BMP Regulation Research Articles

Enhanced BMP-2/BMP-4 ratio in patients with peripheral spondyloarthritis and in cytokine- and stretch-stimulated mouse chondrocytes

BackgroundExcessive bone formation in the entheses is one of the features of peripheral spondyloarthritis (SpA). Complex pathological mechanisms connecting inflammation, mechanical stress, and ossification are probably involved. We focused on bone morphogenetic protein (BMP)-2, -4, and -7 as possible mediators of this process.MethodsBMP-2, -4, and -7 concentration was measured by ELISA in synovial fluids (SFs) of SpA (n = 56) and osteoarthritic (n = 21) patients. Mouse organotypic ankle cultures were challenged by a pro-inflammatory cocktail. Mouse primary chondrocytes, osteoblasts, or tenocytes were treated with TNF-α, interleukin (IL)-17, or IL-22 and/or subjected to cyclic stretch, or with recombinant BMP-2 or -4.ResultsIn SpA SFs, if BMP-7 was barely detectable, BMP-2 concentration was higher and BMP-4 was lower than in osteoarthritic samples, so that BMP-2/BMP-4 ratio augmented 6.5 folds (p < 0.001). In SpA patients, TNF-α, IL-6, and IL-17 levels correlated this ratio (n = 21). Bmp-2/Bmp-4 ratio was similarly enhanced by cytokine treatment in explant and cell cultures, at mRNA level. In particular, simultaneous application of TNF-α and cyclical stretch induced a 30-fold increase of the Bmp-2/Bmp-4 ratio in chondrocytes (p = 0.027). Blockade of prostaglandin E2 and IL-6 production had almost no effect on the stretch-induced regulation of Bmp-2 or -4. Osteoinductive effects of BMP-4, and to a lesser extend BMP-2, were identified on cultured chondrocytes and tenocytes.ConclusionsOur results first settle that BMP factors are locally deregulated in the SpA joint. An unexpected decrease in BMP-4 could be associated to an increase in BMP-2, possibly in response to mechanical and/or cytokine stimulations.

Enhanced BMP-2/BMP-4 ratio in patients with peripheral spondyloarthritis and in cytokine- and stretch-stimulated mouse chondrocytes

Excessive bone formation in the entheses is one of the features of peripheral spondyloarthritis (SpA). Complex pathological mechanisms connecting inflammation, mechanical stress, and ossification are probably involved. We focused on bone morphogenetic protein (BMP)-2, -4, and -7 as possible mediators of this process. BMP-2, -4, and -7 concentration was measured by ELISA in synovial fluids (SFs) of SpA (n = 56) and osteoarthritic (n = 21) patients. Mouse organotypic ankle cultures were challenged by a pro-inflammatory cocktail. Mouse primary chondrocytes, osteoblasts, or tenocytes were treated with TNF-α, interleukin (IL)-17, or IL-22 and/or subjected to cyclic stretch, or with recombinant BMP-2 or -4. In SpA SFs, if BMP-7 was barely detectable, BMP-2 concentration was higher and BMP-4 was lower than in osteoarthritic samples, so that BMP-2/BMP-4 ratio augmented 6.5 folds (p < 0.001). In SpA patients, TNF-α, IL-6, and IL-17 levels correlated this ratio (n = 21). Bmp-2/Bmp-4 ratio was similarly enhanced by cytokine treatment in explant and cell cultures, at mRNA level. In particular, simultaneous application of TNF-α and cyclical stretch induced a 30-fold increase of the Bmp-2/Bmp-4 ratio in chondrocytes (p = 0.027). Blockade of prostaglandin E2 and IL-6 production had almost no effect on the stretch-induced regulation of Bmp-2 or -4. Osteoinductive effects of BMP-4, and to a lesser extend BMP-2, were identified on cultured chondrocytes and tenocytes. Our results first settle that BMP factors are locally deregulated in the SpA joint. An unexpected decrease in BMP-4 could be associated to an increase in BMP-2, possibly in response to mechanical and/or cytokine stimulations.

Endothelial BMP4 Promotes Leukocyte Rolling and Adhesion and Is Elevated in Patients After Survived Out-of-Hospital Cardiac Arrest.

