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

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.     

Smad3-deficient chondrocytes have enhanced BMP signaling and accelerated differentiation.

Smad3 deficiency accelerates chondrocyte maturation and leads to osteoarthritis. Primary chondrocytes without Smad3 lack compensatory increases of TGF-beta signaling factors, but BMP-related gene expression is increased. Smad2 or Smad3 overexpression and BMP blockade abrogate accelerated maturation in Smad3-/- chondrocytes. BMP signaling is increased in TGF-beta deficiency and is required for accelerated chondrocyte maturation. INTRODUCTION: Disruption of TGF-beta signaling results in accelerated chondrocyte maturation and leads to postnatal dwarfism and premature osteoarthritis. The mechanisms involved in this process were studied using in vitro murine chondrocyte cultures. MATERIALS AND METHODS: Primary chondrocytes were isolated from the sterna of neonatal wildtype and Smad3-/- mice. Expressions of maturational markers, as well as genes involved in TGF-beta and BMP signaling were examined. Chondrocytes were treated with TGF-beta and BMP-2, and effects on maturation-related genes and BMP/TGF-beta responsive reporters were examined. Recombinant noggin or retroviral vectors expressing Smad2 or Smad3 were added to the cultures. RESULTS: Expression of colX and other maturational markers was markedly increased in Smad3-/- chondrocytes. Smad3-/- chondrocytes lacked compensatory increases in Smad2, Smad4, TGFRII, Sno, or Smurf2 and had reduced expression of TGF-beta1 and TGFRI. In contrast, Smad1, Smad5, BMP2, and BMP6 expression was increased, suggesting a shift from TGF-beta toward BMP signaling. In Smad3-/- chondrocytes, alternative TGF-beta signaling pathways remained responsive, as shown by luciferase assays. These non-Smad3-dependent TGF-beta pathways reduced colX expression and alkaline phosphatase activity in TGF-beta-treated Smad3-/- cultures, but only partially. In contrast, Smad3-/- chondrocytes were more responsive to BMP-2 treatment and had increased colX expression, phosphoSmads 1, 5, and 8 levels, and luciferase reporter activity. Overexpression of both Smad2 and Smad3 blocked spontaneous maturation in Smad3-deficient chondrocytes. Maturation was also abrogated by the addition of noggin, an extracellular BMP inhibitor. CONCLUSIONS: These findings show a key role for BMP signaling during the chondrocyte maturation, occurring with loss of TGF-beta signaling with important implications for osteoarthritis and cartilage diseases.     

Bone morphogenetic protein activities are enhanced by 3',5'-cyclic adenosine monophosphate through suppression of Smad6 expression in osteoprogenitor cells

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor (TGF)-beta superfamily, and some display potent osteogenic activity both in vivo and in vitro. The BMP signaling cascade involving BMP receptors at the cell membrane and intracellular messengers (Smads) has been elucidated, but the regulatory mechanisms of BMP signaling have not been clarified. We previously found that pentoxifyline (PeTx), a nonspecific inhibitor of phosphodiesterase (PDE), and rolipram, a PDE-4-specific inhibitor, enhance BMP-4-induced osteogenic differentiation of mesenchymal cells, probably through the elevation of intracellular cyclic adenosine monophosphate (cAMP) accumulation and modulation of BMP signaling pathways as enhanced BMP-4 action was reproduced by addition of dibutylyl-cAMP (dbcAMP). However, the precise mechanisms underlying the enhancing effects of those agents on BMP signaling were not completely revealed. As already reported, BMPs utilize a specific intracellular signaling cascade to target genes via R-Smads (Smad1,5,8), Co-Smad (Smad4) and I-Smads (Smad6,7). One possibility for cAMP-mediated effects on BMP signaling might be suppression of I-Smads expression since these proteins form a negative feedback loop in BMP signaling. To examine this possibility, changes in I-Smad (Smad6) expression on addition of dbcAMP or PeTx were examined in a bone-marrow-derived osteogenic cell line (ST2). Alkaline phosphatase activity in ST2 cells was consistently induced by BMP-4 treatment (300 ng/ml), and Smad6 mRNA expression was also induced by BMP-4 treatment. Although concurrent treatment of ST2 cells with BMP-4 and dbcAMP elicited further activation of alkaline phosphatase, addition of dbcAMP reduced BMP-4-induced Smad6 expression in a dose-dependent manner. Furthermore, detection of phosphorylated Smad1/5/8 on Western blotting analysis was prolonged, suggesting prolonged kinase activity of BMP receptors through suppressed expression of Smad6. Elevated intracellular cAMP might thus enhance BMP signaling by suppressing Smad6 induction and prolonging intracellular BMP signaling.     

