Cross‐talk between EGF and BMP9 signalling pathways regulates the osteogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent progenitors, which give rise to several lineages, including bone, cartilage and fat. Epidermal growth factor (EGF) stimulates cell growth, proliferation and differentiation. EGF acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell surface and stimulating the intrinsic protein tyrosine kinase activity of its receptor, which initiates a signal transduction cascade causing a variety of biochemical changes within the cell and regulating cell proliferation and differentiation. We have identified BMP9 as one of the most osteogenic BMPs in MSCs. In this study, we investigate if EGF signalling cross‐talks with BMP9 and regulates BMP9‐induced osteogenic differentiation. We find that EGF potentiates BMP9‐induced early and late osteogenic markers of MSCs in vitro, which can be effectively blunted by EGFR inhibitors Gefitinib and Erlotinib or receptor tyrosine kinase inhibitors AG‐1478 and AG‐494 in a dose‐ and time‐dependent manner. Furthermore, EGF significantly augments BMP9‐induced bone formation in the cultured mouse foetal limb explants. In vivo stem cell implantation experiment reveals that exogenous expression of EGF in MSCs can effectively potentiate BMP9‐induced ectopic bone formation, yielding larger and more mature bone masses. Interestingly, we find that, while EGF can induce BMP9 expression in MSCs, EGFR expression is directly up‐regulated by BMP9 through Smad1/5/8 signalling pathway. Thus, the cross‐talk between EGF and BMP9 signalling pathways in MSCs may underline their important roles in regulating osteogenic differentiation. Harnessing the synergy between BMP9 and EGF should be beneficial for enhancing osteogenesis in regenerative medicine.     

Roles of Wnt signalling in bone growth, remodelling, skeletal disorders and fracture repair

Wnt signalling has an essential role in regulating bone formation and remodelling during embryonic development and throughout postnatal and adult life. Specifically, Wnt signalling regulates bone formation by controlling embryonic cartilage development and postnatal chondrogenesis, osteoblastogenesis, osteoclastogenesis, endochondral bone formation, and bone remodelling. Abnormalities in the function of Wnt genes give rise to or contribute to the development of several pathological bone conditions, including abnormal bone mass, osteosarcomas and bone loss in multiple myeloma. Furthermore, Wnt signalling is activated during bone fracture repair and plays a crucial role in regulating bone regeneration.     

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 Proteins

Bone Morphogenetic Protein (BMP) refers to an activity derived from bone that induces the formation of cartilage and bone in vivo. This activity leads to a series of developmental processes including chemotaxis, proliferation, and differentiation, which result in the transient formation of cartilage and the production of living bone tissue, complete with hematopoietic marrow. The determination of the factor or factors responsible for this activity has clear significance both for our understanding of bone biology and for the clinical application of cartilage and bone replacement. Several newly discovered factors, BMP-1, BMP-2 (BMP-2A), BMP-3 (osteogenin), BMP-4 (BMP-2B), BMP-5, BMP-6, BMP-7, and osteoinductive factor (OIF) have been implicated in the BMP process. BMP-2 through BMP-7 are all in the TGF-β superfamily of molecules, and are closely related to two factors (Vg1 and dpp) which are involved in a variety of developmental processes during embryogenesis. A recently discovered factor, OIF, exhibits BMP activity only in the presence of TGF-β. BMP-2, expressed as a recombinant protein, is the only molecule described to date that has been shown to clearly induce by itself the entire cartilage and bone formation process seen with bone-derived BMP. Evidence is accumulating that the BMP effect is a result of the combined actions of a set of BMP-2-like molecules. Definitive examination of the activities of the other factors will require expression of the recombinant proteins and testing of these in vivo alone and in combinations.     

A Lox/CHOP‐10 crosstalk governs osteogenic and adipogenic cell fate by MSCs

Accelerated marrow adipogenesis has been associated with ageing and osteoporosis and is thought to be because of an imbalance between adipogenic and osteogenic differentiation of mesenchymal stem cell (MSCs). We have previously found that lysyl oxidase (Lox) inhibition disrupts BMP4‐induced adipocytic lineage commitment and differentiation of MSCs. In this study, we found that lox inhibition dramatically up‐regulates BMP4‐induced expression of CCAAT/enhancer binding protein (C/EBP) homologous protein 10 (CHOP‐10), which then promotes BMP4‐induced osteogenesis of MSCs both in vitro and in vivo. Specifically, Lox inhibition or CHOP‐10 up‐regulation activated Wnt/β‐catenin signalling to enhance BMP4‐induced osteogenesis, with pro‐adipogenic p38 MAPK and Smad signalling suppressed. Together, we demonstrate that Lox/CHOP‐10 crosstalk regulates BMP4‐induced osteogenic and adipogenic fate determination of MSCs, presenting a promising therapeutic target for osteoporosis and other bone diseases.     

