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• W2739264605 abstract "Bone morphogenetic proteins (BMPs) regulate diverse cellular responses during embryogenesis and in adulthood including cell differentiation, proliferation, and death in various tissues. In the adult pituitary, BMPs participate in the control of hormone secretion and cell proliferation, suggesting a potential endocrine/paracrine role for BMPs, but some of the mechanisms are unclear. Here, using a bioactivity test based on embryonic cells (C3H10T1/2) transfected with a BMP-responsive element, we sought to determine whether pituitary cells secrete BMPs or BMP antagonists. Interestingly, we found that pituitary-conditioned medium contains a factor that inhibits action of BMP-2 and -4. Combining surface plasmon resonance and high-resolution mass spectrometry helped pinpoint this factor as thrombospondin-1 (TSP-1). Surface plasmon resonance and co-immunoprecipitation confirmed that recombinant human TSP-1 can bind BMP-2 and -4 and antagonize their effects on C3H10T1/2 cells. Moreover, TSP-1 inhibited the action of serum BMPs. We also report that the von Willebrand type C domain of TSP-1 is likely responsible for this BMP-2/4-binding activity, an assertion based on sequence similarity that TSP-1 shares with the von Willebrand type C domain of Crossveinless 2 (CV-2), a BMP antagonist and member of the chordin family. In summary, we identified for the first time TSP-1 as a BMP-2/-4 antagonist and presented a structural basis for the physical interaction between TSP-1 and BMP-4. We propose that TSP-1 could regulate bioavailability of BMPs, either produced locally or reaching the pituitary via blood circulation. In conclusion, our findings provide new insights into the involvement of TSP-1 in the BMP-2/-4 mechanisms of action. Bone morphogenetic proteins (BMPs) regulate diverse cellular responses during embryogenesis and in adulthood including cell differentiation, proliferation, and death in various tissues. In the adult pituitary, BMPs participate in the control of hormone secretion and cell proliferation, suggesting a potential endocrine/paracrine role for BMPs, but some of the mechanisms are unclear. Here, using a bioactivity test based on embryonic cells (C3H10T1/2) transfected with a BMP-responsive element, we sought to determine whether pituitary cells secrete BMPs or BMP antagonists. Interestingly, we found that pituitary-conditioned medium contains a factor that inhibits action of BMP-2 and -4. Combining surface plasmon resonance and high-resolution mass spectrometry helped pinpoint this factor as thrombospondin-1 (TSP-1). Surface plasmon resonance and co-immunoprecipitation confirmed that recombinant human TSP-1 can bind BMP-2 and -4 and antagonize their effects on C3H10T1/2 cells. Moreover, TSP-1 inhibited the action of serum BMPs. We also report that the von Willebrand type C domain of TSP-1 is likely responsible for this BMP-2/4-binding activity, an assertion based on sequence similarity that TSP-1 shares with the von Willebrand type C domain of Crossveinless 2 (CV-2), a BMP antagonist and member of the chordin family. In summary, we identified for the first time TSP-1 as a BMP-2/-4 antagonist and presented a structural basis for the physical interaction between TSP-1 and BMP-4. We propose that TSP-1 could regulate bioavailability of BMPs, either produced locally or reaching the pituitary via blood circulation. In conclusion, our findings provide new insights into the involvement of TSP-1 in the BMP-2/-4 mechanisms of action. Bone morphogenetic proteins (BMPs), 3The abbreviations used are: BMPbone morphogenetic proteinTGFβtransforming growth factor βGnRHgonadotropin releasing hormoneCMconditioned mediaALKactivin receptor-like kinaseBMPR-IBMP receptor type IBMPR-IIBMP receptor type IIActR-IIactivin receptor type IIBREBMP-responsive elementLucluciferase reporterSPRsurface plasmon resonanceTSP-1thrombospondin 1rhrecombinant humanVWCvon Willebrand type C domainCV-2Crossveinless 2RUresonance unit. members of the transforming growth factor β (TGFβ) superfamily, were originally identified by their ability to induce endochondral bone formation (1Wozney J.M. Rosen V. Celeste A.J. Mitsock L.M. Whitters M.J. Kriz R.W. Hewick R.M. Wang E.A. Novel regulators of bone formation: molecular clones and activities.Science. 