Autocrine Bone Morphogenetic Protein Research Articles

A chemically adjustable BMP6-IL6 axis in mesenchymal stem cells drives acute myeloid leukemia cell differentiation

Chemotherapy alone or in combination with allogeneic stem cell transplantation has been the standard of care for acute myeloid leukemia (AML) for decades. Leukemia relapse with limited treatment options remains the main cause of treatment failure. Therefore, an effective and safe approach to improve treatment outcomes is urgently needed for most AML patients. Mesenchymal stem cells (MSCs) have been reported to efficiently induce apoptosis and shape the fate of acute myeloid leukemia cells. Here, we identified LG190155 as a potent compound that enhances the antileukemia efficiency of MSCs. Pretreatment of MSCs with LG190155 significantly provoked differentiation in both AML patient-derived primary leukemia cells and AML cell lines and reduced the tumor burden in the AML mouse model. Using the quantitative proteomic technique, we discovered a pivotal mechanism that mediates AML cell differentiation, in which autocrine bone morphogenetic protein 6 (BMP6) in MSCs boosted IL-6 secretion and further acted on leukemic cells to trigger differentiation. Furthermore, the activity of the BMP6-IL6 axis was dramatically enhanced by activating vitamin D receptor (VDR) in MSCs. Our data illustrated an effective preactivated approach to reinforcing the antileukemia effect of MSCs, which could serve as an effective therapeutic strategy for AML.

Role of autocrine bone morphogenetic protein signaling in trophoblast stem cells†.

The Bone Morphogenetic Protein (BMP) pathway is involved in numerous developmental processes, including cell growth, apoptosis, and differentiation. In mouse embryogenesis, BMP signaling is a well-known morphogen for both mesoderm induction and germ cell development. Recent evidence points to a potential role in development of the extraembryonic compartment, including trophectoderm-derived tissues. In this study, we investigated the effect of BMP signaling in both mouse and human trophoblast stem cells (TSC) in vitro, evaluating the expression and activation of the BMP signaling response machinery, and the effect of BMP signaling manipulation during TSC maintenance and differentiation. Both mouse trophoblast stem cells (mTSC) and human trophoblast stem cells (hTSC) expressed various BMP ligands and the receptors BMPR1A and BMPR2, necessary for BMP response, and displayed maximal active BMP signaling when undifferentiated. We also observed a conserved modulatory role of BMP signaling during trophoblast differentiation, whereby maintenance of active BMP signaling blunted differentiation of TSC in both species. Conversely, the effect of BMP signaling on the undifferentiated state of TSC appeared to be species-specific, with SMAD-independent signaling important in maintenance of mTSC, and a more subtle role for both SMAD-dependent and -independent BMP signaling in hTSC. Altogether, these data establish an autocrine role for the BMP pathway in the trophoblast compartment. As specification and correct differentiation of the extraembryonic compartment are fundamental for implantation and early placental development, insights on the role of the BMP signaling in early development might prove useful in the setting of in vitro fertilization as well as targeting trophoblast-associated placental dysfunction.

The Wnt Effector TCF7l2 Promotes Oligodendroglial Differentiation by Repressing Autocrine BMP4-Mediated Signaling.

