Cellular Signalling

Epidermal Growth factor (EGF) signaling is associated with (oncogenic) proliferation. Conversely, EGF-family ligands are able to trigger a differentiation program in cultured cells, an effect attributed to ligand affinity and EGFR phosphorylation. How EGF/EGFR driven proliferation-differentiation dynamics underlie tissue self-renewal has not been addressed. We show that culturing mouse small intestinal organoids (mSIOs) without EGF enhanced EGFR expression and base phosphorylation while maintaining a balanced development of proliferative crypts and differentiated villi. Addition of EGF or EREG triggers receptor endocytosis, reducing cell-surface and expression levels. While EGF promoted crypt proliferation, EREG promoted both proliferation and villus differentiation compared to untreated controls. Removal or re-introduction of EGF or EREG proved sufficient to induce development comparable to constant presence of ligands over 96h. Sub-saturating concentrations of EGF led to increased villus differentiation, resembling EREG treatments, suggesting that control over EGFR endocytic cycle ultimately regulates the balance of proliferation and differentiation in mSIOs SummaryExpression and signaling competency at the plasma membrane of EGFR drives crypt proliferation vs villus differentiation by medium ligand-composition, aiding mouse intestinal organoids self-renewal and regeneration.

Astrocytes play a key role in neuronal homeostasis and in various neural disorders. The generation of astrocytes from neural progenitor cells (NPCs) and its functions are under a complex control of several signaling networks and transcription factors. In this study, we demonstrate that the transcription factor, GLIS similar 3 (GLIS3), which has been implicated in several neurodegenerative diseases, is highly expressed in astrocytes, and is required for the efficient differentiation of human NPCs into astrocytes. Loss of GLIS3 function greatly impairs astrocytes differentiation, resulting in reduced expression of astrocyte markers, whereas expression of exogenous GLIS3 restores the induction of astrocyte specific genes indicating a critical role for GLIS3 in astrocyte differentiation. Integrated transcriptomic and cistromic analyses revealed that GLIS3 directly regulates the transcription of several astrocyte-associated genes, including GFAP, SLC1A2, NFIA, and ATF3, in coordination with lineage-determining factors, such as STAT3, NFIA, and SOX9. We hypothesize that GLIS3 dysfunction disrupts this transcriptional network thereby contributing to astrocyte-associated neurological disorders. Identification of GLIS3 as a key regulator of astrocyte differentiation and gene expression will advance our understanding of its role in neurodegenerative diseases and may provide a new therapeutic target.

Long noncoding RNAs (LncRNAs) have emerged as a class of important regulatory ncRNAs and are known to fine-tune numerous cellular processes including proliferation, differentiation and development; however, their role in quiescence still remains largely unexplored. A miRNA host gene lncRNA, MIR503HG, has been reported to play important role in cancer development. Here, we demonstrate the role of MIR503HG lncRNA in regulating cellular quiescence. MIR503HG displays elevated levels in human diploid fibroblasts induced to undergo quiescence. Depletion of MIR503HG in HDFs affects the entry of cells into quiescence but has no effect on cell cycle progression, suggesting its role in quiescence attainment and/or maintenance. Additionally, MIR503HG depletion led to a drastic decrease in the levels of miR508 target, PTEN with a concomitant increase in pAkt levels, indicating its role in negative regulation of miR508. Further, we demonstrate that the lncRNA MIR503HG regulates PTEN levels by acting as a ceRNA for miR508 to maintain cellular quiescence. Our studies illustrate that MIR503HG can function synergistically with miR503 to maintain cells under quiescence and both the miRNA-HG and the miRNA encoded by its gene locus synergistically control the same biological process in different ways by regulating different downstream genes.

