Full-Length spp24, but Not Its 18.5-kDa Proteolytic Fragment, Inhibits Bone-Healing in a Rodent Model of Spine Fusion

Sintuu, Chananit; Simon, Robert R.; Miyazaki, Masashi; Morishita, Yuichiro; Hymanson, Henry J.; Taghavi, Cyrus E.; Brochmann, Elsa J.; Murray, Samuel S.; Wang, Jeffrey C.

doi:10.2106/jbjs.j.00081

Cited by 8 publications

(19 citation statements)

References 13 publications

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“…At the concentrations that we employed, roughly a 30-fold molar excess, Spp24 inhibited the TGF-β2 induced bone formation but Spp14.5 did not. These results are similar to our previously reported findings with respect to BMP-2 in which we demonstrated that full-length Spp24 more strenuously inhibited the activity of BMP than did the smaller forms of Spp24 [16,20]. In general, we propose that Spp24 binds to TGF-β2 and inhibits its activity through a mechanism of extracellular sequestration.…”

Section: Discussionsupporting

confidence: 92%

“…TGF-β induced bone formation was inhibited by FL-Spp24, but enhanced by Spp14.5. We also previously reported that FL-Spp24 inhibits BMP-2 and -7-stimulated bone formation, and C-terminal truncation reduces its inhibitory effects [19,20]. …”

Section: Discussionmentioning

confidence: 99%

“…At the highest dose of Spp (2.5 mg), Spp24 completely inhibited BMP-2 induced bone formation, whereas Spp18.1 and Spp16.0 inhibited bone formation by about 30% and Spp14.5 inhibited bone formation by about 70% [16]. In a rat model of BMP-2 enhanced spine fusion, Spp24 reduced “bone area” by a much greater degree than did “spp18.5” (Spp18.1) [20]. In that study the “manual palpation score” (0 to 7) was 7.0 for 10 μg of BMP-2 alone, 6.0 for BMP-2 plus spp18.5, and 1.5 for BMP-2 plus full-length Spp24 [20].…”

Section: Discussionmentioning

confidence: 99%

“…In a rat model of BMP-2 enhanced spine fusion, Spp24 reduced “bone area” by a much greater degree than did “spp18.5” (Spp18.1) [20]. In that study the “manual palpation score” (0 to 7) was 7.0 for 10 μg of BMP-2 alone, 6.0 for BMP-2 plus spp18.5, and 1.5 for BMP-2 plus full-length Spp24 [20]. Therefore, it appeared that Spp24 is a more potent inhibitor of BMP-2 activity than is Spp18.1…”

Section: Discussionmentioning

confidence: 99%

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Secreted Phosphoprotein 24 kD (Spp24) and Spp14 Affect TGF-β Induced Bone Formation Differently

Tian

et al. 2013

PLoS ONE

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Transforming growth factor-β (TGF-β) and bone morphogenetic proteins (BMPs) have opposing but complementary functions in directing bone growth, repair, and turnover. Both are found in the bone matrix. Proteins that bind to and affect the activity of these growth factors will determine the relative abundance of the growth factors and, therefore, regulate bone formation. Secreted phosphoprotein 24 kD (Spp24) is a bone matrix protein that has been demonstrated to bind to and affect the activity of BMPs. The arginine-rich carboxy terminus of Spp24 is proteolytically processed to produce three other predictable truncation products (Spp18.1, Spp16.0, and Spp14.5). In this work, we report that kinetic data obtained by surface plasmon resonance demonstrate that Spp24 and the three C-terminal truncation products all bind to TGF-β1 and TGF-β2 with a similar but somewhat less affinity than they bind BMP-2; that, as in the case of BMP-2, the full-length (FL) form of Spp24 binds TGF-β with greater affinity than do the truncation products; that FL-Spp24 inhibits TGF-β2 induced bone formation in vivo, but Spp14.5 does not; and that co-administration of FL-Spp24 or Spp14.5 with TGF-β2 in vivo is associated with a reduction in the amount of cartilage, relative to new bone, present at the site of injection. This finding is consistent with the observation that low-dose TGF-β administration in vivo is associated with greater bone formation than high-dose TGF-β administration, and suggests that one function of Spp24 and its truncation products is to down-regulate local TGF-β activity or availability during bone growth and development. The similarities and differences of the interactions between Spp24 proteins and TGF-β compared to the interaction of the Spp24 proteins and BMPs have significant implications with respect to the regulation of bone metabolism and with respect to engineering therapeutic proteins for skeletal disorders.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Secreted Phosphoprotein 24 kD (Spp24) and Spp14 Affect TGF-β Induced Bone Formation Differently

Tian

et al. 2013

PLoS ONE

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“…After sacrifice, the spines were explanted from each animal and subjected to manual assessment as we have previously described (15, 29-31). Gross evaluation and manual assessment results were similar to radiographic findings at 8 weeks.…”

Section: Resultsmentioning

confidence: 99%

A Novel Osteogenic Oxysterol Compound for Therapeutic Development to Promote Bone Growth: Activation of Hedgehog Signaling and Osteogenesis Through Smoothened Binding

