Bone Morphogenetic Protein Activity Research Articles

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 Role of Bioceramics for Bone Regeneration: History, Mechanisms, and Future Perspectives.

Osteoporosis is a skeletal disorder marked by compromised bone integrity, predisposing individuals, particularly older adults and postmenopausal women, to fractures. The advent of bioceramics for bone regeneration has opened up auspicious pathways for addressing osteoporosis. Research indicates that bioceramics can help bones grow back by activating bone morphogenetic protein (BMP), mitogen-activated protein kinase (MAPK), and wingless/integrated (Wnt)/β-catenin pathways in the body when combined with stem cells, drugs, and other supports. Still, bioceramics have some problems, such as not being flexible enough and prone to breaking, as well as difficulties in growing stem cells and discovering suitable supports for different bone types. While there have been improvements in making bioceramics better for healing bones, it is important to keep looking for new ideas from different areas of medicine to make them even better. By conducting a thorough scrutiny of the pivotal role bioceramics play in facilitating bone regeneration, this review aspires to propel forward the rapidly burgeoning domain of scientific exploration. In the end, this appreciation will contribute to the development of novel bioceramics that enhance bone regrowth and offer patients with bone disorders alternative treatments.

Abstract 151: BMP signaling determines neuroblastoma cell fate and sensitivity to retinoic acid

Abstract While retinoic acid (RA) has been successfully used for leukemia treatment for decades, the attempt to treat solid tumors with RA remains challenging, with less than 10% of neuroblastoma (NB) patients achieving complete remission when treated with RA alone. It has been believed the anti-cancer activity of retinoids is due to retinoid-induced terminal differentiation, however, what determines cell response or whether differentiation is the main effect of RA has never been fully understood. To better understand RA’s activity, we conducted genome-wide CRISPR modifier screens in RA-treated hyper-sensitive NB cell lines. Surprisingly, we found that, in these cells, RA primarily decreased cell viability via apoptosis or senescence, rather than differentiation—activities in which the CRISPR screens strongly implicated bone morphogenetic protein (BMP) signaling. BMP activation promoted apoptosis/senescence and sensitized cells to RA. Conversely, BMP inhibitors and SMAD9 (a critical transcription factor of the BMP pathway) knockout enhanced RA’s ability to induce differentiation but reduced cell sensitivity to RA. Our ChIP-seq and RNA-seq data showed these behaviors were mediated by the coordinated gene regulatory actions of RARA and BMP-family SMAD transcription factors. Furthermore, in a panel of 19 cell lines we screened with RA, we assessed the correlations between RA IC50 and the expression of all (~20,000) genes (publicly available data from the GDSC and Depmap). Remarkably, SMAD9 was the number 1 and number 10 most correlated gene, respectively, with RA IC50 (ranked by Pearson correlation coefficient; GDSC R = -0.92, P = 4.2 × 10−6; Depmap R = -0.81, P = 4.8 × 10−4). We also performed comprehensive large-scale drug screens in these cell lines with combinations of RA and a BMP activator FK506. RA exhibited a synergistic effect with FK506 in all the NB cell lines, very strikingly in some cell lines. All these data suggest BMP signaling is generally required for RA sensitivity and BMP activators are promising candidates in combination with RA to treat NB. Using published bulk and single cell RNA-seq data from NB patient samples, we found BMP signaling activity is relatively low in primary tumors, but well maintained in disseminated NB cells derived from bone marrow. This explains why RA is clinically used as a maintenance therapy and can only successfully treat the minimal residual disease and suggests that tumor microenvironment is a critical factor in determining cell response to RA. Overall, our study revealed that BMP controls NB cell fate and sensitivity in RA treatment. Notably, interactions between BMP signaling and RA are well established in developmental biology, where BMP signaling can tip cell fate decisions between differentiation, apoptosis, and senescence upon exposure to endogenous RA. Our data suggest that this developmental process can be maintained in NB cells, unveiling a novel mode of anti-neoplastic action. Citation Format: Min Pan, Yinwen Zhang, William C. Wright, Hyeong-Min Lee, Richard H. Chapple, Xueying Liu, Jonathan Low, Duane Currier, Allister J. Loughran, Dyer A. Dyer, Shondra M. Pruett, Burgess Freeman, Taosheng Chen, Brian J. Abraham, Elizabeth Stewart, John Easton, Paul Geeleher. BMP signaling determines neuroblastoma cell fate and sensitivity to retinoic acid [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 151.

