Oxidative stress promotes autophagic cell death in human neuroblastoma cells with ectopic transfer of mitochondrial PPP2R2B (Bβ2)

Background

Bone morphogenetic proteins (BMPs) are pleiotropic members of the TGF-beta superfamily which regulate many biological processes during development and adult tissue homeostasis and are implicated in the pathogenesis of a number of human diseases. Their involvement in both normal and aberrant physiology creates a need for rapid, sensitive and methodologically simple assays to evaluate their activity from a variety of biological samples. Previously alkaline phosphatase based assays, ELISA and luciferase based bioassays have been developed to evaluate either individual or total BMP activity. In this paper, we describe a highly sensitive, rapid and specific cell based assay for the simultaneous quantification of total and isoform specific BMP activity from biological samples.

Results

A C2C12 cell line stably transfected with a reporter plasmid consisting of the BMP response element (BRE) from the Id1 promoter fused to a luciferase reporter gene was generated. Exposure of this cell line to human recombinant BMP2, BMP4, BMP6, BMP7, BMP9 and BMP10 induced the expression of luciferase which was quantified using a luminometer. This assay was specific for BMP activity as the other TGF-β superfamily members TGF-β 1, Nodal and Mullerian Inhibiting Substance (MIS) did not induce the reporter. Pretreatment of samples with isoform specific BMP blocking antibodies coupled with isoform specific titration analysis allowed the simultaneous identification and quantification of BMP4, BMP6 and BMP9 in serum samples.

Conclusion

The assay is rapid (<48 hours) and can be used to simultaneously measure isoform specific and total BMP activity in complex solutions.     

Masked mRNA is stored with aggregated nuclear speckles and its asymmetric redistribution requires a homolog of mago nashi

Background

The capacity of muscle to grow or to regenerate after damage is provided by adult stem cells, so called satellite cells, which are located under the basement lamina of each myofiber. Upon activation satellite cells enter the cell cycle, proliferate and differentiate into myoblasts, which fuse to injured myofibers or form new fibers. These processes are tightly controlled by many growth factors.

Results

Here we investigate the role of bone morphogenetic proteins (BMPs) during satellite cell differentiation. Unlike the myogenic C2C12 cell line, primary satellite cells do not differentiate into osteoblasts upon BMP signaling. Instead BMP signaling inhibits myogenic differentiation of primary satellite cells ex vivo. In contrast, inhibition of BMP signaling results in cell cycle exit, followed by enhanced myoblast differentiation and myotube formation. Using an in vivo trauma model we demonstrate that satellite cells respond to BMP signals during the regeneration process. Interestingly, we found the BMP inhibitor Chordin upregulated in primary satellite cell cultures and in regenerating muscles. In both systems Chordin expression follows that of Myogenin, a marker for cells committed to differentiation.

Conclusion

Our data indicate that BMP signaling plays a critical role in balancing proliferation and differentiation of activated satellite cells and their descendants. Initially, BMP signals maintain satellite cells descendants in a proliferating state thereby expanding cell numbers. After cells are committed to differentiate they upregulate the expression of the BMP inhibitor Chordin thereby supporting terminal differentiation and myotube formation in a negative feedback mechanism.     

Chordin knockdown enhances the osteogenic differentiation of human mesenchymal stem cells

Introduction

Bone morphogenetic proteins (BMPs) are critical growth factors in the osteogenic differentiation of progenitor cells during development in embryos and fracture repair in adults. Although recombinant BMPs are in use clinically, their clinical efficiency needs to be improved. The biological activities of BMPs are naturally regulated by extracellular binding proteins. The specific hypotheses tested in this study were as follows: the BMP inhibitor chordin is produced endogenously during the osteogenic differentiation of human mesenchymal stem cells (MSCs); and blockade of the activity of the BMP inhibitor increases the rate of osteogenic differentiation of human MSCs in vitro.

Methods

Human MSCs were derived from bone marrow from an iliac crest aspirate and from patients undergoing hip hemiarthroplasty. The MSCs were induced down the osteogenic pathway using standard osteogenic differentiation media, and expressions of BMP-2 and chordin were determined by gene expression analysis. During osteogenic differentiation, chordin knockdown was induced using RNA interference. Osteogenic differentiation was assessed by measuring the expression of alkaline phosphatase and calcium deposition. The differences in expression of osteogenic makers between groups were compared by analysis of variance, followed by Gabriel post hoc test.

