The biological function of type I receptors of bone morphogenetic protein in bone

Shuxian Lin,Kathy KH Svoboda,Jian Q Fengand Xinquan Jiang

The biological function of type I receptors of bone morphogenetic protein in bone

Shuxian Lin1,2,Kathy KH Svoboda2,Jian Q Feng2and Xinquan Jiang1

Bone morphogenetic proteins(BMPs)have multiple roles in skeletal development,homeostasis and regeneration.BMPs signal via type I and type II serine/threonine kinase receptors(BMPRI and BMPRII).In recent decades,genetic studies in humans and mice have demonstrated that perturbations in BMP signaling via BMPRI resulted in various diseases in bone,cartilage,and muscles.In this review,we focus on all three types of BMPRI,which consist of activin-like kinase 2(ALK2,also called type IA activin receptor),activinlike kinase 3(ALK3,also called BMPRIA),and activin-like kinase 6(ALK6,also called BMPRIB).The research areas covered include the current progress regarding the roles of these receptors during myogenesis, chondrogenesis,and osteogenesis.Understanding the physiological and pathological functions of these receptors at the cellular and molecular levels will advance drug development and tissue regeneration for treating musculoskeletal diseases and bone defects in the future.

INTRODUCTION

Belonging to the transforming growth factor-β super family,1bone morphogenetic proteins(BMPs)were discovered and named in 1965 by Marshall Urist,who initially identi f i ed their ability to induce ectopic bones in muscles.2In the last 50 years,the potent osteogenic activity in vitro of BMPs has been well characterized,3as well as their constitutive activation or exogenous application,which can induce ectopic bone formation in vivo.4–5BMPs signal through cellsurface receptor complexes that consist of two distinct transmembrane serine/threonine kinase receptors,type I (BMPRI)and type II(BMPRII).6Initially,BMP ligands bind with high af f i nity to BMPRI,followed by heterodimerization with BMPRII,which allows the BMPRII to phosphorylate a short stretch of amino acids in the BMPRI and activate kinase activity.6Classically,after the activation of BMPRI,intracellular signaling is initiated through the phosphorylation of the C-terminal SSXS motif of speci f i c receptor-regulated Smads,including Smad1,5,and 8.7–10After being released from the receptor,the phosphorylated Smads form heteromeric complexes with common partner Smad,that is, Smad4.This complex is then translocated into the nucleus to regulate the transcription of genes,broadly in f l uencing growth and differentiation.9

Three type I receptors have been shown to effectively bind BMP ligands during mammalian skeletal development—types IA and IB BMP receptors(BMPRIA or ALK3 and BMPRIB or ALK6),as well as type IA activin receptor (ACVRI or ALK2).11–12In recent decades,studies of clinical patients,genetic animal models and cell lines have consistently demonstrated that all three type I receptors are essential for osteolineage and chondrolineage proliferation,differentiation and function.It has become clear that alterations in the intensity,location,and duration of BMPRI activity lead to heterotopic bone formation, skeleton,and cartilage deformation,as well as bone metabolism disorders.Here we provide an updated review that speci f i cally focuses on the biological function of BMPRI in bone formation.Emphasis is placed on murine genetic studies(Table 1)that have assessed the requirement for and the roles of different types of BMPRI, including ALK2,ALK3,and ALK6 during osteogenesis, chondrogenesis and osteoclastogenesis,as summarized in Figure 1.

BIOLOGICAL FUNCTIONS OF ALK2 IN BONE FORMATION

ALK2 is widely expressed in many tissues during embryonic development and is highly present in bones during postnatal development.13Several mesenchymal stem cell(MSC)lines show high expression levels of ALK2,11,14and its constitutive activation in myoblasts induces heterotopic bone during the endochondral bone formation process,suggesting that ALK2 has essential roles in both osteogenesis and chondrogenesis.

