BMP Activity Research Articles

Regulation of WNT5A and WNT11 during MSC in vitro chondrogenesis: WNT inhibition lowers BMP and hedgehog activity, and reduces hypertrophy.

Re-directing mesenchymal stromal cell (MSC) chondrogenesis towards a non-hypertrophic articular chondrocyte-(AC)-like phenotype is important for improving articular cartilage neogenesis to enhance clinical cartilage repair strategies. This study is the first to demonstrate that high levels of non-canonical WNT5A followed by WNT11 and LEF1 discriminated MSC chondrogenesis from AC re-differentiation. Moreover, β-catenin seemed incompletely silenced in differentiating MSCs, which altogether suggested a role for WNT signaling in hypertrophic MSC differentiation. WNT inhibition with the small molecule IWP-2 supported MSC chondrogenesis according to elevated proteoglycan deposition and reduced the characteristic upregulation of BMP4, BMP7 and their target ID1, as well as IHH and its target GLI1 observed during endochondral differentiation. Along with the pro-hypertrophic transcription factor MEF2C, multiple hypertrophic downstream targets including IBSP and alkaline phosphatase activity were reduced by IWP-2, demonstrating that WNT activity drives BMP and hedgehog upregulation, and MSC hypertrophy. WNT inhibition almost matched the strong anti-hypertrophic capacity of pulsed parathyroid hormone-related protein application, and both outperformed suppression of BMP signaling with dorsomorphin, which also reduced cartilage matrix deposition. Yet, hypertrophic marker expression under IWP-2 remained above AC level, and in vivo mineralization and ectopic bone formation were reduced but not eliminated. Overall, the strong anti-hypertrophic effects of IWP-2 involved inhibition but not silencing of pro-hypertrophic BMP and IHH pathways, and more advanced silencing of WNT activity as well as combined application of IHH or BMP antagonists should next be considered to install articular cartilage neogenesis from human MSCs.

New roles for Wnt and BMP signaling in neural anteroposterior patterning.

During amphibian development, neural patterning occurs via a two-step process. Spemann's organizer secretes BMP antagonists that induce anterior neural tissue. A subsequent caudalizing step re-specifies anterior fated cells to posterior fates such as hindbrain and spinal cord. The neural patterning paradigm suggests that a canonical Wnt-signaling gradient acts along the anteroposterior axis to pattern the nervous system. Wnt activity is highest in the posterior, inducing spinal cord, at intermediate levels in the trunk, inducing hindbrain, and is lowest in anterior fated forebrain, while BMP-antagonist levels are constant along the axis. Our results in Xenopus laevis challenge this paradigm. We find that inhibition of canonical Wnt signaling or its downstream transcription factors eliminates hindbrain, but not spinal cord fates, an observation not compatible with a simple high-to-low Wnt gradient specifying all fates along the neural anteroposterior axis. Additionally, we find that BMP activity promotes posterior spinal cord cell fate formation in an FGF-dependent manner, while inhibiting hindbrain fates. These results suggest a need to re-evaluate the paradigms of neural anteroposterior pattern formation during vertebrate development.

Orexin A modulates prolactin production by regulating BMP-4 activity in rat pituitary lactotorope cells

The impact of orexins on anterior pituitary function has yet to be clarified. We studied the effects of orexin A and its interaction with the bone morphogenetic protein (BMP) system on the regulatory role of prolactin synthesis using rat lactotrope GH3 cells expressing BMP-4. Orexin type 1 receptor (OX1R), but not type 2 receptor (OX2R), was predominantly expressed in GH3 cells. Orexin A suppressed forskolin-induced, but not basal, prolactin mRNA expression without reducing cAMP levels. Of note, orexin A suppressed BMP-4-induced prolactin mRNA and cAMP synthesis. Impairment of the effects of orexin by chemical inhibitors suggested involvement of the P38 pathway in the OX1R activity that suppresses BMP-4-induced PRL expression. Given that inhibition of BMP-receptor signaling reduced prolactin mRNA levels, endogenous BMP action is likely to be linked to the activation of prolactin synthesis by GH3 cells. Orexin A was revealed to suppress Smad1/5/9 phosphorylation and Id-1 transcription induced by BMP-4, which was restored in the presence of orexin-receptor antagonists, suggesting that the inhibitory effect of orexin A occurred via OX1R. Orexin A also reduced ALK-3 expression but increased inhibitory Smad6/7 expression, while BMP-4 treatment downregulated OX1R expression. These results indicated that orexin A plays an inhibitory role in prolactin production through suppression of endogenous BMP activity in GH3 cells, suggesting that a new functional role of the interaction between orexin and BMP-4 is modulation of prolactin levels in lactotrope cells.