Results

We demonstrate the expression of BMP-2 and chordin in human MSCs during osteogenic differentiation. Knockdown of chordin by RNA interference in vitro resulted in a significant increase in the expression of the osteogenic marker alkaline phosphatase and the deposition of extracellular mineral, in response to osteogenic stimulation.

Conclusion

We conclude that endogenously produced chordin constrains the osteogenic differentiation of human MSCs. The targeting of BMP inhibitors, such as chordin, may provide a novel strategy for enhancing bone regeneration.     

Identification of Small Molecule Activators of BMP Signaling

Bone Morphogenetic Proteins (BMPs) are morphogens that play a major role in regulating development and homeostasis. Although BMPs are used for the treatment of bone and kidney disorders, their clinical use is limited due to the supra-physiological doses required for therapeutic efficacy causing severe side effects. Because recombinant BMPs are expensive to produce, small molecule activators of BMP signaling would be a cost-effective alternative with the added benefit of being potentially more easily deliverable. Here, we report our efforts to identify small molecule activators of BMP signaling. We have developed a cell-based assay to monitor BMP signaling by stably transfecting a BMP-responsive human cervical carcinoma cell line (C33A) with a reporter construct in which the expression of luciferase is driven by a multimerized BMP-responsive element from the Id1 promoter. A BMP-responsive clone C33A-2D2 was used to screen a bioactive library containing ∼5,600 small molecules. We identified four small molecules of the family of flavonoids all of which induced luciferase activity in a dose-dependent manner and ventralized zebrafish embryos. Two of the identified compounds induced Smad1, 5 phosphorylation (P-Smad), Id1 and Id2 expression in a dose-dependent manner demonstrating that our assays identified small molecule activators of BMP signaling.     

Lenalidomide overcomes suppression of human natural killer cell anti-tumor functions by neuroblastoma microenvironment-associated IL-6 and TGFβ1

Background

Treatment for children with high-risk neuroblastoma with anti-disialoganglioside mAb ch14.18, IL-2, and GM-CSF plus 13-cis-retinoic acid after myeloablative chemotherapy improves survival, but 40 % of patients still relapse during or after this therapy. The microenvironment of high-risk neuroblastoma tumors includes macrophages, IL-6, and TGFβ1. We hypothesized that this microenvironment suppresses anti-tumor functions of natural killer (NK) cells and that lenalidomide, an immune-modulating drug, could overcome suppression.

Methods

Purified NK cells were cultured with IL-2, neuroblastoma/monocyte-conditioned culture medium (CM), IL-6, TGFβ1, and lenalidomide in various combinations and then characterized using cytotoxicity (direct and antibody-dependent cell-mediated cytotoxicity), cytokine, flow cytometry, and Western blotting assays. Anti-tumor activity of NK cells with lenalidomide, ch14.18, or both was evaluated with a xenograft model of neuroblastoma.

Results

CM from neuroblastoma/monocyte co-cultures contains IL-6 and TGFβ1 that suppress IL-2 activation of NK cell cytotoxicity and IFNγ secretion. IL-6 and TGFβ1 activate the STAT3 and SMAD2/3 pathways in NK cells and suppress IL-2 induction of cytotoxicity, granzymes A and B release, perforin expression, and IFNγ secretion. Lenalidomide blocks IL-6 and TGFβ1 activation of these signaling pathways and inhibits their suppression of NK cells. Neuroblastoma cells in NOD/SCID mice exhibit activated STAT3 and SMAD2/3 pathways. Their growth is most effectively inhibited by co-injected peripheral blood mononuclear cells (PBMC) containing NK cells when mice are treated with both ch14.18 and lenalidomide.

Conclusion

Immunotherapy with anti-tumor cell antibodies may be improved by lenalidomide, which enhances activation of NK cells and inhibits their suppression by IL-6 and TGFβ1.     

The TGFβ superfamily in stem cell biology and early mammalian embryonic development

Background

Members of the Transforming Growth Factor-beta (TGFβ) superfamily of cytokines are essential for early embryonic development and play crucial roles in pluripotency and differentiation of embryonic stem cells in vitro.

Scope of review

In this review, we discuss how TGFβ family signals are read by cells and how they are modulated by the cellular context. Furthermore, we review recent advances in our understanding of TGFβ function in embryonic stem cells and point out hot topics at the intersection of TGFβ signaling and stem cell biology fields.

Major conclusion

TGFβ family signals are essential for early mammalian development and the importance of this pathway is reflected in pluripotent stem cells derived from the mammalian embryo.

