Inhibition of the RPS6KA1/FoxO1 signaling axis by hydroxycitric acid attenuates HFD-induced obesity through MCE suppression.

BACKGROUND: Because obesity is associated with a hyperplasia-mediated increase in adipose tissue, inhibiting cell proliferation during mitotic clonal expansion (MCE) is a leading strategy for preventing obesity. Although (-)-hydroxycitric acid (HCA) is used to control obesity, the molecular mechanisms underlying its effects on MCE are poorly understood. PURPOSE: This study aimed to investigate the potential effects of HCA on MCE and underlying molecular mechanisms affecting adipogenesis and obesity improvements. METHODS: Preadipocyte cell line, 3T3-L1, were treated with HCA; oil red O, cell proliferation, cell cycle, and related alterations in signaling pathways were examined. High-fat diet (HFD)-fed mice were administered HCA for 12 weeks; body and adipose tissues weights were evaluated, and the regulation of signaling pathways in epidydimal white adipose tissue were examined in vivo. RESULTS: Here, we report that during MCE, HCA attenuates the proliferation of the preadipocyte cell line, 3T3-L1, by arresting the cell cycle at the G0/G1 phase. In addition, HCA markedly inhibits Forkhead Box O1 (FoxO1) phosphorylation, thereby inducing the expression of cyclin-dependent kinase inhibitor 1B and suppressing the levels of cyclin-dependent kinase 2, cyclin E1, proliferating cell nuclear antigen, and phosphorylated retinoblastoma. Importantly, we found that ribosomal protein S6 kinase A1 (RPS6KA1) influences HCA-mediated inactivation of FoxO1 and its nuclear exclusion. An animal model of obesity revealed that HCA reduced high-fat diet-induced obesity by suppressing adipocyte numbers as well as epididymal and mesenteric white adipose tissue mass, which is attributed to the regulation of RPS6KA1, FoxO1, CDKN1B and PCNA that had been consistently identified in vitro. CONCLUSIONS: These findings provide novel insights into the mechanism by which HCA regulates adipogenesis and highlight the RPS6KA1/FoxO1 signaling axis as a therapeutic target for obesity.

Protective Autophagy in 5-Fluorouracil-Resistant Colorectal Cancer Cells and ERK-RSK-ABCG2 Linkage / Autofagia Protectora en Células de Cáncer Colorrectal Resistentes al 5-Fluorouracilo y Enlace ERK-RSK-ABCG2

SUMMARY: To evaluate the anti-cancer effects of yeast extract on resistant cells, autophagy and necroptosis were investigated in 5-fluorouracil (5-FU)-resistant colorectal cancer cells. Further underlying characteristics on drug resistance were evaluated, focused on ERK-RSK-ABCG2 linkage. SNU-C5 and 5-FU resistant SNU-C5 (SNU-C5/5-FUR) colorectal cancer cells were adopted for cell viability assay and Western blotting to examine the anti-cancer effects of yeast extract. Yeast extract induced autophagy in SNU-C5 cells with increased Atg7, Atg12-5 complex, Atg16L1, and LC3 activation (LC3-II/LC3-I), but little effects in SNU-C5/5-FUR cells with increased Atg12-5 complex and Atg16L1. Both colorectal cancer cells did not show necroptosis after yeast extract treatment. Based on increased ABCG2 and RSK expression after yeast extract treatment, drug resistance mechanisms were further evaluated. As compared to wild type, SNU-C5/5-FUR cells showed more ABCG2 expression, less RSK expression, and less phosphorylation of ERK. ABCG2 inhibitor, Ko143, treatment induces following changes: 1) more sensitivity at 500 mM 5-FU, 2) augmented proliferation, and 3) less phosphorylation of ERK. These results suggest that protective autophagy in SNU-C5/5-FUR cells with increased ABCG2 expression might be candidate mechanisms for drug resistance. As the ERK responses were different from each stimulus, the feasible mechanisms among ERK-RSK-ABCG2 should be further investigated in 5-FU-resistant CRC cells.

