Role Of Bone Morphogenetic Proteins Research Articles

The role of bone morphogenetic proteins in ankylosing spondylitis

Ankylosing spondylitis (AS), the best-known form of spondyloarthritis (SpA), is a remodelling arthritis characterized by chronic inflammation and bone formation. Ankylosis of the axial skeleton and sacroiliac joints leads to an impairment of spinal mobility, progressive spinal fusion and an increased risk of spinal fractures. The nature of the relationship between inflammation and new bone formation in AS has been controversial and questions remain as to whether there is a direct relationship between inflammation and new bone formation. Like others, we have hypothesized that the molecular pathways underlying ankylosis recapitulate the process of endochondral bone formation and that bone morphogenetic proteins (BMPs) play a key role in this process in AS. Furthermore, we discuss the entheseal stress hypothesis, which proposes that inflammation and ankylosis are linked but largely independent processes, and consider observations from mouse models and other human diseases which also imply that biomechanical factors contribute to the pathogenesis of AS. As current therapeutics, such as tumour necrosis factor inhibitors do not impede disease progression and ankylosis in AS, it is the pathways discussed in this review that are the now the focus for the identification of future drug targets.

The WNT Inhibitor Dickkopf 1 and Bone Morphogenetic Protein 4 Rescue Adipogenesis in Hypertrophic Obesity in Humans

Overweight characterized by inappropriate expansion of adipose cells (hypertrophic obesity) is associated with the metabolic syndrome and is caused by an inability to recruit and differentiate new precursor cells. We examined the role of bone morphogenetic protein 4 (BMP4) and WNT activation in the regulation of human adipose cell differentiation. Cluster of differentiation (CD)14+/45+ and CD31+ cells were first removed before the remaining stromal vascular cells of human subcutaneous biopsy specimens were differentiated with/without different WNT inhibitors and/or BMP4. Inhibition of WNT and induction of Dickkopf 1 (DKK1) were markers of precursor cells undergoing excellent differentiation. The addition of DKK1 inhibited WNT activation and promoted adipogenesis in cells with a low degree of differentiation. The positive effect of DKK1, inhibiting cellular WNT activation by binding to the Kremen/LDL receptor–related protein receptors, was not seen with inhibitors of secreted WNT ligands. BMP4 increased differentiation, and BMP4 in the presence of DKK1 produced an additive effect. There was an apparent cross-talk between differentiation and commitment because BMP4 expression increased in differentiating adipocytes, and the addition of the BMP4 inhibitor, Noggin, reduced precursor cell differentiation. Thus, differentiated human adipose cells can promote adipogenesis via endogenous BMP4 activation, and the impaired adipogenesis in hypertrophic obesity is mainly due to an inability to suppress canonical WNT and to induce DKK1.

Bone morphogenetic protein 6 promotes FSH receptor and anti-Müllerian hormone mRNA expression in granulosa cells from hen prehierarchal follicles

A growing body of literature provides evidence of a prominent role for bone morphogenetic proteins (BMPs) in regulating various stages of ovarian follicle development. Several actions for BMP6 have been previously reported in the hen ovary, yet only within postselection (preovulatory) follicles. The initial hypothesis tested herein is that BMP6 increases FSH receptor (FSHR) mRNA expression within the granulosa layer of prehierarchal (6-8 mm) follicles (6-8 GC). BMP6 mRNA is expressed at higher levels within undifferentiated (1-8 mm) follicles compared with selected (≥9 mm) follicles. Recombinant human (rh) BMP6 initiates SMAD1, 5, 8 signaling in cultured 6-8 GC and promotes FSHR mRNA expression in a dose-related fashion. In addition, a 21 h preculture with rhBMP6 followed by a 3 h challenge with FSH increases cAMP accumulation, STAR (StAR) expression, and progesterone production. Interestingly, rhBMP6 also increases expression of anti-Müllerian hormone (AMH) mRNA in cultured 6-8 GC. This related BMP family member has previously been implicated in negatively regulating FSH responsiveness during follicle development. Considering these data, we propose that among the paracrine and/or autocrine actions of BMP6 within prehierarchal follicles is the maintenance of both FSHR and AMH mRNA expression. We predict that before follicle selection, one action of AMH within granulosa cells from 6 to 8 mm follicles is to help suppress FSHR signaling and prevent premature granulosa cell differentiation. At the time of selection, we speculate that the yet undefined signal directly responsible for selection initiates FSH responsiveness. As a result, FSH signaling suppresses AMH expression and initiates the differentiation of granulosa within the selected follicle.