Impaired bone morphogenetic protein (BMP) signaling pathways disrupt decidualization in endometriosis

Endometriosis is linked to increased infertility and pregnancy complications due to defective endometrial decidualization. We hypothesized that identification of altered signaling pathways during decidualization could identify the underlying cause of infertility and pregnancy complications. Our study reveals that transforming growth factor β (TGFβ) pathways are impaired in the endometrium of individuals with endometriosis, leading to defective decidualization. Through detailed transcriptomic analyses, we discovered abnormalities in TGFβ signaling pathways and key regulators, such as SMAD4, in the endometrium of affected individuals. We also observed compromised activity of bone morphogenetic proteins (BMP), a subset of the TGFβ family, that control endometrial receptivity. Using 3-dimensional models of endometrial stromal and epithelial assembloids, we showed that exogenous BMP2 improved decidual marker expression in individuals with endometriosis. Our findings reveal dysfunction of BMP/SMAD signaling in the endometrium of individuals with endometriosis, explaining decidualization defects and subsequent pregnancy complications in these individuals.

Human archetypal pluripotent stem cells differentiate into trophoblast stem cells via endogenous BMP5/7 induction without transitioning through naive state

Primary human trophoblast stem cells (TSCs) and TSCs derived from human pluripotent stem cells (hPSCs) can potentially model placental processes in vitro. Yet, the pluripotent states and factors involved in the differentiation of hPSCs to TSCs remain poorly understood. In this study, we demonstrate that the primed pluripotent state can generate TSCs by activating pathways such as Epidermal Growth Factor (EGF) and Wingless-related integration site (WNT), and by suppressing tumor growth factor beta (TGFβ), histone deacetylases (HDAC), and Rho-associated protein kinase (ROCK) signaling pathways, all without the addition of exogenous Bone morphogenetic protein 4 (BMP4)—a condition we refer to as the TS condition. We characterized this process using temporal single-cell RNA sequencing to compare TS conditions with differentiation protocols involving BMP4 activation alone or BMP4 activation in conjunction with WNT inhibition. The TS condition consistently produced a stable, proliferative cell type that closely mimics first-trimester placental cytotrophoblasts, marked by the activation of endogenous retroviral genes and the absence of amnion expression. This was observed across multiple cell lines, including various primed induced pluripotent stem cell (iPSC) and embryonic stem cell (ESC) lines. Primed-derived TSCs can proliferate for over 30 passages and further specify into multinucleated syncytiotrophoblasts and extravillous trophoblast cells. Our research establishes that the differentiation of primed hPSCs to TSC under TS conditions triggers the induction of TMSB4X, BMP5/7, GATA3, and TFAP2A without progressing through a naive state. These findings propose that the primed hPSC state is part of a continuum of potency with the capacity to differentiate into TSCs through multiple routes.

The TGF-β Family in Glioblastoma.

Members of the transforming growth factor β (TGF-β) family have been implicated in the biology of several cancers. In this review, we focus on the role of TGFβ and bone morphogenetic protein (BMP) signaling in glioblastoma. Glioblastoma (GBM) is the most common malignant brain tumor in adults; it presents at a median age of 64 years, but can occur at any age, including childhood. Unfortunately, there is no cure, and even patients undergoing current treatments (surgical resection, radiotherapy, and chemotherapy) have a median survival of 15 months. There is a great need to identify new therapeutic targets to improve the treatment of GBM patients. TGF-βs signaling promotes tumorigenesis in glioblastoma, while BMPs suppress tumorigenic potential by inducing tumor cell differentiation. In this review, we discuss the actions of TGF-βs and BMPs on cancer cells as well as in the tumor microenvironment, and their use in potential therapeutic intervention.

An osteoinductive surface by adhesive bone morphogenetic protein-2 prepared using the bioorthogonal approach for tight binding of titanium with bone.