General significance

Understanding signaling pathways underlying pluripotency and cell fate specification holds promises for the advent of personalized regenerative medicine. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

The interaction of canonical bone morphogenetic protein- and Wnt-signaling pathways may play an important role in regulating cartilage degradation in osteoarthritis

Bone morphogenetic proteins (BMPs) and Wnts are important signaling protein families with key roles in embryologic, patterning, development, and tissue remodeling in growth. BMP and Wnt-β-catenin are highly evolutionarily conserved pathways that, though often regulating similar cellular events, are independent signaling mechanisms that can have complementary or antagonistic effects depending on various factors, including cell type and developmental stage. Although BMP and Wnt-β-catenin have the ability to act entirely independently, there is a developing body of evidence for specific extra- and intra-cellular molecular interactions and crosstalk that occur between BMP and Wnt-β-catenin signaling and that again this may be cell type-specific. In the previous issue of Arthritis Research & Therapy, Papathanasiou and colleagues provide novel insights into the role and direct interaction of BMP2 and canonical Wnt-β-catenin signaling in regulating chondrocyte hypertrophy and matrix metalloproteinase/a disintegrin like and metalloproteinase with thrombospondin type I motif (MMP/ADAMTS) synthesis in osteoarthritis.In the previous issue of Arthritis Research & Therapy, Papathanasiou and colleagues [1] provide novel insights into the role and direct interaction of bone morphogenetic protein 2 (BMP2) and canonical Wnt-β-catenin signaling in regulating chondrocyte hypertrophy and matrix metalloproteinase (MMP)/aggrecanolytic ADAMTS (a disintegrin like and metalloproteinase with thrombospondin type I motif) synthesis in osteoarthritis (OA). OA is the most common cause of joint pain and disability, and with increasing age and obesity of the population, the already major socioeconomic importance will continue to increase. Currently, in most Western cultures, OA afflicts more than 10% of the entire population and over a third of those over 65; an estimated 25 to 30 million people in the US suffer from this disease. The central pathological feature of OA is often considered to be the progressive destruction of articular cartilage that normally provides the load-bearing surface in the joint. Much has been learned in recent years about the mechanisms that drive cartilage matrix breakdown and loss in OA, and chondrocyte-derived metalloproteinases, particularly the ADAMTS and collagenolytic MMPs, have a key role. It is evident that a phenotypic shift in the mature articular chondrocyte to a cell type that displays many characteristics typical of hypertrophic cells in the lower zones of the growth plate is a typical feature of OA and is associated with the progressive cartilage breakdown observed (reviewed in [2]). Less clearly understood are the specific signaling pathways involved in regulating the chondrocyte phenotype, how they interact, and whether this changes in health and in diseases such as OA.BMPs and Wnts are important signaling protein families with key roles in embryologic, patterning, development, and tissue remodeling in growth. BMP and Wnt-β-catenin are highly evolutionarily conserved pathways that, though often regulating similar cellular events, are independent signaling mechanisms that can have complementary or antagonistic effects depending on various factors, including cell type and developmental stage (reviewed in [3]). Although BMP and Wnt-β-catenin have the ability to act entirely independently, there is a developing body of evidence for specific extra-and intra-cellular molecular interactions and crosstalk that occur between BMP and Wnt-β-catenin signaling and that again may be cell type-specific [3]. In addition to having a key role in development, BMPs and Wnts are emerging as critical regulators of bone and cartilage homeostasis in the adult and, importantly, in the onset and progression of musculoskeletal diseases.BMPs are multi-functional growth factors that belong to the transforming growth factor-β super family. Evidence suggests that BMP signaling is mediated primarily through the canonical BMP-Smad pathway in chondrocytes. BMPs bind the type II receptor and phosphorylate type I serine or threonine receptors, which subsequently phosphorylate Smad1, Smad5, and Smad8. BMPs are known to induce human mesenchymal stem cells to differentiate into chondrocytes, and BMP2 is a crucial local factor for chondrocyte proliferation and maturation during endochondral ossification [4,5]. In their report, Papathanasiou and colleagues