Mechanisms for the control of body size by a G-kinase and a downstream TGFbeta signal pathway in Caenorhabditis elegans

We recently showed that egl-4 mutants in Caenorhabditis elegans have a much larger body size and that the egl-4 gene encodes cyclic GMP-dependent protein kinases (G-kinases). Cell sizes, but not cell numbers, in the major organs are increased in the mutants. Genetic interaction studies suggest that EGL-4 represses the DBL-1/TGFbeta pathway that is known to control body size. To understand the mechanisms of body size control in C. elegans, we analysed sma-2, sma-4 and sma-6 small mutants in the DBL-1 pathway. The volumes of major organs were precisely determined with the method developed by us. They are significantly decreased as compared to those of the wild-type while cell numbers are not, indicating that cell size is decreased. DNA contents in the nuclei of major organs are not significantly changed in the small mutants and in an egl-4 large mutant. Total protein contents are much decreased in the small mutants and slightly increased in the egl-4 mutant. Based on these results, we propose that decreased cell and body size of the small mutants in the DBL-1/TGFbeta pathway is mainly due to decreased levels of protein expression, and that increase in fluid content is a major reason for the increase in cell and body size in egl-4 mutants.     

Mutants carrying two sma mutations are super small in the nematode C. elegans

Body size determination is critical for multicellular organisms; however, the mechanisms remain largely unknown. Mutations that alter body size were studied to solve the mechanisms, for example, in mouse, fruit fly and the nematode Caenorhabditis elegans. In C. elegans, a large mutant and several small body size (sma) mutants are known. Of the latter, sma-2, sma-3, sma-4, sma-6, dbl-1 and daf-4 have a mutation in the components of the DBL-1/TGFbeta signal pathway, and sma-5 in a MAP kinase homologue. We have constructed double mutants carrying two of such small body size mutations, sma-5 and sma-4 or sma-2. They are much smaller than either of the parental single mutants, indicating that the sma-5 gene functions independently of the DBL-1/TGFbeta pathway. We show that their body volumes are as small as 1/10 of that of the wild-type, and that the sizes of major organs are much reduced, by the methods previously developed by us. But the numbers of cells are not changed, suggesting that the cells are very small. These results highlight surprising flexibility of body size and cell size in a multicellular organism, which will give a novel insight into the mechanisms of body size control.     

FMRFamide neuropeptides and acetylcholine synergistically inhibit egg-laying by C. elegans

Egg-laying behavior of the Caenorhabditis elegans hermaphrodite is regulated by G protein signaling pathways. Here we show that the egg laying-defective mutant egl-6(n592) carries an activating mutation in a G protein-coupled receptor that inhibits C. elegans egg-laying motor neurons in a G(o)-dependent manner. Ligands for EGL-6 are Phe-Met-Arg-Phe-NH(2) (FMRFamide)-related peptides encoded by the genes flp-10 and flp-17. flp-10 is expressed in both neurons and non-neuronal cells. The major source of flp-17 peptides is a pair of presumptive sensory neurons, the BAG neurons. Genetic analysis of the egl-6 pathway revealed that the EGL-6 neuropeptide signaling pathway functions redundantly with acetylcholine to inhibit egg-laying. The retention of embryos in the uterus of the C. elegans hermaphrodite is therefore under the control of a presumptive sensory system and is inhibited by the convergence of signals from neuropeptides and the small-molecule neurotransmitter acetylcholine.     

EGL-4/PKG regulates the role of an interneuron in a chemotaxis circuit of C. elegans through mediating integration of sensory signals

Interneurons, innervated by multiple sensory neurons, need to integrate information from these sensory neurons and respond to sensory stimuli adequately. Mechanisms how sensory information is integrated to form responses of interneurons are not fully understood. In Caenorhabditis elegans, loss-of-function mutations of egl-4, which encodes a cGMP-dependent protein kinase (PKG), cause a defect in chemotaxis to odorants. Our genetic and imaging analyses revealed that the response property of AIY interneuron to an odorant is reversed in the egl-4 mutant, while the responses of two upstream olfactory neurons, AWA and AWC, are largely unchanged. Cell- ablation experiments show that AIY in the egl-4 mutant functions to suppress chemotaxis. Furthermore, the reversal of AIY response occurs only in the presence of sensory signals from both AWA and AWC. These results suggest that sensory signals are inadequately integrated in the egl-4 mutant. We also show that egl-4 expression in AWA and another sensory neuron prevents the reversed AIY response and restores chemotaxis in the egl-4 mutants. We propose that EGL-4/PKG, by suppressing aberrant integration of signals from olfactory neurons, converts the response property of an interneuron to olfactory stimuli and maintains the role of the interneuron in the circuit to execute chemotactic behavior.     

clr-1 encodes a receptor tyrosine phosphatase that negatively regulates an FGF receptor signaling pathway in Caenorhabditis elegans

