Understanding the role of thyroid hormone in Sertoli cell development: a mechanistic hypothesis

More than a decade of research has shown that Sertoli cell proliferation is regulated by thyroid hormone. Neonatal hypothyroidism lengthens the period of Sertoli cell proliferation, leading to increases in Sertoli cell number, testis weight, and daily sperm production (DSP) when euthyroidism is re-established. In contrast, the neonatal Sertoli cell proliferative period is shortened under hyperthyroid conditions, but the mechanism by which thyroid hormone is able to negatively regulate Sertoli cell proliferation has been unclear. Recent progress in the understanding of the cell cycle has provided the opportunity to dissect the molecular targets responsible for thyroid-hormone-mediated effects on Sertoli cell proliferation. In this review, we discuss recent results indicating a critical role for the cyclin-dependent kinase inhibitors (CDKI) p27^Kip1 and p21^Cip1 in establishing Sertoli cell number, testis weight, and DSP, and the ability of thyroid hormone to modulate these CDKIs. Based on these recent results, we propose a working hypothesis for the way in which thyroid hormone regulates the withdrawal of the cell cycle by controlling CDKI degradation. Finally, although Sertoli cells have been shown to have two biologically active thyroid hormone receptor (TR) isoforms, TRα1 and TRβ1, experiments with transgenic mice lacking TRα or TRβ illustrate that only one TR mediates thyroid hormone effects in neonatal Sertoli cells. Although significant gaps in our knowledge still remain, advances have been made toward appreciation of the molecular sequence of events that occur when thyroid hormone stimulates Sertoli cell maturation. We gratefully acknowledge the support of this work by the NIH, USDA, the University of Illinois, the Lalor Foundation, and the Thanis A. Field Endowment at the University of Illinois. D.R. Holsberger was supported by postdoctoral fellowships from the Lalor Foundation and Reproductive Biology Research Training Program (NIH grant T32 HD07028), University of Illinois at Urbana–Champaign.     

The Orphan G Protein-coupled Receptor GPR17 Negatively Regulates Oligodendrocyte Differentiation via Gαi/o and Its Downstream Effector Molecules

Recent studies have recognized G protein-coupled receptors as important regulators of oligodendrocyte development. GPR17, in particular, is an orphan G protein-coupled receptor that has been identified as oligodendroglial maturation inhibitor because its stimulation arrests primary mouse oligodendrocytes at a less differentiated stage. However, the intracellular signaling effectors transducing its activation remain poorly understood. Here, we use Oli-neu cells, an immortalized cell line derived from primary murine oligodendrocytes, and primary rat oligodendrocyte cultures as model systems to identify molecular targets that link cell surface GPR17 to oligodendrocyte maturation blockade. We demonstrate that stimulation of GPR17 by the small molecule agonist MDL29,951 (2-carboxy-4,6-dichloro-1H-indole-3-propionic acid) decreases myelin basic protein expression levels mainly by triggering the Gα_i/o signaling pathway, which in turn leads to reduced activity of the downstream cascade adenylyl cyclase-cAMP-PKA-cAMP response element-binding protein (CREB). In addition, we show that GPR17 activation also diminishes myelin basic protein abundance by lessening stimulation of the exchange protein directly activated by cAMP (EPAC), thus uncovering a previously unrecognized role for EPAC to regulate oligodendrocyte differentiation. Together, our data establish PKA and EPAC as key downstream effectors of GPR17 that inhibit oligodendrocyte maturation. We envisage that treatments augmenting PKA and/or EPAC activity represent a beneficial approach for therapeutic enhancement of remyelination in those demyelinating diseases where GPR17 is highly expressed, such as multiple sclerosis.     

Partial purification of two forms of Choline kinase and separation of Choline kinase from sphingosine kinase of rat brain

Choline kinase of rat brain was purified approximately 200,000 fold using acid precipitation, ammonium sulphate fractionation, Q-Sepharose, Octyl-Sepharose and AH-Sepharose chromatography. The ability of this enzyme to catalyze the phosphorylation of choline, ethanolamine (Etn), monomethylethanolamine (MeEtn), dimethylethanolamine (Me₂Etn) and sphingosine was investigated. Choline kinase was separated from sphingosine kinase. The fraction with highly purified choline kinase had four major polypeptides with different molecular masses and possessed activities towards choline, Etn, MeEtn and Me₂Etn. Two forms of choline kinase were obtained when the enzymatically active fractions eluted from the Q-Sepharose column were subjected to a horizontal isoelectrofocusing electrophoresis. One form focused around pH 4.7 and is able to phosphorylate choline, Etn, MeEtn and Me₂Etn. The other form focused around pH 10 and possessed only choline kinase activity. The latter form of choline kinase did not display classical Michaelis-Menten's mechanism but revealed a positive co-operative pattern for two choline binding sites. This form was purified to apparent homogeneity with a approximate molecular mass of 14.4 kDa.Abbreviations Etn ethanolamine - MeEtn N-monomethylethanolamine - Me₂Etn N, N-dimethylethanolamine     

Transcriptional suppression of nephrin in podocytes by macrophages: roles of inflammatory cytokines and involvement of the PI3K/Akt pathway

Expression of nephrin, a crucial component of the glomerular slit diaphragm, is downregulated in patients with proteinuric glomerular diseases. Using conditionally immortalized reporter podocytes, we found that bystander macrophages as well as macrophage-derived cytokines IL-1beta and TNF-alpha markedly suppressed activity of the nephrin gene promoter in podocytes. The cytokine-initiated repression was reversible, observed on both basal and inducible expression, independent of Wilms' tumor suppressor WT1, and caused in part via activation of the phosphatidylinositol-3-kinase/Akt pathway. These results indicated a novel mechanism by which activated macrophages participate in the induction of proteinuria in glomerular diseases.     

