BMP‐2 modulates β‐catenin signaling through stimulation of Lrp5 expression and inhibition of β‐TrCP expression in osteoblasts

Canonical BMP and Wnt signaling pathways play critical roles in regulation of osteoblast function and bone formation. Recent studies demonstrate that BMP‐2 acts synergistically with β‐catenin to promote osteoblast differentiation. To determine the molecular mechanisms of the signaling cross‐talk between canonical BMP and Wnt signaling pathways, we have used primary osteoblasts and osteoblast precursor cell lines 2T3 and MC3T3‐E1 cells to investigate the effect of BMP‐2 on β‐catenin signaling. We found that BMP‐2 stimulates Lrp5 expression and inhibits the expression of β‐TrCP, the F‐box E3 ligase responsible for β‐catenin degradation and subsequently increases β‐catenin protein levels in osteoblasts. In vitro deletion of the β‐catenin gene inhibits osteoblast proliferation and alters osteoblast differentiation and reduces the responsiveness of osteoblasts to the BMP‐2 treatment. These findings suggest that BMP‐2 may regulate osteoblast function in part through modulation of the β‐catenin signaling. J. Cell. Biochem. 108: 896–905, 2009. © 2009 Wiley‐Liss, Inc.     

Osteogenic properties of a short BMP‐2 chimera peptide

Bone morphogenetic proteins (BMPs) play a key role in bone and cartilage formation. For these properties, BMPs are employed in the field of tissue engineering to induce bone regeneration in damaged tissues. To overcome drawbacks due to the use of entire proteins, synthetic peptides derived from their parent BMPs have come out as promising molecules for biomaterial design. On the structural ground of the experimental BMP‐2 receptor complexes reported in the literature, we designed three peptides, reproducing the BMP‐2 region responsible for the binding to the type II receptor, ActRIIB. These peptides were characterized by NMR, and the structural features of the peptide–receptor binding interface were highlighted by docking experiments. Peptide–receptor binding affinities were analyzed by means of ELISA and surface plasmon resonance techniques. Furthermore, cellular assays were performed to assess their osteoinductive properties. A chimera peptide, obtained by combining the sequence portions 73–92 and 30–34 of BMP‐2, shows the best affinity for ActRIIB in the series and represents a good starting point for the design of new compounds able to reproduce osteogenic properties of the parent BMP‐2. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Combined effects of direct current stimulation and immobilized BMP‐2 for enhancement of osteogenesis

Direct current (DC) stimulation has been used to promote bone repair and osteogenesis, but problems associated with the implanted metal electrodes may limit its application and compromise the therapeutic results. The replacement of the metal electrodes with a biodegradable conductive polymer film can potentially overcome these problems. In our work, polypyrrole/chitosan films comprising polypyrrole nanoparticles dispersed in a chitosan matrix were prepared. The polypyrrole/chitosan film meets the requirements for DC delivery, as indicated by its electrical conductivity, biodegradability, and mechanical properties. The film supports osteoblast growth to the same degree as dentine discs (a bone‐like mineralized substrate), confirming that it is non‐cytotoxic. Our results showed that optimal DC stimulation was achieved with 200 µA for 4 h per day, and under this condition, osteoblast metabolic activity on Day 7 increased by 1.8‐fold over that without DC stimulation. To further improve osteogenesis on the polypyrrole/chitosan film, bone morphogenetic protein‐2 (BMP‐2) was covalently immobilized on the film surface. Osteoblasts cultured on the BMP‐2‐functionalized polypyrrole/chitosan film and subjected to the optimal DC stimulation exhibited a significant increase in cellular metabolic activity (2.3‐fold on Day 7), ALP activity (1.7‐fold on Day 21) and mineralization (twofold on Day 21) over those cultured on polypyrrole/chitosan film without DC stimulation. Osteogenic gene expression results showed that BMP‐2 and DC stimulation by itself enhanced osteoblast differentiation, and a combination of these two factors resulted in synergistic effects on osteoblast differentiation and maturation. Biotechnol. Bioeng. 2013; 110: 1466–1475. © 2012 Wiley Periodicals, Inc.     

