Mitogen and Stress-Activated Kinases 1 and 2 Mediate Endothelial Dysfunction.

Inflammation promotes endothelial dysfunction, but the underlying mechanisms remain poorly defined in vivo. Using translational vascular function testing in myocardial infarction patients, a situation where inflammation is prevalent, and knock-out (KO) mouse models we demonstrate a role for mitogen-activated-protein-kinases (MAPKs) in endothelial dysfunction. Myocardial infarction significantly lowers mitogen and stress kinase 1/2 (MSK1/2) expression in peripheral blood mononuclear cells and diminished endothelial function. To further understand the role of MSK1/2 in vascular function we developed in vivo animal models to assess vascular responses to vasoactive drugs using laser Doppler imaging. Genetic deficiency of MSK1/2 in mice increased plasma levels of pro-inflammatory cytokines and promoted endothelial dysfunction, through attenuated production of nitric oxide (NO), which were further exacerbated by cholesterol feeding. MSK1/2 are activated by toll-like receptors through MyD88. MyD88 KO mice showed preserved endothelial function and reduced plasma cytokine expression, despite significant hypercholesterolemia. MSK1/2 kinases interact with MAPK-activated proteins 2/3 (MAPKAP2/3), which limit cytokine synthesis. Cholesterol-fed MAPKAP2/3 KO mice showed reduced plasma cytokine expression and preservation of endothelial function. MSK1/2 plays a significant role in the development of endothelial dysfunction and may provide a novel target for intervention to reduce vascular inflammation. Activation of MSK1/2 could reduce pro-inflammatory responses and preserve endothelial vasodilator function before development of significant vascular disease.

High mobility group box 2 regulates skeletal muscle development through ribosomal protein S6 kinase 1.

HMGB2, a DNA-binding protein, highly expresses during embryogenesis and plays an important role in development of some organs and tissues. However, it remains to be further investigated weather HMGB2 influences muscle development. In this work, we identified HMGB2 as an essential factor in myogenesis. Compared to wild type (WT) mice, body weights of systemic hmgb2 homozygous knockout (hmgb2-/- ) mice especially males were reduced. Diameter and cross-section area of tibialis anterior (TA) muscle fibers as well as expression of Myogenin and MyHC were all decreased in hmgb2-/- mice. CTX injury model revealed that HMGB2 was required for satellite cell proliferation and muscle regeneration. Moreover, HMGB2 interacted with S6K1 and regulated the kinase activity of S6K1 during cell proliferation. Knockdown and inactivation of S6K1 in C2C12 cells both resulted in impaired proliferation and differentiation. Furthermore, expression of cyclin D1 and Myf5 were both decreased when HMGB2 or S6K1 were knocked down and kinase activity of S6K1 was inhibited. These results indicate that HMGB2 is required for skeletal muscle development and regeneration, and HMGB2 maintains proliferation of myoblasts through regulating kinase activity of S6K1.

Tumor dormancy in bone.

Background: Bone marrow is a common site of metastasis for a number of tumor types, including breast, prostate, and lung cancer, but the mechanisms controlling tumor dormancy in bone are poorly understood. In breast cancer, while advances in drug development, screening practices, and surgical techniques have dramatically improved survival rates in recent decades, metastatic recurrence in the bone remains common and can develop years or decades after elimination of the primary tumor. Recent Findings: It is now understood that tumor cells disseminate to distant metastatic sites at early stages of tumor progression, leaving cancer survivors at a high risk of recurrence. This review will discuss mechanisms of bone lesion development and current theories of how dormant cancer cells behave in bone, as well as a number of processes suspected to be involved in the maintenance of and exit from dormancy in the bone microenvironment. Conclusions: The bone is a complex microenvironment with a multitude of cell types and processes. Many of these factors, including angiogenesis, immune surveillance, and hypoxia, are thought to regulate tumor cell entry and exit from dormancy in different bone marrow niches.