Ectopic expression of ALK2 in myoblasts leads to heterotopic endochondral bone formation

The regulatory role of ALK2 in osteogenesis and chondrogenesis did not arouse interest until the discovery of f i brodysplasia ossi f i cans progressiva(FOP,MIM 135100),which is characterized by congenital malformations of the great toes and progressive heterotopic ossi f i cation in muscles,tendons, ligaments,and other connective tissues.15–16Genetic analysis of FOP patients has identi f i ed gain-of-function mutations in ALK2,including c.617G＞A(p.R206H),c.619C＞G(p.Q207E), c.1067G＞A(p.G356D),c.982G＞T(p.G328W),c.983G＞A(p.G328E),c.982G＞A(p.G328R),c.774G＞C/c.774G＞T (P.R258S),c.1124G＞C(p.R375P),c.587T＞C(p.L196P), c.590–592delCTT(p.P197_F198delinsL),and c.605G＞T (p.R202I),among which R206H is the most common mutation and can be found in～90%of FOP patients.17–31The classic, constitutively active ALK2 receptor containing the arti f i cial Q207D mutation or the R206H mutation recaptures the FOP condition in transgenic animal models.32–33Further evaluation of these FOP mutations revealed that the ectopic expression of ALK2 increased Smad-dependent BMP signaling activity, which potentially occurs for both osteogenic and chondrogenic differentiation of myoblasts,thus forming heterotopic bone through an endochondral bone formation process.21,32,34–39In addition,inhibiting the activation of BMP signaling effectors Smad1/5/8 in tissues that constitutively express ALK2 resulted in a reduction of the ectopic ossi f i cation and functional impairment.32The stable in vitro transfection of the Alk2R206Hmutation in C2C12 cells(mouse myoblasts)increased the levels of both osteogenic markers (osterix(Osx),alkaline phosphatase(Alp))and chondrogenic markers(type II collagen(Col2),type X collagen(Col10)).40–41Conversely,knockdown of Alk2 in C2C12 cells potentiated muscle differentiation and repressed BMP6-induced osteoblast differentiation.42These elevated results suggest that Smad-dependent ALK2 signaling is important in heterotopic ossi f i cation and endochondral bone formation of myoblasts.

ALK2 regulates osteogenic and chondrogenic differentiation of MSCs

Studies focusing on Alk2R206Hmutant mice or cells also provide evidence indicating that ALK2 has an important role in the osteogenic differentiation of MSCs.First,the mesenchymal progenitor cells isolated from FOP(R206H) patients or Alk2R206Hmutant mice showed increased Smad-dependent BMP signaling activity with upregulated Alp,runt-related gene 2(Runx2),and osteocalcin(Ocn) genes.39,43Second,the constitutive expression of Alk2 in mesenchymal cells or pre-osteoblasts makes those cells more receptive to exogenous BMPs with respect todifferentiating into functional mineralizing osteoblasts.41In contrast,speci f i cally suppressing Alk2 activity decreased the enhanced osteogenic differentiation to control levels.43Furthermore,in vivo studies found that in heterozygous Alk2R206Hknock-in mice,the Tek/Tie2+progenitor cells could be recruited and differentiated into bone cells in heterotopic ossi f i cation lesions.33Consistently,a recent mouse model study showed that conditional activation of Alk2 in mesodermal lineage cells resulted in ectopic bone formation at distal joints with an elevated number of osteoblast progenitors as well as bone formation activity.44Collectively,Smad-dependent ALK2 signaling in the mesenchymal progenitors has an important role for their speci f i cation toward osteolineage cells.

In addition,the tracking study of Tek/Tie2+progenitor cells in heterozygous Alk2R206Hknock-in mice also identi f i ed a chondrogenic differentiation of this cell population, which was responsible for forming a cartilage template that developed to form endochondral bone.33Further analysis found that ectopic expression of Alk2 increased sensitivity and accelerated chondrogenic differentiation of mouse embryonic f i broblasts and that the loss of Alk2 severely inhibited chondrogenic differentiation,suggesting that ALK2 was required during early chondrogenesis.39Moreover,studying a chick limb bud with constitutive Alk2 expression showed accelerated chondrocyte maturation and induced Indian hedgehog,which is a key factor for chondrocyte maturation.45Moreover,chondrocytes with the Alk2R206Hmutation showed increased expression of both the early chondrocyte-speci f i c markers(sex determining region Y)-box 9(Sox9),Col2,aggrecan (Agg),and the late marker Col10.39Collectively,these results established that ALK2 is an essential enhancer of chondrogenic differentiation.

