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

The pro-form of BMP-2 interferes with BMP-2 signalling by competing with BMP-2 for IA receptor binding Anja Hauburger1, Sabrina von Einem1, Gerburg K Schwaerzer2, Anja Buttstedt1, Matthias Zebisch3, Michael Schraml4, , Peter Hortschansky5, Petra Knaus2 and Elisabeth Schwarz1 ă Institut fur Biochemie und Biotechnologie, Martin-Luther-Universitat Halle-Wittenberg, Germany ă ă Institut fur Chemie Biochemie, Freie Universitat Berlin, Germany ă ă Biotechnologisch-Biomedizinisches Zentrum, Universita Leipzig, Germany ăt Scil Proteins GmbH, Halle, Germany Leibniz-Institut fur Naturstoffforschung und Infektionsbiologie, Hans-Knoll-Institut (HKI), Jena, Germany ă ă Keywords alkaline phosphatase; BMPR-IA; BMPR-II; bone morphogenetic protein-2; pro-domain Correspondence E Schwarz, Institut fur Biochemie and ă Biotechnologie, Martin-Luther-Universitat ă 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 April 2009, revised 18 August 2009, accepted 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 osteoblasts, 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 BMPRIA, 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 surface 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 6386 FEBS Journal 276 (2009) 6386–6398 ª 2009 The Authors Journal compilation ª 2009 FEBS A Hauburger et al The pro form of BMP-2 interferes with BMP-2 signalling l MINT-7261597, MINT-7261613: BMPR-IA (uniprotkb:P36894) binds (MI:0407) to BMP2 (uniprotkb:P12643) by surface plasmon resonance (MI:0107) Introduction 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 association 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 endoplasmic reticulum However, the function of the pro-peptide is presently unknown We showed previously that the pro-peptide is not required for in vitro oxidative folding of the mature part [8] Furthermore, recombinant proBMP-2 induced ectopic bone formation in rats, indicating that the pro-peptide does not significantly 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 binding 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 inhibitory 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-peptide appears not to alter significantly the activity of the mature part [17] For the pro-peptide of BMP-7, a targeting role to the extracellular matrix [18] has been shown The pro-peptide of BMP-4 is responsible for stabilization of the mature part, intracellular trafficking and folding in the endoplasmic reticulum [19–21] Thus, the roles of the pro-peptides appear to be divergent within the TGFb ⁄ BMP family, and appear to modulate the function of the mature part by noncovalent 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 mutations in pro-domains lead to abnormal dorsoventral patterning [23] and skeletal malformations [24,25] In the case of BMP-2, no published information is available 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 spondyloarthropathies [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 propeptide 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 FEBS Journal 276 (2009) 6386–6398 ª 2009 The Authors Journal compilation ª 2009 FEBS 6387 The pro form of BMP-2 interferes with BMP-2 signalling A Hauburger et al 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 A 50 B AP activity (%) (ΔE × min–1 × µg–1) 40 AP activity (%) (ΔE × min–1 × µg–1) In order to determine whether proBMP-2 elicits biological responses similar those elicited by mature BMP-2, induction of alkaline phosphatase (AP) was investigated AP represents a marker enzyme for differentiation 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 EC50 of 18 ± 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 propeptide 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 lm proBMP-2, the AP signal was in a similar range to that obtained after induction with 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 competition 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 increasing concentrations of proBMP-2 A proBMP-2 concentration-dependent inhibition of the BMP-2-induced AP activity was observed (Fig 