Tài liệu Báo cáo khoa học: The pro-form of BMP-2 interferes with BMP-2 signalling by competing with BMP-2 for IA receptor binding pptx

ProBMP-2 was found to bind to the purified extracellular ligand binding domain ECD of BMPR-IA, a high-affinity receptor for mature BMP-2, with a similar affinity as mature BMP-2.. We show t

competing with BMP-2 for IA receptor binding

Anja Hauburger1, Sabrina von Einem1, Gerburg K Schwaerzer2, Anja Buttstedt1, Matthias Zebisch3, Michael Schra¨ml4,, Peter Hortschansky5, Petra Knaus2 and Elisabeth Schwarz1

1 Institut fu¨r Biochemie und Biotechnologie, Martin-Luther-Universita¨t Halle-Wittenberg, Germany

2 Institut fu¨r Chemie ⁄ Biochemie, Freie Universita¨t Berlin, Germany

3 Biotechnologisch-Biomedizinisches Zentrum, Universita¨t Leipzig, Germany

4 Scil Proteins GmbH, Halle, Germany

5 Leibniz-Institut fu¨r Naturstoffforschung und Infektionsbiologie, Hans-Kno¨ll-Institut (HKI), Jena, Germany

Keywords

alkaline phosphatase; BMPR-IA; BMPR-II;

bone morphogenetic protein-2; pro-domain

Correspondence

E Schwarz, Institut fu¨r Biochemie and

Biotechnologie, Martin-Luther-Universita¨t

Halle-Wittenberg, Kurt-Mothes-Str 3, 06120

Halle, Germany

Fax: +49 345 55 27 013

Tel: +49 345 55 24 856

E-mail: elisabeth.schwarz@biochemtech.

uni-halle.de

Present address

Roche Diagnostics GmbH, Nonnenwald 2,

82372 Penzberg, Germany

(Received 2 April 2009, revised 18 August

2009, accepted 3 September 2009)

doi:10.1111/j.1742-4658.2009.07361.x

Pro-forms of growth factors have received increasing attention since it was shown that they can affect both the maturation and functions of mature growth factors Here, we assessed the biological function of the pro-form of bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor b (TGFb)⁄ BLP superfamily The role of the 263 amino acids

of the pro-peptide is currently unclear In order to obtain an insight into the function of the pro-form (proBMP-2), the ability of proBMP-2 to induce alkaline phosphatase (AP), a marker enzyme for cells differentiating into os-teoblasts, was tested Interestingly, in contrast to mature BMP-2, proBMP-2 did not lead to induction of AP Instead, proBMP-2 inhibited the induction

of AP by BMP-2 This result raised the question of whether proBMP-2 may compete with mature BMP-2 for receptor binding ProBMP-2 was found to bind to the purified extracellular ligand binding domain (ECD) of

BMPR-IA, a high-affinity receptor for mature BMP-2, with a similar affinity as mature BMP-2 Binding of proBMP-2 to BMPR-IA was confirmed in cell culture by cross-linking proBMP-2 to BMPR-IA presented on the cell sur-face In contrast to this finding, proBMP-2 did not bind to the ECD

of BMPR-II ProBMP-2 also differed from BMP-2 in its capacity to induce p38 and Smad phosphorylation The data presented here suggest that the pro-domain of BMP-2 can alter the signalling properties of the growth factor

by modulating the ability of the mature part to interact with the receptors

Structured digital abstract

l MINT-7261817 :BMPR-IA (uniprotkb: P36894 ) and proBMP2 (uniprotkb: P12643 ) physically interact ( MI:0915 ) by cross-linking studies ( MI:0030 )

l MINT-7261681 , MINT-7261693 : BMP2 (uniprotkb: P12643 ) binds ( MI:0407 ) to BMPR-IA (uniprotkb: P36894 ) by enzyme linked immunosorbent assay ( MI:0411 )

l MINT-7261751 , MINT-7261794 : proBMP2 (uniprotkb: P12643 ) binds ( MI:0407 ) to BMPR-IA (uniprotkb: P36894 ) by competition binding ( MI:0405 )

l MINT-7261806 , MINT-7261846 : BMPR-IA (uniprotkb: P36894 ) physically interacts ( MI:0915 ) with BMP2 (uniprotkb: P12643 ) by anti bait coimmunoprecipitation ( MI:0006 )

l MINT-7261628 , MINT-7261642 : noggin (uniprotkb: Q13253 ) binds ( MI:0407 ) to proBMP2 (uniprotkb: P12643 ) by surface plasmon resonance ( MI:0107 )

Abbreviations

AP, alkaline phosphatase; BMP-2, bone morphogenetic protein-2; ECD, extracellular domain; GDF-8, growth and differentiation factor-8; HA, haemagglutinin; MBP, maltose binding protein; PFC, pre-formed receptor complex; Smad, small mothers against decapentaplegic;

TGFb, transforming growth factor b.