The secretion of glucagon from the pancreatic alpha () cell within the islets of Langerhans is physiologically regulated by nutrients (glucose, amino acids, fatty acids), neurotransmitters, and paracrine hormones. Insulin and somatostatin form an intra-islet paracrine network to control glucagon secretion through direct inhibitory effects on cell secretory granule exocytosis. In a potential new cellular pathway for the regulation of glucagon secretion, we have previously identified the neuronal trafficking protein Stathmin-2 (Stmn2) as a negative regulator of glucagon trafficking and secretion by directing glucagon to degradative lysosomes. In this study, we examined if insulin and somatostatin direct glucagon to lysosomes in a Stmn2-dependent manner as part of their paracrine mechanisms. Using the TC1-6 glucagon-secreting cell line and confocal microscopy of both fixed and live cells, we show that insulin and somatostatin direct glucagon, glucagon+LAMP1+ vesicles, and LAMP1-RFP to the intracellular region, away from sites of exocytosis. As visualized in live cells, insulin treatment resulted in the rapid retrograde transport of lysosomes from the cell periphery, and this effect was lost under siRNA-mediated silencing of Stmn2. Somatostatin appeared to enhance the intracellular retention of lysosomes, also in a Stmn2-dependent manner. We determined a possible mechanism for Stmn2 in the regulation of lysosome transport in TC1-6 cells through the Arf-like small GTPase Arl8, indicating that Stmn2 may function in lysosomal positioning along microtubules. We propose that Stmn2-mediated lysosomal transport may be a potential new pathway, in addition to inhibition of secretory granule exocytosis, through which insulin and somatostatin regulate glucagon secretion.

Mycoplasma synoviae is an avian pathogen that causes respiratory disease and synovitis, and its hemagglutinin plays a critical role in host cell adhesion. However, the key residues and structural mechanisms underlying hemagglutination remain unclear. In this study, domain analysis of the hemagglutinin family of Mycoplasma synoviae revealed that it contains long-chain and short-chain types, among which LAM HA (VY93_RS01465) was selected as the bait protein due to its complete C-terminal conserved domain. Through yeast two-hybrid screening, 18 host proteins interacting with LAM HA were identified. Furthermore, five key amino acid residues S83, R85, Y88, N124, and K192 were found to mediate hemagglutination activity. Deletion of these residues reduced the hemagglutination titer of LAM HA under acidic conditions. Secondary structure analysis showed that the deletion mutation decreased the -helix content while increasing the proportions of {beta}-sheet and random coil. Molecular dynamics simulations revealed that the mutant exhibited generally higher root mean square deviation and root mean square fluctuation values than the wild-type under different pH conditions, with a marked decrease in structural stability particularly at pH 5.0 and 6.0. These findings indicate that LAM HA, as a critical adhesin, exerts its hemagglutination function dependent on specific key residues and pH-sensitive conformational stability. IMPORTANCEMycoplasma synoviae (M. synoviae) causes significant economic losses to the poultry industry worldwide. Lipid-related membrane protein hemagglutinin (LAM HA) is a surface adhesin essential for host cell attachment, but its precise amino acid residues and structural features have not been defined. In this study, five key residues (S83, R85, Y88, N124, and K192) were identified as critical for LAM HA-mediated hemagglutination activity. Deletion of these residues altered the secondary structure composition, reduced conformational stability under acidic pH conditions, and decreased hemagglutination activity. These findings reveal a previously unknown structure-function relationship of M. synoviae LAM HA, demonstrating that its hemagglutination activity depends on specific residues and pH-sensitive structural integrity. This provides new insights into the molecular mechanisms of M. synoviae adhesion and offers potential targets for the development of novel intervention strategies against avian mycoplasmosis.