Chondroitin Sulfate Glycosaminoglycan Scaffolds for Cell and Recombinant Protein-Based Bone Regeneration.

Bone morphogenetic protein 2 (BMP‐2)‐loaded collagen sponges remain the clinical standard for treatment of large bone defects when there is insufficient autograft, despite associated complications. Recent efforts to negate comorbidities have included biomaterials and gene therapy approaches to extend the duration of BMP‐2 release and activity. In this study, we compared the collagen sponge clinical standard to chondroitin sulfate glycosaminoglycan (CS‐GAG) scaffolds as a delivery vehicle for recombinant human BMP‐2 (rhBMP‐2) and rhBMP‐2 expression via human BMP‐2 gene inserted into mesenchymal stem cells (BMP‐2 MSC). We demonstrated extended release of rhBMP‐2 from CS‐GAG scaffolds compared to their collagen sponge counterparts, and further extended release from CS‐GAG gels seeded with BMP‐2 MSC. When used to treat a challenging critically sized femoral defect model in rats, both rhBMP‐2 and BMP‐2 MSC in CS‐GAG induced comparable bone formation to the rhBMP‐2 in collagen sponge, as measured by bone volume, strength, and stiffness. We conclude that CS‐GAG scaffolds are a promising delivery vehicle for controlling the release of rhBMP‐2 and to mediate the repair of critically sized segmental bone defects. stem cells translational medicine 2019;8:575–585

Structural determinants and genetic modifications enhance BMP2 stability and extracellular secretion.

The short half‐life and use of recombinant bone morphogenetic protein (BMP)2 in large doses poses major limitations in the clinic. Events regulating posttranslational processing and degradation of BMP2 in situ, linked to its secretion, have not been understood. Toward identifying mechanisms regulating intracellular BMP2 stability, we first discovered that inhibiting proteasomal degradation enhances both intracellular BMP2 level and its extracellular secretion. Next, we identified BMP2 degradation occurs through an ubiquitin‐mediated mechanism. Since ubiquitination precedes proteasomal turnover and mainly occurs on lysine residues of nascent proteins, we systematically mutated individual lysine residues within BMP2 and tested them for enhanced stability. Results revealed that substitutions on four lysine residues within the pro‐BMP2 region and three in the mature region increased both BMP2 stability and extracellular secretion. Structural modeling revealed key lysine residues involved in proteasomal degradation occupy a lysine cluster near proprotein convertase cleavage site. Interestingly, mutations within these residues did not affect biological activity of BMP2. These data suggest that preventing intracellular proteasomal loss of BMP2 through genetic modifications can overcome limitations related to its short half‐life.

BMPs, TGFβ, and border security at the interzone.

Synovial joints enable movement and protect the integrity of the articular cartilage. Joints form within skeletal condensations destined to undergo chondrogenesis. The suppression of this chondrogenic program in the interzone is the first morphological sign of joint formation. While we have a fairly good understanding of the essential roles of BMP and TGFβ family members in promoting chondrogenic differentiation in developing skeletal elements, we know very little about how BMP activity is suppressed specifically within the interzone, a crucial step in joint development. The function of the BMP ligand Gdf5 has been especially difficult to decipher. On the one hand, Gdf5 is required to promote chondrogenesis of articular elements. On the other hand, Gdf5 is highly expressed in the joint interzone where chondrogenesis must be suppressed for the formation of many joints. Here we review the evidence that BMP signaling must be suppressed within the joint interzone for joint morphogenesis to progress, and consider how Gdf5 exerts its divergent effects on chondrogenesis and joint formation. We also consider how TGFβ signaling impacts formation of the interzone. Finally, we propose a model whereby Gdf5 exerts distinct effects in the interzone vs. surrounding cartilage based on the repertoire of BMP receptors available in these tissues. Understanding how BMP antagonists and counteracting TGFβ signals intersect with Gdf5 to sculpt the joint interzone is essential for understanding the origin of osteoarthritis and other diseases of joint tissues.

