GSK-3beta Phosphorylation Research Articles

Hyperinsulinemic milieu elicits glomerular podocyte impairment and dysfunction via inducing GSK3beta hyperactivity

Background: Epidemiological evidence suggests that hyperinsulinemia or insulin resistance is a significant risk factor for the development of diabetic complications such as DKD. However, whether hyperinsulinemia per se plays a causative role in the development of diabetic kidney injury is unknown and was explored here.
 Methods: Pre-diabetic db/db mice were examined for serum insulin levels, urinary albumin to creatinine ratios and renal histology. Conditionally immortalized mouse podocytes were cultured under non-permissive conditions and exposed to high ambient insulin conditions, following GSK3beta silencing, ectopic expression of a constitutively active GSK3beta mutant (S9A), or treatment with a small molecule GSK3beta inhibitor tideglusib. Podocyte injury was assessed and signaling pathways examined. 
 Results: In pre-diabetic db/db mice, hyperinsulinemia was evident and associated with microalbuminuria and early signs of podocyte impairment, including diminished expression of homeostatic marker proteins like synaptopodin, as compared with db/m littermates. In vitro, prolonged exposure of differentiated podocytes to high ambient insulin induced podocytopathic changes, including cellular hypertrophy, loss of synaptopodin, and disruption of actin cytoskeleton integrity. This was associated with a desensitized insulin signaling and diminished inhibitory phosphorylation of GSK3beta, denoting GSK3beta hyperactivity. In pre-diabetic db/db mice, GSK3beta hyperactivity was confirmed in glomerular podocytes, correlating with the level of hyperinsulinemia or microalbuminuria. In cultured podocytes, ectopic expression of S9A caused podocyte hypertrophy and podocytopathic changes, reminiscent of the harmful effect of the hyperinsulinemic milieu. Conversely, GSK3beta knockdown mitigates podocyte injury elicited by hyperinsulinemic milieu. This protective effect was mimicked by the small molecule inhibitor tideglusib.
 Conclusions: GSK3beta hyperactivity is required and sufficient for Hyperinsulinemic milieu-elicited glomerular podocyte impairment and dysfunction.

P2Y1 and P2Y2 receptors differ in their role in the regulation of signaling pathways during unloading-induced rat soleus muscle atrophy

The current study aimed to investigate the hypothesis that purinergic receptors P2Y1 and P2Y2 play a regulatory role in gene expression in unloaded muscle. ATP is released from cells through pannexin channels, and it interacts with P2Y1 and P2Y2 receptors, leading to the activation of markers of protein catabolism and a reduction in protein synthesis. To test this hypothesis thirty-two rats were randomly divided into four groups (8 per group): a non-treated control group (C), a group subjected to three days of hindlimb unloading with a placebo (HS), a group subjected to three days of hindlimb unloading treated with a P2Y1 receptor inhibitor, MRS2179 (HSM), and a group subjected to three days of hindlimb unloading treated with a P2Y2 receptor inhibitor, AR-C 118925XX (HSA). This study revealed several key findings following three days of soleus muscle unloading: 1Inhibition of P2Y1 or P2Y2 receptors prevented the accumulation of ATP, the increase in IP3 receptor content, and the decrease in the phosphorylation of GSK-3beta. This inhibition also mitigated the reduction in the rate of protein synthesis. However, it had no significant effect on the markers of mTORC1-dependent signaling. 2Blocking P2Y1 receptors prevented the unloading-induced upregulation of phosphorylated p38MAPK and partially reduced the increase in MuRF1mRNA expression. 3Blocking P2Y2 receptors prevented muscle atrophy during unloading, partially maintained the levels of phosphorylated ERK1/2, reduced the increase in mRNA expression of MAFbx, ubiquitin, and IL-6 receptor, prevented the decrease in phosphorylated AMPK, and attenuated the increase in phosphorylated p70S6K.Taken together, these results suggest that the prevention of muscle atrophy during unloading, as achieved by the P2Y2 receptor inhibitor, is likely mediated through a reduction in catabolic processes and maintenance of energy homeostasis. In contrast, the P2Y1 receptor appears to play a relatively minor role in muscle atrophy during unloading.

The Role of Glycogen Synthase Kinase-3 in the Regulation of Ribosome Biogenesis in Rat Soleus Muscle under Disuse Conditions.

