Casein Kinase 2 β-Subunit Is a Regulator of Bone Morphogenetic Protein 2 Signaling

Bone morphogenetic proteins (BMPs) play a crucial role during embryonic development and regulate processes as diverse as neurogenesis, skeletal formation, and hematopoesis. They signal through a hetero-oligomer complex of BMP receptors. Binding of the ligand to the receptors activates several pathways, including Smad and p38. BMP signaling is controlled in the extracellular space, the plasma membrane, and the intracellular space; however, the mechanism of receptor signaling at the plasma membrane and proteins that regulate this process still need to be identified. The experiments presented here identify the protein kinase casein kinase II (CK2) as a BMP receptor type Ia (BRIa) interacting protein. Fluorescence resonance energy transfer revealed that this interaction occurs at the plasma membrane. BMP2 stimulation of C2C12 cells leads to the release of CK2 from BRIa. Blocking this interaction with specific peptides that inhibit the binding sites for CK2 on BRIa demonstrated a redistribution of BRIa on the plasma membrane. Signaling was initiated once CK2 was released from BRIa, leading to the mineralization of C2C12 cells. These data suggest that CK2 is a negative regulator of BMP signaling and osteoblast differentiation.     

Functional redundancy of type II BMP receptor and type IIB activin receptor in BMP2-induced osteoblast differentiation

Signaling pathways for bone morphogenetic proteins (BMPs) are important in osteoblast differentiation. Although the precise function of type I BMP receptors in mediating BMP signaling for osteoblast differentiation and bone formation has been characterized previously, the role of type II BMP receptors in osteoblasts is to be well clarified. In this study, we investigated the role of type II BMP receptor (BMPR-II) and type IIB activin receptor (ActR-IIB) in BMP2-induced osteoblast differentiation. While osteoblastic 2T3 cells expressed BMPR-II and ActR-IIB, loss-of-function studies, using dominant negative receptors and siRNAs, showed that BMPR-II and ActR-IIB compensated each other functionally in mediating BMP2 signaling and BMP2-induced osteoblast differentiation. This was evidenced by two findings. First, unless there was loss of function of both type II receptors, isolated disruption of either BMPR-II or ActR-IIB did not remove BMP2 activity. Second, in cells with loss of function of both receptors, restoration of function of either BMPR-II or ActR-IIB by transfection of the wild-type forms, restored BMP2 activity. These findings suggest a functional redundancy between BMPR-II and ActR-IIB in osteoblast differentiation. Results from experiments to test the effects of transforming growth factor β (TGF-β), activin, and fibroblast growth factor (FGF) on osteoblast proliferation and differentiation suggest that inhibition of receptor signaling by double-blockage of BMPR-II and ActR-IIB is BMP-signaling specific. The observed functional redundancy of type II BMP receptors in osteoblasts is novel information about the BMP signaling pathway essential for initiating osteoblast differentiation.     

BMP signaling in the control of skin development and hair follicle growth

Bone morphogenetic proteins (BMPs), their antagonists, and BMP receptors are involved in controlling a large number of biological functions including cell proliferation, differentiation, cell fate decision, and apoptosis in many different types of cells and tissues during embryonic development and postnatal life. BMPs exert their biological effects via using BMP-Smad and BMP-MAPK intracellular pathways. The magnitude and specificity of BMP signaling are regulated by a large number of modulators operating on several levels (extracellular, cytoplasmic, nuclear). In developing and postnatal skin, BMPs, their receptors, and BMP antagonists show stringent spatio-temporal expressions patterns to achieve proper regulation of cell proliferation and differentiation in the epidermis and in the hair follicle. Genetic studies assert an essential role for BMP signaling in the control of cell differentiation and apoptosis in developing epidermis, as well as in the regulation of key steps of hair follicle development (initiation, cell fate decision, cell lineage differentiation). In postnatal hair follicles, BMP signaling plays an important role in controlling the initiation of the growth phase and is also involved in the regulation of apoptosis-driven hair follicle involution. However, additional efforts are required to fully understand the mechanisms and targets involved in the realization of BMP effects on distinct cell population in the skin and hair follicle. Progress in this area of research will hopefully lead to the development of new therapeutic approaches for using BMPs and BMP antagonists in the treatment of skin and hair growth disorders.     