Promoting oligodendrocyte (OL) differentiation represents a promising option for remyelination therapy for treating the demyelinating disease multiple sclerosis (MS). The Wnt effector transcription factor 7-like 2 (TCF7l2) was upregulated in MS lesions and had been proposed to inhibit OL differentiation. Recent data suggest the opposite yet underlying mechanisms remain elusive. Here, we unravel a previously unappreciated function of TCF7l2 in controlling autocrine bone morphogenetic protein (BMP)4-mediated signaling. Disrupting TCF7l2 in mice of both sexes results in oligodendroglial-specific BMP4 upregulation and canonical BMP4 signaling activation in vivo Mechanistically, TCF7l2 binds to Bmp4 gene regulatory element and directly represses its transcriptional activity. Functionally, enforced TCF7l2 expression promotes OL differentiation by reducing autocrine BMP4 secretion and dampening BMP4 signaling. Importantly, compound genetic disruption demonstrates that oligodendroglial-specific BMP4 deletion rescues arrested OL differentiation elicited by TCF7l2 disruption in vivo Collectively, our study reveals a novel connection between TCF7l2 and BMP4 in oligodendroglial lineage and provides new insights into augmenting TCF7l2 for promoting remyelination in demyelinating disorders such as MS.SIGNIFICANCE STATEMENT Incomplete or failed myelin repairs, primarily resulting from the arrested differentiation of myelin-forming oligodendrocytes (OLs) from oligodendroglial progenitor cells, is one of the major reasons for neurologic progression in people affected by multiple sclerosis (MS). Using in vitro culture systems and in vivo animal models, this study unraveled a previously unrecognized autocrine regulation of bone morphogenetic protein (BMP)4-mediated signaling by the Wnt effector transcription factor 7-like 2 (TCF7l2). We showed for the first time that TCF7l2 promotes oligodendroglial differentiation by repressing BMP4-mediated activity, which is dysregulated in MS lesions. Our study suggests that elevating TCF7l2 expression may be possible in overcoming arrested oligodendroglial differentiation as observed in MS patients.

The calcium channel subunit \u03b12\u03b4-3 organizes synapses via an activity-dependent and autocrine BMP signaling pathway

Synapses are highly specialized for neurotransmitter signaling, yet activity-dependent growth factor release also plays critical roles at synapses. While efficient neurotransmitter signaling relies on precise apposition of release sites and neurotransmitter receptors, molecular mechanisms enabling high-fidelity growth factor signaling within the synaptic microenvironment remain obscure. Here we show that the auxiliary calcium channel subunit α2δ-3 promotes the function of an activity-dependent autocrine Bone Morphogenetic Protein (BMP) signaling pathway at the Drosophila neuromuscular junction (NMJ). α2δ proteins have conserved synaptogenic activity, although how they execute this function has remained elusive. We find that α2δ-3 provides an extracellular scaffold for an autocrine BMP signal, suggesting a mechanistic framework for understanding α2δ’s conserved role in synapse organization. We further establish a transcriptional requirement for activity-dependent, autocrine BMP signaling in determining synapse density, structure, and function. We propose that activity-dependent, autocrine signals provide neurons with continuous feedback on their activity state for modulating both synapse structure and function.

Load-induced osteogenic differentiation of mesenchymal stromal cells is caused by mechano-regulated autocrine signaling.

Mechanical boundary conditions critically influence the bone healing process. In this context, previous in vitro studies have demonstrated that cyclic mechanical compression alters migration and triggers osteogenesis of mesenchymal stromal cells (MSC), both processes being relevant to healing. However, it remains unclear whether this mechanosensitivity is a direct consequence of cyclic compression, an indirect effect of altered supply or a specific modulation of autocrine bone morphogenetic protein (BMP) signaling. Here, we investigate the influence of cyclic mechanical compression (ε=5% and 10%, f=1Hz) on human bone marrow MSC (hBMSC) migration and osteogenic differentiation in a 3D biomaterial scaffold, an in vitro system mimicking the mechanical environment of the early bone healing phase. The open-porous architecture of the scaffold ensured sufficient supply even without cyclic compression, minimizing load-associated supply alterations. Furthermore, a large culture medium volume in relation to the cell number diminished autocrine signaling. Migration of hBMSCs was significantly downregulated under cyclic compression. Surprisingly, a decrease in migration was not associated with increased osteogenic differentiation of hBMSCs, as the expression of RUNX2 and osteocalcin decreased. In contrast, BMP2 expression was significantly upregulated. Enabling autocrine stimulation by increasing the cell-to-medium ratio in the bioreactor finally resulted in a significant upregulation of RUNX2 in response to cyclic compression, which could be reversed by rhNoggin treatment. The results indicate that osteogenesis is promoted by cyclic compression when cells condition their environment with BMP. Our findings highlight the importance of mutual interactions between mechanical forces and BMP signaling in controlling osteogenic differentiation.

Regulation of Dense-Core Granule Replenishment by Autocrine BMP Signalling in Drosophila Secondary Cells.