It is well-established that prolonged exposure to real or simulated microgravity/disuse conditions results in a significant reduction in the rate of muscle protein synthesis (PS) and loss of muscle mass. Muscle protein synthesis is largely dependent upon translational capacity (ribosome content), the regulation of which is poorly explored under conditions of mechanical unloading. Glycogen synthase kinase-3 (GSK-3) (a negative regulator of PS) is known to be activated in rat soleus muscle under unloading conditions. We hypothesized that inhibition of GSK-3 activity under disuse conditions (hindlimb suspension, HS) would reduce disuse-induced downregulation of ribosome biogenesis in rat soleus muscle. Wistar rats were randomly divided into four groups: (1) vivarium control (C), (2) vivarium control + daily injections (4 mg/kg) of AR-A014418 (GSK-3 inhibitor) for 7 days, (3) 7-day HS, (4) 7-day HS + daily injections (4 mg/kg) of AR-A014418. GSK-3beta and glycogen synthase 1 (GS-1) phosphorylation levels were measured by Western-blotting. The key markers of ribosome biogenesis were assessed via agarose gel-electrophoresis and RT-PCR. The rate of muscle PS was assessed by puromycin-based SUnSET method. As expected, 7-day HS resulted in a significant decrease in the inhibitory Ser9 GSK-3beta phosphorylation and an increase in GS-1 (Ser641) phosphorylation compared to the C group. Treatment of rats with GSK-3 inhibitor prevented HS-induced increase in GS1 (Ser641) phosphorylation, which was indicative of GSK-3 inhibition. Administration of GSK-3 inhibitor partly attenuated disuse-induced downregulation of c-Myc expression as well as decreases in the levels of 45S pre-rRNA and 18S + 28S rRNAs. These AR-A014418-induced alterations in the markers of ribosome biogenesis were paralleled with partial prevention of a decrease in the rate of muscle PS. Thus, inhibition of GSK-3 during 7-day HS is able to partially attenuate the reductions in translational capacity and the rate of PS in rat soleus muscle.

Microenvironmental changes induced by placenta-derived mesenchymal stem cells restore ovarian function in ovariectomized rats via activation of the PI3K-FOXO3 pathway

BackgroundTranslational studies have explored the therapeutic potential and feasibility of mesenchymal stem cells (MSCs) in several degenerative diseases; however, mechanistic studies of the function of these cells have been insufficient. As ovarian failure causes anovulation as well as ovarian steroid hormonal imbalances, the specific aims of this study were to analyze the therapeutic role of placenta-derived MSCs (PD-MSCs) in an ovarian failure ovariectomy (OVX) rat model and evaluate whether PD-MSC transplantation (Tx) improved folliculogenesis and oocyte maturation in the injured ovary through PI3K/Akt and FOXO signaling.MethodsBlood and ovary tissue were collected and analyzed after various PD-MSC Tx treatments in an ovariectomized rat model. Changes in the expression of folliculogenesis- and ovary regeneration-related genes induced by PD-MSC treatments were analyzed by qRT-PCR, Western blotting, and histological analysis.ResultsThe levels of hormones related to ovary function were significantly increased in the PD-MSC Tx groups compared with those in the nontransplantation group (NTx). The follicle numbers in the ovarian tissues were increased along with the increased expression of genes related to folliculogenesis in the PD-MSC Tx groups compared with the NTx groups. Furthermore, Tx PD-MSCs induced follicle maturation by increasing the phosphorylation of GSK3 beta and FOXO3 (p < 0.05) and shifting the balance of growth and apoptosis in oocytes.ConclusionsTaken together, these results show that PD-MSC Tx can restore ovarian function and induce ovarian folliculogenesis via the PI3K/Akt and FOXO signaling pathway.

Abstract 1845: Propranolol reduces hemangiosarcoma cell viability by disrupting lipid homeostatic pathways

