Anke Doller

Signalling pathways regulating nucleo-cytoplasmic shuttling of the mRNA-binding protein HuR.

During the last decade evidence from numerous studies has been accumulated demonstrating that posttranscriptional gene regulation including mRNA turnover and translation is an important paradigm of eukaryotic gene expression contributing to the vast majority of cellular processes including cell growth and differentiation, metabolism, migration, and cellular senescence. Accordingly, a large number of reports have documented that the Human antigen R (HuR), a ubiquitously expressed member of the ELAV protein family, is one of the major actors in this scenario. Consequently, HuR is implicated in a large variety of pathologies in which deregulated stabilisation of many short-lived key mRNAs is causally linked with the onset and course of disease. Since HuR is most abundantly localised within the cell nucleus, export of HuR to the cytoplasm seems a major prerequisite for its stabilising effects on its cognate target adenylate- and uridylate-rich elements (AREs) containing cargo mRNAs. Although, the list of reports demonstrating a critical involvement of different signalling cascades in HuR-triggered mRNA functions is steadily growing, the mechanisms underlying HuR trafficking are not well understood. For this reason, the review will cover the most recent advances of knowledge of signalling cascades involved in the stimulus-dependent nucleo-cytoplasmic HuR shuttling and a special emphasis will be put on the possible regulatory role of posttranslational HuR modification.

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.

Ahnak is critical for cardiac Ca(v)1.2 calcium channel function and its β-adrenergic regulation

Defective L‐type Ca2+ channel (ICaL) regulation is one major cause for contractile dysfunction in the heart. The ICaL is enhanced by sympathetic nervous stimulation: via the activation of β‐adrenergic receptors, PKA phosphorylates the α1C(CaV1.2)‐ and β2‐channel subunits and ahnak, an associated 5643‐amino acid (aa) protein. In this study, we examined the role of a naturally occurring, genetic variant Ile5236Thr‐ahnak on ICaL. Binding experiments with ahnak fragments (wild‐type, Ile5236Thr mutated) and patch clamp recordings revealed that Ile5236Thr‐ahnak critically affected both β2 subunit interaction and ICaL regulation. Binding affinity between ahnak‐C1 (aa 4646‐5288) and β2 subunit decreased by ≈50% after PKA phosphorylation or in the presence of Ile5236Thr‐ahnak peptide. On native cardiomyocytes, intracellular application of this mutated ahnak peptide mimicked the PKA‐effects on ICaL increasing the amplitude by ≈60% and slowing its inactivation together with a leftward shift of its voltage dependency. Both mutated Ile5236Thr‐peptide and Ile5236Thr‐fragment (aa 5215‐5288) prevented specifically the further up‐regulation of ICaL by isoprenaline. Hence, we suggest the ahnak‐C1 domain serves as physiological brake on ICaL. Relief from this inhibition is proposed as common pathway used by sympathetic signaling and Ile5236Thr‐ahnak fragments to increase ICaL. This genetic ahnak variant might cause individual differences in ICaL regulation upon physiological challenges or therapeutic interventions.—Haase, H., Alvarez, J., Petzhold, D., Doller, A., Behlke, J., Erdmann, J., Hetzer, R., Regitz‐Zagrosek, V., Vassort, G., Morano, I. Ahnak is critical for cardiac Ca(v)1.2 calcium channel function and its β‐adrenergic regulation. FASEB J. 19, 1969–1977 (2005)

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.

Tandem Phosphorylation of Serines 221 and 318 by Protein Kinase Cδ Coordinates mRNA Binding and Nucleocytoplasmic Shuttling of HuR

ABSTRACT 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 Cδ (PKCδ)-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, PKCδ mediates the AngII-induced stabilization of prominent HuR target mRNAs, including those of cyclin A, cyclin D1, 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 PKCδ 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.

