Abstract
Although load-induced mechanical signals play a key role in bone formation and maintenance of bone mass and structure, the cellular mechanisms involved in the translation of these signals are still not well understood. Recent identification of a novel flow-induced mechanosignaling pathway involving VEGF in osteoblasts and the known VEGF regulation of actin reorganization in various cell types has led us to hypothesize that fluid shear stress-induced Vegf up-regulation underlies the actin cytoskeleton adaptation observed in osteoblasts during mechanotransduction. Our results show that MC3T3-E1 cells secrete significant VEGF in response to 5 h of pulsatile fluid shear stress (PFSS; 5 dynes/cm(2) at 1 Hz), whereas expression of VEGF receptors (VEGFR-1, VEGFR-2, or NRP1) is unaffected. These receptors, in particular VEGFR-2, participate in PFSS-induced VEGF release. Exposure to flow-conditioned medium or exogenous VEGF significantly induces stress fiber formation in osteoblasts that is comparable with PFSS-induced stress fiber formation, whereas VEGF knockdown abrogates this response to PFSS, thereby providing evidence that flow-induced VEGF release plays a role in actin polymerization. Using neutralizing antibodies against the receptors and VEGF isoforms, we found that soluble VEGFs, in particular VEGF(164), play a crucial role in transient stress fiber formation during osteoblast mechanotransduction, most likely through VEGFR-2 and NRP1. Based on these data we conclude that flow-induced VEGF release from osteoblasts regulates osteoblast actin adaptation during mechanotransduction and that VEGF paracrine signaling may provide potent cross-talk among bone cells and endothelial cells that is essential for fracture healing, bone remodeling, and osteogenesis.
Highlights
VEGF, known as VEGF-A, is a heparin-binding homodimeric glycoprotein that through alternative exon splicing of a single VEGF pre-mRNA results in the generation of four different isoforms (VEGF121, VEGF165, VEGF189, and VEGF206) in man [3, 4] and three major distinct isoforms (VEGF120, VEGF164, and VEGF188) in mouse [5]
We show that actin cytoskeleton adaptation in response to pulsatile fluid shear stress (PFSS) is abrogated by knocking down VEGF expression in MC3T3-E1 cells, which demonstrates that VEGF is the mechanosignaling molecule mediating flow-induced actin polymerization in osteoblasts
The average VEGF concentration secreted from osteoblasts in the flow media in response to fluid shear stress was 30 pg/ml, which was ϳ7-fold higher than that measured in the static, no flow condition
Summary
Flt-1, Fms-like tyrosine kinase 1; PFSS, pulsatile fluid shear stress; VEGFR, VEGF receptor; NRP1, neuropilin-1; ␣-MEM, ␣-minimal essential medium; ANOVA, analysis of variance; HIF, hypoxia-inducible factor. Flow-induced VEGF Regulates Actin Adaptation in Osteoblasts vate focal adhesion kinases in both endothelial cells and osteoblasts [13,14,15,16], in a manner very similar to changes observed when these cells are submitted to mechanical stimulation [17,18,19,20]. Based on these similarities and our recent findings indicating that Vegf expression in osteoblasts is upregulated by mechanical stimulation [2], we hypothesized that fluid shear stress regulates actin cytoskeleton adaptation during osteoblast mechanotransduction through VEGF release and autocrine signaling. We conclude that flow-induced VEGF regulates actin adaptation in an autocrine manner during osteoblast mechanotransduction, whereas paracrine signaling may provide potent cross-talk among bone cells and endothelial cells that is essential for the processes of bone remodeling, fracture healing, and osteogenesis
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