A cAMP receptor-like G protein-coupled receptor with roles in growth regulation and development

Dictyostelium discoideum uses G protein-mediated signal transduction for many vegetative and developmental functions, suggesting the existence of G protein-coupled receptors (GPCRs) other than the four known cyclic adenosine monophosphate (cAMP) receptors (cAR1-4). Sequences of the cAMP receptors were used to identify Dictyostelium genes encoding cAMP receptor-like proteins, CrlA-C. Limited sequence identity between these putative GPCRs and the cAMP receptors suggests the Crl receptors are unlikely to be receptors for cAMP. The crl genes are expressed at various times during growth and the developmental life cycle. Disruption of individual crl genes did not impair chemotactic responses to folic acid or cAMP or alter cAMP-dependent aggregation. However, crlA⁻ mutants grew to a higher cell density than did wild-type cells and high-copy-number crlA expression vectors were detrimental to cell viability, suggesting that CrlA is a negative regulator of cell growth. In addition, crlA⁻ mutants produce large aggregates with delayed anterior tip formation indicating a role for the CrlA receptor in the development of the anterior prestalk cell region. The scarcity of GFP-expressing crlA⁻ mutants in the anterior prestalk cell region of chimeric organisms supports a cell-autonomous role for the CrlA receptor in prestalk cell differentiation.     

Six family genes control the proliferation and differentiation of muscle satellite cells

Muscle satellite cells are essential for muscle growth and regeneration and their morphology, behavior and gene expression have been extensively studied. However, the mechanisms involved in their proliferation and differentiation remain elusive. Six1 and Six4 proteins were expressed in the nuclei of myofibers of adult mice and the numbers of myoblasts positive for Six1 and Six4 increased during regeneration of skeletal muscles. Six1 and Six4 were expressed in quiescent, activated and differentiated muscle satellite cells isolated from adult skeletal muscle. Overexpression of Six4 and Six5 repressed the proliferation and differentiation of satellite cells. Conversely, knockdown of Six5 resulted in augmented proliferation, and that of Six4 inhibited differentiation. Muscle satellite cells isolated from Six4^+/−Six5^−/− mice proliferated to higher cell density though their differentiation was not altered. Meanwhile, overproduction of Six1 repressed proliferation and promoted differentiation of satellite cells. In addition, Six4 and Six5 repressed, while Six1 activated myogenin expression, suggesting that the differential regulation of myogenin expression is responsible for the differential effects of Six genes. The results indicated the involvement of Six genes in the behavior of satellite cells and identified Six genes as potential target for manipulation of proliferation and differentiation of muscle satellite cells for therapeutic applications.     

Glutathione initiates the development of Dictyostelium discoideum through the regulation of YakA

Reduced glutathione (GSH) is an essential metabolite that performs multiple indispensable roles during the development of Dictyostelium. We show here that disruption of the gene (gcsA¯) encoding γ-glutamylcysteine synthetase, an essential enzyme in GSH biosynthesis, inhibited aggregation, and that this developmental defect was rescued by exogenous GSH, but not by other thiols or antioxidants. In GSH-depleted gcsA¯ cells, the expression of a growth-stage-specific gene (cprD) was not inhibited, and we did not detect the expression of genes that encode proteins required for early development (cAMP receptor, carA/cAR1; adenylyl cyclase, acaA/ACA; and the catalytic subunit of protein kinase A, pkaC/PKA-C). The defects in gcsA¯ cells were not restored by cAMP stimulation or by cAR1 expression. Further, the expression of yakA, which initiates development and induces the expression of PKA-C, ACA, and cAR1, was regulated by the intracellular concentration of GSH. Constitutive expression of YakA in gcsA¯ cells (YakA^OE/gcsA¯) rescued the defects in developmental initiation and the expression of early developmental genes in the absence of GSH. Taken together, these findings suggest that GSH plays an essential role in the transition from growth to development by modulating the expression of the genes encoding YakA as well as components that act downstream in the YakA signaling pathway.     

Disruption of Stard10 gene alters the PPARα-mediated bile acid homeostasis

STARD10, a member of the steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) protein family, is highly expressed in the liver and has been shown to transfer phosphatidylcholine. Therefore it has been assumed that STARD10 may function in the secretion of phospholipids into the bile. To help elucidate the physiological role of STARD10, we produced Stard10 knockout mice (Stard10^−/−) and studied their phenotype. Neither liver content nor biliary secretion of phosphatidylcholine was altered in Stard10^−/− mice. Unexpectedly, the biliary secretion of bile acids from the liver and the level of taurine-conjugated bile acids in the bile were significantly higher in Stard10^−/− mice than wild type (WT) mice. In contrast, the levels of the secondary bile acids were lower in the liver of Stard10^−/− mice, suggesting that the enterohepatic cycling is impaired. STARD10 was also expressed in the gallbladder and small intestine where the expression level of apical sodium dependent bile acid transporter (ASBT) turned out to be markedly lower in Stard10^−/− mice than in WT mice when measured under fed condition. Consistent with the above results, the fecal excretion of bile acids was significantly increased in Stard10^−/− mice. Interestingly, PPARα-dependent genes responsible for the regulation of bile acid metabolism were down-regulated in the liver of Stard10^−/− mice. The loss of STARD10 impaired the PPARα activity and the expression of a PPARα-target gene such as Cyp8b1 in mouse hepatoma cells. These results indicate that STARD10 is involved in regulating bile acid metabolism through the modulation of PPARα-mediated mechanism.     