Bone morphogenetic protein-2 (BMP-2) belongs to the

transforming growth factor b (TGFb) superfamily

Structural features of proteins in this family include

the arrangement of disulfide bridges in a cystine knot

and the anti-parallel association of the two monomers,

which are linked by an intermolecular disulfide bond

[1,2] The capacity of BMP-2 to induce bone formation

has been exploited for therapeutic application [3]

Signal transduction involves a BMP-2 dimer in

asso-ciation with two type I and two type II receptors Two

binding modes for BMP-2 have been reported, which

indicate the existence of different signalling pathways

[4–7] For the sequential mode of binding, two type I

receptor molecules are bound by the dimeric ligand

Subsequently, two type II receptor molecules are

recruited by the ligand–type I receptor complex This

association initiates the p38–MAPK pathway, which

finally leads to the induction of alkaline phosphatase

The second binding mode is characterized by ligand

binding to pre-formed receptor complexes (PFC),

which consist of two type I and two type II receptors

By binding of the ligand to PFCs, the Smad signalling

pathway is activated

BMP-2 is translated as a prepro-protein in vivo The

pre-sequence mediates translocation into the

endoplas-mic reticulum However, the function of the

pro-pep-tide is presently unknown We showed previously that

the pro-peptide is not required for in vitro oxidative

folding of the mature part [8] Furthermore,

recombi-nant proBMP-2 induced ectopic bone formation in

rats, indicating that the pro-peptide does not

signifi-cantly impair the bone-inducing activity of mature

BMP-2 [8] We are interested in the role of the 263

amino acid pro-peptide of BMP-2, because evidence

accumulated over recent years has shown that the

pro-forms of growth factors can modulate the activities

of the mature domains In case of pro-neurotrophins,

for example, they can even elicit completely opposite

effects to those of the mature growth factors by

bind-ing to pro-form-specific receptors [9–11]

The pro-peptide of the related TGFb has been

shown to retard the function of the mature protein by

non-covalent association with the mature part upon

proteolytic processing This retarding role of the

pro-peptide led to it being named latency-associated

polypeptide [12] In addition to regulating activity, at

least in the case of inhibins, which also belong to the TGFb superfamily, the pro-domains appear to play a role in assembly and secretion [13] Similarly, an inhib-itory role of the non-covalently attached pro-peptide has been demonstrated for growth and differentiation factor-8 (GDF-8) [14,15] Furthermore, the pro-peptide

of GDF-8 impairs interaction of the mature part with its receptors [16] In the case of BMP-9, the pro-pep-tide appears not to alter significantly the activity of the mature part [17] For the pro-peptide of BMP-7, a tar-geting role to the extracellular matrix [18] has been shown The pro-peptide of BMP-4 is responsible for stabilization of the mature part, intracellular traffick-ing and foldtraffick-ing in the endoplasmic reticulum [19–21] Thus, the roles of the pro-peptides appear to be diver-gent within the TGFb⁄ BMP family, and appear to modulate the function of the mature part by non-covalent association after proteolytic cleavage by pro-hormone convertases (for review, see [22]) The biological relevance of pro-domains within the TGFb family is highlighted by reports showing that muta-tions in pro-domains lead to abnormal dorsoventral patterning [23] and skeletal malformations [24,25] In the case of BMP-2, no published information is avail-able on the physiological function of the pro-domain Increased levels of unprocessed proBMP-2 have been shown to be present in synovial tissue from patients suffering from rheumatoid arthritis and spondyloarthr-opathies [26] However, the relevance of this finding for disease development is so far unclear

In this work, we attempted to obtain an insight into the function of proBMP-2 In order to obtain more information about the role of the pro-form, proBMP-2, i.e BMP-2 with the covalently attached pro-peptide, was recombinantly produced and compared to the mature form We show that proBMP-2 can compete with mature BMP-2 for binding to BMP receptor IA (BMPR-IA), one of the main receptors of mature BMP-2 [6,27,28] In contrast, the ECD of BMPR-II was not bound by proBMP-2 Furthermore, the free pro-peptide formed a non-covalent complex with mature BMP-2 in vitro, thereby blocking binding of BMP-2 to BMPR-II The finding that proBMP-2 did not induce alkaline phosphatase is consistent with the finding that proBMP-2 does not lead to p38 phosphorylation We conclude that the pro-peptide of BMP-2, although it

l MINT-7261597 , MINT-7261613 : BMPR-IA (uniprotkb: P36894 ) binds ( MI:0407 ) to BMP2 (uniprotkb: P12643 ) by surface plasmon resonance ( MI:0107 )

does not disturb interaction of the mature part with

BMPR-IA, may nonetheless interfere with signal

induction, possibly at the level of receptor interaction

Results

ProBMP-2 inhibits AP induction by BMP-2

In order to determine whether proBMP-2 elicits

biologi-cal responses similar those elicited by mature BMP-2,

induction of alkaline phosphatase (AP) was

investi-gated AP represents a marker enzyme for

differentia-tion into osteoblasts, thus AP activity is usually

measured to test the response to mature BMP-2 [29,30]

