The Hippo tumour suppressor pathway is a conserved signalling pathway that controls organ size. The core of the Hpo pathway is a kinase cascade, which in Drosophila involves the Hpo and Warts kinases that negatively regulate the activity of the transcriptional coactivator Yorkie. Although several additional components of the Hippo pathway have been discovered, the inputs that regulate Hippo signalling are not fully understood. Here, we report that induction of extra F‐actin formation, by loss of Capping proteins A or B, or caused by overexpression of an activated version of the formin Diaphanous, induced strong overgrowth in Drosophila imaginal discs through modulating the activity of the Hippo pathway. Importantly, loss of Capping proteins and Diaphanous overexpression did not significantly affect cell polarity and other signalling pathways, including Hedgehog and Decapentaplegic signalling. The interaction between F‐actin and Hpo signalling is evolutionarily conserved, as the activity of the mammalian Yorkie‐orthologue Yap is modulated by changes in F‐actin. Thus, regulators of F‐actin, and in particular Capping proteins, are essential for proper growth control by affecting Hippo signalling.

Defects in apical-basal cell polarity and abnormal expression of cell polarity determinants are often associated with cancer in vertebrates. In Drosophila , abnormal expression of apical-basal determinants can cause neoplastic phenotypes, including loss of cell polarity and overproliferation. However, the pathways through which apical-basal polarity determinants affect growth are poorly understood. Here, we investigated the mechanism by which the apical determinant Crumbs (Crb) affects growth in Drosophila imaginal discs. Overexpression of Crb causes severe overproliferation, and we found that loss of Crb similarly results in overgrowth of imaginal discs. Crb gain and loss of function caused defects in Hippo signaling, a key signaling pathway that controls tissue growth in Drosophila and mammals. Manipulation of Crb levels caused the up-regulation of Hippo target genes, genetically interacted with known Hippo pathway components, and required Yorkie, a transcriptional coactivator that acts downstream in the Hippo pathway, for target gene induction and overgrowth. Interestingly, Crb regulates growth and cell polarity through different motifs in its intracellular domain. A juxtamembrane FERM domain-binding motif is responsible for growth regulation and induction of Hippo target gene expression, whereas Crb uses a PDZ-binding motif to form a complex with other polarity factors. The Hippo pathway component Expanded, an apically localized adaptor protein, is mislocalized in both crb mutant cells and Crb overexpressing tissues, whereas the other Hippo pathway components, Fat and Merlin, are unaffected. Taken together, our data show that Crb regulates growth through Hippo signaling, and thus identify Crb as a previously undescribed upstream input into the Hippo pathway.

The pharmacological action of salicylate cannot be explained by its inhibition of cyclooxygenase (COX) activity. In this report, the effects of aspirin and sodium salicylate on COX-2 expressions in human umbilical vein endothelial cells and foreskin fibroblasts were evaluated. Aspirin and sodium salicylate at therapeutic concentrations equipotently blocked COX-2 mRNA and protein levels induced by interleukin-1␤ and phorbol 12-myristate 13-acetate. The suppressing effect was more pronounced in cultured cells deprived of fetal bovine serum for 24 h, suggesting that it may be cell cycle related. Salicylate inhibited nascent COX-2 transcript synthesis but had no effect on COX-2 mRNA stability. It inhibited COX-2 promoter activity in a concentration-dependent manner. In mice pretreated with aspirin (10 and 30 mg͞kg), followed by challenge with lipopolysaccharide, COX-2 mRNA expression in peritoneal macrophages was markedly suppressed. These findings suggest that salicylate exerts its antiinf lammatory action in part by suppressing COX-2 induction, thereby reducing the synthesis of proinf lammatory prostaglandins.Since the classic work of Vane (1), it has been widely accepted that the pharmacological action of nonsteroidal antiinflammatory drugs (NSAID) is mediated by inhibiting the activity of cyclooxygenase (COX), a key enzyme in biosynthesis of proinflammatory prostaglandins. Recent studies implicate COX-2 induction as a critical event in inflammation (2). This notion has been supported by effective suppression of inflammatory responses in experimental animals by selective COX-2 inhibitors (3, 4). Aspirin (acetylsalicylic acid) is a nonselective COX inhibitor (5, 6). Moreover, aspirin is rapidly deacetylated in blood to form salicylic acid. Salicylic acid has been a known NSAID for over a century, but its mechanism of action remains a pharmacological enigma. It has virtually no inhibitory activity against purified COX-1 or COX-2, although it inhibits prostaglandin synthesis in intact cells (7). To elucidate the mechanism by which salicylic acid exerts its antiinflammatory action, we evaluated the effects of aspirin and sodium salicylate on COX-2 expression in human umbilical vein endothelial cells (HUVEC) and human foreskin fibroblasts (HFF) induced by inflammatory mediators. We show here that aspirin and sodium salicylate at therapeutic concentrations suppress COX-2 gene transcription. When administered to mice pretreated with lipopolysaccharide, aspirin inhibited COX-2 mRNA levels in peritoneal macrophages. MATERIALS AND METHODSMaterials. Recombinant IL-1␤, phorbol 12-myristate 13-acetate (PMA), sodium salicylate, aspirin, NS398, indomethacin, and lipopolysaccharide (LPS) were obtained from Sigma.Cell Culture. HUVECs were cultured as described (8) in medium 199 containing 20% FBS, 50 g͞ml endothelium cell growth factor, 10 units͞ml heparin, 100 g͞ml streptomycin, and 100 units͞ml penicillin. Only first and second passage cells were used. HFFs were obtained from American Type Culture Collection and were cu...

