Biomechanical stress provides a second hit in the establishment of BMP/TGFβ-related vascular disorders

Cardiovascular disorders are still the leading cause for mortality in the western world and challenge economies with steadily increasing healthcare costs. Understanding the precise molecular pathomechanisms behind and identifying players involved in the early onset of cardiovascular diseases remains crucial for the development of new therapeutic strategies. Taking advantage of CRISPR/Cas9 gene editing in human endothelial cells (ECs), we re-investigated the early molecular steps in a genetic vascular disorder termed pulmonary arterial hypertension (PAH) in our recent study (Hiepen C., Jatzlau J. et al.; PLOS Biol, 2019). Here, mutations in the Bone Morphogenetic Protein type II receptor (BMPR2) prime for the hereditary form (HPAH) with downregulated BMPR2 followed by a characteristic change in SMAD signaling, i.e. gain in both SMAD1/5 and SMAD2/3 responses. Remarkably these cells show increased susceptibility to signaling by TGFβ due to remodeling of the extracellular matrix (ECM) and increased biomechanics acting as a secondary stressor for ECs pathobiology. This clearly places BMPR2 not only as a BMP-signaling receptor, but also as a gatekeeper to protect ECs from excess TGFβ signaling

BMP Stimulation Differentially Affects Phosphorylation and Protein Stability of β-Catenin in Breast Cancer Cell Lines

Increased expression and nuclear translocation of β-CATENIN is frequently observed in breast cancer, and it correlates with poor prognosis. Current treatment strategies targeting β-CATENIN are not as efficient as desired. Therefore, detailed understanding of β-CATENIN regulation is crucial. Bone morphogenetic proteins (BMP) and Wingless/Integrated (WNT) pathway crosstalk is well-studied for many cancer types including colorectal cancer, whereas it is still poorly understood for breast cancer. Analysis of breast cancer patient data revealed that BMP2 and BMP6 were significantly downregulated in tumors. Since mutation frequency in genes enhancing β-CATENIN protein stability is relatively low in breast cancer, we aimed to investigate whether decreased BMP ligand expression could contribute to a high protein level of β-CATENIN in breast cancer cells. We demonstrated that downstream of BMP stimulation, SMAD4 is required to reduce β-CATENIN protein stability through the phosphorylation in MCF7 and T47D cells. Consequently, BMP stimulation reduces β-CATENIN levels and prevents its nuclear translocation and target gene expression in MCF7 cells. Conversely, BMP stimulation has no effect on β-CATENIN phosphorylation or stability in MDA-MB-231 and MDA-MB-468 cells. Likewise, SMAD4 modulation does not alter the response of those cells, indicating that SMAD4 alone is insufficient for BMP-induced β-CATENIN phosphorylation. While our data suggest that considering BMP activity may serve as a prognostic marker for understanding β-CATENIN accumulation risk, further investigation is needed to elucidate the differential responsiveness of breast cancer cell lines

The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A

Mutations in activin-like kinase 2 (ALK2), e.g., ALK2-R206H, induce aberrant signaling to SMAD1/5/8, leading to Fibrodysplasia Ossificans Progressiva (FOP). In spite of extensive studies, the underlying mechanism is still unclear. Here, we quantified the homomeric and heteromeric interactions of ACVR2A, ACVR2B, ALK2-WT, and ALK2-R206H by combining IgG-mediated immobilization of one receptor with fluorescence recovery after photobleaching (FRAP) measurements on the lateral diffusion of a co-expressed receptor. ACVR2B formed stable homomeric complexes that were enhanced by Activin A (ActA), while ACVR2A required ActA for homodimerization. ALK2-WT, but not ALK2-R206H, exhibited homomeric complexes unaffected by ActA. ACVR2B formed ActA-enhanced heterocomplexes with ALK2-R206H or ALK2-WT, while ACVR2A interacted mainly with ALK2-WT. The extent of the homomeric complex formation of ACVR2A or ACVR2B was reflected in their ability to induce the oligomerization of ALK2-R206H and ALK2-WT. Thus, ACVR2B, which forms dimers without ligand, induced ActA-independent ALK2-R206H clustering but required ActA for enhancing the oligomerization of the largely dimeric ALK2-WT. In contrast, ACVR2A, which undergoes homodimerization in response to ActA, required ActA to induce ALK2-R206H oligomerization. To investigate whether these interactions are translated into signaling, we studied signaling by the FOP-inducing hyperactive ALK2-R206H mutant, with ALK2-WT signaling as control. The activation of SMAD1/5/8 signaling in cells expressing ALK2-R206H alone or together with ACVR2A or ACVR2B was measured by blotting for pSMAD1/5/8 and by transcriptional activation assays using BRE-Luc reporter. In line with the biophysical studies, ACVR2B activated ALK2-R206H without ligand, while activation by ACVR2A was weaker and required ActA. We propose that the homodimerization of ACVR2B or ACVR2A dictates their ability to recruit ALK2-R206H into higher complexes, enabling the homomeric interactions of ALK2-R206H receptors and, subsequently, their activation