Roles of ALK2 in regulating osteoblasts and osteoclasts

The endogenous expression level of Alk2 in postnatal bone was found to be much higher than that in heart and skeletal muscles,13suggesting that ALK2 might also be essential in osteolineage cells.Accordingly,Alk2 knockdown in murine osteoblast progenitors(KS483)reduced BMP6-induced osteogenic differentiation.42Interestingly, an in vivo study found that a conditional disruption of Alk2 in bone cells(including immature osteoblasts,mature osteoblasts,and osteocytes)led to an increase in endogenous bone mass during postnatal development.13Analysis of this mouse model indicated that the disturbed bone homeostasis was more likely due to an upregulation of canonical Wnt signaling in conjunction with the downregulation of Wnt inhibitors,scelerostin(SOST)and dickkopf 1(DKK1;Figure 2).13In addition to BMP signaling,Wnt signaling in osteoblasts has been examined for a decade, and there is suf f i cient evidence supporting the hypothesis that canonical Wnt signaling serves as a bone mass inducer that positively regulates osteoblast differentiation and maturation but negatively affects osteoclast activity(see below for a detailed description).46However,the direct regulatory role of ALK2-induced BMP signaling in late osteolineage cells remains unclear,and further evaluations need to be performed for a comprehensive understanding.

To date,no evidence has demonstrated that ALK2 directly regulates osteoclast function.However,recent studies have found that the constitutively active mutation of ALK2 in myoblasts led to the increased formation of osteoclasts from their precursors through transforming growth factor-β signaling.An implantation of Alk2R206H-transfected C2C12 cells with BMP2 in nude mice resulted in robust heterotopic ossi f i cation with increased osteoclast formation in muscle tissues.47–48Furthermore,a co-culture of Alk2R206H-transfected C2C12 cells as well as the conditioned medium from Alk2R206H-transfected C2C12 cells enhanced osteoclast formation in mouse monocytic RAW264.7 cells.47Mechanism analysis suggested that the elevated secretion of transforming growth factor-β from the mutant myoblasts led to the upregulated activation of p38 mitogen-activated protein kinase(MAPK)signaling in the surrounding monocytes,thus contributing to the enhanced osteoclastogenic differentiation.47

BIOLOGICAL FUNCTIONS OF ALK3 IN BONE FORMATION

ALK3 is widely expressed in a variety of tissues during embryonic development,but it is mainly expressed in osteolineage cells and bone marrow cells during postnatal bone formation,based on in vitro studies of different cell lines.49–50Findings have con f i rmed a high expression level of ALK3 in osteolineage cells from MSCs to differentiated bone cells.14,51–54Many studies have consistently indicated that ALK3 is one of the key receptors for conducting BMP signaling during osteogenesis and chondrogenesis.However,the roles of ALK3 in bone biology have remained unclear until recent studies using Alk3 conditional ablation in osteogenic tissues because its conventional deletion in mice is embryonically lethal before bone development.55It is clear that the regulatory role of ALK3 differs depending on distinctive cells,stages,tissues,and ages.ALK3-induced BMP signaling also crosstalks with the Wnt/β-catenin signaling pathway and functions in interactions between osteoblasts and osteoclasts.

Roles of ALK3 in mesenchymal pre-osteolineage cells

In MSCs,the forced expression of Alk3(that is,the overexpression of wild-type Alk3)initiated osteogenic development.On the contrary,downregulating Alk3 activity(that is,overexpressing truncated Alk3)led to decreased expression of Alp and less von Kossa staining.51When speci f i cally deleting Alk3 in the early development of the palatal mesenchyme by E12.5 using Oar2-Ires Cre,the formation of mesenchymal condensation in the palate was delayed as was theconsequent palate bone formation.56This in vivo study, together with the in vitro study,suggested that ALK3-induced BMP signaling was required for the differentiation of MSCs toward the osteolineage.However,other groups came to an opposite conclusion in similar studies.The downregulation of Alk3 in 2T3 cells(characterized as osteoblast precursors)or the conditional deletion of Alk3 in bone marrow mesenchymal cells using Mx1 Cre(Cre is activated in an osteolineage-restricted stem/progenitor cell subset,one speci f i c subset of bone marrow mesenchymal cells57)led to ectopic mineralization via the upregulation of the bone formation activity of osteoblasts,49,54indicating that ALK3 inhibits the osteoblastic lineage commitment of bone marrow stem cells. These contrary results suggest that ALK3 may regulate (promote or inhibit)the differentiation of MSCs in a tissuedependent manner.