Inorganic biomaterials are used in various orthopedic and dental implants. Nevertheless, they cause clinical issues such as loosening of implants and patient morbidity. Therefore, inspired by mussel adhesive proteins, we aimed to design an adhesive and dimer-forming highly active bone morphogenetic protein-2 (BMP-2) using bioorthogonal chemistry, in which recombinant DNA technology was combined with enzymatic modifications, to achieve long-term osseointegration with titanium. The prepared BMP-2 exhibited substantially higher binding activity than wild-type BMP-2, while the adhered BMP-2 was more active than soluble BMP-2. Therefore, the adhesive BMP-2 was immobilized onto titanium wires and screws and implanted into rat bones, and long-term osteogenesis was evaluated. Adhesive BMP-2 promoted the mechanical binding of titanium to bones, enabling efficient bone regeneration and effective stabilization of implants. Thus, such adhesive biosignaling proteins can be used in regenerative medicine.

Spatiotemporal modulation of growth factors directs the generation of multilineage mouse embryonic stem cell-derived mammary organoids

Ectodermal appendages, such as the mammary gland (MG), are thought to have evolved from hair-associated apocrine glands to serve the function of milk secretion. Through the directed differentiation of mouse embryonic stem cells (mESCs), here, we report the generation of multilineage ESC-derived mammary organoids (MEMOs). We adapted the skin organoid model, inducing the dermal mesenchyme to transform into mammary-specific mesenchyme via the sequential activation of Bone Morphogenetic Protein 4 (BMP4) and Parathyroid Hormone-related Protein (PTHrP) and inhibition of hedgehog (HH) signaling. Using single-cell RNA sequencing, we identified gene expression profiles that demonstrate the presence of mammary-specific epithelial cells, fibroblasts, and adipocytes. MEMOs undergo ductal morphogenesis in Matrigel and can reconstitute the MG invivo. Further, we demonstrate that the loss of function in placode regulators LEF1 and TBX3 in mESCs results in impaired skin and MEMO generation. In summary, our MEMO model is a robust tool for studying the development of ectodermal appendages, and it provides a foundation for regenerative medicine and disease modeling.

Essential Role of BMP4 Signaling in the Avian Ceca in Colorectal Enteric Nervous System Development.

The enteric nervous system (ENS) is principally derived from vagal neural crest cells that migrate caudally along the entire length of the gastrointestinal tract, giving rise to neurons and glial cells in two ganglionated plexuses. Incomplete migration of enteric neural crest-derived cells (ENCDC) leads to Hirschsprung disease, a congenital disorder characterized by the absence of enteric ganglia along variable lengths of the colorectum. Our previous work strongly supported the essential role of the avian ceca, present at the junction of the midgut and hindgut, in hindgut ENS development, since ablation of the cecal buds led to incomplete ENCDC colonization of the hindgut. In situ hybridization shows bone morphogenetic protein-4 (BMP4) is highly expressed in the cecal mesenchyme, leading us to hypothesize that cecal BMP4 is required for hindgut ENS development. To test this, we modulated BMP4 activity using embryonic intestinal organ culture techniques and retroviral infection. We show that overexpression or inhibition of BMP4 in the ceca disrupts hindgut ENS development, with GDNF playing an important regulatory role. Our results suggest that these two important signaling pathways are required for normal ENCDC migration and enteric ganglion formation in the developing hindgut ENS.

Proteomics of CKD progression in the chronic renal insufficiency cohort

Progression of chronic kidney disease (CKD) portends myriad complications, including kidney failure. In this study, we analyze associations of 4638 plasma proteins among 3235 participants of the Chronic Renal Insufficiency Cohort Study with the primary outcome of 50% decline in estimated glomerular filtration rate or kidney failure over 10 years. We validate key findings in the Atherosclerosis Risk in the Communities study. We identify 100 circulating proteins that are associated with the primary outcome after multivariable adjustment, using a Bonferroni statistical threshold of significance. Individual protein associations and biological pathway analyses highlight the roles of bone morphogenetic proteins, ephrin signaling, and prothrombin activation. A 65-protein risk model for the primary outcome has excellent discrimination (C-statistic[95%CI] 0.862 [0.835, 0.889]), and 14/65 proteins are druggable targets. Potentially causal associations for five proteins, to our knowledge not previously reported, are supported by Mendelian randomization: EGFL9, LRP-11, MXRA7, IL-1 sRII and ILT-2. Modifiable protein risk markers can guide therapeutic drug development aimed at slowing CKD progression.

Moderate-Affinity Affibodies Modulate the Delivery and Bioactivity of Bone Morphogenetic Protein-2.