1C) The possibility of contamination of the proBMP-2 preparation with endotoxins was excluded by using a chromogenic limulus amoebocyte lysate (LAL) detection kit (Charles River, Wilmington, MA, USA), which showed that endotoxin 30 20 10 50 40 30 20 10 200 400 600 proBMP-2 [nM] D 80 AP activity (%) AP inhibition (%) C 10 100 BMP-2 [nM] 60 40 20 100 80 60 40 20 0 200 400 600 800 1000 I II III IV proBMP-2 [nM] Fig 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 phosphatase in C2C12 cells AP activity was measured by determination of the change in extinction (DE) per minute and microgram protein In (C), 200 nM 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 noggin Ligand concentrations were 10 nM The lower amplitudes of the AP signals are due to the fact that, in this experiment, the signals obtained using 10 nM BMP-2 were set to 100% Data represent means and standard deviations from four independent measurements 6388 FEBS Journal 276 (2009) 6386–6398 ª 2009 The Authors Journal compilation ª 2009 FEBS A Hauburger et al The pro form of BMP-2 interferes with BMP-2 signalling 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 Table 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 constants KD as determined using BIAcore are shown ka (M)1Ỉs)1) kd (s)1) KD (nM) 3.1 ± 1.8 · 105 7.4 ± 2.9 · 104 2.8 ± 1.0 · 10)4 3.0 ± 0.2 · 10)4 0.9 ± 0.8 4.0 ± 1.8 ProBMP-2 binds to the extracellular ligand binding domain of BMPR-IA, but not that of BMPR-II BMP-2 proBMP-2 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 performed 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)9 m) (Fig 2A and Table 1) [29] When proBMP-2 was tested as an analyte, a comparable KD (4 ± 1.8 · 10)9 m) 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 immobilized 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 immobilized 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 experiments (data not shown) B 400 400 nM 200 nM 100 nM 50 nM 25 nM 12.5 nM 6.2 nM 3.1 nM 1.6 nM RU 300 200 100 0 250 200 RU A 400 nM 200 nM 100 nM 50 nM 25 nM 12.5 nM 6.2 nM 3.1 nM 150 100 50 100 200 300 400 500 100 200 300 400 500 Time (s) D 40 400 nM 200 nM 100 nM 50 nM 25 nM 12.5 nM 6.2 nM 3.1 nM nM 30 20 10 40 600 nM 300 nM 200 nM 100 nM 50 nM 25 nM 12.5 nM 6.2 nM nM 30 RU C RU Fig Surface plasmon resonance experiments 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 sensorgrams in (C) and (D) is due to the fact that only 64 resonance units of noggin were immobilized in these experiments Time (s) 20 10 0 100 200 300 400 500 Time (s) FEBS Journal 276 (2009) 6386–6398 ª 2009 The Authors Journal compilation ª 2009 FEBS 100 200 300 400 Time (s) 6389 The pro form of BMP-2 interferes with BMP-2 signalling A B Immobilized BMP-2 Immobilized proBMP-2 50 15 40 Slope at 405 nm Slope at 405 nm 20 10 0.0 0.5 1.0 1.5 2.0 2.5 30 20 10 0.0 0.5 1.0 1.5 2.0 2.5 5.0 BMPR-IA-ECD [µM] C Immobilized BMP-2 D Slope at 405 nm 0 10 12 14 BMP-2 [µM] In the pre-incubation with 500 nM ECD Immobilized proBMP-2 20 18 16 14 12 10 0 10 12 14 BMP-2 [µM] In the pre-incubation with 500 nM ECD Next, competition experiments were performed The ECD at a concentration of 500 nm was pre-incubated for 30 with increasing concentrations of BMP-2 to allow complex formation Subsequently, the pre-incubated 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, BIAcore 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 immobilized ECD chimera of BMPR-II When mature BMP-2 was pre-incubated with increasing concentrations 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 interact with the free pro-peptide could be proven by 6390 5.0 BMPR-IA-ECD [µM] 10 Slope at 405 nm A Hauburger et al Fig 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 nM ECD were pre-incubated with the indicated concentrations of BMP-2 Unbound ECD reacted with immobilized BMP-2 (C) or proBMP-2 (D) Data represent means and standard deviations from three independent measurements 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 calculated 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 experiment to test binding of proBMP-2 to this receptor presented at the