Using BMP-2 as a control, an EC50of 18 ± 4 nm was

calculated (Fig 1A), which corresponds well with the

published EC50 of 19 nm [30] AP activity induced by

BMP-2 was blocked by noggin (Fig 1D) When AP

activity was tested upon addition of the isolated

pro-peptide as a negative control, no signal increase was

observed (Fig S1) Similarly, using proBMP-2 under

identical assay conditions, only a low AP signal increase

and no concentration dependence was recorded

(Fig 1B) Even when cells were stimulated with 1 lm

proBMP-2, the AP signal was in a similar range to that obtained after induction with 2 nm BMP-2 (data not shown) This very small signal increase upon addition of proBMP-2 may be due to slow cleavage of proBMP-2 to BMP-2 over time, possibly by proteases secreted from the C2C12 cells, rather than an AP-inducing activity of proBMP-2 Contamination of the proBMP-2 protein sample with traces of mature BMP-2 could be excluded

as neither staining of SDS–PAGE gels nor western blot analysis using a rhBMP-2 antibody yielded any evidence for the presence of mature BMP-2 during the first 48 h

of incubation (Fig S2)

Next, we tested whether proBMP-2 suppresses induction of AP by mature BMP-2 For the competi-tion experiments, cells were incubated with BMP-2 at

a concentration of 200 nm, which had been proven to elicit the maximal AP response (Fig 1A), and increas-ing concentrations of proBMP-2 A proBMP-2 concen-tration-dependent inhibition of the BMP-2-induced AP activity was observed (Fig 1C) The possibility of con-tamination of the proBMP-2 preparation with endo-toxins was excluded by using a chromogenic limulus amoebocyte lysate (LAL) detection kit (Charles River, Wilmington, MA, USA), which showed that endotoxin

C

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proBMP-2 [nM]

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proBMP-2 [nM]

AP inhibition (%) 20 40 60

0 20 40 60 80 100

Fig 1 Mature BMP-2 but not proBMP-2 leads to induction of AP Effects of BMP-2 (A) and proBMP-2 (B) on the induction of alkaline phos-phatase in C2C12 cells AP activity was measured by determination of the change in extinction (DE) per minute and microgram protein In (C), 200 n M BMP-2 and the indicated concentrations of proBMP-2 were added simultaneously to the cells; the maximal AP activity in the absence of proBMP-2 was set to 100% (D) The AP assay was controlled by endpoint determinations of substrate turnover in the presence

of an equimolar amount (III) or fivefold molar excess (IV) of noggin over BMP-2 (black) or proBMP-2 (grey); (I) no ligand; (II) absence of nog-gin Ligand concentrations were 10 n M The lower amplitudes of the AP signals are due to the fact that, in this experiment, the signals obtained using 10 n M BMP-2 were set to 100% Data represent means and standard deviations from four independent measurements.

levels were below the determination threshold Thus,

based on these data, we conclude that the observed

reversal of BMP-2-elicited AP induction by proBMP-2

may reflect a biological mechanism

ProBMP-2 binds to the extracellular ligand

binding domain of BMPR-IA, but not that of

BMPR-II

To investigate whether inhibition of BMP-2-induced

AP activity by proBMP-2 results from competition of

proBMP-2 with BMP-2 for binding to the main

receptor BMPR-IA, BIAcore experiments were

per-formed For these studies, the ECD of the receptor

was recombinantly produced in Escherichia coli cells,

refolded and purified [31] The ECD was biotinylated

and immobilized on streptavidin-coated BIAcore chips

Ligand binding was first analysed using the mature

growth factor The fast association rate and the very

slow dissociation rate are in accordance with published

results (KD= 0.9 ± 0.8· 10)9m) (Fig 2A and

Table 1) [29] When proBMP-2 was tested as an

ana-lyte, a comparable KD (4 ± 1.8· 10)9m) was

obtained (Fig 2B and Table 1) This result shows that

the pro-peptide moiety does not interfere with binding

of the mature part to BMPR-IA As the BMPR-IA

binding site for BMP-2 partially overlaps with the area

bound by the antagonist noggin [32], we attempted

to verify these findings by testing the binding of

proBMP-2 to noggin Biotinylated noggin was

immo-bilized on streptavidin-coated chips, and proBMP-2 or

BMP-2 were injected at various concentrations

(Fig 2C,D) The sensorgrams reveal that proBMP-2

binds to noggin with a comparable affinity to that for mature BMP-2, a result that confirms indirectly that the pro-peptide moiety does not interfere with binding

of the mature part to the BMPR-IA ECD Due to the very slow release of both analytes from the immobi-lized ligand, KD values based on the association and dissociation rates could not be determined for the interaction with noggin

