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The coagulation protease thrombin triggers fibrin formation, platelet activation, and other cellular responses at sites of tissue injury. We report a role for PAR1, a protease-activated G protein-coupled receptor for thrombin, in embryonic development. Approximately half of Par1-/-mouse embryos died at midgestation with bleeding from multiple sites. PAR1 is expressed in endothelial cells, and a PAR1 transgene driven by an endothelial-specific promoter prevented death of Par1-/-embryos. Our results suggest that the coagulation cascade and PAR1 modulate endothelial cell function in developing blood vessels and that thrombin's actions on endothelial cells-rather than on platelets, mesenchymal cells, or fibrinogen-contribute to vascular development and hemostasis in the mouse embryo.

When blood vessels are injured, bleeding is stopped by clotting factors that form a thrombus of fibrin threads that trap platelet aggregates. Carmeliet discusses emerging evidence which implicates the clotting system in the building and stabilization of new blood vessels during embryonic development.

The effects of transgene delivery by adenoviral vectors were studied by probing a 588 gene, mouse cDNA array with mRNA derived from infected liver. The liver tissues were obtained from naive mice and mice infected with replication-deficient adenovirus, adenovirus expressing transforming growth factor beta1 (TGFbeta1), and adenovirus expressing connective tissue growth factor (CTGF). Expression of 98 genes was detected in the array analysis. The increased expression of the transcripts for Stat1, gamma interferon-induced monokine (MIG) and interferon regulatory factor 1 (IRF1) clearly demonstrated the immune response induced by infection with a first generation, replication-incompetent adenovirus. In vivo expression of TGFbeta1 led to a down-regulation of genes involved in the immune response. The increased expression of u-PAR1, laminin receptor and BMP-1 confirms the importance of CTGF and TGFbeta1 in angiogenesis, and tissue repair. Expression of the serine protease inhibitors, Spi 2.4 and Spi 2, is also increased in response to AdTGFbeta1 and AdCTGF.

Chronic infection with Helicobacter pylori cagA-positive strains is the strongest risk factor for gastric cancer. The cagA gene product, CagA, is delivered into gastric epithelial cells via the bacterial type IV secretion system. Delivered CagA then undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs in its C-terminal region and acts as an oncogenic scaffold protein that physically interacts with multiple host signaling proteins in both tyrosine phosphorylation-dependent and -independent manners. Analysis of CagA using in vitro cultured gastric epithelial cells has indicated that the nonphysiological scaffolding actions of CagA cell-autonomously promote the malignant transformation of the cells by endowing the cells with multiple phenotypic cancer hallmarks: sustained proliferation, evasion of growth suppressors, invasiveness, resistance to cell death, and genomic instability. Transgenic expression of CagA in mice leads to in vivo oncogenic action of CagA without any overt inflammation. The in vivo oncogenic activity of CagA is further potentiated in the presence of chronic inflammation. Since Helicobacter pylori infection triggers a proinflammatory response in host cells, a feedforward stimulation loop that augments the oncogenic actions of CagA and inflammation is created in CagA-injected gastric mucosa. Given that Helicobacter pylori is no longer colonized in established gastric cancer lesions, the multistep nature of gastric cancer development should include a “hit-and-run” process of CagA action. Thus, acquisition of genetic and epigenetic alterations that compensate for CagA-directed cancer hallmarks may be required for completion of the “hit-and-run” process of gastric carcinogenesis.