Para evaluar los efectos anticancerígenos del extracto de levadura en células resistentes, se investigaron la autofagia y la necroptosis en células de cáncer colorrectal resistentes al 5-fluorouracilo (5-FU). Además se evaluaron otras características subyacentes de la resistencia a los medicamentos centrándose en el enlace ERK-RSK-ABCG2. Se usaron células de cáncer colorrectal SNU-C5 (SNU-C5/5-FUR) resistentes a SNU-C5 y 5- FU para el ensayo de viabilidad celular y la transferencia Western para examinar los efectos anticancerígenos del extracto de levadura. El extracto de levadura indujo autofagia en células SNU-C5 con mayor activación de Atg7, complejo Atg12-5, Atg16L1 y LC3 (LC3-II/LC3-I), pero pocos efectos en células SNU-C5/5-FUR con aumento de Atg12-5 complejo y Atg16L1. Ambas células de cáncer colorrectal no mostraron necroptosis después del tratamiento con extracto de levadura. Se evaluaron los mecanismos de resistencia a los medicamentos. en base al aumento de la expresión de ABCG2 y RSK después del tratamiento con extracto de levadura.En comparación con las de tipo salvaje, las células SNU-C5/5-FUR mostraron más expresión de ABCG2, menos expresión de RSK y menos fosforilación de ERK. El tratamiento con inhibidor de ABCG2, Ko143, induce los siguientes cambios: 1) más sensibilidad a 5-FU 500 mM, 2) proliferación aumentada y 3) menos fosforilación de ERK. Estos resultados sugieren que la autofagia protectora en células SNU-C5/5-FUR con mayor expresión de ABCG2 podría ser un mecanismo candidato para la resistencia a los medicamentos. Como las respuestas de ERK fueron diferentes de cada estímulo, los mecanismos factibles entre ERK-RSK- ABCG2 deberían investigarse más a fondo en células CCR resistentes a 5-FU.

ASMq protects against early burn wound progression in rats by alleviating oxidative stress and secondary mitochondria­associated apoptosis via the Erk/p90RSK/Bad pathway.

Burn wounds present an evolutionary progression, in which the initial wound tissue deepens and expands following thermal injury. Progressive tissue damage in the zone of stasis may worsen burn injury, which is associated with oxidative stress and secondary apoptosis, and worsen the prognosis of patients with burn wounds. The mitochondrial apoptotic pathway is involved in receiving oxidative signals and regulating tissue apoptosis. Previously, Abnormal Savda Munziq (ASMq), a natural compound of traditional Uyghur Medicine, which includes ten types of herb, has been reported to exhibit a number of effects, including anti­inflammatory, antioxidative and anti­apoptotic activities. The present study demonstrated that ASMq protected against early burn wound progression following thermal injury in rats; this effect may be mediated by its ability to attenuate oxidative stress­induced mitochondria­associated apoptosis. The present study may provide a novel therapeutic method to prevent early burn wound progression following burn injury.

Angelica sinensis extract protects against ischemia-reperfusion injury in the hippocampus by activating p38 MAPK-mediated p90RSK/p-Bad and p90RSK/CREB/BDNF signaling after transient global cerebral ischemia in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Angelica sinensis (Oliv.) Diels, commonly known as Dang Gui (DG), is one of the most popular traditional Chinese herbal medicines for the treatment of stroke. However, the effects of DG on transient global cerebral ischemia (GCI) and its precise mechanisms remain unclear. AIM OF THE STUDY: This study aimed to investigate the effects of the DG extract on ischemia-reperfusion (I/R) injury in the hippocampus 7 d after transient GCI and to identify the potential mitogen-activated protein kinase (MAPK)-related signaling pathway in the hippocampus involved in the effects. MATERIALS AND METHODS: Rats were intragastrically administered DG at doses of 0.25 g/kg (DG-0.25g), 0.5 g/kg (DG-0.5g), or 1 g/kg (DG-1g) 1, 3, and 5 d after GCI. RESULTS: DG-0.5g and DG-1g treatments effectively promoted hippocampal cornu ammonis 1 (CA1) neuronal survival. DG-0.5g and DG-1g treatments markedly increased phospho-p38 MAPK (p-p38 MAPK), phospho-90-kDa ribosomal S6 kinase (p-p90RSK), cytosolic and mitochondrial phospho-Bad (p-Bad), phospho-cAMP response element-binding protein (p-CREB), brain-derived neurotrophic factor (BDNF), and p-CREB/BDNF expression; decreased 4-hydroxy-2-nonenal, cytochrome c (Cytc), and cleaved caspase-3 expression, and inhibited apoptosis in the hippocampal CA1 region. Pretreatment with a specific inhibitor of p38 MAPK, SB203580, completely blocked the effects of DG-1g on the expression of the aforementioned proteins. CONCLUSIONS: DG-0.5g and DG-1g treatments exerted neuroprotective effects on I/R injury by activating p38 MAPK-mediated p90RSK/p-Bad-induced anti-apoptotic-Cytc/caspase-3-related and p90RSK/CREB/BDNF survival signaling in the hippocampus 7 d after transient GCI.