CREM modulates the circadian expression of CYP51, HMGCR and cholesterogenesis in the liver

We show for the first time that isoforms of the cAMP response element modulator Crem, regulate the circadian expression of Cyp51 and other cholesterogenic genes in the mouse liver. In the wild type mice the expression of Cyp51, Hmgs, Fpps, and Sqs is minimal between CT12 and CT16 and peaks between CT20 and CT24. Cyp51, Fpps, and Sqs lost the circadian behavior in Crem−/− livers while Hmgcr is phase advanced from CT20 to CT12. This coincides with a phase advance of lathosterol/cholesterol ratio, as detected by GC-MS. Overexpression of CREMτ and ICER has little effect on the Hmgcr proximal promoter while they influence expression from the CYP51 promoter. Our data indicate that Crem-dependent regulation of Cyp51 in the liver results in circadian expression of this gene. We propose that cAMP signaling might generally be involved in the circadian regulation of cholesterol synthesis on the periphery.     

Intracellular expression of Vitreoscilla hemoglobin improves production of Yarrowia lipolytica lipase LIP2 in a recombinant Pichia pastoris

The Yarrowia lipolytica lipase LIP2 (YlLIP2) gene lip2 and Vitreoscilla hemoglobin gene vgb were co-expressed in Pichia pastoris, both under the control of AOX1 promoter, in order to alleviate respiration limitation under conditions of high cell-density fermentation and enhance YlLIP2 production. The results showed that recombinant P. pastoris strains harboring the lip2 and vgb genes (VHb⁺) displayed higher biomass and YlLIP2 activity than control strains (VHb⁻). Compared with VHb⁻ cells, the expression levels of YlLIP2 in VHb-expressing cells when oxygen was not a limiting factor were improved 31.5% in shake-flask culture and 22% in a 10-L fermentor. Under non-limiting dissolved oxygen (DO) conditions, the maximum YlLIP2 activity of VHb⁺ in a 10-L fermentor reached 33,000 U/mL. Oxygen limitation had a more negative effect on YlLIP2 productivity in VHb⁻ cells than in VHb⁺ cells. The highest YlLIP2 activity of VHb⁺ cells was approximately 1.84-fold higher than that of VHb⁻ cells at lower DO levels. Moreover, the recombinant strain VHb⁺ exhibited a higher specific oxygen uptake rate and achieved higher cell viability under oxygen limiting and non-limiting conditions compared with VHb⁻ cells. Therefore, the above results suggest that intracellular expression of VHb in recombinant P. pastoris has the potential to improve cell growth and industrial enzyme production.     

Tbx18 regulates development of the epicardium and coronary vessels

The epicardium and coronary vessels originate from progenitor cells in the proepicardium. Here we show that Tbx18, a T-box family member highly expressed in the proepicardium, controls critical early steps in coronary development. In Tbx18^−/− mouse embryos, both the epicardium and coronary vessels exhibit structural and functional defects. At E12.5, the Tbx18-deficient epicardium contains protrusions and cyst-like structures overlying a disorganized coronary vascular plexus that contains ectopic structures resembling blood islands. At E13.5, the left and right coronary stems form correctly in mutant hearts. However, analysis of PECAM-1 whole mount immunostaining, distribution of SM22α^lacZ/+ activity, and analysis of coronary vascular casts suggest that defective vascular plexus remodeling produces a compromised arterial network at birth consisting of fewer distributing conduit arteries with smaller lumens and a reduced capacity to conduct blood flow. Gene expression profiles of Tbx18^−/− hearts at E12.5 reveal altered expression of 79 genes that are associated with development of the vascular system including sonic hedgehog signaling components patched and smoothened, VEGF-A, angiopoietin-1, endoglin, and Wnt factors compared to wild type hearts. Thus, formation of coronary vasculature is responsive to Tbx18-dependent gene targets in the epicardium, and a poorly structured network of coronary conduit vessels is formed in Tbx18 null hearts due to defects in epicardial cell signaling and fate during heart development. Lastly, we demonstrate that Tbx18 possesses a SRF/CArG box dependent repressor activity capable of inhibiting progenitor cell differentiation into smooth muscle cells, suggesting a potential function of Tbx18 in maintaining the progenitor status of epicardial-derived cells.     

