External mechanical loading overrules cell-cell mechanical communication in sprouting angiogenesis during early bone regeneration.

Sprouting angiogenesis plays a key role during bone regeneration. For example, insufficient early revascularization of the injured site can lead to delayed or non-healing. During sprouting, endothelial cells are known to be mechano-sensitive and respond to local mechanical stimuli. Endothelial cells interact and communicate mechanically with their surroundings, such as outer-vascular stromal cells, through cell-induced traction forces. In addition, external physiological loads act at the healing site, resulting in tissue deformations and impacting cellular arrangements. How these two distinct mechanical cues (cell-induced and external) impact angiogenesis and sprout patterning in early bone healing remains however largely unknown. Therefore, the aim of this study was to investigate the relative role of externally applied and cell-induced mechanical signals in driving sprout patterning at the onset of bone healing. To investigate cellular self-organisation in early bone healing, an in silico model accounting for the mechano-regulation of sprouting angiogenesis and stromal cell organization was developed. Computer model predictions were compared to in vivo experiments of a mouse osteotomy model stabilized with a rigid or a semirigid fixation system. We found that the magnitude and orientation of principal strains within the healing region can explain experimentally observed sprout patterning, under both fixation conditions. Furthermore, upon simulating the selective inhibition of either cell-induced or externally applied mechanical cues, external mechanical signals appear to overrule the mechanical communication acting on a cell-cell interaction level. Such findings illustrate the relevance of external mechanical signals over the local cell-mediated mechanical cues and could be used in the design of fracture treatment strategies for bone regeneration.

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

Increased expression and nuclear translocation of ß-CATENIN is frequently observed in breast cancer, and it correlates with poor prognosis. Current treatment strategies targeting ß-CATENIN are not as efficient as desired. Therefore, detailed understanding of ß-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 ß-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 ß-CATENIN in breast cancer cells. We demonstrated that downstream of BMP stimulation, SMAD4 is required to reduce ß-CATENIN protein stability through the phosphorylation in MCF7 and T47D cells. Consequently, BMP stimulation reduces ß-CATENIN levels and prevents its nuclear translocation and target gene expression in MCF7 cells. Conversely, BMP stimulation has no effect on ß-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 ß-CATENIN phosphorylation. While our data suggest that considering BMP activity may serve as a prognostic marker for understanding ß-CATENIN accumulation risk, further investigation is needed to elucidate the differential responsiveness of breast cancer cell lines.

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

BACKGROUND: The immunophilin FKBP12 binds to TGF-ß 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. Video Abstract.

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.

BMPR2 acts as a gatekeeper to protect endothelial cells from increased TGFß responses and altered cell mechanics.

Balanced transforming growth factor-beta (TGFß)/bone morphogenetic protein (BMP)-signaling is essential for tissue formation and homeostasis. While gain in TGFß 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/TGFßR1/TGFßR2 that enable enhanced cellular responses toward TGFß. These include canonical TGFß-SMAD2/3 and lateral TGFß-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 ß1-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 TGFß 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 TGFß signaling. In summary, our findings highlight a crucial role for BMPR2 as a gatekeeper of endothelial homeostasis protecting cells from increased TGFß responses and integrin-mediated mechano-transduction.

[Interdisciplinary management of extracranial vascular anomalies]. / Interdisziplinäres Management extrakranieller Gefäßanomalien.

BACKGROUND: A multitude of vascular anomalies exist and can lead to severe complications. Treatment can be complex. OBJECTIVE: This overview aims to provide important information for the management of vascular anomalies. MATERIALS AND METHODS: In addition to current literature, experiences from the interdisciplinary Vascular Anomalies Center in Marburg were included in this review. RESULTS: Hemangiomas at critical sites, arteriovenous malformations, and vascular anomalies of uncertain etiology require particular attention. CONCLUSION: Self-help and support groups, specialized interdisciplinary centers, scientific medical societies, and networks can provide help for the treatment of vascular anomalies.

Optimized expression and purification of a soluble BMP2 variant based on in-silico design.

