From skeletal to non skeletal: The intriguing roles of BMP-9: A literature review

doi:10.4236/abb.2013.410A4004

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Advances in Bioscience and Biotechnology, 2013, 4, 31-46 ABB

http://dx.doi.org/10.4236/abb.2013.410A4004 Published Online October 2013 (http://www.scirp.org/journal/abb/)

From skeletal to non skeletal: The intriguing roles of

BMP-9: A literature review

E. Leblanc1,2,3, G. Drouin3,4, G. Grenier3,4, N. Faucheux5, R. Hamdy1,2

1Department of Surgery, Orthopedic Surgery Division, McGill University, Montreal, Canada

2Shriners Hospital for Children, Montreal, Canada

3Department of Surgery, Orthopedic Surgery Division, Université de Sherbrooke, Sherbrooke, Canada

4Étienne-Lebel Clinical Research Centre, Université de Sherbrooke, Sherbrooke, Canada

5Department of Chemical and Biotechnological Engineering, Université de Sherbrooke, Sherbrooke, Canada

Email: elisabeth.leblanc2@usherbrooke.ca

Received 13 August 2013; revised 14 September 2013; accepted 1 October 2013

permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

In the well-known superfamily of transforming growth

factors beta (TGF-



), bone morphogenetic proteins

(BMPs) are one of the most compelling cytokines for

their major role in regulation of cell growth and dif-

ferentiation in both embryonic and adult tissues. This

subfamily was first described for its ability of poten-

tiating bone formation, but nowadays, the power of

BMPs is well beyond the bone healing scope. Some of

the BMPs have been well studied and described in the

literature, but the BMP9 is still worthy of attention. It

has been shown by many authors that it is the most

potent osteogenic BMP. Moreover, it has been de-

scribed as one of the rare circulating BMPs. In this

paper, we will review the recent literature on BMP9

and the different avenues for future research in that

field. Our primary scope is to review its relation to

bone formation and to elaborate on the available lit-

erature on other systems.

Keywords: BMP9; Review; Osteogenesis;

Chondrogenesis; Angiogenesis; Tumorigenesis;

Neurogenesis; Glucose Metabolism

1. INTRODUCTION

Fifty years ago, Urist et al. first reported that demineral-

ized bone matrix could promote ectopic bone formation

by stimulating transformation of primitive mesenchymal

cells of soft tissues into osteogenic cells [1]. Among the

growth factors with osteoinductive potential within the

bone matrix, the most important seem to be the bone

morphogenetic proteins (BMPs). BMPs belong to the

transforming growth factor beta (TGF-



) superfamily.

This group of proteins is characterized by a polybasic

proteolytic processing site which is cleaved to produce a

mature protein containing seven conserved cysteine re-

sidues [2]. More than 20 BMPs have been identified to

date [3], and most of them homodimerize via disulphide

bridges to be active, although heterodimers have been

described and are prop o sed to be m or e powerful [ 4,5].

Classically, BMPs are subdivided in four classes based

on their sequence homology: BMP 2/4, BMP 5/6/7/8,

BMP 9/10 and BMP 12/13/14 [6,7]. The sequence simi-

larity of the members of these subclasses is more than

50% and is also quite conserved across species [8,9].

BMPs signal through serine/threonine kinase receptors

composed of type I and II subtypes that activate the Smad

pathway and some mitogen activated protein kinases

(MAPKs) (Miyazono, 2005 #94; Bandyopadhyay, 2013

#320). Genetic disru ption of BMP pathways can result in

skeletal and extraskeletal abnormalities by their major role

in cell proliferation and differentiation during develop-

ment [10,11].

BMPs can also be classified by their osteogenic poten-

tial. To date, BMP2/4/6/7/9 have been identified as hav-

ing osteogenic properties, notably for their involvement

in the regulation of osteoblast differentiation [12-16 ] and

bone formation [14,17]. From this BMP group, BMP9

has been shown to be the most osteogenic [14]. This might

be due to the lack of inhibition of BMP9 by noggin, an

extracellular antagonist of BMP-2 or BMP-7 [12,18].

While some BMPs such as BMP 2/3/4/6/7/8 can be pro-

duced by preosteoblasts [19], BMP9 is mainly produced

in the liver [20,21]. BMP9 is also circulating at active

concentrations and is known to be a vascular quiescence

factor [22]. BMP9 is the physiological ligand of the en-

dothelial receptor ALK1 (activin receptor-like kinase 1,

also known as Acvrl1) in association with BMPRII [23-

Published Online October 2013 in SciRes. http://www.scirp.org/journal/abb

E. Leblanc et al. / Advances in Bioscience and Biotechnology 4 (2013) 31-46

25]. ALK1 is one of the seven type I recaptors for TGF-



fa mily memb e rs [26] .

Review articles on BMP9 are needed to better under-

stand its potential. Lamplot et al. have recently reviewed

BMP9 signaling in depth. In this study, the osteogenic

pathways including the Smad and different mediators of

BMP9 were reviewed. They also point out crosstalk with

other factors such as TGF-β1, Wnt/β-catenin signaling,

Growth hormone, Mitogen activated protein kinases

(MAPKs), Hypoxia inducible factor 1 alpha (HIF1α),

Notch, Peroxisome proliferator-activated receptor gamma

(PPAR-γ), insulin-like growth factor (IGF) and retinoid

acid [27]. Here, we propose an overview of the multiple

roles of BMP9 in cell proliferation and differentiation

and its relation to current clinical issues. The compelling

osteogenic potential o f BMP9 will be discussed, but also

its role bone resorption, chondrog enesis, cancer develop-

ment, angiogenesis, neurogenesis, hepatocyte physiology,

glucose metabolism, and myogenesis (Figure 1).

2. MECHANISM OF ACTION

BMPs transduce signal through serine/threonine kinase

heterotetramer receptors composed of two type I and two

type II receptors. Once the BMP binds to the receptor

complex, the type II receptors phosphorylate the type I

receptors. Signal will then transduce via Smad (canonical)

or non-Smad pathways. In the canonical Smad pathway,

type I receptors phosphorylate the receptor regulated-

Smads (R-Smad), i.e. Smad1/5/8 which then recruit the

common mediator-Smad4 (coSmad). The R-Smad/co-

Smad complex translocates to the nucleus and regulates

target gene transcription by binding to Smad elements in

their promoters [6,28]. The Smad-independent pathway

[28,29].

In mice and humans, seven TGF-β family type I re-

ceptors (activin receptor-like kinase; ALK1 to ALK7)

have been identified. Luo et al. have demonstrated by

dominant negative mutations and RNAi studies that on ly

ALK1 and ALK2 mutants inhibited BMP9-induced

Smad activity in bone marrow mesenchymal stem cells

as well as osteogenic differentiation and ectopic bone

formation [30]. Brown et al. found that BMP9 has a

strong relative affinity for ALK1 and for BMPRII, one of

the five different TGF-β family type II receptors that

have been identified (ActRIIA, ActRIIB, BMPRII,

Figure 1. The multiple effects of BMP9. (−): downregulation; (+): upregulation, MPC: mesenchymal progenitor cells.