1988; 242: 1528-1534Crossref PubMed Scopus (3349) Google Scholar, 2Chen D. Zhao M. Mundy G.R. Bone morphogenetic proteins.Growth Factors. 2004; 22: 233-241Crossref PubMed Scopus (1713) Google Scholar). They are now known to regulate diverse cellular responses during embryogenesis and in adulthood including cell differentiation, proliferation, and death in various tissues. The BMP system appears as a critical component of the local regulation in several endocrine tissues including the ovary, pituitary, hypothalamus, and adrenal (3Otsuka F. Multiple endocrine regulation by bone morphogenetic protein system.Endocr. J. 2010; 57: 3-14Crossref PubMed Scopus (57) Google Scholar, 4Otsuka F. Multifunctional bone morphogenetic protein system in endocrinology.Acta Med. Okayama. 2013; 67: 75-86PubMed Google Scholar). At the pituitary level, BMPs not only govern organogenesis but also participate to the control of hormone secretion and/or cell proliferation in different differentiated cell types like lactotropes (5Paez-Pereda M. Giacomini D. Refojo D. Nagashima A.C. Hopfner U. Grubler Y. Chervin A. Goldberg V. Goya R. Hentges S.T. Low M.J. Holsboer F. Stalla G.K. Arzt E. Involvement of bone morphogenetic protein 4 (BMP-4) in pituitary prolactinoma pathogenesis through a SMAD/estrogen receptor crosstalk.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 1034-1039Crossref PubMed Scopus (155) Google Scholar, 6Giacomini D. Páez-Pereda M. Stalla J. Stalla G.K. Arzt E. Molecular interaction of BMP-4, TGF-β, and estrogens in lactotrophs: impact on the PRL promoter.Mol. Endocrinol. 2009; 23: 1102-1114Crossref PubMed Scopus (40) Google Scholar), corticotropes (7Nudi M. Ouimette J.F. Drouin J. Bone morphogenic protein (SMAD)-mediated repression of proopiomelanocortin transcription by interference with Pitx/Tpit activity.Mol. Endocrinol. 2005; 19: 1329-1342Crossref PubMed Scopus (47) Google Scholar, 8Giacomini D. Páez-Pereda M. Theodoropoulou M. Labeur M. Refojo D. Gerez J. Chervin A. Berner S. Losa M. Buchfelder M. Renner U. Stalla G.K. Arzt E. Bone morphogenetic protein-4 inhibits corticotroph tumor cells: involvement in the retinoic acid inhibitory action.Endocrinology. 2006; 147: 247-256Crossref PubMed Scopus (72) Google Scholar), and gonadotropes (9Huang H.J. Wu J.C. Su P. Zhirnov O. Miller W.L. A novel role for bone morphogenetic proteins in the synthesis of follicle-stimulating hormone.Endocrinology. 2001; 142: 2275-2283Crossref PubMed Scopus (0) Google Scholar, 10Faure M.O. Nicol L. Fabre S. Fontaine J. Mohoric N. McNeilly A. Taragnat C. BMP-4 inhibits follicle-stimulating hormone secretion in ewe pituitary.J Endocrinol. 2005; 186: 109-121Crossref PubMed Scopus (85) Google Scholar). In these latter cells BMPs as well as activins, other members of the TGFβ superfamily, participate to the regulation of follicle-stimulating hormone (FSH) synthesis and release in addition to the gonadotropin-releasing hormone (GnRH) and the gonadal steroids. Whereas activins are potent stimulators of FSH secretion (11Carroll R.S. Kowash P.M. Lofgren J.A. Schwall R.H. Chin W.W. In vivo regulation of FSH synthesis by inhibin and activin.Endocrinology. 