Regulated secretion by glands and neurons involves release of signalling molecules and enzymes selectively concentrated in dense-core granules (DCGs). Although we understand how many secretagogues stimulate DCG release, how DCG biogenesis is then accelerated to replenish the DCG pool remains poorly characterised. Here we demonstrate that each prostate-like secondary cell (SC) in the paired adult Drosophila melanogaster male accessory glands contains approximately ten large DCGs, which are loaded with the Bone Morphogenetic Protein (BMP) ligand Decapentaplegic (Dpp). These DCGs can be marked in living tissue by a glycophosphatidylinositol (GPI) lipid-anchored form of GFP. In virgin males, BMP signalling is sporadically activated by constitutive DCG secretion. Upon mating, approximately four DCGs are typically released immediately, increasing BMP signalling, primarily via an autocrine mechanism. Using inducible knockdown specifically in adult SCs, we show that secretion requires the Soluble NSF Attachment Protein, SNAP24. Furthermore, mating-dependent BMP signalling not only promotes cell growth, but is also necessary to accelerate biogenesis of new DCGs, restoring DCG number within 24 h. Our analysis therefore reveals an autocrine BMP-mediated feedback mechanism for matching DCG release to replenishment as secretion rates fluctuate, and might explain why in other disease-relevant systems, like pancreatic β-cells, BMP signalling is also implicated in the control of secretion.

Optimal effector functions in human natural killer cells rely upon autocrine bone morphogenetic protein signaling.

Natural killer (NK) cells are critical for innate tumor immunity due to their specialized ability to recognize and kill neoplastically transformed cells. However, NK cells require a specific set of cytokine-mediated signals to achieve optimal effector function. Th1-associated cytokines promote effector functions that are inhibited by the prototypic Th2 cytokine IL4 and the TGFβ superfamily members TGFβ1 and activin-A. Interestingly, the largest subgroup of the TGFβ superfamily are the bone morphogenetic proteins (BMP), but the effects of BMP signaling on NK cell effector functions have not been evaluated. Here, we demonstrate that blood-circulating NK cells express type I and II BMP receptors, BMP-2 and BMP-6 ligands, and phosphorylated isoforms of Smad-1/-5/-8, which mediate BMP family member signaling. In opposition to the inhibitory effects of TGFβ1 or activin-A, autocrine BMP signaling was supportive to NK cell function. Mechanistic investigations in cytokine and TLR-L-activated NK cells revealed that BMP signaling optimized IFNγ and global cytokine and chemokine production, phenotypic activation and proliferation, and autologous dendritic cell activation and target cytotoxicity. Collectively, our findings identify a novel auto-activatory pathway that is essential for optimal NK cell effector function, one that might be therapeutically manipulated to help eradicate tumors. Cancer Res; 74(18); 5019-31. ©2014 AACR.

Sequential and Opposing Activities of Wnt and BMP Coordinate Zebrafish Bone Regeneration

SUMMARYZebrafish fully regenerate lost bone, including after fin amputation, through a process mediated by dedifferentiated, lineage-restricted osteoblasts. Mechanisms controlling the osteoblast regenerative program from its initiation through reossification are poorly understood. We show that fin amputation induces a Wnt/β-catenin-dependent epithelial to mesenchymal transformation (EMT) of osteoblasts in order to generate proliferative Runx2+ preosteoblasts. Localized Wnt/β-catenin signaling maintains this progenitor population toward the distal tip of the regenerative blastema. As they become proximally displaced, preosteoblasts upregulate sp7 and subsequently mature into re-epithelialized Runx2−/sp7+ osteoblasts that extend preexisting bone. Auto-crine bone morphogenetic protein (BMP) signaling promotes osteoblast differentiation by activating sp7 expression and counters Wnt by inducing Dickkopf-related Wnt antagonists. As such, opposing activities of Wnt and BMP coordinate the simultaneous demand for growth and differentiation during bone regeneration. This hierarchical signaling network model provides a conceptual framework for understanding innate bone repair and regeneration mechanisms and rationally designing regenerative therapeutics.