Mechanosensing involves proteins detecting mechanical changes in the cytoskeleton or at cell adhesion sites. These interactions initiate signaling cascades that produce biochemical effects such as post-translational modifications or cytoskeletal rearrangements. Filamin is a ubiquitous mechanosensing protein that binds actin filaments and senses pulling forces within the cytoskeleton. Drosophila Filamin (Cheerio) is structurally similar to mammalian Filamin, with roles in egg chamber development, embryo cellularization, and integrity of muscle attachment sites and Z discs in Drosophila indirect flight muscles (IFMs). Here we report a potential novel binding partner of Drosophila Filamins: the death-associated protein kinase Drak that functions as a myosin light chain kinase. We found that Drak biochemically bound to an open mutant of Filamin that resembles the mechanically activated form partially bound to wild type Filamin and did not bind to closed mutant of Filamin. The interaction site was mapped to the intrinsically unfolded C-terminal region of Drak. To study the functional role of Drak-Filamin interaction, we studied two developmental events where Drak has been earlier shown to be expressed and where Filamin also functions: early embryonic cellularization and indirect flight muscle development at pupal stages. We found partial colocalization between Drak-GFP and Filamin-mCherry during the initiation of cellularization furrow, and at the time of myotube attachment site maturation in tendon cells. However, functionally we could not show direct correlation between Filamin and Drak. Our studies reveal interesting new expression patterns of Drak during Drosophila development and provide detailed information about Filamin localization during IFM development.

During viral infection, viral replication perturbs endoplasmic reticulum (ER) homeostasis and triggers the unfolded protein response (UPR). XBP1s, a transcription factor generated by one branch of the UPR, is known to potentiate both innate and adaptive immunity, but its role in antiviral responses remains incompletely understood beyond its ability to augment type I interferon (IFN) mRNA induction. Here, we show that XBP1s positively regulates the RIG-I-like receptors (RLRs), ribonuclease L (RNase L), and protein kinase R (PKR) pathways, indicating that it enhances all three major antiviral response pathways. We further show that RNase L activation rapidly decreases XBP1 mRNA levels in an RNase activity-dependent manner, leading to a prompt reduction in XBP1s expression. Consistent with this, RNase L deletion significantly increased both thapsigargin-mediated XBP1s induction and XBP1s expression following Japan encephalitis virus infection. Poly(I:C)-induced IFNB mRNA expression was significantly enhanced in RNase L-knockout cells. This enhancement was completely abolished by RNase L reconstitution. XBP1 knockdown also significantly attenuated IFNB mRNA expression in RNase L-knockout cells. These findings suggest a negative-feedback loop in which RNase L suppresses XBP1s, thereby fine-tuning antiviral responsiveness during viral infection. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/713401v1_ufig1.gif" ALT="Figure 1000"> View larger version (19K): org.highwire.dtl.DTLVardef@112d312org.highwire.dtl.DTLVardef@df79a9org.highwire.dtl.DTLVardef@1ac571borg.highwire.dtl.DTLVardef@18ac610_HPS_FORMAT_FIGEXP M_FIG C_FIG

BACKGROUNDMono-ADP ribosylation is a post-translational modification that regulates various cellular physiological processes, including cell cycle progression, genomic stability, transcription, and cellular protein turnover. PARP16 is an endoplasmic reticulum (ER)-localized mono-ADP-ribosyltransferase that has been shown to regulate the unfolded protein response and maintain ER homeostasis under stress conditions. Despite its established role in ER stress signaling, the functional significance of PARP16 in cardiac pathophysiology, particularly in cardiac hypertrophy and heart failure, remains poorly understood. In this study, we aim to investigate the role of PARP16 in cardiac hypertrophy and heart failure using in vitro and mouse model systems. METHODSWe analysed PARP16 expression in human heart failure samples as well as in heart failure-based mouse models. We evaluated gene expression by RT-PCR, immunoblotting, and confocal microscopy to understand the role of PARP16 in heart failure under phenylephrine- or isoproterenol-treated conditions. We also investigated the role of PARP16 in regulating cardiac function in genetically engineered mouse models, including whole-body PARP16 knockout, cardiac-specific PARP16 knockout, inducible cardiac-specific PARP16 knockout, and cardiac-specific PARP16 Transgenic mice. We performed echocardiography to assess cardiac function. We also used an in vitro primary cardiomyocyte system to knock down and overexpress PARP16. We performed RNA sequencing and mass spectrometry, followed by molecular docking, molecular dynamics simulation, immunoprecipitation, and luciferase assay to characterise the molecular mechanism by which PARP16 regulates cardiac function. RESULTSHuman heart failure samples showed reduced PARP16 expression. PARP16 expression was also significantly reduced in models of heart failure, including the hearts of isoproterenol-treated C57B/L6 mice and phenylephrine-treated primary cardiomyocytes. PARP16-deficient NRCMs showed signs of pathological remodelling. Whole-body, cardiac-specific, and inducible cardiac-specific PARP16 KO mice exhibited cardiac remodelling and dysfunction. In contrast, cardiac-specific PARP16-overexpressing mice were protected from iso-induced cardiac hypertrophy. Mechanistically, several hypertrophic signalling pathway genes are dysregulated in PARP16 knockout mouse hearts concomitant with upregulated NFAT1 transcriptional activity and nuclear translocation. PARP16 binds to and catalytically downregulates NFAT activity, thereby maintaining cardiac function. Mass spectrometry analysis showed that PARP16 is involved in ADP-ribosylation of NFAT1 at E398 and T533. Pharmacological inhibition of NFAT activation attenuates structural and functional abnormalities associated with PARP16 deficiency. CONCLUSIONSPARP16 binds to and inhibits NFAT1 activity to regulate cardiac function in mice, and its downregulation may activate NFAT1 signalling, leading to hypertrophy. In this manner, PARP16 plays a critical role in cardiac hypertrophy and failure and may serve as a potential therapeutic target for the treatment of heart failure.

