Identification of a choroid plexus vascular barrier closing during intestinal inflammation.

Up to 40% of patients with inflammatory bowel disease present with psychosocial disturbances. We previously identified a gut vascular barrier that controls the dissemination of bacteria from the intestine to the liver. Here, we describe a vascular barrier in the brain choroid plexus (PVB) that is modulated in response to intestinal inflammation through bacteria-derived lipopolysaccharide. The inflammatory response induces PVB closure after gut vascular barrier opening by the up-regulation of the wingless-type, catenin-beta 1 (Wnt/ß-catenin) signaling pathway, rendering it inaccessible to large molecules. In a model of genetically driven closure of choroid plexus endothelial cells, we observed a deficit in short-term memory and anxiety-like behavior, suggesting that PVB closure may correlate with mental deficits. Inflammatory bowel disease­related mental symptoms may thus be the consequence of a deregulated gut­brain vascular axis.

BAP1 enhances Polycomb repression by counteracting widespread H2AK119ub1 deposition and chromatin condensation.

BAP1 is mutated or deleted in many cancer types, including mesothelioma, uveal melanoma, and cholangiocarcinoma. It is the catalytic subunit of the PR-DUB complex, which removes PRC1-mediated H2AK119ub1, essential for maintaining transcriptional repression. However, the precise relationship between BAP1 and Polycombs remains elusive. Using embryonic stem cells, we show that BAP1 restricts H2AK119ub1 deposition to Polycomb target sites. This increases the stability of Polycomb with their targets and prevents diffuse accumulation of H2AK119ub1 and H3K27me3. Loss of BAP1 results in a broad increase in H2AK119ub1 levels that is primarily dependent on PCGF3/5-PRC1 complexes. This titrates PRC2 away from its targets and stimulates H3K27me3 accumulation across the genome, leading to a general chromatin compaction. This provides evidence for a unifying model that resolves the apparent contradiction between BAP1 catalytic activity and its role in vivo, uncovering molecular vulnerabilities that could be useful for BAP1-related pathologies.

Colorectal cancer residual disease at maximal response to EGFR blockade displays a druggable Paneth cell-like phenotype.

Blockade of epidermal growth factor receptor (EGFR) causes tumor regression in some patients with metastatic colorectal cancer (mCRC). However, residual disease reservoirs typically remain even after maximal response to therapy, leading to relapse. Using patient-derived xenografts (PDXs), we observed that mCRC cells surviving EGFR inhibition exhibited gene expression patterns similar to those of a quiescent subpopulation of normal intestinal secretory precursors with Paneth cell characteristics. Compared with untreated tumors, these pseudodifferentiated tumor remnants had reduced expression of genes encoding EGFR-activating ligands, enhanced activity of human epidermal growth factor receptor 2 (HER2) and HER3, and persistent signaling along the phosphatidylinositol 3-kinase (PI3K) pathway. Clinically, properties of residual disease cells from the PDX models were detected in lingering tumors of responsive patients and in tumors of individuals who had experienced early recurrence. Mechanistically, residual tumor reprogramming after EGFR neutralization was mediated by inactivation of Yes-associated protein (YAP), a master regulator of intestinal epithelium recovery from injury. In preclinical trials, Pan-HER antibodies minimized residual disease, blunted PI3K signaling, and induced long-term tumor control after treatment discontinuation. We found that tolerance to EGFR inhibition is characterized by inactivation of an intrinsic lineage program that drives both regenerative signaling during intestinal repair and EGFR-dependent tumorigenesis. Thus, our results shed light on CRC lineage plasticity as an adaptive escape mechanism from EGFR-targeted therapy and suggest opportunities to preemptively target residual disease.

PRMT1 Is Recruited via DNA-PK to Chromatin Where It Sustains the Senescence-Associated Secretory Phenotype in Response to Cisplatin.

