Date Published: February 27, 2018
Publisher: Public Library of Science
Author(s): Jason A. Bleedorn, Troy A. Hornberger, Craig A. Goodman, Zhengling Hao, Susannah J. Sample, Ermias Amene, Mark D. Markel, Mary Behan, Peter Muir, Damian Christopher Genetos.
Mechanical signals play an integral role in the regulation of bone mass and functional adaptation to bone loading. The osteocyte has long been considered the principle mechanosensory cell type in bone, although recent evidence suggests the sensory nervous system may play a role in mechanosensing. The specific signaling pathways responsible for functional adaptation of the skeleton through modeling and remodeling are not clearly defined. In vitro studies suggest involvement of intracellular signaling through mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), and mammalian target of rapamycin (mTOR). However, anabolic signaling responses to bone loading using a whole animal in vivo model have not been studied in detail. Therefore, we examined mechanically-induced signaling events at five time points from 0 to 24 hours after loading using the rat in vivo ulna end-loading model. Western blot analysis of bone for MAPK’s, PI3K/Akt, and mTOR signaling, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to estimate gene expression of calcitonin gene-related protein alpha (CGRP-α), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), c-jun, and c-fos in dorsal root ganglion (DRG) of the brachial intumescence were performed. There was a significant increase in signaling through MAPK’s including extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) in loaded limbs at 15 minutes after mechanical loading. Ulna loading did not significantly influence expression of the genes of interest in DRG neurons. Bone signaling and DRG gene expression from the loaded and contralateral limbs was correlated (SR>0.40, P<0.05). However, bone signaling did not correlate with expression of the genes of interest in DRG neurons. These results suggest that signaling through the MAPK pathway may be involved in load-induced bone formation in vivo. Further characterization of the molecular events involved in regulation of bone adaptation is needed to understand the timing and impact of loading events, and the contribution of the neuronal signaling to functional adaptation of bone.
Bone has the remarkable ability to continuously change shape and mass in response to a wide variety of mechanical loads . This process has been described as functional adaptation primarily via a mechanism known as mechanotransduction, where cells sense physical stimuli, convert them into biochemical signals, and ultimately trigger a cellular response [2–4]. Osteocytes play a central role in this process due to their abundant distribution throughout bone, connections with other bone cells, and responses to stimulation or targeted ablation [5–7] (for reviews on osteocyte mechanotransduction [2,8,9]). Strain amplification must form part of the regulatory mechanism since physiological in vivo bone strain (<0.5%) is lower than that described to stimulate bone cells cultured in vitro (1–10% strain) [2,10–15]. Despite this adaptive ability, failure and associated accumulation of fatigue damage from cyclic loading of bone is common [13,16–18]. The precise cellular machinery that senses and enacts an adaptive response remains incompletely understood, thus increasing our knowledge in this area is important for comprehensive understanding of adaptative failure of bone. We developed an in vivo whole animal model that enables determination of the effects of mechanical loading on various signaling events in bone and nerve tissue. Protein extraction from each ulna yielded a concentration of 3.74±1.6μg/μl, which was sufficient to perform western blot analysis for the signaling pathways of interest. Protein extraction from DRG neurons did not yield an adequate quantity of protein (0.5–1.3μg/μl); therefore, gene expression was estimated using qRT-PCR from DRG tissue. We demonstrate the ability to detect signaling events in bone tissue in vivo using the rat ulna end-loading model and Western blot analysis. We also quantified downstream products using qRT-PCR to estimate expression of a panel of candidate genes in spinal cord DRG thought to be involved in bone functional adaptation. Load-induced bone formation was associated with phosphorylation of several protein kinases, evident at different time points after mechanical loading in rats. Activation of MAPK signaling (P-ERK and P-JNK) appears rapidly after loading and diminishes rapidly over time. However, PI3K/Akt/mTOR pathway [P-PKB and P-p70(389)] signaling did not appear to be altered by the loading protocol employed in this study. Similarly, no significant changes were identified in our panel of candidate genes associated with neuronal signaling, or in correlations with bone kinase signaling. Source: http://doi.org/10.1371/journal.pone.0192760