Smad2 Is Essential for Maintenance of the Human and Mouse Primed Pluripotent Stem Cell State

Human embryonic stem cells and mouse epiblast stem cells represent a primed pluripotent stem cell state that requires TGF-β/activin signaling. TGF-β and/or activin are commonly thought to regulate transcription through both Smad2 and Smad3. However, the different contributions of these two Smads to primed pluripotency and the downstream events that they may regulate remain poorly understood. We addressed the individual roles of Smad2 and Smad3 in the maintenance of primed pluripotency. We found that Smad2, but not Smad3, is required to maintain the undifferentiated pluripotent state. We defined a Smad2 regulatory circuit in human embryonic stem cells and mouse epiblast stem cells, in which Smad2 acts through binding to regulatory promoter sequences to activate Nanog expression while in parallel repressing autocrine bone morphogenetic protein signaling. Increased autocrine bone morphogenetic protein signaling caused by Smad2 down-regulation leads to cell differentiation toward the trophectoderm, mesoderm, and germ cell lineages. Additionally, induction of Cdx2 expression, as a result of decreased Smad2 expression, leads to repression of Oct4 expression, which, together with the decreased Nanog expression, accelerates the loss of pluripotency. These findings reveal that Smad2 is a unique integrator of transcription and signaling events and is essential for the maintenance of the mouse and human primed pluripotent stem cell state.

The ventral to dorsal BMP activity gradient in the early zebrafish embryo is determined by graded expression of BMP ligands

In the early zebrafish embryo, a ventral to dorsal gradient of bone morphogenetic protein (BMP) activity is established, which is essential for the specification of cell fates along this axis. To visualise and mechanistically determine how this BMP activity gradient forms, we have used a transgenic zebrafish line that expresses monomeric red fluorescent protein (mRFP) under the control of well-characterised BMP responsive elements. We demonstrate that mRFP expression in this line faithfully reports BMP and GDF signalling at both early and late stages of development. Taking advantage of the unstable nature of mRFP transcripts, we use in situ hybridisation to reveal the dynamic spatio-temporal pattern of BMP activity and establish the timing and sequence of events that lead to the formation of the BMP activity gradient. We show that the BMP transcriptional activity gradient is established between 30% and 40% epiboly stages and that it is preceded by graded mRNA expression of the BMP ligands. Both Dharma and FGF signalling contribute to graded bmp transcription during these early stages and it is subsequently maintained through autocrine BMP signalling. We show that BMP2B protein is also expressed in a gradient as early as blastula stages, but do not find any evidence of diffusion of this BMP to generate the BMP transcriptional activity gradient. Thus, in contrast to diffusion/transport-based models of BMP gradient formation in Drosophila, our results indicate that the establishment of the BMP activity gradient in early zebrafish embryos is determined by graded expression of the BMP ligands.

Expression of BMPRIA on human thymic NK cell precursors: role of BMP signaling in intrathymic NK cell development

AbstractThe bone morphogenetic protein (BMP) signaling pathway regulates survival, proliferation, and differentiation of several cell types in multiple tissues, including the thymus. Previous reports have shown that BMP signaling negatively regulates T-cell development. Here, we study the subpopulation of early human intrathymic progenitors expressing the type IA BMP receptor (BMPRIA) and provide evidence that CD34+CD1a−BMPRIA+ precursor cells mostly express surface cell markers and transcription factors typically associated with NK cell lineage. These CD34+ cells mostly differentiate into functional CD56+ natural killer (NK) cells when they are cocultured with thymic stromal cells in chimeric human-mouse fetal thymic organ cultures and also in the presence of SCF and IL-15. Moreover, autocrine BMP signaling can promote the differentiation of thymic NK cells by regulating the expression of key transcription factors required for NK cell lineage (eg, Id3 and Nfil3) as well as one of the components of IL-15 receptor, CD122. Subsequently, the resulting population of IL-15–responsive NK cell precursors can be expanded by IL-15, whose action is mediated by BMP signaling during the last steps of thymic NK cell differentiation. Our results strongly suggest that BMPRIA expression identifies human thymic NK cell precursors and that BMP signaling is relevant for NK cell differentiation in the human thymus.