Thrombomodulin (TM) is a thrombin receptor on endothelial cells that is involved in promoting activation of the anticoagulant protein C pathway during blood coagulation. TM also exerts protective anti-inflammatory properties through a poorly understood mechanism. In this study, we investigated the importance of TM signaling to cellular functions by deleting it from endothelial cells by CRISPR-Cas9 technology and analyzed the resultant phenotype of TM-deficient (TM −/−) cells. Deficiency of TM in endothelial cells resulted in increased basal permeability and hyperpermeability when stimulated by thrombin and TNF-α. The loss of the basal barrier permeability function was accompanied by increased tyrosine phosphorylation of VE-cadherin and reduced polymerization of F-actin filaments at cellular junctions. A significant increase in basal NF-κB signaling and expression of inflammatory cell adhesion molecules was observed in TM −/− cells that resulted in enhanced adhesion of leukocytes to TM −/− cells in flow chamber experiments. There was also a marked increase in expression, storage, and release of the von Willebrand factor (VWF) and decreased storage and release of angiopoietin-2 in TM −/− cells. In a flow chamber assay, isolated platelets adhered to TM −/− cells, forming characteristic VWF–platelet strings. Increased VWF levels and inflammatory foci were also observed in the lungs of tamoxifentreated ERcre-TMf/f mice. Reexpression of the TM construct in TM −/− cells, but not treatment with soluble TM, normalized the cellular phenotype. Based on these results, we postulate cell-bound TM endows a quiescent cellular phenotype by tightly regulating expression of procoagulant, proinflammatory, and angiogenic molecules in vascular endothelial cells.

C4a is a small protein released from complement component C4 upon activation of the complement system’s classical and lectin pathways, which are important constituents of innate immune surveillance. Despite the structural similarity between C4a and well-described anaphylatoxins C3a and C5a, the binding partner and biological function of C4a have remained elusive. Using a cell-based reporter assay, we screened C4a against a panel of both known and orphan G protein-coupled receptors and now provide evidence that C4a is a ligand for protease-activated receptor (PAR)1 and PAR4. Whereas C4a showed no activity toward known anaphylatoxin receptors, it acted as an agonist for both PAR1 and PAR4 with nanomolar activity. In human endothelial cells, ERK activation by C4a was mediated through both PAR1 and PAR4 in a Gαi-independent signaling pathway. Like other PAR1 activators, C4a induced calcium mobilization through the PAR1/Gαq/PLCβ signaling axis. Moreover, C4a increased stress fiber formation and enhanced endothelial permeability, both of which were reduced by PAR1 antagonists. In sum, our study identifies C4a as an untethered agonist for PAR1 and PAR4 with effects on cellular activation and endothelial permeability, thereby revealing another instance of cross-talk between the complement system and other host defense pathways.

Stimulation of protease-activated receptor 1 (PAR1) on endothelium by activated protein C (APC) is protective in several animal models of disease, and APC has been used clinically in severe sepsis and wound healing. Clinical use of APC, however, is limited by its immunogenicity and its anticoagulant activity. We show that a class of small molecules termed “parmodulins” that act at the cytosolic face of PAR1 stimulates APC-like cytoprotective signaling in endothelium. Parmodulins block thrombin generation in response to inflammatory mediators and inhibit platelet accumulation on endothelium cultured under flow. Evaluation of the antithrombotic mechanism showed that parmodulins induce cytoprotective signaling through Gβγ, activating a PI3K/Akt pathway and eliciting a genetic program that includes suppression of NF-κB–mediated transcriptional activation and up-regulation of select cytoprotective transcripts. STC1 is among the up-regulated transcripts, and knockdown of stanniocalin-1 blocks the protective effects of both parmodulins and APC. Induction of this signaling pathway in vivo protects against thromboinflammatory injury in blood vessels. Small-molecule activation of endothelial cytoprotection through PAR1 represents an approach for treatment of thromboinflammatory disease and provides proof-of-principle for the strategy of targeting the cytoplasmic surface of GPCRs to achieve pathway selective signaling.