Prostaglandin E2 inhibits BMP signaling and delays chondrocyte maturation

While cyclooxygenases are important in endochondral bone formation during fracture healing, mechanisms involved in prostaglandin E2 (PGE2) regulation of chondrocyte maturation are incompletely understood. The present study was undertaken to determine if PGE2 effects on chondrocyte differentiation are related to modulation of the bone morphogenetic protein (BMP) signaling pathway. In primary murine sternal chondrocytes, PGE2 differentially regulated genes involved in differentiation. PGE2 induced type II collagen and MMP-13, had minimal effects on alkaline phosphatase, and inhibited the expression of the maturational marker, type X collagen. In BMP-2–treated cultures, PGE2 blocked the induction of type X collagen. All four EP receptors were expressed in chondrocytes and tended to be inhibited by BMP-2 treatment. RCJ3.1C5.18 chondrocytes transfected with the protein kinase A (PKA) responsive reporter, CRE-luciferase, showed luciferase induction following exposure to PGE2, consistent with activation of PKA signaling and the presence of the EP2 and EP4 receptors. Both PGE2 and the PKA agonist, dibutyryl cAMP, blocked the induction of the BMP-responsive reporter, 12XSBE, by BMP-2 in RCJ3.1C5.18 chondrocytes. In contrast, PGE2 increased the ability of TGF-β to activate the TGF-β-responsive reporter, 4XSBE. Finally, PGE2 down-regulated BMP-mediated phosphorylation of Smads 1, 5, and 8 in RCJ3.1C5.18 cells and in primary murine sternal chondrocytes. Altogether, the findings show that PGE2 regulates chondrocyte maturation in part by targeting BMP/Smad signaling and suggest an important role for PGE2 in endochondral bone formation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 785–792, 2009     

Smad6 is essential to limit BMP signaling during cartilage development

Bone morphogenetic protein (BMP) signaling pathways regulate multiple aspects of endochondral bone formation. The importance of extracellular antagonists as regulators of BMP signaling has been defined. In vitro studies reveal that the intracellular regulators, inhibitory Smads 6 and 7, can regulate BMP‐mediated effects on chondrocytes. Although in vivo studies in which inhibitory Smads were overexpressed in cartilage have shown that inhibitory Smads have the potential to limit BMP signaling in vivo, the physiological relevance of inhibitory Smad activity in skeletal tissues is unknown. In this study, we have determined the role of Smad6 in endochondral bone formation. Loss of Smad6 in mice leads to defects in both axial and appendicular skeletal development. Specifically, Smad6?/? mice exhibit a posterior transformation of the seventh cervical vertebra, bilateral ossification centers in lumbar vertebrae, and bifid sternebrae due to incomplete sternal band fusion. Histological analysis of appendicular bones revealed delayed onset of hypertrophic differentiation and mineralization at midgestation in Smad6?/? mice. By late gestation, however, an expanded hypertrophic zone, associated with an increased pool of proliferating cells undergoing hypertrophy, was evident in Smad6 mutant growth plates. The mutant phenotype is attributed, at least in part, to increased BMP responsiveness in Smad6‐deficient chondrocytes. Overall, our results show that Smad6 is required to limit BMP signaling during endochondral bone formation. © 2011 American Society for Bone and Mineral Research     

BMP signaling components are expressed in human fracture callus

Of the various growth factors involved in the healing response after a fracture, bone morphogenetic proteins (BMPs) are emerging as key modulators. BMPs exert their effects by binding to a complex of type I and type II receptors leading to the phosphorylation of specific downstream effector proteins called Smads. The current study examined the presence of BMP signaling components in human callus obtained from five nascent malunions undergoing fracture fixation. These callus samples represented various stages of bone healing and a mixture of endochondral and intramembraneous bone healing. We performed immunohistochemistry on the callus, using antibodies for BMP (BMP-2,-3,-4,-7), their receptors (BMPR-IA, -IB, -II), and phosphorylated BMP receptor-regulated Smads (pBMP-R-Smads). Active osteoblasts showed fairly consistent positive staining for all BMPs that were examined, with the immunoreactivity most intense for BMP-7 and BMP-3. Immunostaining for BMPs in osteoblasts appeared to colocalize with the expression of BMPR-IA, -IB, and -II. Positive immunostaining for pBMP-R-Smads suggests that the BMP receptors expressed in these cells are activated. Staining for BMPs in cartilage cells was variable. The immunostaining appeared stronger in more mature cells, whereas staining for BMP receptors in cartilage cells was less ubiquitous. However, the expression of pBMP-R-Smads in cartilage cells suggests active signal transduction. Fibroblast-like cells also had a variable staining pattern. Overall, our findings indicate the presence of BMPs, their various receptors, and activated forms of receptor-regulated Smads in human fracture callus. To the best of our knowledge, this is the first study that documents the expression of these proteins in human fracture tissue. Complete elucidation of the roles of BMP in bone formation will hopefully lead to improved fracture healing care.     