cell surface COS-7 cells were transfected with an expression construct for BMPR-IA carrying a haemagglutinin (HA) epitope [4] Transient expression of the receptor was first tested 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 controls to which neither proBMP-2 nor DSS were added Detection of bands of comparable sizes with either FEBS Journal 276 (2009) 6386–6398 ª 2009 The Authors Journal compilation ª 2009 FEBS A Hauburger et al A The pro form of BMP-2 interferes with BMP-2 signalling 700 600 500 RU 400 400 nM 200 nM 100 nM 50 nM 25 nM 12.5 nM nM 300 200 100 0 50 100 150 200 Time (s) B 500 400 RU 300 200 200 nM BMP-2 100 800 nM proBMP-2 50 100 150 200 Time (s) C 300 Req (RU) 250 200 150 100 50 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Pro-peptide in pre-incubation [µM] D 700 600 400 nM 500 RU 400 300 200 nM 200 100 nM 50 nM 25 nM 12.5 nM 100 0 50 100 150 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 depending 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 oligomerization 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 preferentially interacting with PFCs, and thus predominantly activate the Smad pathway When 10 nM BMP-2 was added to C2C12 cells, Smad proteins 1, and were phosphorylated at their C-termini after 15 In contrast, the same concentration of proBMP-2 did not lead to significant phosphorylation even after 120 (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 phosphorylation (Fig 6A,C) To measure Smad activation in long-term experiments, a BRE–luciferase assay was used, in which luciferase (as reporter gene) is under the control of a Smad-responsive element [5] Figure shows that both ligands induce luciferase in a concentration-dependent manner; however, proBMP-2 was less effective than BMP-2 Discussion 200 Time (s) Fig ProBMP-2 does not bind to the immobilized ECD of BMPRII (A) Interaction of BMP-2 with immoblilized BMPR-II–Fc (B) Comparison 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 formation of BMP-2 and the free pro-peptide was tested by BIAcore For the experiments, the pro-peptide was immobilized on a CM5 chip BMP-7, BMP-9, GDF-8 and TGFb are highly homologous to BMP-2 For all these growth factors, noncovalent 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 prohormone processing because precise and detailed information on the levels of BMP-2 and proBMP-2 in the FEBS Journal 276 (2009) 6386–6398 ª 2009 The Authors Journal compilation ª 2009 FEBS 6391 The pro form of BMP-2 interferes with BMP-2 signalling A B WB: anti-BMP-2 A Hauburger et al WB: anti-HA proBMP-2 – + proBMP-2 + + DSS + + DSS – + 160 160 proBMP-2 BMPR-IAcomplex 116 116 97 97 proBMP-2 kDa C proBMP-2BMPR-IAcomplex kDa D WB: anti-BMP-2 WB: anti-BMP-2 BMP-2 + + proBMP-2 + + DSS – + DSS – + BMP-2 BMPR-IAdimer complex 97 66 kDa 160 116 proBMP-2BMPR-IAcomplex BMP-2 BMPR-IAcomplex 116 97 proBMP-2 kDa 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, BMPRIA-positive cells have been detected in synovial tissue A proBMP-2 15 30 Fig 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 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 BMP-2 60 120 15 30 60 120 P-Smad 1/5/8 Smad P-p38 Actin P-Smad 1/5/8 signal intensity 120 100 80 60 40 20 P-p38 signal intensity 120 100 80 60 40 20 0 20 40 60 80 Time (min) 6392 C Relative intensity (%) Relative intensity (%) B 100 120 140 20 40 60 80 Time (min) 100 120 140 Fig BMP-2 and proBMP-2 differ in their ability to lead to Smad1 ⁄ ⁄ or p38 phosphorylation C2C12 cells were treated with 10 nM ligand for the indicated time periods (A) After blotting, whole-cell lysates were analysed using antibodies against phosphorylated (P-Smad1 ⁄ ⁄ 8) and total Smad1 ⁄ ⁄ 8, phosphorylated p38 (P-p38), or actin as a loading control (B) Quantification of C-terminally phosphorylated Smad1 ⁄ ⁄ in relation to total Smad1 ⁄ ⁄ (C) Quantification of phosphorylated p38 in relation to actin Circles, BMP-2; squares, proBMP-2 FEBS Journal 276 (2009) 6386–6398 ª 2009 The Authors Journal compilation ª 2009 FEBS RLU A Hauburger et al The pro form of BMP-2 interferes with BMP-2 signalling 18 16 14 12 10 0 0.1 0.5 10 20 50 