The BIAcore experiments that revealed binding of proBMP-2 were corroborated by ELISA studies For these experiments, BMP-2 or proBMP-2 was adsorbed

on to the well surfaces of microtitre plates After blocking free binding sites of the wells, the BMPR-IA ECD was added at various concentrations Growth factor-bound ECD was detected by incubation with BMPR-IA ECD antibody and subsequent detection via a horseradish peroxidase-conjugated antibody The BMPR-IA ECD bound to both immobilized BMP-2 and proBMP-2 (Fig 3A,B) The final signal for proBMP-2 was approximately twice as high as that for BMP-2 Presumably, this effect is due to more efficient coating of proBMP-2 to the well surface than with BMP-2, as has also been observed in other experi-ments (data not shown)

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Fig 2 Surface plasmon resonance

experi-ments demonstrate binding of proBMP-2 to

the ECD of BMPR-IA Interaction of BMP-2

(A) and proBMP-2 (B) with immobilized ECD

of BMPR-IA Interaction of BMP-2 (C) and

proBMP-2 (D) with immobilized noggin are

indicated The higher scattering of the

sen-sorgrams in (C) and (D) is due to the fact

that only 64 resonance units of noggin were

immobilized in these experiments.

Table 1 Kinetic constants for interaction of the ECD of BMPR-IA with the growth factors Association (ka) and dissociation (kd) rates for the ligands with the ECD and the apparent dissociation con-stants KD as determined using BIAcore are shown.

ka ( M )1Æs)1) kd(s)1) KD(nM) BMP-2 3.1 ± 1.8 · 10 5 2.8 ± 1.0 · 10)4 0.9 ± 0.8 proBMP-2 7.4 ± 2.9 · 10 4

3.0 ± 0.2 · 10)4 4.0 ± 1.8

Next, competition experiments were performed The

ECD at a concentration of 500 nm was pre-incubated

for 30 min with increasing concentrations of BMP-2 to

allow complex formation Subsequently, the

pre-incu-bated samples were added to wells that had been

coated with BMP-2 or proBMP-2 ECD binding to

immobilized BMP-2 or proBMP-2 decreased with

increasing concentrations of the growth factors in the

pre-incubations (Fig 3C,D) These data confirmed that

proBMP-2 binds specifically to the ECD of BMPR-IA

Furthermore, the results indicate that both proteins

interact with the same epitope on the ECD because

proBMP-2 binding can be inhibited by BMP-2

To assess binding of proBMP-2 to BMPR-II,

BIA-core experiments were performed using the ECD of

BMPR-II linked to a Fc domain of human IgG

(BMPR-II-Fc) After immobilization of the chimeric

protein on a CM5 chip, binding of BMP-2 as a

positive control (Fig 4A) and of proBMP-2 (Fig 4B)

were recorded ProBMP-2 did not bind to the

immo-bilized ECD chimera of BMPR-II When mature

BMP-2 was pre-incubated with increasing

concentra-tions of separately produced, free pro-peptide,

decreased signals were observed (Fig 4C), which

confirms that the pro-peptide inhibits association of

mature BMP-2 with the ECD of BMPR-II, probably

by masking binding sites of BMP-2 Consistently,

maximal inhibition was observed by using equimolar

concentrations (0.4 lm) of both pro-peptide and

BMP-2 Furthermore, the ability of BMP-2 to

inter-act with the free pro-peptide could be proven by

BIAcore experiments (Fig 4D) From these studies,

a KD of 28 ± 16 nm for the non-covalent complex

of mature BMP-2 and the pro-peptide was calcu-lated

BMP-2 and proBMP-2 bind to BMPR-IA at the cell surface

After demonstrating that proBMP-2 binds to the ECD

of BMPR-IA in vitro, we performed an in vivo experi-ment to test binding of proBMP-2 to this receptor presented at the cell surface COS-7 cells were trans-fected with an expression construct for BMPR-IA carrying a haemagglutinin (HA) epitope [4] Transient expression of the receptor was first tested 2 days after transfection by examination of whole-cell extracts using SDS–PAGE, western blotting and decoration with HA antibodies (data not shown)

COS-7 cells transiently expressing BMPR-IA were incubated with BMP-2 or proBMP-2 After removal of unbound ligands, cell-bound growth factors were chemically cross-linked using disuccinimidylsuberate (DSS) Ligand–receptor complexes were detected directly in whole-cell lysates after western blotting Detection of the proBMP-2 moiety was done using a BMP-2 antibody (Fig 5A) and the BMPR-IA part by

a HA antibody (Fig 5B) A band at the expected size

of approximately 140 kDa was detected using each antibody, but was never observed in the negative con-trols to which neither proBMP-2 nor DSS were added Detection of bands of comparable sizes with either

