Balanced transforming growth factor-beta (TGFβ)/bone morphogenetic protein (BMP)-signaling is essential for tissue formation and homeostasis. While gain in TGFβ signaling is often found in diseases, the underlying cellular mechanisms remain poorly defined. Here we show that the receptor BMP type 2 (BMPR2) serves as a central gatekeeper of this balance, highlighted by its deregulation in diseases such as pulmonary arterial hypertension (PAH). We show that BMPR2 deficiency in endothelial cells (ECs) does not abolish pan-BMP-SMAD1/5 responses but instead favors the formation of mixed-heteromeric receptor complexes comprising BMPR1/TGFβR1/TGFβR2 that enable enhanced cellular responses toward TGFβ. These include canonical TGFβ-SMAD2/3 and lateral TGFβ-SMAD1/5 signaling as well as formation of mixed SMAD complexes. Moreover, BMPR2-deficient cells express genes indicative of altered biophysical properties, including up-regulation of extracellular matrix (ECM) proteins such as fibrillin-1 (FBN1) and of integrins. As such, we identified accumulation of ectopic FBN1 fibers remodeled with fibronectin (FN) in junctions of BMPR2-deficient ECs. Ectopic FBN1 deposits were also found in proximity to contractile intimal cells in pulmonary artery lesions of BMPR2-deficient heritable PAH (HPAH) patients. In BMPR2-deficient cells, we show that ectopic FBN1 is accompanied by active β1-integrin highly abundant in integrin-linked kinase (ILK) mechano-complexes at cell junctions. Increased integrin-dependent adhesion, spreading, and actomyosin-dependent contractility facilitates the retrieval of active TGFβ from its latent fibrillin-bound depots. We propose that loss of BMPR2 favors endothelial-to-mesenchymal transition (EndMT) allowing cells of myo-fibroblastic character to create a vicious feed-forward process leading to hyperactivated TGFβ signaling. In summary, our findings highlight a crucial role for BMPR2 as a gatekeeper of endothelial homeostasis protecting cells from increased TGFβ responses and integrin-mediated mechano-transduction.

Understanding information flow in sensory pathways requires cell-selective approaches to manipulate the activity of defined neurones. Primary afferent nociceptors, which detect painful stimuli, are enriched in specific voltage-gated sodium channel (VGSC) subtypes. Toxins derived from venomous animals can be used to dissect the contributions of particular ion currents to cell physiology. Here we have used a transgenic approach to target a membrane-tethered isoform of the conotoxin MrVIa (t-MrVIa) only to nociceptive neurones in mice. T-MrVIa transgenic mice show a 44 ± 7% reduction of tetrodotoxin-resistant (TTX-R) VGSC current densities. This inhibition is permanent, reversible and does not result in functional upregulation of TTX-sensitive (TTX-S) VGSCs, voltage-gated calcium channels (VGCCs) or transient receptor potential (TRP) channels present in nociceptive neurones. As a consequence of the reduction of TTX-R VGSC currents, t-MrVIa transgenic mice display decreased inflammatory mechanical hypersensitivity, cold pain insensitivity and reduced firing of cutaneous C-fibres sensitive to noxious cold temperatures. These data validate the use of genetically encoded t-toxins as a powerful tool to manipulate VGSCs in specific cell types within the mammalian nervous system. This novel genetic methodology can be used for circuit mapping and has the key advantage that it enables the dissection of the contribution of specific ionic currents to neuronal function and to behaviour.

Interleukin 17-producing helper T (Th17) cells have been widely defined by the lineage transcription factor retinoid-related orphan receptor (ROR)γt. Pathophysiologically, these cells play a crucial role in autoimmune diseases and have been linked to dysregulated germinal center (GC) reactions and autoantibody production. In this study, we used gene expression and flow cytometric analyses for the characterization of Rorγt(-/-) and Rorγt(-/-)Il21(RFP/+) mice to demonstrate a previously unknown transcriptional flexibility in the development of IL-17-producing Th-cell subsets. We found an accumulation of follicular Th (Tfh) cells by 5.2-fold, spontaneous 13-fold higher GC formation, decreased frequency of follicular Foxp3(+) T-regulatory (Treg) cells (50%), and a 3.4-fold increase in the number of proliferating follicular B cells in RORγt-deficient vs. wild-type mice. Dysregulated B-cell responses were associated with enhanced production of IL-17 (6.4-fold), IL-21 (2.2-fold), and B-cell-activating factor (BAFF) (2-fold) and were partially rescued by adoptive transfer of Treg cells. In an unexpected finding, we detected RORγt-independent IL-17 expression in ICOS(+)CXCR5(+)Tfh and in ICOS(+)CXCR5(-)Th cells. Based on the observed high Irf4 and Batf gene expression, we suggest that CD4(+) T-cell transcription factors other than RORγt can cooperatively induce differentiation of IL-17-producing Th cells, including Th17-like Tfh-cell subsets. We conclude that the occurrence of aberrant Tfh and follicular Treg cells support spontaneous GC formation and dysregulated B-cell responses in RORγt-deficient mice.

Balanced signal transduction is crucial in tissue patterning, particularly in the vasculature. Heterotopic ossification (HO) is tightly linked to vascularization with increased vessel number in hereditary forms of HO, such as Fibrodysplasia ossificans progressiva (FOP). FOP is caused by mutations in the BMP type I receptor ACVR1 leading to aberrant SMAD1/5 signaling in response to ActivinA. Whether observed vascular phenotype in human FOP lesions is connected to aberrant ActivinA signaling is unknown. Blocking of ActivinA prevents HO in FOP mice indicating a central role of the ligand in FOP. Here, we established a new FOP endothelial cell model generated from induced pluripotent stem cells (iECs) to study ActivinA signaling. FOP iECs recapitulate pathogenic ActivinA/SMAD1/5 signaling. Whole transcriptome analysis identified ActivinA mediated activation of the BMP/NOTCH pathway exclusively in FOP iECs, which was rescued to WT transcriptional levels by the drug candidate Saracatinib. We propose that ActivinA causes transcriptional pre-patterning of the FOP endothelium, which might contribute to differential vascularity in FOP lesions compared to non-hereditary HO. Graphical abstract