Agmatine Protects Against the Progression of Sepsis Through the Imidazoline I2 Receptor-Ribosomal S6 Kinase 2-Nuclear Factor-κB Signaling Pathway.

OBJECTIVES: The knowledge that agmatine is found in the human body has existed for several years; however, its role in sepsis has not yet been studied. In the present study, we investigate the role of agmatine in the progression and treatment of sepsis. DESIGN: Clinical/laboratory investigations. SETTING: Medical centers/University-based research laboratory. SUBJECTS: Elective ICU patients with severe sepsis and healthy volunteers; C57BL/6 mice weighing 18-22 g. INTERVENTIONS: Serum agmatine level and its associations with inflammatory markers were assessed in patients with sepsis. Agmatine was administered intraperitoneally to mice before a lipopolysaccharide challenge. Human peripheral blood mononuclear cells and murine macrophages were pretreated with agmatine followed by lipopolysaccharide stimulation. MEASUREMENTS AND MAIN RESULTS: Serum agmatine levels were significantly decreased in patients with sepsis and lipopolysaccharide-induced mice, and correlated with Acute Physiology and Chronic Health Evaluation II score, procalcitonin, tumor necrosis factor-α, and interleukin-6 levels. In a therapeutic experiment, exogenous agmatine attenuated the cytokine production of peripheral blood mononuclear cells from patients with sepsis and healthy controls. Agmatine also exerted a significant beneficial effect in the inflammatory response and organ damage and reduced the death rate in lipopolysaccharide-induced mice. Imidazoline I2 receptor agonist 2-benzofuran-2-yl blocked the pharmacological action of agmatine; whereas, other imidazoline receptor ligands did not. Furthermore, agmatine significantly impaired the inflammatory response by inactivating nuclear factor-κB, but not protein 38 mitogen-activated protein kinase, c-Jun N-terminal kinase, extracellular signal-regulated kinase, and inducible nitric oxide synthase signaling in macrophages. Activation of imidazoline I2 receptor or knockdown of ribosomal S6 kinase 2 counteracted the effects of agmatine on phosphorylation and degradation of inhibitor of nuclear factor-κBα. CONCLUSIONS: Endogenous agmatine metabolism correlated with the progression of sepsis. Supplemental exogenous agmatine could ameliorate the lipopolysaccharide-induced systemic inflammatory responses and multiple organ injuries through the imidazoline I2 receptor-ribosomal S6 kinase 2-nuclear factor-κB pathway. Agmatine could be used as both a clinical biomarker and a promising pharmaconutrient in patients with severe sepsis.

Protective Effect of Mitogen- and Stress-Activated Protein Kinase on the Rats with Focal Ischemia-Reperfusion Injury.

Mitogen- and stress-activated protein kinase (MSK) is a recently identified nuclear cAMP-regulated enhancer B (CREB) and histone H3 kinase that responds to both mitogen- and stress-activated protein kinases. This study was designed to investigate the protective effect of MSK on the rats with focal ischemia-reperfusion injury. The rat model was established by inserting thread into the middle cerebral artery. The protein expression was measured by immunoblotting. The localization of MSK was measured by immunofluorescence assay. Highly-differentiated pheochromocytoma 12 (PC12) is used as a sympathetic neuron-like cell line and treated with glutamate to induce neurotoxicity. MSK was knocked down and overexpressed by siRNA and MSK over-expressing vector, respectively. The cell viability was measured by cell counting kit (CCK-8) assay. The coronal sections were isolated and stained with 2, 3, 5-triphenyltetrazolium chloride (TTC) to determine infarct volume. Finally, astrocytes were separated from cerebral cortexes of normal rats to analyze the effects of MSK on inflammatory response. In the rats with focal ischemia-reperfusion injury, the expression of MSK was reduced, reaching the lowest level at 3 d after ischemia-reperfusion, and then recovered gradually. MSK was found mainly localized in neurons and astrocytes. The expression levels of caspase-3, caspase-8, caspase-9, and INOS showed the opposite trend with respect to MSK. Further analysis showed that overexpression of MSK exerted a protective effect on glutamate-induced neurotoxicity through inhibiting apoptosis of PC12 cells, as well as decreased the infarct size in rat with focal ischemia-reperfusion injury. On the contrary, knockdown of MSK showed opposite results. Finally, MSK suppressed LPS-induced inflammatory response by decreasing the expression of inducible nitric oxide synthase (INOS) and increasing the expression of interleukin-10 (IL-10) in astrocytes from cerebral cortexes of normal rats. In conclusion, MSK exerted a protective effect on rat with focal ischemia-reperfusion injury through its anti-apoptotic effect on neurons and anti-inflammatory effect on astrocytes.