PakD,a Putative p21-Activated Protein Kinase in Dictyostelium discoideum,Regulates Actin

Proper regulation of the actin cytoskeleton is essential for cell function and ultimately for survival. Tight control of actin dynamics is required for many cellular processes, including differentiation, proliferation, adhesion, chemotaxis, endocytosis, exocytosis, and multicellular development. Here we describe a putative p21-activated protein kinase, PakD, that regulates the actin cytoskeleton in Dictyostelium discoideum. We found that cells lacking pakD are unable to aggregate and thus unable to develop. Compared to the wild type, cells lacking PakD have decreased membrane extensions, suggesting defective regulation of the actin cytoskeleton. pakD⁻ cells show poor chemotaxis toward cyclic AMP (cAMP) but normal chemotaxis toward folate, suggesting that PakD mediates some but not all chemotaxis responses. pakD⁻ cells have decreased polarity when placed in a cAMP gradient, indicating that the chemotactic defects of the pakD⁻ cells may be due to an impaired cytoskeletal response to cAMP. In addition, while wild-type cells polymerize actin in response to global stimulation by cAMP, pakD⁻ cells exhibit F-actin depolymerization under the same conditions. Taken together, the results suggest that PakD is part of a pathway coordinating F-actin organization during development.     

Gene Promoter Evolution Targets the Center of the Human Protein Interaction Network

Assessing the contribution of promoters and coding sequences to gene evolution is an important step toward discovering the major genetic determinants of human evolution. Many specific examples have revealed the evolutionary importance of cis-regulatory regions. However, the relative contribution of regulatory and coding regions to the evolutionary process and whether systemic factors differentially influence their evolution remains unclear. To address these questions, we carried out an analysis at the genome scale to identify signatures of positive selection in human proximal promoters. Next, we examined whether genes with positively selected promoters (Prom⁺ genes) show systemic differences with respect to a set of genes with positively selected protein-coding regions (Cod⁺ genes). We found that the number of genes in each set was not significantly different (8.1% and 8.5%, respectively). Furthermore, a functional analysis showed that, in both cases, positive selection affects almost all biological processes and only a few genes of each group are located in enriched categories, indicating that promoters and coding regions are not evolutionarily specialized with respect to gene function. On the other hand, we show that the topology of the human protein network has a different influence on the molecular evolution of proximal promoters and coding regions. Notably, Prom⁺ genes have an unexpectedly high centrality when compared with a reference distribution (P = 0.008, for Eigenvalue centrality). Moreover, the frequency of Prom⁺ genes increases from the periphery to the center of the protein network (P = 0.02, for the logistic regression coefficient). This means that gene centrality does not constrain the evolution of proximal promoters, unlike the case with coding regions, and further indicates that the evolution of proximal promoters is more efficient in the center of the protein network than in the periphery. These results show that proximal promoters have had a systemic contribution to human evolution by increasing the participation of central genes in the evolutionary process.     

A protein with similarity to PTEN regulates aggregation territory size by decreasing cyclic AMP pulse size during Dictyostelium discoideum development

An interesting but largely unanswered biological question is how eukaryotic organisms regulate the size of multicellular tissues. During development, a lawn of Dictyostelium cells breaks up into territories, and within the territories the cells aggregate in dendritic streams to form groups of ~20,000 cells. Using random insertional mutagenesis to search for genes involved in group size regulation, we found that an insertion in the cnrN gene affects group size. Cells lacking CnrN (cnrN⁻) form abnormally small groups, which can be rescued by the expression of exogenous CnrN. Relayed pulses of extracellular cyclic AMP (cAMP) direct cells to aggregate by chemotaxis to form aggregation territories and streams. cnrN⁻ cells overaccumulate cAMP during development and form small territories. Decreasing the cAMP pulse size by treating cnrN⁻ cells with cAMP phosphodiesterase or starving cnrN⁻ cells at a low density rescues the small-territory phenotype. The predicted CnrN sequence has similarity to phosphatase and tensin homolog (PTEN), which in Dictyostelium inhibits cAMP-stimulated phosphatidylinositol 3-kinase signaling pathways. CnrN inhibits cAMP-stimulated phosphatidylinositol 3,4,5-trisphosphate accumulation, Akt activation, actin polymerization, and cAMP production. Our results suggest that CnrN is a protein with some similarities to PTEN and that it regulates cAMP signal transduction to regulate territory size.     