Screening of Drugs That Suppress Ste11 MAPKKK Activation in Yeast Identified a c-Abl Tyrosine Kinase Inhibitor

The yeast MAPKKK Ste11 activates three MAP kinase pathways, including pheromone signaling, osmosensing, and pseudohyphal/invasive growth pathways. We identified two chemical compounds, BTB03006 and GK03225, that suppress growth defects induced by Ste11 activation in diploid yeast cells. BTB03006, but not GK03225, was found to suppress growth defects induced by both α-factor and Ste4 G_β overexpression in the pheromone signaling pathway, suggesting that GK03225 is an osmosensing pathway-specific inhibitor. We also performed genome-wide suppressor analysis for Ste11 activation, using a yeast deletion strains collection, and identified PBS2 and HOG1, and several genes associated with chaperone functions, which represent potential target proteins of the drugs screened from Ste11 activation. GK03225 possesses an Iressa-like quinazoline ring structure, and its chemical analog, 11N-078, suppresses c-Abl human tyrosine kinase activity. These results suggest that drug screening in yeast can identify human tyrosine kinase inhibitors and other drugs for human diseases.     

Monoamine oxidases (MAO) in the pathogenesis of heart failure and ischemia/reperfusion injury

Recent evidence highlights monoamine oxidases (MAO) as another prominent source of oxidative stress. MAO are a class of enzymes located in the outer mitochondrial membrane, deputed to the oxidative breakdown of key neurotransmitters such as norepinephrine, epinephrine and dopamine, and in the process generate H₂O₂. All these monoamines are endowed with potent modulatory effects on myocardial function. Thus, when the heart is subjected to chronic neuro-hormonal and/or peripheral hemodynamic stress, the abundance of circulating/tissue monoamines can make MAO-derived H₂O₂ production particularly prominent. This is the case of acute cardiac damage due to ischemia/reperfusion injury or, on a more chronic stand, of the transition from compensated hypertrophy to overt ventricular dilation/pump failure. Here, we will first briefly discuss mitochondrial status and contribution to acute and chronic cardiac disorders. We will illustrate possible mechanisms by which MAO activity affects cardiac biology and function, along with a discussion as to their role as a prominent source of reactive oxygen species. Finally, we will speculate on why MAO inhibition might have a therapeutic value for treating cardiac affections of ischemic and non-ischemic origin. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

Structural Basis for Auto-Inhibition of the NDR1 Kinase Domain by an Atypically Long Activation Segment

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Rapid increase in endothelial nitric oxide production by bradykinin is mediated by protein kinase A signaling pathway

Bradykinin (BK) acutely increases endothelial nitric oxide (NO) production by activating endothelial NO synthase (eNOS), and this increase is in part correlated with enhanced phosphorylation/dephosphorylation of eNOS by several protein kinases and phosphatases. However, the signaling mechanisms producing this increase are still controversial. In an attempt to delineate the acute effect of BK on endothelial NO production, confluent bovine aortic endothelial cells were incubated with BK, and NO production was measured by NO-specific chemiluminescence. Significant increase in NO levels was detected as early as 1 min after BK treatment, with concomitant increase in the phosphorylation of Ser(1179) (bovine sequence) site of eNOS (eNOS-Ser(1179)). This acute effect of BK on both increases was blocked only by treatment of protein kinase A inhibitor H-89, but not by the inhibitors of calmodulin-dependent kinase II and protein kinase B, suggesting that the rapid increase in NO production by BK is mediated by the PKA-dependent phosphorylation of eNOS-Ser(1179).     

PLD activation in Chinese hamster ovary (CHO) cells transfected with PGF2α receptor cDNA

Pertussis toxin-insensitive GTP-binding protein was observed to be involved in prostaglandin F2α(PGF2α)-induced phosphoinositide metabolism in Chinese hamster ovary (CHO) cells transfected with PGF2α receptor cDNA (CHO-PGF2α·R cells) (Ito, S. et al. Biochem. Biophys. Res. Commun. 200: 756, 1994). In the present study, we investigated PGF2α-induced PLD activation in CHO-PGF2α·R cells. PLD activation was examined by measuring the production of [³H]phosphatidylbutanol ([³H]PBut), a specific product of the PLD-catalyzed transphosphatidylation reaction. PGF2α-induced [³H]PBut formation was concentration-dependent with the maximal level obtained at 1 μM PGF2α. The maximal [³H]PBut formation was observed at 2 min after addition of PGF2α. Depletion of extracellular Ca²⁺ with EGTA suppressed PGF2α-induced PLD activation by 50%. PKC inhibitors Ro31–8425 and calphostin C inhibited PGF2α-induced [³H]PBut formation by 50%. PTK inhibitors genistein and herbimycin A failed to inhibit PGF2α-induced PLD activation. A combination of maximal effective concentrations of PGF2α (1 μM) and PMA (100 nM) enhanced PLD activation in an additive manner. Pretreatment of the cells with PMA for 2 h down-regulated PKCα and decreased PGF2α-induced PLD activation. These results suggest that PLD activation by PGF2α is mediated by both PKC-dependent and -independent pathways and that PKCα is involved in the former pathway.