Montgomery

Nargizyan

Meliton

et al. 2014

Journal of Bone and Mineral Research

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Osteogenic factors are often used in orthopedics to promote bone growth, improve fracture healing, and induce spine fusion. Osteogenic oxysterols are naturally occurring molecules that were shown to induce osteogenic differentiation in vitro and promote spine fusion in vivo. The purpose of this study was to identify an osteogenic oxysterol more suitable for clinical development than those previously reported, and evaluate its ability to promote osteogenesis in vitro and spine fusion in rats in vivo. Among more than 100 oxysterol analogues synthesized, Oxy133 induced significant expression of osteogenic markers Runx2, osterix (OSX), alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OCN) in C3H10T1/2 mouse embryonic fibroblasts and in M2-10B4 mouse marrow stromal cells. Oxy133-induced activation of an 8×-Gli luciferase reporter, its direct binding to Smoothened, and the inhibition of Oxy133-induced osteogenic effects by the Hedgehog (Hh) pathway inhibitor, cyclopamine, demonstrated the role of Hh pathway in mediating osteogenic responses to Oxy133. Oxy133 did not stimulate osteogenesis via BMP or Wnt signaling. Oxy133 induced the expression of OSX, BSP, and OCN and stimulated robust mineralization in primary human mesenchymal stem cells. In vivo, bilateral spine fusion occurred through endochondral ossification and was observed in animals treated with Oxy133 at the fusion site on xray after 4 weeks and confirmed with manual assessment, micro CT (μCT), and histology after 8 weeks, with equal efficiency to recombinant human bone morphogenetic protein-2 (rhBMP-2). Unlike rhBMP-2, Oxy133 did not induce adipogenesis in the fusion mass and resulted in denser bone evidenced by greater BV/TV ratio and smaller trabecular separation. Findings here suggest that Oxy133 has significant potential as an osteogenic molecule with greater ease of synthesis and improved time to fusion compared to previously studied oxysterols. Small molecule osteogenic oxysterols may serve as the next generation of bone anabolic agents for therapeutic development.

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Secreted phosphoprotein‐24 kDa (Spp24) attenuates BMP‐2‐stimulated Smad 1/5 phosphorylation and alkaline phosphatase induction and was purified in a protective complex with alpha₂‐Macroglobulins From Serum

Zhao

Murray

2012

J of Cellular Biochemistry

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Secreted phosphoprotein-24 kDa (Spp24) binds cytokines of the bone morphogenetic protein/transforming growth factor-β (BMP/TGFβ) superfamily and is one of the most abundant serum phosphoproteins synthesized by the liver. Little is known about how Spp24 binding affects BMP signal transduction and osteoblastic differentiation or how this labile protein is transported from the liver to remote tissues, such as bone. When Spp24 was administered to W-20-17 mesenchymal stem cells with rhBMP-2, short-term Smad1/5 phosphorylation was inhibited, intermediate-term alkaline phosphatase (ALP) induction was blunted, and long-term mineralization was unaffected. This supports the hypothesis that Spp24 proteolysis restricts the duration of its regulatory effects, but offers no insight into how Spp24 is transported intact from the liver to bone. When Spp24 was immunopurified from serum and subjected to native PAGE and Western blotting, a high molecular weight band of >500 kDa was found. Under reducing SDS-PAGE, a 24 kDa band corresponding to monomeric Spp24 was liberated, suggesting that Spp24 is bound to a complex linked by disulfide bonds. However, such a complex cannot be disrupted by 60 mM EDTA under non-reducing condition or in purification buffers containing 600 mM NaCl and 0.1% Tween-20 at pH 2.7-8.5. LC-MS/MS analysis of affinity-purified, non-reducing SDS-PAGE separated, and trypsin digested bands showed that the Spp24 was present in a complex with three α(2) -macroglobulins (α(2) -macroglobulin [α(2) M], pregnancy zone protein [PZP] and complement C3 [C3]), as well as ceruloplasmin and the protease inhibitor anti-thrombin III (Serpin C1), which may protect Spp24 from proteolysis.

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Full-Length spp24, but Not Its 18.5-kDa Proteolytic Fragment, Inhibits Bone-Healing in a Rodent Model of Spine Fusion

Cited by 8 publications

References 13 publications

Secreted Phosphoprotein 24 kD (Spp24) and Spp14 Affect TGF-β Induced Bone Formation Differently

Secreted Phosphoprotein 24 kD (Spp24) and Spp14 Affect TGF-β Induced Bone Formation Differently

A Novel Osteogenic Oxysterol Compound for Therapeutic Development to Promote Bone Growth: Activation of Hedgehog Signaling and Osteogenesis Through Smoothened Binding

Secreted phosphoprotein‐24 kDa (Spp24) attenuates BMP‐2‐stimulated Smad 1/5 phosphorylation and alkaline phosphatase induction and was purified in a protective complex with alpha₂‐Macroglobulins From Serum

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Full-Length spp24, but Not Its 18.5-kDa Proteolytic Fragment, Inhibits Bone-Healing in a Rodent Model of Spine Fusion

Cited by 8 publications

References 13 publications

Secreted Phosphoprotein 24 kD (Spp24) and Spp14 Affect TGF-β Induced Bone Formation Differently

Secreted Phosphoprotein 24 kD (Spp24) and Spp14 Affect TGF-β Induced Bone Formation Differently

A Novel Osteogenic Oxysterol Compound for Therapeutic Development to Promote Bone Growth: Activation of Hedgehog Signaling and Osteogenesis Through Smoothened Binding

Secreted phosphoprotein‐24 kDa (Spp24) attenuates BMP‐2‐stimulated Smad 1/5 phosphorylation and alkaline phosphatase induction and was purified in a protective complex with alpha2‐Macroglobulins From Serum

Contact Info

Product

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Secreted phosphoprotein‐24 kDa (Spp24) attenuates BMP‐2‐stimulated Smad 1/5 phosphorylation and alkaline phosphatase induction and was purified in a protective complex with alpha₂‐Macroglobulins From Serum