E. Leblanc et al. / Advances in Bioscience and Biotechnology 4 (2013) 31-46 33

TGFBRII and AMHRII) [23 ]. BMP9 also has a good af-

finity for Activin RIIA, another type II receptor [23].

More recently, Townson et al. analyzed the kinetic and

thermodynamic properties of BMP9 with the different

type II receptors and they noticed that ActRIIB had the

strongest affinity for BMP9 followed by BMPRII. The

affinity of BMP9 for ActRIIA was 300-fold weaker than

for ActRIIB [31].

To date, amongst all BMPs, only BMP2/4/6/7 and 9

have been identified to have osteogenic properties and

BMP9 seems to be the one with the strongest osteogenic

potential [14{Li, 2003 #7]}. Few studies tried to under-

stand why BMP9 has this strong osteogenic potential.

First, Bergeron et al. have shown that the Smad phos-

phorylation induced by BMP9 was not prevented by

noggin, an extracellular antagonist of the BMP pathway

[12]. These data were recently confirmed by two studies

by Wang et al. and Luther et al. and reinforced the hy-

pothesis that the high osteogenic capacity of BMP9 is

probably augmented by its noggin resistance [18,32].

Kang et al. also showed that BMP3 inhibited the osteo-

genic potential of BMP2 and BMP7 but had no effect on

BMP9 [14]. To date, no BMP9 inhibitor has been identi-

fied. Moreover, BMP9 is a secreted protein that has a

pro-region but this pro-region does not seem to function-

ally inhibit BMP9; quite opposite, the pro-region may

stabilize and protect BMP9 in vivo [23]. Finally, BMP9

has synergistic partners that would enhance its effects.

Growth hormones have been shown to be upregulated by

BMP9 and act synergistically to promote endochondral

ossification in mesenchymal progenitor cells (MPC) [33].

EGF also demonstrated an increase in BMP9-induced

bone formation in cultured mouse fœtal limb explants. In

MPCs, BMP9 expression was up-regulated when EGF

was present. Furthermore, EGF receptor expression was

directly up-regulated by BMP9 [34]. These specific fea-

tures could, in part, explain the strong osteogenic poten-

tial of BMP9 as compared to other osteogenic BMPs.

Production of endogenous BMP9 is mainly located in

the liver [20,21]. Indeed , BMP9 transcripts were lower in

the human lung (400 fold less), brain (470 fold less) and

bone marrow (3400 fold less) than in the liver [21]. Us-

ing Harlan Sprague Dawley rats, Miller et al. have first

identified the non-parenchymal liver cells (Kupffer and

endothelial cells) as BMP9 producers, no BMP9 being

detectable in hepatocytes [20]. In contrast, Bidart et al.

(2012) have recently found, using human liver biopsies,

that BMP9 is present in hepatocytes and intrahepatic

biliary epithelial cells [21]. In their experimental condi-

tions, BMP9 was not detected in blood vessel walls and

mesenchyme [21]. BMP9 mRNA was also detected by

semi quantitative reverse transcription polymerase chain

reaction (RT-PCR) in both fresh human endochondral

(iliac bone) and intramembranous (mandible) bones [35].

Primary cultures of osteoblastic cells extracted from hu-

man intramembranous and endochondral bones seem to

be able to synthesize BMP9 [35 ]. BMP9 is also found in

human circulation (2 - 12 ng/mL) under two forms: an

active (60%) and inactive form (40%) that can be further

activated by a serine endoprotease to a mature and fully

active form [21]. A mouse development study revealed

that BMP9 circulating levels peak within 3 weeks after

birth (with a peak at about 6 ng/mL) and decrease to 2

ng/mL in adulthood [21].

3. BMP9 AND BONE FORMATION

The first report about BMP9 as an osteogenic factor is in

the mid 90’s by Celeste et al. [36]. In this study, the au-

thors describe the molecular cloning and biological ac-

tivity of this new member of the TGF-



superfamily [36].

The osteogenic potential of BMP9 was then mostly test-

ed through viral vectors to promote bone formation in

many clinical settings.

Hence, Alden et al. [37] showed that they could fill a

cranio-facial critical bone defect in rats with an adenovi-

ral human BMP9 vector. Radiologic and histologic ana-

lyses of the mandibular bones demonstrated significant

bony healing compared to adenoviral



-galactosidase

controls. Also, the mechanism through which bone was

formed during the critical defect healing was proven to

be endochondral ossification [37].

In the spinal region, BMPs have been shown to pro-

mote spinal arthrodesis at a higher rate than autologous

bone alone, and to BMP2 and 7, which are actually ap-

proved for this specific indication [38]. Another group

has evaluated the efficacy of the use of a direct percuta-

neous injection of BMP9 expressing adenovirus to pro-

mote spinal fusion in rats [39]. The animals were directly

injected with viral v ectors in the lumbar paraspinal mus-

culature and were sacrificed after 16 weeks. Computer-

ized tomography studies and histological analysis dem-

onstrated bone induction at the injection sites, leading to

solid spinal arthrodesis. The authors did not have any

evidence of pseudarthroses, nerve root compression, or

systemic side effects. This study, while strongly support-

ing the advancement of BMP gene therapy techniques

toward clinical use, has also shown that the use of BMP9

through gene therapy could be an interesting avenue to

explore [39].

In a similar experimental design, ex vivo BMP9 gene

therapy on human mesenchymal stem cells (hMSCs) has

been proven to be efficacious for spinal fusion in rats.

Indeed, Dumont et al. infected ex vivo hMSCs by using

adenovirus (Ad) containing BMP9’s chimeric sequence

composed of the murine BMP9 proregion and human

mature region under the control of cytomegalovirus

(CMV) promoter [40]. The infected hMSCs by AdBMP9

or Ad β-galactosidase used as control were then injected

E. Leblanc et al. / Advances in Bioscience and Biotechnology 4 (2013) 31-46

in paraspinal muscles of nude athymic rats. After only 8

weeks, rats treated with BMP9 expressing cells, showed

radiographically and histologically massive ectopic bone

formation leading to spinal fusion [40]. The results of

this study suggested that gene therapy on hMSCs ex vivo

using CMV driven BMP9 expression was possible and

may be useful to induce sp inal f usion . While show ing the

effect of BMP9 on hMSCs, this study also brought a

more attractive way of using BMP gene therapy. The

year after, the same team showed that a similar injection

of AdBMP9 ex vivo treated hMSCs was able to produce

ectopic bone formation in the hindlimbs musculature of

athymic nude rats [41].

Heterotopic bone formation by itself can tu rn out to be

useful if the muscle is used as a vascularised bone graft.