Abstract Angiosarcoma is a highly aggressive cancer of blood vessel-forming cells with few effective treatment options and high patient mortality. Recent studies have shown that the beta-adrenergic antagonist propranolol, a drug historically used for the treatment of cardiac disease, promotes regression of angiosarcomas when combined with standard chemotherapies and increases the overall survival of angiosarcoma patients. Because angiosarcomas are rare, undertaking studies to understand the susceptibility of these tumors to propranolol remains a challenge. In contrast, hemangiosarcoma is a common cancer in dogs that closely models the genetic landscape and the pathogenesis of human angiosarcoma, making it a powerful tool for investigating the susceptibility of angiosarcomas to propranolol. Treatment of an angiosarcoma and hemangiosarcoma cell line panel with propranolol or its receptor active S-(-), and inactive R-(+) enantiomers similarly reduced cell viability in all lines in a concentration dependent manner, suggesting the effects of propranolol are independent of adrenergic receptor activity. Lipin-1, an enzyme responsible for the conversion of phosphatidic acid (PA) to diacylglycerol (DAG), is a known target of propranolol. Inhibition of lipin-1 by propranolol rapidly increases the levels PA, which plays a key role in phospholipid synthesis, intracellular vesicle transport, and signaling pathways involved in regulation of metabolism. Hence, we tested the hypothesis that propranolol induces its detrimental effects in angiosarcomas by disrupting lipid metabolic pathways and vesicular transport. We found that propranolol induced synthesis of cholesterol and fatty acid pathways within 30 minutes via the activation of SREBP-1. Propranolol also blocked endocytosis of extracellular cholesterol, suggesting the rapid induction of lipid synthesis was used by cells to maintain lipid homeostasis. The rapid induction of lipid synthesis activated an ER stress response, contributing to a loss of cell viability. Propranolol also blocked autophagy, proteasomal degradation pathways, and vesicular transport of cholesterol, limiting access to essential metabolites and further exacerbating ER stress. Because mTORC1 is a key regulator of SREBP-1 and mTORC1 is stabilized by PA, we evaluate the effects of propranolol on mTORC1 activity. While propranolol rapidly increased PA levels, it modestly increased the phosphorylation of 4E-BP1, a downstream target of mTORC1, and results were not consistent across all cell lines. Instead, propranolol rapidly induced the phosphorylation and inhibition of GSK-3beta, a known inhibitor of SREBP-1, providing a mechanism for the activation of lipid synthesis pathways by propranolol. Our data suggest propranolol induces sarcoma cell death by disrupting lipid homeostatic pathways and inducing ER stress. Importantly, our findings identify a mechanistic pathway that can be targeted in combination with propranolol to further improve patient responses. Citation Format: Derek M. Korpela, Jhuma Saha, Ali Khammanivong, Shee Kwan Phung, David R. Brown, Erin B. Dickerson. Propranolol reduces hemangiosarcoma cell viability by disrupting lipid homeostatic pathways [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1845.

The Protective Effect of Insulin on Rat Cortical Neurons in Oxidative Stress and Its Dependence on the Modulation of Akt, GSK-3beta, ERK1/2, and AMPK Activities.

Insulin is a promising drug for the treatment of diseases associated with brain damage. However, the mechanism of its neuroprotective action is far from being understood. Our aim was to study the insulin-induced protection of cortical neurons in oxidative stress and its mechanism. Immunoblotting, flow cytometry, colorimetric, and fluorometric techniques were used. The insulin neuroprotection was shown to depend on insulin concentration in the nanomolar range. Insulin decreased the reactive oxygen species formation in neurons. The insulin-induced modulation of various protein kinase activities was studied at eight time-points after neuronal exposure to prooxidant (hydrogen peroxide). In prooxidant-exposed neurons, insulin increased the phosphorylation of GSK-3beta at Ser9 (thus inactivating it), which resulted from Akt activation. Insulin activated ERK1/2 in neurons 5–30 min after cell exposure to prooxidant. Hydrogen peroxide markedly activated AMPK, while it was for the first time shown that insulin inhibited it in neurons at periods of the most pronounced activation by prooxidant. Insulin normalized Bax/Bcl-2 ratio and mitochondrial membrane potential in neurons in oxidative stress. The inhibitors of the PI3K/Akt and MEK1/2/ERK1/2 signaling pathways and the AMPK activator reduced the neuroprotective effect of insulin. Thus, the protective action of insulin on cortical neurons in oxidative stress appear to be realized to a large extent through activation of Akt and ERK1/2, GSK-3beta inactivation, and inhibition of AMPK activity increased by neuronal exposure to prooxidant.

Increased PD-L1 expression in radioresistant HNSCC cell lines after irradiation affects cell proliferation due to inactivation of GSK-3beta.

At present, targeting PD-1/PD-L1 axis for immune checkpoint inhibition has improved treatment of various tumor entities, including head and neck squamous cell carcinoma (HNSCC). However, one part of the patient cohort still shows little improvement or even hyperprogression. We established three radioresistant (RR) and three radiosensitive (RS) HNSCC cell lines. RR cells showed prolonged survival as well as delayed and diminished apoptosis after irradiation with vimentin expression but no E-cadherin expression, whereas RS cell lines died early and exhibited early apoptosis after irradiation and high vimentin expression. Here, we present results demonstrating differential basal PD-L1 gene and protein expression in RR and RS HNSCC cell lines. Moreover, we observed a radiation dose dependent increase of total PD-L1 protein expression in RR cell lines up to 96h after irradiation compared to non-irradiated (non-IRR) cells. We found a significant GSK-3beta phosphorylation, resulting in an inactivation, after irradiation of RR cell lines. Co-immunoprecipitation experiments revealed decreased interaction of GSK-3beta with PD-L1 in non-IRR compared to irradiated (IRR) RR cells leading to PD-L1 stabilization in RR cells. PD-L1 knockdown in RR cells showed a strong decrease in cell survival. In summary, our results suggest an irradiation dependent increase in basal PD-L1 expression in RR HNSCC cell lines via GSK-3beta inactivation.