Temporal and spatial expression of bone morphogenetic proteins in extracorporeal shock wave-promoted healing of segmental defect

Extracorporeal shock wave (ESW) is a noninvasive acoustic wave, which has recently been demonstrated to promote bone repair. The actual healing mechanism triggered by ESW has not yet been identified. Bone morphogenetic proteins (BMP) have been implicated as playing an important role in bone development and fracture healing. In this study, we aimed to examine the involvement of BMP-2, BMP-3, BMP-4, and BMP-7 expression in ESW promotion of fracture healing. Rats with a 5-mm segmental femoral defect were given ESW treatment using 500 impulses at 0.16 mJ/mm(2). Femurs and calluses were subjected to immunohistochemistry and RT-PCR assay 1, 2, 4, and 8 weeks after treatment. Histological observation demonstrated that fractured femurs received ESW treatment underwent intensive mesenchymal cell aggregation, hypertrophic chondrogenesis, and endochondral/intramembrane ossification, resulting in the healing of segmental defect. Aggregated mesenchymal cells at the defect, chondrocytes at the hypertrophic cartilage, and osteoblasts adjunct to newly formed woven bone showed intensive proliferating cell nuclear antigen expression. ESW treatment significantly promoted BMP-2, BMP-3, BMP-4, and BMP-7 mRNA expression of callus as determined by RT-PCR, and BMP immunoreactivity appeared throughout the bone regeneration period. Mesenchymal cells and immature chondrocytes showed intensive BMP-2, BMP-3, and BMP-4 immunoreactivity. BMP-7 expression was evident on osteoblasts located at endochondral ossification junction. Our findings suggest that BMP play an important role in signaling ESW-activated cell proliferation and bone regeneration of segmental defect.     

Activation of mitogen-activated protein kinase cascades is involved in regulation of bone morphogenetic protein-2-induced osteoblast differentiation in pluripotent C2C12 cells

Bone morphogenetic protein (BMP)-2, a member of the transforming growth factor-beta (TGF-beta) superfamily, is able to induce osteoblastic differentiation of C2C12 cells. Both Smad and mitogen-activated protein kinase (MAPK) pathways are essential components of the TGF-beta superfamily signaling machinery. Although Smads have been demonstrated to participate in the BMP-2-induced osteoblastic differentiation of C2C12 cells, the role of MAPK has not been addressed. This report shows that BMP-2 activates ERK and p38, but not JNK, in C2C12 cells. Pretreatment of cells with the p38 inhibitor, SB203580, dramatically reduced BMP-2-induced expression of the osteoblast markers alkaline phosphatase (ALP) and osteocalcin (OC). Nevertheless, overexpression of MKK3, a protein kinase that phosphorylates and activates p38, failed to induce ALP or OC expression in the absence of BMP-2, indicating that p38 activation is necessary but not sufficient for the acquisition of the osteoblast phenotype by these cells. Although ALP induction was increased slightly in the presence of PD-98059, a selective inhibitor of the ERK cascade, this compound significantly inhibited both steady-state and BMP-2-induced OC RNA levels. Our results indicate that p38 and ERK cascades play a crucial role in the osteoblast differentiation of C2C12 cells mediated by BMP-2.     

