Form Of Glycogen Synthase Kinase-3 Research Articles

Glycogen Synthase Kinase 3 activation status in soleus and EDL muscles from space‐flown mice aboard the BION‐M1 biosatellite

Background A significant limitation to long-term spaceflight and space habitation is the deterioration of human musculoskeletal health. It is well known that prolonged exposure to microgravity leads to pronounced muscle and bone loss, with consequences mimicking several major effects observed in human aging (i.e. osteoporosis and sarcopenia). It is therefore critical to elucidate the underlying molecular mechanisms of these processes to develop meaningful intervention strategies. Glycogen synthase kinase 3 (GSK3) is a serine/threonine protein kinase initially defined in its role of regulating glycogen homeostasis, though it's now known to interact with more than 100 target proteins. GSK3 has notably proven to negatively regulate muscle mass by inhibiting hypertrophy-related mechanisms, myoblast fusion, and muscle regeneration, while also promoting ubiquitin-mediated protein degradation and muscle inflammation. In this study, we explored GSK3 expression and its serine phosphorylation status, known to control its activation/inactivation, in soleus and extensor digitorum longus (EDL) muscles obtained from the BION-M1 mission. Methods Male C57BL6/J mice (3 months old) were flown in space for 1 month aboard the BION-M1 biosatellite (F) and were compared to a ground control (GC, space housing and diet conditions) and a flight control (FC, standard housing conditions) group. Soleus and EDL muscles were extracted and homogenized in protein extraction buffer. Western blotting was used to examine total and phosphorylated (i.e. inactivated) forms of GSK3 (ser21 GSK3α; ser9 GSK3β). Results In the soleus muscle, total GSK3α and GSK3β were reduced in the F group. Though we did not observe any changes in ser21 (GSK3α) or ser9 (GSK3β) phosphorylation, the ratio of phosphorylated:total GSK3β was significantly elevated in the F group compared with GC and FC. We did not observe any differences in soleus phosphorylated:total GSK3α. Further, we did not observe any differences in total, phosphorylated or phosphorylated:total GSK3α/β content in the EDL F group compared to GC and FC. Conclusions Altogether, our data suggest that in response to 1 month of spaceflight, GSK3 is less active in the soleus muscle through changes in expression and phosphorylation. GSK3 may therefore not be involved in the later stages of soleus atrophy in flown mice. This may be explained by a slow-to-fast fibre shift in soleus muscle, as GSK3 presence and activity is typically reduced in fast-twitch muscle fibres compared to slow-twitch. Future studies with soleus muscles from two other NASA-flown missions (RR1 and RR9) will be conducted to confirm these effects.

(4-[6-(4-isopropoxyphenyl)pyrazolo [1,5-a]pyrimidin-3-yl] quinoline) is a novel inhibitor of autophagy.

Autophagy is an important intracellular degradation system, which is related to various diseases. In preliminary experiments we found that D4-[6-(4-isopropoxyphenyl)pyrazolo [1,5-a]pyrimidin-3-yl] quinoline (DMH1) inhibited autophagy responses. However DMH1 also inhibits the signalling pathway activated by bone morphogenetic protein-4 (BMP4). The aim of the present study was to elucidate the inhibitory effects of DMH1 on autophagy and the underlying mechanisms. The effects of DMH1 on autophagy responses were evaluated in cultures of different cell types and with different stimuli to induce autophagy, using Western blots, transmission electron microscopy and fluorescent microscopy. DMH1 inhibited starvation-induced autophagy in cardiomyocytes, HeLa and MCF-7 cells, without involving the signalling pathway of BMP4. DMH1 inhibited aminoimidazole carboxamide ribonucleotide (AICAR)- and rapamycin-induced autophagy in HeLa and MCF-7 cells. DMH1 reversed starvation- and AICAR-induced inhibition of Akt, mammalian target of rapamycin (mTOR) and p70S6 kinase (S6K), and reversed rapamycin-induced inhibition of mTOR and S6K. DMH1 reversed starvation-induced decrease of the phosphorylated form of glycogen synthase kinase-3 in MCF-7 and HT29 cells. Activation of Akt and inhibition of autophagy induced by DMH1 were antagonized by an Akt specific inhibitor or by small interfering RNA for Akt in HeLa cells. DMH1 inhibited cellular autophagy responses in a range of cell types and the underlying mechanisms include activation of the Akt pathway.

Expression of pGSK-3α/β Tyr279/216 and XIAP proteins in cholangiocarcinoma and their clinical significance