Bone morphogenetic protein signaling in articular chondrocyte differentiation

Articular chondrocytes progressively undergo dedifferentiation into a spindle-shaped mesenchymal cellular phenotype in monolayers. Chondrocyte dedifferentiation is stimulated by retinoic acid. On the other hand, bone morphogenic proteins (BMPs) stimulate differentiation of chondrocytes. We examined the mechanism of effects of BMP in chondrocyte differentiation with use of a recombinant adenovirus vector system. Constitutively active forms of BMP type I receptors (BMPR-IA and BMPR-IB) and those of activin receptor-like kinase (ALK)-1 and ALK-2 maintained differentiation of chondrocytes in the presence of retinoic acid. The BMP receptor-regulated signaling substrates, Smad1/5, weakly induced chondrocyte differentiation; the effects of Smad1/5 were enhanced by BMP-7 treatment. Inhibitory Smad, Smad6, blocked increase of expression of chondrocyte markers by BMP-7 in a dose-dependent manner. SB202190, a p38 mitogen-activated protein kinase inhibitor, inhibited this effect of BMP-7; however, since SB202190 suppressed phosphorylation of Smad1/5, this may be due to blockade of BMP receptor activation. These results together strongly suggest that induction of chondrocyte differentiation by BMP-7 is regulated by Smad pathways.     

TGF-β and BMP signaling in osteoblast differentiation and bone formation

Transforming growth factor-beta (TGF-β)/bone morphogenic protein (BMP) signaling is involved in a vast majority of cellular processes and is fundamentally important throughout life. TGF-β/BMPs have widely recognized roles in bone formation during mammalian development and exhibit versatile regulatory functions in the body. Signaling transduction by TGF-β/BMPs is specifically through both canonical Smad-dependent pathways (TGF-β/BMP ligands, receptors and Smads) and non-canonical Smad-independent signaling pathway (e.g. p38 mitogen-activated protein kinase pathway, MAPK). Following TGF-β/BMP induction, both the Smad and p38 MAPK pathways converge at the Runx2 gene to control mesenchymal precursor cell differentiation. The coordinated activity of Runx2 and TGF-β/BMP-activated Smads is critical for formation of the skeleton. Recent advances in molecular and genetic studies using gene targeting in mice enable a better understanding of TGF-β/BMP signaling in bone and in the signaling networks underlying osteoblast differentiation and bone formation. This review summarizes the recent advances in our understanding of TGF-β/BMP signaling in bone from studies of genetic mouse models and human diseases caused by the disruption of TGF-β/BMP signaling. This review also highlights the different modes of cross-talk between TGF-β/BMP signaling and the signaling pathways of MAPK, Wnt, Hedgehog, Notch, and FGF in osteoblast differentiation and bone formation.     

The role of bone morphogenetic proteins in endochondral bone formation

Bone morphogenetic proteins (BMPs) were originally identified as proteins capable of inducing endochondral bone formation when implanted at extraskeletal sites. BMPs have diverse biological activities during early embryogenesis and various aspects of organogenesis. BMPs bind to BMP receptors on the cell surface, and these signals are transduced intracellularly by Smad proteins. BMP signal pathways can be inhibited by both extra- and intracellular mechanisms. As for skeletal development, genetic studies suggest that BMPs are skeletal mesoderm inducers. Recent studies of tissue-specific activation and inactivation of BMP signals have revealed that BMP signals control proliferation and differentiation of chondrocytes, differentiation of osteoblasts and bone quality. These findings may contribute not only to understanding of bone biology and pathology, but also to improvement of the clinical efficacy of BMPs.     

Bone morphogenetic proteins and their receptors in the eye

The human genome encodes at least 42 different members of the transforming growth factor-beta superfamily of growth factors. Bone morphogenetic proteins (BMPs) are the largest subfamily of proteins within the transforming growth factor-beta superfamily and are involved in numerous cellular functions including development, morphogenesis, cell proliferation, apoptosis, and extracellular matrix synthesis. This article first reviews BMPs and BMP receptors, BMP signaling pathways, and mechanisms controlling BMP signaling. Second, we review BMP and BMP receptor expression during embryonic ocular development/ differentiation and in adult ocular tissues. Lastly, future research directions with respect to BMP, BMP receptors, and ocular tissues are suggested.     