Peptide drugs accelerate BMP‐2‐induced calvarial bone regeneration and stimulate osteoblast differentiation through mTORC1 signaling

Both W9 and OP3‐4 were known to bind the receptor activator of NF‐κB ligand (RANKL), inhibiting osteoclastogenesis. Recently, both peptides were shown to stimulate osteoblast differentiation; however, the mechanism underlying the activity of these peptides remains to be clarified. A primary osteoblast culture showed that rapamycin, an mTORC1 inhibitor, which was recently demonstrated to be an important serine/threonine kinase for bone formation, inhibited the peptide‐induced alkaline phosphatase activity. Furthermore, both peptides promoted the phosphorylation of Akt and S6K1, an upstream molecule of mTORC1 and the effector molecule of mTORC1, respectively. In the in vivo calvarial defect model, W9 and OP3‐4 accelerated BMP‐2‐induced bone formation to a similar extent, which was confirmed by histomorphometric analyses using fluorescence images of undecalcified sections. Our data suggest that these RANKL‐binding peptides could stimulate the mTORC1 activity, which might play a role in the acceleration of BMP‐2‐induced bone regeneration by the RANKL‐binding peptides.     

Adiponectin increases BMP‐2 expression in osteoblasts via AdipoR receptor signaling pathway

Adiponectin is a protein hormone secreted predominantly by differentiated adipocytes and involved in energy homeostasis. Bone morphogenetic protein (BMP) plays important roles in osteoblastic differentiation and bone formation. However, the effects of adiponectin on BMPs expression in cultured osteoblasts are largely unknown. Here we found that adiponectin increased mRNA expression of BMP‐2 but not other BMPs in cultured osteoblastic cells. Stimulation of osteoblasts with adiponectin also increased protein levels of BMP‐2 by Western blot and ELISA assay. Adiponectin‐mediated BMP‐2 expression was attenuated by 5′‐AMP‐activated protein kinase (AMPK) small interference RNA and AMPK inhibitor (araA and compound C). Activations of p38 and NF‐κB pathways after adiponectin treatment were demonstrated, and adiponectin‐induced expression of BMP‐2 was inhibited by the specific inhibitor and mutant of p38 and NF‐κB cascades. Taken together, our results provide evidence that adiponectin enhances BMP‐2 expression in osteoblastic cells, and AdipoR1 receptor, AMPK, p38 and NF‐κB signaling pathways may be involved in increasing BMP‐2 expression by adiponectin. J. Cell. Physiol. 224: 475–483, 2010. © 2010 Wiley‐Liss, Inc.     

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

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 α₂‐macroglobulins (α₂‐macroglobulin [α₂M], 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. J. Cell. Biochem. 114: 378–387, 2013. © 2012 Wiley Periodicals, Inc.     

2H‐Pyran‐2‐one and 2H‐Furan‐2‐one Derivatives from the Plant Endophytic Fungus Pestalotiopsis fici

Two new α‐pyrones (=2H‐pyran‐2‐ones), ficipyrones A and B ( 1 and 2 , resp.), and two new α‐furanones (=2H‐furan‐2‐ones), ficifuranones A and B ( 3 and 4 , resp.), together with three known metabolites, antibiotic F 0368 ( 5 ), hydroxyseiridin ( 6 ), and hydroxyisoseiridin ( 7 ), were isolated from solid cultures of the plant endophytic fungus Pestalotiopsis fici. Their structures were elucidated primarily by NMR spectroscopy, and the absolute configuration of 1 was deduced from the circular‐dichroism (CD) data. Compound 1 showed antifungal activity against the plant pathogen Gibberella zeae (CGMCC 3.2873) with an IC₅₀ value of 15.9 μM .