DNA-Dependent Protein Kinase Mediates YB-1 (Y-Box Binding Protein)-Induced Double Strand Break Repair.

BACKGROUND: DNA-PK (DNA-dependent protein kinase) is a stress-activated serine/threonine kinase that plays a central role in vascular smooth muscle cell proliferation and vascular proliferative disease processes such as neointimal formation. In this study, we link the activation of DNA-PK to the function of the transcription factor YB-1 (Y-box binding protein). METHODS: To identify YB-1 phosphorylation by DNA-PK, we generated different YB-1-expressing vectors. YB-1 nuclear translocation was investigated using immunoblotting and immunofluorescence staining. For YB-1 activity, luciferase assays were performed. RESULTS: We show by mutational analysis and kinase assay that the transcriptional regulator YB-1 is a substrate of DNA-PK. Blockade of DNA-PK by specific inhibitors revealed its critical involvement in YB-1phosphorylation as demonstrated by inhibition of an overexpressed YB-1 reporter construct. Using DNA-PK-deficient cells, we demonstrate that the shuttling of YB-1 from the cytoplasm to the nucleus is dependent on DNA-PK and that the N-terminal domain of YB-1 is phosphorylated at threonine 89. Point mutation of YB-1 at this residue abrogated the translocation of YB-1 into the nucleus. The phosphorylation of YB-1 by DNA-PK increased cellular DNA repair after exposure to ionizing radiation. Atherosclerotic tissue specimens were analyzed by immunohistochemistry. The DNA-PK subunits and YB-1 phosphorylated at T89 were found colocalized suggesting their in vivo interaction. In mice, the local application of the specific DNA-PK inhibitor NU7026 via thermosensitive Pluronic F-127 gel around dilated arteries significantly reduced the phosphorylation of YB-1. CONCLUSIONS: DNA-PK directly phosphorylates YB-1 and, this way, modulates YB-1 function. This interaction could be demonstrated in vivo, and colocalization in human atherosclerotic plaques suggests clinical relevance of our finding. Phosphorylation of YB-1 by DNA-PK may represent a novel mechanism governing atherosclerotic plaque progression.

IKKγ/NEMO Localization into Multivesicular Bodies.

The NF-κB pathway is central pathway for inflammatory and immune responses, and IKKγ/NEMO is essential for NF-κB activation. In a previous report, we identified the role of glycogen synthase kinase-3ß (GSK-3ß) in NF-κB activation by regulating IKKγ/NEMO. Here, we show that NEMO phosphorylation by GSK-3ß leads to NEMO localization into multivesicular bodies (MVBs). Using the endosome marker Rab5, we observed localization into endosomes. Using siRNA, we identified the AAA-ATPase Vps4A, which is involved in recycling the ESCRT machinery by facilitating its dissociation from endosomal membranes, which is necessary for NEMO stability and NF-κB activation. Co-immunoprecipitation studies of NEMO and mutated NEMO demonstrated its direct interaction with Vps4A, which requires NEMO phosphorylation. The transfection of cells by a mutated and constitutively active form of Vps4A, Vps4A-E233Q, resulted in the formation of large vacuoles and strong augmentation in NEMO expression compared to GFP-Vps4-WT. In addition, the overexpression of the mutated form of Vps4A led to increased NF-κB activation. The treatment of cells with the pharmacologic V-ATPase inhibitor bafilomycin A led to a dramatic downregulation of NEMO and, in this way, inhibited NF-κB signal transduction. These results reveal an unexpected role for GSK-3ß and V-ATPase in NF-κB signaling activation.

DNA-PK: gatekeeper for IKKγ/NEMO nucleocytoplasmic shuttling in genotoxic stress-induced NF-kappaB activation.