Hence, the latissimus dorsi muscle from nude rat was

infected with an adenoviral vector of BMP9 to make a

vascularised bone flap using heterotopic ossification as

an advantage [42]. With weekly biopsies and using RT-

PCR and immunohistochemistry, they found that the mu-

scle expressed chondrogenic and then osteogenic mark-

ers through the 3 weeks following the injection. They

estimated that the flap harvest sh ould be after 2 weeks to

be used as a vascularised moldable bone graft [42].

Gene therapy has been shown to be efficient, but the

transfer in humans is problematic because of its viral na-

ture and the existing lack of control over that kind of

technology. To work around that scientific and ethical

issue, nucleofecting hMSCs with nonviral plasmid ex-

pressing hBMP9 was developed [43]. Nonviral gene

transfer is safer, can be controlled and is reproducible.

hBMP9-nucleofected hMSCs in culture demonstrated an

increase in calcium deposition compared to control

EGFP (enhanced green fluorescent protein) nucleofected

hMSCs. Moreover, hBMP9-nucleofected hMSCs trans-

planted in ectopic sites in mice induced bone formation

28 days post-injection [43]. This safe and attractive al-

ternative was a breakthrough in the field of regenerative

medicine at the time, however this technique is demand-

ing.

In the late 70’s, Boyer’s team synthetized the first

functional polypeptide product from a gene of chemi-

cally synthesized origin [44]. This turning point in bio-

technology opened the door to the use of recombinant

proteins for clinical purpose. The use of recombinant

proteins is safer than viral gene transfer and yet more

accessible than nucleofection. Furthermore, the avail-

ability of recombinant BMP9 has liberalized its use and

led to more accessible research on its osteogenic poten-

tial. Even if recombinant human BMP9 (rhBMP9) was

used by Song and Celeste in the mid 90’s, reports on its

use for bone formation assays appear 10 years later in the

mid 2000’s [12,36,45,46]. The interest of using rhBMP9

is also related to the controlled dosag e and an easier cor-

relation to its effect. Thus rhBMP9 produced by E. coli at

500 ng/mL was shown to induce after 5 days th e expres-

sion of early markers of bone differentiation such as

smad-1/5, run × 2, dl × 5, osterix, osteopontin, bone sia-

loprotein and alkaline phosphatase in C2C12 cells [47].

In the same manner, Bergeron et al. have shown that a

peptide derived from BMP9, pBMP9 is able to induce

osteoblastic differentiation in pre-osteoblasts [12,48].

The osteoinductive potential of rhBMP9 was especial-

ly determined in the context of muscular heterotopic os-

sification (HO). Indeed, HO is a common model to study

the mechanism of ossification process in animals [49,50].

On the other hand, this model can be useful in order to

obtain a better understanding of the ossification process-

es occuring in muscle and to treat that condition [50].

Some papers have shown that adult muscle regeneration

could be impaired by BMPs [51,52]. Myogenesis was re-

ported to be inhibited by BMPs by their down-regulation

on satellite cells fusion [53]. Besides, mesenchymal pro-

genitor cells contained into the muscle are also respon-

sive to BMPs [54,55]. Overall, it was shown that both

myogenic and mesenchymal cell populations could be al-

tered by BMP and that tends to go from myogenesis to

osteogenesis [16].

Since the most common traumatic form of HO occurs

generally at sites of damaged muscles, we hypothesized a

relation between damaged muscle microenvironment and

BMP9 induced HO [56]. In this paper, we show for the

first times that BMP9 induces HO only in damaged mus-

cle. This paper highlighted the importance of cell micro-

environment for HO. We also showed that muscle resi-

dent stromal cells (mrSCs) are sensitive to BMP9 sig-

naling and express bone specific markers when exposed

to BMP9. Interestingly, mrSCs isolated from damaged

muscle are more responsive to BMP9. During a trans-

plantation experiment using labeled cells, we demon-

strated that mrSCs contribute to HO. Importantly, the

addition of a fusion protein containing the receptor

ALK1 significantly in hibited the osteoinductiv e potential

of BMP9 in mrSCs in vitro as well as HO in damaged

muscles [56].

Our experimental results show that a better under-

standing of the ossification processes and BMP implica-

tion is essential for developing new bone healing and HO

treatments. They also revealed that the osteoinductive

potential of rhBMP9 is highly dependent on the micro-

environment [56]. Therefore, delivery systems have to be

developed to protect the protein against breakdown. The

type of carrier, its concentration as well as the BMP dose

used in these delivery systems greatly influence the

growth factor release over time and its osteoinductive

potential [57]. In vitro and in vivo studies using different

delivery systems have been therefore studied throughout

E. Leblanc et al. / Advances in Bioscience and Biotechnology 4 (2013) 31-46 35

the years for rhBMP9 and/or its less expensive derived

peptide (pBMP9) [48,58,59].

Delivery systems slow-down degradation and enhance

the effect of growth factors like BMP9. Bessa et al. has

recently developed BMP loaded silk fibroin microparti-

cles as delivery systems of BMP2, 9, or 14 [59]. The

encapsulation efficiency of the BMPs loaded at 0.5 or 5

µg varied from 68% to 98% and BMP14 was better re-

tained over time than BMP4 and BMP9 in this silk fib-

roin carrier [59].

The combination of collagen Type I gel, BMP (100

ng/mL) and bioactive glass microspheres (45S5 Bio-

glass®, GM) has been shown to in crease osteoblastic dif-

ferentiation by Bergeron et al. [48]. For this assay, a pep-

tide derived from BMP9 (pBMP9) to treat MC3T3-E1

preosteoblasts was used. Alkaline phosphatase staining

showed that pBMP9 induced osteoblast differentiation

and that bioactive GM increased this effect. Since pre-

osteoblasts secrete matrix metalloproteinases (MMPs),

the influence of the delivery system on MMPs expres-

sion was then studied. pBMP9 with bioactive GM gener-

ated less MMP than did rhBMP-2 [48]. Thus, this deliv-

ery system using a combination of bioactive GM and

BMP9 derived peptide seems to be promising.

In vivo experiments also highlighted the importance of

the carrier choice. For example, rhBMP9 (169.2 ng/ml)

embedded in chitosan can induce endochondral ossifica-

tion 24 days after injection in quadriceps of mice, while

BMP9 entrapped into collagen seems less efficient [58].

pBMP9, a peptide derived from the knuckle epitope of

BMP9, is also able to induce bone formation in quadric-

ceps when injected embedded in chitosan [58]. This un-

derlines the importance to optimize carrier and the in-

fluence of microenvironment, to improve BMP9 in vivo

bone induction. Together, these delivery systems can

offer promising approaches for the sustained delivery of

BMPs, such as BMP9 for tissue engineering applications

and for improving bone formation.