The Ser/Thr kinase p90RSK promotes kidney fibrosis by modulating fibroblast-epithelial crosstalk.

Healthy kidney structure and environment rely on epithelial integrity and interactions between epithelial cells and other kidney cells. The Ser/Thr kinase 90 kDa ribosomal protein S6 kinase 1 (p90RSK) belongs to a protein family that regulates many cellular processes, including cell motility and survival. p90RSK is predominantly expressed in the kidney, but its possible role in chronic kidney disease (CKD) remains largely unknown. Here, we found that p90RSK expression is dramatically activated in a classic mouse obstructive chronic kidney disease model, largely in the interstitial FSP-1-positive fibroblasts. We generated FSP-1-specific p90RSK transgenic mouse (RSK-Tg) and discovered that these mice, after obstructive injury, display significantly increased fibrosis and enhanced tubular epithelial damage compared with their wt littermates (RSK-wt), indicating a role of p90RSK in fibroblast-epithelial communication. We established an in vitro fibroblast-epithelial coculture system with primary kidney fibroblasts from RSK-Tg and RSK-wt mice and found that RSK-Tg fibroblasts consistently produce excessive H2O2 causing epithelial oxidative stress and inducing nuclear translocation of the signaling protein ß-catenin. Epithelial accumulation of ß-catenin, in turn, promoted epithelial apoptosis by activating the transcription factor forkhead box class O1 (FOXO1). Of note, blockade of reactive oxygen species (ROS) or ß-catenin or FOXO1 activity abolished fibroblast p90RSK-mediated epithelial apoptosis. These results make it clear that p90RSK promotes kidney fibrosis by inducing fibroblast-mediated epithelial apoptosis through ROS-mediated activation of ß-catenin/FOXO1 signaling pathway.

p90 ribosomal S6 kinase (RSK) phosphorylates myosin phosphatase and thereby controls edge dynamics during cell migration.

Cell migration is essential to embryonic development, wound healing, and cancer cell dissemination. Cells move via leading-edge protrusion, substrate adhesion, and retraction of the cell's rear. The molecular mechanisms by which extracellular cues signal to the actomyosin cytoskeleton to control these motility mechanics are poorly understood. The growth factor-responsive and oncogenically activated protein extracellular signal-regulated kinase (ERK) promotes motility by signaling in actin polymerization-mediated edge protrusion. Using a combination of immunoblotting, co-immunoprecipitation, and myosin-binding experiments and cell migration assays, we show here that ERK also signals to the contractile machinery through its substrate, p90 ribosomal S6 kinase (RSK). We probed the signaling and migration dynamics of multiple mammalian cell lines and found that RSK phosphorylates myosin phosphatase-targeting subunit 1 (MYPT1) at Ser-507, which promotes an interaction of Rho kinase (ROCK) with MYPT1 and inhibits myosin targeting. We find that by inhibiting the myosin phosphatase, ERK and RSK promote myosin II-mediated tension for lamella expansion and optimal edge dynamics for cell migration. These findings suggest that ERK activity can coordinately amplify both protrusive and contractile forces for optimal cell motility.

Signalling of ERK1/2, P38MAPK and P90RSK in HIV-associated pre-eclampsia.

