Mob as tumor suppressor is activated by Hippo kinase for growth inhibition in Drosophila

Tissue growth and organ size are determined by coordinated cell proliferation and apoptosis in development. Recent studies have demonstrated that Hippo (Hpo) signaling plays a crucial role in coordinating these processes by restricting cell proliferation and promoting apoptosis. Here we provide evidence that the Mob as tumor suppressor protein, Mats, functions as a key component of the Hpo signaling pathway. We found that Mats associates with Hpo in a protein complex and is a target of the Hpo serine/threonine protein kinase. Mats phosphorylation by Hpo increases its affinity with Warts (Wts)/large tumor suppressor (Lats) serine/threonine protein kinase and ability to upregulate Wts catalytic activity to target downstream molecules such as Yorkie (Yki). Consistently, our epistatic analysis suggests that mats acts downstream of hpo. Coexpression analysis indicated that Mats can indeed potentiate Hpo-mediated growth inhibition in vivo. Our results support a model in which Mats is activated by Hpo through phosphorylation for growth inhibition, and this regulatory mechanism is conserved from flies to mammals.     

Akt is negatively regulated by the MULAN E3 ligase

The serine/threonine kinase Akt functions in multiple cellular processes, including cell survival and tumor development. Studies of the mechanisms that negatively regulate Akt have focused on dephosphorylation-mediated inactivation. In this study, we identified a negative regulator of Akt, MULAN, which possesses both a RING finger domain and E3 ubiquitin ligase activity. Akt was found to directly interact with MULAN and to be ubiquitinated by MULAN in vitro and in vivo. Other molecular assays demonstrated that phosphorylated Akt is a substantive target for both interaction with MULAN and ubiquitination by MULAN. The results of the functional studies suggest that the degradation of Akt by MULAN suppresses cell proliferation and viability. These data provide insight into the Akt ubiquitination signaling network.     

Engulfment of apoptotic cells is negatively regulated by Rho-mediated signaling

The rapid and efficient phagocytosis of apoptotic cells plays a critical role in preventing secondary necrosis, inflammation as well as in tissue remodeling and regulating immune responses. However, the molecular details of engulfment are just beginning to be elucidated. Among the Rho family GTPases, previous studies have implicated a role for Rac and Cdc42 in the uptake of apoptotic cells by phagocytes, yet the role of Rho has remained unclear. Here, we present evidence that Rho-GTP levels decrease during engulfment. RhoA seems to negatively affect basal engulfment, such that inhibition of Rho-mediated signaling in phagocytes enhanced the uptake of apoptotic targets. Activation of endogenous Rho or overexpression of constitutively active forms of Rho also inhibited engulfment. By testing mutants of RhoA that selectively activate downstream effectors, the Rho-kinase seemed to be primarily responsible for this inhibitory effect. Taken together, these data suggest that inhibition of Rho- and Rho-kinase-mediated signaling might be important during engulfment, which could have important implications for several clinical trials involving inhibition of the Rho kinase.     

The pre-B cell receptor signaling for apoptosis is negatively regulated by Fc gamma RIIB.

Many studies have shown that FcgammaRIIB is a negative regulator of B cell receptor signaling, and even though FcgammaRIIB is expressed through all developmental stages of the B cell lineage, its involvement in pre-B cell receptor (pre-BCR) signaling has not been examined. To investigate FcgammaRIIB function at the pre-B cell stage, we have established pre-BCR positive pre-B cell lines from normal mice and FcgammaRIIB-deficient mice, named PreBR and Fcgamma(-/-)PreBR, respectively. These cell lines are able to differentiate into immature B cells in vitro by removal of IL-7. In PreBR, apoptosis was moderately induced by F(ab')(2) anti-mu Ab, but not by intact anti-mu Ab. Phosphorylation of SH2-containing inositol 5-phosphatase (SHIP) and Dok, which are involved in FcgammaRIIB signaling, was induced by anti-mu cross-linking in PreBR. In contrast, apoptosis was strongly induced by both the F(ab')(2) and intact anti-mu Abs in Fcgamma(-/-)PreBR, and the level of phosphorylation of SHIP or Dok was much lower in Fcgamma(-/-)PreBR than those observed in PreBR. Restoration of FcgammaRIIB to Fcgamma(-/-)PreBR followed by anti-mu cross-linking blocked severe apoptosis, and up-regulated SHIP and Dok phosphorylation. The results demonstrate that FcgammaRIIB negatively regulates pre-BCR-mediated signaling for apoptosis.     

The Drosophila caspase inhibitor DIAP1 is essential for cell survival and is negatively regulated by HID.