Another alternative to improve bone formation is to

develop biomimetic materials that favor cell adhesion

and promote their response to rhBMP9 or pBMP9. For

example, the functionalization of material by adhesive

peptides that target specific integrins can strongly influ-

ence the bone cell response to BMP9 and pBMP9 [60,

61]. The peptide derived from the bone sialoprotein Ac-

CGGNGERPRGDTYRAY-NH2 that is recognized by the

avb3 integrins promote the early differentiation of

MC3T3-E1 preosteoblasts induced by pBMP9. In con-

trast, pBMP9 had no effect on cells attached through the

a2b1 integrins to the peptides Ac-CGGDGEA-NH2 de-

rived from type I collagen [61]. The selection of adhesive

peptides to develop biomimetic materials promoting

bone cell response to BMPs is therefore critical.

4. BMP9 AND CHONDROGENESIS

Chondrogenesis is a multistep process that leads to the

formation of cartilage [62]. Understanding chondrogene-

sis is essential when addressing bone formation since

cartilage is part of bone development and repair [62].

The first step of chondrogenesis is the aggregation and

condensation of mensenchymal progenitor cells (MPCs)

to form chondroprogenitors [63,64]. Then, chondropro-

genitors differentiate into chondrocytes that proliferate

and produce matrix containing collagen II, IX, XI and

aggrecan [63,65]. The n ewly formed cartilage serves as a

template for the formation of long bone, a process called

endochondral ossification [63,66]. Indeed, chondrocytes

undergo a terminal differentiation to hypertrophic phe-

notype, followed by calcification of the cartilage matrix,

which leads to its vascularisation and its ossification [63].

Both intramembranous and endochondral bone formation

are associated to BMPs, but BMP9 was proposed to be

more related to endochondral bone formation [37]. Its

proposed implication in endochondral ossification, is

highlighting its major role in skeleton development,

maintenance and repair [35].

BMP signaling is essential during chon drogenesis; it is

required for the condensation of MPCs and their differ-

entiation into chondrocytes [64-66]. Kramer et al. have

shown that BMP2 (2 ng/ml; 10 ng/ml) and BMP-4 (10

ng/ml) are able to induce cho ndrogenic differentiation of

embryonic stem cells via embryonic bodies [67]. More

interesting ly, an increased mRNA expression of BMP1/2/

5/6 have been reported in human chondrocytes through-

out chondrogenic differentiation [68,69] and that adult

articular chondrocytes was able to express BMP7 [70].

BMP9 has been shown to induce ectopic bone formation

into skeletal muscle [56,71]. This occurs via an endo-

chondral bone process as shown by Varady et al. [71].

They noticed that six days after an intramuscular injec-

tion of AdBMP9 vector, MPCs had begun a differentia-

tion into primitive chondroblasts that secreted loose ex-

tracellular matrix [71]. Thirteen days post-injection, they

also observed hypertrophied chondrocytes. Three weeks

post-injection, woven bone appeared and turned into

lamellar bone within three months [71]. Majumdar et al.

showed that Sox9, a nuclear transcription factor required

to undergo chondrogenesis, was upregulated in BMP9

treated MPCs [72]. Furthermore, BMP9 treatment (100

ng/ml) also induced overexpression of Col2a1 and ag-

grecan after 2 weeks, two important components of the

cartilage matrix [72]. Finally, Blunk et al. have shown

that BMP9 was able to stimulate the proliferation of pri-

mary bovine chondrocytes and extracellular matrix de-

position at a concentration of more than 1 ng/ml and to

induce chondrocytes hypertrophy and mineralization

when the concentration was above 10 ng/ml [73]. All

these results strongly suggest that BMP9 is a modulator

E. Leblanc et al. / Advances in Bioscience and Biotechnology 4 (2013) 31-46

of chondrocytes activity.

5. BMP9 AND BONE RESORPTION

BMP9, as described above, is involved in chondrogene-

sis and can strongly promote bone formation. In addition,

Fong et al. recently demonstrated in vitro that rhBMP9

can also augment bone resorption [74]. This increase was

shown to be functional and not related to osteoclast for-

mation. Furthermore, rhBMP9 could alter the intrinsic

apoptosis pathway and increase survival of osteoclasts.

The effect of rhBMP9 on osteoclast was explained by the

presence of ALK1 and BMPRII co-receptors and their

activation of the Smad 1/5/8 and non-smad MAPK/ERK

pathways. These results show for the first time that

BMP9 can directly affect human osteoclasts, acting on

their function and th eir survival [74].

6. BMP9 AND CANCER DEVELOPMENT

There are still discrepancies in studies concerning BMP9

and cancer development. Although some studies suggest

that BMP9 promoted differentiation and decreased pro-

liferation, whereas some others have shown the opposite.

Indeed, different studies have found that BMP9 restricts

tumor growth including prostate cancer, osteosarcoma

and colon adenocarcinoma [75-77]. The relation between

cancer and BMP9 seems to be mostly through two sepa-

rate mechanisms: cancer cell migration and invasion as

well as angiogenesis process [75-79].

Recent studies analyzed the effect of BMP9 (rhBMP9

or AdBMP9) in different human osteosarcoma cell lines

such as MG-63 cells [80,81]. For example, BMP9 ex-

pression was obtained through infection of human os-

teosarcoma MG-63 and 143B cells by a BMP9 express-

ing adenovirus [82]. Migration and invasion in vitro po-

tential were significantly lower (about 50% - 55%) in the

infected cells [82]. In a sub-cutaneous nude mice model,

the BMP9 overexpressing tumors were found to be sig-

nificantly smaller [77]. Then, BMP9 was found to induce

apoptosis in osteosarcoma cells [77]. Moreover matrix

metalloproteinases (MMP), specifically MMP-9, were

shown to be decreased significantly by the overexpres-

sion of BMP9 in osteosarcoma cells [82]. The loss of

migration and invasion potential was suggested to be

through a Smad-dependent pathway by downregulating

the expression and activity of MMP9 [77,82].

Furthermore, BMPs have been implicated in the pro-

duction of bone metastases in prostate cancer [83]. How-

ever, based on the observation that the expression of

BMP9 was decreased or absent in higher grade prostate

cancer, Ye et al. investigated the influence of BMP9 on

the biology of prostate cancer cells [76]. They showed

that the overexpression of BMP9 reduced cell growth,

matrix adhesion, invasion, and migration of prostate can-

cer PC-3 cells. They also showed that BMP9 induced

apoptosis in those particular cells through the overex-

pression of the prostate apoptosis response-4. The effect

of BMP9 on PC-3 cells relied on Smad-dependent signal

transduction. Thus, knockdown of BMPR-IB or BMPR-

II could promote PC-3 growth in vitro, but it had no sig-

nificant effect on their invasion potential. This suggests

that BMP9 may function as a tumor suppressor and

apoptosis regulator in prostate cancer through diverse

mechanisms [76].

Regarding angiogenesis, BMP9 has been shown to be

linked to vascular maturation [22]. However, contradic-

tory studies about the role of TGF-



and other family

members with reported vascular functions, such as BMP9,

in physiological and pathological angiogenesis make the

need for more comprehensive studies.