RNA-Binding Proteins Heterogeneous Nuclear Ribonucleoprotein A1, E1, and K Are Involved in Post-Transcriptional Control of Collagen I and III Synthesis

Collagen types I and III, coded by COL1A1/COL1A2 and COL3A1 genes, are the major fibrillar collagens produced by fibroblasts, including cardiac fibroblasts of the adult heart. Characteristic for different cardiomyopathies is a remodeling process associated with an upregulation of collagen synthesis, which leads to fibrosis. We report identification of three mRNA-binding proteins, heterogeneous nuclear ribonucleoprote (hnRNP) A1, E1, and K, as positive effectors of collagen synthesis acting at the post-transcriptional level by interaction with the 3′-untranslated regions (3′-UTRs) of COL1A1, 1A2, and 3A1 mRNAs. In vitro, binding experiments (electromobility shift assay and UV cross-linking) reveal significant differences in binding to CU- and AU-rich binding motifs. Reporter gene cell transfection experiments and RNA stability assays show that hnRNPs A1, E1, and K stimulate collagen expression by stabilizing mRNAs. Collagen synthesis is activated via the angiotensin II type 1 (AT1) receptor. We demonstrate that transforming growth factor-&bgr;1, a major product of stimulated AT1 receptor, does not activate solely collagen synthesis but synergistically the synthesis of hnRNP A1, E1, and K as well. Thus, post-transcriptional control of collagen synthesis at the mRNA level may substantially be caused by alteration of the expression of RNA-binding proteins. The pathophysiological impact of this finding was demonstrated by screening the expression of hnRNP E1 and K in cardiovascular diseases. In the heart muscle of patients experiencing aortic stenosis, ischemic cardiomyopathy, or dilatative cardiomyopathy, a significant increase in the expression of hnRNP E1, A1, and K was found between 1.5- and 4.5-fold relative to controls.

Transforming growth factor-β2 upregulates sphingosine kinase-1 activity, which in turn attenuates the fibrotic response to TGF-β2 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.

Nitric Oxide Induces TIMP-1 Expression by Activating the Transforming Growth Factor β-Smad Signaling Pathway

Excessive accumulation of the extracellular matrix is a hallmark of many inflammatory and fibrotic diseases, including those of the kidney. This study addresses the question whether NO, in addition to inhibiting the expression of MMP-9, a prominent metalloprotease expressed by mesangial cells, additionally modulates expression of its endogenous inhibitor TIMP-1. We demonstrate that exogenous NO has no modulatory effect on the extracellular TIMP-1 content but strongly amplifies the early increase in cytokine-induced TIMP-1 mRNA and protein levels. We examined whether transforming growth factor β (TGFβ), a potent profibrotic cytokine, is involved in the regulation of NO-dependent TIMP-1 expression. Experiments utilizing a pan-specific neutralizing TGFβ antibody demonstrate that the NO-induced amplification of TIMP-1 is mediated by extracellular TGFβ. Mechanistically, NO causes a rapid increase in Smad-2 phosphorylation, which is abrogated by the addition of neutralizing TGFβ antisera. Similarly, the NO-dependent increase in Smad-2 phosphorylation is prevented in the presence of an inhibitor of TGFβ-RI kinase, indicating that the NO-dependent activation of Smad-2 occurs via the TGFβ-type I receptor. Furthermore, activation of the Smad signaling cascade by NO is corroborated by the NO-dependent increase in nuclear Smad-4 level and is paralleled by increased DNA binding of Smad-2/3 containing complexes to a TIMP-1-specific Smad-binding element (SBE). Reporter gene assays revealed that NO activates a 0.6-kb TIMP-1 gene promoter fragment as well as a TGFβ-inducible and SBE-driven control promoter. Chromatin immunoprecipitation analysis also demonstrated DNA binding activity of Smad-3 and Smad-4 proteins to the TIMP-1-specific SBE. Finally, by enzyme-linked immunosorbent assay, we demonstrated that NO causes a rapid increase in TGFβ1 levels in cell supernatants. Together, these experiments demonstrate that NO by induction of the Smad signaling pathway modulates TIMP-1 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.

High-constitutive HuR phosphorylation at Ser 318 by PKCδ 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.