Osteogenic Protein-1 (OP-1) Expression and Processing in Chinese Hamster Ovary Cells: Isolation of a Soluble Complex Containing the Mature and Pro-Domains of OP-1

Jones, W. Kinzy; Richmond, Emilie A.; White, K.H.; Sasak, Halina; Kusmik, William F.; Smart, James L.; Oppermann, Hermann; Rueger, David C.; Tucker, Ronald F.

doi:10.3109/08977199409046919

Cited by 81 publications

(58 citation statements)

References 25 publications

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“…Consistent with published studies, the prodomain of BMP7 coimmunoprecipitated with mature BMP7 homodimers (Fig. 5, lane 9) (19). Also consistent with published studies (20), the S1 only cleaved prodomain from BMP4 S2G coimmunoprecipitated with mature BMP4 homodimers (lane 4) whereas the S1 and S2 cleaved prodomain from wild-type BMP4 did not (lane 3), thus providing internal positive and negative controls for these experiments.…”

Section: Bmp4 and Bmp7 Prodomains Remain Associated With Mature Bmp4/7supporting

confidence: 80%

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The prodomain of BMP4 is necessary and sufficient to generate stable BMP4/7 heterodimers with enhanced bioactivity in vivo

Neugebauer¹,

Kwon

Kim³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Bone morphogenetic proteins 4 and 7 (BMP4 and BMP7) are morphogens that signal as either homodimers or heterodimers to regulate embryonic development and adult homeostasis. BMP4/7 heterodimers exhibit markedly higher signaling activity than either homodimer, but the mechanism underlying the enhanced activity is unknown. BMPs are synthesized as inactive precursors that dimerize and are then cleaved to generate both the bioactive ligand and prodomain fragments, which lack signaling activity. Our study reveals a previously unknown requirement for the BMP4 prodomain in promoting heterodimer activity. We show that BMP4 and BMP7 precursor proteins preferentially or exclusively form heterodimers when coexpressed in vivo. In addition, we show that the BMP4 prodomain is both necessary and sufficient for generation of stable heterodimeric ligands with enhanced activity and can enable homodimers to signal in a context in which they normally lack activity. Our results suggest that intrinsic properties of the BMP4 prodomain contribute to the relative bioactivities of homodimers versus heterodimers in vivo. These findings have clinical implications for the use of BMPs as regenerative agents for the treatment of bone injury and disease.B one morphogenetic proteins (BMPs) are members of the TGFβ superfamily that were originally isolated as boneinducing morphogens and were subsequently found to play central roles during embryogenesis and in adult homeostasis (1). BMPs are clinically important therapeutic agents that are used to reverse bone loss caused by trauma, disease, and tumor resection (2). Their use as regenerative agents is limited, however, by their short half-life and low specific activity when implanted in vivo. Understanding how BMP activity is regulated is important for the development of more effective therapeutic agents for the treatment of bone injury and disease.BMPs bind to and activate a receptor complex consisting of type I and type II transmembrane serine/threonine kinases. Following ligand binding, activated receptors propagate their signal by phosphorylating one of the SMADs that is specific for the BMP pathway (SMAD1, -5, or -8). The phosphorylated Smads then form heterooligomers with the common Smad, Smad4, and this complex translocates into the nucleus where it binds to BMP response elements and activates transcription of target genes (1).BMPs are classified into subfamilies based on sequence homology. They signal as either homodimers, or as heterodimers from different subfamilies. For example, class I BMPs, which consist of BMP2 and BMP4, can heterodimerize with class II BMPs, consisting of BMP5-8 (3). Heterodimers composed of distinct BMP family members show a higher specific activity than do homodimers of either subunit. Homodimers of BMP2, -4, or -7, for example, can all induce bone formation, but heterodimers of BMP2 plus BMP7, or BMP4 plus BMP7 are significantly more potent (5-to 20-fold) than any of the homodimers in osteogenic differentiation assays (4-6). BMP2/7 and BMP4/7 heterodimers also sho...

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Section: Bmp4 and Bmp7 Prodomains Remain Associated With Mature Bmp4/7supporting

confidence: 80%

“…1A). BMP7 is cleaved at a single conserved furin site to generate a noncovalently associated prodomain/ligand complex (18,19). By contrast, BMP4 is sequentially cleaved at two sites: initially at a site adjacent to the ligand domain (the S1 site) and subsequently at an upstream site (the S2 site) within the prodomain (Fig.…”

Section: Significancementioning

confidence: 99%

The prodomain of BMP4 is necessary and sufficient to generate stable BMP4/7 heterodimers with enhanced bioactivity in vivo

Neugebauer¹,

Kwon

Kim³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…The 1.9 kb open-reading frame encodes a deduced protein of 440 amino acids. BMP7 is expressed in an inactive pro-form and proteolytic cleavage by Furin proteases is required to release the 139-amino acid mature peptide (Jones et al, 1994). For the generation of Bmp7 null alleles by gene targeting in ES cells different strategies have been used.…”

Section: Resultsmentioning

confidence: 99%

Generation and functional characterization of mice with a conditional BMP7 allele

Zouvelou

Passa²,

Segklia³

et al. 2009

Int. J. Dev. Biol.