Leukocyte recruitment is a fundamental step in the inflammatory response during ischemia/reperfusion injury (IRI). Rolling and adhesion of leukocytes to activated endothelium promote tissue inflammation after IRI and require presentation of adhesion molecules E-selectin and ICAM-1 on the endothelial surface. Bone morphogenetic protein (BMP) 4 is a prominent member of the BMP family expressed and secreted by endothelial cells. BMP4 derived from endothelial cells has important functions in vascular disease but its influence on the leukocyte adhesion cascade during inflammation is incompletely understood. In the present study, we challenged mice with an inducible endothelial-specific BMP4 deletion (referred to as EC-BMP4-/- mice) and their control littermates (EC-BMP4+/+) with thioglycollate i.p. and assessed extravasation of different leukocyte subsets during peritonitis. Peritoneal lavages were performed and peritoneal cells were counted. Total cell count in lavages of EC-BMP4-/- mice was markedly reduced compared with lavages of EC-BMP4+/+ mice. FACS analyses of thioglycollate-elicited peritoneal cells revealed that diverse leukocyte subsets were reduced in EC-BMP4-/- mice. Intravital microscopy of cremaster venules demonstrated that rolling and adhesion of leukocytes were significantly diminished in EC-BMP4-/- mice in comparison with control mice in response to TNFα. These observations indicate that endothelial BMP4 is essential for rolling, adhesion, and extravasation of leukocytes in vivo. To understand the underlying mechanisms, levels of endothelial adhesion molecules E-selectin and ICAM-1 were quantified in EC-BMP4-/- and EC-BMP4+/+ mice by quantitative PCR and Western blotting. Interestingly, ICAM-1 and E-selectin expressions were reduced in the hearts of EC-BMP4-/- mice. Next we confirmed pro-inflammatory properties of BMP4 in a gain of function experiments and found that administration of recombinant BMP4 in male C57BL/6 mice increased leukocyte rolling and adhesion in cremaster venules in vivo. To assess the regulation of BMP4 in inflammatory disease in humans, we collected plasma samples of patients from day 0 to day 7 after survived out-of-hospital cardiac arrest (OHCA, n = 42). Remarkably, plasma of OHCA patients contained significantly higher BMP4 protein levels compared with patients with coronary artery disease (CAD, n = 12) or healthy volunteers (n = 11). Subgroup analysis revealed that elevated plasma BMP4 levels after ROSC are associated with decreased survival and unfavorable neurological outcome. Collectively, endothelial BMP4 is a potent activator of inflammation in vivo that promotes rolling, adhesion, and extravasation of leukocyte subsets by induction of E-selectin and ICAM-1. Elevation of plasma BMP4 levels in the post-resuscitation period suggests that BMP4 contributes to pathophysiology and poor outcome of post-cardiac arrest syndrome.

Bmp2 regulates Serpinb6b expression via cAMP/PKA/Wnt4 pathway during uterine decidualization.

Serpinb6b is a novel member of Serpinb family and found in germ and somatic cells of mouse gonads, but its physiological function in uterine decidualization remains unclear. The present study revealed that abundant Serpinb6b was noted in decidual cells, and advanced the proliferation and differentiation of stromal cells, indicating a creative role of Serpinb6b in uterine decidualization. Further analysis found that Serpinb6b modulated the expression of Mmp2 and Mmp9. Meanwhile, Serpinb6b was identified as a target of Bmp2 regulation in stromal differentiation. Treatment with rBmp2 resulted in an accumulation of intracellular cAMP level whose function in this differentiation program was mediated by Serpinb6b. Addition of PKA inhibitor H89 impeded the Bmp2 induction of Serpinb6b, whereas 8‐Br‐cAMP rescued the defect of Serpinb6b expression elicited by Bmp2 knock‐down. Attenuation of Serpinb6b greatly reduced the induction of constitutive Wnt4 activation on stromal cell differentiation. By contrast, overexpression of Serpinb6b prevented this inhibition of differentiation process by Wnt4 siRNA. Moreover, blockage of Wnt4 abrogated the up‐regulation of cAMP on Serpinb6b. Collectively, Serpinb6b mediates uterine decidualization via Mmp2/9 in response to Bmp2/cAMP/PKA/Wnt4 pathway.