Ligand concentration [nM] Fig Activation of the Smad1 ⁄ ⁄ 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 non-covalent complex of the pro-peptide and BMP-9 can bind to the type I receptor Alk1 [17], the latencyassociated polypeptide of TGFb inhibits the interaction 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 BMP7 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 complex 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 covalent peptide linkage, as the peptide region between the pro-peptide and the mature part may be flexible and thus allow sufficient conformational freedom to discharge 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 implications 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 quantitative terms, the pro-peptide moiety probably does not obstruct the key residues of BMP-2 that mediate binding 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 covers part of the BMPR-II binding site of BMP-2, an interaction of noggin with proBMP-2 is counter-intuitive given that proBMP-2 did not bind to BMPR-II Consequently, the pro-peptide masks at least the interaction 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 latencyassociated polypeptide has been reported [44], the finding 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 factorinduced 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 analysed 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 consistent with a slow action of proBMP-2 The Smad pathway is predominantly induced by ligand binding to PFCs Possibly, proBMP-2 signalling occurs predominantly 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 Recombinant production and purification of BMP-2, 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 as described previously [31] The BMPR-II ⁄ Fc chimera (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 FEBS Journal 276 (2009) 6386–6398 ª 2009 The Authors Journal compilation ª 2009 FEBS 6393 The pro form of BMP-2 interferes with BMP-2 signalling A Hauburger et al overcome codon usage limitations, cells were additionally transformed with plasmid pUBS520, which carries the gene dnaY that encodes a rare tRNA for arginine codons in E coli [45] Cultivation in LB medium was performed in shake flasks at 37 °C Noggin cDNA expression was induced using mm isopropyl thio-b-d-galactoside when the cells had reached an attenuance at 600 nm of 0.5–0.8 Cells were harvested h after induction A volume of 10 mL buffer A (20 mm Tris ⁄ HCl pH 7.4, 200 mm NaCl, mm EDTA) was added per gram of cell pellet Cells were disrupted by high-pressure cell dispersion After sedimentation of cell debris at 9000 g for 30 at °C, the supernatant was diluted at a ratio of : in buffer A, and loaded at a flow rate of mLỈmin)1 on a 15 mL bed size amylose 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 m with solid urea, and renaturation of the fusion protein was achieved by fourfold dilution into refolding buffer (50 mm Tris ⁄ HCl pH 8.5, m NaCl, mm EDTA, mm phenylmethanesulfonyl fluoride, 25 mm Chaps, mm oxidized glutathione and 0.2 mm reduced glutathione) The protein concentration during renaturation was 100 lgỈmL)1 After days of renaturation, the protein solution was concentrated using a Vivaflow 200 ultrafiltration tube (Sartorius Vivascience, Gottingen, Germany), and was then dialysed against 20 mm ă Tris ⁄ HCl pH 8.5, 150 mm NaCl For proteolytic MBP removal, the protein solution (0.35 mgỈml)1) was incubated at room temperature for 24 h with 0.5% w ⁄ w tobacco etch virus (TEV) protease related to the fusion protein content Upon proteolysis, noggin precipitated and was recovered by a 20 centrifugation at 48 000 g Precipitated protein was further purified by reverse-phase high-performance liquid chromatography Protein resuspended in solvent A (0.1% v ⁄ v trifluoroacetic acid in 20% v ⁄ v aqueous acetonitrile) was loaded at a flow rate of 0.7 mLỈmin)1 onto a SOURCE 15 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 of 0–37.5% solvent B (0.1% v ⁄ v trifluoroacetic acid in 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 maintained in RPMI-1640 medium (PAA Laboratories