Fungal genes related to calcium homeostasis and signalling are upregulated in symbiotic arbuscular mycorrhiza interactions

Fluctuations in intracellular calcium levels generate signalling events and regulate different cellular processes. Whilst the implication of Ca²⁺ in plant responses during arbuscular mycorrhiza (AM) interactions is well documented, nothing is known about the regulation or role of this secondary messenger in the fungal symbiont. The spatio-temporal expression pattern of putatively Ca²⁺-related genes of Glomus intraradices BEG141 encoding five proteins involved in membrane transport and one nuclear protein kinase, was investigated during the AM symbiosis. Expression profiles related to successful colonization of host roots were observed in interactions of G. intraradices with roots of wild-type Medicago truncatula (line J5) compared to the mycorrhiza-defective mutant dmi3/Mtsym13. Symbiotic fungal activity was monitored using stearoyl-CoA desaturase and phosphate transporter genes. Laser microdissection based-mapping of fungal gene expression in mycorrhizal root tissues indicated that the Ca²⁺-related genes were differentially upregulated in arbuscules and/or in intercellular hyphae. The spatio-temporal variations in gene expression suggest that the encoded proteins may have different functions in fungal development or function during symbiosis development. Full-length cDNA obtained for two genes with interesting expression profiles confirmed a close similarity with an endoplasmic reticulum P-type ATPase and a Vcx1-like vacuolar Ca²⁺ ion transporter functionally characterized in other fungi and involved in the regulation of cell calcium pools. Possible mechanisms are discussed in which Ca²⁺-related proteins G. intraradices BEG141 may play a role in mobilization and perception of the intracellular messenger by the AM fungus during symbiotic interactions with host roots.     

Evidence for complete epistasis of null mutations in murine Fanconi anemia genes Fanca and Fancg

Fanconi anemia (FA) is a heritable disease characterized by bone marrow failure, congenital abnormalities, and cancer predisposition. The 15 identified FA genes operate in a molecular pathway to preserve genomic integrity. Within this pathway the FA core complex operates as an ubiquitin ligase that activates the complex of FANCD2 and FANCI to coordinate DNA repair. The FA core complex is formed by at least 12 proteins. However, only the FANCL subunit displays ubiquitin ligase activity. FANCA and FANCG are members of the FA core complex for which no other functions have been described than to participate in protein interactions. In this study we generated mice with combined null alleles for Fanca and Fancg to identify extended functions for these genes by characterizing the double mutant mice and cells.Double mutant a^−/−/g^−/− mice were born at near Mendelian frequencies without apparent developmental abnormalities. Histological analysis of a^−/−/g^−/− mice revealed a Leydig cell hyperplasia and frequent vacuolization of Sertoli cells in testes, while ovaries were depleted from developing follicles and displayed an interstitial cell hyperplasia. These gonadal aberrations were associated with a compromised fertility of a^−/−/g^−/− males and females. During the first year of life a^−/−/g^−/− did not develop malignancies or bone marrow failure. At the cellular level a^−/−/g^−/−, Fanca^−/−, and Fancg^−/− cells proved equally compromised in DNA crosslink and homology-directed repair. Overall the phenotype of a^−/−/g^−/− double knockout mice and cells appeared highly similar to the phenotype of Fanca or Fancg single knockouts. The lack of an augmented phenotype suggest that null mutations in Fanca or Fancg are fully epistatic, making additional important functions outside of the FA core complex highly unlikely.     

α2-3 Sialic acid glycoconjugate loss and its effect on infection with Toxoplasma parasites

Recognition of sialylated glycoconjugates is important for host cell invasion by Apicomplexan parasites. Toxoplasma gondii parasites penetrate host cells via interactions between their microneme proteins and sialylated glycoconjugates on the surface of host cells. However, the role played by sialic acids during infection with T. gondii is not well understood. Here, we focused on the role of α2-3 sialic acid linkages as they appear to be widely expressed in vertebrates. Removal of α2-3 sialic acid linkages on macrophages by neuraminidase treatment did not influence the rate of infection or growth of T. gondii, nor did it affect phagocytosis in vitro. Sialyltransferase ST3Gal-I deficient mice (ST3Gal-I^−/− mice) lost α2-3 sialic acid linkages in macrophages and spleen cells. The numbers of T. gondii-infected CD11b⁺ cells in peritoneal cavities of the infected ST3Gal-I^−/− mice were relatively lower than those of the infected wild type animals. In addition, CD8⁺ T cell populations and numbers in the spleens and peritoneal cavities of the ST3Gal-I^−/− mice were significantly lower than those in the wild type animals before and after the T. gondii infection. ST3Gal-I^−/− mice had severe liver damage and reduced survival rates following peritoneal infection with T. gondii. Furthermore, adoptive transfer of immune CD8⁺ cells from wild type mice to ST3Gal-I^−/− mice increased their survival during infection with T. gondii. Our data show that parasite invasion via α2-3 sialic acid linkages might not contribute on host survival and indicate the impact that loss of α2-3 sialic acid linkages has on CD8⁺ T cell populations, which are necessary for effective immune responses against infection with T. gondii.