The dose-time effects of fluoride on the expression and DNA methylation level of the promoter region of BMP-2 and BMP-7 in rats

Skeletal fluorosis is a chronic metabolic bone disease caused by excessive exposed to fluoride. Recent studies have shown that fluoride causes abnormal bone metabolism through disrupting the expression of Bone Morphogenetic Proteins (BMPs). However, the relationship between fluoride and BMPs is not fully understood, and the mechanism of fluoride on BMPs expression is still unclear. This study investigated the dose-time effects of fluoride on BMP-2 and BMP-7 levels and DNA methylation status of the promoter regions of these two genes in peripheral blood of rats. Eighty Wistar male rats were randomly divided into four groups and treated for 1 month and 3 months with distilled water (control), 25 mg/L, 50 mg/L or 100 mg/L of sodium fluoride (NaF). Rats exposed to fluoride had higher protein expression of BMP-2 and BMP-7 in plasma at 1 month and 3 months. An increase in BMP-2 expression was also observed with an increase of fluoride exposure time. Significant hypomethylation was observed in 2 CpG sites (CpGs) of BMP-2 and 1 CpG site of BMP-7 promoter regions in the fluoride treatment groups. It concludes that fluoride has a dose-response effect on BMP-2 in fluorosis rats, and fluoride-induced hypomethylation of specific CpGs may play an essential role in the regulation of BMP-2 and BMP-7 expression in rats.

Skin Stem Cells in Silence, Action, and Cancer.

In studying how stem cells make and maintain tissues, nearly every chapter of a cell biology textbook takes on special interest. The field even allows us to venture where no chapters have yet been written. In studying this basic problem, we are continually bombarded by nature's surprises and challenges. Stem cell biology has captured my interest for nearly my entire scientific career. Below, I focus on my laboratory's contributions to this fascinating field, to which so many friends and colleagues have made seminal discoveries equally deserving of this award.

Prostaglandin E2 Is Required for BMP4-Induced Mesoderm Differentiation ofHuman Embryonic Stem Cells.

SummaryThe accurate control of early cell fate specification during differentiation of human embryonic stem cells (hESCs) is critical for acquiring pure therapeutic cell populations of interest. Bone morphogenetic protein 4 (BMP4) is a key mesoderm inducer from ESCs. However, the molecular mechanism of the mesodermal cell fate decision induced by BMP4 remains unclear. Here, we demonstrate the requirement of a bioactive lipid, prostaglandin E2 (PGE2), for the mesoderm specification from hESCs by BMP4 induction. We show that BMP4 directly regulates the expression of the key enzyme for PGE2 synthesis, COX-1, and promotes PGE2 production. More importantly, in the absence of BMP4, forced COX-1 expression or PGE2 treatment is sufficient to initiate mesoderm specification of hESCs by activation of EP2-PKA signaling and modulation of nuclear translocation of β-catenin. Together, our findings provide insights into the critical role of BMP regulation of PGE2 synthesis and its downstream signaling in initiating mesoderm commitment of hESCs.

Homeobox protein MSX-1 inhibits expression of bone morphogenetic protein 2, bone morphogenetic protein 4, and lymphoid enhancer-binding factor 1 via Wnt/β-catenin signaling to prevent differentiation of dental mesenchymal cells during the late bell stage.

Homeobox protein MSX-1 (hereafter referred to as MSX-1) is essential for early tooth-germ development. Tooth-germ development is arrested at bud stage in Msx1 knockout mice, which prompted us to study the functions of MSX-1 beyond this stage. Here, we investigated the roles of MSX-1 during late bell stage. Mesenchymal cells of the mandibular first molar were isolated from mice at embryonic day (E)17.5 and cultured invitro. We determined the expression levels of β-catenin, bone morphogenetic protein 2 (Bmp2), Bmp4, and lymphoid enhancer-binding factor 1 (Lef1) after knockdown or overexpression of Msx1. Our findings suggest that knockdown of Msx1 promoted expression of Bmp2, Bmp4, and Lef1, resulting in elevated differentiation of odontoblasts, which was rescued by blocking the expression of these genes. In contrast, overexpression of Msx1 decreased the expression of Bmp2, Bmp4, and Lef1, leading to a reduction in odontoblast differentiation. The regulation of Bmp2, Bmp4, and Lef1 by Msx1 was mediated by the Wnt/β-catenin signaling pathway. Additionally, knockdown of Msx1 impaired cell proliferation and slowed S-phase progression, while overexpression of Msx1 also impaired cell proliferation and prolonged G1-phase progression. We therefore conclude that MSX-1 maintains cell proliferation by regulating transition of cells from G1-phase to S-phase and prevents odontoblast differentiation by inhibiting expression of Bmp2, Bmp4, and Lef1 at the late bell stage via the Wnt/β-catenin signaling pathway.