We recently demonstrated that the LKB1 tumour suppressor kinase, in complex with the pseudokinase STRAD and the scaffolding protein MO25, phosphorylates and activates AMP‐activated protein kinase (AMPK). A total of 12 human kinases (NUAK1, NUAK2, BRSK1, BRSK2, QIK, QSK, SIK, MARK1, MARK2, MARK3, MARK4 and MELK) are related to AMPK. Here we demonstrate that LKB1 can phosphorylate the T‐loop of all the members of this subfamily, apart from MELK, increasing their activity >50‐fold. LKB1 catalytic activity and the presence of MO25 and STRAD are required for activation. Mutation of the T‐loop Thr phosphorylated by LKB1 to Ala prevented activation, while mutation to glutamate produced active forms of many of the AMPK‐related kinases. Activities of endogenous NUAK2, QIK, QSK, SIK, MARK1, MARK2/3 and MARK4 were markedly reduced in LKB1‐deficient cells. Neither LKB1 activity nor that of AMPK‐related kinases was stimulated by phenformin or AICAR, which activate AMPK. Our results show that LKB1 functions as a master upstream protein kinase, regulating AMPK‐related kinases as well as AMPK. Between them, these kinases may mediate the physiological effects of LKB1, including its tumour suppressor function.

Purpose Triple-negative breast cancer (TNBC) is a subtype of breast cancer with high risk of distant metastasis, in which the intercellular communication between tumor cells also plays a role. Exosomes can be released by tumor cells and promote distant metastasis through intercellular communication or changes in tumor microenvironment, it is an optimized transportation facility for biologically active payloads. This was a hypothesis-generating research on role of exosomal payload in TNBC distant metastasis. Methods Exosomes isolated from supernatant of MDA-MB-231 and MDA-MB-231-HM (a highly pulmonary metastatic variant of parental MDA-MB-231 cells) were characterized. MMP-1 level was detected using mass spectrometry and western blot. Transwell assay, wound healing and CCK-8 assay were employed to explore the effect of exosomal MMP-1 on the metastatic capability of TNBC cells in vitro. Human breast cancer lung metastasis model in nude mice was established to observe the effect of exosomal MMP-1 in vivo. Tissue microarray and blood samples of TNBC patients were applied to analyze the relevance between MMP-1 with metastasis. Results MDA-MB-231-HM cells secrete exosomes enriched MMP-1, which can be taken up and enhance invasion and migration activities of TNBC cells, including MDA-MB-231, MDA-MB-468 and BT549. After ingesting exosomes enriched with MMP-1, cells secret more MMP-1, which may interact with membrane G protein receptor protease activated receptor 1 (PAR1), thereby initiating epithelial-mesenchymal transition (EMT) to enhance capability of migration and invasion. The lung colonization model shows that the expressions of MMP-1 and PAR1 in the metastases of the 231-HM-exo treated mice were both upregulated. Clinically, the enrichment of MMP-1 can be detected in exosomes extracted from serum of patients with metastasis at higher concentration than that in pre-operative patients. Moreover, in patients with multiple distant metastases, the level of MMP-1 in exosomes is also higher than that in patients with single lesion. Conclusion MMP-1 from TNBC cells of high metastasis potential can promote the distant metastasis of transform those with low metastasis potential through PAR1-mediated EMT and is likely to be a potential molecular marker.

The G-protein coupled receptors (GPCRs) belong to a large family of diverse receptors that are well recognized as pharmacological targets. However, very few of these receptors have been pursued as oncology drug targets. The Protease-activated receptor 1 (PAR1), which is a G-protein coupled receptor, has been shown to act as an oncogene and is an emerging anti-cancer drug target. In this paper, we provide an overview of PAR1’s biased signaling role in metastatic cancers of the breast, lungs, and ovaries and describe the development of PAR1 inhibitors that are currently in clinical use to treat acute coronary syndromes. PAR1 inhibitor PZ-128 is in a Phase II clinical trial and is being developed to prevent ischemic and thrombotic complication of patients undergoing cardiac catheterization. PZ-128 belongs to a new class of cell-penetrating, membrane-tethered peptides named pepducins that are based on the intracellular loops of receptors targeting the receptor G-protein interface. Application of PZ-128 as an anti-metastatic and anti-angiogenic therapeutic agent in breast, lung, and ovarian cancer is being reviewed.