Smads及其相关转录因子与骨形态发生蛋白诱导成骨的信号传导

目的 综述有关骨形态发生蛋白(BMPs)诱导成骨的信号传导机制，深入了解BMPs基础及应用研究的理论依据。方法 查阅近期相关文献，了解BMPs相关蛋白(Smads)及其相关转录因子在BMPs诱导成骨的信号传导中的作用。结果 BMPs的信号传导过程是：BMPs首先与Ⅱ型受体和Ⅰ型受体结合，Ⅰ型受体磷酸化Smads，Smads进入核内与转录因子相互作用影响相关蛋白的转录。Smads可分为受体调节Smads(R—Smads：Smadl、2、3、5、8和9)、共同介导者Smad(Co—SmadlSmad4)和抑制性Smads(Ⅰ—Smads：Smad6、7)。Smadl、Smad5和Smad8，可能还有Smad9涉及BMPs的信号传导。多种激酶如局部粘连激酶(FAK)、Ras-细胞外信号调节激酶(ERK)、磷脂酰肌醇3激酶(P13K)和Akt丝／苏氨酸激酶与Smads的信号传导有关。与Smadl和Smad5相关的转录因子有核心结合蛋白A1(CB-FA1)、Smad相互作用蛋白1(SIPl)、鸟氨酸脱羧酶拮抗酶(OAZ)、激活蛋白-1(AP-1)、蟾蜍腹侧形成同源框蛋白-2(Xvent-2)、生长抑素(Ski)、抗增殖蛋白(Tob)、同源结构域转录因子-8(Hoxc-8)等。CBFAI与转化生长因子-β和BMP-2信号传导有关，能与Smadl、2、3、5相互作用，在骨形成中起重要作用。锁骨、颅骨发育不良被认为是CBFAI的杂合型突变引起的。CBFAI基因敲除的小鼠缺乏膜内和软骨内成骨，软骨的成熟及分化也受到了严重干扰。结论 Smads及其相关转录因子尤其是Smadl、5、8和CBFAI在BMPs诱导成骨的信号传导中起重要作用。     

ALK2 functions as a BMP type I receptor and induces Indian hedgehog in chondrocytes during skeletal development.

Growth plate chondrocytes integrate multiple signals during normal development. The type I BMP receptor ALK2 is expressed in cartilage and expression of constitutively active (CA) ALK2 and other activated type I BMP receptors results in maturation-independent expression of Ihh in chondrocytes in vitro and in vivo. The findings suggest that BMP signaling modulates the Ihh/PTHrP signaling pathway that regulates the rate of chondrocyte differentiation. INTRODUCTION: Bone morphogenetic proteins (BMPs) have an important role in vertebrate limb development. The expression of the BMP type I receptors BMPR-IA (ALK3) and BMPR-IB (ALK6) have been more completely characterized in skeletal development than ALK2. METHODS: ALK2 expression was examined in vitro in isolated chick chondrocytes and osteoblasts and in vivo in the developing chick limb bud. The effect of overexpression of CA ALK2 and the other type I BMP receptors on the expression of genes involved in chondrocyte maturation was determined. RESULTS: ALK2 was expressed in isolated chick osteoblasts and chondrocytes and specifically mediated BMP signaling. In the developing chick limb bud, ALK2 was highly expressed in mesenchymal soft tissues. In skeletal elements, expression was higher in less mature chondrocytes than in chondrocytes undergoing terminal differentiation. CA ALK2 misexpression in vitro enhanced chondrocyte maturation and induced Ihh. Surprisingly, although parathyroid hormone-related peptide (PTHrP) strongly inhibited CA ALK2 mediated chondrocyte differentiation, Ihh expression was minimally decreased. CA ALK2 viral infection in stage 19-23 limbs resulted in cartilage expansion with joint fusion. Enhanced periarticular expression of PTHrP and delayed maturation of the cartilage elements were observed. In the cartilage element, CA ALK2 misexpression precisely colocalized with the expression with Ihh. These findings were most evident in partially infected limbs where normal morphology was maintained. In contrast, BMP-6 had a normal pattern of differentiation-related expression. CA BMPR-IA and CA BMPR-IB overexpression similarly induced Ihh and PTHrP. CONCLUSIONS: The findings show that BMP signaling induces Ihh. Although the colocalization of the activated type I receptors and Ihh suggests a direct BMP-mediated signaling event, other indirect mechanisms may also be involved. Thus, while BMPs act directly on chondrocytes to induce maturation, this effect is counterbalanced in vivo by induction of the Ihh/PTHrP signaling loop. The findings suggest that BMPs are integrated into the Ihh/PTHrP signaling loop and that a fine balance of BMP signaling is essential for normal chondrocyte maturation and skeletal development.     

Bone morphogenetic proteins (BMP-2 and BMP-3) promote growth and expression of the differentiated phenotype of rabbit chondrocytes and osteoblastic MC3T3-E1 cells in vitro.