7. BMP9 AND ANGIOGENESIS

The effect of BMP9 on angiogenesis remains controver-

sial as both pro [79] and anti-angiogenic [22] effects

have been demonstrated. This discrepancy can be ex-

plained by different dosages, but also microenvironment

and the subtle interplay between vascular sprouting and

maturation [84]. The interest of studying BMP9 in an-

giogenesis resides in two principal pathologic angio-

genesis conditions: cancer angiogenesis and vascular

diseases per se (Hereditary Hemorrhagic Telangiectasia

(HHT), pre-eclampsia, pulmonary arterial hypertension

(PAH)).

BMP9 has been reported to bind to ALK1 in endothe-

lial cells [24]. It was also shown using rat aortic cross

sections that circulating BMP9 induces a constitutive

Smad1/5/8 phosphorylation in endothelial cells [21].

However, the roles of BMP9-ALK1 signaling in the

regulation of endothelial cells have not yet been fully

elucidated. Suzuki et al. examined the effects of BMP9

on the proliferation of endothelial cells. Vascular tube

formation from ex vivo mouse embryonic allantois ex-

plants and proliferation of in vitro cultured mouse stem

cell-derived endothelial cells (MESECs) was both pro-

moted by BMP9 [79]. This effect was shown to be re-

lated to ALK1 signaling , supported by the mimicked pro-

angiogenic effect of the expression of constitutively ac-

tive ALK1 in MESECs. Furthermore, in vivo angiogene-

sis was promoted by BMP9 in a Matrigel plug assay and

in a mouse xenograft model after subcutaneous injection

of BxPC3 human pancreatic adenocarcinoma cells in-

fected with lentiviruses enco ding BMP9. Taken together,

these results showed that BMP9 enhanced the prolifera-

tion of normal endoth elial cells and tumor-associated en-

dothelial cells [79].

Through genetic and pharmacological means, Cunha

demonstrated that ALK1 represents a new therapeutic

target for tumor angiogenesis [78]. Using a soluble fu-

E. Leblanc et al. / Advances in Bioscience and Biotechnology 4 (2013) 31-46 37

sion protein of ALK1 with an immunoglobulin (ALK1-

Fc), they have been able to inhibit the BMP9 -ALK1 sig-

naling. Systemic treatment with the ALK1-Fc fusion pro-

tein RAP-041 (12 mg/kg), which acts as a competitive

inhibitor of BMP9, delayed pancreatic tumor growth and

progression, as diminution of ALK 1 ge ne d o s age also di d.

This delayed growth was thought to be related to the

inhibition of angiogenesis. Furthermore, the use of RAP-

041 significantly impaired angiogenic response toward

vascular endothelial growth factor A (VEGFA). In that

study, they found that the observed effect was related to

an unexpected synergy between TGF-β and BMP9. In

fact, the combined action of the two factors augmented

the response to angiogenic stimuli o f the endo thelial cells

[78]. This finding reinforces the idea that BMP9 can

have a dual effect on angiogenesis and that its pro-an-

giogenic effect may be dependent on TGF-



synergy.

The same year, another team showed a diminution of

vessel formation in a chick chorioallantoic assay and a

reduced tumor burden in both melanoma explants and

mammary adenocarcinomas with ALK1-Fc treatment

[85].

Later, Cunha et al. presented another paper on the

critical role for ALK1, reviewing its effect on the endo-

thelial phenotype in vitro and in vivo [25]. These findings

have led to the development of new ALK1 pathway in-

hibitors that target different malignant diseases. In the

same vein, another group used soluble endoglin ECD as

an inhibitor in the regulation of angiogenesis and high-

lighted the efficacy of this fusion protein as a potential

anti-angiogenic drug [75]. They showed that this inhibi-

tor reduced the VEGF-induced angiogenesis and the tu-

mor burden in a mouse colon cancer model.

Angiogenesis requires a finely tuned balance between

numerous signals and although BMP9 has been shown to

have pro-angiogenic potential (above), other studies have

shown the opposite. Indeed, a team has been working a

lot on the relationship between endothelial cells and

ALK1 [21,24,86]. Using NIH3T3 fibroblasts, found that

BMP9 signal transduction involved ALK1 and both

BMPRII and ActRIIA. Moreover, by overexpressing en-

doglin in NIH3T3 fibroblasts, they promoted the BMP9

stimulation of ALK1. BMP9 could inhibit endothelial

cell migration and growth, and activate the expression of

genes encoding proteins related to vascular maturation

such as Id1, Id2, Smad6, Smad7, endoglin and BMPRII

[24].

A year after, they reviewed the presence of BMP9 in

human serum to verify whether in fact it could be im-

plied in vascular quiescence. First, they found that hu-

man serum alone could indu ce Smad1/5 phosphorylatio n

in NIH3T3 transfected by ALK1 expression plasmid.

Second, they identified the active factor by using anti-

bodies agains t all BMP members and found that only the

anti-BMP9 inhibited this Smad1/5 phosphorylation [22].

These results were refined by data from Herrera and

Inman (2009) where they identified circulating BMP4

and 9 as active BMPs in human sera with a luciferase

reporter fused with the promoter BMP response element

Id1, transfected in a C2C12 line [87]. Present at a con-

centration between 2 and 12 ng/mL in human plasma,

BMP9 is circulating at a biologically active concentra-

tion (EC50 = 50 pg/mL). BMP9 was then tested in vivo in

angiogenic assays. Both in vivo mouse cellulose sponge

angiogenesis and chicken chorioallantoic membrane as-

says showed that BMP9 could negatively affect angio-

genesis may play a role in vascular quiescence [22]. In-

terestingly, the role of BMPs in angiogenesis opened the

door to a better understanding of genetic vascular dis-

eases caused by mutations in genes encoding for recap-

tors of this pathway (endoglin, ALK1 and BMPRII). This

leads to an important review in which the authors pro-

posed a model to distinguish BMP2, BMP7 and GDF5

from BMP9 in their functional implication in vessel for-

mation and maturation [84].

Hereditary Hemorrhagic Telangiectasia (HHT), also

known as Rendu-Osler-Weber, is a disease that causes

frequent nose bleeds, mucocutaneous and visceral te-

langiectasia [88]. This pathology follows an autosomal-

dominant inheritance pattern which has been shown to be

linked in 80% to a mutation in ALK1 or the co-receptor

endoglin [89]. Endoglin is a tran smembrane glycoprotein

on human vascular endothelial cells and is a co-receptor

implicated in this dominant vascular dysplasia as well as

in preeclampsia [90]. The alteration of ALK1 or endoglin

affects the maturation phase of angiogenesis, leading to

the absence of capillaries connecting the arterial and ve-

nous networks [89]. As BMP9 acts as the specific ligand

of the receptor ALK1 and to endoglin as its co-receptor,

a defect in either members of the receptor complex

means a disrupted BMP9 signaling pathway. Heterozy-

gous mice for endoglin or ALK1 supports the involve-

ment of endothelial hyperproliferation in the pathogene-

sis of the disease while the maturation is disrupted [91].

