) via protein kinase Cdelta (PKCdelta)-triggered serine phosphorylation of HuR. By testing different point-mutated Flag-tagged HuR proteins, we found that Ser 318 within RNA recognition motif 3 (RRM3) is essential for AngII-induced binding to ARE-bearing mRNA but irrelevant for nucleocytoplasmic HuR shuttling. Conversely, mutation at Ser 221 within the HuR hinge region prevents AngII-triggered HuR export without affecting mRNA binding of HuR. Using phosphorylation state-specific antibodies, we found a transient increase in HuR phosphorylation at both serines by AngII. Functionally, PKCdelta mediates the AngII-induced stabilization of prominent HuR target mRNAs, including those of cyclin A, cyclin D(1), and cyclooxygenase-2 (COX-2), and is indispensable for AngII-triggered migration and wound healing of HMC. Our data suggest a regulatory paradigm wherein a simultaneous phosphorylation at different domains by PKCdelta coordinates mRNA binding and nucleocytoplasmic shuttling of HuR, both of which events are essentially involved in the stabilization of HuR target mRNAs and relevant cell functions.

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.

Transforming growth factor-beta2 upregulates sphingosine kinase-1 activity, which in turn attenuates the fibrotic response to TGF-beta2 by impeding CTGF expression.

Transforming growth factor-beta2 (TGF-beta2) stimulates the expression of pro-fibrotic connective tissue growth factor (CTGF) during the course of renal disease. Because sphingosine kinase-1 (SK-1) activity is also upregulated by TGF-beta, we studied its effect on CTGF expression and on the development of renal fibrosis. When TGF-beta2 was added to an immortalized human podocyte cell line we found that it activated the promoter of SK-1, resulting in upregulation of its mRNA and protein expression. Further, depletion of SK-1 by small interfering RNA or its pharmacological inhibition led to accelerated CTGF expression in the podocytes. Over-expression of SK-1 reduced CTGF induction, an effect mediated by intracellular sphingosine-1-phosphate. In vivo, SK-1 expression was also increased in the podocytes of kidney sections of patients with diabetic nephropathy when compared to normal sections of kidney obtained from patients with renal cancer. Similarly, in a mouse model of streptozotocin-induced diabetic nephropathy, SK-1 and CTGF were upregulated in podocytes. In SK-1 deficient mice, exacerbation of disease was detected by increased albuminuria and CTGF expression when compared to wild-type mice. Thus, SK-1 activity has a protective role in the fibrotic process and its deletion or inhibition aggravates fibrotic disease.

CXCL16 and oxLDL are induced in the onset of diabetic nephropathy.

Diabetic nephropathy (DN) is a major cause of end-stage renal failure worldwide. Oxidative stress has been reported to be a major culprit of the disease and increased oxidized low density lipoprotein (oxLDL) immune complexes were found in patients with DN. In this study we present evidence, that CXCL16 is the main receptor in human podocytes mediating the uptake of oxLDL. In contrast, in primary tubular cells CD36 was mainly involved in the uptake of oxLDL. We further demonstrate that oxLDL down-regulated alpha(3)-integrin expression and increased the production of fibronectin in human podocytes. In addition, oxLDL uptake induced the production of reactive oxygen species (ROS) in human podocytes. Inhibition of oxLDL uptake by CXCL16 blocking antibodies abrogated the fibronectin and ROS production and restored alpha(3) integrin expression in human podocytes. Furthermore we present evidence that hyperglycaemic conditions increased CXCL16 and reduced ADAM10 expression in podocytes. Importantly, in streptozotocin-induced diabetic mice an early induction of CXCL16 was accompanied by higher levels of oxLDL. Finally immunofluorescence analysis in biopsies of patients with DN revealed increased glomerular CXCL16 expression, which was paralleled by high levels of oxLDL. In summary, regulation of CXCL16, ADAM10 and oxLDL expression may be an early event in the onset of DN and therefore all three proteins may represent potential new targets for diagnosis and therapeutic intervention in DN.

Angiotensin II induces renal plasminogen activator inhibitor-1 and cyclooxygenase-2 expression post-transcriptionally via activation of the mRNA-stabilizing factor human-antigen R.

Angiotensin (Ang) II-induced fibrosis of the kidney is characterized by the enhanced expression of profibrotic and proinflammatory genes, including the serine protease inhibitor plasminogen activator inhibitor-1 (PAI-1) and cyclooxygenase-2 (COX-2). In addition to transcriptional regulation, both genes are subject to post-transcriptional control by AU-rich destabilizing elements that reside within the 3' untranslated region of the mRNA. We demonstrated that the continuous infusion of AngII in rats induced fibrosis concomitant with a significant increase in glomerular PAI-1 and COX-2 expression levels. Using RNA pull-down assays and electromobility shift assays, we demonstrated the increased binding of the ubiquitous RNA-binding protein human-antigen R (HuR) to the mRNAs of both PAI-1 and COX-2 in the cytoplasmic fractions of renal homogenates from AngII-treated rats. Actinomycin D experiments in rat mesangial cells revealed that AngII stabilizes both mRNAs via HuR as proven by small interfering RNA. Mechanistically, AngII promotes an increase in nucleo-cytoplasmic HuR shuttling, which was blocked by the PKC inhibitor rottlerin and the type-I AngII (AT(1)) receptor antagonist valsartan but was unaffected by both AT(2) receptor antagonists PD123319 and CGP42112. Co-immunoprecipitation revealed that AngII treatment caused an increase in nuclear PKC-delta concomitant with binding to nuclear HuR both in vitro and in vivo. The post-transcriptional regulation of PAI-1 and COX-2 by PKC-delta-dependent HuR shuttling may contribute to the pathogenesis of hypertensive nephrosclerosis triggered by AngII.

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.