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Fig 3 Binding of proBMP-2 to the ECD was confirmed by ELISA Binding of the BMPR-IA ECD to immobilized BMP-2 (A) and proBMP-2 (B) For the competition experiments, 500 n M ECD were pre-incu-bated with the indicated concentrations of BMP-2 Unbound ECD reacted with immobi-lized BMP-2 (C) or proBMP-2 (D) Data represent means and standard deviations from three independent measurements.

antibody indicates that this signal represents the complex of proBMP-2 with BMPR-IA Complexes

of BMP-2 or proBMP-2 with BMPR-IA were also immunoprecipitated using BMPR-IA ECD antibody (Fig 5C,D) After immuno-precipitation, for both ligands, signals corresponding to the positions of the ligand-receptor complexes were detected (Fig 5C,D)

ProBMP-2 differs from BMP-2 in its ability to induce Smad and p38 phosphorylation BMP-2 can induce two signalling pathways depend-ing on its mode of interaction with surface receptors [5,6,33]: upon BMP-2 binding to PFCs, the Smad pathway is induced, or BMP-2-induced receptor olig-omerization triggers phosphorylation of p38, resulting

in AP induction [33,34], unless Smad proteins are overexpressed [35,36] The inability of proBMP-2

to induce AP prompted the question of whether proBMP-2 might induce cellular signals by preferen-tially interacting with PFCs, and thus predominantly activate the Smad pathway When 10 nM BMP-2 was added to C2C12 cells, Smad proteins 1, 5 and 8 were phosphorylated at their C-termini after 15 min

In contrast, the same concentration of proBMP-2 did not lead to significant phosphorylation even after

120 min (Fig 6A,B) Only at high concentrations (200 nm) did proBMP-2 led to instantaneous Smad phosphorylation (Fig S3) When the effect of BMP-2 and proBMP-2 on the phosphorylation status

of p38 was tested, addition of BMP-2 resulted in maximal phosphorylation of p38 after 60 min, while proBMP2 showed no induction of p38 phosphoryla-tion (Fig 6A,C)

To measure Smad activation in long-term experi-ments, a BRE–luciferase assay was used, in which luciferase (as reporter gene) is under the control of a Smad-responsive element [5] Figure 7 shows that both ligands induce luciferase in a concentration-dependent manner; however, proBMP-2 was less effective than BMP-2

Discussion

BMP-7, BMP-9, GDF-8 and TGFb are highly homol-ogous to BMP-2 For all these growth factors, non-covalent association of the pro-peptides with the mature domains after proteolytic processing has been demonstrated [12,15,17,18,37] In case of BMP-2, it is not clear whether the pro-peptide moiety remains associated with the mature part of BMP-2 after pro-hormone processing because precise and detailed infor-mation on the levels of BMP-2 and proBMP-2 in the

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Fig 4 ProBMP-2 does not bind to the immobilized ECD of

BMPR-II (A) Interaction of BMP-2 with immoblilized BMPR-II–Fc (B)

Com-parison of proBMP-2- and BMP-2 interaction with immobilized

BMPR-II–Fc (C) Inhibition of BMP-2 binding to BMPR-II–Fc by the

isolated pro-peptide Signal reduction is dependent on the

concentrations of pro-peptide in the pre-incubation (D) Complex

for-mation of BMP-2 and the free pro-peptide was tested by BIAcore.

For the experiments, the pro-peptide was immobilized on a CM5 chip.

extracellular space is not available In fact, only one

such study has been performed, which found that a

small amount of unprocessed proBMP-2 is secreted

upon recombinant expression in CHO cells [38] Lories

et al [26] described accumulation of the pro-form of

BMP-2 in human tissue, with high levels of proBMP-2

being found in tissue from patients with rheumatoid

arthritis or spondyloarthropathy Interestingly,

BMPR-IA-positive cells have been detected in synovial tissue

from arthritic patients [39], and a role of the receptor

in the development of the arthritis has been discussed [26]

The data presented here provide clear evidence that the pro-form of BMP-2 can interact with BMPR-IA However, receptor binding of pro-forms of the TGFb⁄ BMP family appears to be complex, depending

on both the receptor type and the individual pro-form While indirect evidence has been obtained that a

97 116

kDa

proBMP-2 DSS

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+ –

+ +

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BMP-2

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proBMP-2

–

+

+ +

proBMP-2

WB: anti-BMP-2

C WB: anti-BMP-2 D WB: anti-BMP-2

WB: anti-HA

BMP2 - BMPR-IA-dimer complex BMP2 - BMPR-IA-complex

Fig 5 ProBMP-2 can be cross-linked to recombinantly expressed BMPR-IA COS-7 cells expressing BMPR-IA were incubated with proBMP-2 (A,B,D) or BMP-2 (C) After blotting, cell extracts (A,B) were decorated with BMP-2 antibody (A) or with HA antibody (B) Cross-linked ligand–receptor complexes were immunoprecipitated using BMPR-IA ECD antibody (C,D) and analysed with BMP-2 antibody after blotting.