PO-095 Effects of dynamic foot stimulation on GSK-3 beta signaling pathway in rat soleus muscle under hindlimb unloading

Objective It is known that under simulated microgravity GSK-3 beta phosphorylation (Ser 9) is decreased [Mirzoev et al., 2016] which is associated with the activation of its kinase activity. GSK-3 beta activation may lead to NFATc1 export from myonuclei resulting in a slow-to-fast myosin shift in soleus muscle and may cause a decrease in muscle oxidative capacity and protein synthesis [Glass, 2003, Theeuwes et al, 2017].&#x0D; It was demonstrated that GSK-3 beta Ser 9 phosphorylation is decreased in response to nitric oxide synthase inhibition [Martins et al., 2011]. Under unloading conditions, the content of NO as well as nitric oxide synthase is attenuated in rat soleus muscle [Tidball et al., 1998; Lomonosova et al, 2011]. Dynamic foot stimulation (DFS) of the soles of the feet results in an increase of neuromuscular activation [Muller et al., 2005] of the lower limb muscles and prevents nitric synthase content decrease during 7-day exposure to dry immersion [Moukhina et al, 2004; Shenkman et al., 2004]. The aim of our study was to analyze the effect of rat dynamic foot stimulation during early unloading on GSK-3 beta phosphorylation and some of its downstream targets.&#x0D; Methods Male Wistar rats were randomly assigned to vivarium control, 1-day unloading and 1-day&#x0D; unloading with DFS. The pressure stimulation protocol mimicked the normal animal walking (104 mm Hg pressure, 4 Hz frequency and 250 ms signal duration) for a total of 20 min followed by a 10-min rest interval for 4 hours.&#x0D; Results We found that 1-day unloading caused a significant decrease (p˂ 0.025) in NFATc1 nuclear content as well as slow myosin (MHC I (beta) isoform) mRNA expression. In the 1-day unloading group, the level of glycogen synthase 1 phosphorylation (Ser 641), which is a direct GSK-3 beta target, was significantly higher than that in the control group, although a decrease in GSK-3 beta Ser 9 phosphorylation in the 1-day unloaded group was not statistically significant compared to the control group. However, in the DFS-treated unloaded rats the level of GSK-3 beta phosphorylation was significantly higher than in the untreated unloaded rats. NFATc1 nuclear content, slow myosin mRNA expression and glycogen synthase 1 phosphorylation (Ser 641) did not differ from the control group. The glycogen content in soleus muscles of both unloaded groups was higher than in the control group, which is in accordance with previous studies [Henriksen and Tischler, 1988], but in DFS subjected unloaded group the glycogen content was higher compared to the untreated unloaded group.&#x0D; Conclusions Thus, we found that dynamic foot stimulation during 1-day hindlimb unloading leads to GSK-3 beta inactivation and prevention of both NFATc1 myonuclei export and the decrease in MyHC I beta expression.&#x0D; The study was supported by the Russian Foundation for Basic Research grant #17-29-1029 and Basic Research Program of the IBMP, RAS. The authors express their deep gratitude to Prof. Inessa B. Kozlovskaya for useful recommendations concerning the design of the study.

PO-064 A potential role of stretch-activated channels in anabolic mechanotransduction in the atrophied rat soleus muscle

Objective Prolonged immobilization or unloading of skeletal muscle causes muscle disuse atrophy, which is characterized by a reduction in muscle cross-sectional area and compromised contractile function. To date, the mechanisms of anabolic mechanotransduction in the atrophied mammalian skeletal muscle remain poorly understood. The aim of the present study was to assess a possible role of stretch-activated ion channels (SAC) in the propagation of a mechanical signal to anabolic signaling and protein synthesis (PS) in an isolated rat soleus muscle following mechanical unloading.&#x0D; Methods The mechanical unloading was performed via hindlimb suspension (HS). Twenty-eight male Wistar rats weighing were randomly assigned to the following 4 groups (n=7/group): 1) vivarium control (C), 2) control + SAC inhibitor (gadolinium) (C+Gd3+), 3) 7-day HS (HS), 4) 7-day HS + SAC inhibitor (HS+Gd3+). Following unloading, an isolated rat soleus was placed in an organ culture medium and subjected to a bout of eccentric contractions (EC). Upon completion of the EC, muscles were collected for Western blot analyses to determine the content of the key anabolic markers. The rate of PS was measured by SUnSET technique.&#x0D; Results EC-induced increase in PS was significantly less in the HS and HS+ Gd3+ groups vs. the C group. There was no statistically significant difference between the HS and HS+ Gd3+ groups in terms of EC-induced increase in muscle PS. A decrease in EC-induced phosphorylation of p70S6K, 4E-BP1, RPS6 and GSK-3beta in the 7-day unloaded soleus treated with SAC inhibitor did not differ from that of the 7-day unloaded soleus without SAC blockade. Thus, the inhibition of SAC with gadolinium did not lead to further decline in EC-induced phosphorylation of the key anabolic markers and muscle PS.&#x0D; Conclusions The results of the study suggest that attenuation of mTORC1-signaling and PS in response to EC in unloaded soleus muscle may be associated with inactivation of SAC. The study was supported by the RFBR grant # 16-34-60055.