The role of bone morphogenetic proteins in ankylosing spondylitis

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta superfamily and were originally identified as proteins that induce a full cascade of endochondral bone formation in vivo and ectopically. We hypothesized that the BMP signaling pathway could also play a role in the process of ankylosis that characterizes ankylosing spondylitis and related spondyloarthritides. By over expression of noggin, a BMP antagonist, in a dedicated mouse model of joint ankylosis, we provided evidence for this hypothesis. Our current studies focus on the relationship between stromal cell activation and inflammation, a link that is essential in ankylosing spondylitis. Emerging functional and genetic evidence further corroborates the essential role of BMPs in these processes. BMPs can trigger different downstream signaling cascades, in particular Smad and p38 signaling. Inhibition of p38 signaling in vivo in the spontaneous ankylosing enthesitis model appears to accelerate ankylosis while inhibiting in vitro chondrogenesis. Genetic analysis of intercrosses between the susceptible DBA/1 strain and the H2 identical Balb/c strain point towards a region containing the BMP type Ib receptor as a factor determining genetic susceptibility. Recent clinical data suggest that levels of BMPs are higher in patients with progressive ankylosis in comparison with patients that show no structural progression of disease. Taken together, these data support our hypothesis that BMP signaling is a therapeutic target in ankylosing spondylitis.

Palmitate Promotes the Paracrine Effects of Macrophages on Vascular Smooth Muscle Cells: The Role of Bone Morphogenetic Proteins

Saturated fatty acids are known to activate macrophages and induce vascular inflammation. Although cytokines from activated macrophage influence other vascular cells, the influence of saturated fatty acids on the paracrine effect of macrophages is not fully understood yet. Here we examined the impact of palmitate on the effect of macrophages on vascular smooth muscle cells (SMCs) and their mediators. SMCs proliferation increased significantly after treatment with conditioned media from palmitate-stimulated RAW264.7 cells. SMC migration was found to be greater after treatment with palmitate-conditioned media. SM α-actin and SM22α were decreased in SMCs treated with palmitate-conditioned media. When stimulated with palmitate, RAW264.7 cells secreted more bone morphogenetic protein (BMP)2 and BMP4 into the cell culture media. SMC proliferation, migration, and phenotypic changes were attenuated after treatment of neutralizing antibodies against BMPs or knockdown of BMPs with siRNA. The influences of these proteins were further confirmed by direct treatment of recombinant BMP2 and BMP4 on SMCs. Particularly, the effects of BMPs on SMC migration on phenotypic change were obvious, whereas their effect on SMC proliferation seemed not significant or modest. In conclusion, palmitate promoted macrophages' paracrine effects on SMC proliferation, migration, and phenotypic change. The effect of stimulated macrophages was mediated, at least in part, by BMP2 and BMP4. These results suggest a novel mechanism linking saturated fatty acids and the progression of vascular diseases that is possibly mediated by BMPs from macrophages.