Receptor tyrosine phosphatases have been implicated in playing important roles in cell signaling events by their ability to regulate the level of protein tyrosine phosphorylation. Although the catalytic activity of their phosphatase domains has been well established, the biological roles of these molecules are, for the most part, not well understood. Here we show that the Caenorhabditis elegans protein CLR-1 (CLeaR) is a receptor tyrosine phosphatase (RTP) with a complex extracellular region and two intracellular phosphatase domains. Mutations in clr-1 result in a dramatic Clr phenotype that we have used to study the physiological requirements for the CLR-1 RTP. We show that the phosphatase activity of the membrane-proximal domain is essential for the in vivo function of CLR-1. By contrast, we present evidence that the membrane-distal domain is not required to prevent the Clr phenotype in vivo. The Clr phenotype of clr-1 mutants is mimicked by activation of the EGL-15 fibroblast growth factor receptor (FGFR) and is suppressed by mutations that reduce or eliminate the activity of egl-15. Our data strongly indicate that CLR-1 attenuates the action of an FGFR-mediated signaling pathway by dephosphorylation.     

Trio's Rho-specific GEF domain is the missing Galpha q effector in C. elegans

The Galpha(q) pathway is essential for animal life and is a central pathway for driving locomotion, egg laying, and growth in Caenorhabditis elegans, where it exerts its effects through EGL-8 (phospholipase Cbeta [PLCbeta]) and at least one other effector. To find the missing effector, we performed forward genetic screens to suppress the slow growth and hyperactive behaviors of mutants with an overactive Galpha(q) pathway. Four suppressor mutations disrupted the Rho-specific guanine-nucleotide exchange factor (GEF) domain of UNC-73 (Trio). The mutations produce defects in neuronal function, but not neuronal development, that cause sluggish locomotion similar to animals lacking EGL-8 (PLCbeta). Strains containing null mutations in both EGL-8 (PLCbeta) and UNC-73 (Trio RhoGEF) have strong synthetic phenotypes that phenocopy the arrested growth and near-complete paralysis of Galpha(q)-null mutants. Using cell-based and biochemical assays, we show that activated C. elegans Galpha(q) synergizes with Trio RhoGEF to activate RhoA. Activated Galpha(q) and Trio RhoGEF appear to be part of a signaling complex, because they coimmunoprecipitate when expressed together in cells. Our results show that Trio's Rho-specific GEF domain is a major Galpha(q) effector that, together with PLCbeta, mediates the Galpha(q) signaling that drives the locomotion, egg laying, and growth of the animal.     

Abnormalities of the transforming growth factor-beta pathway in ocular melanoma

The majority of ocular melanomas occur in the uveal tract. Chemotherapy is generally ineffective and large tumours requiring enucleation have a greater than 50% mortality at 5 years. Monosomy for chromosome 3 is common in uveal melanoma and it is known that there is loss of responsiveness to transforming growth factor beta (TGFbeta) in melanoma cell lines. Since the gene for TGFbeta receptor II (TGFbetaR2) is located on chromosome 3p22, this study investigates the possibility that the TGFbeta pathway, and TGFbetaR2 in particular, might be involved in the pathogenesis of this rare eye tumour. To this end, the expression of molecules in the pathway has been examined by immunocytochemistry (TGFbeta, TGFbetaR2, SMAD2, SMAD3, SMAD4, and p27), backed up by a cell culture assay of TGFbeta-mediated growth suppression, RT-PCR for SMAD4, and loss of heterozygosity (LOH) on 3p22. There was LOH at 3p22 in 6/19 tumours and loss of TGFbetaR2 expression in 10/27 tumours. Immunohistochemistry for SMADs 2, 3, and 4 showed potential loss of signal transduction in 14/27 tumours. The results indicate abnormality of the TGFbeta pathway in 61% of tumours for which unequivocal results were obtained and suggest that abrogation of control of melanocyte growth by the TGFbeta pathway may be important in the formation of uveal melanoma.     

Antagonism between Goα and Gqα in Caenorhabditis elegans: the RGS protein EAT-16 is necessary for Goα signaling and regulates Gqα activity

To elucidate the cellular role of the heterotrimeric G protein G(o), we have taken a molecular genetic approach in Caenorhabditis elegans. We screened for suppressors of activated GOA-1 (G(o)alpha) that do not simply decrease its expression and found mutations in only two genes, sag-1 and eat-16. Animals defective in either gene display a hyperactive phenotype similar to that of goa-1 loss-of-function mutants. Double-mutant analysis indicates that both sag-1 and eat-16 act downstream of, or parallel to, G(o)alpha and negatively regulate EGL-30 (G(q)alpha) signaling. eat-16 encodes a regulator of G protein signaling (RGS) most similar to the mammalian RGS7 and RGS9 proteins and can inhibit endogenous mammalian G(q)/G(11) in COS-7 cells. Animals defective in both sag-1 and eat-16 are inviable, but reducing function in egl-30 restores viability, indicating that the lethality of the eat-16; sag-1 double mutant is due to excessive G(q)alpha activity. Analysis of these mutations indicates that the G(o) and G(q) pathways function antagonistically in C. elegans, and that G(o)alpha negatively regulates the G(q) pathway, possibly via EAT-16 or SAG-1. We propose that a major cellular role of G(o) is to antagonize signaling by G(q).     