More recently, Choi et al. demonstrated that ALK1 defi-

cient endothelial cells have high migratory and invasion

properties both in vitro and in vivo. This new data con-

firms the importance of BMP9-ALK1 for angiogenesis

and into the differentiation and maturation of neocapil-

laries [92]. This BMP9-ALK1 signaling in angiogenesis

was shown to be transmitted downstream through Tmem

100, a gene encoding a previously uncharacterized intra-

cellular transmembrane protein [93].

On the other hand, pulmonary arterial hypertension

(PAH) is characterized by dysregulation in pulmonary

artery endothelial cell proliferation, apoptosis and per-

meability. The most common cause of inheritable PAH is

a mutation in the BMPR-II, leading to a loss of its func-

E. Leblanc et al. / Advances in Bioscience and Biotechnology 4 (2013) 31-46

tion [94]. In the same way as the defective signaling of

ALK1-endoglin in HHT, a deficient BMPRII signaling

leads to an absent function of BMP9 and to an anarchic

development of blood vessels [94].

Recently, it has been shown that chloroquine, an anti-

malarial drug, is able to indirectly increase cell surface

expression of BMPRII in pulmonary artery endothelial

cells [95]. This increase seemed to be independent from

transcription since chloroquine enhanced BMPRII ex-

pression even after siRNA knockdown of the BMPRII

transcript. Using BMPRII deficient cells from PAH sick

patients, BMP9 signaling was also shown to be decreas-

ed and treatment with chloroquine significantly increased

expression of BMP9-BMPRII signaling gene targets.

These findings prov ide ne w insigh ts for the restor atio n of

cell surface receptors in treating hereditary vascular dis-

eases, specifically BMPR-II with chloroquine for treat-

ment of PAH [95].

The effect of BMP9 on angiogenesis remains contro-

versial as both pro [79] and anti-angiogenic [22] effects

have been demonstrated. This discrepancy can be ex-

plained by different dosages, but also microenvironment

and the subtle interplay between vascular sprouting and

maturation [84]. The interest of studying BMP9 in an-

giogenesis resides in two principal pathologic angioge-

nesis conditions: cancer angiogenesis and vascular dis-

eases per se (Hereditary Hemorrhagic Telangiectasia

(HHT), pre-eclampsia, pulmonary arterial hypertension

(PAH)).

BMP9 has been reported to bind to ALK1 in endothe-

lial cells [24]. It was also shown using rat aortic cross

sections that circulating BMP9 induces a constitutive

Smad1/5/8 phosphorylation in endothelial cells [21].

However, the roles of BMP9-ALK1 signaling in the

regulation of endothelial cells have not yet been fully

elucidated. Suzuki et al. examined the effects of BMP9

on the proliferation of endothelial cells. Vascular tube

formation from ex vivo mouse embryonic allantois ex-

plants and proliferation of in vitro cultured mouse stem

cell-derived endothelial cells (MESECs) was both pro-

moted by BMP9 [79]. This effect was shown to be re-

lated to ALK1 signaling , supported by the mimicked pro-

angiogenic effect of the expression of constitutively ac-

tive ALK1 in MESECs. Furthermore, in vivo angiogene-

sis was promoted by BMP9 in a Matrigel plug assay and

in a mouse xenograft model after subcutaneous injection

of BxPC3 human pancreatic adenocarcinoma cells in-

fected with lentiviruses enco ding BMP9. Taken together,

these results showed that BMP9 enhanced the prolifera-

tion of normal endoth elial cells and tumor-associated en-

dothelial cells [79].

Through genetic and pharmacological means, Cunha

demonstrated that ALK1 represents a new therapeutic

target for tumor angiogenesis [78]. Using a soluble fu-

sion protein of ALK1 with an immunoglobulin (ALK1-

Fc), they have been able to inhibit the BMP9 -ALK1 sig-

naling. Systemic treatment with the ALK1-Fc fusion pro-

tein RAP-041 (12 mg/kg), which acts as a competitive

inhibitor of BMP9, delayed pancreatic tumor growth and

progression, as diminution of ALK 1 ge ne d o s age also di d.

This delayed growth was thought to be related to the in-

hibition of angiogenesis. Furthermore, the use of RAP-

041 significantly impaired angiogenic response toward

vascular endothelial growth factor A (VEGFA). In that

study, they found that the observed effect was related to

an unexpected synergy between TGF-β and BMP9. In

fact, the combined action of the two factors augmented

the response to angiogenic stimuli o f the endo thelial cells

[78]. This finding reinforces the idea that BMP9 can

have a dual effect on angiogenesis and that its pro-an-

giogenic effect may be dependent on TGF-b synergy.

The same year, another team showed a diminution of

vessel formation in a chick chorioallantoic assay and a

reduced tumor burden in both melanoma explants and

mammary adenocarcinomas with ALK1-Fc treatment

[85].

Later, Cunha et al. presented another paper on the

critical role for ALK1, reviewing its effect on the endo-

thelial phenotype in vitro and in vivo [25]. These findings

have led to the development of new ALK1 pathway in-

hibitors that target different malignant diseases. In the

same vein, another group used soluble endoglin ECD as

an inhibitor in the regulation of angiogenesis and high-

lighted the efficacy of this fusion protein as a potential

anti-angiogenic drug [75]. They showed that this inhibi-

tor reduced the VEGF-induced angiogenesis and the tu-

mor burden in a mouse colon cancer model.

Angiogenesis requires a finely tuned balance between

numerous signals and although BMP9 has been shown to

have pro-angiogenic potential (above), other studies have

shown the opposite. Indeed, a team has been working a

lot on the relationship between endothelial cells and

ALK1 [21,24,86]. Using NIH3T3 fibroblasts, found that

BMP9 signal transduction involved ALK1 and both

BMPRII and ActRIIA. Moreover, by overexpressing en-

doglin in NIH3T3 fibroblasts, they promoted the BMP9

stimulation of ALK1. BMP9 could inhibit endothelial

cell migration and growth, and activate the expression of

genes encoding proteins related to vascular maturation

such as Id1, Id2, Smad6, Smad7, endoglin and BMPRII

[24].

A year after, they reviewed the presence of BMP9 in

human serum to verify whether in fact it could be im-

plied in vascular quiescence. First, they found that hu-

man serum alone could indu ce Smad1/5 phosphorylatio n

in NIH3T3 transfected by ALK1 expression plasmid. Se-

cond, they identified th e active factor by using an tibod ies

against all BMP members and found that only the anti-

E. Leblanc et al. / Advances in Bioscience and Biotechnology 4 (2013) 31-46 39

BMP9 inhibited this Smad1/5 phosphorylation [22].

These results were refined by data from Herrera and

Inman (2009) where they identified circulating BMP4

and 9 as active BMPs in human sera with a luciferase re-

porter fused with the promoter BMP response element

Id1, transfected in a C2C12 line [87]. Present at a con-

centration between 2 and 12 ng/mL in human plasma,

BMP9 is circulating at a biologically active concentra-

tion (EC50 = 50 pg/mL). BMP9 was then tested in vivo

in angiogenic assays. Both in vivo mouse cellulose

sponge angiogenesis and chicken chorioallantoic mem-

brane assays showed that BMP9 could negatively affect

angiogenesis may play a role in vascular quiescence [22].