Isl1 mediates mesenchymal expansion in the developing external genitalia via regulation of Bmp4, Fgf10 and Wnt5a.

Genital malformations are among the most common human birth defects, and both genetic and environmental factors can contribute to these malformations. Development of the external genitalia in mammals relies on complex signaling networks, and disruption of these signaling pathways can lead to genital defects. Islet-1 (ISL1), a member of the LIM/Homeobox family of transcription factors, has been identified as a major susceptibility gene for classic bladder exstrophy in humans, a common form of the bladder exstrophy-epispadias complex (BEEC), and is implicated in a role in urinary tract development. We report that deletion of Isl1 from the genital mesenchyme in mice led to hypoplasia of the genital tubercle and prepuce, with an ectopic urethral opening and epispadias-like phenotype. These mice also developed hydroureter and hydronephrosis. Identification of ISL1 transcriptional targets via ChIP-Seq and expression analyses revealed that Isl1 regulates several important signaling pathways during embryonic genital development, including the BMP, WNT, and FGF cascades. An essential function of Isl1 during development of the external genitalia is to induce Bmp4-mediated apoptosis in the genital mesenchyme. Together, these studies demonstrate that Isl1 plays a critical role during development of the external genitalia and forms the basis for a greater understanding of the molecular mechanisms underlying the pathogenesis of BEEC and urinary tract defects in humans.

BMP-2 induced Dspp transcription is mediated by Dlx3/Osx signaling pathway in odontoblasts

Dentin sialophosphoprotein (Dspp) as a differentiation marker of odontoblasts is regulated by BMP-2. However, the intimate mechanism is still unknown. Transcription factors Dlx3 and Osx are essential for odontoblasts differentiation. We hypothesized that BMP-2 regulation of Dspp transcription was mediated by Dlx3 and/or Osx in odontoblasts. In the present investigation, we found that BMP-2 stimulated expression and nuclear translocation of Dlx3 and Osx in odontoblasts both in vitro and in vivo. Osx was a downstream target of Dlx3 and both of them stimulated Dsp expression. Both Dlx3 and Osx were able to activate Dspp promoter from nucleotides (nt) −318 to +54 by transfections of luciferase reports containing different lengths of mouse Dspp promoters. The binding of Dlx3 and Osx with nt −318 to +54 of Dspp promoter was verified by chromatin immunoprecipitation in vivo. Two Dlx3 binding sites and one Osx binding site on Dspp promoter were found by EMSA. Furthermore, the exact biological function of these binding sites was confirmed by site-directed mutagenesis. At last, the protein-protein interaction between Dlx3 and Osx in odontoblasts was detected by co-immunoprecipitation. In conclusion, in this study we found a novel signaling pathway in which BMP-2 activates Dspp gene transcription via Dlx3/Osx pathway.

Differential involvement of Wnt signaling in Bmp regulation of cancellous versus periosteal bone growth