P4754Pivotal role of the region-specific phosphorylation of GSK3beta in cardioprotection in anesthetized mice

P4754Pivotal role of the region-specific phosphorylation of GSK3beta in cardioprotection in anesthetized mice

Loss of Sprouty2 enhances IL‐33 expression and protects against experimental colitis.

OBJECTIVESprouty2 (Spry2) is an intracellular modulator of MAPK signaling. It is present in the intestinal tract, but its function there is unknown. Aberrant MAPK signaling is associated with altered cytokine production in disorders such as inflammatory bowel disease, raising the possibility that Spry2 may affect the response to colitis. In particular, MAPK regulates IL‐33 expression in response to bacterial infection, and IL‐33 has been shown to be protective in some forms of colitis. Furthermore, both MAPK and IL‐33 regulate secretory cell differentiation. Here we tested the hypothesis that Spry2 control of MAPK signaling alters cytokine production and/or secretory cell development, thus altering the intestinal response to injury.METHODSMice that lack Spry2 in the intestinal epithelium (VillinCre;Spry2flox/flox, hereafter Spry2KOIE) and littermate controls were given 3% dextran sodium sulfate (DSS) in drinking water for 4 days followed by 3 days without DSS, and colitis severity was assessed. Cytokine levels and epithelial marker expression were determined by qPCR in challenged and unchallenged mice. GSK3beta phosphorylation was determined by western blot. In vitro, IEC6 and YAMC intestinal epithelial cells were treated with GSK3beta and MAPK inhibitors and cytokine expression was assessed.RESULTSColonic Spry2 levels were reduced in DSS colitis by 59% (p&lt;0.05). Spry2KOIE mice subjected to DSS had lower levels of pro‐inflammatory cytokines versus wildtype littermates, and were protected from histological damage (injury score reduced by 30%, p=0.037), weight loss, and colon shortening. Unchallenged Spry2KOIE mice displayed elevated GSK3beta phosphorylation on the inactivating Ser9 site in epithelial cells (88% increase, p&lt;0.01). These mice also had increased IL‐33 expression (80% increase, p&lt;0.05) and more DCLK1+ tuft cells in the colon (2.2‐fold increase, p&lt;0.01). In vitro, inactivation of GSK3beta in both IEC6 and YAMC cells mimicked the increased IL‐33 observed in vivo; this effect was attenuated by ERK or p38 MAPK inhibitors. Expression of the epithelial‐derived cytokine CXCL2 was unaffected by GSK3beta inhibition, suggesting an IL‐33 specific response.CONCLUSIONColonic epithelial Spry2 is downregulated in response to injury and inflammation. This may represent an integration point of environmentally‐driven MAPK signals that leads to altered Wnt signaling, elevated IL‐33 expression, and promotion of intestinal tuft cell development as a protective or regenerative mechanism.Support or Funding InformationNIH R01DK095004This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Connexin 43 Modulates Osteogenic Differentiation of Bone Marrow Stromal Cells Through GSK-3beta/Beta-Catenin Signaling Pathways