show not only that human end-stage OA chondrocytes produce BMP2 and BMP4 but also, importantly, that BMP2, but not BMP4, can drive expression of low-density lipoprotein receptor 5 (LRP5). LRP5 is one of the most important co-receptors in the canonical Wnt-β-catenin signaling pathway; binding of Wnt ligands to the frizzled/LRP co-receptor complex leads to β-catenin stabilization, nuclear translocation, and activation of target genes.There is a large body of evidence demonstrating the central role for Wnt signaling in regulating adult bone turnover; increased β-catenin activity inducing bone production and inhibition of soluble antagonists is an emerging therapeutic approach for osteoporotic and inflammatory bone loss [6,7]. In cartilage, Wnt-β-catenin signaling plays a dual role; activity is essential for chondrocyte proliferation and maintenance of their phenotypic characteristics [8], but excessive activity increases chondrocyte hypertrophy and expression of cartilage degrades metalloproteinases [9]. The effect may be cell type- specific, and Wnt-β-catenin activation is essential for maintenance of the superficial zone chondrocyte phenotype and proteoglycan 4 (lubricin) expression [8]. Inhibition of β-catenin rapidly leads to downregulation of lubricin and increased collagen × expression in superficial zone chondrocytes. In chondrocytes from human end-stage OA cartilage, activation of canonical Wnt-β-catenin signaling by Wnt-2B and Wnt-16 can drive MMP and aggrecanase production [9]. Understanding the mechanisms that regulate Wnt signaling in chondrocytes in OA may provide keys to controlling cartilage degradation.One of the most important findings by Papathanasiou and colleagues is the demonstration of a new and unique function of BMP2 in chondrocytes in acting as a regulator of canonical Wnt-β-catenin signaling. Treatment of both normal and OA primary human chondrocytes with BMP2 for 12 hours enhanced total β-catenin expression while diminishing the degradation of β-catenin (phospho-β-catenin). This was accompanied by significant increases in mRNA for key cartilage-degrading enzymes MMP-13 and ADAMTS-5 in concert with a shift toward a hypertrophic chondrocyte phenotype as measured by increased collagen × expression. This effect was absent in LRP5 small interfering RNA (siRNA) pretreated chondrocytes and did not occur with BMP4, suggesting the unique function of BMP2 in specifically upregulating LRP5 and augmenting Wnt-β-catenin signaling. The BMP2-driven increase in LRP5 mRNA was mediated through Smad1/5/8 binding to the LRP5 promoter.The paper by Papathanasiou and colleagues adds to the accumulating evidence that increased or perhaps excessive activation of canonical Wnt-β-catenin signaling in chondrocytes is detrimental and contributes to OA cartilage degradation. Therapeutic approaches to block or suppress canonical Wnt-β-catenin signaling may protect cartilage damage in end-stage OA. There are many naturally occurring Wnt-β-catenin signaling antagonists, including dickkopf 1 (DKK1), secreted frizzled-related proteins (sFRPs), and sclerostin (SOST). Evidence suggests that circulating DKK1 levels negatively correlate with biomarkers of cartilage breakdown in patients with OA [10]; sFRP3 knockout mice have augmented cartilage proteoglycan loss in a collagenase-induced instability model of arthritis [11], and co-treatment of SOST with pro-inflammatory cytokines can attenuate cartilage matrix breakdown [12]. The role of SOST is interesting in light of the interaction between BMP2 and Wnt signaling pathways reported by Papathanasiou and colleagues. It appears that SOST can also function as a BMP antagonist in osteoblast and osteocytes by binding intra-cellularly to BMP7 and targeting the growth factor for proteosomal degradation [13]. This provides yet another mechanism by which BMP and Wnt signaling pathways may directly interact; it will be interesting to see whether this effect of SOST on BMP7 (and possibly other BMPs) also occurs in chondrocytes, particularly in OA, where chondrocyte SOST expression is increased [12].The BMP and Wnt signaling pathways are critical in regulating chondrocytes and maintaining the health and integrity of cartilage matrix. In other cell types/organs such as those in bone, it is the combinatorial integration and complex crosstalk between these two pathways that are emerging as significant regulators of development and tissue homeostasis [3]. The findings by Papathanasiou and colleagues suggest that similar signaling pathway interactions may be important in chondrocytes and could play a role in the development and progression of OA. A better appreciation of chondrocyte regulatory mechanisms may provide new avenues for development of therapeutic approaches for the treatment of OA.     