Bone morphogenetic proteins (Bmp) are well-known to induce bone formation following chondrogenesis, but the direct role of Bmp signaling in the osteoblast lineage is not completely understood. We have recently shown that deletion of the receptor Bmpr1a in the osteoblast lineage with Dmp1-Cre reduces osteoblast activity in general but stimulates proliferation of preosteoblasts specifically in the cancellous bone region, resulting in diminished periosteal bone growth juxtaposed with excessive cancellous bone formation. Because expression of sclerostin (SOST), a secreted Wnt antagonist, is notably reduced in the Bmpr1a-deficient osteocytes, we have genetically tested the hypothesis that increased Wnt signaling might mediate the increase in cancellous bone formation in response to Bmpr1a deletion. Forced expression of human SOST from a Dmp1 promoter fragment partially rescues preosteoblast hyperproliferation and cancellous bone overgrowth in the Bmpr1a mutant mice, demonstrating functional interaction between Bmp and Wnt signaling in the cancellous bone compartment. To test whether increased Wnt signaling can compensate for the defect in periosteal growth caused by Bmpr1a deletion, we have generated compound mutants harboring a hyperactive mutation (A214V) in the Wnt receptor Lrp5. However, the mutant Lrp5 does not restore periosteal bone growth in the Bmpr1a-deficient mice. Thus, Bmp signaling restricts cancellous bone accrual partly through induction of SOST that limits preosteoblast proliferation, but promotes periosteal bone growth apparently independently of Wnt activation.

Characterization of new bone morphogenetic protein (Bmp)‐2 regulatory alleles

Bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) is a classical morphogen; a molecule that acts at a distance and whose concentration influences cell proliferation, differentiation, and apoptosis. Key events requiring precise Bmp2 regulation include heart specification and morphogenesis and neural development. In mesenchymal cells, the concentration of BMP2 influences myogenesis, adipogenesis, chondrogenesis, and osteogenesis. Because the amount, timing, and location of BMP2 synthesis influence pattern formation and organogenesis, the mechanisms that regulate Bmp2 are crucial. A sequence within the 3'UTR of the Bmp2 mRNA termed the "ultra-conserved sequence" (UCS) has been largely unchanged since fishes and mammals diverged. Cre-lox mediated deletion of the UCS in a reporter transgene revealed that the UCS may repress Bmp2 in proepicardium, epicardium, and epicardium-derived cells (EPDC) and in tissues with known epicardial contributions (coronary vessels and valves). The UCS also repressed the transgene in the aorta, outlet septum, posterior cardiac plexus, cardiac and extra-cardiac nerves, and neural ganglia. We used homologous recombination and conditional deletion to generate three new alleles in which the Bmp2 3'UTR was altered as follows: a UCS flanked by loxP sites with or without a neomycin resistance targeting vector, or a deleted UCS. Deletion of the UCS was associated with elevated Bmp2 mRNA and BMP signaling levels, reduced fitness, and embryonic malformations.

Semipermeable Capsules Wrapping a Multifunctional and Self-regulated Co-culture Microenvironment for Osteogenic Differentiation.

A new concept of semipermeable reservoirs containing co-cultures of cells and supporting microparticles is presented, inspired by the multi-phenotypic cellular environment of bone. Based on the deconstruction of the “stem cell niche”, the developed capsules are designed to drive a self-regulated osteogenesis. PLLA microparticles functionalized with collagen I, and a co-culture of adipose stem (ASCs) and endothelial (ECs) cells are immobilized in spherical liquified capsules. The capsules are coated with multilayers of poly(L-lysine), alginate, and chitosan nano-assembled through layer-by-layer. Capsules encapsulating ASCs alone or in a co-culture with ECs are cultured in endothelial medium with or without osteogenic differentiation factors. Results show that osteogenesis is enhanced by the co-encapsulation, which occurs even in the absence of differentiation factors. These findings are supported by an increased ALP activity and matrix mineralization, osteopontin detection, and the up regulation of BMP-2, RUNX2 and BSP. The liquified co-capsules also act as a VEGF and BMP-2 cytokines release system. The proposed liquified capsules might be a valuable injectable self-regulated system for bone regeneration employing highly translational cell sources.

PRMT5 is essential for the maintenance of chondrogenic progenitor cells in the limb bud.

During embryonic development, undifferentiated progenitor cells balance the generation of additional progenitor cells with differentiation. Within the developing limb, cartilage cells differentiate from mesodermal progenitors in an ordered process that results in the specification of the correct number of appropriately sized skeletal elements. The internal pathways by which these cells maintain an undifferentiated state while preserving their capacity to differentiate is unknown. Here, we report that the arginine methyltransferase PRMT5 has a crucial role in maintaining progenitor cells. Mouse embryonic buds lacking PRMT5 have severely truncated bones with wispy digits lacking joints. This novel phenotype is caused by widespread cell death that includes mesodermal progenitor cells that have begun to precociously differentiate into cartilage cells. We propose that PRMT5 maintains progenitor cells through its regulation of Bmp4 Intriguingly, adult and embryonic stem cells also require PRMT5 for maintaining pluripotency, suggesting that similar mechanisms might regulate lineage-restricted progenitor cells during organogenesis.