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Bone Morphogenetic Proteins (BMPs) play multiple and important roles in embryonic development as well as in homeostasis and tissue repair in the adult. Bmp7 has been implicated in developmental disorders and in a variety of diseases, but functional studies to elucidate its role so far have been hampered, since mice deficient in BMP7 die around or just after birth. To facilitate such studies, we generated mice in which the Bmp7 gene has been rendered conditional-null by flanking its first coding exon with loxP sites. To this end, we adapted the two-loxP site strategy to Bacterial Homologous Recombination to create a Bacterial Artificial Chromosome-based vector for direct targeting in mouse embryonic stem cells. Functional analysis showed that in vivo, the conditional-null Bmp7 flx/flx mice are phenotypically wild type, whereas post Cre-mediated recombination, the resulting Bmp7 ∆/∆ mice are phenotypically null. Thus, this study validates the usefulness of the Bmp7 flx/flx mouse which in turn should empower in vivo studies aimed at elucidating the roles of Bmp7 in postnatal development, homeostasis and disease.

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“…Animals were left untreated for 3 weeks following surgery to await the development of osteopenia, and then divided into the following groups: (1) sham (n= 10), (2) OVX (n=10), (3) OVX + BMP-6 (10 μg/kg intravenously, 3 times per week) (n=10). Human mature BMP-6 was produced in CHO cells and purified as previously described [6,20]. Therapy started 3 weeks following OVX and continued for the next 9 weeks for bone densitometry measurements (n=15), or 4 weeks for gene expression analysis (n=15) when the animals were sacrificed by cervical dislocation.…”

Section: Animalsmentioning

confidence: 99%

“…Alkaline phosphatase (AP) activity was measured over a 28 day period at specific time points (6,10,14,19,23 and 28 days) as previously described [22] and expressed as product produced/min/μg protein. Adherent cell cultures from day 19 through day 28 were stained for the presence of minerals using the method of Von Kossa as previously described [4].…”

Section: Cell Culturementioning

confidence: 99%

BMP-6 exerts its osteoinductive effect through activation of IGF-I and EGF pathways

Grasser

Orlić

Borovečki

et al. 2007

International Orthopaedics (SICO

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We have recently shown that human recombinant BMP-6 (rhBMP-6), given systematically, can restore bone in animal models of osteoporosis. To further elucidate the underlying mechanisms of new bone formation following systemic application of BMPs, we conducted gene expression profiling experiments using bone samples of oophrectomised mice treated with BMP-6. Gene set enrichment analysis revealed enrichment of insulin-like growth factor-I and epidermal growth factor related pathways in animals treated with BMP-6. Significant upregulation of IGF-I and EGF expression in bones of BMP-6 treated mice was confirmed by quantitative PCR. To develop an in vitro model for evaluation of the effects of BMP-6 on cells of human origin, we cultured primary human osteoblasts. Treatment with rhBMP-6 accelerated cell differentiation as indicated by the formation of mineralised nodules by day 18 of culture versus 28-30 days in vehicle treated cultures. In addition, alkaline phosphatase gene expression and activity were dramatically increased upon BMP-6 treatment. Expression of IGF-I and EGF was upregulated in human osteoblast cells treated with BMP-6. These results collectively indicate that BMP-6 exerts its osteoinductive effect, at least in part, through IGF-I and EGF pathways, which can be observed both in a murine model of osteopenia and in human osteoblasts.Résumé Nous avons récemment pu mettre en évidence que la BMP-6 (rhBMP-6), administrée de façon systématique, pouvait améliorer la restauration du capital osseux de modèles animaux avec ostéoporose. Nous avons conduit une expérimentation utilisant des souris ovarieactomisées traitées par BMP-6. L'analyse a montré qu'il y avait un apport d'insulin like growth factor et d'épidermal growth factor chez les animaux traités par BMP-6. Pour développer un modèle in vitro nous avons étudié l'effet de la BMP-6 sur les cellules de type ostéoplasties d'origine humaine. Le traitement par BMP-6 accélère la différenciation cellulaire au 18ème jour alors que normalement cette différence est notée aux alentours du 28ème et 30ème jour. De plus, l'expression du gène de la phosphatase alkaline et l'activité sont augmentées par le traitement par la BMP-6, de même en ce qui concerne l'IGF-1 et l'EGF. Ces résultats nous permettent de penser que le BMP-6 a un effet ostéo conducteur notamment pour les pathologies intéressant IGF-1 et EGF. Nous avons observé ces effets dans un modèle animal avec ostéoplastie et sur les ostéoblastes humains.

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Osteogenic Protein-1 (OP-1) Expression and Processing in Chinese Hamster Ovary Cells: Isolation of a Soluble Complex Containing the Mature and Pro-Domains of OP-1

Cited by 81 publications

References 25 publications

The prodomain of BMP4 is necessary and sufficient to generate stable BMP4/7 heterodimers with enhanced bioactivity in vivo

The prodomain of BMP4 is necessary and sufficient to generate stable BMP4/7 heterodimers with enhanced bioactivity in vivo

Generation and functional characterization of mice with a conditional BMP7 allele

BMP-6 exerts its osteoinductive effect through activation of IGF-I and EGF pathways

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