Distinct and Overlapping Patterns of Localization of Bone Morphogenetic Protein (BMP) Family Members and a BMP Type II Receptor During Fracture Healing in Rats

doi:10.1016/S8756-3282(98)00056-8

Bone

Volume 22, Issue 6, June 1998, Pages 605-612

https://doi.org/10.1016/S8756-3282(98)00056-8 Get rights and content

Abstract

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) are thought to play an important role in bone morphogenesis. The purpose of this study was to determine the locations of BMP-2/-4, osteogenic protein-1 (OP-1, also termed BMP-7), and BMP type II receptor (BMPR-II) during rat fracture healing by immunostaining, and thereby elucidate the possible roles of the BMPs and BMPR-II in intramembranous ossification and endochondral ossification. In the early stage of fracture repair, the expression of BMP-2/-4 and OP-1 was strongly induced in the thickened periosteum near the fracture ends, and coincided with an enhanced expression of BMPR-II. On day 7 after fracture, staining for BMP-2/-4 and OP-1 immunostaining was increased in various types of chondrocytes, and was strong in fibroblast-like spindle cells and proliferating chondrocytes in endochondral bone. On day 14 after fracture, staining with OP-1 antibody disappeared in proliferating and mature chondrocytes, while BMP-2/-4 staining continued in various types of chondrocytes until the late stage. In the newly formed trabecular bone, BMP-2/-4 and OP-1 were present at various levels. BMPR-II was actively expressed in both intramembranous ossification and endochondral ossification. Additionally, immunostaining for BMP-2/-4 and OP-1 was observed in multinucleated osteoclast-like cells on the newly formed trabecular bone, along with BMPR-II. In reference to our previous study of BMP type I receptors (BMPR-IA and BMPR-IB), BMPR-II was found to be co-localized with BMPR-IA and BMPR-IB. BMP-2/-4 and OP-1 antibodies exhibited distinct and overlapping immunostaining patterns during fracture repair. OP-1 may act predominantly in the initial phase of endochondral ossification, while BMP-2/-4 acts throughout this process. Thus, these findings suggested that BMPs acting through their BMP receptors may play major roles in modulating the sequential events leading to bone formation.

Introduction

Fracture healing involves the coordinated effects of many local and systemic regulatory factors, including growth and differentiation factors, hormones, cytokines, and extracellular matrix components. This complex interaction of local mediators causes primitive, undifferentiated mesenchymal cells to migrate, proliferate, and differentiate at the fracture site.3., 6., 44. Recent molecular studies have deepened our knowledge of cellular proliferation, chondrogenesis, and osteogenesis during fracture healing, and revealed that certain peptide growth factors including bone morphogenetic proteins (BMPs) may play important roles in this process.3., 20. BMPs constitute a unique subfamily within the transforming growth factor-β (TGF-β) superfamily, and many of them exhibit a potent cartilage- and bone-inducing activity in vivo. BMPs may provoke a sequential multistep cascade of events, i.e., migration of progenitor cells, proliferation of mesenchymal cells, differentiation to chondrogenic or osteogenic cells, vascular invasion, and remodeling of bone.37., 38. Several BMPs are known to be produced during bone formation with different spatial and temporal profiles,38., 42., 45., 51. and to play distinct or related functional roles in this process.23., 29. During embryonic endochondral ossification, BMP-2, BMP-4, osteogenic protein-1 (OP-1), and growth/differentiation factor-5 (GDF-5) are expressed in the early stage of embryonic development, whereas BMP-6, OP-1, and GDF-6 may function in the later stage.7., 12., 19., 30., 50. Chondro/osteoprogenitor cells have, in general, been shown to migrate and proliferate in response to treatment with BMPs. BMPs induce the differentiation of mesenchymal cells into several phenotypes and increase the expression of markers associated with phenotypes of committed cells.1., 8., 22., 24., 31., 53.