1991; 129: 3299-3304Crossref PubMed Scopus (96) Google Scholar, 12Weiss J. Harris P.E. Halvorson L.M. Crowley Jr., W.F. Jameson J.L. Dynamic regulation of follicle-stimulating hormone-β messenger ribonucleic acid levels by activin and gonadotropin-releasing hormone in perifused rat pituitary cells.Endocrinology. 1992; 131: 1403-1408Crossref PubMed Scopus (0) Google Scholar13Weiss J. Guendner M.J. Halvorson L.M. Jameson J.L. Transcriptional activation of the follicle-stimulating hormone β-subunit gene by activin.Endocrinology. 1995; 136: 1885-1891Crossref PubMed Google Scholar), BMPs modulate FSH secretion with a species-dependent effect. In rat pituitary cells and murine LβT2 gonadotrope cell lines, recombinant human (rh) BMP-6 and BMP-7 at high concentrations or rhBMP-2 and rhBMP15 at lower concentrations stimulate basal FSH secretion and FSHβ promoter activity (9Huang H.J. Wu J.C. Su P. Zhirnov O. Miller W.L. A novel role for bone morphogenetic proteins in the synthesis of follicle-stimulating hormone.Endocrinology. 2001; 142: 2275-2283Crossref PubMed Scopus (0) Google Scholar, 14Lee K.B. Khivansara V. Santos M.M. Lamba P. Yuen T. Sealfon S.C. Bernard D.J. Bone morphogenetic protein 2 and activin A synergistically stimulate follicle-stimulating hormone β subunit transcription.J Mol Endocrinol. 2007; 38: 315-330Crossref PubMed Scopus (50) Google Scholar, 15Otsuka F. Shimasaki S. A novel function of bone morphogenetic protein-15 in the pituitary: selective synthesis and secretion of FSH by gonadotropes.Endocrinology. 2002; 143: 4938-4941Crossref PubMed Scopus (125) Google Scholar). Moreover, rhBMP-4 increased the release of FSH in response to activin and activin plus GnRH (16Nicol L. Faure M.O. McNeilly J.R. Fontaine J. Taragnat C. McNeilly A.S. Bone morphogenetic protein-4 interacts with activin and GnRH to modulate gonadotrophin secretion in LβT2 gonadotrophs.J. Endocrinol. 2008; 196: 497-507Crossref PubMed Scopus (39) Google Scholar). In contrast, in ovine pituitary cells, rhBMP-4 inhibits FSH secretion and antagonizes the effects of activin (10Faure M.O. Nicol L. Fabre S. Fontaine J. Mohoric N. McNeilly A. Taragnat C. BMP-4 inhibits follicle-stimulating hormone secretion in ewe pituitary.J Endocrinol. 2005; 186: 109-121Crossref PubMed Scopus (85) Google Scholar, 17Young J.M. Juengel J.L. Dodds K.G. Laird M. Dearden P.K. McNeilly A.S. McNatty K.P. Wilson T. The activin receptor-like kinase 6 Booroola mutation enhances suppressive effects of bone morphogenetic protein 2 (BMP-2), BMP-4, BMP-6 and growth and differentiation factor-9 on FSH release from ovine primary pituitary cell cultures.J. Endocrinol. 2008; 196: 251-261Crossref PubMed Scopus (25) Google Scholar). Concerning lactotropes and corticotropes, previous studies reported that BMP-4 induces prolactin secretion in lactotropes (6Giacomini D. Páez-Pereda M. Stalla J. Stalla G.K. Arzt E. Molecular interaction of BMP-4, TGF-β, and estrogens in lactotrophs: impact on the PRL promoter.Mol. Endocrinol. 2009; 23: 1102-1114Crossref PubMed Scopus (40) Google Scholar, 18Tsukamoto N. Otsuka F. Miyoshi T. Inagaki K. Nakamura E. Suzuki J. Ogura T. Iwasaki Y. Makino H. Activities of bone morphogenetic proteins in prolactin regulation by somatostatin analogs in rat pituitary GH3 cells.Mol. Cell. Endocrinol. 2011; 332: 163-169Crossref PubMed Scopus (12) Google Scholar) meanwhile inhibiting ACTH secretion in corticotropes (7Nudi M. Ouimette J.F. Drouin J. Bone morphogenic protein (SMAD)-mediated repression of proopiomelanocortin transcription by interference with Pitx/Tpit activity.Mol. Endocrinol. 2005; 19: 1329-1342Crossref PubMed Scopus (47) Google Scholar, 8Giacomini D. Páez-Pereda M. Theodoropoulou M. Labeur M. Refojo D. Gerez J. Chervin A. Berner S. Losa M. Buchfelder M. Renner U. Stalla G.K. Arzt E. Bone morphogenetic protein-4 inhibits corticotroph tumor cells: involvement in the retinoic acid inhibitory action.Endocrinology. 