SCUBE1-enhanced bone morphogenetic protein signaling protects against renal ischemia-reperfusion injury

We previously reported that the membrane-bound SCUBE1 (signal peptide-CUB-epithelial growth factor domain-containing protein 1) forms a complex with bone morphogenetic protein 2 (BMP2) ligand and its receptors, thus acting as a BMP co-receptor to augment BMP signal activity. However, whether SCUBE1 can bind to and facilitate signaling activity of BMP7, a renal protective molecule for ischemia-reperfusion (I/R) insult, and contribute to the proliferation and repair of renal tubular cells after I/R remains largely unknown. In this study, we first showed that I/R-induced SCUBE1 was expressed in proximal tubular cells, which coincided with the expression of renoprotective BMP7. Molecular and biochemical analyses revealed that SCUBE1 directly binds to BMP7 and its receptors, functioning as a BMP co-receptor to promote BMP7 signaling. Furthermore, we used a new Scube1 deletion (Δ2) mouse strain to further elucidate the renal pathophysiological function of SCUBE1 after I/R injury. As compared with wild-type littermates, Δ2 mice showed severe renal histopathologic features (extensive loss of brush border, tubular necrosis, and tubular dilation) and increased inflammation (neutrophil infiltrate and induction of monocyte chemoattractant protein-1, tumor necrosis factor-α and interleukin-6) during the resolution of I/R damage. They also showed reduced BMP signaling (phosphorylated Smad1/5/8) along with decreased proliferation and increased apoptosis of renal tubular cells. Importantly, lentivirus-mediated overexpression of SCUBE1 enhanced BMP signaling and conferred a concomitant survival outcome for Δ2 proximal tubular epithelial cells after hypoxia–reoxygenation treatment. The protective BMP7 signaling may be facilitated by stress-inducible SCUBE1 after renal I/R, which suggests potential targeted approaches for acute kidney injury.

Differentiation of Human Embryonic Stem Cells to Sympathetic Neurons: A Potential Model for Understanding Neuroblastoma Pathogenesis.

Background and Aims Previous studies modelling human neural crest differentiation from stem cells have resulted in a low yield of sympathetic neurons. Our aim was to optimise a method for the differentiation of human embryonic stem cells (hESCs) to sympathetic neuron-like cells (SN) to model normal human SNS development. Results Using stromal-derived inducing activity (SDIA) of PA6 cells plus BMP4 and B27 supplements, the H9 hESC line was differentiated to neural crest stem-like cells and SN-like cells. After 7 days of PA6 cell coculture, mRNA expression of SNAIL and SOX-9 neural crest specifier genes and the neural marker peripherin (PRPH) increased. Expression of the pluripotency marker OCT 4 decreased, whereas TP53 and LIN28B expression remained high at levels similar to SHSY5Y and IMR32 neuroblastoma cell lines. A 5-fold increase in the expression of the catecholaminergic marker tyrosine hydroxylase (TH) and the noradrenergic marker dopamine betahydroxylase (DBH) was observed by day 7 of differentiation. Fluorescence-activated cell sorting for the neural crest marker p75, enriched for cells expressing p75, DBH, TH, and PRPH, was more specific than p75 neural crest stem cell (NCSC) microbeads. On day 28 post p75 sorting, dual immunofluorescence identified sympathetic neurons by PRPH and TH copositivity cells in 20% of the cell population. Noradrenergic sympathetic neurons, identified by copositivity for both PHOX2B and DBH, were present in 9.4% ± 5.5% of cells. Conclusions We have optimised a method for noradrenergic SNS development using the H9 hESC line to improve our understanding of normal human SNS development and, in a future work, the pathogenesis of neuroblastoma.

BMP7 antagonizes proliferative vitreoretinopathy through retinal pigment epithelial fibrosis in vivo and in vitro.