Modulation of hypothalamic S6K1 and S6K2 alters feeding behavior and systemic glucose metabolism.

The mTOR/S6Ks signaling is one of the intracellular pathways important for metabolic control, acting both peripherally and centrally. In the hypothalamus, mTOR/S6Ks axis mediates the action of leptin and insulin and can modulate the expression of neuropeptides. We analyzed the role of different S6Ks isoforms in the hypothalamic regulation of metabolism. We observed decreased food intake and decreased expression of agouti-related peptide (AgRP) following intracerebroventricular (icv) injections of adenoviral-mediated overexpression of three different S6Ks isoforms. Moreover, mice overexpressing p70-S6K1 in undefined periventricular hypothalamic neurons presented changes in glucose metabolism, as an increase in gluconeogenesis. To further evaluate the hypothalamic role of a less-studied S6K isoform, p54-S6K2, we used a Cre-LoxP approach to specifically overexpress it in AgRP neurons. Our findings demonstrate the potential participation of S6K2 in AgRP neurons regulating feeding behavior.

Identification of PP2A and S6 Kinase as Modifiers of Leucine-Rich Repeat Kinase-Induced Neurotoxicity.

Mutations in LRRK2 are currently recognized as the most common monogenetic cause of Parkinsonism. The elevation of kinase activity of LRRK2 that frequently accompanies its mutations is widely thought to contribute to its toxicity. Accordingly, many groups have developed LRRK2-specific kinase inhibitors as a potential therapeutic strategy. Given that protein phosphorylation is a reversible event, we sought to elucidate the phosphatase(s) that can reverse LRRK2-mediated phosphorylation, with the view that targeting this phosphatase(s) may similarly be beneficial. Using an unbiased RNAi phosphatase screen conducted in a Drosophila LRRK2 model, we identified PP2A as a genetic modulator of LRRK2-induced neurotoxicity. Further, we also identified ribosomal S6 kinase (S6K), a target of PP2A, as a novel regulator of LRRK2 function. Finally, we showed that modulation of PP2A or S6K activities ameliorates LRRK2-associated disease phenotype in Drosophila.

Anti-epileptic effect of 16-O-acetyldigitoxigenin via suppressing mTOR signaling pathway.

Epilepsy is a common chronic disease of the central nervous system that can last for years or even decades, causing serious adverse effects on the body, mind, and psychology of patients. Traditional antiepileptic drugs can effectively control seizures, but because of large individual differences, serious adverse reactions, narrow therapeutic window and other shortcomings, more effective, new treatment drugs are looked for. Streptocaulon griffithii is a plant of Asclepiadaceae. 16-O-acetyldigitoxigenin (ACE) is a strong cardiac glycoside isolated from methanol extract of Streptocaulon griffithii. The aim of this study was to investigate the antiepileptic effect of ACE on Pilocarpine (Pilo) induced epilepsy in mice, and to explore the effect of mTOR signaling pathway on its antiepileptic effect. The results showed that ACE had antiepileptic and neuroprotective effects on Pilo induced epilepsy mice. ACE attenuates Pilo induced seizures by inhibiting the activation of p-mTOR/p-70S6K pathway, and inhibits Pilocarpine induced brain damage by inhibiting mTOR signaling pathway. These results suggest that ACE has a promising future in the treatment of epilepsy and other nervous system diseases.

Lapachol is a novel ribosomal protein S6 kinase 2 inhibitor that suppresses growth and induces intrinsic apoptosis in esophageal squamous cell carcinoma cells.