2006; 147: 247-256Crossref PubMed Scopus (72) Google Scholar). Moreover, recent studies also revealed a role of BMP-4 in the pituitary pathogenesis. It promotes pituitary prolactinoma while it inhibits corticotrope pathogenesis in Cushing's disease (5Paez-Pereda M. Giacomini D. Refojo D. Nagashima A.C. Hopfner U. Grubler Y. Chervin A. Goldberg V. Goya R. Hentges S.T. Low M.J. Holsboer F. Stalla G.K. Arzt E. Involvement of bone morphogenetic protein 4 (BMP-4) in pituitary prolactinoma pathogenesis through a SMAD/estrogen receptor crosstalk.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 1034-1039Crossref PubMed Scopus (155) Google Scholar, 8Giacomini D. Páez-Pereda M. Theodoropoulou M. Labeur M. Refojo D. Gerez J. Chervin A. Berner S. Losa M. Buchfelder M. Renner U. Stalla G.K. Arzt E. Bone morphogenetic protein-4 inhibits corticotroph tumor cells: involvement in the retinoic acid inhibitory action.Endocrinology. 2006; 147: 247-256Crossref PubMed Scopus (72) Google Scholar). bone morphogenetic protein transforming growth factor β gonadotropin releasing hormone conditioned media activin receptor-like kinase BMP receptor type I BMP receptor type II activin receptor type II BMP-responsive element luciferase reporter surface plasmon resonance thrombospondin 1 recombinant human von Willebrand type C domain Crossveinless 2 resonance unit. BMP signaling occurs through heteromeric receptor complexes composed of type 1 and type 2 transmembrane serine/threonine kinase receptors. Three type 1 receptors are known to bind BMPs, which include the activin-receptor-like kinase 2 (ALK2), ALK3 (also known as BMPR-IA), and ALK6 (BMPR-IB) (19Kawabata M. Imamura T. Miyazono K. Signal transduction by bone morphogenetic proteins.Cytokine Growth Factor Rev. 1998; 9: 49-61Crossref PubMed Scopus (455) Google Scholar, 20Miyazono K. Kusanagi K. Inoue H. Divergence and convergence of TGF-β/BMP signaling.J. Cell. Physiol. 2001; 187: 265-276Crossref PubMed Scopus (455) Google Scholar). Similarly, three type 2 receptors possess binding affinity for BMPs, including BMPR-II, activin type IIA receptor (ActR-IIA), and ActR-IIB. The ligand binding induces the trans-phosphorylation of the type 1 receptor by the type 2 receptor. Consequently, the activated BMPR-I phosphorylates intracellular receptor-activated SMADs, SMAD1, SMAD5, and SMAD8, which then interacts with SMAD4 (Co-SMAD). The receptor-activated SMAD–Co-SMAD complex translocates to the nucleus and acts as transcription factors, either activating or repressing gene expression (21Zwijsen A. Verschueren K. Huylebroeck D. New intracellular components of bone morphogenetic protein/SMAD signaling cascades.FEBS Lett. 2003; 546: 133-139Crossref PubMed Scopus (95) Google Scholar). BMP signaling is modulated by several extra- and intracellular modulators acting at multiple levels. A large number of extracellular antagonists, such as noggin, the chordin family, gremlin, twisted gastrulation protein, and the Dan family (22Vitt U.A. Hsu S.Y. Hsueh A.J. Evolution and classification of cystine knot-containing hormones and related extracellular signaling molecules.Mol. Endocrinol. 2001; 15: 681-694Crossref PubMed Scopus (0) Google Scholar, 23Avsian-Kretchmer O. Hsueh A.J. Comparative genomic analysis of the eight-membered ring cystine knot-containing bone morphogenetic protein antagonists.Mol. Endocrinol. 2004; 18: 1-12Crossref PubMed Scopus (192) Google Scholar) bind BMPs and block their interaction with the receptors, thus inhibiting BMP signaling (24Yanagita M. BMP antagonists: their roles in development and involvement in pathophysiology.Cytokine Growth Factor Rev. 2005; 16: 309-317Crossref PubMed Scopus (126) Google Scholar). Moreover, non-signaling membrane pseudoreceptors or intracellular inhibitory SMADs are also able to block the BMP signaling (25Gazzerro E. Canalis E. Bone morphogenetic proteins and their antagonists.Rev. Endocr. Metab. Disord. 