Molecular characterization of the effects of Y-27632

Many key cellular functions, such as cell motility and cellular differentiation are mediated by Rho-associated protein kinases (ROCKs). Numerous studies have been conducted to examine the ROCK signal transduction pathways involved in these motile and contractile events with the aid of pharmacological inhibitors such as Y-27632. However the molecular mechanism of action of Y-27632 has not been fully defined. To assess the relative contribution of these Rho effectors to the effects of Y-27632, we compared the cytoskeletal phenotype, wound healing and neurite outgrowth in cells treated with Y-27632 or subjected to knockdown with ROCK-I, ROCK-II or PRK-2- specific siRNAs. Reduction of ROCK-I enhances the formation of thin actin-rich membrane extensions, a phenotype that closely resembles the effect of Y-27632. Knockdown of ROCK II or PRK-2, leads to the formation of disc-like extensions and thick actin bundles, respectively. The effect of ROCK-I knockdown also mimicked the effect of Y-27632 on wound closer rates. ROCK-I knockdown and Y-27632 enhanced wound closure rates, while ROCK-II and PRK-2 were not appreciably different from control cells. In neurite outgrowth assays, knockdown of ROCK-I, ROCK-II or PRK-2 enhances neurite lengths, however no individual knockdown stimulated neurite outgrowth as robustly as Y-27632. We conclude that several kinases contribute to the global effect of Y-27632 on cellular responses.     

USP11-dependent selective cIAP2 deubiquitylation and stabilization determine sensitivity to Smac mimetics