Lapachol is a 1,4-naphthoquinone that is isolated from the Bignoniaceae family. It has been reported to exert anti-inflammatory, antibacterial, and anticancer activities. However, the anticancer activity of lapachol and its molecular mechanisms against esophageal squamous cell carcinoma (ESCC) cells have not been fully investigated. Herein, we report that lapachol is a novel ribosomal protein S6 kinase 2 (RSK2) inhibitor that suppresses growth and induces intrinsic apoptosis in ESCC cells. We found that lapachol strongly attenuates downstream signaling molecules of RSK2 in ESCC cells and also directly inhibits RSK2 activity in vitro. The RSK protein is highly activated in ESCC cells and knockdown of RSK2 significantly suppresses anchorage-dependent and anchorage-independent growth of ESCC cells. Additionally, lapachol inhibits anchorage-dependent and anchorage-independent growth of ESCC cells, and the inhibition of cell growth by lapachol is dependent on the expression of RSK2. We also found that lapachol induces mitochondria-mediated cellular apoptosis by activating caspases-3, -7, and PARP, inducing the expression of cytochrome c and BAX by inhibiting downstream molecules of RSK2. Overall, lapachol is a potent RSK2 inhibitor that might be used for chemotherapy against ESCC.

Isobavachalcone exerts anti­proliferative and pro­apoptotic effects on human liver cancer cells by targeting the ERKs/RSK2 signaling pathway.

Aberrant activation of the extracellular signal­regulated kinases (ERKs)/ribosomal S6 kinase 2 (RSK2) signaling pathway is frequently determined in various human tumor types, including liver cancer, and has been considered as a promising target for cancer chemoprevention and therapy. In the present study, using computer­aided virtual screening and molecular docking, isobavachalcone (IBC), a natural chalcone compound, was identified to be an ATP­competitive inhibitor targeting ERK1/2 and RSK2. Cell Counting Kit­8, EdU incorporation and colony formation assays were used to detect the effects of IBC on cell viability and proliferation, and the results demonstrated that IBC effectively inhibited the proliferation of liver cancer HepG2 and Hep3B cells, whereas it had no notable cytotoxic effect on immortal liver L02 cells. Flow cytometric analysis and western blotting further revealed that IBC caused significant levels of apoptosis on liver cancer cells via the caspase­dependent mitochondria pathway. The computer prediction was confirmed with pull­down and in vitro kinase assays, in which IBC directly bound with ERK1/2 and RSK2, and dose­dependently blocked RSK2 kinase activity in liver cancer cells. Treatment of HepG2 or Hep3B cells with IBC significantly attenuated epidermal growth factor­induced phosphorylation of RSK2 and resulted in the reduced activation of its downstream substrates including cAMP response element­binding protein, activating transcription factor 1, histone H3 and activating protein­1. Enforced RSK2 expression in L02 cells could increase the effect of IBC on suppressing cell growth. Conversely, knockdown of RSK2 reduced the inhibitory effect of IBC on HepG2 cell proliferation. Overall, the present data indicated that ERKs/RSK2 signaling serves a pivotal role in IBC­induced suppression of liver cancer cells and that IBC may be a potential therapeutic candidate for human cancer with elevated ERKs/RSK2 activity.

Epimagnolin targeting on an active pocket of mammalian target of rapamycin suppressed cell transformation and colony growth of lung cancer cells.

Mammalian target of rapamycin (mTOR) has a pivotal role in carcinogenesis and cancer cell proliferation in diverse human cancers. In this study, we observed that epimagnolin, a natural compound abundantly found in Shin-Yi, suppressed cell proliferation by inhibition of epidermal growth factor (EGF)-induced G1/S cell-cycle phase transition in JB6 Cl41 cells. Interestingly, epimagnolin suppressed EGF-induced Akt phosphorylation strongly at Ser473 and weakly at Thr308 without alteration of phosphorylation of MAPK/ERK kinases (MEKs), extracellular signal-regulated kinase (ERKs), and RSK1, resulting in abrogation of the phosphorylation of GSK3ß at Ser9 and p70S6K at Thr389. Moreover, we found that epimagnolin suppressed c-Jun phosphorylation at Ser63/73, resulting in the inhibition of activator protein 1 (AP-1) transactivation activity. Computational docking indicated that epimagnolin targeted an active pocket of the mTOR kinase domain by forming three hydrogen bonds and three hydrophobic interactions. The prediction was confirmed by using in vitro kinase and adenosine triphosphate-bead competition assays. The inhibition of mTOR kinase activity resulted in the suppression of anchorage-independent cell transformation. Importantly, epimagnolin efficiently suppressed cell proliferation and anchorage-independent colony growth of H1650 rather than H460 lung cancer cells with dependency of total and phosphorylated protein levels of mTOR and Akt. Inhibitory signaling of epimagnolin on cell proliferation of lung cancer cells was observed mainly in mTOR-Akt-p70S6K and mTOR-Akt-GSK3ß-AP-1, which was similar to that shown in JB6 Cl41 cells. Taken together, our results indicate that epimagnolin potentiates as chemopreventive or therapeutic agents by direct active pocket targeting of mTOR kinase, resulting in sensitizing cancer cells harboring enhanced phosphorylation of the mTORC2-Akt-p70S6k signaling pathway.