Protein arginine methyltransferase 1 (PRMT1) is overexpressed in various human cancers and linked to poor response to chemotherapy. Various PRMT1 inhibitors are currently under development; yet, we do not fully understand the mechanisms underpinning PRMT1 involvement in tumorigenesis and chemoresistance. Using mass spectrometry-based proteomics, we identified PRMT1 as regulator of arginine methylation in ovarian cancer cells treated with cisplatin. We showed that DNA-dependent protein kinase (DNA-PK) binds to and phosphorylates PRMT1 in response to cisplatin, inducing its chromatin recruitment and redirecting its enzymatic activity toward Arg3 of histone H4 (H4R3). On chromatin, the DNA-PK/PRMT1 axis induces senescence-associated secretory phenotype through H4R3me2a deposition at pro-inflammatory gene promoters. Finally, PRMT1 inhibition reduces the clonogenic growth of cancer cells exposed to low doses of cisplatin, sensitizing them to apoptosis. While unravelling the role of PRMT1 in response to genotoxic agents, our findings indicate the possibility of targeting PRMT1 to overcome chemoresistance in cancer.

Stimulation of mTORC2 by integrin αIIbß3 is required for PI3Kß-dependent activation of Akt but is dispensable for platelet spreading on fibrinogen.

Phosphatidylinositol 3 kinase (PI3K) is a major player in platelet activation and regulates thrombus formation and stabilization. The ß isoform of PI3K is implicated in integrin αIIbß3 outside-in signaling, is required for the phosphorylation of Akt, and controls efficient platelet spreading upon adhesion to fibrinogen. In this study we found that during integrin αIIbß3 outside-in signaling PI3Kß-dependent phosphorylation of Akt on Serine473 is mediated by the mammalian target of rapamycin complex 2 (mTORC2). The activity of mTORC2 is stimulated upon platelet adhesion to fibrinogen, as documented by increased autophosphorylation. However, mTORC2 activation downstream of integrin αIIbß3 is PI3Kß-independent. Inhibition of mTORC2, but not mTORC1, also prevents Akt phosphorylation of Threonine308 and affects Akt activity, resulting in the inhibition of GSK3α/ß phosphorylation. Nevertheless, mTORC2 or Akt inhibition does not alter PI3Kß-dependent platelet spreading on fibrinogen. The activation of the small GTPase Rap1b downstream of integrin αIIbß3 is regulated by PI3Kß but is not affected upon inhibition of either mTORC2 or Akt. Altogether, these results demonstrate for the first time the activation of mTORC2 and its involvement in Akt phosphorylation and stimulation during integrin αIIbß3 outside-in signaling. Moreover, the results demonstrate that the mTORC2/Akt pathway is dispensable for PI3Kß-regulated platelet spreading on fibrinogen.

Histone H2AK119 Mono-Ubiquitination Is Essential for Polycomb-Mediated Transcriptional Repression.

Polycomb group proteins (PcGs) maintain transcriptional repression to preserve cellular identity in two distinct repressive complexes, PRC1 and PRC2, that modify histones by depositing H2AK119ub1 and H3K27me3, respectively. PRC1 and PRC2 exist in different variants and show a complex regulatory cross-talk. However, the contribution that H2AK119ub1 plays in mediating PcG repressive functions remains largely controversial. Using a fully catalytic inactive RING1B mutant, we demonstrated that H2AK119ub1 deposition is essential to maintain PcG-target gene repression in embryonic stem cells (ESCs). Loss of H2AK119ub1 induced a rapid displacement of PRC2 activity and a loss of H3K27me3 deposition. This preferentially affected PRC2.2 variant with respect to PRC2.1, destabilizing canonical PRC1 activity. Finally, we found that variant PRC1 forms can sense H2AK119ub1 deposition, which contributes to their stabilization specifically at sites where this modification is highly enriched. Overall, our data place H2AK119ub1 deposition as a central hub that mounts PcG repressive machineries to preserve cell transcriptional identity.

Loss of PRC1 activity in different stem cell compartments activates a common transcriptional program with cell type-dependent outcomes.

Polycomb repressive complexes are evolutionarily conserved complexes that maintain transcriptional repression during development and differentiation to establish and preserve cell identity. We recently described the fundamental role of PRC1 in preserving intestinal stem cell identity through the inhibition of non-lineage-specific transcription factors. To further elucidate the role of PRC1 in adult stem cell maintenance, we now investigated its role in LGR5+ hair follicle stem cells during regeneration. We show that PRC1 depletion severely affects hair regeneration and, different from intestinal stem cells, derepression of its targets induces the ectopic activation of an epidermal-specific program. Our data support a general role of PRC1 in preserving stem cell identity that is shared between different compartments. However, the final outcome of the ectopic activation of non-lineage-specific transcription factors observed upon loss of PRC1 is largely context-dependent and likely related to the transcription factors repertoire and specific epigenetic landscape of different cellular compartments.