A nucleostemin family GTPase, NS3, acts in serotonergic neurons to regulate insulin signaling and control body size

Growth and body size are regulated by the CNS, integrating the genetic developmental program with assessments of an animal's current energy state and environmental conditions. CNS decisions are transmitted to all cells of the animal by insulin/insulin-like signals. The molecular biology of the CNS growth control system has remained, for the most part, elusive. Here we identify NS3, a Drosophila nucleostemin family GTPase, as a powerful regulator of body size. ns3 mutants reach <60% of normal size and have fewer and smaller cells, but exhibit normal body proportions. NS3 does not act cell-autonomously, but instead acts at a distance to control growth. Rescue experiments were performed by expressing wild-type ns3 in many different cells of ns3 mutants. Restoring NS3 to only 106 serotonergic neurons rescued global growth defects. These neurons are closely apposed with those of insulin-producing neurons, suggesting possible communication between the two neuronal systems. In the brains of ns3 mutants, excess serotonin and insulin accumulate, while peripheral insulin pathway activation is low. Peripheral insulin pathway activation rescues the growth defects of ns3 mutants. The findings suggest that NS3 acts in serotonergic neurons to regulate insulin signaling and thus exert global growth control.     

Modification of intracellular cAMP and cGMP concentration in yeast wild strains and in selected mutants from Saccharomyces cerevisiae as a regulation model for higher eukaryotes]

The addition of D(+)-glucose (final concentration 50 mM) to a cell suspension of yeasts (wild type and several mutants of the cell cycle, the cAMP-dependent protein kinase system, and a mutant of the adenylate cyclase gene) triggers a rapid increase in the concentrations of cAMP and cGMP in the wild strain. In contrast to cAMP, an increase of cGMP was also found in the mutants. cAMP and cGMP have been characterized as second messengers in eucaryotic cells. Cyclic nucleotide activation of the protein kinases enables them to perform their only known function in eukaryotes, the phosphorylation of substrate proteins. The results, described here by using selected yeast mutants as a model for higher eukaryotes, indicate that there exist two different regulatory systems for the control of the cAMP and cGMP levels.     

The Axin-like protein PRY-1 is a negative regulator of a canonical Wnt pathway in C. elegans

Axin, APC, and the kinase GSK3 beta are part of a destruction complex that regulates the stability of the Wnt pathway effector beta-catenin. In C. elegans, several Wnt-controlled developmental processes have been described, but an Axin ortholog has not been found in the genome sequence and SGG-1/GSK3 beta, and the APC-related protein APR-1 have been shown to act in a positive, rather than negative fashion in Wnt signaling. We have shown previously that the EGL-20/Wnt-dependent expression of the homeobox gene mab-5 in the Q neuroblast lineage requires BAR-1/beta-catenin and POP-1/Tcf. Here, we have investigated how BAR-1 is regulated by the EGL-20 pathway. First, we have characterized a negative regulator of the EGL-20 pathway, pry-1. We show that pry-1 encodes an RGS and DIX domain-containing protein that is distantly related to Axin/Conductin. Our results demonstrate that despite its sequence divergence, PRY-1 is a functional Axin homolog. We show that PRY-1 interacts with BAR-1, SGG-1, and APR-1 and that overexpression of PRY-1 inhibits mab-5 expression. Furthermore, pry-1 rescues the zebrafish axin1 mutation masterblind, showing that it can functionally interact with vertebrate destruction complex components. Finally, we show that SGG-1, in addition to its positive regulatory role in early embryonic Wnt signaling, may function as a negative regulator of the EGL-20 pathway. We conclude that a highly divergent destruction complex consisting of PRY-1, SGG-1, and APR-1 regulates BAR-1/beta-catenin signaling in C. elegans.     

Effects of L-arginine and sodium nitroprusside on the spontaneous contractility of human non-pregnant uterus

BACKGROUND: The aim of this study was to investigate, in isolated human non-gravid myometrium, the involvement of the cyclic guanosine monophosphate (cGMP) pathway in nitric oxide (NO) induced relaxation. METHODS: Strips of human myometrium from hysterectomized women were suspended in organ baths for recording of isometric tension. Cumulative concentration-response curves for L-arginine and sodium nitroprusside were performed in the presence of methylene blue (10 micromol/l) or vehicle (control). The effect of increasing concentrations of 8-bromo-cGMP on uterine spontaneous contraction was also studied. RESULTS: L-arginine and sodium nitroprusside induced a concentration-dependent decrease in the amplitude of the myometrial spontaneous contractions. Pre-treatment with methylene blue enhanced the inhibitory effect of L-arginine and sodium nitroprusside on myometrial spontaneous contractions. In addition, 8-bromo-cGMP had no effect on spontaneous contractions in human myometrium. CONCLUSIONS: These data provide evidence that L-arginine and sodium nitroprusside inhibit the spontaneous contractions of the non-pregnant human uterus through a cGMP independent pathway.