Abstract B35: BMP regulation of cancer stem cell quiescence is responsible for chemotherapeutic resistance in glioblastoma

Abstract Introduction: Cancer stem cells (CSCs) represent a distinct cellular population that has been implicated in tumor recurrence following chemotherapy. Accumulating evidence indicates that BMP signaling plays an important role in the regulation of CSCs, and may be involved in the resistance of CSCs to cytotoxic drugs. The role of BMP pathway regulation of the CSC phenotype responsible for treatment resistance in glioblastoma (GBM) has not been explored. Methods: Paraffin-embedded (FFPE) samples from GBM patients were used to evaluate the expression of markers for BMP signaling (pSMAD1/5/8), proliferation (PCNA), and stemness (SOX2) by immunostaining. We tested the self-renewal capacity of primary patient-derived glioma stem cell (GSC) lines using a sphere formation assay. Expression of stemness markers SOX2 and Bmi-1 in BMP4 treated and untreated GSCs, was analyzed by Western blotting. We also performed xenograft transplantation, whereby GSCs were injected intracranially into immunedeficient mice. When tumor formation was confirmed post-injection, mice were administrated EdU and sacrificed at successive time points thereafter. The frequency of all EdU positive cells as well as ID-1positive/ EdU positive cells was quantified from the primary tumor sections throughout the labeling and chase. To study chemoresistance in glioblastoma, GSC lines were treated with graded concentrations of temozolomide (TMZ). Results: We found that pSMAD1/5/8 positive cells in primary patient tumors were largely PCNA-negative. BMP4 treatment inhibited GSC proliferation and self-renewal (sphere-formation), but did not abolish expression of markers of stemness, such as Sox2 and Bmi-1, or tumorigenicity. In our in vivo model, we found ID-1 positive cells retain EdU following a long-term chase. Taken together, these results demonstrate that BMP4-enriched cells possess the characteristics of a quiescent stem cell population. BMP4 treatment protects GSCs against TMZ-induced growth inhibition. Furthermore, pSMAD1/5/8-expressing cells do not possess the DNA damage marker gamma-H2AX following TMZ treatment, suggesting that activation of the BMP pathway protects GSCs against TMZ-induced DNA damage. Conclusion: This study establishes that the BMP signaling pathway is involved in the maintenance of GSC quiescence and may play a role in glioblastoma chemoresistance. Citation Format: Megan YiJun Wu, Angela Celebre, Jeffrey Chan, Jennifer Guan, Karan Dhand, James Loukides, Jason Karamchandani, Sunit Das. BMP regulation of cancer stem cell quiescence is responsible for chemotherapeutic resistance in glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B35.

Pluripotent Conversion of Muscle Stem Cells Without Reprogramming Factors or Small Molecules.

Muscle derived stem cells (MDSCs) are multipotent stem cells that can differentiate into several lineages including skeletal muscle precursor cells. Here, we show that MDSCs from myostatin null mice (Mstn (-/-) ) can be readily induced into pluripotent stem cells without using reprogramming factors. Microarray studies revealed a strong upregulation of markers like Leukemia Inhibitory factor (LIF) and Leukemia Inhibitory factor receptor (LIFR) in Mstn (-/-) MDSCs as compared to wild type MDSCs (WT-MDSCs). Furthermore when cultured in mouse embryonic stem cell media with LIF for 95days, Mstn (-/-) MDSCs formed embryonic stem cell (ES) like colonies. We termed such ES like cells as the culture-induced pluripotent stem cells (CiPSC). CiPSCs from Mstn (-/-) MDSCs were phenotypically similar to ESCs, expressed high levels of Oct4, Nanog, Sox2 and SSEA-1, maintained a normal karyotype. Furthermore, CiPSCs formed embryoid bodies and teratomas when injected into immunocompromised mice. In addition, CiPSCs differentiated into somatic cells of all three lineages. We further show that culturing in ES cell media, resulted in hypermethylation and downregulation of BMP2 in Mstn(-/-) MDSCs. Western blot further confirmed a down regulation of BMP2 signaling in Mstn (-/-) MDSCs in supportive of pluripotent reprogramming. Given that down regulation of BMP2 has been shown to induce pluripotency in cells, we propose that lack of myostatin epigenetically reprograms the MDSCs to become pluripotent stem cells. Thus, here we report the successful establishment of ES-like cells from adult stem cells of the non-germline origin under culture-induced conditions without introducing reprogramming genes.