Uncontrolled bone morphogenetic protein-2 (BMP-2) release can lead to off-target bone growth and other adverse events. To tackle this challenge, yeast surface display is used to identify unique BMP-2-specific protein binders known as affibodies that bind to BMP-2 with different affinities. Biolayer interferometry reveals an equilibrium dissociation constant of 10.7 nm for the interaction between BMP-2 and high-affinity affibody and 34.8 nm for the interaction between BMP-2 and the low-affinity affibody. The low-affinity affibody-BMP-2 interaction also exhibits an off-rate constant that is an order of magnitude higher. Computational modeling of affibody-BMP-2 binding predicts that the high- and low-affinity affibodies bind to two distinct sites on BMP-2 that function as different cell-receptor binding sites. BMP-2 binding to affibodies reduces expression of the osteogenic marker alkaline phosphatase (ALP) in C2C12 myoblasts. Affibody-conjugated polyethylene glycol-maleimide hydrogels increase uptake of BMP-2 compared to affibody-free hydrogels, and high-affinity hydrogels exhibit lower BMP-2 release into serum compared to low-affinity hydrogels and affibody-free hydrogels over four weeks. Loading BMP-2 into affibody-conjugated hydrogels prolongs ALP activity of C2C12 myoblasts compared to soluble BMP-2. This work demonstrates that affibodies with different affinities can modulate BMP-2 delivery and activity, creating a promising approach for controlling BMP-2 delivery in clinical applications.

Bringing gene therapy to regenerative medicine

Bringing gene therapy to regenerative medicine

Inhibitory SMAD6 interferes with BMP-dependent generation of muscle progenitor cells and perturbs proximodistal pattern of murine limb muscles.

The mechanism of pattern formation during limb muscle development remains poorly understood. The canonical view holds that naïve limb muscle progenitor cells (MPCs) invade a pre-established pattern of muscle connective tissue, thereby forming individual muscles. Here, we show that early murine embryonic limb MPCs highly accumulate pSMAD1/5/9, demonstrating active signaling of bone morphogenetic proteins (BMP) in these cells. Overexpression of inhibitory human SMAD6 (huSMAD6) in limb MPCs abrogated BMP signaling, impaired their migration and proliferation, and accelerated myogenic lineage progression. Fewer primary myofibers developed, causing an aberrant proximodistal muscle pattern. Patterning was not disturbed when huSMAD6 was overexpressed in differentiated muscle, implying that the proximodistal muscle pattern depends on BMP-mediated expansion of MPCs before their differentiation. We show that limb MPCs differentially express Hox genes, and Hox-expressing MPCs displayed active BMP signaling. huSMAD6 overexpression caused loss of HOXA11 in early limb MPCs. In conclusion, our data show that BMP signaling controls expansion of embryonic limb MPCs as a prerequisite for establishing the proximodistal muscle pattern, a process that involves expression of Hox genes.

Direct BMP signaling to chordoblasts is required for the initiation of segmented notochord sheath mineralization in zebrafish vertebral column development.

The vertebral column, with the centra as its iteratively arranged building blocks, represents the anatomical key feature of the vertebrate phylum. In contrast to amniotes, where vertebrae are formed from chondrocytes and osteoblasts deriving from the segmentally organized neural crest or paraxial sclerotome, teleost vertebral column development is initiated by chordoblasts of the primarily unsegmented axial notochord, while sclerotomal cells only contribute to later steps of vertebrae formation. Yet, for both mammalian and teleostean model systems, unrestricted signaling by Bone Morphogenetic Proteins (BMPs) or retinoic acid (RA) has been reported to cause fusions of vertebral elements, while the interplay of the two signaling processes and their exact cellular targets remain largely unknown. Here, we address this interplay in zebrafish, identifying BMPs as potent and indispensable factors that, as formerly shown for RA, directly signal to notochord epithelial cells/chordoblasts to promote entpd5a expression and thereby metameric notochord sheath mineralization. In contrast to RA, however, which promotes sheath mineralization at the expense of further collagen secretion and sheath formation, BMP defines an earlier transitory stage of chordoblasts, characterized by sustained matrix production/col2a1 expression and concomitant matrix mineralization/entpd5a expression. BMP-RA epistasis analyses further indicate that RA can only affect chordoblasts and their further progression to merely mineralizing cells after they have received BMP signals to enter the transitory col2a1/entpd5a double-positive stage. This way, both signals ensure consecutively for proper mineralization of the notochord sheath within segmented sections along its anteroposterior axis. Our work sheds further light onto the molecular mechanisms that orchestrate early steps of vertebral column segmentation in teleosts. Similarities and differences to BMP's working mechanisms during mammalian vertebral column formation and the pathomechanisms underlying human bone diseases such as Fibrodysplasia Ossificans Progressiva (FOP) caused by constitutively active BMP signaling are discussed.