FAM122A regulates cell cycle progression through inhibition of the PP2A-B55 phosphoprotein phosphatase. Recent structural work has uncovered helical elements in the N-terminus of FAM122A as binding determinants for PP2A-B55 but whether FAM122A inhibition towards PP2A-B55 is regulated is presently unclear. To address this we performed a systematic analysis of the PP2A-B55 interaction with FAM122A in cells uncovering a novel region in the C-terminus of FAM122A, spanning residues 150-170, required for binding. This C-terminal region and the N-terminal helices are both required for efficient binding to PP2A-B55 suggesting a bipartite binding mechanism. We perform amino acid resolution scans of FAM122A 150-170 uncovering several residues in this region contributing to binding including the conserved Ser158, a reported phosphorylation site. We show that Ser158 is important for PP2A-B55 inhibition in human cells as well as efficient stimulation of mitotic entry in Xenopus laevis egg extracts. In human cells and in Xenopus laevis Ser158 phosphorylation is regulated with increased occupancy correlating with cell cycle stages requiring PP2A-B55 inhibition. Collectively our work uncovers novel aspects of FAM122A interaction with PP2A-B55 and provides a possible mechanism for how the inhibitory activity of FAM122A can be regulated during the cell cycle.

Right ventricular failure (RVF) is a serious disease with a high mortality but no effective pharmacologic treatments. We reported RVF was reversed by chronic treatment with an 1A-adrenergic receptor (1A-AR) agonist. Recent studies suggest mitochondrial dysfunction contributes to RVF. Therefore, we investigated if reversal of RVF by chronic 1A-AR agonist treatment involved improved mitochondrial function. A mouse model of RVF caused by pulmonary artery constriction (PAC) for 2 wk was chronically treated for a further 2 wk. with a low dose of the 1A-AR agonist A61603 (10 ng/kg/day) or vehicle (no drug control). RV dysfunction was assessed from the fractional shortening of the RV outflow tract (RVOT FS). RVOT FS for sham controls (46.5 {+/-} 1.3 %, n = 9) was reduced 4 wk after PAC (27.6 {+/-} 1.5 %, n = 13, P < 0.0001), but was higher after PAC plus 2 wk A61603 treatment (34.5 {+/-} 0.6 %, n = 14, P < 0.001). RV myocardial respiration rate (O2 consumption) for sham controls (776 {+/-} 51 pM/s/mg, n = 9) was reduced 4 wk after PAC (493 {+/-} 28 pM/s/mg, n = 15, P <0.0001), but was higher after PAC plus 2 wk A61603 treatment (634 {+/-} 30 pM/s/mg, n = 11, P <0.05). RV myocardial ATP level for sham controls (3.3 {+/-} 0.1 mM, n = 10) was reduced 4 wk after PAC (1.9 {+/-} 0.1 mM, n = 6, P < 0.0001), but was higher after PAC plus 2 wk A61603 treatment (2.6 {+/-} 0.13 mM, n = 7, P < 0.01). In conclusion, reversal of RVF after chronic A61603 treatment involved reversal of mitochondrial dysfunction. Consistent with our previous studies, this study suggests that the 1A-AR is a therapeutic target to treat RVF. HighlightsRV failure is reported to involve mitochondrial dysfunction which might impair RV contraction by decreasing cardiomyocyte ATP level. Using the pulmonary artery constriction model of RV failure, we found that chronic treatment with an 1A-adrenergic receptor agonist increased RV myocardial respiration rate, increased RV myocardial ATP level, and increased RV function. These findings suggest that the 1A-adrenergic receptor is a therapeutic target for treating RV failure, and that the mechanism involves improved RV cardiomyocyte bioenergetic status.