The transcription factors of the nuclear factor κB (NF-κB) family play a pivotal role in the cellular response to DNA damage. Genotoxic stress-induced activation of NF-κB differs from the classical canonical pathway by shuttling of the NF-κB Essential Modifier (IKKγ/NEMO) subunit through the nucleus. Here, we show that DNA-dependent protein kinase (DNA-PK), an enzyme involved in DNA double-strand break (DSB) repair, triggers the phosphorylation of NEMO by genotoxic stress, thereby enabling shuttling of NEMO through the nucleus with subsequent NF-κB activation. We identified serine 43 of NEMO as a DNA-PK phosphorylation site and point mutation of this serine to alanine led to a complete block of NF-κB activation by ionizing radiation (IR). Blockade of DNA-PK by a specific shRNA or by DNA-PKcs-deficient cells abrogated NEMO entry into the nucleus, as well. Accordingly, SUMOylation of NEMO, a prerequisite of nuclear NEMO, was abolished. Based on these observations, we propose a model in which NEMO phosphorylation by DNA-PK provides the first step in the nucleocytoplasmic trafficking of NEMO.

Left atrial enlargement and clinical considerations in patients with or without a residual interatrial shunt after closure of the left atrial appendage with the WATCHMAN™-device.

BACKGROUND: Interventional closure of the left atrial appendage (LAA) in patients with non-valvular atrial fibrillation, high thromboembolic and bleeding risk or bleeding history is an alternative therapeutic strategy to oral anticoagulation. It is not known if the exclusion of the LAA from the blood circulation affects the left atrial volume (LAV) and consequently its prognostic value or the circulatory performance of the heart in humans. METHODS: We aimed to prospectively assess potential changes in baseline LAV, left ventricular ejection fraction (LVEF), NT-proBNP-level and the covered distance in the 6-min walk-test 6 weeks and 6 months after LAA closure with the WATCHMAN™ device. We used serial 3-dimensional transthoracic and transesophageal echocardiography to assess LAV, residual interatrial shunt and device performance in 58 consecutive patients with successful LAA closure. RESULTS: Accurate 3D-echocardiographic data for LAV measurements were evaluable for 51 (91%) patients. Maximum LAV (LAVmax) at baseline was 102.8 ± 30.8 ml and increased significantly to 107.7 ± 32.8 ml after 6 weeks (p < 0.01) and 113.5 ± 34.2 ml after 6 months (p < 0.01). Minimal LAV (LAVmin) increased from 76.9 ± 29.5 ml at baseline to 81.8 ± 30.2 ml after 45 days (p < 0.01) and 82.1 ± 33.3 ml after 6 months (p < 0.01). Similarly, their indexes to BSA (LAVImax and LAVImin) increased significantly, as well. Patients without a residual left-to-right interatrial shunt showed a significantly higher increase in LAVmax or LAVmin. Baseline LVEF, NT-proBNP-level or the distance covered at the 6-min walk test did not significantly change 6 weeks or 6 months after LAA closure. CONCLUSIONS: LAVmax and LAVmin increase significantly after interventional LAA closure. LA enlargement does not correlate with clinical progression of heart failure. Persistent left-to-right interatrial shunt counteracts the LA enlargement. A reduced LA compliance after exclusion of the LAA from the blood circulation with consecutive increase in LA pressure may be a potential cause of LA enlargement and warrants further investigation. TRIAL REGISTRATION: German Clinical Trials Register ID: DRKS00010768 ; Registration Date 07.07.2016.

Transcriptional activation of DNA-dependent protein kinase catalytic subunit gene expression by oestrogen receptor-alpha.

The cellular response to DNA double-strand break (DSB) occurs through an integrated sensing and signalling network that maintains genomic stability. Oestrogen (E2), among its many functions, is known to have a positive effect on global genomic DNA repair; however, the mechanism by which it functions is unclear. A central enzyme involved in DNA DSB repair in mammalian cells is the DNA-dependent protein kinase (DNA-PK). Here, we show that E2 enhances DNA-PK catalytic subunit (DNA-PKcs) promoter activity with subsequent transcriptional and translational upregulation of DNA-PKcs in a breast cancer cell line. We identify two potential E2 receptor-alpha (ERalpha)-binding sites in a region upstream from the DNA-PKcs initiation site. By using small interfering RNA and the specific E2 receptor antagonist ICI 182,780, we demonstrate that ERalpha knockdown reduces E2-induced upregulation of DNA-PKcs expression and activity in breast carcinoma cells. E2-induced DNA-PK transactivation results in an increased ability of the cells to repair DNA DSB. This previously unknown mechanism of DNA-PK regulation sheds new light on tumour biology and reveals new possibilities for the prevention and therapy of E2-sensitive proliferative diseases.