Drosophila Reaper (RPR), Head Involution Defective (HID), and GRIM induce caspase-dependent cell death and physically interact with the cell death inhibitor DIAP1. Here we show that HID blocks DIAP1's ability to inhibit caspase activity and provide evidence suggesting that RPR and GRIM can act similarly. Based on these results, we propose that RPR, HID, and GRIM promote apoptosis by disrupting productive IAP-caspase interactions and that DIAP1 is required to block apoptosis-inducing caspase activity. Supporting this hypothesis, we show that elimination of DIAP1 function results in global early embryonic cell death and a large increase in DIAP1-inhibitable caspase activity and that DIAP1 is still required for cell survival when expression of rpr, hid, and grim is eliminated.     

Drosophila PI4KIIIalpha is required in follicle cells for oocyte polarization and Hippo signaling

In a genetic screen we isolated mutations in CG10260, which encodes a phosphatidylinositol 4-kinase (PI4KIIIalpha), and found that PI4KIIIalpha is required for Hippo signaling in Drosophila ovarian follicle cells. PI4KIIIalpha mutations in the posterior follicle cells lead to oocyte polarization defects similar to those caused by mutations in the Hippo signaling pathway. PI4KIIIalpha mutations also cause misexpression of well-established Hippo signaling targets. The Merlin-Expanded-Kibra complex is required at the apical membrane for Hippo activity. In PI4KIIIalpha mutant follicle cells, Merlin fails to localize to the apical domain. Our analysis of PI4KIIIalpha mutants provides a new link in Hippo signal transduction from the cell membrane to its core kinase cascade.     

CYLD negatively regulates Hippo signaling by limiting Hpo phosphorylation in Drosophila

Cylindromatosis (CYLD), a deubiquitinase and regulator of microtubule dynamics, has important roles in the regulation of inflammation, immune response, apoptosis, mitosis, cell migration and tumorigenesis. Although great progress has been made in the biochemical and cellular functions of CYLD, its role in animal development remains elusive. In this study, we identified Drosophila CYLD (dCYLD) as a negative regulator of the Hippo pathway in vivo. dCYLD associates and colocalizes with Hpo, a core component of the Hippo pathway, in the cytoplasm, and decreases Hpo activity through limiting its phosphorylation at T195. We also showed that dCYLD limits Hippo signal transduction as evidenced by decreasing phosphorylation and thereby increasing activity of Yki, the key downstream effector of the Hippo pathway. These findings uncover dCYLD as a negative regulator of the Hippo pathway and provide new insights into the physiological function of dCYLD in animal development.     

Murine folliculogenesis in vitro is stage-specifically regulated by insulin via the Akt signaling pathway

Folliculogenesis is a critical process during mammalian development, but little is known about the mechanisms underlying primordial follicular formation, growth, and activation. The objective of this study was to assess the effect of continuous insulin exposure on folliculogenesis in 16.5 dpc and 3 dpp mouse ovaries in vitro. The results demonstrated that 5 μg/ml insulin promotes the phosphorylation of Akt and the primordial follicular assembly and activation process via the InsR/PI3K/Akt signaling pathway in the 16.5 dpc fetal mouse ovaries cultured in vitro. However, the presence of 5 μg/ml insulin represses the phosphorylation of Akt and the follicular assembly and activation process in the 3 dpp mouse ovaries cultured in vitro. Furthermore, in vitro cultures of 3 dpp mouse ovaries with insulin can repress the expression of Pten. In conclusion, folliculogenesis was stage-specifically regulated by insulin via the Akt signaling pathway in vitro.     

Suppression of Patronin deficiency by altered Hippo signaling in Drosophila organ development

The microtubule network is crucial for cell structure and function. Patronin is a conserved protein involved in protecting the minus end of microtubules. Conversely, Klp10A is a kinesin-like microtubule depolymerase. Here we report the role of Drosophila Patronin and Klp10A for cell survival in developing organs. Loss of Patronin reduces the size of organs by activation of a caspase in imaginal discs. Reduced wing by Patronin RNAi is suppressed by knockdown of Spastin (Spas) but not Katanin 60, suggesting that Patronin is inhibitory to the severing function of Spas at the minus end. Patronin RNAi phenotype is also recovered by overexpressing Death-associated inhibitor of apoptosis 1 (Diap1), a Yorkie target gene. Heterozygote mutations in Hippo pathway genes, including hippo and warts (wts), suppress the Patronin RNAi wing phenotypes. Furthermore, Patronin physically interacts with Merlin and Expanded while reducing their function. Patronin and Klp10A antagonistically regulate their levels. Wing phenotypes of Patronin RNAi are rescued by knockdown of Klp10A, consistent with their antagonistic interaction. Klp10A overexpression also causes organ size reduction that is partially suppressed by Diap1 overexpression or wts heterozygote mutation. Taken together, this study suggests that the antagonistic interaction between Patronin and Klp10A is required for controlling cell survival and organ size by modulating microtubule stability and Hippo components.Subject terms: Development, Genetic interaction     