2006; 7: 51-65Crossref PubMed Scopus (284) Google Scholar). Several BMP mRNAs are present in pituitary. For example, BMP-2, BMP-4, BMP-7, and GDF9 (growth and differentiation factor 9) mRNAs were detected in ewe pituitary (10Faure M.O. Nicol L. Fabre S. Fontaine J. Mohoric N. McNeilly A. Taragnat C. BMP-4 inhibits follicle-stimulating hormone secretion in ewe pituitary.J Endocrinol. 2005; 186: 109-121Crossref PubMed Scopus (85) Google Scholar, 26Sallon C. Faure M.O. Fontaine J. Taragnat C. Dynamic regulation of pituitary mRNAs for BMP-4, BMP receptors and activin/inhibin subunits in the ewe during the oestrous cycle and in cultured pituitary cells.J. Endocrinol. 2010; 207: 55-65Crossref PubMed Scopus (8) Google Scholar). Furthermore, ALK3, ALK6, and BMPR-II are found on different cell types including gonadotropes and corticotropes (10Faure M.O. Nicol L. Fabre S. Fontaine J. Mohoric N. McNeilly A. Taragnat C. BMP-4 inhibits follicle-stimulating hormone secretion in ewe pituitary.J Endocrinol. 2005; 186: 109-121Crossref PubMed Scopus (85) Google Scholar). These data suggest that pituitary BMPs can exert paracrine/autocrine actions on hormone synthesis and release. Alternatively, BMPs can act as endocrine factors supplied by the blood. Indeed, BMP-4, BMP-6, and BMP-9 were found in bovine serum (27Herrera B. Inman G.J. A rapid and sensitive bioassay for the simultaneous measurement of multiple bone morphogenetic proteins. Identification and quantification of BMP-4, BMP-6 and BMP9 in bovine and human serum.BMC Cell Biol. 2009; 10: 20Crossref PubMed Scopus (112) Google Scholar), suggesting a potential endocrine role for BMPs at pituitary level. In this context the first aim of the present study was to investigate whether pituitary cells produced BMPs and/or BMP inhibitors as well as whether the serum conveys BMPs. In the absence of available antibodies and protein assays for ovine BMPs, a sensitive in vitro bioassay based on mouse C3H-B12 cells was used. These cells are mesenchymal embryonic C3H10T1/2 cells stably transfected with an expression construct (BRE-Luc) containing a BMP-responsive element fused to firefly luciferase reporter gene (28Logeart-Avramoglou D. Bourguignon M. Oudina K. Ten Dijke P. Petite H. An assay for the determination of biologically active bone morphogenetic proteins using cells transfected with an inhibitor of differentiation promoter-luciferase construct.Anal. Biochem. 2006; 349: 78-86Crossref PubMed Scopus (46) Google Scholar). This assay presents the advantage to monitor the bioactivity of the protein and is not isoform-specific. It can also allow to detect factors that inhibit BMP action. Interestingly, we found that pituitary cell-conditioned media exhibited an inhibitory activity for BMP-induced luciferase activity. We then conducted the identification of the putative inhibitory factor combining surface plasmon resonance and high resolution tandem mass spectrometry. Last, based on sequence and structure analysis, we provide insights into the molecular basis of interaction between BMP-4 and this inhibitor. First, the BMP effect on the BRE-Luc construct was determined by treating C3H-B12 cells with increasing concentrations of BMP-2, BMP-4, BMP-6, or BMP-7 (0–50 ng/ml) overnight and monitoring changes in the luciferase activity. BMPs stimulated luciferase activity in a dose-dependent manner (Fig. 1A). BMP-4 was the most potent inducer of luciferase activity with an ED50 of 1.2 ng/ml and detection threshold of 0.25 ng/ml, whereas BMP-7 was the least potent inducer with a