Gossypin inhibits gastric cancer growth by direct targeting of AURKA and RSK2.

Gossypin is a flavone extracted from Hibiscus vitifolius, which has been reported to exhibit anti-inflammatory, antioxidant, and anticancer activities. However, the anticancer properties of gossypin and its molecular mechanism of action against gastric cancer have not been fully investigated. In the present study, we report that gossypin is an Aurora kinase A (AURKA) and RSK2 inhibitor that suppresses gastric cancer growth. Gossypin attenuated anchorage-dependent and anchorage-independent gastric cancer cell growth as well as cell migration. Based on the results of in vitro screening and cell-based assays, gossypin directly binds to and inhibits AURKA and RSK2 activities and their downstream signaling proteins. Gossypin decreased S phase and increased G2/M phase cell cycle arrest by reducing the expression of cyclin A2 and cyclin B1 and the phosphorylation of the CDC protein. Additionally, gossypin also induced intrinsic apoptosis by activating caspases and PARP and increasing the expression of cytochrome c. Our results demonstrate that gossypin is an AURKA and RSK2 inhibitor that could be useful for treating gastric cancer.

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.

A natural ent­kaurane diterpenoid induces antiproliferation in ovarian cancer cells via ERK1/2 regulation and inhibition of cellular migration and invasion.

Ovarian cancer is one of the most common causes of female mortalities from gynecological tumors. An ent­kaurane diterpenoid compound CRT1 (ent­18­acetoxy­7ß­hydroxy kaur­15­oxo­16­ene), mainly isolated from the Vietnamese herb Croton tonkinesis has been used in folk medicine in Vietnam for cancer treatment. However, the effect of this compound on human ovarian cancer cells has not yet been reported. The objective of the present study was to determine the effect of CRT1 on the cell viability, apoptosis and metastasis of SKOV3 human ovarian cancer cells using a Cell Counting Kit­8 assay, flow cytometric analysis of Annexin V­fluorescein isothiocyanate/propidium iodide staining, western blot analysis, soft agar colony forming assay, wound healing assay and Matrigel invasion assay. The results revealed that CRT1 possessed significant anti­proliferative effects on SKOV3 cells. CRT1 treatment at 25 and 50 µM induced apoptosis, enhanced the percentage of Annexin V­positive cells, increased the expression of pro­apoptotic protein B­cell lymphoma 2 (Bcl­2)­associated X protein, cytochrome c release from the mitochondria to the cytosol, cleaved caspase­3, caspase­7, caspase­9, and poly (adenosine diphosphate­ribose) polymerase. However, it decreased the expression of Bcl­2 in a dose­dependent manner. The percentage of necrotic cells increased following CRT1 treatment at <10 µM. CRT1 at 50 µM significantly induced the phosphorylation of extracellular signal­regulated kinase (ERK). Growth inhibition and the apoptotic effects of CRT1 could be reversed by PD98059, an ERK inhibitor. Additionally, CRT1 inhibited cell migration and invasion via ERK1/2 activation in SKOV3 cells. These results indicated that CRT1, an ent­kaurane diterpenoid, may be a potential inhibitor of ovarian cancer by the activating ERK1/2/p90 ribosomal S6 kinase signaling pathway.

EGb761 Ameliorates Neuronal Apoptosis and Promotes Angiogenesis in Experimental Intracerebral Hemorrhage via RSK1/GSK3ß Pathway.