The major pathogenesis of proliferative vitreoretinopathy (PVR) is that retinal pigment epithelial (RPE) cells undergo epithelial-mesenchymal transition (EMT) because of disordered growth factors, such as TGF-β, in the vitreous humor. Bone morphogenetic proteins (BMPs) are pluripotent growth factors. In this study, we identified the antifibrotic activity of BMP7 in a PVR model both in vivo and in vitro. BMP7 expression was confirmed on the PVR proliferative membranes. BMP7 was down-regulated in the PVR vitreous humor and TGF-β-induced RPE cell EMT. In the in vivo studies, BMP7 injection attenuated PVR progression in the eyes of the rabbit model. Additionally, BMP7 treatment maintained RPE cell phenotypes and relieved TGF-β2-induced EMT, migration, and gel contraction in vitro. BMP7 inhibited the TGF-β2-induced up-regulation of fibronectin and α-smooth muscle actin and the down-regulation of E-cadherin and zona occludens-1 by balancing the TGF-β2/Smad2/3 and BMP7/Smad1/5/9 pathways. These findings provide direct evidence of the ability of BMP7 in PVR inhibition and the potential of BMP7 for use in PVR therapeutic intervention.-Yao, H., Ge, T., Zhang, Y., Li, M., Yang, S., Li, H., Wang, F. BMP7 antagonizes proliferative vitreoretinopathy through retinal pigment epithelial fibrosis in vivo and in vitro.

Gremlin, Noggin, Chordin and follistatin differentially modulate BMP induced suppression of androgen secretion by bovine ovarian theca cells.

Bone morphogenetic proteins (BMP) are firmly implicated as intra-ovarian regulators of follicle function and steroidogenesis but information is lacking regarding the regulation of BMP signalling by extracellular binding proteins co-expressed in the ovary. In this study we compared the abilities of four BMP binding proteins (gremlin, noggin, chordin, follistatin) to antagonize the action of four different BMPs (BMP2 BMP4, BMP6, BMP7) on LH-induced androstenedione secretion by bovine theca cells in primary culture. Expression of the four BMP binding proteins and BMPs investigated here has previously been documented in bovine follicles. All four BMPs suppressed androstenedione secretion by >85%. Co-treatment with gremlin antagonized BMP2- and, less potently, BMP4-induced suppression of androgen secretion but did not affect responses to BMP6 and BMP7. Noggin antagonized the effects of three BMPs (rank order: BMP4 > BMP2 > BMP7) but did not affect the response to BMP6. Follistatin partially reversed the suppressive effects of BMP6 on androgen secretion but did not affect BMP2, BMP4 and BMP7 action. Chordin had no effect on the response to any of the four BMPs. BMP6 treatment upregulated thecal expression of GREM1, NOG, CHRD and SMAD6 mRNA whilst inhibiting expression of the four BMPs. Taken together with previous work documenting the intra-ovarian expression of different BMPs, BMP binding proteins and signalling receptors, these observations reinforce the conclusion that extracellular binding proteins selectively modulate BMP-dependent alterations in thecal steroidogenesis. As such they likely constitute an important regulatory component of this, and other intra-ovarian actions of BMPs.

The polyphenol resveratrol promotes skeletal growth in mice through a sirtuin 1-bone morphogenic protein 2 longevity axis.

Background and PurposeThe polyphenol resveratrol (RSV) exists in high quantities in certain foods (e.g. grapes and nuts). However, the capacity of RSV to confer physiological health benefits and a biological mechanism through which this might occur remains unclear.Experimental ApproachAged, RSV‐treated (300 mg·kg−1·day−1) and genetically modified [endothelial NOS (eNOS−/−)] female mice were assessed using histomorphometric and μCT analysis. Alongside in vivo analysis, molecular siRNA knockdown and pharmacological manipulation of eNOS, BMP2 and sirtuin 1 (SIRT1) and functional cellular assays in an osteoblast cell line panel, explored the mechanism through which RSV might impact overall bone volume.Key ResultsRSV promoted osteoblast activity and bone growth in vivo. RSV dose‐dependently and simultaneously increased alkaline phosphatase (ALP) and eNOS levels. Similarly, NO‐donor treatment increased ALP, runt homology transcription factor 2, BMP2 and stimulated bone formation, whilst eNOS‐deficient mice displayed a bone loss phenotype. Moreover, RSV‐induced increase in ALP and BMP2 expression was blocked in eNOS−/− osteoblasts and by BMP‐inhibitor noggin. The longevity‐linked SIRT1 enzyme was positively regulated by RSV and SIRT1 deletion reduced eNOS, BMP2 and ALP. Like eNOS deletion, loss of SIRT1 blocked RSV‐induced osteoblast activity; however, SIRT1 levels remained unchanged in eNOS−/− mice, indicating RSV activation of SIRT1 stimulates BMP2 release via eNOS. This signalling axis is supported by decreased SIRT1, eNOS and BMP2 confirmed in old versus young bone.Conclusions and ImplicationsThese findings suggest a new mechanism of action in bone remodelling and the ageing skeleton, where RSV positively impacts bone homeostasis via SIRT1 activation of BMP2.

