Signal Transduction of Mechanical Stimuli Is Dependent on Microfilament Integrity: Identification of Osteopontin as a Mechanically Induced Gene in Osteoblasts

Abstract

Mechanical perturbation has been shown to modulate a wide variety of changes in second message signals and patterns of gene expression in osteoblasts. Embryonic chick osteoblasts were subjected to a dynamic spatially uniform biaxial strain (1.3% applied strain) at 0.25 Hz for a single 2-h period, and osteopontin (OPN), an Arg-Gly-Asp (RGD)-containing protein, was shown to be a mechanoresponsive gene. Expression of opn mRNA reached a maximal 4-fold increase 9 h after the end of the mechanical perturbation that was not inhibited by cycloheximide, thus demonstrating that mechanoinduction of opn expression is a primary response through the activation of pre-existing transcriptional factors. The signal transduction pathways, which mediated the increased expression of opn in response to mechanical stimuli, were shown to be dependent on the activation of a tyrosine kinase(s) and protein kinase A (PKA) or a PKA-like kinase. Selective inhibition of protein kinase C (PKC) had no effect on the mechanoinduction of osteopontin even though opn has been demonstrated to be an early response gene to phorbol 12-myristate 13-acetate (PMA) stimulation. Mechanotransduction was dependent on microfilament integrity since cytochalasin-D blocked the up-regulation of the opn expression; however, microfilament disruption had no effect on the PMA induction of the gene. The microtubule component of the cytoskeleton was not related to the mechanism of signal transduction involved in controlling opn expression in response to mechanical stimulation since colchicine did not block opn expression. Mechanical stimulus was shown to activate focal adhesion kinase (FAK), which specifically became associated with the cytoskeleton after mechanical perturbation, and its association with the cytoskeleton was dependent on tyrosine kinase activity. In conclusion, these results demonstrate that the signal transduction pathway for mechanical activation of opn is uniquely dependent on the structural integrity of the microfilament component of the cytoskeleton. In contrast, the PKC pathway, which also activates this gene in osteoblasts, acts independently of the cytoskeleton in the transduction of its

Mechanical loading stimulates chondrogenesis via the PKA/CREB-Sox9 and PP2A pathways in chicken micromass cultures

“…In chondrocytes, integrins play important roles also in cartilage remodelling and chondrogenesis through interactions with the focal adhesion kinase and the cytoskeleton [57]. Mechanically induced alterations in the cytoskeleton can directly initiate the formation and/or activation of specific signal transducers within the focal adhesion complex [58]. Based on our data, the next component of mechanotransduction pathways can be the activation of adenylate cyclase which, by producing cAMP triggers PKA.…”

“…Based on our data, the next component of mechanotransduction pathways can be the activation of adenylate cyclase which, by producing cAMP triggers PKA. Such a putative link has been established in primary embryonic osteoblasts, where signal transduction pathways to mediate the upregulation of osteopontin in response to mechanical stimuli were shown to be dependent on the activation of PKA [58]. Further, our data suggest that the dramatically increased PKA activity following mechanical stimulation could be accounted for the observed loadinginduced changes in HDC including enhanced cartilage ECM production, elevated Sox9 and CREB phosphorylation as well as stronger nuclear signals for these factors, since most of these effects could be prevented by the PKA-inhibitor H89.…”

Role of Irisin on the bone–muscle functional unit

“…19 In several studies, OPN expression was shown to be modulated in response to mechanical stimulation 30,31 and, therefore, it has been defined as a mechanically responsive gene. 32 Consistently with the loading-mimetic function of Irisin, this myokine might be the mediator of loading-induced increase in OPN expression.…”

Bone tissue engineering: A review in bone biomimetics and drug delivery strategies

“…68,69 In vitro studies have shown that osteoblasts reorganize their cytoskeleton, upregulate transmembrane focal adhesion proteins, and express osteoblast-specific proteins involved in ECM adhesion such as osteopontin under fluid flow stimulation. [70][71][72] These known mechanisms of mechanical stimulate in bone cells and tissue have significant implications in synthetic bone scaffold design. Most importantly, to simulate the dynamic mechanical physiological environment, a synthetic scaffold must have similar mechanical properties (specifically elastic modulus) compared to native bone.…”