Characterization of Fibrodysplasia Ossificans Progessiva relevant Acvr1/Acvr2 Activin receptors in medaka (Oryzias latipes)

Activin and Bone Morphogenetic Protein (BMP) signaling plays crucial roles in vertebrate organ formation, including osteo- and angiogenesis, and tissue homeostasis, such as neuronal maintenance. Activin and BMP signaling needs to be precisely controlled by restricted expression of shared receptors, stoichiometric composition of receptor-complexes and presence of regulatory proteins. A R206H mutation in the human (hs) BMP type I receptor hsACVR1, on the other hand, leads to excessive phosphorylation of Sons of mothers against decapentaplegic (SMAD) 1/5/8. This in turn causes increased inflammation and heterotopic ossification in soft tissues of patients suffering from Fibrodysplasia Ossificans Progressiva (FOP). Several animal models have been established to understand the spontaneous and progressive nature of FOP, but often have inherent limitations. The Japanese medaka (Oryzias latipes, ola) has recently emerged as popular model for bone research. To assess whether medaka is suitable as a potential FOP animal model, we determined the expression of Activin receptor type I (ACVR1) orthologs olaAcvr1 and olaAcvr1l with that of Activin type II receptors olaAcvr2ab, olaAcvr2ba and olaAcvr2bb in embryonic and adult medaka tissues by in situ hybridization. Further, we showed that Activin A binding properties are conserved in olaAcvr2, as are the mechanistic features in the GS-Box of both olaAcvr1 and olaAcvr1l. This consequently leads to FOP-typical elevated SMAD signaling when the medaka type I receptors carry the R206H equivalent FOP mutation. Together, this study therefore provides experimental groundwork needed to establish a unique medaka model to investigate mechanisms underlying FOP

Differential Impact of Fluid Shear Stress and YAP/TAZ on BMP/TGF‐β Induced Osteogenic Target Genes

Bone is a remarkable dynamic structure, which integrates mechanical and biochemical signaling inputs. Interstitial fluid in the intramedullary space transmits signals derived from compression‐induced fluid shear stress (FSS) to stimulate osteoblasts for bone formation. Using a flow system and human osteoblasts, this study demonstrates how BMP/TGF‐β signaling integrates stimuli derived from FSS and YAP/TAZ and confirms these findings by transcriptome analyses. Here, FSS positively affects the phosphorylation of both SMAD1/5 and SMAD2/3, the respective BMP‐ and TGFβ‐R‐SMADs. Increase in phosphorylated SMAD1/5 levels affects distinct target genes, which are susceptible to low levels of phosphorylated SMADs (such as ID1–3) or dependent on high levels of phosphorylated SMAD1/5 (NOG, noggin). Thus, FSS lowers the threshold for genes dependent on high levels of phosphorylated SMAD1/5 when less BMP is available. While the impact of FSS on direct BMP target genes is independent of YAP/TAZ, FSS acts cooperatively with YAP/TAZ on TGF‐β target genes, which are shared by both pathways (such as CTGF). As mechanical stimuli are key in bone regeneration, their crosstalk to biochemical signaling pathways such as BMP and TGF‐β and YAP/TAZ acts on different levels, which allows now to think about new and more specified intervention strategies for age‐related bone loss

IRS4, a novel modulator of BMP/Smad and Akt signalling during early muscle differentiation

Elaborate regulatory networks of the Bone Morphogenetic Protein (BMP) pathways ensure precise signalling outcome during cell differentiation and tissue homeostasis. Here, we identified IRS4 as a novel regulator of BMP signal transduction and provide molecular insights how it integrates into the signalling pathway. We found that IRS4 interacts with the BMP receptor BMPRII and specifically targets Smad1 for proteasomal degradation consequently leading to repressed BMP/Smad signalling in C2C12 myoblasts while concomitantly activating the PI3K/Akt axis. IRS4 is present in human and primary mouse myoblasts, the expression increases during myogenic differentiation but is downregulated upon final commitment coinciding with Myogenin expression. Functionally, IRS4 promotes myogenesis in C2C12 cells, while IRS4 knockdown inhibits differentiation of myoblasts. We propose that IRS4 is particularly critical in the myoblast stage to serve as a molecular switch between BMP/Smad and Akt signalling and to thereby control cell commitment. These findings provide profound understanding of the role of BMP signalling in early myogenic differentiation and open new ways for targeting the BMP pathway in muscle regeneration

A versatile Halo- and SNAP-tagged BMP/TGFβ receptor library for quantification of cell surface ligand binding