Turning Bone Morphogenetic Protein 2 (BMP2) on and off in Mesenchymal Cells.

The concentration, location, and timing of bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) gene expression must be precisely regulated. Abnormal BMP2 levels cause congenital anomalies and diseases involving the mesenchymal cells that differentiate into muscle, fat, cartilage, and bone. The molecules and conditions that influence BMP2 synthesis are diverse. Understandably, complex mechanisms control Bmp2 gene expression. This review includes a compilation of agents and conditions that can induce Bmp2. The currently known trans-regulatory factors and cis-regulatory elements that modulate Bmp2 expression are summarized and discussed. Bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) is a classical morphogen; a molecule that acts at a distance and whose concentration influences cell behavior. In mesenchymal cells, the concentration of BMP2 influences myogenesis, adipogenesis, chondrogenesis, and osteogenesis. Because the amount, timing, and location of BMP2 synthesis influence the allocation of cells to muscle, fat, cartilage, and bone, the mechanisms that regulate the Bmp2 gene are crucial. Key early mesodermal events that require precise Bmp2 regulation include heart specification and morphogenesis. Originally named for its osteoinductive properties, healing fractures requires BMP2. The human Bmp2 gene also has been linked to osteoporosis and osteoarthritis. In addition, all forms of pathological calcification in the vasculature and in cardiac valves involve the pro-osteogenic BMP2. The diverse tissues, mechanisms, and diseases influenced by BMP2 are too numerous to list here (see OMIM: 112261). However, in all BMP2-influenced pathologies, changes in the behavior and differentiation of pluripotent mesenchymal cells are a recurring theme. Consequently, much effort has been devoted to identifying the molecules and conditions that influence BMP2 synthesis and the complex mechanisms that control Bmp2 gene expression. This review begins with an overview of the Bmp2 gene's chromosomal neighborhood and then summarizes and evaluates known regulatory mechanisms and inducers.

Abstract 3823: Dysregulation of bone morphogenetic protein 2 serves as a candidate molecular marker in human renal cell carcinoma

Abstract Introduction and Objective Bone morphogenetic protein-2 (BMP-2) has been shown to function as a tumor suppressor with several different cancers, while its expression level is down-regulated by CpG hypermethylation. We hypothesized that impaired regulation of BMP-2 through epigenetic pathways is associated with the pathogenesis of renal cell carcinoma (RCC). Methods To test our hypothesis, CpG methylation of the BMP-2 gene was analyzed using 2 RCC cell lines, and 96 matched RCC and normal renal tissues. We also performed functional analysis using BMP-2 restored RCC cells. Results BMP-2 mRNA expression was significantly enhanced in the RCC cells by 5-aza-2′-deoxycitidie treatment. The prevalence of BMP-2 promoter methylation was significantly higher, while BMP-2 mRNA expression was significantly lower in RCC samples as compared to normal kidney samples. Furthermore, a significant correlation was found between BMP-2 promoter methylation and mRNA transcription in the tumors. There was also a significant association of BMP-2 mRNA expression with tumor stage and lymph node involvement, while lower BMP-2 mRNA expression was significantly associated with poor overall survival after a radical nephrectomy. In RCC cells, BMP-2 restoration significantly inhibited cell proliferation, migration, invasion, and colony formation. In addition, BMP-2 overexpression effectively induced p21WAF1/CIP1 and p27KIP1 expression, and cellular apoptosis in RCC cells. Conclusion This is the first study to show inactivation of the BMP-2 gene via epigenetic pathways in RCC. Aberrant BMP-2 methylation and the resultant loss of BMP-2 expression may be useful as a molecular marker for designing improved diagnostic and therapeutic strategies for RCC. Citation Format: Yozo Mitsui, Hiroshi Hirata, Naoko Arichi, Miho Hiraki, Hiroaki Yasumono, Inik Chang, Shinichiro Fukuhara, Soichiro Yamamura, Varahram Shahryari, Guoren Deng, Sharanjot Saini, Shahana Majid, Yuichiro Tanaka, Rajvir Dahiya, Hiroaki Shiina. Dysregulation of bone morphogenetic protein 2 serves as a candidate molecular marker in human renal cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3823. doi:10.1158/1538-7445.AM2015-3823