Background/Aims: Bone marrow stromal cells (BMSCs) are multipotent precursors that give rise to osteoblasts, and contribute directly to bone formation. Connexin 43 (Cx43) is the most ubiquitous gap junction protein expressed in bone cell types, and plays crucial roles in regulating intercellular signal transmission for bone development, differentiation and pathology. However, the precise role and mechanism of Cx43 in BMSCs are less known. Here, we investigate the function of Cx43 in osteogenic differentiation of BMSCs in vitro. Methods: BMSCs were isolated by whole bone marrow adherent culture. Knock down of Cx43 was performed by using lentiviral transduction of Cx43 shRNA. BMSCs were induced to differentiate by culturing in a-MEM, 10% FBS, 50 µM ascorbic acid, 10 mM beta-glycerophosphate, and 100 nM dexamethasone. Alkaline phosphatase (ALP) activity and alizarin red S staining were used to evaluate osteogenic differentiation in calcium nodules. Target mRNAs and proteins were analyzed by using real-time quantitative PCR (qPCR) and western blotting. Results: Cx43 expression markedly increased during osteogenic differentiation. Osteogenic differentiation was suppressed following lentiviral-mediated knockdown of Cx43 expression, as judged by decreased levels of Runt-related transcription factor 2 (Runx2), bone sialoprotein (BSP), osteocalcin (Bglap), Osterix (Osx), alkaline phosphatase (ALP) activity and the number of calcium nodules in response to osteogenic differentiation stimuli. Knock down of Cx43 reduced the level of phosphorylation of GSK-3beta at Ser9 (p-GSK-3beta), resulting in decreased beta-catenin expression and activation. Furthermore, treatment of Cx43-knockdown cells with lithium chloride (LiCl), a GSK-3beta inhibitor, reduced osteogenic differentiation and decreased GSK-3beta levels, as well as partially rescued levels of both total and activated beta-catenin. Conclusion: These findings indicate that Cx43 positively modulates osteogenic differentiation of BMSCs by up-regulating GSK-3beta/beta-catenin signaling pathways, suggesting a potential role for Cx43 in determining bone mass and bone mineral density by modulating osteogenesis.

THE VAPB-PTPIP51 ER-MITOCHONDRIA TETHERS IN FRONTOTEMPORAL DEMENTIA AND RELATED AMYOTROPHIC LATERAL SCLEROSIS

Signaling between the ER and mitochondria regulates many of the neurophysiological functions that are damaged in FTD/ALS. This signaling involves close physical contacts between the two organelles. We showed that the integral ER protein VAPB binds to the outer mitochondrial membrane protein PTPIP51 to mediate these contacts. VAPB and PTPIP51 thus act as molecular scaffolds to tether the two organelles. We have investigated how FTD/ALS insults affect ER-mitochondria contacts and signaling, and the VAPB-PTPIP51 tethers. ER-mitochondria contacts and the VAPB-PTPIP51 interaction were quantified in cell and transgenic mouse models of FTD/ALS. Contacts were quantified by EM and super-resolution microscopy. The VAPB-PTPIP51 interaction was monitored by proximity ligation, immunoprecipitation and dimerization-dependent EGFP fusion assays. Ca2+ imaging was used to determine ER-mitochondria Ca2+ exchange. Signal transduction mechanisms linking FTD/ALS insults with damage to the VAPB-PTPIP51 tethers involved mass spectrometry. FTD/ALS associated TDP-43 and FUS both disrupt ER-mitochondria contacts, Ca2+ exchange and linked mitochondrial ATP production. This disruption involved activation of GSK3beta and breaking of the VAPB-PTPIP51 tethers. Potential GSK3beta phosphorylation sites were identified in both VAPB and PTPIP51. The Sigma-1 receptor was identified as a regulator of the VAPB-PTPIP51 interaction that may be targeted for damage by TDP-43 and FUS. The identification of VAPB and PTPIP51 as tethering proteins enabled the construction of novel drug screens. Damage to ER-mitochondria signaling and the VAPB-PTPIP51 ER-mitochondria tethering proteins is a feature of FTD/ALS. Together these findings identify a new pathogenic process in FTD/ALS that can explain many disease features and which represents a novel therapeutic target.

Divergent Anabolic Signalling responses of Murine Soleus and Tibialis Anterior Muscles to Chronic 2G Hypergravity

The purpose of the study was to assess the rate of protein synthesis (PS) and elucidate signalling pathways regulating PS in mouse soleus (Sol) and tibialis anterior (TA) muscles following chronic hypergravity (30-day centrifugation at 2G). The content of the key signalling proteins of the various anabolic signalling pathways was determined by Western-blotting. The rate of PS was assessed using in-vivo SUnSET technique. An exposure to 2G centrifugation did not induce any significant changes in the rate of PS as well as phosphorylation status of the key anabolic markers (AKT, p70s6k, 4E-BP1, GSK-3beta, eEF2) in Sol. On the contrary, a significant 55% increase in PS (p < 0.05) was found in TA. The cause of such a rise in PS could be associated with an increase in AKT (+72%, p < 0.05), GSK-3beta (+60%, p < 0.05) and p70s6k (+40%, p < 0.05) phosphorylation, as well as a decrease in eEF2 phosphorylation (−46%, p < 0.05) as compared to control values. Thus, the results of our study indicate that 30-day 2G centrifugation induces a distinct anabolic response in mouse Sol and TA muscles. The activation of the PS rate in TA could be linked to an up-regulation of both mTORC1-dependent and mTORC1-independent signalling pathways.

L-F001, a Multifunction ROCK Inhibitor Prevents 6-OHDA Induced Cell Death Through Activating Akt/GSK-3beta and Nrf2/HO-1 Signaling Pathway in PC12 Cells and Attenuates MPTP-Induced Dopamine Neuron Toxicity in Mice.