Due to their significance in trophoblast differentiation and survival, we evaluated the expression of the cell signalling molecules; Extracellular signal-regulated kinase 1/2 (ERK1/2), Mitogen Activated Protein Kinase 38 (MAPK38) and p90 ribosomal protein S6 kinase (p90 RSK) in buffy coat samples. Eighty pregnant women attending a large hospital in Durban, South Africa were assigned into normotensive and pre-eclamptic groups and further stratified by their HIV status. The degree of phosphorylation of the analytes was determined using the Bio-Plex ProTM Cell Signalling Immunoassay. There was a significantly lower protein concentration of the analytes in the pre-eclamptic versus the normotensive patients, irrespective of HIV status (p < .0001). Also, there was no significant difference in expression of ERK1/2 (p = .4369), p38MAPK (p = .4720) and p90 RSK (p = .0188), according to HIV status. This study demonstrates a down-regulation of ERK1/2, p38MAPK and p90RSK prosurvival markers in pre-eclampsia. This implicates the involvement of the MAPK pathway in the pathogenesis of preeclampsia. Activation of these pathways may prove useful in increasing the body of evidence on prevention of placenta dysfunction and apoptosis. Impact statement What is already known on this subject? Preeclampsia occurring in co-morbidity with HIV is a public health problem among pregnant, black South-African women. There have been conflicting theories regarding the predisposition to the development of preeclampsia as a result of compromised immune response due to HIV infection. In normal pregnancies, the MAPK pathway plays a significant role in molecular processes involved in the cells including survival and differentiation of the placental trophoblast. ERK1/2, p38MAPK and p90RSK are members of the MAPK family, which are pro-apoptotic. Inhibition in the signalling of MAPKs has been found to result in oxidative stress, a process which contributes to the defective trophoblast invasion seen in preeclampsia. What do the results of this study add? The results from this study showed that there is no relationship between HIV infection and an increased predisposition to the development of preeclampsia. In addition, this study highlights a downregulation in the expression of ERK1/2, p38 MAPK and p90RSK in preeclampsia. What are the implications of these findings for clinical practice and/or further research? These findings demonstrate the potential of these analytes as biomarkers for the diagnosis of preeclampsia. Also, this may serve as a framework for further research in the prevention of preeclampsia by elucidating more on the pathway.

Inactivation of Sirtuin2 protects mice from acetaminophen-induced liver injury: possible involvement of ER stress and S6K1 activation.

Acetaminophen (APAP) overdose can cause hepatotoxicity by inducing mitochondrial damage and subsequent necrosis in hepatocytes. Sirtuin2 (Sirt2) is an NAD+-dependent deacetylase that regulates several biological processes, including hepatic gluconeogenesis, as well as inflammatory pathways. We show that APAP decreases the expression of Sirt2. Moreover, the ablation of Sirt2 attenuates APAP-induced liver injuries, such as oxidative stress and mitochondrial damage in hepatocytes. We found that Sirt2 deficiency alleviates the APAP-mediated endoplasmic reticulum (ER) stress and phosphorylation of the p70 ribosomal S6 kinase 1 (S6K1). Moreover, Sirt2 interacts with and deacetylates S6K1, followed by S6K1 phosphorylation induction. This study elucidates the molecular mechanisms underlying the protective role of Sirt2 inactivation in APAP-induced liver injuries. [BMB Reports 2019; 52(3): 190-195].

Dimethyl fumarate is an allosteric covalent inhibitor of the p90 ribosomal S6 kinases.

Dimethyl fumarate (DMF) has been applied for decades in the treatment of psoriasis and now also multiple sclerosis. However, the mechanism of action has remained obscure and involves high dose over long time of this small, reactive compound implicating many potential targets. Based on a 1.9 Å resolution crystal structure of the C-terminal kinase domain of the mouse p90 Ribosomal S6 Kinase 2 (RSK2) inhibited by DMF we describe a central binding site in RSKs and the closely related Mitogen and Stress-activated Kinases (MSKs). DMF reacts covalently as a Michael acceptor to a conserved cysteine residue in the αF-helix of RSK/MSKs. Binding of DMF prevents the activation loop of the kinase from engaging substrate, and stabilizes an auto-inhibitory αL-helix, thus pointing to an effective, allosteric mechanism of kinase inhibition. The biochemical and cell biological characteristics of DMF inhibition of RSK/MSKs are consistent with the clinical protocols of DMF treatment.