Normoxic HIF-1α Stabilization Caused by Local Inflammatory Factors and Its Consequences in Human Coronary Artery Endothelial Cells.

Knowledge about normoxic hypoxia-inducible factor (HIF)-1α stabilization is limited. We investigated normoxic HIF-1α stabilization and its consequences using live cell imaging, immunoblotting, Bio-Plex multiplex immunoassay, immunofluorescence staining, and barrier integrity assays. We demonstrate for the first time that IL-8 and M-CSF caused HIF-1α stabilization and translocation into the nucleus under normoxic conditions in both human coronary endothelial cells (HCAECs) and HIF-1α-mKate2-expressing HEK-293 cells. In line with the current literature, our data show significant normoxic HIF-1α stabilization caused by TNF-α, INF-γ, IL-1ß, and IGF-I in both cell lines, as well. Treatment with a cocktail consisting of TNF-α, INF-γ, and IL-1ß caused significantly stronger HIF-1α stabilization in comparison to single treatments. Interestingly, this cumulative effect was not observed during simultaneous treatment with IL-8, M-CSF, and IGF-I. Furthermore, we identified two different kinetics of HIF-1α stabilization under normoxic conditions. Our data demonstrate elevated protein levels of HIF-1α-related genes known to be involved in the development of atherosclerosis. Moreover, we demonstrate an endothelial barrier dysfunction in HCAECs upon our treatments and during normoxic HIF-1α stabilization comparable to that under hypoxia. This study expands the knowledge of normoxic HIF-1α stabilization and activation and its consequences on the endothelial secretome and barrier function. Our data imply an active role of HIF-1α in vivo in the vasculature in the absence of hypoxia.

Glycogen synthase kinase-3 protects estrogen receptor alpha from proteasomal degradation and is required for full transcriptional activity of the receptor.

Glycogen synthase kinase-3 (GSK-3) plays a key role in the regulation of transcription factors including steroid receptors. Having identified estrogen receptor-alpha (ERalpha) as substrate for GSK-3, the impact of GSK-3 on ERalpha function and activity upon 17beta-estradiol (E2)-dependent activation remains to be clarified. Here we show by using small interfering technology in combination with immunoblot, gene expression analysis, and luciferase reporter assays that silencing of GSK-3alpha or GSK-3beta results in the reduction of ERalpha levels and transcriptional activity in ERalpha-positive breast cancer cells. Using MCF-7 cells we demonstrate that reduction of ERalpha levels upon GSK-3 silencing was due to increased proteasomal degradation of ERalpha rather than inhibition of ERalpha protein synthesis. Indeed, under this condition, ERalpha protein was rescued using the proteasome inhibitor MG132 in presence of the protein synthesis inhibitor cycloheximide. In addition, strong accumulation of ubiquitinated ERalpha was obtained after GSK-3 silencing in the presence of MG132. We conclude that GSK-3 protects ERalpha from proteasomal degradation and plays a crucial role in ERalpha protein stabilization and turnover. Furthermore, in vitro kinase assay depicted that GSK-3beta phosphorylates ERalpha at Ser-118. GSK-3 silencing resulted in decrease of E2-induced nuclear ERalpha phosphorylation at Ser-118 and E2-induced estrogen response element-dependent luciferase reporter gene expression. Neither Ser-118 phosphorylation nor luciferase activity was restored by use of MG132. Moreover, the expression of estrogen-responsive genes (pS2 and progesterone receptor) was decreased upon GSK-3 silencing. These findings demonstrated that GSK-3 is required for E2-induced ERalpha phosphorylation at Ser-118 and full transcriptional activity of the receptor upon E2 stimulation.