BMP-2 Induced Expression of Alx3 That Is a Positive Regulator of Osteoblast Differentiation

Bone morphogenetic proteins (BMPs) regulate many aspects of skeletal development, including osteoblast and chondrocyte differentiation, cartilage and bone formation, and cranial and limb development. Among them, BMP-2, one of the most potent osteogenic signaling molecules, stimulates osteoblast differentiation, while it inhibits myogenic differentiation in C2C12 cells. To evaluate genes involved in BMP-2-induced osteoblast differentiation, we performed cDNA microarray analyses to compare BMP-2-treated and -untreated C2C12 cells. We focused on Alx3 (aristaless-like homeobox 3) which was clearly induced during osteoblast differentiation. Alx3, a homeobox gene related to the Drosophila aristaless gene, has been linked to developmental functions in craniofacial structures and limb development. However, little is known about its direct relationship with bone formation. In the present study, we focused on the mechanisms of Alx3 gene expression and function during osteoblast differentiation induced by BMP-2. In C2C12 cells, BMP-2 induced increase of Alx3 gene expression in both time- and dose-dependent manners through the BMP receptors-mediated SMAD signaling pathway. In addition, silencing of Alx3 by siRNA inhibited osteoblast differentiation induced by BMP-2, as showed by the expressions of alkaline phosphatase (Alp), Osteocalcin, and Osterix, while over-expression of Alx3 enhanced osteoblast differentiation induced by BMP-2. These results indicate that Alx3 expression is enhanced by BMP-2 via the BMP receptors mediated-Smad signaling and that Alx3 is a positive regulator of osteoblast differentiation induced by BMP-2.     

Thyroid hormone treatment of cultured chondrocytes mimics in vivo stimulation of collagen X mRNA by increasing BMP 4 expression

During endochondral bone formation, chondrocytes undergo terminal differentiation, during which the rate of proliferation decreases, cells become hypertrophic, and the extracellular matrix is altered by production of collagen X, as well as proteins required for matrix mineralization. This maturation process is responsible for most longitudinal bone growth, both during embryonic development and in postnatal long bone growth plates. Among the major signaling molecules implicated in regulation of this process are the positive regulators thyroid hormone (T3) and bone morphogenetic proteins (BMPs). Both T3 and BMPs are essential for endochondral bone formation and cannot compensate for each other, suggesting interaction of the two signaling pathways. We have analyzed the temporal and spatial expression patterns of numerous genes believed to play a role in chondrocyte maturation. Our results show that T3 stimulates collagen X gene expression in cultured chondrocytres with kinetics and magnitude similar to those observed in vivo. Stimulation of collagen X gene expression by T3 occurs only after a significant delay, implying that this hormone may act indirectly. We show further that T3 rapidly stimulates production of BMP 4, concomitant with a decrease in the BMP inhibitor Noggin, potentially resulting in a net increase in BMP signaling. Finally, inhibition of BMP signaling with exogenous Noggin prevents T3 stimulation of collagen X expression, indicating that BMP signaling is essential for this process. These data position thyroid hormone at the top of a T3/BMP cascade, potentially explaining why both pathways are essential for chondrocyte maturation. J. Cell. Physiol. 219: 595–605, 2009. © 2009 Wiley‐Liss, Inc.     

The BMP signaling and in vivo bone formation

Bone morphogenetic proteins (BMPs) are multi-functional growth factors that belong to the transforming growth factor beta (TGFbeta) superfamily. The roles of BMPs in embryonic development and cellular functions in postnatal and adult animals have been extensively studied in recent years. Signal transduction studies have revealed that Smads 1, 5 and 8 are the immediate downstream molecules of BMP receptors and play a central role in BMP signal transduction. Studies from transgenic and knockout mice and from animals and humans with naturally occurring mutations in BMPs and their signaling molecules have shown that BMP signaling plays critical roles in bone and cartilage development and postnatal bone formation. BMP activities are regulated at different molecular levels. Tissue-specific knockout of a specific BMP ligand, a subtype of BMP receptors or a specific signaling molecule is required to further determine the specific role of a BMP ligand, receptor or signaling molecule in a particular tissue.     

Bone morphogenetic protein receptor signaling is necessary for normal murine postnatal bone formation

Functions of bone morphogenetic proteins (BMPs) are initiated by signaling through specific type I and type II serine/threonine kinase receptors. In previous studies, we have demonstrated that the type IB BMP receptor (BMPR-IB) plays an essential and specific role in osteoblast commitment and differentiation. To determine the role of BMP receptor signaling in bone formation in vivo, we generated transgenic mice, which express a truncated dominant-negative BMPR-IB targeted to osteoblasts using the type I collagen promoter. The mice are viable and fertile. Tissue-specific expression of the truncated BMPR-IB was demonstrated. Characterization of the phenotype of these transgenic mice showed impairment of postnatal bone formation in 1-mo-old homozygous transgenic mice. Bone mineral density, bone volume, and bone formation rates were severely reduced, but osteoblast and osteoclast numbers were not significantly changed in the transgenic mice. To determine whether osteoblast differentiation is impaired, we used primary osteoblasts isolated from the transgenic mice and showed that BMP signaling is blocked and BMP2-induced mineralized bone matrix formation was inhibited. These studies show the effects of alterations in BMP receptor function targeted to the osteoblast lineage and demonstrate a necessary role of BMP receptor signaling in postnatal bone growth and bone formation in vivo.     