The conserved Hippo tumor suppressor pathway is a key signaling pathway that controls organ size in Drosophila. To date a signal transduction cascade from the Cadherin Fat at the plasma membrane into the nucleus has been discovered. However, how the Hippo pathway is regulated by extracellular signals is poorly understood. Fat not only regulates growth but also planar cell polarity, for which it interacts with the Dachsous (

Although it has been shown that mast cell-deficient mice have diminished innate immune responses against bacteria, the most important immunoprotective factors secreted from activated mast cells have not been identified. Mouse mast cell protease 6 is a tetramer-forming tryptase. This serine protease is abundant in the secretory granules and is exocytosed upon bacterial challenge. Here we have described the generation of a mast cell protease-6-null mouse. Our discovery that mice lacking this neutral protease cannot efficiently clear Klebsiella pneumoniae from their peritoneal cavities reveals an essential role for this serine protease, and presumably its human ortholog, in innate immunity.Approximately 50% of the weight of a mature tissue mast cell (MC) 2 consists of protease-serglycin proteoglycan complexes stored in the secretory granules. In humans, ␤ tryptases are the most abundant MC-restricted neutral proteases (1-3). The corresponding tryptases in mice are mouse MC protease (mMCP)-6 (4, 5) and mMCP-7 (6), with mMCP-6 being the most similar in amino acid sequence and substrate specificity to human tryptase (hTryptase) ␤1 (7-9). MCs are the only cells that express mMCP-6, and this serine protease is particularly abundant in those MCs that reside in the peritoneal cavity, skin, and lung (4,5,10).Numerous biochemical studies have been carried out to understand the biosynthesis and substrate preference of mMCP-6. This tryptase is initially translated as a zymogen with a 245-mer mature domain. When the signal and propeptides are proteolytically removed, the mature protease spontaneously forms tetramers with the active site of each monomer facing the central core of the tetramer unit, as first described for its human ortholog (11). A positively charged face forms on the surface of each monomer, thereby allowing mature mMCP-6 to interact with negatively charged serglycin proteoglycans in the Golgi complex. The resulting tryptase-serglycin macromolecular complexes are then targeted and packaged in the cell secretory granules. When exocytosed, these complexes are retained in connective tissues for hours because of their large sizes (12). Protease inhibitors are abundant in blood. Nevertheless, no circulating protease inhibitor has been identified that rapidly inactivates mMCP-6 or hTryptase ␤1. Substrate specificity studies carried out using varied peptide combinatorial libraries revealed that recombinant mMCP-6 (7) and hTryptase ␤1 (8, 9) prefer to cleave peptides having a Pro at residues P2 to P5 and a Lys or Arg at residue P1. However, due to the unique structural constraints of the tetramer unit, the abilities of mMCP-6 and hTryptase ␤1 to cleave large-sized proteins are very limited. Thus, the importance of these evolutionally conserved enzymes in MC-dependent reactions remains to be determined.MC development in vivo is highly dependent on the cytokine kit ligand/stem cell factor on the surface of mesenchymal cells and its tyrosine kinase receptor c-Kit/CD117 on the surface of MC-committed progenitors. Signaling...