Activation of phosphatidylinositol 3-kinase ß by the platelet collagen receptors integrin α2ß1 and GPVI: The role of Pyk2 and c-Cbl.

Phosphatidylinositol 3-kinaseß (PI3Kß) plays a predominant role in integrin outside-in signaling and in platelet activation by GPVI engagement. We have shown that the tyrosine kinase Pyk2 mediates PI3Kß activation downstream of integrin αIIbß3, and promotes the phosphorylation of the PI3K-associated adaptor protein c-Cbl. In this study, we compared the functional correlation between Pyk2 and PI3Kß upon recruitment of the two main platelet collagen receptors, integrin α2ß1 and GPVI. PI3Kß-mediated phosphorylation of Akt was inhibited in Pyk2-deficient platelets adherent to monomeric collagen through integrin α2ß1, but occurred normally upon GPVI ligation. Integrin α2ß1 engagement led to Pyk2-independent association of c-Cbl with PI3K. However, c-Cbl was not phosphorylated in adherent platelets, and phosphorylation of Akt occurred normally in c-Cbl-deficient platelets, indicating that the c-Cbl is dispensable for Pyk2-mediated PI3Kß activation. Stimulation of platelets with CRP, a selective GPVI ligand, induced c-Cbl phosphorylation in the absence of Pyk2, but failed to promote its association with PI3K. Pyk2 activation was completely abrogated in PI3KßKD, but not in PI3KγKD platelets, and was strongly inhibited by Src kinases and phospholipase C inhibitors, and by BAPTA-AM. The absence of PI3Kß activity also hampered GPVI-induced tyrosine-phosphorylation and activation of PLCγ2, preventing intracellular Ca2+ increase and phosphorylation of pleckstrin. Moreover, GPVI-induced intracellular Ca2+ increase and pleckstrin phosphorylation were also strongly inhibited in human platelets treated with the PI3Kß inhibitor TGX-221. These results outline important differences in the regulation of PI3Kß by GPVI and integrin α2ß1 and suggest that inhibition of Pyk2 may target PI3Kß activation in a selective context of platelet stimulation.

The focal adhesion kinase Pyk2 links Ca2+ signalling to Src family kinase activation and protein tyrosine phosphorylation in thrombin-stimulated platelets.

In blood platelets, stimulation of G protein-coupled receptors (GPCRs) by thrombin triggers the activation of Src family kinases (SFKs), resulting in the tyrosine-phosphorylation of multiple substrates, but the mechanism underlying this process is still poorly understood. In the present study, we show that the time-dependent protein-tyrosine phosphorylation triggered by thrombin in human or murine platelets was totally suppressed only upon concomitant chelation of intracellular Ca(2+) and inhibition of SFKs. Thrombin-induced activation of SFKs was regulated by intracellular Ca(2+) and accordingly the Ca(2+) ionophore A23187 was sufficient to stimulate SFKs. A23187 also triggered the phosphorylation and activation of the Ca(2+)-dependent focal adhesion kinase Pyk2 and Pyk2 activation by thrombin was Ca(2+)-dependent. Stimulation of SFKs by thrombin or A23187 was strongly reduced in platelets from Pyk2 knockout (KO) mice, as was the overall pattern of protein-tyrosine phosphorylation. By immunoprecipitation experiments, we demonstrate that Lyn and Fyn, but not Src, were activated by Pyk2. Inhibition of SFKs by PP2 also reduced the phosphorylation of Pyk2 in thrombin or A23187-stimulated platelets. Analysis of KO mice demonstrated that Fyn, but not Lyn, was required for complete Pyk2 phosphorylation by thrombin. Finally, PP2 reduced aggregation of murine platelets to a level comparable to that of Pyk2-deficient platelets, but did not have further effects in the absence of Pyk2. These results indicate that in thrombin-stimulated platelets, stimulation of Pyk2 by intracellular Ca(2+) initiates SFK activation, establishing a positive loop that reinforces the Pyk2/SFK axis and allows the subsequent massive tyrosine phosphorylation of multiple substrates required for platelet aggregation.