The Ube2J1 enzyme that mediates the ubiquitination and proteasomal degradation of misfolded proteins at the ER is phosphorylated at serine S184. Following anisomycin treatment of HEK293T cells, we observed an inverse relationship between phosphorylation and dephosphorylation at this site. This suggested a dynamic interchange between the two forms, and we show that S184 is a target for protein phosphatase 2A. The S184-phosphorylated protein is known to exhibit increased sensitivity to proteasomal degradation, and we found that mutation at K186R increased the ratio of S184-phosphorylated to S184-dephosphorylated protein. Although the K186R mutant retained some sensitivity to proteasomal inhibition, our results show that Ube2J1 steady state expression can be exercised at multiple levels, and can involve dynamic phosphorylation and dephosphorylation at S184.

This retrospective study aims to explore the interactive effects of biological maturation and relative age effect (RAE) on talent identification. 56 male elite soccer players matched for chronological age (15.08{+/-}0.41 years) were studied. Test items included anthropometry (height, body mass, sitting height, leg length, BMI and Quetelet index), physiology (power, speed, agility, speed endurance and aerobic performance), soccer-specific skills (passing, shooting and dribbling), psychology (achievement motivation, orientation and resilience) and biological maturation (APHV) tests. The test results were analyzed independent sample t-test, Pearson correlation analysis, and stratified regression. Conclusion: Biological maturation significantly influences anthropometry (height, weight and Quetelet index), lower limb explosive, and speed (single-leg jump, standing triple jump, and 30-m sprint) in U16 male elite soccer players in Shanghai. The relative age effect shows no significant impact on talent selection indicators, which is attributed to the accumulated training load effect. The mechanisms of biological maturation and RAE in youth soccer talent selection are distinct and operate independently.

Metabolic dysfunction-associated steatohepatitis (MASH) is associated with increased expression of peroxisome proliferator-activated receptor gamma (PPAR{gamma}, Pparg) and reduced expression of genes involved in methionine metabolism in the liver. The nuclear receptor PPAR{gamma} is activated by fatty acids, and the knockout of Pparg in hepatocytes (Pparg{Delta}Hep) reduced the negative effects of MASH on methionine metabolism. Here, we sought to determine whether hepatocyte Pparg is required for the transcriptional regulation of genes involved in hepatic methionine metabolism in conditions with altered fatty acid flux to the liver: fasting, refeeding, and high-fat diet (HFD)-induced obesity/steatosis. Fasting induced liver steatosis and increased the expression of key genes involved in the methionine metabolism in the liver, while 6h-refeeding reversed these effects and reduced the expression of phosphatidylethanolamine N-methyltransferase (Pemt) and cystathionine beta synthase (Cbs). Overall, fasting and refeeding did not alter hepatocyte Pparg expression nor Pparg{Delta}Hep affected fasting and refeeding-mediated regulation of methionine metabolism gene expression. Diet-induced steatosis reduced hepatic Pemt expression in control (Pparg-intact) mice, and the thiazolidinedione (TZD)-mediated activation of PPAR{gamma} in diet-induced obese control (Pparg-intact) mice reduced the expression of betaine homocysteine S-methyltransferase (Bhmt) and Cbs. However, diet-induced steatosis increased hepatocyte Pparg expression, and Pparg{Delta}Hep blocked the negative effects of HFD and TZD on hepatic methionine metabolism. The PPAR{gamma}-dependent reduction of hepatic Bhmt and Cbs expression was confirmed in mouse primary hepatocytes. Taken together, hepatocyte Pparg may serve as a negative regulator of hepatic methionine metabolism in diet-induced obese mice and these actions could contribute to promoting the onset of MASH.