Impact of PKCdelta on estrogen receptor localization and activity in breast cancer cells.

Regulation of estrogen receptor (ER) function in breast cancer cells is a complex process involving different signalling mechanisms. One signal transduction component that appears to influence ER signalling is protein kinase C (PKC). PKCdelta is a particular isoenzyme of the novel PKC subfamily that plays a role in growth control, differentiation and apoptosis. The aim of the present study was to investigate the impact of PKCdelta on the regulation of the transcriptional activity of the human ERalpha. By using 12-O-tetradecanoylphorbol-13-acetate (TPA), Bryostatin1 and Rottlerin, we show that active PKCdelta is a proproliferative factor in estrogen-dependent breast cancer cells. Furthermore, activation of PKCdelta by TPA resulted in activation and nuclear translocation of ERalpha and in an increase of ER-dependent reporter gene expression. Transfection and expression of the regulatory domain RDdelta of PKCdelta, which is inhibitory to PKCdelta, inhibited the TPA-induced ERalpha activation and translocation. ERalpha was not phosphorylated by PKCdelta; however, glycogen synthase kinase-3 (GSK3) was identified as a substrate of PKCdelta. The expression of RDdelta resulted in a decrease of TPA-induced GSK3 phosphorylation and translocation into the nucleus. We suggest that GSK3 plays a role in the PKCdelta-related nuclear translocation of ERalpha.

Calcium-binding proteins S100A8 and S100A9 as novel diagnostic markers in human prostate cancer.

PURPOSE: S100 proteins comprise a family of calcium-modulated proteins that have recently been associated with epithelial tumors. We examined the expression of two members of this family, S100A8 and S100A9, together with the S100 receptor RAGE (receptor for advanced glycation end products) in human prostate adenocarcinomas and in prostatic intraepithelial neoplasia. EXPERIMENTAL DESIGN: Tissue specimens of 75 patients with organ-confined prostate cancer of different grades were analyzed by immunohistochemistry for expression of S100A8, S100A9, and RAGE. In addition, in situ hybridization of S100A8 and S100A9 was done for 20 cases. An ELISA was applied to determine serum concentrations of S100A9 in cancer patients compared with healthy controls or to patients with benign prostatic hyperplasia (BPH). RESULTS: S100A8, S100A9, and RAGE were up-regulated in prostatic intraepithelial neoplasia and preferentially in high-grade adenocarcinomas, whereas benign tissue was negative or showed weak expression of the proteins. There was a high degree of overlap of S100A8 and S100A9 expression patterns and of S100A8 or S100A9 and RAGE, respectively. Frequently, a gradient within the tumor tissue with an increased expression toward the invaded stroma of the prostate was observed. S100A9 serum levels were significantly elevated in cancer patients compared with BPH patients or healthy individuals. CONCLUSION: Our data suggest that enhanced expression of S100A8, S100A9, and RAGE is an early event in prostate tumorigenesis and may contribute to development and progression or extension of prostate carcinomas. Furthermore, S100A9 in serum may serve as useful marker to discriminate between prostate cancer and BPH.

GSK-3ß controls NF-kappaB activity via IKKγ/NEMO.

The NF-κB signaling pathway is central for the innate immune response and its deregulation is found in multiple disorders such as autoimmune, chronic inflammatory and metabolic diseases. IKKγ/NEMO is essential for NF-κB activation and NEMO dysfunction in humans has been linked to so-called progeria syndromes, which are characterized by advanced ageing due to age-dependent inflammatory diseases. It has been suggested that glycogen synthase kinase-3ß (GSK-3ß) participates in NF-κB regulation but the exact mechanism remained incompletely understood. In this study, we identified NEMO as a GSK-3ß substrate that is phosphorylated at serine 8, 17, 31 and 43 located within its N-terminal domain. The kinase forms a complex with wild-type NEMO while point mutations of NEMO at the specific serines abrogated GSK-3ß binding and subsequent phosphorylation of NEMO resulting in its destabilization. However, K63-linked polyubiquitination was augmented in mutated NEMO explaining an increased binding to IKKα and IKKß. Even IκBα was found degraded. Still, TNFα-stimulated NF-κB activation was impaired pointing towards an un-controlled signalling process. Our data suggest that GSK-3ß is critically important for ordered NF-κB signalling through modulation of NEMO phosphorylation.