Roles of ALK3 in osteoblasts

Deletion of Alk3 speci f i cally in immature osteoblasts using 2.3 kb Col1 Cre or 3.2 kb Col1 Cre indicated that the maturation progress of these osteoblasts was suppressed because both the bone formation rate and the mineral apposition rate were downregulated.58–60An analysis of these mutant osteoblasts found an enhanced proliferation with decreased expression of several speci f i c osteoblast markers,including Runx2,Osx,bone sialoprotein (Bsp),and Alp.58–60Mice with a speci f i c disruption of Alk3 in differentiated osteoblasts(using Og2 Cre)were born normally and did not exhibit overt bone changes,except for a slight decrease in bone mass at 3 months,which may be caused by a mild downregulation of osteoblast function(lower bone formation rate with decreased BV/ TV,but no change in the expression levels of osteopontin (Opn)and Ocn),61suggesting that ALK3 promotes mature osteoblast function and osteoblast–osteocyte transition in a relatively mild way.However,the decreased bone mass in these mutant mice later increased and was con f i rmed to have an even higher bone volume compared with that of wild-type mice at 10 months,which mainly resulted from the downregulated bone resorption caused by reduced osteoclast activity.61These results suggest that ALK3 expressed in mature osteoblasts has diverse effects on bone mass and homeostasis in an age-dependent manner.

Roles of ALK3 in the interaction between osteoblasts and osteoclasts

For decades,more and more studies have con f i rmed that there is a communication between osteoblasts and osteoclasts,which has an exquisite and important role in bone modeling and remodeling.The discoveries of the biological functions of ALK3 imply that this factor may be one of the key molecules in this process.

Osteoblasts have critical roles in bone resorption by regulating osteoclastogenesis due to their ability to produce nuclear factor kappa-B ligand(RANKL),which is essential for promoting osteoclast differentiation and function,and its decoy receptor osteoprotegerin (OPG).62–63Several studies have con f i rmed that ALK3-induced signaling in osteoblasts regulates osteoclastogenesis via the RANKL-OPG mechanism.The earliest direct evidence came from a report by Wan et al.,64in which they found that the transfection of constitutively active Alk3 stimulated the OPG promoter and that two homeobox C8(Hoxc-8)-binding sites in the OPG promoter responded to the ALK3 activation.In accordance with these results,several in vivo studies have con f i rmed that conditional deletion of osteoblastic Alk3 in distinct cell differentiation stages,59–61or in different developmental periods,58,60–61,65led to decreased osteoclast numbers and decreased expression of bone resorption markers(matrix metallopeptidase 9(Mmp9),tartrate-resistant acid phosphatase(Trap),and cathepsin K(CatK),among others). Furthermore,accumulating evidence suggests that crosstalk between BMP and Wnt signaling in bone may also be involved in osteoblast-regulated osteoclastogenesis through the RANKL-OPG pathway.Kamiya et al.60,65recognized that a disruption of ALK3-induced signaling, including both Smad and non-Smad signaling(such as p38 MAPK),in osteoblasts resulted in upregulated Wnt/ β-catenin activity due to decreased production of its downstream targets,DKK1 and SOST.60,65It is widely reported that canonical Wnt signaling in osteoblasts negatively regulates their supporting function in osteoclastogenesis by affecting RANKL and OPG expression,thus inhibiting osteoclast differentiation and activity as well as suppressing osteoclast-mediated bone resorption.66–67Taken together,in osteoblasts,ALK3 activates SOST and DKK1,while both SOST and DKK1 inhibit canonical Wnt signaling and maintain the activity of Wnt/β-catenin signaling at a certain level.68–71As a result,ALK3-induced BMP signaling and Wnt signaling contribute not only to osteoblast proliferation,differentiation,and maturation,but also to the regulation of osteoclastogenesis(mainly via the RANKL-OPG pathway;Figure 2).

