Beyond osteogenesis: an in vitro comparison of the potentials of six bone morphogenetic proteins

Bone morphogenetic proteins (BMPs) other than the clinically available BMP-2 and BMP-7 may be useful for improving fracture healing through both increasing osteogenesis and creating a favorable healing environment by altering cytokine release by endogenous cells. Given the spectrum of potential applications for BMPs, the objective of this study was to evaluate various BMPs under a variety of conditions to provide further insight into their therapeutic capabilities. The alkaline phosphatase (ALP) activity of both C(2)C(12) and human adipose-derived stem cells (hASCs) was measured after exposure of increasing doses of recombinant human BMP-2, -4, -5, -6, -7, or -9 for 3 and 7 days. BMPs-2, -4, -5, -6, -7, and -9 were compared in terms of their ability to affect the release of stromal derived factor-1 (SDF-1), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (b-FGF) from human bone marrow stromal cells (hBMSCs). Gene expression of ALP, osteocalcin, SDF-1, VEGF, and b-FGF following shRNA-mediated knockdown of BMP-2 and BMP-6 in hBMSCs or human osteoblasts under osteogenic differentiation conditions was also evaluated. Collectively, BMPs-6 and -9 produced the greatest osteogenic differentiation of C(2)C(12) and hASCs as determined by ALP. The hBMSC secretion of SDF-1 was most affected by BMP-5, VEGF by BMP-4, and b-FGF by BMP-2. The knockdown of BMP-2 in BMSCs had no effect on any of the genes measured whereas BMP-6 knockdown in hBMSCs caused a significant increase in VEGF gene expression. BMP-2 and BMP-6 knockdown in human osteoblasts caused significant increases in VEGF gene expression and trends toward decreases in osteocalcin expression. These findings support efforts to study other BMPs as potential bone graft supplements, and to consider combined BMP delivery for promotion of multiple aspects of fracture healing

The family-specific α4-helix of the kinesin-13, MCAK, is critical to microtubule end recognition

Kinesins that influence the dynamics of microtubule growth and shrinkage require the ability to distinguish between the microtubule end and the microtubule lattice. The microtubule depolymerizing kinesin MCAK has been shown to specifically recognize the microtubule end. This ability is key to the action of MCAK in regulating microtubule dynamics. We show that the a4-helix of the motor domain is crucial to microtubule end recognition. Mutation of the residues K524, E525 and R528, which are located in the C-terminal half of the a4-helix, specifically disrupts the ability of MCAK to recognize the microtubule end. Mutation of these residues, which are conserved in the kinesin-13 family and discriminate members of this family from translocating kinesins, impairs the ability of MCAK to discriminate between the microtubule lattice and the microtubule end

The role of resveratrol on skeletal muscle cell differentiation and myotube hypertrophy during glucose restriction

Glucose restriction (GR) impairs muscle cell differentiation and evokes myotube atrophy. Resveratrol treatment in skeletal muscle cells improves inflammatory-induced reductions in skeletal muscle cell differentiation. We therefore hypothesised that resveratrol treatment would improve muscle cell differentiation and myotube hypertrophy in differentiating C2C12 myoblasts and mature myotubes during GR. Glucose restriction at 0.6 g/L (3.3 mM) blocked differentiation and myotube hypertrophy versus high-glucose (4.5 g/L or 25 mM) differentiation media (DM) conditions universally used for myoblast culture. Resveratrol (10 μM) treatment increased SIRT1 phosphorylation in DM conditions, yet did not improve differentiation when administered to differentiating myoblasts in GR conditions. Resveratrol did evoke increases in hypertrophy of mature myotubes under DM conditions with corresponding elevated Igf-I and Myhc7 gene expression, coding for the ‘slow’ type I MYHC protein isoform. Inhibition of SIRT1 via EX-527 administration (100 nM) also reduced myotube diameter and area in DM conditions and resulted in lower gene expression of Myhc 1, 2 and 4 coding for ‘intermediate’ and ‘faster’ IIx, IIa and IIb protein isoforms, respectively. Resveratrol treatment did not appear to modulate phosphorylation of energy-sensing protein AMPK or protein translation initiator P70S6K. Importantly, in mature myotubes, resveratrol treatment was able to ameliorate reduced myotube growth in GR conditions over an acute 24-h period, but not over 48–72 h. Overall, resveratrol evoked myotube hypertrophy in DM conditions while favouring ‘slower’ Myhc gene expression and acutely ameliorated impaired myotube growth observed during glucose restriction