DNA-dependent protein kinase (DNA-PK) permits vascular smooth muscle cell proliferation through phosphorylation of the orphan nuclear receptor NOR1.

AIMS: Being central part of the DNA repair machinery, DNA-dependent protein kinase (DNA-PK) seems to be involved in other signalling processes, as well. NOR1 is a member of the NR4A subfamily of nuclear receptors, which plays a central role in vascular smooth muscle cell (SMC) proliferation and in vascular proliferative processes. We determined putative phosphorylation sites of NDA-PK in NOR1 and hypothesized that the enzyme is able to modulate NOR1 signalling and, this way, proliferation of SMC. METHODS AND RESULTS: Cultured human aortic SMC were treated with the specific DNA-PK inhibitor NU7026 (or siRNA), which resulted in a 70% inhibition of FCS-induced proliferation as measured by BrdU incorporation. Furthermore, FCS-stimulated up-regulation of NOR1 protein as well as the cell-cycle promoting proteins proliferating cell nuclear antigen (PCNA), cyclin D1, and hyperphosphorylation of the retinoblastoma protein were prevented by DNA-PK inhibition. Co-immunoprecipitation studies from VSM cell lysates demonstrated that DNA-PK forms a complex with NOR1. Mutational analysis and kinase assays demonstrated that NOR1 is a substrate of DNA-PK and is phosphorylated in the N-terminal domain. Phosphorylation resulted in post-transcriptional stabilization of the protein through prevention of its ubiquitination. Active DNA-PK and NOR1 were found predominantly expressed within the neointima of human atherosclerotic tissue specimens. In mice, inhibition of DNA-PK significantly attenuated neointimal lesion size 3 weeks after wire-injury. CONCLUSION: DNA-PK directly phosphorylates NOR-1 and, this way, modulates SMC proliferation. These data add to our understanding of vascular remodelling processes and opens new avenues for treatment of vascular proliferative diseases.

Insulin-like growth factor binding protein-4 and -5 modulate ligand-dependent estrogen receptor-α activation in breast cancer cells in an IGF-independent manner.

Insulin-like growth factor binding proteins (IGFBPs) are modulators of numerous cellular processes including cell proliferation. Although IGFBPs classically act by sequestration of extracellular insulin-like growth factors (IGFs), thereby contributing to the fine-tuning of growth factor signals, IGF-independent actions of IGFBPs have also been described. In the breast, growth factor signaling in association with estradiol (E2)-stimulated estrogen receptor function is organized in a complex cross-talk. The importance of phosphatidylinositol 3-kinase/protein kinase B (Akt/PKB) pathway components for the E2-induced activation of estrogen receptor-alpha (ERα) is well accepted. Here we show that in the absence of IGFs, IGFBP-4 or IGFBP-5, either overexpressed in MCF-7 breast cancer cells or added exogenously, decreased the capability of E2 to induce ERα transcriptional activity. In addition, overexpression or addition of recombinant IGFBP-4 or IGFBP-5 resulted in reduction of E2-induced phosphorylation of Akt/PKB, GSK-3α/ß and ERα in MCF-7 cells. The activation of the Akt/PKB-pathway describes a non-genomic effect of E2, which did not involve activation/phosphorylation of the IGF-I receptor (IGF-IR). Furthermore, knockdown of the IGF-IR did not affect the inhibition of E2-induced ERα phosphorylation by IGFBP-4 and 5. Moreover, IGFBP-4 and IGFBP-5 strongly decreased E2-triggered growth of MCF-7 cells. Our data suggest that IGFBPs interfere with the E2-induced activation of the Akt/PKB-pathway and prevent full hormone-dependent activation of ERα and breast cancer cell growth in an IGF- and IGF-IR-independent manner.