BMP-Id pathway targeted by cholesterol myristate suppresses the apoptosis of PC12 cells

Identifying small molecules that suppress apoptosis is promising for the therapy of brain diseases. We recently showed that autocrine bone morphogenetic protein (BMP) signaling involves the effects of cholesterol myristate present in traditional Chinese medicine on mesenchymal stem cells. The present study evaluated the effects of cholesterol myristate on the apoptosis and BMP signaling of PC12 cells. PC12 cells transfected by the inhibitor of differentiation (Id1) promoter reporter construct target gene of BMP4 signaling; cholesterol myristate increases the activity of Id1 promoter. However, structurally related steroids such as cholesterol, β-sitosterol and cholesten-3-one, lack of the myristate, did not affect the activity of Id1 promoter, suggesting that myristate is essential for the effect of cholesterol myristate. These effects depend on BMP signaling. Apoptosis analysis indicated that cholesterol myristate inhibited the apoptosis of PC12 cells induced in serum-free condition. Cholesterol myristate significantly increases the expression of BMP4, BMPRIA, p-Smad1/5/8, Id1 and its antiapoptotic target gene Bcl-xL in PC12 cells treated in serum-free condition. Moreover, BMP antagonist reduced the anti-apoptotic effect of cholesterol myristate. Thus, this study is to provide evidence that BMP-Id pathway targeted by cholesterol myristate suppresses the apoptosis of PC12 cells. Our findings are therefore of considerable therapeutic significance and provide the potential of newly exploiting cholesterol myristate and clinically in brain disease therapies.

Lack of Noggin Expression by Cancer Cells Is a Determinant of the Osteoblast Response in Bone Metastases

Prostate and mammary cancer bone metastases can be osteoblastic or osteolytic, but the mechanisms determining these features are unclear. Bone morphogenetic and Wnt proteins are osteoinductive molecules. Their activity is modulated by antagonists such as noggin and dickkopf-1. Differential expression analysis of bone morphogenetic and Wnt protein antagonists in human prostate and mammary cancer cell lines showed that osteolytic cell lines constitutively express in vitro noggin and dickkopf-1 and at least one of the osteolytic cytokines parathyroid hormone-related protein, colony-stimulating factor-1, and interleukin-8. In contrast, osteoinductive cell lines express neither noggin nor dickkopf-1 nor osteolytic cytokines in vitro. The noggin differential expression profile observed in vitro was confirmed in vivo in prostate cancer cell lines xenografted into bone and in clinical samples of bone metastasis. Forced noggin expression in an osteoinductive prostate cancer cell line abolished the osteoblast response induced in vivo by its intraosseous xenografts. Basal bone resorption and tumor growth kinetics were marginally affected. Lack of noggin and possibly dickkopf-1 expression by cancer cells may be a relevant mechanism contributing to the osteoblast response in bone metastases. Concomitant lack of osteolytic cytokines may be permissive of this effect. Noggin is a candidate drug for the adjuvant therapy of bone metastasis.

Spatio-temporal activation of Smad1 and Smad5 in vivo: monitoring transcriptional activity of Smad proteins.

Signaling by bone morphogenetic proteins is essential for a wide variety of developmental processes. Receptor-regulated Smad proteins, Smads 1 and 5, are intracellular mediators of bone morphogenetic protein signaling. Together with Smad4, these proteins translocate to the nucleus and modulate transcription by binding to specific sequences on the promoters of target genes. We sought to map transcriptional Smad1/5 activity in development by generating embryonic stem cell lines carrying a Smad1/5-specific response element derived from the Id1 promoter coupled to beta-galactosidase or luciferase as reporters. Three independent lines (BRE-lac1, BRE-lac2 and BRE-luc) have shown the existence of an autocrine bone morphogenetic protein signaling pathway in mouse embryonic stem cells. Reporter activity was detected in chimeric embryos, suggesting sensitivity to physiological concentrations of bone morphogenetic protein. Reporter activity in embryos from transgenic mouse lines was detected in tissues where an essential role for active bone morphogenetic protein signaling via Smads 1 or 5 had been previously established. We have thus generated, for the first time, an in vivo readout for studying the role of Smad1/5-mediated transcriptional activity in development.