No abstract

The Hippo signaling pathway and its two downstream effectors, the YAP and TAZ transcriptional coactivators, are drivers of tumor growth in experimental models. Studying mouse models, we show that YAP and TAZ can also exert a tumor-suppressive function. We found that normal hepatocytes surrounding liver tumors displayed activation of YAP and TAZ and that deletion of Yap and Taz in these peritumoral hepatocytes accelerated tumor growth. Conversely, experimental hyperactivation of YAP in peritumoral hepatocytes triggered regression of primary liver tumors and melanoma-derived liver metastases. Furthermore, whereas tumor cells growing in wild-type livers required YAP and TAZ for their survival, those surrounded by Yap- and Taz-deficient hepatocytes were not dependent on YAP and TAZ. Tumor cell survival thus depends on the relative activity of YAP and TAZ in tumor cells and their surrounding tissue, suggesting that YAP and TAZ act through a mechanism of cell competition to eliminate tumor cells.

The anti-inflammatory actions of salicylates cannot be explained by inhibition of cyclooxygenase (COX) activity. This study demonstrates that sodium salicylate at a therapeutic concentration suppressed COX-2 gene transcription induced by phorbol 12-myristate 13-acetate and interleukin 1␤ by inhibiting the binding of CCAAT/ enhancer-binding protein ␤ to its promoter region of COX-2. By contrast, salicylate did not inhibit nuclear factor B-dependent COX-2 induction by tumor necrosis factor ␣. The inhibitory effect of sodium salicylate was restricted to serum-deprived quiescent cells. These findings indicate that contrary to the current view that salicylate acts via inhibition of nuclear factor B the pharmacological actions of aspirin and salicylates are mediated by inhibiting CCAAT/enhancer-binding protein ␤ binding and transactivation. These findings have a major impact on the conceptual understanding of the mechanism of action of salicylates and on new drug discovery and design.Salicylic acid is arguably the oldest anti-inflammatory drug preparation, and its acetylated form, acetylsalicylic acid (aspirin), is the most commonly used non-steroidal anti-inflammatory drug. The groundbreaking work of Vane (1) demonstrated that non-steroidal anti-inflammatory drugs, such as aspirin, owe their anti-inflammatory effects to the inhibition of prostaglandin synthesis. The last 30 years has seen major developments in prostanoid biology, not least the identification of cyclooxygenase-2 (COX-2), 1 the inducible COX isoform that is believed to play a major role in inflammation (2-4) and tumorigenesis (5-8). Despite such advances, salicylate remains a pharmacological enigma, as it inhibits prostanoid synthesis in intact cells (9 -11) but has little effect on purified COX-1 or COX-2 activity (10). Recent studies have suggested that salicylate owes its anti-inflammatory effects to the inhibition of nuclear factor B (NF-B)-mediated gene expression (12, 13). However, inhibition of NF-B is exerted only at suprapharmacological concentrations of sodium salicylate (NaS) (Ͼ5 mM) with no effects at pharmacological concentrations (14). Salicylate at such high concentrations inhibits numerous cellular kinases nonspecifically (15). Our group recently demonstrated that NaS and aspirin at pharmacologically relevant concentrations equipotently inhibited COX-2 transcription (16) and that the maximal inhibitory effect occurs in cells deprived of serum for 24 h. Because aspirin is rapidly deacetylated and converted to salicylate in vivo its action on COX-2 expression is attributed to salicylate. To elucidate the mechanism by which salicylate suppresses COX-2 transcription we evaluated the effects of NaS, at a representative therapeutic concentration, on COX-2 expression in serum-deprived G 0 and serum-driven cycling human foreskin fibroblasts (HFF). The results show that the inhibitory effect of salicylate on phorbol 12-myristate 13-acetate (PMA)-and interleukin 1␤ (IL-1␤)-induced COX-2 transcription was confined to G 0 cells. Salicylate suppress...