Signaling by members of the TGF-β superfamily occurs through heterodimer complex formation of the type I and type II receptors.32., 46. BMP type I (BMPR-IA, BMPR-IB, and ActR-I) and type II receptors (BMPR-II, ActR-II, and ActR-IIB), which are serine/threonine kinase receptors, have recently been identified.25., 28., 34., 41., 47., 54. Ligand binding to both of these receptors results in the phosphorylation of type I receptors by type II receptor kinase. Activated type I receptors then transduce signals by phosphorylating intracellular targets, including members of the Smad family.14., 27. Members of the BMP family exhibit different affinities for the various combinations of type I and type II receptors, e.g., BMP-4 binds to BMPR-IA and BMPR-IB with a high affinity, but does not bind to ActR-I. OP-1 binds to BMPR-IB more efficiently than to BMPR-IA and ActR-I.47 These differences in ligand binding affinities to type I and type II receptor complexes may be related to the overlapping but also distinct functional effects of BMPs.

The expression of BMP type I receptors was found to increase during bone formation in fracture repair17., 33. and pathological ectopic ossification in the spinal ligament, such as ossification of the posterior longitudinal ligament (OPLL) and ossification of the ligamentum flavum (OLF).11., 55. In this study, the expression of BMP-2/-4, OP-1, and BMPR-II was examined immunohistochemically during rat fracture repair, and their roles in intramembranous and endochondral ossification are discussed.

Section snippets

Experimental fracture models and preparation of specimens

Thirty-two Wistar rats between 12 and 14 weeks of age were used. Under intraperitoneal anesthesia using pentobarbiturate, a closed femoral fracture was induced using a method described previously.4 After inserting a Kirschner wire 0.8 mm in diameter along the femoral medullar cavity, a closed fracture of the femur was induced at the center. The rats were then kept free and fed in the cages without external fixation, and in a warm environment until they were killed. The experimental procedures

Histomorphological findings for fracture healing

Morphological findings for fracture healing were described in our previous report.17 In brief, the first step of the sequence of histomorphological changes in the site of fracture is early inflammatory change, which occurs as an immediate response to the injury. On days 1–3 after fracture, a hematoma forms between and around the fracture gap, and inflammatory cells invade the connective tissue around the fracture site (Figure 1A).

Intramembranous ossification occurs adjacent to the fracture

Discussion

During development, members of the BMP family of proteins have been shown to induce mesenchymal migration, proliferation, and differentiation, leading to cartilage and bone formation.39 Among members of this family, BMP-2, BMP-4, and OP-1 have been shown to have potent ectopic and orthotopic bone-induction activities,2., 10., 26., 43., 48., 49., 51. while individual BMPs may have differences in dose on exhibiting bone inducing activities. BMPs commit mesenchymal progenitor cells to

Acknowledgements

The authors express deep gratitude to Dr. Kohei Miyazono for valuable discussions.

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Original ArticlesDistinct and Overlapping Patterns of Localization of Bone Morphogenetic Protein (BMP) Family Members and a BMP Type II Receptor During Fracture Healing in Rats

Abstract

Introduction

Section snippets

Experimental fracture models and preparation of specimens

Histomorphological findings for fracture healing

Discussion

Acknowledgements

J Biol Chem

J Biol Chem

Exp Cell Res

Bone

Biochem Biophys Res Commun

Cell

Bone

Orthop Clin North Am

Biochem Biophys Res Commun

Biochem Biophys Res Commun

Trends Genet

Trends Cell Biol

J Biol Chem

J Biol Chem

Cytokine Growth Factor Rev

J Biol Chem

J Biol Chem

Biochem Biophys Res Commun

Human osteogenic protein-1 induces both chondroblastic and osteoblastic differentiation of osteoprogenitor cells derived from newborn rat calvaria

J Cell Biol

Regulation of fracture repair by growth factors

Proc Soc Exp Biol Med

Production of a standard closed fracture in laboratory animal bone

J Orthop Res

Immunolocalization and expression of bone morphogenetic protein 2 and 4 in fracture healing

J Orthop Res

Growth factors and the regulation of bone remodeling

J Clin Invest

Distinct spatial and temporal expression patterns of two type I receptors for bone morphogenetic proteins during mouse embryogenesis

Endocrinology

Bone-inducing activity of mature BMP-2b produced from a hybrid BMP-2a/2b precursor

Mol Endocrinol

Expression pattern of osteogenic protein-1 (bone morphogenetic protein-7) in human and mouse development

J Histochem Cytochem

Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniquesA comparison between ABC and unlabeled antibody (PAP) procedures

J Histochem Cytochem

Original Articles
Distinct and Overlapping Patterns of Localization of Bone Morphogenetic Protein (BMP) Family Members and a BMP Type II Receptor During Fracture Healing in Rats