GmbH, Colbe, Germany) supplemented with 10% fetal bovine ă serum at 37 C in 5% CO2 To allow differentiation and induction of AP, the serum concentration was reduced to 2% 6394 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 were grown in six-well plates (10 cm2 per well) in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum Transfection with RotifectÔ (Roth, Karlsruhe, Germany) was performed according to the supplier’s instructions To each well, 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 buffer W (50 mm Hepes pH 7.5, 128 mm NaCl, 1.2 mm CaCl2, mm MgSO4, mm KCl, mgỈmL)1 BSA) Subsequently, 10 nm BMP-2 or proBMP-2 in buffer W were added and allowed to interact with surface receptors for h at °C Cross-linking was performed as described previously [47] After cross-linking, cells were solubilized in 100 lL lysis buffer per well [NaCl ⁄ Pi pH 7.4, 0.5% Triton X-100, mm EDTA and protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany)] at °C for 40 Cell extracts were analysed by western blotting or after immunoprecipitation Receptor complexes were immunoprecipitated using BMPR-IA ECD antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a ratio of lgỈmL)1 cell extract for h at °C Then 25 lL of the solution was added to 25 lL protein A–Sepharose slurry (GE Healthcare) in lysis buffer for h at °C After washing the beads three times with NaCl ⁄ Pi, SDS–PAGE sample buffer containing b-mercaptoethanol was added, and the samples were heated for at 95 °C All protein samples were run on 4–12% 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 incubated with 10 lgỈmL)1 HA antibody (Covance, Emeryville, CA, USA) or 0.4 lgỈmL)1 BMP-2 antibody (Dianova, Hamburg, Germany) Detection was achieved using horseradish peroxidase-coupled secondary antibody with the ECL system (GE Healthcare) Alkaline phosphatase (AP) assay and luciferase-based reporter gene assay C2C12 cells (2 · 103 per well) were seeded into 96-well 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 days, the medium was removed, cells were washed with NaCl ⁄ Pi and lysed in 100 lL lysis buffer [100 mm glycine ⁄ Na+, mm MgCl2, mm ZnCl2 pH 9.6, 1% Nonidet P-40 (Applichem, Darmstadt, Germany)] by gentle shaking at room temperature for 2–3 h 20 lL lysate was transferred to a new 96-well plate Then 200 lL substrate solution (9 mm FEBS Journal 276 (2009) 6386–6398 ª 2009 The Authors Journal compilation ª 2009 FEBS A Hauburger et al p-nitrophenyl phosphate in lysis buffer) was added per well [29] Changes in absorption at 405 nm were followed over 30 using an ELISA plate reader For determination of inhibition by noggin, the AP assay was slightly modified: · 104 cells were seeded and tests were performed after days with 100 lL substrate solution per well For the luciferase-based reporter gene assay, C2C12 cells were seeded into 48-well plates at a density of 104 cells per well under normal growth conditions After 24 h, cells were transfected with a construct containing firefly luciferase driven by the BMP response element (pBRE-Luc) [48] and a constitutive active Renilla luciferase (pRLTK) as an internal control using Lipofectamine 2000 (Invitrogen, Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions The next day, cells were starved for 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 were seeded in six-well plates (2 · 105 cells per well) After 24 h, cells were starved for 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 NaCl ⁄ Pi and lysed in 200 lL SDS–PAGE sample buffer (60 mm Tris ⁄ HCl pH 6.8, 2% SDS, 10% glycerol, 15% b-mercaptoethanol, 0.01% bromophenol blue) per well After boiling at 95 °C for min, protein was separated by SDS– 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 Membranes were washed three times for 10–15 in TBST For antibody staining, the primary antibodies (antiphospho-p38 and anti-phospho-Smad1 ⁄ ⁄ 8, Cell Signaling Technology Inc., Beverly, MA, USA) were diluted : 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 quantification, the maximum phosphorylation level was set to 100% Surface plasmon resonance Binding of BMP-2 and proBMP-2 to the immobilized