However,ALK3 signaling in osteoclasts also negatively regulates osteoblast functions.For example,conditionally disturbed Alk3 expression in osteoclasts using CatK Cre not only inhibited osteoclast function but also enhanced osteoblast function,that is,increased osteoblast number and bone formation rate.59Furthermore,it has been reported that several factors(including plateletderived growth factor BB,v-ATPase V0 subunit d2 (Atp6v0d2),CatK and osteoclast inhibitory lectin(OCIL))produced by osteoclasts negatively affect osteoblast functions.72–75Among these osteoclast-derived osteoblastic inhibitors,both CatK and ATV6v0d2 were signi f i cantly increased after a stimulation of Smad-dependent ALK3 signaling in osteoclasts.76–78Collectively,BMPs might bind to ALK3 on the surface of osteoclasts and activate BMP signaling,leading to the upregulation of factors such as CatK and ATV6v0d2,which suppress osteoblast activity and downregulate the rate of bone formation.76–77,79

Roles of ALK3 in chondrogenesis

Although ALK3 mainly functions in osteogenesis,it also has an important role in chondrogenesis.For instance, a forced expression of Alk3 in MSCs(C3H10T1/2)51or pre-chondrocytes80induced chondrogenic differentiation, while downregulated Alk3 suppressed this process.51The regulatory function of ALK3 in chondrogenesis has been further supported by in vivo studies.81–86Initially,the role of ALK3 in chondrogenesis,such as regulating chondrocyte proliferation,survival,and differentiation,was thought to be associated with ALK6 during chondrogenesis.81Soon after,it was demonstrated that ALK3 itself has a unique and broad role during chondrogenesis.First,overexpressing a constitutively active ALK3 in chondrocytes in vivo stimulated the differentiation of pre-chondrocytes and proliferating chondrocytes,promoting their maturation toward hypertrophy.82Second,the conditional deletion of Alk3 speci f i cally in developing joints resulted in the downregulation of proteoglycans and extracellular matrix cartilage genes,including Col2,Col10,and Agg,leading to articular cartilage f i brosis and degeneration during postnatal development.83Third,Alk3 ablation in postnatal chondrocytes caused arrested chondrogenesis and endochondral ossi f i cation,with diminished chondrocyte proliferation and little expression of cartilage markers,such as SOX9,Indian hedgehog,Col II,Col X,AGG,and glycoproteins,among others.84–86Taken together,these studies support the notion that ALK3 is one of the key factors for regulating the speci f i cation of pre-chondrogenic mesenchyme as well as chondrolineage differentiation and maturation,postnatal chondrogenesis and the maintenance of articular cartilage.

BIOLOGICAL FUNCTIONS OF ALK6 IN BONE FORMATION

The expression of ALK6 is primarily restricted to mesenchymal pre-cartilage condensations during mouse development,but it has also been identi f i ed in differentiated chondrocytes and osteoblasts in adult mice.50,87–89Accordingly,a study of different cell lines suggested that Alk6 was expressed at a low level or was undetectable in MSCs;however,it was speci f i cally upregulated during osteoblastic differentiation.14,51,53These ALK6 expression patterns suggest that it may participate in chondrogenesis and in f l uence late osteolineage cells.

ALK6 in regulating chondrogenesis

Patients with a homozygous mutation in ALK6 have severe limb deformations consisting of a short stature,aplasia of the f i bula,severe brachydactyly and ulnar deviation of the hands,which mainly result from chondrodysplasia during skeletal development.90In addition,constitutive expression of Alk6 in pre-chondrocytes signi f i cantly increases the induction of chondrocyte differentiation.80,91Accordingly, several in vivo studies have consistently demonstrated that ALK6 is required for the proliferation and chondrogenic differentiation of pre-chondrogenic and proliferating chondrocytes.82,88However,null mutations of Alk6 only exhibited mild limb abnormalities that were largely restricted to the appendicular skeleton.82,88,92Soyun Yi88posited that ALK6 had broadly overlapping functions with other BMP receptors because a Alk6-Bmp7 double-mutant exhibited more-severe skeletal defects than did an Alk6 single-knockout.Later,it was suggested that ALK3 and ALK6 display functionally redundant aspects during early chondrogenesis81because ALK6 signaling could be replaced by constitutively active ALK3.82In summary,these results indicate that ALK6,rather than having a unique role, may have overlapping functions with other BMP receptors, especially ALK3,in supporting pre-chondrogenic mesenchyme as well as chondrocyte proliferation,differentiation, and maturation.