Neuronal apoptosis after intracerebral hemorrhage (ICH) plays an essential role in neurological deterioration. Preclinical studies have shown that EGb761, an extract of Ginkgo biloba, is neuroprotective in some other neurological diseases with apoptosis. This study was conducted to investigate the potential neuroprotective effect of EGb761 on neuronal apoptosis in experimental ICH. A model of ICH was induced in C57BL/6 mice by injecting collagenase. EGb761 was administered for 21 days and neurologic behaviors were assessed at 1, 3, 7, 14, and 21 days after ICH. RNAi-mediated knockdown of p90 ribosomal S6 kinase 1 (RSK1) was used to further investigate the role of RSK1 in EGb761-induced neuroprotective effects. Neuronal death was determined by TUNEL staining. The image datasets of neurovascular networks were acquired via micro-optical sectioning tomography (MOST). The glycogen synthase kinase-3ß (GSK3ß) activity was assayed using commercial kit. Primary cultured cortical neurons were exposed to ferrous iron and treated with EGb761. Apoptotic neurons were counted by flow cytometry. RSK1, GSK3ß, phosphorylated-GSK3ß (pGSK3ß), Bcl2, Bax, cleaved-caspase3 (CC3), and VEGF were measured by Western blot. The pGSK3ß was also detected by immunofluorescence staining. We found that mice in EGb761 group performed better on rotarod test. Reduced TUNEL-positive neurons and richer microvascular networks were observed in mice treated with EGb761. EGb761 attenuates neuronal apoptosis induced by ferrous iron counted by flow cytometry in vitro. Decreased GSK3ß activity was observed in EGb761-treated mice compared with mice with ICH. EGb761 increased the expression of pGSK3ß (Ser9), RSK1 and the Bcl2/Bax ratio, and VEGF and decreased CC3 expression. In conclusion, EGb761 reduces neuronal apoptosis and promotes angiogenesis in experimental intracerebral hemorrhage via RSK1/GSK3ß pathway.

Muscle-specific deletion of BDK amplifies loss of myofibrillar protein during protein undernutrition.

Branched-chain amino acids (BCAAs) are essential amino acids for mammals and play key roles in the regulation of protein metabolism. However, the effect of BCAA deficiency on protein metabolism in skeletal muscle in vivo remains unclear. Here we generated mice with lower BCAA concentrations by specifically accelerating BCAA catabolism in skeletal muscle and heart (BDK-mKO mice). The mice appeared to be healthy without any obvious defects when fed a protein-rich diet; however, bolus ingestion of BCAAs showed that mTORC1 sensitivity in skeletal muscle was enhanced in BDK-mKO mice compared to the corresponding control mice. When these mice were fed a low protein diet, the concentration of myofibrillar protein was significantly decreased (but not soluble protein) and mTORC1 activity was reduced without significant change in autophagy. BCAA supplementation in drinking water attenuated the decreases in myofibrillar protein levels and mTORC1 activity. These results suggest that BCAAs are essential for maintaining myofibrillar proteins during protein undernutrition by keeping mTORC1 activity rather than by inhibiting autophagy and translation. This is the first report to reveal the importance of BCAAs for protein metabolism of skeletal muscle in vivo.

Pulsatile delivery of a leucine supplement during long-term continuous enteral feeding enhances lean growth in term neonatal pigs.

Neonatal pigs are used as a model to study and optimize the clinical treatment of infants who are unable to maintain oral feeding. Using this model, we have shown previously that pulsatile administration of leucine during continuous feeding over 24 h via orogastric tube enhanced protein synthesis in skeletal muscle compared with continuous feeding alone. To determine the long-term effects of leucine pulses, neonatal piglets (n = 11-12/group) were continuously fed formula via orogastric tube for 21 days, with an additional parenteral infusion of either leucine (CON + LEU; 800 µmol·kg-1·h-1) or alanine (CON + ALA) for 1 h every 4 h. The results show that body and muscle weights and lean gain were ∼25% greater, and fat gain was 48% lower in CON + LEU than CON + ALA; weights of other tissues were unaffected by treatment. Fractional protein synthesis rates in longissimus dorsi, gastrocnemius, and soleus muscles were ∼30% higher in CON + LEU compared with CON + ALA and were associated with decreased Deptor abundance and increased mTORC1, mTORC2, 4E-BP1, and S6K1 phosphorylation, SNAT2 abundance, and association of eIF4E with eIF4G and RagC with mTOR. There were no treatment effects on PKB, eIF2α, eEF2, or PRAS40 phosphorylation, Rheb, SLC38A9, v-ATPase, LAMTOR1, LAMTOR2, RagA, RagC, and LAT1 abundance, the proportion of polysomes to nonpolysomes, or the proportion of mRNAs encoding rpS4 or rpS8 associated with polysomes. Our results demonstrate that pulsatile delivery of a leucine supplement during 21 days of continuous enteral feeding enhances lean growth by stimulating the mTORC1-dependent translation initiation pathway, leading to protein synthesis in skeletal muscle of neonates.

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.