VEGF-D Promotes Tumor Metastasis by Regulating Prostaglandins Produced by the Collecting Lymphatic Endothelium

Lymphatic metastasis is facilitated by lymphangiogenic growth factors VEGF-C and VEGF-D that are secreted by some primary tumors. We identified regulation of PGDH, the key enzyme in prostaglandin catabolism, in endothelial cells of collecting lymphatics, as a key molecular change during VEGF-D-driven tumor spread. The VEGF-D-dependent regulation of the prostaglandin pathway was supported by the finding that collecting lymphatic vessel dilation and subsequent metastasis were affected by nonsteroidal anti-inflammatory drugs (NSAIDs), known inhibitors of prostaglandin synthesis. Our data suggest a control point for cancer metastasis within the collecting lymphatic endothelium, which links VEGF-D/VEGFR-2/VEGFR-3 and the prostaglandin pathways. Collecting lymphatics therefore play an active and important role in metastasis and may provide a therapeutic target to restrict tumor spread.

Role of Bone Morphogenetic Proteins and Their Antagonists during Fracture Healing

Role of Bone Morphogenetic Proteins and Their Antagonists during Fracture Healing

The application of bone morphogenetic proteins to periodontal and peri-implant tissue regeneration: A literature review

Progress in understanding the role of bone morphogenetic proteins (BMPs) in craniofacial and tooth development and the demonstration of stem cells in periodontal ligament have set the stage for periodontal regenerative therapy and tissue engineering. Furthermore, recent approval by the Food and Drug Administration of recombinant human BMPs for accelerating bone fusion in slow-healing fractures indicates that this protein family may prove useful in designing regenerative treatments in periodontics. In the near term, these advances are likely to be applied to periodontal surgery; ultimately, they may facilitate approaches to regenerating whole lost periodontal structures.

BMP2 Induces PANC-1 cell invasion by MMP-2 overexpression through ROS and ERK

The emerging roles of bone morphogenetic proteins (BMPs) in the initiation and progression of multiple cancers have drawn great attention in cancer research. We hypothesized that BMP2 promotes cancer metastasis by modulating MMP-2 secretion and activity through intracellular ROS regulation and ERK activation in human pancreatic cancer. Our data show that stimulation of PANC-1 cells with BMP2 induced MMP-2 secretion and activation, associated with decreased E-cadherin expression, resulting in epithelial-to-mesenchymal transformation (EMT) and cell invasion. Blockade of ROS by the ROS scavenger, 2-MPG, abolished cell invasion, inhibited the EMT process and decreased MMP-2 expression, suggesting ROS accumulation caused an increase in MMP-2 expression in BMP2-stimulated PANC-1 cell invasion. Furthermore, treatment of PANC-1 cells with 2-MPG or ERK inhibitor PD98059 reduced the phosphorylation of ERK, resulting in attenuation of BMP2-induced cell invasion and MMP-2 activation. Taken together, these results suggest that BMP2 induces the cell invasion of PANC-1 cells by enhancing MMP-2 secretion and acting through ROS accumulation and ERK activation.

Outcomes for Single-Level Lumbar Fusion: The Role of Bone Morphogenetic Protein

Outcomes for Single-Level Lumbar Fusion: The Role of Bone Morphogenetic Protein

BMP-7/TGF-β1 signalling in myoblasts: Components involved in signalling and BMP-7-dependent blockage of TGF-β-mediated CTGF expression

We and others have recently described the antagonistic role of Bone morphogenetic protein-7 (BMP-7) in TGF-β signalling and myogenic differentiation. To specify the underlying mechanism(s), we here analysed the expression and function of the individual components mediating TGF-β1 and BMP-7 responses. We found that BMP-7 at a concentration of 25ng/ml induces signalling exclusively via ALK2 and ALK3 leading to the activation of Smad1 and Smad5 and subsequent expression of Id proteins. In contrast, low doses of TGF-β1 (0.1ng/ml) lead to an exclusive activation of ALK5 and phosphorylation of Smad2 and Smad3 that regulate specific target genes including connective tissue growth factor (CTGF). CTGF is rapidly induced by TGF-β1 already 1h after stimulation and reduced by BMP-7 application. Smad1/Smad5 or Id1/2 overexpression reduced the TGF-β1-mediated expression of CTGF. However, although siRNA-mediated knock down of Alk2/3 or Smad1/5 counteracts the BMP-7 effect on basal CTGF expression there was no consistent reversion of the observed BMP-7 effect on TGF-β1-mediated CTGF expression. Moreover, ALK5 inhibition using the SB431542 inhibitor significantly affected CTGF expression only at later time points whereas ERK1/2 inhibition completely abrogated CTGF expression. These findings point towards a regulatory role of BMP-7 that relies on modulation of Mitogen-activated protein kinases rather than mechanisms that are exclusively driven by differential Smad activation.