proteins was examined using the BIA2000 system (BIAcore, Uppsala, Sweden) Biotinylated BMPR-IA ECD or noggin (sulfo-NHS-LC-biotin, Pierce ⁄ Thermo Fisher, Rockford, IL, USA) were immobilized on streptavidin-coated chips at a density of approximately 300 resonance units for the ECD or The pro form of BMP-2 interferes with BMP-2 signalling 64 resonance units for noggin For BMPR-II–Fc, 1000 resonance 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 concentrations Sensorgrams were recorded at a flow rate of 30 lLỈmin)1 at 25 °C Regeneration of the ECD-coated chip surface was achieved by perfusion with 10 mm glycine pH 2, and regeneration of the noggin-coupled chip surface by m guanidinium chloride ⁄ acetic acid pH 2.0, m NaCl Correction for background signals was performed by subtraction 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 : interaction Although one BMP-2 dimer is known to interact with two ECD monomers [42], a : interaction has been used previously for the assessment of relative binding affinities [29] Association and dissociation rate constants were obtained from 7–9 analyte concentrations Mean values with a standard deviation were deduced from five independent measurements Apparent dissociation constants, KD, were calculated as KD = kd ⁄ ka 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 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 equilibrium 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-ImmunoÔ Maxisorp, Nunc, Wiesbaden, Germany) at room temperature Washing was performed with 200 lL 10 mm NaCl ⁄ Pi pH 7.4 Wells were coated with 100 lL of either 100 nm BMP-2 or 100 nm proBMP-2 in 50 mm carbonate buffer, pH 8, at °C overnight Free sites were blocked for h at room temperature using 200 lL blocking buffer per well [5% BSA (Sigma), 10 mm NaCl ⁄ Pi pH 7.4, 0.05% Tween-20] After washing, the BMPR-IA ECD was added at the indicated concentrations in blocking buffer and incubated for 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 0.2 lgỈmL)1 Detection was performed using goat IgG horseradish peroxidase-conjugated antibody (Perbio Science, Bonn, Germany) at a concentration of 0.4 lgỈmL)1 in blocking buffer Photometric detection was achieved using 0.5 mgặmL)1 2,2Â-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS; Roche Diagnostics) in ABTS buffer FEBS Journal 276 (2009) 6386–6398 ª 2009 The Authors Journal compilation ª 2009 FEBS 6395 The pro form of BMP-2 interferes with BMP-2 signalling A Hauburger et al Changes of absorption at 405 nm were followed every 30 s for h using a microtitre plate reader (MR 7000, Dynex Technologies, Denkendorf, Germany), and the slopes were calculated Acknowledgements This work was funded by the Land Sachsen-Anhalt through the program ‘Structures and Mechanisms of Biological 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characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter J Biol Chem 277, 4883–4891 49 Kanzaki S, Takahashi T, Kanno T, Ariyoshi W, Shinmyouzu K, Tujisawa T & Nishihara T (2008) Heparin inhibits BMP-2 osteogenic bioactivity by binding to both BMP-2 and BMP receptor J Cell Physiol 216, 844–850 50 Wendler J, Hoffmann A, Gross G, Weich HA & Bilitewski U (2005) Development of an enzyme-linked immunoreceptor assay (ELIRA) for quantification of the biological activity of recombinant human bone morphogenetic protein-2 J Biotechnol 119, 425–435 6398 Supporting information The following supplementary material is available: Fig S1 The pro-peptide does not cause AP induction Fig S2 ProBMP-2 is maintained stably in cell culture over 49 h Fig S3 At high concentrations, proBMP-2 induces immediate Smad phosphorylation This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 276 (2009) 6386–6398 ª 2009 The Authors Journal compilation ª 2009 FEBS ... competition of proBMP-2 with BMP-2 for binding to the main receptor BMPR -IA, BIAcore experiments were performed For these studies, the ECD of the receptor was recombinantly produced in Escherichia coli... 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 propeptide... 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