S6 kinase 1 plays a key role in mitochondrial morphology and cellular energy flow.

Mitochondrial morphology, which is associated with changes in metabolism, cell cycle, cell development and cell death, is tightly regulated by the balance between fusion and fission. In this study, we found that S6 kinase 1 (S6K1) contributes to mitochondrial dynamics, homeostasis and function. Mouse embryo fibroblasts lacking S6K1 (S6K1-KO MEFs) exhibited more fragmented mitochondria and a higher level of Dynamin related protein 1 (Drp1) and active Drp1 (pS616) in both whole cell extracts and mitochondrial fraction. In addition, there was no evidence for autophagy and mitophagy induction in S6K1 depleted cells. Glycolysis and mitochondrial respiratory activity was higher in S6K1-KO MEFs, whereas OxPhos ATP production was not altered. However, inhibition of Drp1 by Mdivi1 (Drp1 inhibitor) resulted in higher OxPhos ATP production and lower mitochondrial membrane potential. Taken together the depletion of S6K1 increased Drp1-mediated fission, leading to the enhancement of glycolysis. The fission form of mitochondria resulted in lower yield for OxPhos ATP production as well as in higher mitochondrial membrane potential. Thus, these results have suggested a potential role of S6K1 in energy metabolism by modulating mitochondrial respiratory capacity and mitochondrial morphology.

Fibroblast growth factor signalling induces loss of progesterone receptor in breast cancer cells.

We have recently demonstrated that, fibroblast growth factor 2 (FGFR2), signalling via ribosomal S6 kinase 2 (RSK2), promotes progression of breast cancer (BCa). Loss of progesterone receptor (PR), whose activity in BCa cells can be stimulated by growth factor receptors (GFRs), is associated with poor patient outcome. Here we showed that FGF7/FGFR2 triggered phosphorylation of PR at Ser294, PR ubiquitination and subsequent receptor`s degradation via the 26S proteasome pathway in BCa cells. We further demonstrated that RSK2 mediated FGF7/FGFR2-induced PR downregulation. In addition, a strong synergistic effect of FGF7 and progesterone (Pg), reflected in the enhanced anchorage-independent growth and cell migration, was observed. Analysis of clinical material demonstrated that expression of PR inversely correlated with activated RSK (RSK-P) (p = 0.016). Patients with RSK-P(+)/PR(-) tumours had 3.629-fold higher risk of recurrence (p = 0.002), when compared with the rest of the cohort. Moreover, RSK-P(+)/PR(-) phenotype was shown as an independent prognostic factor (p = 0.006). These results indicate that the FGF7/FGFR2-RSK2 axis promotes PR turnover and activity, which may sensitize BCa cells to stromal stimuli and contribute to the progression toward steroid hormone negative BCa.

RSK2 activity mediates glioblastoma invasiveness and is a potential target for new therapeutics.

In glioblastoma (GBM), infiltration of primary tumor cells into the normal tissue and dispersal throughout the brain is a central challenge to successful treatment that remains unmet. Indeed, patients respond poorly to the current therapies of tumor resection followed by chemotherapy with radiotherapy and have only a 16-month median survival. It is therefore imperative to develop novel therapies. RSK2 is a kinase that regulates proliferation and adhesion and can promote metastasis. We demonstrate that active RSK2 regulates GBM cell adhesion and is essential for cell motility and invasion of patient-derived GBM neurospheres. RSK2 control of adhesion and migration is mediated in part by its effects on integrin-Filamin A complexes. Importantly, inhibition of RSK2 by either RSK inhibitors or shRNA silencing impairs invasion and combining RSK2 inhibitors with temozolomide improves efficacy in vitro. In agreement with the in vitro data, using public datasets, we find that RSK2 is significantly upregulated in vivo in human GBM patient tumors, and that high RSK2 expression significantly correlates with advanced tumor stage and poor patient survival. Together, our data provide strong evidence that RSK inhibitors could enhance the effectiveness of existing GBM treatment, and support RSK2 targeting as a promising approach for novel GBM therapy.