BMP3 Suppresses Osteoblast Differentiation of Bone Marrow Stromal Cells via Interaction with Acvr2b

Enhancing bone morphogenetic protein (BMP) signaling increases bone formation in a variety of settings that target bone repair. However, the role of BMP in the maintenance of adult bone mass is not well understood. Targeted disruption of BMP3 in mice results in increased trabecular bone formation, whereas transgenic overexpression of BMP3 in skeletal cells leads to spontaneous fracture, consistent with BMP3 having a negative role in bone mass regulation. Here we investigate the importance of BMP3 as a mediator of BMP signaling in the adult skeleton. We find that osteoblasts (OBL) and osteocytes are the source of BMP3 in adult bone. Using in vitro cultures of primary bone marrow stromal cells, we show that overexpression of BMP3 suppresses OBL differentiation, whereas loss of BMP3 increases colony-forming unit fibroblasts and colony-forming unit OBL. The ability of BMP3 to affect OBL differentiation is due to its interaction with activin receptor type 2b (Acvr2b) because knockdown of endogenous Acvr2b in bone marrow stromal cells reduces the suppressive effect of BMP3 on OBL differentiation. These findings best fit a model in which BMP3, produced by mature bone cells, acts to reduce BMP signaling through Acvr2b in skeletal progenitor cells, limiting their differentiation to mature OBL. Our data further support the idea that endogenous BMPs have a physiological role in regulating adult bone mass.

A Role for Bone Morphogenetic Protein-4 in Lymph Node Vascular Remodeling and Primary Tumor Growth

Lymph node metastasis, an early and prognostically important event in the progression of many human cancers, is associated with expression of VEGF-D. Changes to lymph node vasculature that occur during malignant progression may create a metastatic niche capable of attracting and supporting tumor cells. In this study, we sought to characterize molecules expressed in lymph node endothelium that could represent therapeutic or prognostic targets. Differential mRNA expression profiling of endothelial cells from lymph nodes that drained metastatic or nonmetastatic primary tumors revealed genes associated with tumor progression, in particular bone morphogenetic protein-4 (BMP-4). Metastasis driven by VEGF-D was associated with reduced BMP-4 expression in high endothelial venules, where BMP-4 loss could remodel the typical high-walled phenotype to thin-walled vessels. VEGF-D expression was sufficient to suppress proliferation of the more typical BMP-4-expressing high endothelial venules in favor of remodeled vessels, and mechanistic studies indicated that VEGF receptor-2 contributed to high endothelial venule proliferation and remodeling. BMP-4 could regulate high endothelial venule phenotype and cellular function, thereby determining morphology and proliferation responses. Notably, therapeutic administration of BMP-4 suppressed primary tumor growth, acting both at the level of tumor cells and tumor stromal cells. Together, our results show that VEGF-D-driven metastasis induces vascular remodeling in lymph nodes. Furthermore, they implicate BMP-4 as a negative regulator of this process, suggesting its potential utility as a prognostic marker or antitumor agent.