proBMP-2

A

BMP-2

P-Smad 1/5/8

Smad 1

P-p38

Actin

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Fig 6 BMP-2 and proBMP-2 differ in their ability to lead to Smad1 ⁄ 5 ⁄ 8 or p38 phos-phorylation C2C12 cells were treated with

10 n M ligand for the indicated time periods (A) After blotting, whole-cell lysates were analysed using antibodies against phosphor-ylated (P-Smad1 ⁄ 5 ⁄ 8) and total Smad1 ⁄ 5 ⁄ 8, phosphorylated p38 (P-p38), or actin as a loading control (B) Quantification of C-termi-nally phosphorylated Smad1 ⁄ 5 ⁄ 8 in relation

to total Smad1 ⁄ 5 ⁄ 8 (C) Quantification of phosphorylated p38 in relation to actin Circles, BMP-2; squares, proBMP-2.

non-covalent complex of the pro-peptide and BMP-9

can bind to the type I receptor Alk1 [17], the

latency-associated polypeptide of TGFb inhibits the

interac-tion of TGFb isoforms with type II and III receptors

[40] An even more complex situation has very recently

been described for the non-covalent complex of

BMP-7 and pro-peptides, which binds to type I receptors

and type II receptors, depending on the experimental

set-up [41] Upon binding to the complex of mature

part and pro-domain(s), type II receptors are able to

dissociate the pro-domains from the complex [41] As

we showed here that the ECD of BMPR-II is not

bound by proBMP-2, displacement of the covalently

bound pro-domain is unlikely Moreover, our results

on the inhibitory effect of the free pro-peptide on

com-plex formation between BMPR-II and BMP-2 indicate

that, in this case, BMPR-II per se cannot rearrange

the pro-peptide Generally, however, displacement of

the pro-peptide cannot be excluded although in

cova-lent peptide linkage, as the peptide region between the

pro-peptide and the mature part may be flexible and

thus allow sufficient conformational freedom to

dis-charge the pro-peptide part from a receptor binding

interface Thus, the slower association kinetics of

proBMP-2 with noggin could be due to such a

displacement, induced by noggin

In addition to the potential physiological

implica-tions of our findings, the interaction of proBMP-2 with

BMPR-IA and noggin allows indirect conclusions

about the position of the pro-peptide with respect to

the mature part As neither the interaction with

BMPR-IA nor with noggin was affected in

quantita-tive terms, the pro-peptide moiety probably does not

obstruct the key residues of BMP-2 that mediate

bind-ing to either the receptor or noggin This observation

is consistent with crystallographic results, which

revealed that the BMPR-IA binding site of the related BMP-7 largely coincides with the noggin-binding area [32,42] Thus, the proBMP-2 binding to noggin detected here indirectly confirms the interaction of proBMP-2 with BMPR-IA Conversely, as noggin cov-ers part of the BMPR-II binding site of BMP-2, an interaction of noggin with proBMP-2 is counter-intui-tive given that proBMP-2 did not bind to BMPR-II Consequently, the pro-peptide masks at least the inter-action site in BMP-2 for BMPR-II binding On the other hand, a recent NMR study showed that both the ECD of BMPR-IA and BMP-2 undergo structural transitions upon association [43], indicating that discussions on ligand–receptor interactions have to consider the high inherent flexibility of probably all the partners involved In what way BMPR-IA binding

by proBMP-2 may rearrange the pro-peptide moiety as discussed above remains to be tested

Although suppression of TGFb effects by latency-associated polypeptide has been reported [44], the find-ing that proBMP-2 inhibits AP induction by mature BMP-2 was unexpected, as our previous results had shown ectopic bone formation in response to proBMP-2 In these studies, however, growth factor-induced bone formation was assessed at a single time point (30 days), and neither the pharmacokinetics of proBMP-2 nor the kinetics of bone formation were anal-ysed Thus, the fate of administered proBMP-2 in the animal over time is unclear and it cannot be excluded that proBMP-2 becomes converted to mature BMP-2 by extracellular proteases with time

The fact that proBMP-2 elicited Smad-mediated luciferase transcription in cell culture after 16 h is con-sistent with a slow action of proBMP-2 The Smad pathway is predominantly induced by ligand binding

to PFCs Possibly, proBMP-2 signalling occurs pre-dominantly via this binding mode However, how proBMP-2 transmits signals despite being unable to bind BMPR-II is unclear, and is the next issue to be addressed

Experimental procedures

Recombinant proteins

proBMP-2 and the pro-domain of BMP-2 were performed

as described previously [8] The extracellular ligand binding domain (ECD) of BMPR-IA was prepared and biotinylated

(ECD) was purchased from R&D Systems (Minneapolis,

MN, USA) Noggin was produced in E coli BL21 (DE3) as

a soluble maltose binding protein (MBP) fusion protein To

Ligand concentration [nM]

10

0

2

4

6

8

10

12

14

16

18

Fig 7 Activation of the Smad1 ⁄ 5 ⁄ 8 pathway by BMP2 and

proBMP2 as measured by reporter gene assay (BRE-luciferase).