Bone morphogenetic protein 2 (BMP21) is a highly interesting therapeutic growth factor due to its strong osteogenic/osteoinductive potential. However, its pronounced aggregation tendency renders recombinant and soluble production troublesome and complex. While prokaryotic expression systems can provide BMP2 in large amounts, the typically insoluble protein requires complex denaturation-renaturation procedures with medically hazardous reagents to obtain natively folded homodimeric BMP2. Based on a detailed aggregation analysis of wildtype BMP2, we designed a hydrophilic variant of BMP2 additionally containing an improved heparin binding site (BMP2-2Hep-7M). Consecutive optimization of BMP2-2Hep-7M expression and purification enabled production of soluble dimeric BMP2-2Hep-7M in high yield in E. coli. This was achieved by a) increasing protein hydrophilicity via introducing seven point mutations within aggregation hot spots of wildtype BMP2 and a longer N-terminus resulting in higher affinity for heparin, b) by employing E. coli strain SHuffle® T7, which enables the structurally essential disulfide-bond formation in BMP2 in the cytoplasm, c) by using BMP2 variant characteristic soluble expression conditions and application of l-arginine as solubility enhancer. The BMP2 variant BMP2-2Hep-7M shows strongly attenuated although not completely eliminated aggregation tendency.

Impaired proteoglycan glycosylation, elevated TGF-ß signaling, and abnormal osteoblast differentiation as the basis for bone fragility in a mouse model for gerodermia osteodysplastica.

Gerodermia osteodysplastica (GO) is characterized by skin laxity and early-onset osteoporosis. GORAB, the responsible disease gene, encodes a small Golgi protein of poorly characterized function. To circumvent neonatal lethality of the GorabNull full knockout, Gorab was conditionally inactivated in mesenchymal progenitor cells (Prx1-cre), pre-osteoblasts (Runx2-cre), and late osteoblasts/osteocytes (Dmp1-cre), respectively. While in all three lines a reduction in trabecular bone density was evident, only GorabPrx1 and GorabRunx2 mutants showed dramatically thinned, porous cortical bone and spontaneous fractures. Collagen fibrils in the skin of GorabNull mutants and in bone of GorabPrx1 mutants were disorganized, which was also seen in a bone biopsy from a GO patient. Measurement of glycosaminoglycan contents revealed a reduction of dermatan sulfate levels in skin and cartilage from GorabNull mutants. In bone from GorabPrx1 mutants total glycosaminoglycan levels and the relative percentage of dermatan sulfate were both strongly diminished. Accordingly, the proteoglycans biglycan and decorin showed reduced glycanation. Also in cultured GORAB-deficient fibroblasts reduced decorin glycanation was evident. The Golgi compartment of these cells showed an accumulation of decorin, but reduced signals for dermatan sulfate. Moreover, we found elevated activation of TGF-ß in GorabPrx1 bone tissue leading to enhanced downstream signalling, which was reproduced in GORAB-deficient fibroblasts. Our data suggest that the loss of Gorab primarily perturbs pre-osteoblasts. GO may be regarded as a congenital disorder of glycosylation affecting proteoglycan synthesis due to delayed transport and impaired posttranslational modification in the Golgi compartment.

Cofilin-1 phosphorylation catalyzed by ERK1/2 alters cardiac actin dynamics in dilated cardiomyopathy caused by lamin A/C gene mutation.

Hyper-activation of extracellular signal-regulated kinase (ERK) 1/2 contributes to heart dysfunction in cardiomyopathy caused by mutations in the lamin A/C gene (LMNA cardiomyopathy). The mechanism of how this affects cardiac function is unknown. We show that active phosphorylated ERK1/2 directly binds to and catalyzes the phosphorylation of the actin depolymerizing factor cofilin-1 on Thr25. Cofilin-1 becomes active and disassembles actin filaments in a large array of cellular and animal models of LMNA cardiomyopathy. In vivo expression of cofilin-1, phosphorylated on Thr25 by endogenous ERK1/2 signaling, leads to alterations in left ventricular function and cardiac actin. These results demonstrate a novel role for cofilin-1 on actin dynamics in cardiac muscle and provide a rationale on how increased ERK1/2 signaling leads to LMNA cardiomyopathy.

BMPR2 inhibits activin and BMP signaling via wild-type ALK2.

TGF-ß/BMP superfamily ligands require heteromeric complexes of type 1 and 2 receptors for ligand-dependent downstream signaling. Activin A, a TGF-ß superfamily member, inhibits growth of multiple myeloma cells, but the mechanism for this is unknown. We therefore aimed to clarify how activins affect myeloma cell survival. Activin A activates the transcription factors SMAD2/3 through the ALK4 type 1 receptor, but may also activate SMAD1/5/8 through mutated variants of the type 1 receptor ALK2 (also known as ACVR1). We demonstrate that activin A and B activate SMAD1/5/8 in myeloma cells through endogenous wild-type ALK2. Knockdown of the type 2 receptor BMPR2 strongly potentiated activin A- and activin B-induced activation of SMAD1/5/8 and subsequent cell death. Furthermore, activity of BMP6, BMP7 or BMP9, which may also signal via ALK2, was potentiated by knockdown of BMPR2. Similar results were seen in HepG2 liver carcinoma cells. We propose that BMPR2 inhibits ALK2-mediated signaling by preventing ALK2 from oligomerizing with the type 2 receptors ACVR2A and ACVR2B, which are necessary for activation of ALK2 by activins and several BMPs. In conclusion, BMPR2 could be explored as a possible target for therapy in patients with multiple myeloma.This article has an associated First Person interview with the first author of the paper.