Interestingly, the role of BMPs in angiogenesis opened

the door to a better understanding of genetic vascular

diseases caused by mutations in genes encoding for re-

ceptors of this pathway (endoglin, ALK1 and BMPRII).

This lead to an important review in which the authors

proposed a model to distinguish BMP2, BMP7 and

GDF5 from BMP9 in their functional implication in ves-

sel formation and maturation [84].

Hereditary Hemorrhagic Telangiectasia (HHT), also

known as Rendu-Osler-Weber, is a disease that causes

frequent nose bleeds, mucocutaneous and visceral te-

langiectasia [88]. This pathology follows an autosomal-

dominant inheritance pattern which has been shown to be

linked in 80% to a mutation in ALK1 or the co-receptor

endoglin [89]. Endoglin is a tran smembrane glycoprotein

on human vascular endothelial cells and is a co-receptor

implicated in this dominant vascular dysplasia as well as

in preeclampsia [90]. The alteration of ALK1 or endoglin

affects the maturation phase of angiogenesis, leading to

the absence of capillaries connecting the arterial and ve-

nous networks [89]. As BMP9 acts as the specific ligand

of the receptor ALK1 and to endoglin as its co-receptor,

a defect in either members of the receptor complex

means a disrupted BMP9 signaling pathway. Heterozy-

gous mice for endoglin or ALK1 supports the involve-

ment of endothelial hyperproliferation in the pathogene-

sis of the disease while the maturation is disrupted [91].

More recently, Choi et al. demonstrated that ALK1 defi-

cient endothelial cells have high migratory and invasion

properties both in vitro and in vivo. This new data con-

firms the importance of BMP9-ALK1 for angiogenesis

and into the differentiation and maturation of neocapil-

laries [92]. This BMP9-ALK1 signaling in angiogenesis

was shown to be transmitted downstream through Tmem

100, a gene encoding a previously uncharacterized intra-

cellular transmembrane protein [93].

On the other hand, pulmonary arterial hypertension

(PAH) is characterized by dysregulation in pulmonary

artery endothelial cell proliferation, apoptosis and per-

meability. The most common cause of inheritable PAH is

a mutation in the BMPR-II, leading to a loss of its func-

tion [94]. In the same way as the defective signaling of

ALK1-endoglin in HHT, a deficient BMPRII signaling

leads to an absent function of BMP9 and to an anarchic

development of blood vessels [94].

Recently, it has been shown that chloroquine, an anti-

malarial drug, is able to indirectly increase cell surface

expression of BMPRII in pulmonary artery endothelial

cells [95]. This increase seemed to be independent from

transcription since chloroquine enhanced BMPRII ex-

pression even after siRNA knockdown of the BMPRII

transcript. Using BMPRII deficient cells from PAH sick

patients, BMP9 signaling was also shown to be decreas-

ed and treatment with chloroquine significantly increased

expression of BMP9-BMPRII signaling gene targets.

These findings prov ide ne w insigh ts for the restor atio n of

cell surface receptors in treating hereditary vascular dis-

eases, specifically BMPR-II with chloroquine for treat-

ment of PAH [95].

Indeed, there still remain s contro v ersy on pro and an ti-

angiogenic effects of BMP9. Recent literature highlights

the importance of understanding its role in vessel matu-

ration as opposed to formation and also the role of the

microenvironment and synergy with TGF-b. On the other

hand, it is clear that the signaling pathway is through re-

ceptor and co-receptor ALK1-endoglin, and that this can

be inhibited by the use of ALK1 or endoglin fusion pro-

teins. Certainly, it will be interesting to test whether

some drugs derived from inhibitors like ALK1-Fc could

be used experimentally to treat cancer and on the other

hand develop ALK1 pathway enhancers that could help

to treat hereditary vascular diseases.

8. BMP9 AND NEUROGENESIS

BMPs are critical in neurogenesis, forebrain formation

and more globally in the dorsov entral axis determination

[96,97]. BMPs promote cell survival and differentiation

of neurons in both peripheral and central nervous sys-

tems (CNS) [98] Lopez-Coviella et al. have been work-

ing specifically on the cholinergic neurons of the fore-

brain [99]. Basal forebrain cholinergic neurons (BFCN)

participate in processes of learning, memory, attention

and their degeneration is involved in several human dis-

orders such as Alzheimer’s disease [100]. They have

shown that 14 days old mice embryos express BMP9

mRNA principally in the forebrain and spine [99]. This

developmental stage is also the one where the cholinergic

neurons are most sensitive to BMP9. Thus, cultured pri-

mary cells derived from the septal area increase their

acetylcholine (Ach) expression by 20 fold when exposed

to BMP9 during that period. Compared to 5 other BMPs

(BMP2-4-6-7-12), BMP9 is the most potent in inducing

Ach expression by septal cells. Moreover, when injected

into mouse embryo cerebral ventricles, BMP9 is able to

increase cerebral Ach level [99]. The genes responsive to

E. Leblanc et al. / Advances in Bioscience and Biotechnology 4 (2013) 31-46

this BMP9 induction of basal forebrain neurons include

cell-cycle related proteins, transcription factors, signal

transduction molecules, ex tracellular matrix proteins, ad-

hesion molecules, enzymes, transporters, and chaperon-

ins. The set of genes induced with BMP9 treatment sug-

gests that BMP9 creates a trophic environment for basal

forebrain neurons and contributes to the maturation of

this part of the brain in vivo [ 101].

During the development of cholinergic neurons of the

basal forebrain, BMP9 activates the canonical BMP sig-

naling pathway, through phosphorylation of Smad1 and

Smad5 which associate to co-Smad4 and translocate into

the nucleus [98]. Finally, this team demonstrated that

BMP9 administration can prevent lesion-evoked impair-

ment of the cholinergic hippocampal neurons in adult

mice by inducing a favorable environment for these cells

[102].

Interestingly, Bissonnette et al. have been able to gen-

erate BFCN from human embryonic stem cells (hESCs)

using relevant ligands of the developing forebrain [103].

hESCs respond to BMP9 signaling after sonic hedge-

hog/fibroblast growth factor 8 pretreatment and differen-

tiate the progenitors toward a BFCN phenotype. These

derived neurons express only markers characteristic of

BFCN and have their functional characteristics (action

potentials, functional cholinergic synapses). The ability

to derive BFCN from hESCs is a significant step for the

development of therapeutic interventions in diseases af-

fecting the forebrain (e.g. Alzheimer’s disease) [103].