ALK6 in regulating osteogenesis

In vitro studies have found that the expression of a constitutively active Alk6 induced the formation of mineralized bone matrix,while the overexpression of truncated Alk6 or the inhibition of endogenous Alk6 completely blocked BMP2-induced osteoblast differentiation and mineralized bone matrix formation.54These results suggest that the osteoblastic ALK6 is required for osteoblast differentiation and bone formation.Transgenic mice that expressed a truncated dominant-negative Alk6 in targeted osteoblasts using the 2.3 kb Col1 promoter exhibited impaired postnatal bone formation,including severely reduced bone mineral density,bone volume,and bone formation rates.These characteristics indicate that the osteoblastic ALK6 has a necessary role during postnatal bone modeling and remodeling via regulating osteoblast/ osteocyte maturation.93However,in Alk6 null mice,the defects largely resulted from the disturbed chondrogenesis,and there was little in f l uence on osteogenesis,88implying that the regulatory role of ALK6 is mildly involved in bone ossi f i cation.

CONCLUSION

In conclusion,all three types of BMPRI have distinct but important roles during chondrogenesis,osteogenesis,and osteoclastogenesis.They might not only directly regulate the chondrogenic or osteogenic differentiation of bone cells and in f l uence osteoclast activity through the RANKL-OPG pathway but also crosstalk with Wnt signaling by altering their downstream molecules,including DKK1 and SOST,during bone development and homeostasis. Despite some knowledge gaps,much has been learned over recent decades about the functions of BMPRI in a variety of cell types,including MSCs,chondrocytes, osteoblasts,osteoclasts,and myoblasts,using genetic animal models.However,its regulatory role in osteocytes remains unknown.Although osteocytes,which compose 90%–95%of all bone cells in adult bone,have recently been demonstrated to be crucial for bone biology because of their functions in inducing osteoclasts,regulating mineral metabolism and matrix remodeling,and reacting to mechanical loading.94Furthermore,studies of BMPRI have been fueled by the desire to understand the molecular underpinnings of rare bone diseases or the mechanisms of clinical applications for BMPs in common diseases,such as bone fracture healing and spinal surgery, and these studies now might contribute to the development of new therapies for congenital or age-related bone diseases.Recently,Marc Baud’huin et al.95developed a soluble mBMPRIA(ALK3)-mFc fusion protein and found that mBMPRIA(ALK3)-mFc treatment could successfully downregulate Smad-dependent ALK3 signaling,thus increasing bone mass in both young(7–10 weeks)and old (14–18 weeks)mice or preventing bone loss induced by estrogen de f i ciency in ovariectomized mice.This work set an example showing that regulation of signaling through BMPRI may have therapeutic bene f i ts.Hence, continuing the bedside-to-bench exchange of information about BMPRI will help to provide novel,therapeutically useful strategies for skeletal physiology,pathology,and regeneration.

Acknowledgements

This work was supported by the National Natural Science Foundation of China(No.81500814)(SXL);the National Basic Research Program of China(973 Program,2012CB933604),the National Science Fund for Distinguished Young Scholars of China(No.81225006)and the National Natural Science Foundation of China(No.81430012 and No.81170939)(XJ); and the National Institutes of Health Grants DE025014 and R56DE022789 (JQF).We would like to thank Jeanne Santa Cruz for her assistance in preparing the manuscript.

Competing interests

The authors declare no con f l ict of interest.

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©The Author(s)2016

Research(2016)4,16005;

10.1038/boneres.2016.5;published online:5 April 2016

1Department of Prosthodontics,Ninth People’s Hospital Af f i liated with Shanghai Jiao Tong University,School of Medicine,Shanghai,China and2Department of Biomedical Sciences,Texas A&M Baylor College of Dentistry,Dallas,TX,USA

Correspondence:Jian Q Feng and Xinquan Jiang(Jfeng@bcd.tamhsc.edu and xinquanj@aliyun.com)

Received:7 December 2015;Revised:4 February 2016;Accepted:20 February 2016