Repulsive Guidance Molecule (RGM) Family Proteins Exhibit Differential Binding Kinetics for Bone Morphogenetic Proteins (BMPs)

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor beta superfamily that exert their effects via type I and type II serine threonine kinase receptors and the SMAD intracellular signaling pathway to regulate diverse biologic processes. Recently, we discovered that the repulsive guidance molecule (RGM) family, including RGMA, RGMB, and RGMC/hemojuvelin (HJV), function as co-receptors that enhance cellular responses to BMP ligands. Here, we use surface plasmon resonance to quantitate the binding kinetics of RGM proteins for BMP ligands. We show that among the RGMs, HJV exhibits the highest affinity for BMP6, BMP5, and BMP7 with K_D 8.1, 17, and 20 nM respectively, versus 28, 33, and 166 nM for RGMB, and 55, 83, and 63 nM for RGMA. Conversely, RGMB exhibits preferential binding to BMP4 and BMP2 with K_D 2.6 and 5.5 nM respectively, versus 4.5 and 9.4 nM for HJV, and 14 and 22 nM for RGMA, while RGMA exhibits the lowest binding affinity for most BMPs tested. Among the BMP ligands, RGMs exhibit the highest relative affinity for BMP4 and the lowest relative affinity for BMP7, while none of the RGMs bind to BMP9. Thus, RGMs exhibit preferential binding for distinct subsets of BMP ligands. The preferential binding of HJV for BMP6 is consistent with the functional role of HJV and BMP6 in regulating systemic iron homeostasis. Our data may help explain the mechanism by which BMPs exert cell-context specific effects via a limited number of type I and type II receptors.     

Diverse Bone Morphogenetic Protein Expression Profiles and Smad Pathway Activation in Different Phenotypes of Experimental Canine Mammary Tumors

Background

BMPs are currently receiving attention for their role in tumorigenesis and tumor progression. Currently, most BMP expression studies are performed on carcinomas, and not much is known about the situation in sarcomas.

Methodology/Principal Findings

We have investigated the BMP expression profiles and Smad activation in clones from different spontaneous canine mammary tumors. Spindle cell tumor and osteosarcoma clones expressed high levels of BMPs, in particular BMP-2, -4 and -6. Clones from a scirrhous carcinoma expressed much lower BMP levels. The various clones formed different tumor types in nude mice but only clones that expressed high levels of BMP-6 gave bone formation. Phosphorylated Smad-1/5, located in the nucleus, was detected in tumors derived from clones expressing high levels of BMPs, indicating an active BMP signaling pathway and BMP-2 stimulation of mammary tumor cell clones in vitro resulted in activation of the Smad-1/5 pathway. In contrast BMP-2 stimulation did not induce phosphorylation of the non-Smad pathway p38 MAPK. Interestingly, an increased level of the BMP-antagonist chordin-like 1 was detected after BMP stimulation of non-bone forming clones.

Conclusions/Significance

We conclude that the specific BMP expression repertoire differs substantially between different types of mammary tumors and that BMP-6 expression most probably has a biological role in bone formation of canine mammary tumors.     

Extracellular cyclophilin-A stimulates ERK1/2 phosphorylation in a cell-dependent manner but broadly stimulates nuclear factor kappa B

Background

Although the peptidyl-prolyl isomerase, cyclophilin-A (peptidyl-prolyl isomerase, PPIA), has been studied for decades in the context of its intracellular functions, its extracellular roles as a major contributor to both inflammation and multiple cancers have more recently emerged. A wide range of activities have been ascribed to extracellular PPIA that include induction of cytokine and matrix metalloproteinase (MMP) secretion, which potentially underlie its roles in inflammation and tumorigenesis. However, there have been conflicting reports as to which particular signaling events are under extracellular PPIA regulation, which may be due to either cell-dependent responses and/or the use of commercial preparations recently shown to be highly impure.

Methods

We have produced and validated the purity of recombinant PPIA in order to subject it to a comparative analysis between different cell types. Specifically, we have used a combination of multiple methods such as luciferase reporter screens, translocation assays, phosphorylation assays, and nuclear magnetic resonance to compare extracellular PPIA activities in several different cell lines that included epithelial and monocytic cells.

Results

Our findings have revealed that extracellular PPIA activity is cell type-dependent and that PPIA signals via multiple cellular receptors beyond the single transmembrane receptor previously identified, Extracellular Matrix MetalloPRoteinase Inducer (EMMPRIN). Finally, while our studies provide important insight into the cell-specific responses, they also indicate that there are consistent responses such as nuclear factor kappa B (NFκB) signaling induced in all cell lines tested.

Conclusions

We conclude that although extracellular PPIA activates several common pathways, it also targets different receptors in different cell types, resulting in a complex, integrated signaling network that is cell type-specific.