Identification of GPI-anchored protein LYPD1 as an essential factor for odontoblast differentiation in tooth development

Lipid rafts are membrane microdomains rich in cholesterol, sphingolipids, glycosylphosphatidylinositol-anchored proteins (GPI-APs), and receptors. These lipid raft components are localized at the plasma membrane and are essential for signal transmission and organogenesis. However, few reports have been published on the specific effects of lipid rafts on tooth development. Using microarray and single-cell RNA sequencing methods, we found that a GPI-AP, lymphocyte antigen-6/Plaur domain-containing 1 (Lypd1), was specifically expressed in preodontoblasts. Depletion of Lypd1 in tooth germ using an exvivo organ culture system and in mouse dental pulp (mDP) cells resulted in the inhibition of odontoblast differentiation. Activation of bone morphogenetic protein (BMP) signaling by BMP2 treatment in mDP cells promoted odontoblast differentiation via phosphorylation of Smad1/5/8, while this BMP2-mediated odontoblast differentiation was inhibited by depletion of Lypd1. Furthermore, we created a deletion construct of the C terminus containing the omega site in LYPD1; this site is necessary for localizing GPI-APs to the plasma membrane and lipid rafts. We identified that this site is essential for odontoblast differentiation and morphological change of mDP cells. These findings demonstrated that LYPD1 is a novel marker of preodontoblasts in the developing tooth; in addition, they suggest that LYPD1 is important for tooth development and that it plays a pivotal role in odontoblast differentiation by regulating Smad1/5/8 phosphorylation through its effect as a GPI-AP in lipid rafts.

Lack of evidence that fibrillin1 regulates bone morphogenetic protein 4 activity in kidney or lung.

The Bone morphogenetic protein 4 (BMP4) precursor protein is cleaved at two sites to generate an active ligand and inactive prodomain. The ligand and prodomain form a noncovalent complex following the first cleavage, but dissociate after the second cleavage. Transient formation of this complex is essential to generate a stable ligand. Fibrillins (FBNs) bind to the prodomains of BMPs, and can regulate the activity of some ligands. Whether FBNs regulate BMP4 activity is unknown. Mice heterozygous for a null allele of Bmp4 showed incompletely penetrant kidney defects and females showed increased mortality between postnatal day 6 and 8. Removal of one copy of Fbn1 did not rescue or enhance kidney defects or lethality. The lungs of Fbn1+/- females had enlarged airspaces that were unchanged in Bmp4+/- ;Fbn1+/- mice. Additionally, removal of one or both alleles of Fbn1 had no effect on steady state levels of BMP4 ligand or on BMP activity in postnatal lungs. These findings do not support the hypothesis that FBN1 plays a role in promoting BMP4 ligand stability or signaling, nor do they support the alternative hypothesis that FBN1 sequesters BMP4 in a latent form, as is the case for other BMP family members.

Current molecular dynamics opinions on interactions between bone morphogenetic protein-2 and inorganic materials

Understanding atomic interactions of bone morphogenetic protein-2 (BMP-2)/inorganic surface is of great significance to the rational design of new products in tissue engineering, regenerative medicine, and materiobiology for bone repair. Molecular dynamics (MD) simulations could provide complementary approaches for experimental studies at the atomic level, which have been widely applied for studying the interactions between BMP-2 and inorganic materials, exploring the binding mechanisms of BMP-2 upon biomaterial surfaces, and deciphering the thermodynamics kinetics of adsorption and desorption of BMP-2. In this review, recent MD opinions of simulated parameters and affecting factors of BMP-2 upon inorganic materials were summarized. The effects of surface properties and protein characteristics on the binding energy, binding site, secondary structure, and biological activity of BMP-2 upon hydroxyapatite and graphene materials were discussed in detail. This review will release atomistic- and molecular-level insights on the factors stabilizing the structure and bioactivity of BMP-2 upon biomaterials, which could provide theoretical guidelines for immobilization of BMP-2 upon implants and scaffolds for bone regenerative medicine.

