Yuichi Nishi

Neural-specific Sox2 input and differential Gli-binding affinity provide context and positional information in Shh-directed neural patterning

In the vertebrate neural tube, regional Sonic hedgehog (Shh) signaling invokes a time- and concentration-dependent induction of six different cell populations mediated through Gli transcriptional regulators. Elsewhere in the embryo, Shh/Gli responses invoke different tissue-appropriate regulatory programs. A genome-scale analysis of DNA binding by Gli1 and Sox2, a pan-neural determinant, identified a set of shared regulatory regions associated with key factors central to cell fate determination and neural tube patterning. Functional analysis in transgenic mice validates core enhancers for each of these factors and demonstrates the dual requirement for Gli1 and Sox2 inputs for neural enhancer activity. Furthermore, through an unbiased determination of Gli-binding site preferences and analysis of binding site variants in the developing mammalian CNS, we demonstrate that differential Gli-binding affinity underlies threshold-level activator responses to Shh input. In summary, our results highlight Sox2 input as a context-specific determinant of the neural-specific Shh response and differential Gli-binding site affinity as an important cis-regulatory property critical for interpreting Shh morphogen action in the mammalian neural tube.

Global Transcriptional Repression in C. elegans Germline Precursors by Regulated Sequestration of TAF-4

In C. elegans, four asymmetric divisions, beginning with the zygote (P0), generate transcriptionally repressed germline blastomeres (P1-P4) and somatic sisters that become transcriptionally active. The protein PIE-1 represses transcription in the later germline blastomeres but not in the earlier germline blastomeres P0 and P1. We show here that OMA-1 and OMA-2, previously shown to regulate oocyte maturation, repress transcription in P0 and P1 by binding to and sequestering in the cytoplasm TAF-4, a component critical for assembly of TFIID and the pol II preinitiation complex. OMA-1/2 binding to TAF-4 is developmentally regulated, requiring phosphorylation by the DYRK kinase MBK-2, which is activated at meiosis II after fertilization. OMA-1/2 are normally degraded after the first mitosis, but ectopic expression of wild-type OMA-1 is sufficient to repress transcription in both somatic and later germline blastomeres. We propose that phosphorylation by MBK-2 serves as a developmental switch, converting OMA-1/2 from oocyte to embryo regulators.

Exendin-4 Suppresses Src Activation and Reactive Oxygen Species Production in Diabetic Goto-Kakizaki Rat Islets in an Epac-Dependent Manner

OBJECTIVE Reactive oxygen species (ROS) is one of most important factors in impaired metabolism secretion coupling in pancreatic β-cells. We recently reported that elevated ROS production and impaired ATP production at high glucose in diabetic Goto-Kakizaki (GK) rat islets are effectively ameliorated by Src inhibition, suggesting that Src activity is upregulated. In the present study, we investigated whether the glucagon-like peptide-1 signal regulates Src activity and ameliorates endogenous ROS production and ATP production in GK islets using exendin-4. RESEARCH DESIGN AND METHODS Isolated islets from GK and control Wistar rats were used for immunoblotting analyses and measurements of ROS production and ATP content. Src activity was examined by immunoprecipitation of islet lysates followed by immunoblotting. ROS production was measured with a fluorescent probe using dispersed islet cells. RESULTS Exendin-4 significantly decreased phosphorylation of Src Tyr416, which indicates Src activation, in GK islets under 16.7 mmol/l glucose exposure. Glucose-induced ROS production (16.7 mmol/l) in GK islet cells was significantly decreased by coexposure of exendin-4 as well as PP2, a Src inhibitor. The Src kinase–negative mutant expression in GK islets significantly decreased ROS production induced by high glucose. Exendin-4, as well as PP2, significantly increased impaired ATP elevation by high glucose in GK islets. The decrease in ROS production by exendin-4 was not affected by H-89, a PKA inhibitor, and an Epac-specific cAMP analog (8CPT-2Me-cAMP) significantly decreased Src Tyr416 phosphorylation and ROS production. CONCLUSIONS Exendin-4 decreases endogenous ROS production and increases ATP production in diabetic GK rat islets through suppression of Src activation, dependently on Epac.

Polo kinases regulate C. elegans embryonic polarity via binding to DYRK2-primed MEX-5 and MEX-6.

Polo kinases are known key regulators of cell divisions. Here we report a novel, non-cell division function for polo kinases in embryonic polarity of newly fertilized Caenorhabditis elegans embryos. We show that polo kinases, via their polo box domains, bind to and regulate the activity of two key polarity proteins, MEX-5 and MEX-6. These polo kinases are asymmetrically localized along the anteroposterior axis of newly fertilized C. elegans embryos in a pattern identical to that of MEX-5 and MEX-6. This asymmetric localization of polo kinases depends on MEX-5 and MEX-6, as well as genes regulating MEX-5 and MEX-6 asymmetry. We identify an amino acid of MEX-5, T186, essential for polo binding and show that T186 is important for MEX-5 function in vivo. We also show that MBK-2, a developmentally regulated DYRK2 kinase activated at meiosis II, primes T186 for subsequent polo kinase-dependent phosphorylation. Prior phosphorylation of MEX-5 at T186 greatly enhances phosphorylation of MEX-5 by polo kinases in vitro. Our results provide a mechanism by which MEX-5 and MEX-6 function is temporally regulated during the crucial oocyte-to-embryo transition.