BMP9 expression in the CNS seems to be related to

the development and maintenance of the cholinergic phe-

notype. The cited studies give new insights on a clearer

comprehension of the role of BMP9 during early embry-

ology and the later ability of damage repair. The proven

expression of BMP9 in CNS is also interesting for dif-

ferent reasons: the relation of brain/spinal cord injury

and increased ossification potential. As shown in many

papers, CNS injury (brain and spinal cord) is associated

with an increase in ossification potential (bone healing

and HO) [104-109]. However, no specific growth factor

has been identified to date [105]. BMP9 would be an in-

teresting candidate to evaluate.

9. BMP9 AND HEPATOCYTES

Since BMP9 was first described to be expressed in non

parenchymal cells of the liver [20,45], studies have been

conducted to determine the effect of BMP9 on liver cells

and hepatocellular regeneration [110]. Miller et al. have

determined the binding affinity of BMP9 and its number

of receptors on rat hepatic endothelial cells (Kd of ~85

pM, 3600 receptors/cell) and Kupffer cells (Kd of ~25

pM, 1100 receptors/cell), cells identified as main BMP9

producers. They also found that the binding of BMP9 on

these cells was reversible and BMP9/recep tor complexes

can be internalized [20].

However, while Miller et al. did not detect BMP9 in

rat hepatocytes [20], Bidart et al. recently found that

BMP9 expression (mRNA and protein) was mainly lo-

cated in human h epatocytes and biliary hepatic ep ithelial

cells [21]. The effect of BMP9 on hepatocytes was first

evaluated by Song et al. [45]. They found that rhBMP9

bind to human HepG2 liver tumor cells with high affinity.

They also found that HepG2 cells express two BMP9

receptors similar in size to the Type I and Type II recap-

tors reported by others for TGF-



members, but specific

for BMP9 [45]. Increasing doses (0.1 - 10 ng/mL) of

BMP9 stimulated human HepG2 and primary rat heap-

tocytes cell proliferation. In contrast, TGF-



(0.1 - 100

ng/mL) had no effect on HepG2 cell proliferation but

inhibited proliferation in primary hepatocytes [45]. This

study was the first to report the effect of BMP9 on heap-

tocyte proliferation. The direct effect of BMP9 on he-

patic progenitor cells was also studied through infection s

of progenitors with a recombinant adenovirus containing

human BMP9 gene (AdBMP9) by Gao et al. (2011). The

maturation and differentiation of progenitor cells were

examined. AdBMP9 enhanced glycogen storage as well

as albumin production in hepatic progenitor cells, a ma-

ture hepatic cell behavior [111].

BMP9 is expressed in the liver and seems to have an

effect on hepatocytes, but more studies are needed to cla-

rify its differential effect over proliferation [45] and dif-

ferentiation [111] as well as its neoplastic influence on

liver cells [110].

10. BMP9 AND GLUCOSE METABOLISM

Insulin resistance is a systemic multifactorial impair ment

of glucose uptake. Muscle, the most important glucose

consuming organ, need Akt2 to be able to activate insu-

lin-induced glucose uptake and this pathway seems to be

severely impaired in insulin resistance [112,113]. Inter-

estingly, a combination of bioinformatic and high-

throughput functional analyses have shown BMP9 to be

the first hepatic factor to regulate blood glucose concen-

tration [46]. Moreover, this effect was thought to be me-

diated by activation of Akt kinase in differentiated myo-

tubes [46]. Then, it has been demonstrated that recombi-

nant BMP9 (1 and 5 mg/kg) improves glucose homeo-

stasis in vivo in diabetic and non-diabetic rodents [46].

The mechanism relied on the upregulation of Smad5 and

Akt2 in differentiated rats myotubes [114]. On the oppo-

site side, Smad5 was downregulated in myotubes by de-

xamethasone, a well known hyperglycemia inducer and

Smad5 knockdown in rats decreased Akt2 expression and

phosphorylation leading to a decrease in insulin-induced

glucose uptake by myotubes. It was then hypothesized

that Smad5 regulated glucose uptake in skeletal muscle

through Akt2 expression and phosphorylation. These

E. Leblanc et al. / Advances in Bioscience and Biotechnology 4 (2013) 31-46 41

findings also revealed Smad5 as a potential target for the

treatment of type 2 diabetes. Hence, BMP9 could be seen

as a potential activator of Smad5 for that purpose [114].

This promising effect of BMP9 could lead to a better

control on hepatic glucose, furthermore on diabetes. In-

deed, BMP9 expression is severely reduced in the liver

of insulin-resistant rats [115]. In this study, the authors

have been reporting several assays showing that BMP9

directly controls glucose homeostasis [115]. Direct in

situ exposition of normal fasting rats-liver to glucose an d

insulin leads to BMP9 expression. Moreover, neutraliza-

tion of BMP9 with an anti-BMP9 antibody induces glu-

cose intolerance and insulin resistance. Analyzed toge-

ther, these results reinforce the eligibility of BMP9 as a

hepatic insulin-sensitizing substance (HISS) in glucose

homeostasis physiology [115].

11. CONCLUSION

BMP9 is a powerful osteogenic growth factor and was

shown to be able to activate bone differentiation in many

animal studies. The osteogenic potential of BMP9 was

first studied through gene therapy, but nowadays it is

used through its recombinant human form, rhBMP9. The

study of BMP9 bone formation has led to a better under-

standing on endochondral bone healing, but also of the

new insights in delivery systems that can enhance its

effect as well as improved comprehension of the trau-

matic HO and its relation to BMP9. As bone formation is

always associated with resorption and remodeling, it has

also been shown that BMP9 can directly affect human

osteoclasts, acting on their function and their survival. In

cancer development, BMP9 has two major effects. First

of all, its anti-proliferative/pro-differentiation potential

seems to be protective for many neoplastic conditions.

On the other hand, its inhibition through ALK1 and en-

doglin fusion proteins seemed to decrease tumor angio-

genesis. Therefore, more studies are warranted in order

to clarify its influence on tumorigenesis. On the angio-

genic side, we have discussed the sometimes confusing

contradictions about the pro and anti-angiogenic effects

of BMP9. We have presented the importance of under-

standing BMP9’s role in blood vessel maturation as op-

posed to formation. The importance of BMP9 in the cen-

tral nervous system resides in its ability to promote cho-

linergic neurons proliferation and survival. Looking for

new innovative treatment paths in Alzheimer’s disease,

BMP9 could be an interesting candidate. The relation

between liver homeostasis and BMP9 is still not clear,

but it is now to our knowledge that it is expressed and

produced in that organ. Some studies have tried to show

its hepatocellular regeneration potential, but still, more

studies are needed. Finally, we have discussed its com-

pelling effect in glucose metabolism, principally pro-

moting sensitivity to insuline in skeletal muscle. Thus,

BMP9 is a maj or memb er of th e TGF-



superfamily that

is implied in many fundamental developmental and pa-

thologic processes. Future research will certainly bring

answers to the many questions left open, and those an-

swers will unquestionably lead to clinical applications.

12. ACKNOWLEDGEMENTS

We are grateful to Dr Juan Sebastian Rendon who did a critical reading

of this review anf helped for corrections.

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