Differentiation of murine preosteoblastic KS483 cells depends on autocrine bone morphogenetic protein signaling during all phases of osteoblast formation

In this study, we examine the role of bone morphogenetic protein (BMP) signaling during differentiation of the murine preosteoblastic KS483 cell line, which formed alkaline phosphatase (ALP)-positive and mineralized nodules during a 3 week culture period. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) demonstrated the presence of various BMPs (BMP-2, -3, -4, -6, -7, and -8A and -8B), BMP type I and II receptors (ALK2, ALK3, ALK4, BMPR-II, and ActR-IIA and -IIB), BMP antagonists (DAN, gremlin, chordin, cerberus, noggin, and tsg), and Smads 1–8. mRNA expression of these genes did not change during differentiation, except for BMP-3, BMP-8a, and noggin. BMP-3 increased gradually, particularly in the matrix formation phase; BMP-8a was induced from the onset of matrix maturation and mineralization, in parallel to the expression of osteocalcin; and noggin tended to decline during the mineralization phase. Treatment of KS483 cells with the BMP antagonists noggin or soluble truncated BMPR-IA, either continuously or during distinct periods of osteoblast differentiation; that is, matrix formation or matrix maturation and mineralization phase, decreased ALP-positive and mineralized nodule area independent of the phase of osteoblast differentiation. Notably, the antagonists inhibited mineralization of already existing nodules. Similarly, BMP-4 stimulated differentiation not only at the beginning of the culture period, but also at late stages of differentiation. These data indicate that autocrine BMP signaling is involved in KS483 osteoblastic differentiation not only during the early phase of differentiation, but also during matrix maturation and mineralization. The different expression patterns of components of BMP signaling in the KS483 cells suggest distinct functions of individual BMPs during osteoblast differentiation. In summary, our data suggest that BMP activity is required not only for initiation of osteoblast differentiation and further development of early osteoblasts, but is also involved in late-stage osteoblast differentiation and matrix mineralization.

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Promoting oligodendrocyte (OL) differentiation represents a promising option for remyelination therapy for treating the demyelinating disease multiple sclerosis (MS). The Wnt effector transcription factor 7-like 2 (TCF7l2) was upregulated in MS lesions and had been proposed to inhibit OL differentiation. Recent data suggest the opposite yet underlying mechanisms remain elusive. Here, we unravel a previously unappreciated function of TCF7l2 in controlling autocrine bone morphogenetic protein (BMP)4-mediated signaling. Disrupting TCF7l2 in mice of both sexes results in oligodendroglial-specific BMP4 upregulation and canonical BMP4 signaling activation <i>in vivo</i>. Mechanistically, TCF7l2 binds to <i>Bmp4</i> gene regulatory element and directly represses its transcriptional activity. Functionally, enforced TCF7l2 expression promotes OL differentiation by reducing autocrine BMP4 secretion and dampening BMP4 signaling. Importantly, compound genetic disruption demonstrates that oligodendroglial-specific BMP4 deletion rescues arrested OL differentiation elicited by TCF7l2 disruption <i>in vivo</i>. Collectively, our study reveals a novel connection between TCF7l2 and BMP4 in oligodendroglial lineage and provides new insights into augmenting TCF7l2 for promoting remyelination in demyelinating disorders such as MS. <b>SIGNIFICANCE STATEMENT</b> Incomplete or failed myelin repairs, primarily resulting from the arrested differentiation of myelin-forming oligodendrocytes (OLs) from oligodendroglial progenitor cells, is one of the major reasons for neurologic progression in people affected by multiple sclerosis (MS). Using <i>in vitro</i> culture systems and <i>in vivo</i> animal models, this study unraveled a previously unrecognized autocrine regulation of bone morphogenetic protein (BMP)4-mediated signaling by the Wnt effector transcription factor 7-like 2 (TCF7l2). We showed for the first time that TCF7l2 promotes oligodendroglial differentiation by repressing BMP4-mediated activity, which is dysregulated in MS lesions. Our study suggests that elevating TCF7l2 expression may be possible in overcoming arrested oligodendroglial differentiation as observed in MS patients.