MSK1 triggers the expression of the INK4AB/ARF locus in oncogene-induced senescence.

The tumor suppressor proteins p15(INK4B), p16(INK4A), and p14(ARF), encoded by the INK4AB/ARF locus, are crucial regulators of cellular senescence. The locus is epigenetically silenced by the repressive Polycomb complexes in growing cells but is activated in response to oncogenic stress. Here we show that the mitogen- and stress-activated kinase (MSK1) is up-regulated after RAF1 oncogenic stress and that the phosphorylated (activated) form of MSK1 is significantly increased in the nucleus and recruited to the INK4AB/ARF locus. We show that MSK1 mediates histone H3S28 phosphorylation at the INK4AB/ARF locus and contributes to the rapid transcriptional activation of p15(INK4B) and p16(INK4A) in human cells despite the presence of the repressive H3K27me3 mark. Furthermore, we show that upon MSK1 depletion in oncogenic RAF1-expressing cells, H3S28ph presence at the INK4 locus and p15(INK4B) and p16(INK4A) expression are reduced. Finally, we show that H3S28-MSK-dependent phosphorylation functions in response to RAF1 signaling and that ERK and p38α contribute to MSK1 activation in oncogene-induced senescence.

[IL-33 promotes airway remodeling in a mouse model of asthma via ERK1/2 signaling pathway].

OBJECTIVE: To explore the role of IL-33 in asthmatic airway remodeling. METHODS: Male BALB/c mice were randomly divided into 3 groups: a control group, an ovalbumin (OVA) group, and an anti-IL-33 antibody combined with OVA group. The airway remodeling features in mice were observed by HE staining. In addition, the expressions of IL-33, alpha smooth muscle actin (α-SMA), and type 1 collagen (Col1) in the airway of mice were detected by immunohistochemistry and Western blotting. Finally, Western blotting was used to determine the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and mitogen- and stress-activated protein kinase 1 (MSK1) in the lungs of mice. In vitro, human lung fibroblasts (HLF-1) were pretreated with the ERK1/2 inhibitor U0126 or the MSK1 inhibitor H89 respectively, and then treated with the human recombinant IL-33 (rIL-33). Then real-time quantitative PCR and Western blotting were used to test the expressions of α-SMA and Col1. Immunofluorescence cytochemistry and Western blotting were also used to observe the phosphorylation of ERK1/2 and MSK1 in HLF-1 cells. RESULTS: The pre-treatment with the ERK1/2 inhibitor U0126 or anti-IL-33 antibody significantly abolished the OVA-induced airway remodeling, increased expressions of IL-33, α-SMA, Col1, and phosphorylation of ERK1/2 and MSK1 in the airway of mice. In vitro, the increased expressions of α-SMA and Col1 and the phosphorylation of ERK1/2 and MSK1 induced by rIL-33 in HLF-1 cells were markedly inhibited by the pre-treatment with U0126 or H89. CONCLUSION: IL-33 promotes airway remodeling in asthmatic mice via the ERK1/2-MSK1 signaling pathway.

Rsk2, the Kinase Mutated in Coffin-Lowry Syndrome, Controls Cementum Formation.