Given their crucial role in apoptosis suppression, inhibitor of apoptosis proteins (IAPs) have recently become attractive targets for cancer therapy. Here, we report that cellular IAP2 (cIAP2) is specifically stabilized in several cancer cell lines, leading to resistance to Smac mimetics, such as BV6 and birinapant. In particular, our results showed that cIAP2 depletion, but not cIAP1 depletion, sensitized cancer cells to Smac mimetic-induced apoptosis. Ubiquitin-specific protease 11 (USP11) is a deubiquitylase that directly stabilizes cIAP2. USP11 overexpression is frequently found in colorectal cancer and melanoma and is correlated with poor survival. In our study, cancer cell lines expressing high levels of USP11 exhibited strong resistance to Smac mimetic-induced cIAP2 degradation. Furthermore, USP11 downregulation sensitized these cells to apoptosis induced by TRAIL and BV6 and suppressed tumor growth in a xenograft model. Finally, the TNFα/JNK pathway induced USP11 expression and maintained cIAP2 stability, suggesting an alternative TNFα-dependent cell survival pathway. Collectively, our data suggest that USP11-stabilized cIAP2 may serve as a barrier against IAP-targeted clinical approaches.Apoptosis is an inherent cell death program that is crucial for various physiological processes such as development, the immune response, and tumorigenesis.¹ This process is finely tuned by numerous cellular signaling pathways involving hundreds of pro-apoptotic and anti-apoptotic factors.^{2, 3} Inhibitor of apoptosis proteins (IAPs) are a conserved protein family containing the baculoviral IAP repeat (BIR) domain.⁴ There are eight human IAP proteins, including cellular IAP1 (cIAP1/BIRC2), cIAP2/BIRC3, X chromosome-linked IAP (XIAP/BIRC4), and melanoma IAP (ML-IAP/BIRC7).⁵ IAPs such as XIAP can exert their anti-apoptotic function through the BIR domain, which directly interacts with caspases.⁵ In addition, several IAPs contain a RING domain with E3 ubiquitin ligase activities, which are crucial for apoptosis suppression. In particular, the E3 ligase activities of cIAP1/2 are necessary to regulate tumor necrosis factor receptor (TNFR) signaling.⁶ Upon TNFR activation, cIAP1/2 is recruited to TNFR through TNFα receptor-associated factor 2 (TRAF2), leading to K63-linked polyubiquitylation of receptor interacting protein kinase 1 (RIPK1), which is essential for NF-κB-mediated cell survival.⁷ The lack of RIPK1 polyubiquitylation via cIAP1/2 depletion or the presence of CYLD deubiquitylase triggers TNFR complex IIa formation, thereby inducing caspase-8-dependent apoptosis.⁸ In addition, cIAP1/2 prevents the formation of the RIPK1-containing death complex ripoptosome in response to several stimuli including CD95, TNFα-related apoptosis-inducing ligand (TRAIL), genotoxic stress, and Toll-like receptor (TLR) activation.^{9, 10, 11, 12, 13}IAPs are frequently overexpressed in various human cancers, and their expression is associated with chemoresistance and poor clinical outcome.⁶ Therefore, inhibiting IAP function is an attractive strategy to treat cancer through the induction of apoptosis.^{5, 14} Upon apoptotic stimuli, IAPs are inhibited by the second mitochondria-derived activator of caspases (Smac),⁵ and this discovery led to the development of Smac mimetic peptides using the IAP binding motif containing four amino acids (Ala-Val-Pro-Ile). These peptides were shown to sensitize cells to apoptotic stimuli and efficiently suppress tumor growth in a xenograft model.^{15, 16} Subsequently, a number of small-molecule compounds mimicking the Smac mimetic peptide (Smac mimetics) were developed with improved pharmacological properties and IAP-binding affinity. Interestingly, Smac mimetics, such as BV6 and compound A, were found to induce autoubiquitylation and degradation of cIAP1/2.^{17, 18} Furthermore, cIAP1/2 depletion with Smac mimetics activates the non-canonical NF-κB pathway to induce autocrine TNFα production, which is essential for Smac mimetic-induced apoptotic cell death.^{18, 19}Because cIAP1 and cIAP2 show functional redundancy in TNFα-mediated survival, the depletion of both proteins is usually required for effective induction of cell death upon TNFα treatment.^{20, 21} However, there are several reports showing that cIAP2 expression, but not cIAP1 expression, renders cells resistant to Smac mimetic-induced cell death.^{20, 21} For example, cIAP2 upregulation via phosphoinositide 3-kinase (PI3K) upon compound 3 treatment in certain cell lines was shown to facilitate apoptosis evasion.²² In addition, treatment with compounds A and C led to cIAP1 dimerization, without cIAP2 dimerization, resulting in the autoubiquitylation and subsequent degradation of cIAP1. These findings may explain why cIAP1 is degraded more efficiently than cIAP2 upon treatment with Smac mimetics.²³ Alternatively, because cIAP2 degradation requires cIAP1, cIAP2 may become more stable when cIAP1 is depleted using Smac mimetics.²⁴ Direct cIAP deubiquitylation by OTUB1 or USP19 has been suggested to be responsible for cIAP stabilization;^{25, 26} however, these previous studies did not focus on the difference in stabilization between cIAP1 and cIAP2 and only provided general deubiquitylation-dependent mechanisms.^{25, 26}While several studies have supported hypotheses for how cIAP2 survives in the presence of Smac mimetics, numerous independent studies have also shown that cIAP2 can be efficiently degraded by Smac mimetics in various cell lines.^{27, 28, 29, 30, 31, 32} These observations suggest the existence of other factors that specifically regulate cIAP2 stability upon Smac mimetic treatment. In this study, we propose a new mechanism involving USP11-mediated cIAP2 regulation. We found that the differential destabilization of cIAP1 and cIAP2 is dependent on the presence of the cIAP2-specific deubiquitylase USP11. Mechanistically, USP11 can protect cIAP2 from Smac mimetic-mediated degradation, rendering cell lines with high USP11 expression unresponsive to Smac mimetic treatment. However, USP11 downregulation sensitized these cells to TNFα- or TRAIL-induced apoptosis in the presence of Smac mimetic and further suppressed tumor growth in a xenograft model. Corroborating these data, USP11 overexpression was observed in colon cancer and melanoma patients with poor clinical outcome. Finally, the TNFα/c-Jun N-terminal kinase (JNK) pathway induced USP11 expression, which was necessary for cIAP2 protein stabilization and its anti-apoptotic function. Thus, the identification of cIAP2-specific deubiquitylation indicates that more elaborate strategies should be developed for pharmaceutical therapies targeting cIAPs.     

Functional characterisation of the regulation of CAAT enhancer binding protein alpha by GSK-3 phosphorylation of Threonines 222/226

Background  

Glycogen Synthase Kinase-3 (GSK3) activity is repressed following insulin treatment of cells. Pharmacological inhibition of GSK3 mimics the effect of insulin on Phosphoenolpyruvate Carboxykinase (PEPCK), Glucose-6 Phosphatase (G6Pase) and IGF binding protein-1 (IGFBP1) gene expression. CAAT/enhancer binding protein alpha (C/EBPα) regulates these gene promoters in liver and is phosphorylated on two residues (T222/T226) by GSK3, although the functional outcome of the phosphorylation has not been established. We aimed to establish whether CEBPα is a link between GSK3 and these gene promoters.     