Minimum structural requirements for BMP-2-binding of heparin oligosaccharides

Bone morphogenetic proteins (BMPs) are essential during tissue repair and remodeling after injury. Glycosaminoglycan (GAG) sugars are known to enhance BMP activity in vitro and in vivo; here the interactions of BMP-2 with various glycosaminoglycan classes were compared and shown to be selective for heparin over other comparable saccharides. The minimal chain lengths and specific sulfate moieties required for heparin-derived oligosaccharide binding to BMP-2, and the ability of such oligosaccharides to promote BMP-2-induced osteogenic differentiation in vitro were then determined. BMP-2 could bind to heparin hexasaccharides (dp6) and octasaccharides (dp8), but decasaccharides (dp10) were the minimum chain length required for both efficient binding of BMP-2 and consequent heparin-dependent cell responses. N-sulfation is the most important, and 6-O-sulfation moderately important for BMP-2 binding and activity, whereas 2-O-sulfation was much less critical. Bone formation assays in vivo further confirmed that dp10, N-sulfated heparin oligosaccharides were the minimal requirement for effective enhancement of BMP-2-induced bone formation. Such information is necessary for the rational design of the next generations of heparan-based devices for bone tissue repair.

Notch, BMP and WNT/β-catenin network is impaired in endothelial cells of the patients with thoracic aortic aneurysm

Cellular and molecular mechanisms of thoracic aortic aneurysm are still not clear and therapeutic approaches are mostly absent. The role of endothelial cells in aortic wall integrity is emerging from recent studies. Although Notch pathway ensures endothelial development and integrity, and NOTCH1 mutations have been associated with thoracic aortic aneurysms, the role of this pathway in aneurysm remains elusive. The purpose of the present work was to study functions of Notch genes in endothelial cells of patients with sporadic thoracic aortic aneurysm.Aortic endothelial cells were isolated from aortic tissue of patients with thoracic aortic aneurysm and healthy donors. Gene expression of Notch and related BMP and WNT/β-catenin pathways was estimated by qPCR; WNT/β-catenin signaling was studied by TCF-luciferase reporter. To study the stress-response the cells were subjected to laminar shear stress and the expression of corresponding genes was estimated by qPCR.Analyses of mRNA expression of Notch genes, Notch target genes and Notch related pathways showed that endothelial cells of aneurysm patients have dysregulated Notch/BMP/WNT pathways compared to donor cells. Activity of Wnt pathway was significantly elevated in endothelial cells of the patients. Cells from patients had attenuated activation of DLL4, SNAIL1, DKK1 and BMP2 in response to shear stress.In conclusion endothelial cells of the patients with thoracic aortic aneurysm have dysregulated Notch, BMP and WNT/β-catenin related signaling. Shear stress-response and cross-talk between Notch and Wnt pathways that normally ensures aortic integrity and resistance of endothelial cells to stress is impaired in aneurysmal patients.

Bone marrow stromal cells from low-turnover osteoporotic mouse model are less sensitive to the osteogenic effects of fluvastatin.