Peripheral joint ankylosis in the spontaneous model of arthritis in DBA/1 mice is genetically associated with BMP signaling

Progression of ankylosis in patients with ankylosing spondylitis is highly variable. This suggests that ankylosis is at least partially due to genetic factors. We used the ankylosing enthesits model in DBA/1 mice to search for associated genes. Male DBA/1 were crossed with female BALB/c mice. Male F2 mice from different litters were studied by histomorphology at 26 weeks. 159 markers with sequence-length polymorphisms on the autosomes were selected. Median spacing between the markers was 6.9 cM. 162 F2 male mice were studied. Genes in regions of interests were linked to skeletal development, bone morphogenetic protein (BMP) and Wnt signaling pathways with the Gene Ontology database. The association was evaluated by Chi-square tests with a False Discovery Rate (FDR) algorithm. Incidence of ankylosing enthesitis was lower in the F2 generation as compared to wild-type DBA/1 males (42% vs. 72%; p<0.0001). When applying the FDR algorithm for 159 markers, associations with D3MIT199 and D3MIT160 were significant (p<0.016). Adjacent markers were additionally genotyped. In the associated region between markers D3MIT42 and D3MIT129, 162 genes were found among which Bmpr1b, Cxxc4, Lef1, Papss1, Pitx2, and Ube2d3. Only BMP receptor type 1b (BMPr1b) was specifically upregulated in mice with spontaneous arthritis as demonstrated by quantitative RT-PCR. By using F2 mouse genetics in the analysis of ankylosis, we identified a locus on chromosome 3 that shows association. Within this locus, several genes could play a role in ankylosis but the expression profile suggests that BMP receptor 1b is involved, further supporting a critical role for BMPs in this process.

BMP signals and the transcriptional repressor BLIMP1 during germline segregation in the mammalian embryo

Molecular factors and tissue compartments involved in the foundation of the mammalian germline have been mainly described in the mouse so far. To find mechanisms applicable to mammals in general, we analyzed temporal and spatial expression patterns of the transcriptional repressor BLIMP1 (also known as PRDM1) and the signaling molecules BMP2 and BMP4 in perigastrulation and early neurulation embryos of the rabbit using whole-mount in situ hybridization and high-resolution light microscopy. Both BMP2 and BMP4 are expressed in annular domains at the boundary of the embryonic disc, which—in contrast to the situation in the mouse—partly belong to intraembryonic tissues. While BMP2 expression begins at (pregastrulation) stage 1 in the hypoblast, BMP4 expression commences—distinctly delayed compared to the mouse—diffusely at (pregastrulation) stage 2; from stage 3 onwards, BMP4 is expressed peripherally in hypoblast and epiblast and in the mesoderm at the posterior pole of the embryonic disc. BLIMP1 expression begins throughout the hypoblast at stage 1 and emerges in single primordial germ cell (PGC) precursors in the posterior epiblast at stage 2 and then in single mesoderm cells at positions identical to those identified by PGC-specific antibodies. These expression patterns suggest that function and chronology of factors involved in germline segregation are similar in mouse and rabbit, but higher temporal and spatial resolution offered by the rabbit demonstrates a variable role of bone morphogenetic proteins and makes “blimping” a candidate case for lateral inhibition without the need for an allantoic germ cell niche.

Expression of cyclin A and bone morphogenetic protein receptors and response to induction therapy in patients with acute leukemias

Bone morphogenetic proteins (BMPs) are multifunctional cytokines that belong to the transforming growth factor β (TGFβ) family. They participate in the regulation of growth, differentiation and apoptosis in a variety of cell types including hematopoietic lineages. To date, the role of BMPs in carcinogenesis has not been well known. Cyclin A is a cell cycle regulatory protein which plays the role of a parameter of cell proliferation in various types of carcinomas including hematological malignancies. The role of BMPRIA, BMPRIB, BMPRI and cyclin A in the pathogenesis of acute leukemias remains unclear. The aim of this study was to evaluate the expression of BMP receptors and cyclin A on blast cells and their possible relationship with clinical outcome. Seventy patients with acute leukemias (28 female and 42 male) and 10 aged-matched healthy controls were studied. All patients were examined before cytostatic treatment. The expression of BMP receptors and cyclin A was detected by flow cytometry. The results show that higher expression of BMPRIA, BMPRIB, BMPRII and cyclin A is related with a higher complete response (CR) rate, higher overall survival (OS) and lower relapse risk. The expressions of BMPRIA, BMPRIB, BMPRII and cyclin A could be useful as prognostic parameters of the proliferation status of acute leukemia cells, but further studies are needed to assess this phenomenon.