The lysosomal transfer of LDL/cholesterol from macrophages into vascular smooth muscle cells induces their phenotypic alteration.

AIMS: Macrophages (MPs) and vascular smooth muscle cells (VSMCs) closely interact within the growing atherosclerotic plaque. An in vitro co-culture model was established to study how MPs modulate VSMC behaviour. METHODS AND RESULTS: MPs were exposed to fluorescence-labelled-acetylated LDL (FL-acLDL) prior to co-culture with VSMCs. Fluorescence microscopy visualized first transport of FL-acLDL within 6 h after co-culture implementation. When MPs had been fed with FL-acLDL in complex with fluorescence-labelled cholesterol (FL-Chol), these complexes were also transferred during co-culture and resulted in cholesterol positive lipid droplet formation in VSMCs. When infected with a virus coding for a fusion protein of Rab5a and fluorescent protein reporter (FP) to mark early endosomes, no co-localization between Rab5a-FP and the transported FL-acLDL within VSMCs was detected implying a mechanism independent of phagocytosis. Next, expression of lysosome-associated membrane glycoprotein 1 (LAMP1)-FP, marking all lysosomes in VSMCs, revealed that the FL-acLDL was located in non-acidic lysosomes. MPs infected with virus encoding for LAMP1-FP prior to co-culture demonstrated that intact fluorescence-marked lysosomes were transported into the VSMC, instead. Xenogenic cell composition (rat VSMC, human MP) and subsequent quantitative RT-PCR with rat-specific primers rendered induction of genes typical for MPs and down-regulation of the cholesterol sensitive HMG-CoA reductase. CONCLUSION: Our results demonstrate that acLDL/cholesterol-loaded lysosomes are transported from MPs into VSMCs in vitro. Lysosomal transfer results in a phenotypic alteration of the VSMC towards a foam cell-like cell. This way VSMCs may lose their plaque stabilizing properties and rather contribute to plaque destabilization and rupture.

The impact of rapid atrial pacing on ADMA and endothelial NOS.

BACKGROUND: The endothelial nitric oxide synthase (eNOS) inhibitor asymmetric dimethylarginine (ADMA) is a well-established risk factor for oxidative stress, vascular dysfunction, and congestive heart failure. The aim of the present study was to determine the impact of rapid atrial pacing (RAP) on ADMA levels and eNOS expression. METHODS AND RESULTS: ADMA levels were studied in 60 age- and gender-matched patients. Thirty five patients had persistent atrial fibrillation (AF)≥ 4months. In AF-patients, parameters were studied before and 24h after electrical cardioversion. Moreover, ADMA, eNOS expression, and calcium-handling proteins were studied in pigs subjected to RAP as well as in endothelial cell (EC) cultures. ADMA level was significantly higher in AF compared to sinus rhythm patients (p=0.024). ADMA was highest in AF-patients, who also showed elevated troponin T (TnT) levels. Moreover, ADMA showed a significant linear correlation to TnT (r=0.47; p<0.01). After electrical cardioversion ADMA returned to normal within 24h. In pigs, RAP for 7h increased ADMA levels (p=0.018) and TnI (p<0.05), and reduced mRNA expression of ventricular and aortic eNOS (-80%; p<0.05) compared to sham-control. However, ADMA per se did not affect eNOS mRNA level in EC cultures. CONCLUSION: The current study shows that acute and persistent episodes of atrial tachyarrhythmia are associated with elevated ADMA levels accompanied by increased ischemic myocardial markers. Moreover, RAP increases ADMA and down-regulates eNOS expression in an ADMA-independent manner. We conclude that the combination of these two separate and potentially synergistic mechanisms may contribute to long-term vascular injury during atrial tachyarrhythmia.