Curcumin inhibits glucose production in isolated mice hepatocytes

Curcumin is a compound derived from the spice turmeric, and is a potent anti-oxidant, anti-carcinogenic, and anti-hepatotoxic agent. We have investigated the acute effects of curcumin on hepatic glucose production. Gluconeogenesis and glycogenolysis in isolated hepatocytes, and gluconeogenetic enzyme activity after 120 min exposure to curcumin were measured. Hepatic gluconeogenesis from 1 mM pyruvate was inhibited in a concentration-dependent manner, with a maximal decrease of 45% at the concentration of 25 microM. After 120 min exposure to 25 microM curcumin, hepatic gluconeogenesis from 2mM dihydroxyacetone phosphate and hepatic glycogenolysis were inhibited by 35% and 20%, respectively. Insulin also inhibited hepatic gluconeogenesis from 1mM pyruvate and inhibited hepatic glycogenolysis in a concentration-dependent manner. Curcumin (25 microM) showed an additive inhibitory effect with insulin on both hepatic gluconeogenesis and glycogenolysis, indicating that curcumin inhibits hepatic glucose production in an insulin-independent manner. After 120 min exposure to 25 microM curcumin, hepatic glucose-6-phosphatase (G6Pase) activity and phosphoenolpyruvate carboxykinase (PEPCK) activity both were inhibited by 30%, but fructose-1,6-bisphosphatase (FBPase) was not reduced. After 120 min exposure to 25 microM curcumin, phosphorylation of AMP kinase alpha-Thr(172) was increased. Thus, the anti-diabetic effects of curcumin are partly due to a reduction in hepatic glucose production caused by activation of AMP kinase and inhibition of G6Pase activity and PEPCK activity.

Essential role for ligand-dependent feedback antagonism of vertebrate hedgehog signaling by PTCH1, PTCH2 and HHIP1 during neural patterning

Hedgehog (HH) signaling is essential for vertebrate and invertebrate embryogenesis. In Drosophila, feedback upregulation of the HH receptor Patched (PTC; PTCH in vertebrates), is required to restrict HH signaling during development. By contrast, PTCH1 upregulation is dispensable for early HH-dependent patterning in mice. Unique to vertebrates are two additional HH-binding antagonists that are induced by HH signaling, HHIP1 and the PTCH1 homologue PTCH2. Although HHIP1 functions semi-redundantly with PTCH1 to restrict HH signaling in the developing nervous system, a role for PTCH2 remains unresolved. Data presented here define a novel role for PTCH2 as a ciliary localized HH pathway antagonist. While PTCH2 is dispensable for normal ventral neural patterning, combined removal of PTCH2- and PTCH1-feedback antagonism produces a significant expansion of HH-dependent ventral neural progenitors. Strikingly, complete loss of PTCH2-, HHIP1- and PTCH1-feedback inhibition results in ectopic specification of ventral cell fates throughout the neural tube, reflecting constitutive HH pathway activation. Overall, these data reveal an essential role for ligand-dependent feedback inhibition of vertebrate HH signaling governed collectively by PTCH1, PTCH2 and HHIP1.

A low resistance microfluidic system for the creation of stable concentration gradients in a defined 3D microenvironment

The advent of microfluidic technology allows control and interrogation of cell behavior by defining the local microenvironment with an assortment of biochemical and biophysical stimuli. Many approaches have been developed to create gradients of soluble factors, but the complexity of such systems or their inability to create defined and controllable chemical gradients has limited their widespread implementation. Here we describe a new microfluidic device which employs a parallel arrangement of wells and channels to create stable, linear concentration gradients in a gel region between a source and a sink well. Pressure gradients between the source and sink wells are dissipated through low resistance channels in parallel with the gel channel, thus minimizing the convection of solute in this region. We demonstrate the ability of the new device to quantitate chemotactic responses in a variety of cell types, yielding a complete profile of the migratory response and representing the total number of migrating cells and the distance each cell has migrated. Additionally we show the effect of concentration gradients of the morphogen Sonic hedgehog on the specification of differentiating neural progenitors in a 3-dimensional matrix.