TGFβs, BMPs and Activins regulate numerous developmental and homeostatic processes and signal through hetero-tetrameric receptor complexes composed of two types of serine/threonine kinase receptors. Each of the 33 different ligands possesses unique affinities towards specific receptor types. However, the lack of specific tools hampered simultaneous testing of ligand binding towards all BMP/TGFβ receptors. Here we present a N-terminally Halo- and SNAP-tagged TGFβ/BMP receptor library to visualize receptor complexes in dual color. In combination with fluorescently labeled ligands, we established a Ligand Surface Binding Assay (LSBA) for optical quantification of receptor-dependent ligand binding in a cellular context. We highlight that LSBA is generally applicable to test (i) binding of different ligands such as Activin A, TGFβ1 and BMP9, (ii) for mutant screens and (iii) evolutionary comparisons. This experimental set-up opens opportunities for visualizing ligand-receptor binding dynamics, essential to determine signaling specificity and is easily adaptable for other receptor signaling pathways

Atheroprone fluid shear stress-regulated ALK1-Endoglin-SMAD signaling originates from early endosomes

Background Fluid shear stress enhances endothelial SMAD1/5 signaling via the BMP9-bound ALK1 receptor complex supported by the co-receptor Endoglin. While moderate SMAD1/5 activation is required to maintain endothelial quiescence, excessive SMAD1/5 signaling promotes endothelial dysfunction. Increased BMP signaling participates in endothelial-to-mesenchymal transition and inflammation culminating in vascular diseases such as atherosclerosis. While the function of Endoglin has so far been described under picomolar concentrations of BMP9 and short-term shear application, we investigated Endoglin under physiological BMP9 and long-term pathophysiological shear conditions. Results We report here that knock-down of Endoglin leads to exacerbated SMAD1/5 phosphorylation and atheroprone gene expression profile in HUVECs sheared for 24 h. Making use of the ligand-trap ALK1-Fc, we furthermore show that this increase is dependent on BMP9/10. Mechanistically, we reveal that long-term exposure of ECs to low laminar shear stress leads to enhanced Endoglin expression and endocytosis of Endoglin in Caveolin-1-positive early endosomes. In these endosomes, we could localize the ALK1-Endoglin complex, labeled BMP9 as well as SMAD1, highlighting Caveolin-1 vesicles as a SMAD signaling compartment in cells exposed to low atheroprone laminar shear stress. Conclusions We identified Endoglin to be essential in preventing excessive activation of SMAD1/5 under physiological flow conditions and Caveolin-1-positive early endosomes as a new flow-regulated signaling compartment for BMP9-ALK1-Endoglin signaling axis in atheroprone flow conditions

FKBP12 is a major regulator of ALK2 activity in multiple myeloma cells

Background The immunophilin FKBP12 binds to TGF-β family type I receptors, including the BMP type I receptor ALK2. FKBP12 keeps the type I receptor in an inactive state and controls signaling activity. Removal of FKBP12 with drugs such as the FKBP-ligand FK506 enhances BMP activity in various cell types. In multiple myeloma cells, activation of SMAD1/5/8 leads to apoptosis. We hypothesized that removing FKBP12 from ALK2 in myeloma cells would potentiate BMP-induced ALK2-SMAD1/5/8 activity and in consequence cell death. Methods Multiple myeloma cell lines were treated with FK506, or other FKBP-binding compounds, combined with different BMPs before analyzing SMAD1/5/8 activity and cell viability. SMAD1/5/8 activity was also investigated using a reporter cell line, INA-6 BRE-luc. To characterize the functional signaling receptor complex, we genetically manipulated receptor expression by siRNA, shRNA and CRISPR/Cas9 technology. Results FK506 potentiated BMP-induced SMAD1/5/8 activation and apoptosis in multiple myeloma cell lines. By using FKBP-binding compounds with different affinity profiles, and siRNA targeting FKBP12, we show that the FK506 effect is mediated by binding to FKBP12. Ligands that typically signal via ALK3 in myeloma cells, BMP2, BMP4, and BMP10, did not induce apoptosis in cells lacking ALK3. Notably, BMP10 competed with BMP6 and BMP9 and antagonized their activity via ALK2. However, upon addition of FK506, we saw a surprising shift in specificity, as the ALK3 ligands gained the ability to signal via ALK2 and induce apoptosis. This indicates that the receptor complex can switch from an inactive non-signaling complex (NSC) to an active one by adding FK506. This gain of activity was also seen in other cell types, indicating that the observed effects have broader relevance. BMP2, BMP4 and BMP10 depended on BMPR2 as type II receptor to signal, which contrasts with BMP6 and BMP9, that activate ALK2 more potently when BMPR2 is knocked down. Conclusions In summary, our data suggest that FKBP12 is a major regulator of ALK2 activity in multiple myeloma cells, partly by switching an NSC into an active signaling complex. FKBP12 targeting compounds devoid of immunosuppressing activity could have potential in novel treatment strategies aiming at reducing multiple myeloma tumor load