Interaction of the double-strand break repair kinase DNA-PK and estrogen receptor-alpha.

Estrogens are suggested to play a role in the development and progression of proliferative diseases such as breast cancer. Like other steroid hormone receptors, the estrogen receptor-alpha (ERalpha) is a substrate of protein kinases, and phosphorylation has profound effects on its function and activity. Given the importance of DNA-dependent protein kinase (DNA-PK) for DNA repair, cell cycle progression, and survival, we hypothesized that it modulates ERalpha signaling. Here we show that, upon estrogen stimulation, DNA-PK forms a complex with ERalpha in a breast cancer cell line (MELN). DNA-PK phosphorylates ERalpha at Ser-118. Phosphorylation resulted in stabilization of ERalpha protein as inhibition of DNA-PK resulted in its proteasomal degradation. Activation of DNA-PK by double-strand breaks or its inhibition by siRNA technology demonstrated that estrogen-induced ERalpha activation and cell cycle progression is, at least, partially dependent on DNA-PK.

S100A8 and S100A9 activate MAP kinase and NF-kappaB signaling pathways and trigger translocation of RAGE in human prostate cancer cells.

S100 proteins, a multigenic family of calcium-binding proteins, have been linked to human pathologies in recent years. Deregulated expression of S100 proteins, including S100A8 and S100A9, was reported in association with neoplastic disorders. In a previous study, we identified enhanced expression of S100A8 and S100A9 in human prostate cancer. To investigate potential functional implications of S100A8 and S100A9 in prostate cancer, we examined the influence of over-expressed and of purified recombinant S100A8 and S100A9 proteins in different prostate epithelial cell lines. S100A8 and S100A9 were secreted by prostate cancer cells, a finding which prompted us to analyze a possible function as extracellular ligands. S100A8/A9 induced the activation of NF-kappaB and an increased phosphorylation of p38 and p44/42 MAP kinases. In addition, extracellular S100A8/A9 stimulated migration of benign prostatic cells in vitro. Furthermore, in immunofluorescence experiments, we found a strong speckled co-localization of intracellular S100A8/A9 with RAGE after stimulating cells with recombinant S100A8/A9 protein or by increasing cytosolic Ca2+ levels. In summary, our findings show that S100A8 and S100A9 are linked to the activation of important features of prostate cancer cells.

Glycogen synthase kinase-3 interacts with and phosphorylates estrogen receptor alpha and is involved in the regulation of receptor activity.

Like other steroid hormone receptors, estrogen receptor-alpha (ERalpha) is a substrate for protein kinases, and phosphorylation has profound effects on the function and activity of this receptor. A number of different kinases have been implicated in ERalpha regulation. In this report we show by mutational analysis and in vitro kinase assays that ERalpha is a substrate for glycogen synthase kinase-3 (GSK-3) in vitro and is phosphorylated on two sites, the Ser-102, -104, and -106 motif and Ser-118, both located in the N-terminal transcription activation function (AF-1) domain. GSK-3 forms a complex with ERalpha in vivo as demonstrated by co-immunoprecipitation from cell lysates. The GSK-3 inhibitor lithium chloride was used to determine the role of GSK-3 in phosphorylation of Ser-102, -104, and -106 and Ser-118 in vivo and to explore the role of these serines in the regulation of ERalpha function. Treatment of cells with lithium chloride resulted in dephosphorylation of Ser-104 and -106 and Ser-118, which suggests these serine residues as targets for GSK-3 in vivo. Our results further suggest that ERalpha phosphorylation by GSK-3 stabilizes ERalpha under resting conditions and modulates ERalpha transcriptional activity upon ligand binding. Inhibition and constitutive activation of GSK-3, both, resulted in inhibition of ERalpha transcriptional activity, indicating a function of active as well as inactive GSK-3 in ERalpha regulation. These findings uncover a novel mechanism for the regulation of ERalpha-mediated estrogen signaling controlled by a dual action of GSK-3.