Interaction between gonadotropin-releasing hormone and bone morphogenetic protein-6 and -7 signaling in LβT2 gonadotrope cells

It is known that bone morphogenetic proteins (BMPs) regulate gonadotropin transcription and production by pituitary gonadotrope cells. However, the role of BMPs in gonadotropin-releasing hormone (GnRH)-induced FSH production remains uncertain. Here, we describe a functional link between BMP-6 and BMP-7 signals and FSH transcriptional activity induced by GnRH using mouse gonadotrope LβT2 cells. In LβT2 cells, BMP-6 and BMP-7 increased mouse FSHβ-promoter activity in a concentration-dependent manner. The induction by BMP-6 and BMP-7 was inhibited by treatment with extracellular domains of ActRII but not BMPRII. These findings suggest that the type II receptor ActRII participates in BMP-induced FSHβ transcription regulation. Notably, BMP-6, but not BMP-7, enhanced GnRH-induced FSHβ-promoter activity in LβT2 cells. Since GnRH stimulated MAPK phosphorylation in LβT2 cells, a functional link between MAPK and FSHβ transcription was examined. Inhibition of the ERK pathway, but not that of p38 or SAPK/JNK signaling, suppressed GnRH-induced FSHβ transcription, suggesting that ERK is functionally involved in GnRH-induced FSHβ transcription. Co-treatment with BMP-7, but not with BMP-6, suppressed GnRH-induced MAPK phosphorylation in LβT2 cells. Thus, the difference between BMP-6 and BMP-7 in enhancing GnRH-induced FSHβ transcription may be due to the differential effects of BMP ligands on GnRH-induced ERK signaling. On the other hand, GnRH reduced Smad1/5/8 phosphorylation but increased Smad6/7 expression. These findings imply the presence of a functional link between GnRH action, MAPK signaling and the BMP system in pituitary gonadotropes for fine-tuning of FSH gene expression.

Dmitriev et al. respond

We appreciate the interest in our work by Salisbury et al. The role of bone morphogenetic protein-2 (BMP-2) in bone formation has been extensively characterized over the last 30 years [ 1 Boden S.D. Bioactive factors for bone tissue engineering. Clin Orthop Relat Res. 1999; : S84-S94 Crossref PubMed Scopus (136) Google Scholar , 2 Axelrad T.W. Einhorn T.A. Bone morphogenetic proteins in orthopaedic surgery. Cytokine Growth Factor Rev. 2009; 20: 481-488 Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar ]. The neuroscience field has also found significant involvement of BMPs in development, maturation, and repair of the peripheral nervous system (PNS) and central nervous system (CNS) [ 3 Hall A.K. Miller R.H. Emerging roles for bone morphogenetic proteins in central nervous system glial biology. J Neurosci Res. 2004; 76: 1-8 Crossref PubMed Scopus (61) Google Scholar , 4 Setoguchi T. Yone K. Matsuoka E. et al. Traumatic injury-induced BMP7 expression in the adult rat spinal cord. Brain Res. 2001; 921: 219-225 Crossref PubMed Scopus (105) Google Scholar , 5 Fuller M.L. Dechant A.K. Rothstein B. et al. Bone morphogenetic proteins promote gliosis in demyelinating spinal cord lesions. Ann Neurol. 2007; 62: 288-300 Crossref PubMed Scopus (105) Google Scholar ]. Although the direct influence of the PNS on bone homeostasis is well described, the interactions through which peripheral neurons may influence bone formation or repair remain to be fully defined [ 6 Togari A. Arai M. Pharmacological topics of bone metabolism: the physiological function of the sympathetic nervous system in modulating bone resorption. J Pharmacol Sci. 2008; 106: 542-546 Crossref PubMed Scopus (69) Google Scholar , 7 Li J. Kreicbergs A. Bergström J. et al. Site-specific CGRP innervation coincides with bone formation during fracture healing and modeling: a study in rat angulated tibia. J Orthop Res. 2007; 25: 1204-1212 Crossref PubMed Scopus (69) Google Scholar , 8 Li J. Ahmed M. Bergstrom J. Ackermann P. et al. Occurrence of substance P in bone repair under different load comparison of straight and angulated fracture in rat tibia. J Orthop Res. 2010; 28: 1643-1650 Crossref PubMed Scopus (20) Google Scholar ]. We would agree that further study of neuron-bone interaction could be highly significant for the orthopedic world. However, the potential role of BMPs in this interaction is largely unexplored. Adverse events and bone morphogenetic protein-2The Spine JournalVol. 11Issue 8PreviewRecently, a number of adverse events have been reported in the use of recombinant bone morphogenetic protein-2 (BMP-2) in spinal fusion [1–4]. We have been studying new bone formation after delivery of physiologic levels of BMP-2 using a cell therapy approach and have found this to rely on very selective neurogenic inflammation. We would like to suggest that this is actually a normal physiologic event in bone formation and fracture repair rather than an adverse event associated with delivery of this protein. Full-Text PDF