C2C12 cells were stimulated with the indicated concentrations of

proBMP-2 and BMP-2 for 16 h Black columns, BMP-2; grey

columns, proBMP-2.

overcome codon usage limitations, cells were additionally

transformed with plasmid pUBS520, which carries the gene

induced using 1 mm isopropyl thio-b-d-galactoside when the

cells had reached an attenuance at 600 nm of 0.5–0.8 Cells

were harvested 3 h after induction A volume of 10 mL

EDTA) was added per gram of cell pellet Cells were

dis-rupted by high-pressure cell dispersion After sedimentation

was diluted at a ratio of 1 : 5 in buffer A, and loaded at a

column (New England Biolabs, Beverly, MA, USA) The

column was washed with 10 column volumes of buffer A

Elution of the MBP–noggin fusion protein was achieved

using buffer A containing 10 mm maltose Pooled elution

fractions were adjusted to a concentration of 8 m with solid

urea, and renaturation of the fusion protein was achieved by

8.5, 1 m NaCl, 2 mm EDTA, 1 mm phenylmethanesulfonyl

fluoride, 25 mm Chaps, 2 mm oxidized glutathione and

0.2 mm reduced glutathione) The protein concentration

rena-turation, the protein solution was concentrated using a

Vivaflow 200 ultrafiltration tube (Sartorius Vivascience,

Go¨ttingen, Germany), and was then dialysed against 20 mm

virus (TEV) protease related to the fusion protein content

Upon proteolysis, noggin precipitated and was recovered by

a 20 min centrifugation at 48 000 g Precipitated protein was

further purified by reverse-phase high-performance liquid

chromatography Protein resuspended in solvent A (0.1%

reverse phase chromatography column (GE Healthcare,

Munich, Germany) The column was washed with three

column volumes of solvent A Elution of noggin was

achieved using a non-linear gradient of four column volumes

aqueous acetonitrile) and 1.5 column volumes of 37.5–100%

solvent B For some experiments, noggin from R&D Systems

was used as a reference, and was found to behave identically

Cell culture

C2C12 cells (DSMZ, Braunschweig, Germany) were

main-tained in RPMI-1640 medium (PAA Laboratories GmbH,

Co¨lbe, Germany) supplemented with 10% fetal bovine

induction of AP, the serum concentration was reduced

to 2%

Cross-linking of ligands to transiently transfected COS-7 cells

Transfection and cross-linking of ligand–receptor complexes were performed as described previously [4] COS-7 cells

Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum Transfection with Rotifect (Roth, Karlsruhe, Ger-many) was performed according to the supplier’s instruc-tions To each well, 2 lg pcDNA-3.1-BRIA-HA [46] was added for expression of HA-tagged BMPR-IA Forty-eight hours after transfection, cells were washed twice in

10 nm BMP-2 or proBMP-2 in buffer W were added and

Cross-linking was performed as described previously [47] After cross-linking, cells were solubilized in 100 lL lysis

EDTA and protease inhibitor cocktail (Roche Diagnostics,

were analysed by western blotting or after immunoprecipi-tation Receptor complexes were immunoprecipitated using BMPR-IA ECD antibodies (Santa Cruz Biotechnology,

25 lL protein A–Sepharose slurry (GE Healthcare) in lysis

b-mer-captoethanol was added, and the samples were heated for

Bis–Tris pre-cast gradient gels (Invitrogen, Karlsruhe, Germany) Western blotting was performed according to standard protocols After blocking (10 mm Tris pH 7.9,

150 mm NaCl, 0.5% Tween-20, 3% BSA), blots were

Hamburg, Germany) Detection was achieved using horse-radish peroxidase-coupled secondary antibody with the ECL system (GE Healthcare)

Alkaline phosphatase (AP) assay and luciferase-based reporter gene assay

plates Cells were allowed to attach overnight, and then complete medium (10% fetal bovine serum) was replaced

by 200 lL differentiation medium (2% fetal bovine serum) supplemented with the growth factors After 4 days, the