Lessons from LIMK1 enzymology and their impact on inhibitor design.

LIM domain kinase 1 (LIMK1) is a key regulator of actin dynamics. It is thereby a potential therapeutic target for the prevention of fragile X syndrome and amyotrophic lateral sclerosis. Herein, we use X-ray crystallography and activity assays to describe how LIMK1 accomplishes substrate specificity, to suggest a unique 'rock-and-poke' mechanism of catalysis and to explore the regulation of the kinase by activation loop phosphorylation. Based on these findings, a differential scanning fluorimetry assay and a RapidFire mass spectrometry activity assay were established, leading to the discovery and confirmation of a set of small-molecule LIMK1 inhibitors. Interestingly, several of the inhibitors were inactive towards the closely related isoform LIMK2. Finally, crystal structures of the LIMK1 kinase domain in complex with inhibitors (PF-477736 and staurosporine, respectively) are presented, providing insights into LIMK1 plasticity upon inhibitor binding.

Cell-specific responses to the cytokine TGFß are determined by variability in protein levels.

The cytokine TGFß provides important information during embryonic development, adult tissue homeostasis, and regeneration. Alterations in the cellular response to TGFß are involved in severe human diseases. To understand how cells encode the extracellular input and transmit its information to elicit appropriate responses, we acquired quantitative time-resolved measurements of pathway activation at the single-cell level. We established dynamic time warping to quantitatively compare signaling dynamics of thousands of individual cells and described heterogeneous single-cell responses by mathematical modeling. Our combined experimental and theoretical study revealed that the response to a given dose of TGFß is determined cell specifically by the levels of defined signaling proteins. This heterogeneity in signaling protein expression leads to decomposition of cells into classes with qualitatively distinct signaling dynamics and phenotypic outcome. Negative feedback regulators promote heterogeneous signaling, as a SMAD7 knock-out specifically affected the signal duration in a subpopulation of cells. Taken together, we propose a quantitative framework that allows predicting and testing sources of cellular signaling heterogeneity.

VE-cadherin facilitates BMP-induced endothelial cell permeability and signaling.

Several vascular disorders, such as aberrant angiogenesis, atherosclerosis and pulmonary hypertension, have been linked to dysfunctional BMP signaling. Vascular hyperpermeability via distortion of endothelial cell adherens junctions is a common feature of these diseases, but the role of BMPs in this process has not been investigated. BMP signaling is initiated by binding of ligand to, and activation of, BMP type I (BMPRI) and type II (BMPRII) receptors. Internalization of VE-cadherin as well as c-Src kinase-dependent phosphorylation have been implicated in the loosening of cell-cell contacts, thereby modulating vascular permeability. Here we demonstrate that BMP6 induces hyperpermeabilization of human endothelial cells by inducing internalization and c-Src-dependent phosphorylation of VE-cadherin. Furthermore, we show BMP-dependent physical interaction of VE-cadherin with the BMP receptor ALK2 (BMPRI) and BMPRII, resulting in stabilization of the BMP receptor complex and, thereby, the support of BMP6-Smad signaling. Our results provide first insights into the molecular mechanism of BMP-induced vascular permeability, a hallmark of various vascular diseases, and provide the basis for further investigations of BMPs as regulators of vascular integrity, both under physiological and pathophysiological conditions.

Role of bone morphogenetic proteins in sprouting angiogenesis: differential BMP receptor-dependent signaling pathways balance stalk vs. tip cell competence.