We studied the effects of highly purified bone morphogenetic protein 2 and 3 (BMP-2 and -3) on growth plate chondrocytes and osteoblastic cells in vitro and compared to TGF-beta. A mixture of BMP-2 and 3 (BMPs) strongly stimulated DNA synthesis of chondrocytes in the presence of fibroblast growth factor (FGF). BMPs induced rapid maturation of chondrocytes at a growing stage: BMPs transformed the cells into rounded cells and induced marked accumulation of cartilage matrix; TGF-beta slightly reduced matrix accumulation and changed cell morphology into spindle-like in the presence of FGF. Moreover, exposure of chondrocytes to BMPs resulted in a dramatic increase of the putative approximately 80 kD PTH receptors expressed on the cell surface. In multilayered chondrocytes at the calcifying stage, BMPs stimulated alkaline phosphatase (ALPase) activity but TGF-beta inhibited it. In osteoblastic MC3T3-E1 cells, BMPs were found to be the most potent stimulator of ALPase activity thus far described: ALPase in the cells treated with approximately 100 ng/ml of BMPs reached 5- to 20-fold over the basal, whereas TGF-beta inhibited expression of ALPase activity in these cells. The stimulatory action of BMPs overrode the inhibition of ALPase activity by TGF-beta when the cells were incubated with TGF-beta and BMPs. BMPs also upregulated expression of the approximately 80 kD PTH receptor on the cells. These results suggest that BMPs have unique biologic activities in vitro that lead to growth and phenotypic expression of cells playing a critical role in endochondral bone formation.     

Bone morphogenetic protein-7 expression and activity in the human adult normal kidney is predominantly localized to the distal nephron

Bone morphogenetic protein-7 (BMP)-7 plays an important role during fetal kidney development. In the adult, BMP-7 is most strongly expressed in the kidney compared to other organs, but the exact expression pattern as well as the function of BMP-7 is unclear. The major aim of the present study was to define which parts of the human kidney do physiologically express BMP-7 and which cells appear to be targets of BMP activity by showing phosphorylated BMP-receptor-associated Smads 1, 5, or 8 and inhibitor of differentiation factor 1 (ID1) expression. BMP-7 expression was localized by immunohistology to the epithelia of the distal tubule as well as the collecting ducts (CDs). Phospho-Smads 1/5/8 and ID1 expression largely colocalized with BMP-7 and was also localized in the epithelia of the distal tubule and the CDs. This was confirmed by polymerase chain reaction-based mRNA expression analysis. In vitro, proximal tubular cells (PTCs) expressed BMP receptors and BMP-receptor-associated Smads and were reactive to BMP-7. Our data indicate that BMP-7 expression in the adult human kidney appears to be more restricted than in the fetal situation and predominantly found in the distal nephron. Also, evidence of in vivo BMP signalling (i.e. phospho-Smads and ID1 expression) was found there. These findings suggest that BMP-7 plays a physiological role mostly in this part of the kidney. Still, as reported previously, PTCs are responsive to BMP-7, but presumably not in an autocrine or paracrine mode in normal adult kidneys.     

Protein phosphatase magnesium‐dependent 1A–mediated inhibition of BMP signaling is independent of Smad dephosphorylation

Phosphorylation of Smad1/5/8 at carboxyl‐terminal serine residues by type I receptors activates downstream bone morphogenetic protein (BMP) signaling. Protein phosphatase magnesium‐dependent 1A (PPM1A) has been shown to suppress BMP activity by dephosphorylating phospho‐Smads. We report here that PPM1A suppresses BMP signaling via a novel mechanism. PPM1A inhibited a constitutively activated Smad1 mutant lacking BMP receptor phosphorylation sites. PPM1A reduced the protein levels not only of Smad1 but also of Smad5 and Smad8. A proteasome inhibitor blocked the inhibitory effects of PPM1A on Smad1, but the Smurf‐binding motif in the Smad1 linker region was not involved in this inhibition. The phosphatase activity of PPM1A is essential for inhibition. Taken together, these findings suggest that through the dephosphorylation of unidentified substrate(s), PPM1A inhibits BMP signaling by decreasing Smad protein levels via the proteasome pathway. Moreover, knockdown of endogenous PPM1A stimulated osteoblastic differentiation, suggesting that PPM1A may physiologically suppress BMP signaling via Smads. © 2010 American Society for Bone and Mineral Research