The ribosomal S6 kinase RSK2 is essential for osteoblast function, and inactivating mutations of RSK2 cause osteopenia in humans with Coffin-Lowry syndrome (CLS). Alveolar bone loss and premature tooth exfoliation are also consistently reported symptoms in CLS patients; however, the pathophysiologic mechanisms are unclear. Therefore, aiming to identify the functional relevance of Rsk2 for tooth development, we analyzed Rsk2-deficient mice. Here, we show that Rsk2 is a critical regulator of cementoblast function. Immunohistochemistry, histology, micro-computed tomography imaging, quantitative backscattered electron imaging, and in vitro assays revealed that Rsk2 is activated in cementoblasts and is necessary for proper acellular cementum formation. Cementum hypoplasia that is observed in Rsk2-deficient mice causes detachment and disorganization of the periodontal ligament and was associated with significant alveolar bone loss with age. Moreover, Rsk2-deficient mice display hypomineralization of cellular cementum with accumulation of nonmineralized cementoid. In agreement, treatment of the cementoblast cell line OCCM-30 with a Rsk inhibitor reduces formation of mineralization nodules and decreases the expression of cementum markers. Western blot analyses based on antibodies against Rsk1, Rsk2, and an activated form of the 2 kinases confirmed that Rsk2 is expressed and activated in differentiating OCCM-30 cells. To discriminate between periodontal bone loss and systemic bone loss, we additionally crossed Rsk2-deficient mice with transgenic mice overexpressing the osteoanabolic transcription factor Fra1. Fra1 overexpression clearly increases systemic bone volume in Rsk2-deficient mice but does not protect from alveolar bone loss. Our results indicate that cell autonomous cementum defects are causing early tooth loss in CLS patients. Moreover, we identify Rsk2 as a nonredundant regulator of cementum homeostasis, alveolar bone maintenance, and periodontal health, with all these features being independent of Rsk2 function in systemic bone formation.

Kinase analysis in alcoholic hepatitis identifies p90RSK as a potential mediator of liver fibrogenesis.

OBJECTIVE: Alcoholic hepatitis (AH) is often associated with advanced fibrosis, which negatively impacts survival. We aimed at identifying kinases deregulated in livers from patients with AH and advanced fibrosis in order to discover novel molecular targets. DESIGN: Extensive phosphoprotein analysis by reverse phase protein microarrays was performed in AH (n=12) and normal human livers (n=7). Ribosomal S6 kinase (p90RSK) hepatic expression was assessed by qPCR, Western blot and immunohistochemistry. Kaempferol was used as a selective pharmacological inhibitor of the p90RSK pathway to assess the regulation of experimentally-induced liver fibrosis and injury, using in vivo and in vitro approaches. RESULTS: Proteomic analysis identified p90RSK as one of the most deregulated kinases in AH. Hepatic p90RSK gene and protein expression was also upregulated in livers with chronic liver disease. Immunohistochemistry studies showed increased p90RSK staining in areas of active fibrogenesis in cirrhotic livers. Therapeutic administration of kaempferol to carbon tetrachloride-treated mice resulted in decreased hepatic collagen deposition, and expression of profibrogenic and proinflammatory genes, compared to vehicle administration. In addition, kaempferol reduced the extent of hepatocellular injury and degree of apoptosis. In primary hepatic stellate cells, kaempferol and small interfering RNA decreased activation of p90RSK, which in turn regulated key profibrogenic actions. In primary hepatocytes, kaempferol attenuated proapoptotic signalling. CONCLUSIONS: p90RSK is upregulated in patients with chronic liver disease and mediates liver fibrogenesis in vivo and in vitro. These results suggest that the p90RSK pathway could be a new therapeutic approach for liver diseases characterised by advanced fibrosis.

Rsk2 controls synovial fibroblast hyperplasia and the course of arthritis.