Feeding and territorial behaviour in juveniles of three co-existing triggerfishes

Of three coexisting triggerfishes, in the intertidal and immediate subtidal zones of a coral reef within the Kenting National Park, southern Taiwan, Balistoides viridescens was observed only in the subtidal zone and foraged preferentially by nipping directly at small invertebrates among algal covered substrata. They fed also, although at a lower frequency, by using a water-jet technique to uncover prey on sandy substrata. Territory size ranged from 61·05 to 161·57 m² but was not related to rates of feeding or defence. Feeding frequency was, however, positively related to the area of algae present in the territory, and inversely related to the frequency of defence. In contrast to B. viridescens, Rhinecanthus verrucosus and R. aculeatus foraged mainly in the intertidal zone when the tide was high. They fed preferentially off rocky substrata and used only nipping (not water-jetting) to secure prey.     

Neuronal responses to myelin are mediated by rho kinase

CNS myelin inhibits axon growth due to the expression of several growth-inhibitory proteins, including myelin-associated glycoprotein, oligodendrocyte myelin glycoprotein and Nogo. Myelin-associated inhibitory proteins activate rho GTPase in responsive neurons. Rho kinase (ROCK) has been implicated as a critical rho effector in this pathway due to the ability of the pharmacological inhibitor Y-27632 to circumvent myelin-dependent inhibition. Y-27632, however, inhibits the activity of additional kinases. Using three independent approaches, we provide direct evidence that ROCKII is activated in response to the myelin-associated inhibitor Nogo. We demonstrate that Nogo treatment enhances ROCKII translocation to the cellular membrane in PC12 cells and enhances ROCKII kinase activity towards an in vitro substrate. In addition, Nogo treatment enhances phosphorylation of myosin light chain II, a known ROCK substrate. Further, we demonstrate that primary dorsal root ganglia neurons can be rendered insensitive to the inhibitory effects of myelin via infection with dominant negative ROCK. Together these data provide direct evidence for a rho-ROCK-myosin light chain-II signaling cascade in response to myelin-associated inhibitors.     

PKC-dependent ERK phosphorylation is essential for P2X7 receptor-mediated neuronal differentiation of neural progenitor cells

Purinergic receptors have been shown to be involved in neuronal development, but the functions of specific subtypes of P2 receptors during neuronal development remain elusive. In this study we investigate the distribution of P2X₇ receptors (P2X₇Rs) in the embryonic rat brain using in situ hybridization. At E15.5, P2X₇R mRNA was observed in the ventricular zone and subventricular zone, and colocalized with nestin, indicating that P2X₇R might be expressed in neural progenitor cells (NPCs). P2X₇R mRNA was also detected in the subgranular zone and dentate gyrus of the E18.5 and P4 brain. To investigate the roles of P2X₇R and elucidate its mechanism, we established NPC cultures from the E15.5 rat brain. Stimulation of P2X₇Rs induced Ca²⁺ influx, inhibited proliferation, altered cell cycle progression and enhanced the expression of neuronal markers, such as TUJ1 and MAP2. Similarly, knockdown of P2X₇R by shRNA nearly abolished the agonist-stimulated increases in intracellular Ca²⁺ concentration and the expression of TUJ1 and NeuN. Furthermore, stimulation of P2X₇R induced activation of ERK1/2, which was inhibited by the removal of extracellular Ca²⁺ and treatment with blockers for P2X₇R and PKC activity. Stimulation of P2X₇R also induced translocation of PKCα and PKCγ, but not of PKCβ, whereas knockdown of either PKCα or PKCγ inhibited ERK1/2 activation. Inhibition of PKC or p-ERK1/2 also caused a decrease in the number of TUJ1-positive cells and a concomitant increase in the number of GFAP-positive cells. Taken together, the activation of P2X₇R in NPCs induced neuronal differentiation through a PKC-ERK1/2 signaling pathway.     