detection threshold of 25 ng/ml. To determine whether CM from ovine pituitary cells exhibited BMP activity, C3H-B12 cells were exposed to CM from cultured pituitary cells, which were treated or not with 10−8 m GnRH for 6 h (CM GnRH 6 h) or with either 10−9 m activin for 48 h (CM activin 48 h). Luciferase activity from C3H-B12 was not modified compared with C3H-B12 cells exposed to Dulbecco's modified Eagle's medium (DMEM-0.1% bovine serum albumin (BSA) i.e. non-conditioned media; Fig. 1B). This result suggests that conditioned media exhibits no or low amounts of bioactive BMP whatever the treatment and the incubation period of the pituitary cells. To evaluate the potential presence of BMP inhibitors in CM, we supplemented pituitary cell media conditioned for 48 h with increasing doses of BMP-4 and incubated them with C3H-B12 cells. A dose-dependent increase in the luciferase activity was observed (Fig. 1C, gray spots). However, this increase was impaired relative to that obtained with DMEM, 0.1% BSA supplemented with similar doses of BMP-4 (Fig. 1C, black spots). A dose of 50 ng/ml BMP-4 in CM was necessary to recover full activity of BMP-4 on C3H-B12 cells compared with 5 ng/ml in DMEM, 0.1% BSA (Fig. 1C). These results suggest that an inhibitory activity of BMP action is present in CM. Moreover, the inhibitory activity of BMP action was more elevated in the medium conditioned for 48 h (CM basal 48 h) than for 6 h (CM basal 6 h) (72% of inhibition and 17%, respectively, versus DMEM + BMP-4) (Fig. 1D). As comparison, we analyzed CM basal 48 h from adrenocortical cells cultured in the same conditions as the pituitary cells. The C3H-B12 cell luciferase activity induced by BMP-4 (10 ng/ml) supplemented in adrenal cell CM was similar to that of BMP-4 diluted in DMEM, 0.1% BSA indicating that the CM from adrenal cells did not exhibit a detectable inhibitory activity of BMP action (Fig. 1D, inset). To determine whether FSH regulatory factors, such as GnRH and activin, were capable of modulating the production of the inhibitory activity, CM from pituitary cells treated with these factors in dose and time conditions known to affect FSH secretion were supplemented with 10 ng/ml rhBMP-4 and incubated with C3H-B12 cells overnight. In the presence of BMP-4, CM from pituitary cells treated with GnRH for 6 h (CM GnRH 6 h) impaired the increase in luciferase activity of C3H-B12 cells versus DMEM + BMP-4 (77% inhibition) (p < 0.01) more than did CM from non-treated cells (CM basal 6 h) (17% inhibition) (p > 0.05) (Fig. 1D). This suggests that GnRH increased the CM inhibitory activity on BMP action. When the CM from pituitary cells treated with activin for 48 h (CM activin 48 h) were added with BMP-4, they tended to impair the increase in luciferase activity versus DMEM + BMP-4 more than did CM from non treated cells (CM basal 48 h) (83% of inhibition versus 72%), although the difference was not statistically different (Fig. 1D). The treatment of C3H-B12 cells with pituitary CM, BMP-4, GnRH, or activin did not affect cell proliferation compared with DMEM, 0.1% BSA (Fig. 1E). Moreover, we tested the ability of the pituitary CM to decrease the effect of another BMP, BMP-2. Fig. 1F shows that luciferase activity was impaired when BMP-2 was added to CM conditioned for 48 h compared with the addition in DMEM, similarly to the effect observed with BMP-4. To explore the hypothesis that the CM factor(s) responsible for the inhibition of BMP action can be the BMP-4-binding protein(s), interaction between conditioned media and BMP-4 was analyzed using surface plasmon resonance (Biacore). The injection of CM (1/10 diluted) resulted in binding to high density immobilized rhBMP-4, whereas the injection of DMEM, 0.1% BSA led to a low nonspecific binding