OBJECTIVE: Arthritis is a chronic inflammatory disease characterised by immune cell infiltration and mesenchymal cell expansion in the joints. Although the role of immune cells in arthritis is well characterised, the development of mesenchymal cell hyperplasia needs to be better defined. Here, we analysed the role of the ribosomal S6 kinase Rsk2, which we found to be highly activated in joints of patients with arthritis, in the development of mesenchymal cell hyperplasia. METHODS: We genetically inactivated Rsk2 in the tumour necrosis factor (TNF)-α transgenic (TNFtg) mice, an animal model for human inflammatory arthritis. Clinical and histological signs of arthritis as well as molecular markers of inflammation and joint destruction were quantified. Fibroblast-like synoviocytes (FLS) were characterised in vitro and the effect of Rsk2 deletion on the pattern of gene expression was determined. RESULTS: Rsk2 deficiency in TNFtg mice results in earlier and exacerbated inflammation as well as increased bone and cartilage destruction. The production of inflammatory cytokines, matrix metalloproteinases and osteoclastogenic molecules was significantly increased in vivo upon Rsk2 inactivation. Bone marrow deficient in Rsk2 could not transfer this phenotype, indicating that Rsk2 expression in mesenchymal cells controls the course of arthritis. Indeed, Rsk2 deficiency was associated with a more activated phenotype and higher proliferative capacity of FLS, thereby increasing cytokines and production of matrix proteinases. CONCLUSIONS: Rsk2 emerges as a key regulator of mesenchymal cell numbers in the joint and thereby could be targeted to control the inflammatory and tissue-destructive feature of joints in arthritis.

S6 Kinase- and ß-TrCP2-Dependent Degradation of p19Arf Is Required for Cell Proliferation.

The kinase mTOR (mammalian target of rapamycin) promotes translation as well as cell survival and proliferation under nutrient-rich conditions. Whereas mTOR activates translation through ribosomal protein S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4E-BP), how it facilitates cell proliferation has remained unclear. We have now identified p19(Arf), an inhibitor of cell cycle progression, as a novel substrate of S6K that is targeted to promote cell proliferation. Serum stimulation induced activation of the mTOR-S6K axis and consequent phosphorylation of p19(Arf) at Ser(75). Phosphorylated p19(Arf) was then recognized by the F-box protein ß-TrCP2 and degraded by the proteasome. Ablation of ß-TrCP2 thus led to the arrest of cell proliferation as a result of the stabilization and accumulation of p19(Arf). The ß-TrCP2 paralog ß-TrCP1 had no effect on p19(Arf) stability, suggesting that phosphorylated p19(Arf) is a specific substrate of ß-TrCP2. Mice deficient in ß-TrCP2 manifested accumulation of p19(Arf) in the yolk sac and died in utero. Our results suggest that the mTOR pathway promotes cell proliferation via ß-TrCP2-dependent p19(Arf) degradation under nutrient-rich conditions.

S6K1 controls pancreatic ß cell size independently of intrauterine growth restriction.

Type 2 diabetes mellitus (T2DM) is a worldwide heath problem that is characterized by insulin resistance and the eventual loss of ß cell function. As recent studies have shown that loss of ribosomal protein (RP) S6 kinase 1 (S6K1) increases systemic insulin sensitivity, S6K1 inhibitors are being pursued as potential agents for improving insulin resistance. Here we found that S6K1 deficiency in mice also leads to decreased ß cell growth, intrauterine growth restriction (IUGR), and impaired placental development. IUGR is a common complication of human pregnancy that limits the supply of oxygen and nutrients to the developing fetus, leading to diminished embryonic ß cell growth and the onset of T2DM later in life. However, restoration of placental development and the rescue of IUGR by tetraploid embryo complementation did not restore ß cell size or insulin levels in S6K1-/- embryos, suggesting that loss of S6K1 leads to an intrinsic ß cell lesion. Consistent with this hypothesis, reexpression of S6K1 in ß cells of S6K1-/- mice restored embryonic ß cell size, insulin levels, glucose tolerance, and RPS6 phosphorylation, without rescuing IUGR. Together, these data suggest that a nutrient-mediated reduction in intrinsic ß cell S6K1 signaling, rather than IUGR, during fetal development may underlie reduced ß cell growth and eventual development of T2DM later in life.