G protein-coupled receptors rely on dynamic conformational changes to coordinate G protein activation and recruitment of regulatory transducers such as G protein-coupled receptor kinases and {beta}-arrestins. The chemotactic receptor GPR183 has been implicated in a context-dependent role in hematological malignancies. Here, we investigated the impact of A338V mutation located within the C-terminal tail of GPR183. This mutation is associated with acute myeloid leukaemia. Using bioluminescence resonance energy transfer-based assays in HEK293A cells, we assessed receptor-proximal signaling events. The A338V variant displayed preserved agonist potency and comparable agonist-induced Gi activation relative to wild type, although constitutive activity towards Gi was modestly reduced. In contrast, recruitment of GRK2 and {beta}-arrestin2 was consistently impaired across multiple assay configurations. These differences were not attributable to altered receptor abundance, as the C-tail untagged mutant exhibited increased plasma membrane expression despite reduced regulatory transducer engagement. While intramolecular conformational biosensor measurements revealed subtle differences in global receptor conformation between WT and A338V, extensive molecular dynamics simulations supported the altered conformational sampling of the C-terminal tail in the A338V variant. Together, these data support a model in which the A338V substitution selectively alters C-terminal structural dynamics, impairing GRK2 and {beta}-arrestin2 recruitment while preserving G protein activation.

Bone morphogenetic proteins (BMPs) play an important role in dorsal spinal cord patterning. Their presence in the roof plate of the midbrain indicates a role in its development. We examined whether the BMP signaling contributes to dorsal midbrain size expansion in chick embryos by missexpressing pathway activators and inhibitors. Overactivation of BMP4 did not affect midbrain development, whereas GDF7 reduced midbrain growth. In contrast, expression of a truncated dominant-negative BMP receptor type 1b or the extracellular inhibitor Chordin had no detectable effect. Ectopic expression of SMAD6, the intracellular inhibitor of the BMP/ TGF-{beta} pathway, significantly reduced midbrain size, which correlated with decreased proliferation rates of SMAD6-overexpressing cells. In some cases, SMAD6 also disrupted MTN axon trajectory. These results indicate an important role for SMAD-dependent signaling pathways in early dorsal midbrain growth.

Terminal erythroid differentiation involves dramatic cellular remodeling that culminates in the expulsion of the nucleus, a process known as enucleation. While enucleation is conserved across mammals and is crucial for the generation of fully functional erythrocytes, the mechanisms governing this process have remained largely unknown, in part because the absence of genetic material in mature, enucleated red blood cells hinders genetic experimentation. Here, we performed a pooled, forward-genetic CRISPR-Cas9 screen in enucleated red blood cells derived from primary human hematopoietic stem cells to identify genes required for enucleation. We found that Chloride Intracellular Channel 3 (CLIC3) and Vesicle-associated membrane protein 8 (VAMP8) are both necessary for terminal erythroid differentiation, yet likely act through different mechanisms. Knockdown of CLIC3 led to a delay in erythroblast differentiation, culminating in impaired enucleation. We found that the knockdown cells had increased p53 and p21 and exhibited cell cycle alterations, suggesting CLIC3 plays a crucial role in coordinating cell cycle progression during erythropoiesis. In comparison, VAMP8-depleted cells initially appear to undergo accelerated differentiation but then display a specific defect in enucleation. Transcriptional analysis of the VAMP8-knockdown cells suggested dysregulation of pathways for vesicle trafficking and actin binding, and imaging of late-stage erythroblasts revealed impaired nuclear polarization and disorganized actin. This work provides a new approach for functional genomics in enucleated cells and reveals novel factors important for terminal erythroid differentiation and enucleation. Key pointsO_LIA CROPseq-based CRISPR-Cas9 screen enables functional genomics in enucleated primary human red blood cells. C_LIO_LIChloride Intracellular Channel 3 (CLIC3) and Vesicle Associated Membrane Protein 8 (VAMP8) were identified as critical for terminal erythroid differentiation and enucleation, likely acting through two distinct mechanisms. C_LI