CD44-Positive Cells Are Candidates for Astrocyte Precursor Cells in Developing Mouse Cerebellum

Neural stem cells are generally considered to be committed to becoming precursor cells before terminally differentiating into either neurons or glial cells during neural development. Neuronal and oligodendrocyte precursor cells have been identified in several areas in the murine central nervous system. The presence of astrocyte precursor cells (APCs) is not so well understood. The present study provides several lines of evidence that CD44-positive cells are APCs in the early postnatal mouse cerebellum. In developing mouse cerebellum, CD44-positive cells, mostly located in the white matter, were positive for the markers of the astrocyte lineage, but negative for the markers of mature astrocytes. CD44-positive cells were purified from postnatal cerebellum by fluorescence-activated cell sorting and characterized in vitro. In the absence of any signaling molecule, many cells died by apoptosis. The surviving cells gradually expressed glial fibrillary acidic protein, a marker for mature astrocytes, indicating that differentiation into mature astrocytes is the default program for these cells. The cells produced no neurospheres nor neurons nor oligodendrocytes under any condition examined, indicating these cells are not neural stem cells. Leukemia inhibitory factor greatly promoted astrocytic differentiation of CD44-positive cells, whereas bone morphogenetic protein 4 (BMP4) did not. Fibroblast growth factor-2 was a potent mitogen for these cells, but was insufficient for survival. BMP4 inhibited activation of caspase-3 and greatly promoted survival, suggesting a novel role for BMP4 in the control of development of astrocytes in cerebellum. We isolated and characterized only CD44 strongly positive large cells and discarded small and/or CD44 weakly positive cells in this study. Further studies are necessary to characterize these cells to help determine whether CD44 is a selective and specific marker for APCs in the developing mouse cerebellum. In conclusion, we succeeded in preparing APC candidates from developing mouse cerebellum, characterized them in vitro, and found that BMPs are survival factors for these cells.

Mouse auditory organ development required bone morphogenetic protein signaling

To investigate roles of bone morphogenetic protein (BMP) signaling on the development of mouse cochlear sensory organ, we isolated initial auditory epithelium from embryo and cultured with agonist or antagonist of BMP signaling. When BMP signaling was blocked with noggin cochlear epithelia were disordered, with hair cells in abnormal arrangement and disheveled hair bundles. The number of myosin VIIA and SRY (sex-determining region Y)-box 2 (SOX2)-positive cells also decreased dramatically. Conversely, an increase of myosin VIIA and SRY (sex-determining region Y)-box 2-positive cells was observed when auditory epithelia were treated with exogenous BMP4. Cell proliferation and cell death were not changed significantly by BMP signaling. Collectively, our results indicated that BMP signaling is essential to cochlear sensory formation and hair cell differentiation.