Augmentation of BMP Signaling in Cranial Neural Crest Cells Leads to Premature Cranial Sutures Fusion through Endochondral Ossification in Mice.

Craniosynostosis is a congenital anomaly characterized by the premature fusion of cranial sutures. Sutures are a critical connective tissue that regulates bone growth; their aberrant fusion results in abnormal shapes of the head and face. The molecular and cellular mechanisms have been investigated for a long time, but knowledge gaps remain between genetic mutations and mechanisms of pathogenesis for craniosynostosis. We previously demonstrated that the augmentation of bone morphogenetic protein (BMP) signaling through constitutively active BMP type 1A receptor (caBmpr1a) in neural crest cells (NCCs) caused the development of premature fusion of the anterior frontal suture, leading to craniosynostosis in mice. In this study, we demonstrated that ectopic cartilage forms in sutures prior to premature fusion in caBmpr1a mice. The ectopic cartilage is subsequently replaced by bone nodules leading to premature fusion with similar but unique fusion patterns between two neural crest-specific transgenic Cre mouse lines, P0-Cre and Wnt1-Cre mice, which coincides with patterns of premature fusion in each line. Histologic and molecular analyses suggest that endochondral ossification in the affected sutures. Both in vitro and in vivo observations suggest a greater chondrogenic capacity and reduced osteogenic capability of neural crest progenitor cells in mutant lines. These results suggest that the augmentation of BMP signaling alters the cell fate of cranial NCCs toward a chondrogenic lineage to prompt endochondral ossification to prematurely fuse cranial sutures. By comparing P0-Cre;caBmpr1a and Wnt1-Cre;caBmpr1a mice at the stage of neural crest formation, we found more cell death of cranial NCCs in P0-Cre;caBmpr1a than Wnt1-Cre;caBmpr1a mice at the developing facial primordia. These findings may provide a platform for understanding why mutations of broadly expressed genes result in the premature fusion of limited sutures. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Chaperone-mediated production of active homodimer human bone morphogenetic protein – 2 in E. coli

Human bone morphogenetic protein 2 (hBMP-2) plays a leading role in the process of osteogenesis and is one of the key components of osteoplastic materials, ensuring their high osteoinduction. In order to obtain a homodimeric form hBMP-2 using the E. coli expression system, a number of problems associated with refolding in vitro and purification from monomer and oligomeric forms must be solved.The developed method for co-expression of the target protein with chaperone proteins makes it possible to obtain the biologically active homodimeric form of hBMP-2 in vivo. Purification with simple ion-exchange sorbents without the use of denaturing reagents affecting the structure of the protein molecule provides a chromatographic purity of the product of at least 97%. The expressed hBMP-2 was identified by Western blotting and the LC-ESI-TOF mass spectrometry confirmed its molecular weight of 26052.72 Da. Circular dichroism spectroscopy showed that recombinant hBMP-2 has a native secondary structure.

Functional comparison of human ACVR1 and zebrafish Acvr1l FOP-associated variants in embryonic zebrafish.

Fibrodysplasia ossificans progressiva (FOP), a rare disease characterized by progressive heterotopic ossification of muscle and connective tissues, is caused by autosomal dominant activating mutations in the type I receptor, ACVR1/ALK2. The classic human FOP variant, ACVR1R206H , shows increased bone morphogenetic protein (BMP) signaling and activation by activins. Here, we performed in vivo functional characterization of human ACVR1R206H and orthologous zebrafish Acvr1lR203H using early embryonic zebrafish dorsoventral patterning as a phenotypic readout for receptor activity. Our results showed that human ACVR1R206H and zebrafish Acvr1lR203H exhibit functional differences in early embryonic zebrafish, and that human ACVR1R206H retained its signaling activity in the absence of a ligand-binding domain (LBD). We also showed, for the first time, that zebrafish Acvr2ba/Acvr2bb receptors are required for human ACVR1R206H signaling in early embryonic zebrafish. Together, these data provide new insight into ACVR1R206H signaling pathways that may facilitate the design of new and effective therapies for FOP patients.