This study aimed to investigate the effects of fluvastatin on the differentiation of bone marrow stromal cells (BMSCs) into osteoblasts in senescence-accelerated mouse prone 6 (SAMP6) compared with that in the normal senescence-accelerated-resistant mouse (SAMR1) model. SAMP strains arose spontaneously from the AKR/J background and display shortened life span and an array of signs of accelerated aging, compared with control SAMR strains. The dose effects of fluvastatin were also evaluated. BMSCs were cultured with/without fluvastatin (0 μM, 0.1 μM, 0.5 μM, and 1.0 μM). WST-1-based colorimetry was performed to evaluate cell proliferation. To evaluate cell differentiation, gene expression levels of bmp2 and runx2 were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR), and protein expression levels were determined using enzyme-linked immunosorbent assay (BMP2) and immunofluorescence staining (BMP2 and Runx2). Alkaline phosphatase (ALP) activity assay and histochemical detection were determined; the effect of noggin, a BMP-specific antagonist, was examined using ALP histochemical detection. To assess for mature osteogenic marker, gene expression levels of bglap2 were determined by qRT-PCR and mineralization was determined by alizarin red staining. RhoA activity was also examined by Western blotting. In SAMP6, BMP2, Runx2 and Bglap2 mRNA and protein expressions were significantly increased by fluvastatin, and ALP activity was increased by BMP2 action. RhoA activity was also inhibited by fluvastatin. The concentration of fluvastatin sufficient to increase BMP2 and Runx2 expression and ALP activity was 0.5 μM in SAMP6 and 0.1 μM in SAMR1. In conclusion, the present study revealed that fluvastatin promoted BMSC differentiation into osteoblasts by RhoA-BMP2 pathway in SAMP6. BMSCs of SAMP6 are less sensitive to the osteogenic effects of fluvastatin than SAMR1.

In Vitro Osteogenic Differentiation and Antibacterial Potentials of Chalcone Derivatives.

Chalcone derivatives have been investigated as therapeutic agents for the anticancer, antioxidant, and anti-inflammatory fields. In this study, we have synthesized four different types of chalcone derivatives and demonstrated in vitro bioactivities. We divided these derivatives into two groups of chalcones on the basis of similar substituents on the aromatic rings, and we tested cell viability and proliferation potentials, which indicated that the methoxy substituent on the A ring could enhance cytotoxicity and antiproliferation potential depending on the chalcone concentration. We also investigated osteogenic differentiation of C2C12 cells by ALP staining, the early marker for osteogenesis, which demonstrated that the chalcones could not only induce activity of BMP-2 but also inhibit the activity of noggin, a BMP antagonist. In addition, chalcone bearing hydroxyl groups at the 2-, 4-, and 6-position on the A ring inhibited treptococcus mutans growth, a major causative agent of dental caries. Therefore, we concluded that the chalcone derivatives synthesized in this research can be good candidates for therapeutic agents promoting bone differentiation, with an expectation of inhibiting S. mutans, in dentistry.

A heterodimer formed by bone morphogenetic protein 9 (BMP9) and BMP10 provides most BMP biological activity in plasma

Bone morphogenetic protein 9 (BMP9) and BMP10 are the two high-affinity ligands for the endothelial receptor activin receptor-like kinase 1 (ALK1) and are key regulators of vascular remodeling. They are both present in the blood, but their respective biological activities are still a matter of debate. The aim of the present work was to characterize their circulating forms to better understand how their activities are regulated in vivo First, by cotransfecting BMP9 and BMP10, we found that both can form a disulfide-bonded heterodimer in vitro and that this heterodimer is functional on endothelial cells via ALK1. Next, we developed an ELISA that could specifically recognize the BMP9-BMP10 heterodimer and which indicated its presence in both human and mouse plasma. In addition to using available Bmp9-KO mice, we generated a conditional Bmp10-KO mouse strain. The plasma from Bmp10-KO mice, similarly to that of Bmp9-KO mice, completely lacked the ability to activate ALK1-transfected 3T3 cells or phospho-Smad1-5 on endothelial cells, indicating that the circulating BMP activity is mostly due to the BMP9-BMP10 heterodimeric form. This result was confirmed in human plasma that had undergone affinity chromatography to remove BMP9 homodimer. Finally, we provide evidence that hepatic stellate cells in the liver could be the source of the BMP9-BMP10 heterodimer. Together, our findings demonstrate that BMP9 and BMP10 can heterodimerize and that this heterodimer is responsible for most of the biological BMP activity found in plasma.

Differential BMP Expression and Activities Impact the Osteogenic Capacities of Equine Bone Marrow and Adipose Stem Cells

Differential BMP Expression and Activities Impact the Osteogenic Capacities of Equine Bone Marrow and Adipose Stem Cells

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

TGF-β/BMP superfamily ligands require heteromeric complexes of type 1 and 2 receptors for ligand-dependent downstream signaling. Activin A, a TGF-β 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.