Before the onset of sprouting angiogenesis, the endothelium is prepatterned for the positioning of tip and stalk cells. Both cell identities are not static, as endothelial cells (ECs) constantly compete for the tip cell position in a dynamic fashion. Here, we show that both bone morphogenetic protein 2 (BMP2) and BMP6 are proangiogenic in vitro and ex vivo and that the BMP type I receptors, activin receptor-like kinase 3 (ALK3) and ALK2, play crucial and distinct roles in this process. BMP2 activates the expression of tip cell-associated genes, such as delta-like ligand 4 (DLL4) and kinase insert domain receptor (KDR), and p38-heat shock protein 27 (HSP27)-dependent cell migration, thereby generating tip cell competence. Whereas BMP6 also triggers collective cell migration via the p38-HSP27 signaling axis, BMP6 induces in addition SMAD1/5 signaling, thereby promoting the expression of stalk cell-associated genes, such as hairy and enhancer of split 1 (HES1) and fms-like tyrosine kinase 1 (FLT1). Specifically, ALK3 is required for sprouting from HUVEC spheroids, whereas ALK2 represses sprout formation. We demonstrate that expression levels and respective complex formation of BMP type I receptors in ECs determine stalk vs. tip cell identity, thus contributing to endothelial plasticity during sprouting angiogenesis. As antiangiogenic monotherapies that target the VEGF or ALK1 pathways have not fulfilled efficacy objectives in clinical trials, the selective targeting of the ALK2/3 pathways may be an attractive new approach.-Benn, A., Hiepen, C., Osterland, M., Schütte, C., Zwijsen, A., Knaus, P. Role of bone morphogenetic proteins in sprouting angiogenesis: differential BMP receptor-dependent signaling pathways balance stalk vs. tip cell competence.

Small molecules dorsomorphin and LDN-193189 inhibit myostatin/GDF8 signaling and promote functional myoblast differentiation.

GDF8, or myostatin, is a member of the TGF-ß superfamily of secreted polypeptide growth factors. GDF8 is a potent negative regulator of myogenesis both in vivo and in vitro. We found that GDF8 signaling was inhibited by the small molecule ATP competitive inhibitors dorsomorphin and LDN-193189. These compounds were previously shown to be potent inhibitors of BMP signaling by binding to the BMP type I receptors ALK1/2/3/6. We present the crystal structure of the type II receptor ActRIIA with dorsomorphin and demonstrate that dorsomorphin or LDN-193189 target GDF8 induced Smad2/3 signaling and repression of myogenic transcription factors. As a result, both inhibitors rescued myogenesis in myoblasts treated with GDF8. As revealed by quantitative live cell microscopy, treatment with dorsomorphin or LDN-193189 promoted the contractile activity of myotubular networks in vitro. We therefore suggest these inhibitors as suitable tools to promote functional myogenesis.

Nanoscale control of surface immobilized BMP-2: toward a quantitative assessment of BMP-mediated signaling events.

In this work we determine the impact of surface density of immobilized BMP-2 on intracellular signal transduction. We use block copolymer micellar nanolithography to fabricate substrates with precisely spaced and tunable gold nanoparticle arrays carrying single BMP-2 molecules. We found that the immobilized growth factor triggers prolonged and elevated Smad signaling pathway activation compared to the same amount of soluble protein. This approach is suitable for achieving controlled and sustained local delivery of BMP-2 and other growth factors.

Antagonism of GxxPG fragments ameliorates manifestations of aortic disease in Marfan syndrome mice.

Marfan syndrome (MFS) is an inherited disorder of connective tissue caused by mutations in the gene for fibrillin-1 (FBN1). The complex pathogenesis of MFS involves changes in transforming growth factor beta (TGF-ß) signaling and increased matrix metalloproteinase (MMP) expression. Fibrillin-1 and elastin have repeated Gly-x-x- Pro-Gly (GxxPG) motifs that can induce a number of effects including macrophage chemotaxis and increased MMP activity by induction of signaling through the elastin-binding protein (EBP). In this work, we test the hypothesis that antagonism of GxxPG fragments can suppress disease progression in the Marfan aorta. Fibrillin-1 underexpressing mgR/mgR Marfan mice were treated with weekly intraperitoneal (i.p.) injections of an antibody directed against GxxPG fragments. The treatment was started at 3 weeks of age and continued for 8 weeks. The treatment significantly reduced MMP-2, MMP-9 and pSmad2 activity, as well as fragmentation and macrophage infiltration in the aorta of the mgR/mgR mice. Additionally, airspace enlargement and increased pSmad2 activity in the lungs of mgR/mgR animals were prevented by the treatment. Our findings demonstrate the important role of secondary cellular events caused by GxxPG-containing fragments and matrix-induced inflammatory activity in the pathogenesis of thoracic aortic aneurysm (TAA) in mgR/mgR mice. Moreover, the results of the current study suggest that antagonism of the effects of GxxPG fragments may be a fruitful therapeutic strategy in MFS.