To investigate the expressions of the active form of glycogen synthase kinase-3(GSK-3)-pGSK-3α/β (Tyr279/216) and its downstream moleculor X-linked inhibitor of apoptosis protein (XIAP) in cholangiocarcinoma and to analyze their correlation with clinicopathological and survival significance. Immunohistoehemistry was used to detect the expressions of the active form of GSK-3- pGSK-3α/β (Tyr279/216) and its downstream moleculor XIAP proteins in 50 cholangiocarcinoma tissues and 20 normal bile duct tissues. The positive rates of pGSK-3α/β (Tyr279/216) and XIAP were 62.0% and 68.0% in cholangiocarcinoma, and 10.0% and 25.0% in normal bile duct tissues, respectively. The intensity of pGSK-3α/β (Tyr279/216) and XIAP expressions in cholangiocarcinoma were significantly higher than that in the normal bile duct tissues (P < 0.001), and there was a significant correlation between pGSK-3α/β (Tyr279/216) and XIAP expressions (r = 0.544, P < 0.001). The expression of pGSK-3α/β(Tyr279/216) protein in cholangiocarcinoma was associated with TNM stage (P = 0.042), histological grade (P = 0.031), whereas the expression of XIAP protein in cholangiocarcinoma was correlated with CEA level (P = 0.006). Patients with positive expression of pGSK-3α/β (Tyr279/216) and XIAP demonstrate a significantly worse prognosis than that of patients with negative expression of pGSK-3α/β (Tyr279/216) and XIAP for overall survival (P = 0.002, P = 0.018). Multivariate survival analysis revealed that positive pGSK-3α/β (Tyr279/216) expression provided significant independent prognostic value for overall survival (P = 0.002). The expressions of pGSK-3α/β(Tyr279/216) and XIAP proteins were significantly associated with the development and progression of cholangiocarcinoma. pGSK-3α/β(Tyr279/216) may be an important prognostic factor for survival of patients with cholangiocarcinoma.

Gene transfer into Purkinje cells using herpesviral amplicon vectors in cerebellar cultures

Purkinje cells play a crucial role in sensory motor coordination since they are the only output projection neurons in the cerebellar cortex and are affected in most spinocerebellar ataxias. They stand out in the central nervous system due to their large size and their profusely branched dendritic arbor. However, molecular and cellular studies on Purkinje cells are often hampered by the difficulty of maintaining these cells in culture. Here we report an easy, robust and reproducible method to obtain Purkinje-enriched mixed cerebellar cell cultures from day 16 mouse embryos using papain digestion and a semi-defined culture medium, being the composition of the culture approximately 20% Purkinje cells, 70% non-Purkinje neurons and 10% glial cells. We demonstrate that efficient gene transfer into Purkinje cells (as well as into other cerebellar populations) is possible using herpes simplex virus-1 (HSV-1)-derived vectors. Indeed, up to 50% of the Purkinje cells can be transduced and gene expression may persist for at least 14 days. As a result, this procedure permits functional gene expression studies to be carried out on cultured Purkinje neurons. To demonstrate this, we show that the expression of a dominant-negative form of glycogen synthase kinase-3 protects Purkinje neurons against cell death triggered by a chemical inhibitor of phosphatidylinositol-3 kinase. In summary, we have established reproducible and reliable cerebellar cell cultures enriched for Purkinje cells which enables gene transfer studies to be carried out using herpesviral vectors.

Variations in endometrial expression of active and inactive forms of glycogen synthase kinase-3 β protein during the menstrual cycle

Objective: Glycogen Synthase Kinase-3β (GSK-3β) is a serine/threonine kinase that plays a role in glycogen synthesis by inhibiting glycogen synthase (GS) through phosphorylation (Figure 1). We have previously reported on the expression of its total form in the female reproductive tract with highest expression being in the premenopausal endometrium. The objective of this investigation was to examine the expression of total and phosphorylated form (inactive) of GSK-3β in the premenopausal endometrium during the menstrual cycle. Based on our previous data we hypothesized that the inactive form of GSK-3β is preferentially expressed in the late proliferative through the secretory phase of the endometrium.

In utero ethanol suppresses cerebellar activator protein-1 and nuclear factor-kappa B transcriptional activation in a rat fetal alcohol syndrome model.

A model of fetal alcohol syndrome was used to investigate prenatal ethanol effects on cerebellar transcription factors. Pregnant Sprague-Dawley rats were divided into three treatment groups: ethanol-exposed (E), calorically matched pair-fed (PF), and freely fed ad libitum (AL) groups. Ethanol exposure was stopped 2 days before parturition. The DNA binding in neonatal cerebella of the redox-sensitive transcription factors nuclear factor-kappa B (NF-kappa B) and activator protein-1 (AP-1) were determined by electrophoretic mobility shift assays. On the first postnatal day (PD1), there was decreased activation of these transcription factors in the E group relative to the control groups. The PD1 transcriptional effects were reversed as the neonate underwent development without further ethanol exposure. Western blot studies showed no corresponding decreases in protein amounts of both AP-1 and NF-kappa B components on PD1. Postnatal glutathione levels and catalase activity, as measures of oxidative stress hypothesized to be a probable cause of the transcriptional effects, showed no statistically significant effects attributable to ethanol. Examination of prenatal cerebella on embryonic day 20 (EM20), a time during ethanol exposure, showed DNA-binding trends similar to those of PD1. EM20 Western blot studies showed decreases in the levels of the active form of glycogen synthase kinase-3 (GSK-3). GSK-3 inhibition was reversed by PD1. Blocking of GSK-3 activity with gestational dietary lithium diminished both AP-1 and NF-kappa B DNA binding. Thus, prenatal ethanol exposure has the effect of diminishing pro-survival transcriptional activation, an effect possibly mediated by changes in GSK-3 activity.