Crosstalk across two major receptor families involved in signal transduction, namely receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs), have been observed at different levels of their signaling cascades. Using newly developed enhanced bystander bioluminescence resonance energy transfer (ebBRET)-based biosensors that monitor the recruitment of SH2 domains to activated RTKs, we assessed the ability of GPCRs to modulate cellular localization of SH2 domains. Receptor-mediated activation of either Gq/11 or G12/13 but not Gs or Gi/o (e.g., thromboxane A2 receptor, TP, and type-2 protease activated receptor, PAR2) resulted in the plasma membrane (PM) dissociation of SH2 domains derived from RTKs effectors such as GRB2, STAT5 and PLC{gamma}1. The role of Gq/11, G12/13, Rho and downstream kinases in the subcellular SH2 domain redistribution was further confirmed using both pharmacological and genetic approaches. BRET imaging and spectrometric analyses showed that the dissociation of SH2 domains from the PM was accompanied by their accumulation in the nucleus and a reduction in RTK signaling activity, as determined using a STAT5 transcriptional assay. The effect of Gq/11 and G12/13 activation on STAT5 transcriptional activity was observed both in engineered systems and in HeLa cells endogenously expressing all the components of the regulatory mechanism. The Gq/11 / G12/13-mediated redistribution of SH2 domain-containing proteins represents an undescribed mechanism through which GPCRs regulate RTKs activity. Significance StatementThis study reveals a novel crosstalk mechanism between G protein coupled receptors and receptor tyrosine kinases showing that Gq/11 and G12/13 activation triggers Rho-dependent translocation of SH2-containing effector proteins, such as GRB2, PLC{gamma}1 and STAT5. This process causes compartmentalization inside the nucleus and thus reduces their availability at the plasma membrane, leading to attenuated RTK responses.

Tubular organs present a common solution to fluid transport in multicellular organisms. They often arise by an initial bulging of flat epithelial progenitor cells, which then undergo branching morphogenesis. Here, we present 3 cooperative programs fully defining the Drosophila airway progenitor field and their roles in early morphogenesis linking the radial pattern of the 2-dimensional (2D) field to the proximo-distally patterning of the 3D tubes. We previously showed that extrinsic Hedgehog (Hh) and intrinsic POU-Homeobox TF Ventral-veinless (Vvl)/Drifter/U-turn dominantly drive the transcriptional program toward the distal airway cell identity at the expense of a proximal program specified by the GATA TF grain (grn). Both programs require the basic-HLH-POU TF trachealess (trh) (Matsuda et. al, 2015). Whereas trh is not essential for primordia invagination, we show that in hh vvl double mutants, the oval-shaped primordia frequently remain at the 2D plane, retaining trh expression in a grn dependent manner. Therefore, hh and vvl are the principal regulators of progenitor invagination independent of trh. Each of the 3 regulators, Trh, Vvl and Grn fulfills only complementary or compensatory functions in transcription and morphogenesis but their combinations functionally define the airway progenitor field. We further provide a comprehensive description for allocating the airway progenitors on the body coordinates, involving dorsal Decapentaplegic/BMP signaling along the dorso-ventral axis and subsequent radial EGFR signaling along the proximo-distal axis. The presence of 3 complementary, regulatory programs in early gene expression and morphogenesis of the simple Drosophila airways may reflect the vital needs for respiration, and their influence on the evolution of various strategies in tubular organ development.