BMP2-induced chemotaxis requires PI3K p55γ/p110α-dependent phosphatidylinositol (3,4,5)-triphosphate production and LL5ß recruitment at the cytocortex.

BACKGROUND: BMP-induced chemotaxis of mesenchymal progenitors is fundamental for vertebrate development, disease and tissue repair. BMP2 induces Smad and non-Smad signalling. Whereas signal transduction via Smads lead to transcriptional responses, non-Smad signalling induces both, transcriptional and immediate/early non-transcriptional responses. However, the molecular mechanisms by which BMP2 facilitates planar cell polarity, cortical actin rearrangements, lamellipodia formation and chemotaxis of mesenchymal progenitors are poorly understood. Our aim was to uncover the molecular mechanism by which BMP2 facilitates chemotaxis via the BMP2-dependent activation of PI3K and spatiotemporal control of PIP3 production important for actin rearrangements at the mesenchymal cell cytocortex. RESULTS: We unveiled the molecular mechanism by which BMP2 induces non-Smad signalling by PI3K and the role of the second messenger PIP3 in BMP2-induced planar cell polarity, cortical actin reorganisation and lamellipodia formation. By using protein interaction studies, we identified the class Ia PI3K regulatory subunit p55γ to act as a specific and non-redundant binding partner for BMP receptor type II (BMPRII) in concert with the catalytic subunit p110α. We mapped the PI3K interaction to a region within the BMPRII kinase. Either BMP2 stimulation or increasing amounts of BMPRI facilitated p55γ association with BMPRII, but BMPRII kinase activity was not required for the interaction. We visualised BMP2-dependent PIP3 production via PI3K p55γ/p110α and were able to localise PIP3 to the leading edge of intact cells during the process of BMP2-induced planar cell polarity and actin dependent lamellipodia formation. Using mass spectrometry, we found the highly PIP3-sensitive PH-domain protein LL5ß to act as a novel BMP2 effector in orchestrating cortical actin rearrangements. By use of live cell imaging we found that knock-down of p55γ or LL5ß or pharmacological inhibition of PI3K impaired BMP2-induced migratory responses. CONCLUSIONS: Our results provide evidence for an important contribution of the BMP2-PI3K (p55γ/p110α)- PIP3-LL5ß signalling axis in mesenchymal progenitor cell chemotaxis. We demonstrate molecular insights into BMP2-induced PI3K signalling on the level of actin reorganisation at the leading edge cytocortex. These findings are important to better understand BMP2-induced cytoskeletal reorganisation and chemotaxis of mesenchymal progenitors in different physiological or pathophysiological contexts.

A two-way street - cellular metabolism and myofibroblast contraction.

Tissue fibrosis is characterised by the high-energy consumption associated with myofibroblast contraction. Although myofibroblast contraction relies on ATP production, the role of cellular metabolism in myofibroblast contraction has not yet been elucidated. Studies have so far only focused on myofibroblast contraction regulators, such as integrin receptors, TGF-ß and their shared transcription factor YAP/TAZ, in a fibroblast-myofibroblast transition setting. Additionally, the influence of the regulators on metabolism and vice versa have been described in this context. However, this has so far not yet been connected to myofibroblast contraction. This review focuses on the known and unknown of how cellular metabolism influences the processes leading to myofibroblast contraction and vice versa. We elucidate the signalling cascades responsible for myofibroblast contraction by looking at FMT regulators, mechanical cues, biochemical signalling, ECM properties and how they can influence and be influenced by cellular metabolism. By reviewing the existing knowledge on the link between cellular metabolism and the regulation of myofibroblast contraction, we aim to pinpoint gaps of knowledge and eventually help identify potential research targets to identify strategies that would allow switching tissue fibrosis towards tissue regeneration.

Protocol for chromatin accessibility profiling of human endothelial cells cultured under fluid shear stress using ATAC-seq.

Chromatin accessibility influences gene regulation and can be quantified using assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). Recapitulating in vivo fluid shear stress (FSS) mechano-regimes in vitro allows the study of atheroprone and atheroprotective mechanisms. In this protocol, we show how to culture and harvest endothelial cells from microfluidic channels for the preparation of ATAC-seq, highlighting optional growth factor stimulation and different FSS rates. This extends the application of ATAC-seq to the analysis of in vitro mechanically stimulated cells. For complete details on the use and execution of this protocol, please refer to Jatzlau et al.1.