Interaction of Ihh and BMP/Noggin signaling during cartilage differentiation.

Bone morphogenetic proteins (BMPs) have been implicated in regulating multiple stages of bone development. Recently it has been shown that constitutive activation of the BMP receptor-IA blocks chondrocyte differentiation in a similar manner as misexpression of Indian hedgehog. In this paper we analyze the role of BMPs as possible mediators of Indian hedgehog signaling and use Noggin misexpression to gain insight into additional roles of BMPs during cartilage differentiation. We show by comparative analysis of BMP and Ihh expression domains that the borders of Indian hedgehog expression in the chondrocytes are reflected in changes of the expression level of several BMP genes in the adjacent perichondrium. We further demonstrate that misexpression of Indian hedgehog appears to directly upregulate BMP2 and BMP4 expression, independent of the differentiation state of the flanking chondrocytes. In contrast, changes in BMP5 and BMP7 expression in the perichondrium correspond to altered differentiation states of the flanking chondrocytes. In addition, Noggin and Chordin, which are both expressed in the developing cartilage elements, also change their expression pattern after Ihh misexpression. Finally, we use retroviral misexpression of Noggin, a potent antagonist of BMP signaling, to gain insight into additional roles of BMP signaling during cartilage differentiation. We find that BMP signaling is necessary for the growth and differentiation of the cartilage elements. In addition, this analysis revealed that the members of the BMP/Noggin signaling pathway are linked in a complex autoregulatory network.     

BMPs regulate multiple aspects of growth-plate chondrogenesis through opposing actions on FGF pathways

Bone morphogenetic protein (BMP) signaling pathways are essential regulators of chondrogenesis. However, the roles of these pathways in vivo are not well understood. Limb-culture studies have provided a number of essential insights, including the demonstration that BMP pathways are required for chondrocyte proliferation and differentiation. However, limb-culture studies have yielded contradictory results; some studies indicate that BMPs exert stimulatory effects on differentiation, whereas others support inhibitory effects. Therefore, we characterized the skeletal phenotypes of mice lacking Bmpr1a in chondrocytes (Bmpr1a(CKO)) and Bmpr1a(CKO);Bmpr1b+/- (Bmpr1a(CKO);1b+/-) in order to test the roles of BMP pathways in the growth plate in vivo. These mice reveal requirements for BMP signaling in multiple aspects of chondrogenesis. They also demonstrate that the balance between signaling outputs from BMP and fibroblast growth factor (FGF) pathways plays a crucial role in the growth plate. These studies indicate that BMP signaling is required to promote Ihh expression, and to inhibit activation of STAT and ERK1/2 MAPK, key effectors of FGF signaling. BMP pathways inhibit FGF signaling, at least in part, by inhibiting the expression of FGFR1. These results provide a genetic in vivo demonstration that the progression of chondrocytes through the growth plate is controlled by antagonistic BMP and FGF signaling pathways.     

The dynamic role of bone morphogenetic proteins in neural stem cell fate and maturation

The bone morphogenetic proteins (BMPs) are a group of powerful morphogens that are critical for development of the nervous system. The effects of BMP signaling on neural stem cells are myriad and dynamic, changing with each stage of development. During early development inhibition of BMP signaling differentiates neuroectoderm from ectoderm, and BMP signaling helps to specify neural crest. Thus modulation of BMP signaling underlies formation of both the central and peripheral nervous systems. BMPs secreted from dorsal structures then form a gradient which helps pattern the dorsal-ventral axis of the developing spinal cord and brain. During forebrain development BMPs sequentially induce neurogenesis and then astrogliogenesis and participate in neurite outgrowth from immature neurons. BMP signaling also plays a critical role in maintaining adult neural stem cell niches in the subventricular zone (SVZ) and subgranular zone (SGZ). BMPs are able to exert such diverse effects through closely regulated temporospatial expression and interaction with other signaling pathways.     