Osteoclastogenesis is negatively regulated by D-serine produced by osteoblasts

We have shown the functional expression by chondrocytes of serine racemase (SR) which is responsible for the synthesis of D-serine (Ser) from L-Ser in cartilage. In this study, we evaluated the possible functional expression of SR by bone-forming osteoblasts and bone-resorbing osteoclasts. Expression of SR mRNA was seen in osteoblasts localized at the cancellous bone surface in neonatal rat tibial sections and in cultured rat calvarial osteoblasts endowed to release D-Ser into extracellular medium, but not in cultured osteoclasts differentiated from murine bone marrow progenitor cells. Sustained exposure to D-Ser failed to significantly affect alkaline phosphatase activity and Ca(2+) accumulation in cultured osteoblasts, but significantly inhibited differentiation and maturation in a concentration-dependent manner at a concentration range of 0.1-1 mM without affecting cellular survival in cultured osteoclasts. By contrast, L-Ser promoted osteoclastic differentiation in a manner sensitive to the inhibition by D-Ser. Matured osteoclasts expressed mRNA for the amino acid transporter B(0,+) (ATB(0,+) ) and the system alanine, serine, and cysteine amino acid transporter-2 (ASCT2), which are individually capable of similarly incorporating extracellular L- and D-Ser. Knockdown of these transporters by siRNA prevented both the promotion by L-Ser and the inhibition by D-Ser of osteoclastic differentiation in pre-osteoclastic RAW264.7 cells. These results suggest that D-Ser may play a pivotal role in osteoclastogenesis through a mechanism related to the incorporation mediated by both ATB(0,+) and ASCT2 of serine enantiomers in osteoclasts after the synthesis and subsequent release from adjacent osteoblasts.     

Drosophila NO-dependent guanylyl cyclase is finely regulated by sequential order of coincidental signaling

We investigate the mechanism of regulation of Drosophila-soluble guanylate cyclase. Multiple putative sites of phosphorylation for the major kinases are present on both subunits of the heterodimer. We show that NO activation after binding to the heme group, is specifically modulated by sequential phosphorylations. PKA increases the NO stimulation at optimum level when both subunits are phosphorylated. Phosphorylation by CK (casein kinase-like) first, inhibits the PKA phosphorylation of the alpha subunit and limits the PKA upregulation of the cyclase activity. However, PKA phosphorylation first didn't prevent CK phosphorylation of the two subunits and the sequence PKA/CK induces higher level of NO activation than CK/PKA. These phosphorylations occur independently of NO binding and the direct inhibitory effect of calcium is observed for all the sCG forms. These data show that the sGC activity is regulated in a complex way, and the well-known asymmetry of the two subunits appears to cause the reading of the sequence of regulatory signals. This qualifies sGC as molecular detector on which converge coincidental and/or sequential neuronal signals. Furthermore, due to the fact that NO induction is huge (more than 600-fold obtained with the mammal counterpart), we might consider that any variation in kinases activation and/or calcium concentration in micro area of neuronal processes, provokes locally significant quantitative difference of cGMP synthesis in presence of diffusing NO.     

Bone morphogenetic protein 2 signaling in osteoclasts is negatively regulated by the BMP antagonist, twisted gastrulation

Bone morphogenetic proteins (BMPs) have been shown to regulate both osteoblasts and osteoclasts. We previously reported that BMP2 could directly enhance RANKL-mediated osteoclast differentiation by increasing the size and number of osteoclasts. Similarly, genetic deletion of the BMP antagonist Twisted gastrulation (TWSG1) in mice, resulted in an enhancement of osteoclast formation, activity and osteopenia. This was accompanied by increased levels of phosphorylated Smad (pSmad) 1/5/8 in Twsg1(-/-) osteoclasts in vitro. The purpose of this study was to develop an adenoviral vector overexpressing Twsg1 as a means of inhibiting osteoclast activity. We demonstrate that overexpressing TWSG1 in primary osteoclasts decreased the size and number of multinuclear TRAP-positive osteoclasts, expression of osteoclast genes, and resorption ability. Overexpression of TWSG1 did not affect osteoclast proliferation or apoptosis. However, overexpression of TWSG1 decreased the levels of pSmad 1/5/8 in osteoclasts. Addition of exogenous BMP2 to osteoclasts overexpressing TWSG1 rescued the size and levels of pSmad 1/5/8 compared to cultures infected with a control virus. Finally, TWSG1 overexpression in osteoclasts isolated from the Twsg1(-/-) mice rescued size of the osteoclasts while further addition of exogenous BMP2 reversed the effect of TWSG1 overexpression and increased the size of the osteoclasts similar to control virus infected cells. Taken together, we demonstrate that overexpressing TWSG1 in osteoclasts via an adenoviral vector results in inhibition of osteoclastogenesis and may provide a potential therapy for inhibiting osteoclast activity in a localized manner.