BackgroundWingless/Int-1 (Wnts) proteins are canonical Frizzled receptor ligands. Recent evidence indicates that some Wnts, including Wnt9b and Wnt5a, bind to polycystin 1 (PKD1), a transmembrane protein which can couple to polycystin 2 (PKD2) to form a non-selective cation channel. The functional significance of Wnts binding to PKD1 is unclear. Here, we tested the hypothesis that Wnts act through PKD1/PKD2 channels on endothelial cells (ECs) to regulate arterial contractility and blood pressure and investigated the cellular source and secretory regulation of vasoactive Wnt proteins. MethodsA wide variety of approaches, including inducible EC-specific PKD1 and PKD2 knockout mice, reverse-transcription polymerase chain reaction, Western blotting, immunofluorescence, pressurized artery myography, blood pressure measurements, patch-clamp electrophysiology, in vivo and in vitro Wnt and nitric oxide assays, and Wnt secretion assays. ResultsIntravascular Wnt9b or Wnt5a administration stimulates an EC PKD1/PKD2-dependent dilation in pressurized resistance-size arteries. Wnt9b and Wnt5a are present in serum and plasma and intravenous infusion rapidly stimulates a blood pressure reduction which requires EC PKD1. Wnts stimulate a PKD1-dependent non-selective cation current in ECs which through Ca2+ signaling activates endothelial nitric oxide synthase (eNOS) and small conductance Ca2+-activated K+ channels to induce vasodilation. Wnt9b acts solely via PKD1/PKD2 channels, whereas Wnt5a stimulates signaling through PKD1/PKD2, Frizzled-7 (Fzd-7), Dishevelled and c-Jun N-terminal kinase (JNK). Intravascular flow stimulates angiotensin II type 1 (AT1) receptors, which through Gq/11 and Porcupine activate Wnt9b and Wnt5a secretion in ECs. Wnts secreted in response to flow activate PKD1/PKD2 signaling in ECs and contribute to flow-mediated vasodilation. ConclusionsIntravascular flow activates AT1 receptors, which through Gq/11 and Porcupine stimulate Wnt9b and Wnt5a secretion in ECs. Wnt9b activates PKD1/PKD2 channels whereas Wnt5a stimulates both PKD1/PKD2 and Fzd-7 in ECs to induce vasodilation. Wnts contribute to flow-mediated autocrine/paracrine dilation and reduce blood pressure. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/712518v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@158bad1org.highwire.dtl.DTLVardef@5113eforg.highwire.dtl.DTLVardef@f3b94eorg.highwire.dtl.DTLVardef@10ab479_HPS_FORMAT_FIGEXP M_FIG C_FIG

SGLT2 inhibitor (SGLT2i)-induced diabetic hyperketonemia is a life-threatening acute complication of diabetes. While Celastrol has been reported to exert beneficial effects on obesity; its potential role in ketogenesis remains unclear. In this study, Celastrol administration significantly attenuates the fasting-induced elevation of blood {beta}-hydroxybutyrate. Moreover, a 7-day course of Celastrol (1 mg/kg/day) leads to reductions in body weight and fat mass. Mechanistically, Celastrol specifically downregulates HMGCS2 expression and suppressess hepatic ketogenesis through inhibiting PPAR expression in the short term ([&le;] 2 days). However, after prolonged treatment for 7 days, Celastrol modulates both PPARand serum free fatty acids (FFAs) levels. Furthermore, anti-ketogenic effect of Celastrol is abolished in Ppar{square} /{square} mice. Importantly, Celastrol effectively ameliorates SGLT2i-induced hyperketonemia. In summary, Celastrol curbs hepatic ketone overproduction in a PPAR-dependent manner, indicating its protective potential against SGLT2i-induced hyperketonemia.