Oligomeric and functional properties of a debranching enzyme (TreX) from the archaeon Sulfolobus solfataricus P2

A gene, treX, encoding a debranching enzyme previously cloned from the trehalose biosynthesis gene cluster of Sulfolobus solfataricus P2 was expressed in Escherichia coli as a His-tagged protein and the biochemical properties were studied. The specific activity of the S. solfataricus debranching enzyme (TreX) was highest at 75°C and pH 5.5. The enzyme exhibited hydrolysing activity toward α-1,6-glycosidic linkages of amylopectin, glycogen, pullulan, and other branched substrates, and glycogen was the preferred substrate. TreX has a high specificity for hydrolysis of maltohexaosyl α-1,6-β-cyclodextrin, indicating the high preference for side chains consisting of 6 glucose residues or more. The enzyme also exhibited 4-α-sulfoxide-glucan transferase activity, catalysing transfer of α-1,4-glucan oligosaccharides from one chain to another. Dimethyl sulfoxide (10%, v/v) increased the hydrolytic activity of TreX. Gel permeation chromatography and sedimentation equilibrium analytical ultracentrifugation revealed that the enzyme exists mostly as a dimer at pH 7.0, and as a mixture of dimers and tetramers at pH 5.5. Interestingly, TreX existed as a tetramer in the presence of DMSO at pH 5.5–6.5. The tetramer showed a 4-fold higher catalytic efficiency than the dimer. The enzyme catalysed not only intermolecular trans-glycosylation of malto-oligosaccharides (disproportionation) to produce linear α-1,4-glucans, but also intramolecular trans-glycosylation of glycogen. The results presented in this study indicated that TreX may be associated with glycogen metabolism by selective cleavage of the outer side chain.     

Interval between preovulatory surges of luteinizing hormone increases late in the reproductive period in turkey hens

In turkey hens, the egg production rate is relatively high early during a reproductive period, but declines as the period progresses. Among lines with different egg production potential, the interval between preovulatory surges of LH is the primary determinant of the egg production rate. The main objective of this study was to determine whether the decline in egg production rate late during an egg production period is also associated with a difference in the interval between LH preovulatory surges. A group of photosensitive turkey hens (Early) were photostimulated with continuous light (24L:0D) at 40 wk of age to induce egg laying, and serial blood samples were collected after about 3 wk of egg production. A second group of hens (Late) were housed in floor pens and photostimulated with 14L:10D at 40 wk of age for a normal 36-wk reproduction period and were then switched to 24L:0D lighting for 2 wk before collection of serial blood samples. Continuous light photostimulation was used for at least 2 wk before and during serial blood sampling to avoid potential masking effects of diurnal lighting on the interval between LH surges. The Early (n = 12) and Late (n = 16) hens were cannulated 3 days before being serially bled hourly for 10 days. The mean interval between preovulatory surges of LH was shorter in the Early hens than in the Late hens (26.1 +/- 2.5 h and 34.7 +/- 3.9 h, respectively). The intra-hen LH surge interval coefficient of variation was lower in the Early hens than in the Late hens (7.2% and 18.6%, respectively). The inter-hen LH surge interval coefficient of variation was similar in the Early and Late hens (9.5% and 11.2%, respectively). The incidence of blind surges of LH (those not retrospectively associated with ovipositions) was not different between Early and Late laying hens (8.4% +/- 15.2% and 7.3% +/- 14.6%, respectively). In conclusion, in turkey hens, longer intervals and greater intra-hen variation between LH surges were associated with a poorer rate of egg production late in the reproductive period relative to early in the reproductive period.     

Isoprenoid depletion by statins antagonizes cytokine-induced down-regulation of endothelial nitric oxide expression and increases NO synthase activity in human umbilical vein endothelial cells.

Endothelial dysfunction and atherosclerosis are associated with an inflammation-induced decrease in endothelial nitric oxide synthase (eNOS) expression. Based on the differences between hydrophobic and hydrophilic statins in their reduction of cardiac events, we analyzed the effects of rosuvastatin and cerivastatin on eNOS and inducible NO synthase (iNOS) expression and NOS activity in TNF-alpha-stimulated human umbilical vein endothelial cells (HUVEC). Both statins reversed down-regulation of eNOS mRNA and protein expression by inhibiting HMG-CoA reductase and isoprenoid synthesis. Cerivastatin tended to a more pronounced effect on eNOS expression compared to rosuvastatin. NOS activity - measured by conversion of [(3)H]-L-arginine to [(3)H]-L-citrulline - was enhanced under treatment with both drugs due to inhibition of HMG-CoA reductase. Statin-treatment reduced iNOS mRNA expression under normal conditions, but had no relevant effects on iNOS mRNA expression in cytokine-treated cells. Rosuvastatin and cerivastatin reverse the detrimental effects of TNF-alpha-induced down-regulation in eNOS protein expression and increase NO synthase activity by inhibiting HMG-CoA reductase and subsequent blocking of isoprenoid synthesis. These results provide evidence that statins have beneficial effects by increasing eNOS expression and activity during the atherosclerotic process.