Requirement for ErbB2/ErbB signaling in developing cartilage and bone

During endochondral ossification, the skeletal elements of vertebrate limbs form and elongate via coordinated control of chondrocyte and osteoblast differentiation and proliferation. The role of signaling by the ErbB family of receptor tyrosine kinases, which consists of ErbB1 (epidermal growth factor receptor or EGFR), ErbB2, ErbB3 and ErbB4, has been little studied during cartilage and bone development. Signaling by the ErbB network generates a diverse array of cellular responses via formation of ErbB dimers activated by distinct ligands that produce distinct signal outputs. Herstatin is a soluble ErbB2 receptor that acts in a dominant negative fashion to inhibit ErbB signaling by binding to endogenous ErbB receptors, preventing functional dimer formation. Here, we examine the effects of Herstatin on limb skeletal element development in transgenic mice, achieved via Prx1 promoter-driven expression in limb cartilage and bone. The limb skeletal elements of Prx1-Herstatin embryos are shortened, and chondrocyte maturation and osteoblast differentiation are delayed. In addition, proliferation by chondrocytes and periosteal cells of Prx1-Herstatin limb skeletal elements is markedly reduced. Our study identifies requirements for ErbB signaling in the maintenance of chondrocyte and osteoblast proliferation involved in the timely progression of chondrocyte maturation and periosteal osteoblast differentiation.     

Conditional Ablation of the Heparan Sulfate-synthesizing Enzyme Ext1 Leads to Dysregulation of Bone Morphogenic Protein Signaling and Severe Skeletal Defects

Increasing evidence indicates that heparan sulfate (HS) is an integral component of many morphogen signaling pathways. However, its mechanisms of action appear to be diverse, depending on the type of morphogen and the developmental contexts. To define the function of HS in skeletal development, we conditionally ablated Ext1, which encodes an essential glycosyltransferase for HS synthesis, in limb bud mesenchyme using the Prx1-Cre transgene. These conditional Ext1 mutant mice display severe limb skeletal defects, including shortened and malformed limb bones, oligodactyly, and fusion of joints. In developing limb buds of mutant mice, chondrogenic differentiation of mesenchymal condensations is delayed and impaired, whereas the area of differentiation is diffusely expanded. Correspondingly, the distribution of both bone morphogenic protein (BMP) signaling domains and BMP2 immunoreactivity in the mutant limb mesenchyme is broadened and diffuse. In micromass cultures, chondrogenic differentiation of mutant chondrocytes is delayed, and the responsiveness to exogenous BMPs is attenuated. Moreover, the segregation of the pSmad1/5/8-expressing chondrocytes and fibronectin-expressing perichondrium-like cells surrounding chondrocyte nodules is disrupted in mutant micromass cultures. Together, our results show that HS is essential for patterning of limb skeletal elements and that BMP signaling is one of the major targets for the regulatory role of HS in this developmental context.     

BMP and Ihh/PTHrP signaling interact to coordinate chondrocyte proliferation and differentiation.

During endochondral ossification, two secreted signals, Indian hedgehog (Ihh) and parathyroid hormone-related protein (PTHrP), have been shown to form a negative feedback loop regulating the onset of hypertrophic differentiation of chondrocytes. Bone morphogenetic proteins (BMPs), another family of secreted factors regulating bone formation, have been implicated as potential interactors of the Ihh/PTHrP feedback loop. To analyze the relationship between the two signaling pathways, we used an organ culture system for limb explants of mouse and chick embryos. We manipulated chondrocyte differentiation by supplementing these cultures either with BMP2, PTHrP and Sonic hedgehog as activators or with Noggin and cyclopamine as inhibitors of the BMP and Ihh/PTHrP signaling systems. Overexpression of Ihh in the cartilage elements of transgenic mice results in an upregulation of PTHrP expression and a delayed onset of hypertrophic differentiation. Noggin treatment of limbs from these mice did not antagonize the effects of Ihh overexpression. Conversely, the promotion of chondrocyte maturation induced by cyclopamine, which blocks Ihh signaling, could not be rescued with BMP2. Thus BMP signaling does not act as a secondary signal of Ihh to induce PTHrP expression or to delay the onset of hypertrophic differentiation. Similar results were obtained using cultures of chick limbs. We further investigated the role of BMP signaling in regulating proliferation and hypertrophic differentiation of chondrocytes and identified three functions of BMP signaling in this process. First we found that maintaining a normal proliferation rate requires BMP and Ihh signaling acting in parallel. We further identified a role for BMP signaling in modulating the expression of IHH: Finally, the application of Noggin to mouse limb explants resulted in advanced differentiation of terminally hypertrophic cells, implicating BMP signaling in delaying the process of hypertrophic differentiation itself. This role of BMP signaling is independent of the Ihh/PTHrP pathway.