Amyloid ß-peptide-dependent activation of human platelets: essential role for Ca2+ and ADP in aggregation and thrombus formation.

Alzheimer's disease is associated with the accumulation of Aß (amyloid ß)-peptides in the brain. Besides their cytotoxic effect on neurons, Aß-peptides are thought to be responsible for the atherothrombotic complications associated with Alzheimer's disease, which are collectively known as cerebrovascular disease. In the present study, we investigated the effect of Aß-peptides on human platelet signal transduction and function. We discovered that the 25-35 domain of Aß-peptides induce an increase in platelet intracellular Ca2+ that stimulates α-granule and dense granule secretion and leads to the release of the secondary agonist ADP. Released ADP acts in an autocrine manner as a stimulant for critical signalling pathways leading to the activation of platelets. This includes the activation of the protein kinases Syk, protein kinase C, Akt and mitogen-activated protein kinases. Ca2+-dependent release of ADP is also the main component of the activation of the small GTPase Rap1b and the fibrinogen receptor integrin αIIbß3, which leads to increased platelet aggregation and increased thrombus formation in human whole blood. Our discoveries complement existing understanding of cerebrovascular dementia and suggest that Aß-peptides can induce vascular complications of Alzheimer's disease by stimulating platelets in an intracellular Ca2+-dependent manner. Despite a marginal ADP-independent component suggested by low levels of signalling activity in the presence of apyrase or P2Y receptor inhibitors, Ca2+-dependent release of ADP by Aß-peptides clearly plays a critical role in platelet activation. Targeting ADP signalling may therefore represent an important strategy to manage the cerebrovascular component of Alzheimer's disease.

Immobilized amyloid Aß peptides support platelet adhesion and activation.

Accumulation of amyloidogenic Aß peptides in the brain contributes to the onset of Alzheimer disease. Aß peptide deposits are also present in blood vessel walls, mainly deriving from circulating platelets. However, their effect on platelet function is unclear. We demonstrate that immobilized Aß peptides induce platelet adhesion and spreading through metalloproteinase-sensitive surface receptors. Aß peptides also fasten platelet spreading on collagen, and support the time- and ADP-dependent activation of adherent platelets, leading to stimulation of several signalling proteins. Our results indicate a potential role for peripheral Aß peptides in promoting platelet adhesion and activation in the initiation of thrombus formation.

Phosphorylation of the guanine-nucleotide-exchange factor CalDAG-GEFI by protein kinase A regulates Ca(2+)-dependent activation of platelet Rap1b GTPase.

In blood platelets the small GTPase Rap1b is activated by cytosolic Ca2+ and promotes integrin αIIbß3 inside-out activation and platelet aggregation. cAMP is the major inhibitor of platelet function and antagonizes Rap1b stimulation through a mechanism that remains unclear. In the present study we demonstrate that the Ca2+-dependent exchange factor for Rap1b, CalDAG-GEFI (calcium and diacylglycerol-regulated guanine-nucleotide-exchange factor I), is a novel substrate for the cAMP-activated PKA (protein kinase A). CalDAG-GEFI phosphorylation occurred in intact platelets treated with the cAMP-increasing agent forskolin and was inhibited by the PKA inhibitor H89. Purified recombinant CalDAG-GEFI was also phosphorylated in vitro by the PKA catalytic subunit. By screening a panel of specific serine to alanine residue mutants, we identified Ser116 and Ser586 as PKA phosphorylation sites in CalDAG-GEFI. In transfected HEK (human embryonic kidney)-293 cells, as well as in platelets, forskolin-induced phosphorylation of CalDAG-GEFI prevented the activation of Rap1b induced by the Ca2+ ionophore A23187. In platelets this effect was associated with the inhibition of aggregation. Moreover, cAMP-mediated inhibition of Rap1b was lost in HEK-293 cells transfected with a double mutant of CalDAG-GEFI unable to be phosphorylated by PKA. The results of the present study demonstrate that phosphorylation of CalDAG-GEFI by PKA affects its activity and represents a novel mechanism for cAMP-mediated inhibition of Rap1b in platelets.