TGF-, BMPs, and their signal transducing mediators, Smads, in rat fracture healing Yan Yu, Jia-Lin Yang, Philip J. Chapman-Sheath, Williams R. Walsh Orthopaedic Research Laboratories, Division of Surgery, Prince of Wales Hospital, Faculty of Medicine, University of New South Wales, Sydney, NSW 2031, Australia Received 20 April 2001; revised 22 June 2001; accepted 3 July 2001 Abstract: Smads are cytoplasmic signal transducers of transforming growth factor- (TGF-) and bone morphoge- netic proteins (BMPs). Their relation to fracture healing is unknown. This study examined the temporal protein ex- pression of Smads, together with TGF- and BMPs, using immunohistochemistry in a rodent fracture model. Over- expression of TGF-, BMPs-2, 4, and 7, common-mediator Smad (Smad4), and receptor-regulated Smads (Smads1, 2, 3, and 5) versus lower levels of inhibitory Smad (Smad6), were detected at day 3 in osteogenic cells in the thickened peri- osteum and bone marrow at the fracture sites. At day 10, Smad6 increased dramatically, Smad2, Smad3, and Smad4 remained elevated while Smad1 and Smad5 decreased in the fracture callus. Smad7 was expressed only in vascular en- dothelial cells. By day 28, when new bone had replaced the fracture callus, all the protein regulators decreased, ap- proaching control levels. During fracture healing, the ex- pression patterns of Smads1 and 5 were similar to that of BMPs-2 and 7 whereas the expression of Smads2 and 3 was parallel with that of TGF-. The Smad family, associated with BMPs and TGF-, may play an important role in the early stage of rat fracture healing. © 2002 Wiley Periodicals, Inc. J Biomed Mater Res 60: 392–397, 2002; DOI 10.1002/ jbm.1289 Key words: Smads; BMPs; TGF-; fracture healing; immu- nohistochemistry INTRODUCTION Fracture healing is a complex biologic cascade. Fol- lowing immediate formation of hematoma, there are two separate but simultaneous events: (a) intramem- branous ossification via proliferation of osteoprogeni- tor cells from the cambial layer of periosteum; and (b) endochondral ossification from the chondrogenesis of undifferentiated mesenchymal cells in the soft tissues adjacent to the fracture site and in the bone marrow. Many local and systemic regulators are involved in fracture healing, including growth and differentiation factors, cytokines, hormones, and extracellular matrix components. 1–6 Among these factors, the transforming growth factor- (TGF-)/activin/bone morphoge- netic protein (BMP) cytokine family plays an im- portant role. Some BMPs (BMP-2, 4, 6, and 7) have demonstrated ectopic bone formation in vivo and in- fluence mesenchymal cell proliferation, differentia- tion, chondrogenesis, and osteogenesis in vitro 7–10 while TGF- is involved in wound healing and tissue repair (through regulation of extracellular matrix for- mation), cell migration, and angiogenesis. 11 Both fac- tors initiate their biologic effects from the cell surface through two related serine-threonine kinase receptors, the type I and II receptors. Upon ligand binding, the activated type II receptor phosphorylates/activates the type I receptor. 12,13 In turn, the activated type I receptor propagates the signal to downstream sub- strates. 14 Progress in identifying signaling events down- stream of the type I receptors has been made with the discovery of a novel family of proteins, the Smads, as signal transducers for TGF-/activins/BMPs. 15–17 Smads are the vertebrate homologues of Mad (Moth- ers against decapentaplegic) genes from Drosophila and/or Smad genes from Caenorhabditis elegans. They are categorized into three classes based on their dif- ferent functions. Smad1, Smad2, Smad3, Smad5, and Smad8 belong to the receptor-regulated class (R- Smads), which are phosphorylated by the type I re- ceptors. 15–17 Smad2 and Smad3 are initiated by TGF- /activin and induce the responses to their ligands, while Smad1, Smad5, and Smad8 display BMP speci- ficities. 15–17 This class of Smads forms heteromers with the common-mediator class (Co-Smad): Smad4, mov- ing from cell surface to nucleus, positively regulates target gene transcription. 18 Smad6 and Smad7 (inhibi- Correspondence to: W.R. Walsh; e-mail: w.walsh@unsw. edu.au © 2002 Wiley Periodicals, Inc.