LIM Mineralization Protein-1 Enhances Bone Morphogenetic Protein-2-Mediated Osteogenesis Through Activation of ERK1/2 MAPK Pathway and Upregulation of Runx2 Transactivity.

LIM mineralization protein-1 (LMP-1) is an intracellular regulator of bone formation. Upregulation of bone morphogenetic proteins (BMPs) and stabilization of BMP/Smad signaling have been proven to be the key mechanisms through which LMP-1 enhances osteogenesis. However, how LMP-1 regulates BMPs expression and related bone formation remains unclear. In this study, a LMP-1-induced osteogenesis cell model was used to study the molecular action of LMP-1 on BMP-2 expression and bone formation. The results show that overexpression of LMP-1 significantly increases, whereas downregulation of endogenous LMP-1 decreases BMP-2 expression and bone formation. Antagonism of BMP-2 with noggin or short hairpin BMP-2 significantly attenuates the osteoinductive effect of LMP-1, suggesting that the osteoinductive effect of LMP-1 is mediated by BMP-2. LMP-1 regulation of BMP-2 is found to occur at the transcription level using a luciferase reporter assay with a reporter construct containing a BMP-2 promoter. A promoter deletion assay reveals that -1000/-500 bp is the key regulated region by LMP-1. A Runx2-binding site is then located at -934/-920 bp and confirmed by luciferase assay using a reporter construct containing repeats of this Runx2-binding site and the site-directed mutagenesis analysis. Overexpression of LMP-1 significantly increases Runx2 expression. Downregulation of Runx2 expression significantly decreases BMP-2 promoter activity and BMP-2 expression. A ChIP assay demonstrates that LMP-1 increases the interaction between Runx2 and BMP-2 promoter. A luciferase reporter assay using the OSE2 promoter containing a Runx2-binding site confirms that Runx2 transactivity can be upregulated by LMP-1. Moreover, inhibiting the activation of different pathways with specific pathway inhibitors reveals that ERK1/2 MAPK activation is essential for LMP-1-induced upregulation of Runx2 transactivity and subsequent BMP-2 expression. In conclusion, our novel findings describe a positive regulatory effect of LMP-1 on BMP-2 expression and BMP-2-mediated osteogenesis. This effect occurs through activation of ERK1/2 pathway and subsequent upregulation of Runx2 transactivity.

An essential role for Ewing sarcoma gene (EWS) in early white adipogenesis.

White adipose tissue is important for mammalian energy homeostasis and metabolism. It was previously demonstrated that Ewing sarcoma gene (EWS) is essential for early classical brown fat lineage determination, but its role in white adipocyte differentiation is not known. Mouse embryonic fibroblasts (MEFs) lacking Ews and shRNA-mediated silencing of Ews in 3T3L1 preadipocytes were used to investigate the role of EWS in adipogenesis. White fat differentiation was determined by analyzing the expression of key adipogenic genes and by Oil red O staining. Following adipogenic stimulation, Ews expression arose rapidly in 3T3L1 cells during early induction period. Ews-null MEFs and 3T3L1 cells with reduced Ews expression failed to undergo adipogenesis. This was accompanied by significant reduction in the expression of critical early adipogenic regulators, Bmp2, Bmp4 (bone morphogenic protein 2 and 4), Cebpβ, and Cebpδ (CCAAT/enhancer binding protein β and δ). Complementation of recombinant BMP2 or BMP4 partially rescued adipogenesis in Ews-depleted 3T3L1 cells. These results demonstrate that EWS is essential during the early steps of white adipocyte differentiation, at least in part through its regulation of BMP2 and BMP4 expression.