Darmstadt, Germany)] by gentle shaking at room tempera-ture for 2–3 h 20 lL lysate was transferred to a new 96-well plate Then 200 lL substrate solution (9 mm

p-nitrophenyl phosphate in lysis buffer) was added per well

[29] Changes in absorption at 405 nm were followed over

30 min using an ELISA plate reader For determination of

inhibition by noggin, the AP assay was slightly modified:

cells were seeded and tests were performed after

3 days with 100 lL substrate solution per well For the

luciferase-based reporter gene assay, C2C12 cells were

under normal growth conditions After 24 h, cells were

transfected with a construct containing firefly luciferase

dri-ven by the BMP response element (pBRE-Luc) [48] and a

constitutive active Renilla luciferase (pRLTK) as an internal

control using Lipofectamine 2000 (Invitrogen, Life

Technol-ogies, Carlsbad, CA, USA) according to the manufacturer’s

instructions The next day, cells were starved for 5 h and

stimulated with ligand in medium containing 0.5% fetal

bovine serum for 16 h Luciferase activity was measured

using the dual luciferase reporter assay system (Promega,

Madison, WI, USA) and a Mithras LB 940 luminometer

(Berthold Detection Systems, Pforzheim, Germany)

Test for phosphorylated p38 and Smad

The test was performed as described previously [49] C2C12

cells per well)

After 24 h, cells were starved for 3 h and then incubated in

the absence or presence of BMP-2 (10 nm) or proBMP-2

(10 nm) for the indicated time Cells were washed with

b-mercaptoethanol, 0.01% bromophenol blue) per well After

PAGE and blotted to poly(vinylidene difluoride) membranes

Membranes were blocked with TBST (TBS containing 0.1%

Tween-20) containing 5% BSA at room temperature for

60 min Membranes were washed three times for 10–15 min

in TBST For antibody staining, the primary antibodies

Technology Inc., Beverly, MA, USA) were diluted 1 : 1000 in

TBST containing 5% BSA Detection was performed using a

secondary horseradish peroxidase-coupled antibody using the

ECL system and ChemiSmart 5000 (Peqlab, Erlangen,

Germany) Phosphorylation was quantified relative to total

Smad (anti-Smad, Cell Signaling Technology Inc.) or actin

(anti-b-actin, Sigma-Aldrich, St Louis, MO, USA) For

quanti-fication, the maximum phosphorylation level was set to 100%

Surface plasmon resonance

Binding of BMP-2 and proBMP-2 to the immobilized

pro-teins was examined using the BIA2000 system (BIAcore,

Uppsala, Sweden) Biotinylated BMPR-IA ECD or noggin

USA) were immobilized on streptavidin-coated chips at a

density of approximately 300 resonance units for the ECD or

64 resonance units for noggin For BMPR-II–Fc, 1000 reso-nance units were immobilized on a CM5 sensorchip (GE Healthcare) by amine coupling BMP-2 or proBMP-2 (in

10 mm Hepes pH 7.4, 500 mm NaCl, 3.4 mm EDTA, 0.005% Tween-20) were injected at the indicated concen-trations Sensorgrams were recorded at a flow rate of

surface was achieved by perfusion with 10 mm glycine pH 2, and regeneration of the noggin-coupled chip surface by 6 m

Correc-tion for background signals was performed by subtracCorrec-tion of the signals from the control flow cell Non-specific binding was negligible The sensorgrams were evaluated using the software BIAevaluation version 2.0 (biacore) assuming a

1 : 1 interaction Although one BMP-2 dimer is known to interact with two ECD monomers [42], a 1 : 1 interaction has been used previously for the assessment of relative bind-ing affinities [29] Association and dissociation rate constants were obtained from 7–9 analyte concentrations Mean values with a standard deviation were deduced from five

The influence of the free pro-peptide on BMP-2 binding

to BMPR-II was tested using 400 nm BMP-2 that had been pre-incubated for 2 h at room temperature with increasing pro-peptide concentrations Binding to BMPR-II was then detected by recording equilibrium binding via BIAcore To this end, the time interval for the association was prolonged until a constant maximal response representing an equilib-rium was observed Signals were then plotted against the pro-peptide concentrations

ELISA

For the assays, a previously published protocol [50] was modified Tests were performed in 96-well microtitre plates (Nunc-Immuno Maxisorp, Nunc, Wiesbaden, Germany)

at room temperature Washing was performed with 200 lL

either 100 nm BMP-2 or 100 nm proBMP-2 in 50 mm

blocked for 2 h at room temperature using 200 lL blocking

0.05% Tween-20] After washing, the BMPR-IA ECD was added at the indicated concentrations in blocking buffer and incubated for 1 h Tests for non-specific binding were performed using blocking buffer alone After three washing steps, goat human BMPR-IA ECD antibody (Santa Cruz Biotechnology) was added to a final concentration of

horseradish peroxidase-conjugated antibody (Perbio

blocking buffer Photometric detection was achieved using

2,2¢-azino-bis(3-ethylbenzthiazoline-6-sulpho-nic acid (ABTS; Roche Diagnostics) in ABTS buffer