Amounting evidences demonstrated that Rho/Rho-associated kinase (ROCK) might be a novel target for the therapy of Parkinson's disease (PD). Recently, we synthesized L-F001 and revealed it was a potent ROCK inhibitor with multifunctional effects. Here we investigated the effects of L-F001 in PD models. We found that L-F001 potently attenuated 6-OHDA-induced cytotoxicity in PC12 cells and significantly decreased intracellular reactive oxygen species (ROS), prevented the 6-OHDA-induced decline of mitochondrial membrane potential and intracellular GSH levels. In addition, L-F001 increased Akt and GSK-3beta phosphorylation and induced the nuclear Nrf2 and HO-1 expression in a time- and concentration-dependent manner. Moreover, L-F001 restored the levels of p-Akt and p-GSK-3beta (Ser9) as well as HO-1 expression reduced by 6-OHDA. Those effects were blocked by the specific PI3K inhibitor, LY294002, indicating the involvement of Akt/GSK-3beta pathway in the neuroprotective effect of L-F001. In addition, L-F001 significantly attenuated the tyrosinehydroxylase immunoreactive cell loss in 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP)-induced mice PD model. Together, our findings suggest that L-F001 prevents 6-OHDA-induced cell death through activating Akt/GSK-3beta and Nrf2/HO-1 signaling pathway and attenuates MPTP-induced dopaminergic neuron toxicity in mice. L-F001 might be a promising drug candidate for PD.

Proteomic Analysis Reveals a New Benefit of Periodic Mechanical Stress on Chondrocytes

Background/Aims: In recent years, a variety of studies have been performed to investigate the cellular responses of periodic mechanical stress. In our previous studies, we found that periodic mechanical stress can promote proliferation and matrix synthesis through the integrin beta 1-mediated ERK1/2 pathway, and we used proteomic analysis to detect quantitative changes in chondrocytes under periodic mechanical stress. Despite these results, the effects and mechanisms of periodic mechanical stress are still not fully understood, so in this study we extended our study using phosphoproteomic techniques. Methods: We used phosphoproteomic techniques to detect phosphorylation changes in chondrocytes under periodic mechanical stress and combined the results with the quantitative proteomic data to further explore the underlying mechanisms. Data were obtained by phosphorylation inhibition, quantitative real-time PCR (qPCR) analysis, western blot analysis and immunofluorescence assay. Results: From phosphoproteomic analysis, a total of 1073 phosphorylated proteins and 2054 phosphopeptides were identified. The number of significant differentially expressed proteins and phosphopeptides was 97 and 108, respectively (ratio >1.20 or <0.83 at p<0.05). Periodic mechanical stress increased glycogen synthase kinase 3-beta (GSK3-beta) phosphorylation at Y216, promoted the phosphorylation of beta-catenin, decreased beta-catenin levels and suppressed the expression of type I collagen. In contrast, inhibition of GSK3-beta by TWS119, which specifically inhibits the phosphorylation of Y216, suppressed the phosphorylation of beta-catenin, which resulted in the accumulation of beta-catenin and an increase in the expression of type I collagen. Conclusions: We successfully constructed differentially expressed phosphoproteomic profiles of rat chondrocytes under periodic mechanical stress, and discovered a potential new therapeutic benefit in which periodic mechanical stress suppressed the formation of type I collagen in the matrix of chondrocytes via phosphorylation of GSK3-beta and beta-catenin.

Developmental Programming: Prenatal Testosterone Excess and Insulin Signaling Disruptions in Female Sheep.

Women with polycystic ovary syndrome often manifest insulin resistance. Using a sheep model of polycystic ovary syndrome-like phenotype, we explored the contribution of androgen and insulin in programming and maintaining disruptions in insulin signaling in metabolic tissues. Phosphorylation of AKT, ERK, GSK3beta, mTOR, and p70S6K was examined in the liver, muscle, and adipose tissue of control and prenatal testosterone (T)-, prenatal T plus androgen antagonist (flutamide)-, and prenatal T plus insulin sensitizer (rosiglitazone)-treated fetuses as well as 2-yr-old females. Insulin-stimulated phospho (p)-AKT was evaluated in control and prenatal T-, prenatal T plus postnatal flutamide-, and prenatal T plus postnatal rosiglitazone-treated females at 3 yr of age. GLUT4 expression was evaluated in the muscle at all time points. Prenatal T treatment increased mTOR, p-p70S6K, and p-GSK3beta levels in the fetal liver with both androgen antagonist and insulin sensitizer preventing the mTOR increase. Both interventions had partial effect in preventing the increase in p-GSK3beta. In the fetal muscle, prenatal T excess decreased p-GSK3beta and GLUT4. The decrease in muscle p-GSK3beta was partially prevented by insulin sensitizer cotreatment. Both interventions partially prevented the decrease in GLUT4. Prenatal T treatment had no effect on basal expression of any of the markers in 2-yr-old females. At 3 yr of age, prenatal T treatment prevented the insulin-stimulated increase in p-AKT in liver and muscle, but not in adipose tissue, and neither postnatal intervention restored p-AKT response to insulin stimulation. Our findings provide evidence that prenatal T excess changes insulin sensitivity in a tissue- and development-specific manner and that both androgens and insulin may be involved in the programming of these metabolic disruptions.