Rapamycin impairs metabolism-secretion coupling in rat pancreatic islets by suppressing carbohydrate metabolism

Rapamycin, an immunosuppressant used in human transplantation, impairs beta-cell function, but the mechanism is unclear. Chronic (24 h) exposure to rapamycin concentration dependently suppressed 16.7 mM glucose-induced insulin release from islets (1.65+/-0.06, 30 nM rapamycin versus 2.35+/-0.11 ng/islet per 30 min, control, n=30, P<0.01) without affecting insulin and DNA contents. Rapamycin also decreased alpha-ketoisocaproate-induced insulin release, suggesting reduced mitochondrial carbohydrate metabolism. ATP content in the presence of 16.7 mM glucose was significantly reduced in rapamycin-treated islets (13.42+/-0.47, rapamycin versus 16.04+/-0.46 pmol/islet, control, n=30, P<0.01). Glucose oxidation, which indicates the velocity of metabolism in the Krebs cycle, was decreased by rapamycin in the presence of 16.7 mM glucose (30.1+/-2.7, rapamycin versus 42.2+/-3.3 pmol/islet per 90 min, control, n=9, P<0.01). Immunoblotting revealed that the expression of complex I, III, IV, and V was not affected by rapamycin. Mitochondrial ATP production indicated that the respiratory chain downstream of complex II was not affected, but that carbohydrate metabolism in the Krebs cycle was reduced by rapamycin. Analysis of enzymes in the Krebs cycle revealed that activity of alpha-ketoglutarate dehydrogenase (KGDH), which catalyzes one of the slowest reactions in the Krebs cycle, was reduced by rapamycin (10.08+/-0.82, rapamycin versus 13.82+/-0.84 nmol/mg mitochondrial protein per min, control, n=5, P<0.01). Considered together, these findings indicate that rapamycin suppresses high glucose-induced insulin secretion from pancreatic islets by reducing mitochondrial ATP production through suppression of carbohydrate metabolism in the Krebs cycle, together with reduced KGDH activity.

zif-1 translational repression defines a second, mutually exclusive OMA function in germline transcriptional repression.

Specification of primordial germ cells requires global repression of transcription. In C. elegans, primordial germ cells are generated through four rounds of asymmetric divisions, starting from the zygote P0, each producing a transcriptionally repressed germline blastomere (P1-P4). Repression in P2-P4 requires PIE-1, which is provided maternally in oocytes and segregated to all germline blastomeres. We have shown previously that OMA-1 and OMA-2 repress global transcription in P0 and P1 by sequestering TAF-4, an essential component of TFIID. Soon after the first mitotic cycle, OMA proteins undergo developmentally regulated degradation. Here, we show that OMA proteins also repress transcription in P2-P4 indirectly, through a completely different mechanism that operates in oocytes. OMA proteins bind to both the 3′ UTR of the zif-1 transcript and the eIF4E-binding protein, SPN-2, repressing translation of zif-1 mRNA in oocytes. zif-1 encodes the substrate-binding subunit of the E3 ligase for PIE-1 degradation. Inhibition of zif-1 translation in oocytes ensures high PIE-1 levels in oocytes and germline blastomeres. The two OMA protein functions are strictly regulated in both space and time by MBK-2, a kinase activated following fertilization. Phosphorylation by MBK-2 facilitates the binding of OMA proteins to TAF-4 and simultaneously inactivates their function in repressing zif-1 translation. Phosphorylation of OMA proteins displaces SPN-2 from the zif-1 3′ UTR, releasing translational repression. We propose that MBK-2 phosphorylation serves as a developmental switch, converting OMA proteins from specific translational repressors in oocytes to global transcriptional repressors in embryos, together effectively repressing transcription in all germline blastomeres.

Src activation generates reactive oxygen species and impairs metabolism–secretion coupling in diabetic Goto–Kakizaki and ouabain-treated rat pancreatic islets

Aims/hypothesisNa+/K+-ATPase inhibition by ouabain suppresses ATP production by generating reactive oxygen species (ROS) and impairs glucose-induced insulin secretion from pancreatic islets. To clarify the signal-transducing function of Na+/K+-ATPase in decreasing ATP production by the generation of ROS in pancreatic islets, the involvement of Src was examined. In addition, the significance of Src activation in diabetic islets was examined.MethodsIsolated islets from Wistar rats and diabetic Goto–Kakizaki (GK) rats (a model for diabetes) were used. ROS was measured by 5-(and 6)-chloromethyl-2′,7′-dichlorofluorescein fluorescence using dispersed islet cells. After lysates were immunoprecipitated by anti-Src antibody, immunoblotting was performed.ResultsOuabain caused a rapid Tyr418 phosphorylation, indicating activation of Src in the presence of high glucose. The specific Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) restored the ouabain-induced decrease in ATP content and the increase in ROS production. Both PP2 and ROS scavenger restored the impaired insulin release and impaired ATP elevation in GK islets, but had no such effect in control islets. PP2 reduced the high glucose-induced increase in ROS generation in GK islet cells but had no effect on that in control islet cells. Moreover, ouabain had no effect on ATP content and ROS production in the presence of high glucose in GK islets.Conclusions/interpretationThese results indicate that Src plays a role in the signal-transducing function of Na+/K+-ATPase, in which ROS generation decreases ATP production in control islets. Moreover, ROS generated by Src activation plays an important role in impaired glucose-induced insulin secretion in GK islets, in which Src is endogenously activated independently of ouabain.