signal (Fig. 2). Moreover, the interaction signal was more elevated with media conditioned for 48 h compared with media conditioned for 6 h. To concentrate the binding factor and eliminate small molecules, the CM volumes were 10-fold reduced using high molecular mass polyethylene glycol (PEG) dialysis. The concentrated media exhibited an increased interaction signal compared with crude CM (Fig. 2). Collectively, these results demonstrated that an interaction occurs between pituitary CM and BMP-4. Note that the differences in interaction signal observed between media conditioned for 6 h and 48 h are consistent with the changes observed in the biological effect of the corresponding CM on CH3-B12 cells (Fig. 1D). The CM fraction bound to BMP-4 on CM5 sensorchip was eluted and analyzed by on-line nanoflow liquid chromatography tandem mass spectrometry after tryptic digestion. The only three detectable peptides allowed the identification of the predicted thrombospondin-1 isoform 1 (TSP-1) (Table 1), a 450-kDa secreted homotrimeric protein that regulates a wide range of functions (29Myszka D.G. Improving biosensor analysis.J. Mol. Recognit. 1999; 12: 279-284Crossref PubMed Scopus (651) Google Scholar). These peptides were not detected when elution was performed after injection of DMEM, 0.1% BSA on CM5 sensorchip instead of conditioned media. These results demonstrated that BMP-4 chip acts as a specific and efficient affinity separation method.Table 1Protein and peptides identified by tandem mass spectrometry in the fraction from CM bound to BMP-4 coated CM5 sensorchipIdentified peptidesProtein descriptionAccession number (nrNCBI)TaxonomyGeneTheoretical molecular weightTGDEWTVDSCTECRPredicted thrombospondin-1gi 426232958Ovis ariesTSP-1129 kDaCENTDPGYNCLPCPPRQVTQSYWDTNPTR Open table in a new tab To confirm the interaction between TSP-1 and BMP-4, surface plasmon resonance analysis was conducted. The injection of rhTSP-1 on low density BMP-4-immobilized CM5 sensorchip led to dose-dependent binding (Fig. 3A). To determine affinity data, we used the method of steady-state analysis. The equilibrium dissociation constant (KD) for the interaction was calculated at 10−8 m in two independent experiments. BMP-4 binding to TSP-1 was further assayed using TSP-1-immobilized CM5 sensorchip. As expected, the injection of BMP-4 resulted in a dose-dependent binding signal (Fig. 3B). BMP-2 binding to TSP-1 was also studied on TSP-1-immobilized CM5 sensorchip. Dose-dependent binding signal was observed (Fig. 3C) with a kinetics slightly different from that of BMP-4. The dissociation constants (KD) were: 5.95 × 10−7 m for BMP-4 and 1.26 × 10−7 for BMP-2. Evaluation of the kinetic parameters showed faster association and dissociation rates for BMP-2 (Table 2).Table 2Kinetic parameters of the interaction between BMP-4 and TSP-1 analysed by surface plasmon resonanceInteracting proteinskakdKDRmaxχ2m−1 s−1s−1mRU2BMP4-TSP16.9 × 1040.0375.95 × 10−7421.762.58BMP2-TSP11.23 × 1060.1541.26 × 10−7609.84.52 Open table in a new tab In contrast, no interaction between TSP-1 and activin, chosen as control, was observed (Fig. 3D). To add more experimental data showing the interaction between BMP-4 and TSP-1, co-immunoprecipitation experiments were performed. When BMP-4 and TSP-1 are incubated together, precipitation with anti-BMP-4 antibody co-precipitates TSP-1 as detected by Western blot analysis (Fig. 3E, fourth lane). In contrast, when the anti-BMP-4 antibody was omitted or replaced by a nonspecific IgG, TSP-1 was not detected (Fig. 3E, second and third lanes, respectively). These observations confirm the interaction between BMP-4 and TSP-1. Furthermore, to assess the biological relevance of the BMP-4 and TSP-1 interaction, we studied the ability of TSP-1 to block BMP-4 