Silkworm Thorn Stem Extract Targets RSK2 and Suppresses Solar UV-Induced Cyclooxygenase-2 Expression.

Excessive exposure to solar UV (sUV) is associated with numerous human skin disorders, such as carcinogenesis, skin photoaging and skin inflammation. Silkworm Thorn (Cudraniatricuspidata, SW) is a plant belonging to the Moraceae family and widely present throughout Korea, China, and Japan. Most parts of the tree (including the fruit, leaf, stem, root, and bark) is consumable as a functional food or tea. In this study, we found that SW extract (SWE) inhibited the elevated expression of sUV-induced cyclooxygenase (COX)-2 levels in both HaCaT and JB6 cells. Levels of nuclear factor-κB and activator protein-1, two crucial transcription factors involved in COX-2 expression, were elevated by sUV treatment. Treatment with SWE abolished this activation. SWE also inhibited sUV-induced histone H3 phosphorylation. However, sUV-induced phosphorylation of Akt, c-Jun N-terminal kinase and p38 kinase remained unchanged in the presence of SWE. SWE inhibited RSK2 activity, and pull-down assays using SWE-Sepharose beads revealed that SWE binds directly with RSK2 in an ATP-competitive manner. These results suggest a potential for SWE to be developed as a cosmeceutical material and functional food constituent for the promotion of skin health.

UVB Stimulates the Expression of Endothelin B Receptor in Human Melanocytes via a Sequential Activation of the p38/MSK1/CREB/MITF Pathway Which Can Be Interrupted by a French Maritime Pine Bark Extract through a Direct Inactivation of MSK1.

Melanogenesis is the physiological process by which melanin is synthesized to protect the skin from UV damage. While paracrine interactions between keratinocytes and melanocytes are crucial for regulating epidermal pigmentation, the endothelin (EDN)-endothelin B-receptor (EDNRB) interaction is one of the key linkages. In this study, we found that a single exposure of normal human melanocytes (NHMs) with UVB stimulates the expression of EDNRB and its upstream transcription factor microphthalmia-associated transcription factor (MITF) at the transcriptional and translational levels. That stimulation can be abrogated by post-irradiation treatment with a French maritime pine bark extract (PBE). UVB stimulated the phosphorylation of p38 and c-jun N-terminal kinase (JNK), but not ERK, followed by the increased phosphorylation of MSK1 and CREB. The post-irradiation treatment with PBE did not affect the increased phosphorylation of p38 and JNK, but distinctly abrogated the phosphorylation of MSK1 and CREB. Post-irradiation treatment with the MSK1 inhibitor H89 significantly down-regulated the increased gene expression of MITF and EDNRB in UVB-exposed NHMs. Our findings indicate for the first time that the increased expression of MITF that leads to the up-regulation of melanocyte-specific proteins in UVB-exposed NHMs is mediated via activation of the p38/MSK1/CREB pathway but not the ERK/RSK/CREB pathway. The mode of action by PBE demonstrates that interrupting MSK1 activation is a new target for antioxidants including PBE which can serve as anti-pigmenting agents in a reactive oxygen species-depletion-independent manner.

Computational and Biochemical Discovery of RSK2 as a Novel Target for Epigallocatechin Gallate (EGCG).

The most active anticancer component in green tea is epigallocatechin-3-gallate (EGCG). Protein interaction with EGCG is a critical step for mediating the effects of EGCG on the regulation of various key molecules involved in signal transduction. By using computational docking screening methods for protein identification, we identified a serine/threonine kinase, 90-kDa ribosomal S6 kinase (RSK2), as a novel molecular target of EGCG. RSK2 includes two kinase catalytic domains in the N-terminal (NTD) and the C-terminal (CTD) and RSK2 full activation requires phosphorylation of both terminals. The computer prediction was confirmed by an in vitro kinase assay in which EGCG inhibited RSK2 activity in a dose-dependent manner. Pull-down assay results showed that EGCG could bind with RSK2 at both kinase catalytic domains in vitro and ex vivo. Furthermore, results of an ATP competition assay and a computer-docking model showed that EGCG binds with RSK2 in an ATP-dependent manner. In RSK2+/+ and RSK2-/- murine embryonic fibroblasts, EGCG decreased viability only in the presence of RSK2. EGCG also suppressed epidermal growth factor-induced neoplastic cell transformation by inhibiting phosphorylation of histone H3 at Ser10. Overall, these results indicate that RSK2 is a novel molecular target of EGCG.