Aberrant activity of NKL homeobox gene NKX3-2 in a T-ALL subset.

T-cell acute lymphoblastic leukemia (T-ALL) is a hematopoietic malignancy originating from T-cell progenitors in which differentiation is blocked at early stages. Physiological expression of specific NKL homeobox genes obeys a hematopoietic NKL-code implicated in the process of lymphopoiesis while in differentiated T-cells these genes are silenced. We propose that this developmental expression pattern underlies the observation that NKL homeobox genes are the most ubiquitous group of transcription factors deregulated in T-ALL, including TLX1, TLX3, NKX2-5 and NKX3-1. Here, we describe a novel member of the NKL homeobox gene subclass, NKX3-2 (BAPX1), which is aberrantly activated in 18% of pediatric T-ALL patients analyzed while being normally expressed in developing spleen. Identification of NKX3-2 expression in T-ALL cell line CCRF-CEM qualified these cells to model its deregulation and function in a leukemic context. Genomic and chromosomal analyses demonstrated normal configuration of the NKX3-2 locus at chromosome 4p15, thus excluding cytogenetic dysregulation. Comparative expression profiling analysis of NKX3-2 patient data revealed deregulated activity of BMP- and MAPK-signalling. These candidate pathways were experimentally confirmed to mediate aberrant NKX3-2 expression. We also show that homeobox gene SIX6, plus MIR17HG and GATA3 are downstream targets of NKX3-2 and plausibly contribute to the pathogenesis of this malignancy by suppressing T-cell differentiation. Finally, NKL homeobox gene NKX2-5 was activated by NKX3-2 in CCRF-CEM and by FOXG1 in PEER, representing mutually inhibitory activators of this translocated oncogene. Together, our findings reveal a novel oncogenic NKL homeobox gene subclass member which is aberrantly expressed in a large subset of T-ALL patients and participates in a deregulated gene network likely to arise in developing spleen.

Design of hydrogels to stabilize and enhance bone morphogenetic protein activity by heparin mimetics

Although bone morphogenetic protein-2 (BMP-2) is known to be the most potent stimulator available for bone formation, a major barrier to widespread clinical use is its inherent instability and absence of an adequate delivery system. Heparin is being widely used in controlled release systems due to its strong binding ability and protective effect for many growth factor proteins. In this work, we developed a hydrogel surface that can mimic heparin to stabilize BMP-2 and to enhance osteogenesis by introducing heparin-mimicking sulfonated molecules such as poly-vinylsulfonic acid (PVSA) or poly-4-styrenesulfonic acid (PSS), into photo-crosslinkable hydrogel. Bioactivity of BMP-2 was well preserved in the presence of polysulfonates during exposure to various therapeutically relevant stressors. The heparin-mimicking sulfonated hydrogels were effective to bind BMP-2 compared to unmodified MeGC hydrogel and significantly enhanced osteogenic differentiation of encapsulated bone marrow stromal cells (BMSCs) without the addition of exogenous BMP-2. The sulfonated hydrogels were effective in delivering exogenous BMP-2 with reduced initial burst and increased BMSCs osteogenesis induced by BMP-2. These findings suggest a novel hydrogel platform for sequestering and stabilizing BMP-2 to enhance osteoinductive activity in bone tissue engineering. Statement of SignificanceAlthough bone morphogenetic protein-2 (BMP-2) is believed to be the most potent cytokine for bone regeneration, its clinical applications require supraphysiological BMP dosage due to its intrinsic instability and fast enzymatic degradation, leading to worrisome side effects. This study demonstrates a novel hydrogel platform that mimics a natural protector of BMPs, heparin, to sequester and stabilize BMP-2 for increased osteoinductive signaling. This study will achieve the stabilization of BMPs with prolonged bioactivity by a synthetic heparin mimic that has not been examined previously. Moreover, the heparin mimetic hydrogel surface can augment endogenous BMP activity by sequestering and localizing the cell-produced BMPs. The additional knowledge gained from this study may suggest basis for future development of material-based therapeutics for tissue engineering.