or BMP-2 action. As shown in Fig. 4, A and B, incubation of TSP-1 with BMP-4 or BMP-2 led to antagonize BMP action as demonstrated in CH3-B12 cell bioassay where rhTSP-1 inhibited the activity of BMP-4 or BMP-2 (2.5 ng/ml, i.e. 10−10 m) on luciferase transcription in a dose-dependent manner. Dose-response curves were fitted using the Hill equation (Fig. 4, E and F). Half-maximal inhibitory concentration (IC50) values were estimated at 11.4 ± 6.3 and 8.5 ± 3.9 nm, respectively, with Hill slope values of 0.36 ± 0.1 and 0.61 ± 0.13. These values are in agreement with a negative cooperative interaction. TSP-1 alone did not affect luciferase transcription (Fig. 4A). As a comparison, noggin, a well-known BMP-2/4-binding protein, inhibited the activity of BMP-4 as well as BMP-2 (2.5 ng/ml, i.e. 10−10 m) (Fig. 4, C and D). When the data were fitted with the Hill equation, estimated IC50 values were 0.023 ± 0.001 and 0.021 ± 0.003 nm, respectively, with Hill slope values of 7.4 ± 0.6 and 6.9 ± 0.8 (Fig. 4, E and F). These values suggest a positive cooperative interaction. To confirm our findings from mass spectrometry identifying TSP-1 as a factor present in pituitary cell CM, the expression of its mRNA was analyzed by RT-PCR in cells collected at the time of CM recovery (48 h). Fig. 5A shows the presence of a single PCR product at the expected size for TSP-1 mRNA, i.e. 98 bp, whereas no product was detected when reverse transcriptase was omitted. Western blot analysis confirmed the presence of the protein in CM with an apparent Mr of 150 or 450 in reducing or non-reducing conditions, respectively, corresponding to TSP-1 (Fig. 5B). As expected, the level of TSP-1 was increased in CM 48 h compared with CM 6 h. This result is consistent with the increase of the inhibition of BMP-4 action on luciferase activity from B12 cells observed with media from pituitary cells conditioned for 48 h compared with 6 h (Fig. 1D). TSP-1 level also tended to increase in the presence of CM from pituitary cells treated with GnRH for 6 h compared with CM basal 6 h, although the difference was not significant (p < 0.1, Fig. 5B). To validate that a high molecular mass factor like ovine TSP-1 present in pituitary CM is able to bind BMP-4 and inhibit its action, conditioned media were fractionated using 100-kDa cut-off membranes. The presence of TSP-1 in the retentate (CM >100 kDa) but not in the filtrate (CM <100 kDa) was confirmed by Western blotting (Fig. 5C). Fig. 5D shows that retentate (CM >100 kDa), but not filtrate (CM <100 kDa), bound to BMP-4-immobilized sensorchip. Consistent with this result is the inhibition of BMP-4 (10 ng/ml) action observed with the retentate but not with the filtrate on luciferase transcription in CH3-B12 cells (Fig. 5E). The above data showed that pituitary cells produced TSP-1 and that TSP-1 was able to bind BMPs. We made the hypothesis that pituitary TSP-1 could bind BMPs reaching the pituitary through the blood. We first asked whether ovine serum contains BMPs. To address this question, C3H-B12 cells were exposed to dilutions of serum from adult ewes. Fig. 6A showed that serum induced a dose-dependent increase of luciferase activity in C3H-B12 cells, suggesting that BMP-like activity was present. Serum did not affect cell proliferation or morphology in our assay conditions when diluted ½ or more (data not shown). To test whether the increas" @default.
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• W2739264605 title "Thrombospondin-1 (TSP-1), a new bone morphogenetic protein-2 and -4 (BMP-2/4) antagonist identified in pituitary cells" @default.
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• W2739264605 doi "https://doi.org/10.1074/jbc.m116.736207" @default.
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