Abstract B36: Rab25 aggravates cancer cell invasion through Snail expression

Abstract Background: Rab25 is a member of the Rab11 subfamily and exclusively expressed in epithelial cells. As a small GTP-binding protein, Rab25 has been known to mediate recycling of proteins from the endosome to the plasma membrane. Cancer metastasis is multi-step events including epithelial-to-mesenchymal transition (EMT). Various transcription factors mediate EMT through downregulation of E-cadherin. In the present study, we investigate whether Rab25 induces EMT transcription factors, thereby increasing cancer invasiveness. Methods: Immunoblotting, quantitative RT-PCR and immunofluorescence assay were used to examine the expression of Rab25 and EMT factors. siRNAs of Rab25 and Snail were transfected to determine their role in Rab25-induced cancer cell EMT and invasiveness. Transwell invasion assay were used to determine cancer cell invasiveness. Results: Ectopic expression of Rab25 marked reduced E-cadherin expression, while Snail expression was upregulated. In addition, Rab25 significantly aggravated cancer cell invasiveness. However, silencing Snail expression by utilizing specific siRNA significantly attenuated cancer cell invasion. In addition, Rab25 induced phosphorylation of Akt and GSK-3beta. However, pharmacological inhibition of Akt and GSK-3beta marked attenuated Rab25-induced Snail expression. Further, we observed that a beta1integrin located upstream of an Akt/GSK-3beta signaling is important for Rab25-induced Snail expression and cancer cell invasion. Conclusion: Our results show for the first time that Snail mediates Rab25-induced cancer cell EMT and invasiveness through a beta1 integrin /Akt/GSK-3beta signaling cascade, providing novel biomarkers, and potential therapeutic targets for deadly cancers. Citation Format: Bo Young Jeong, Kyung Hwa Cho, Seung Hwa Kim, Yu Na Kim, Chang Gyo Park, Hoi Young Lee. Rab25 aggravates cancer cell invasion through Snail expression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B36.

Up-regulation of SRPK1 in non-small cell lung cancer promotes the growth and migration of cancer cells.

Dys-regulation of serine-arginine protein kinase 1 (SRPK1) has been reported in non-small cell lung cancer (NSCLC). However, its functions in the progression of NSCLC remain poorly understood. In this study, the expression of SRPK1 in NSCLC tissues was determined using real-time PCR, and the roles of SRPK1 in the progression of NSCLC were investigated. It was found that both the mRNA level and the protein level of SRPK1 were up-regulated in NSCLC tissues. Forced expression of SRPK1 promoted the growth and migration of NSCLC cells, while knocking down the expression of SRPK1 inhibited the growth, migration, and tumorigenicity of NSCLC cells. Mechanism studies showed that SRPK1 activated the transcriptional activity of beta-catenin/T-cell factor (TCF) complex, and knocking down the expression of SRPK1 attenuated the expression of target genes of beta-catenin/T-cell factor (TCF) complex. In addition, silencing the expression of SRPK1 down-regulated the phosphorylation of GSK3beta. Taken together, SRPK1 might play an oncogenic role in NSCLC, and SRPK1 might be a therapeutic target for NSCLC.

Serum and glucocorticoid kinase 1 promoted the growth and migration of non-small cell lung cancer cells

Serum and glucocorticoid kinase 1 (SGK1) has been reported to be up-regulated in non-small cell lung cancer (NSCLC). However, its functions in NSCLC remained unclear. Here, SGK1 was found to be up-regulated in NSCLC samples. Over-expression of SGK1 promoted the growth and migration of NSCLC cells, while down-regulation of SGK1 inhibited the growth, migration and metastasis of NSCLC cells. SGK1 promoted the phosphorylation of GSK3 beta and the accumulation of beta-catenin, up-regulation of the target genes downstream of beta-catenin/TCF signaling, and activating the transcriptional activity of beta-catenin/TCF complex. Collectively, SGK1 might promote the progression of NSCLC through activating beta-catenin/TCF signaling.