A vertebrate crossveinless 2 homologue modulates BMP activity and neural crest cell migration

Previous work has revealed that proteins that bind to bone morphogenetic proteins (BMPs) and inhibit their signalling have a crucial role in the spatial and temporal regulation of cell differentiation and cell migration by BMPs. We have identified a chick homologue of crossveinless 2, a Drosophila gene that was identified in genetic studies as a promoter of BMP-like signalling. Chick Cv-2 has a conserved structure of five cysteine-rich repeats similar to those found in several BMP antagonists, and a C-terminal Von Willebrand type D domain. Cv-2 is expressed in the chick embryo in a number of tissues at sites at which elevated BMP signalling is required. One such site of expression is premigratory neural crest, in which at trunk levels threshold levels of BMP activity are required to initiate cell migration. We show that, when overexpressed, Cv-2 can weakly antagonise BMP4 activity in Xenopus embryos, but that in other in vitro assays Cv-2 can increase the activity of co-expressed BMP4. Furthermore, we find that increased expression of Cv-2 causes premature onset of trunk neural crest cell migration in the chick embryo, indicative of Cv-2 acting to promote BMP activity at an endogenous site of expression. We therefore propose that BMP signalling is modulated both by antagonists and by Cv-2 that acts to elevate BMP activity.     

BMP4 promotes chondrocyte proliferation and hypertrophy in the endochondral cranial base

Defects in the growth and development of the endochondral bones that comprise the cranial base contribute to several craniofacial dysmorphic syndromes. Since Bone Morphogenetic Protein (BMP) signaling regulates chondrocyte differentiation and endochondral ossification in developing long bones, we have tested the hypothesis that BMP signaling also participates in regulating development of the cranial base. During in vivo developmental progression of the cranial base in mice, a burst of skeletal growth and chondrocyte maturation was identified in the perinatal period. Using a novel serum-free organ culture system, cranial base structures were cultured as explants in the presence of BMP4 or noggin, and analyzed for morphological and molecular changes. Growth of perinatal cranial base explants was inhibited by treatment with noggin, a BMP inhibitor. Exogenous BMP4 promoted cartilage growth, matrix deposition and chondrocyte proliferation in a dose dependent manner. Correspondingly, expression level of the cartilage markers Sox9 and collagen type II were also increased. Alkaline phosphatase and collagen type X expression were up-regulated and expressed in ectopic hypertrophic chondrocytes after treatment of the cultures with 100 ng/ml BMP4 for seven days. This increase in chondrocyte hypertrophy was accompanied by increased indian hedgehog (Ihh) and parathyroid hormone/parathyroid hormone related peptide (PTH/PTHrP) receptor (PPR) expression, but not increased PTHrP expression. We conclude that endogenous BMPs are required to maintain cartilage growth, and exogenous BMP4 can enhance cartilage maturation and induce ectopic chondrocyte hypertrophy in the cranial base. Therefore, appropriate levels of BMP signaling are important for normal cranial base development.     

Dynamic 3D culture: Models of chondrogenesis and endochondral ossification

The formation of cartilage from stem cells during development is a complex process which is regulated by both local growth factors and biomechanical cues, and results in the differentiation of chondrocytes into a range of subtypes in specific regions of the tissue. In fetal development cartilage also acts as a precursor scaffold for many bones, and mineralization of this cartilaginous bone precursor occurs through the process of endochondral ossification. In the endochondral formation of bones during fetal development the interplay between cell signalling, growth factors, and biomechanics regulates the formation of load bearing bone, in addition to the joint capsule containing articular cartilage and synovium, generating complex, functional joints from a single precursor anlagen. These joint tissues are subsequently prone to degeneration in adult life and have poor regenerative capabilities, and so understanding how they are created during development may provide useful insights into therapies for diseases, such as osteoarthritis, and restoring bone and cartilage lost in adulthood. Of particular interest is how these tissues regenerate in the mechanically dynamic environment of a living joint, and so experiments performed using 3D models of cartilage development and endochondral ossification are proving insightful. In this review, we discuss some of the interesting models of cartilage development, such as the chick femur which can be observed in ovo, or isolated at a specific developmental stage and cultured organotypically in vitro. Biomaterial and hydrogel‐based strategies which have emerged from regenerative medicine are also covered, allowing researchers to make informed choices on the characteristics of the materials used for both original research and clinical translation. In all of these models, we illustrate the essential importance of mechanical forces and mechanotransduction as a regulator of cell behavior and ultimate structural function in cartilage. Birth Defects Research (Part C) 105:19–33, 2015. © 2015 Wiley Periodicals, Inc.