MECHANICAL LOAD INDUCES ZIF-268 AND C-FOS BUT NOT MSX-2 EARLY GENE EXPRESSION IN AN IN VIVO MODEL OF DE NOVO BONE FORMATION

Participants: M.R. Moalli, P.V. Patil, M. Machwate*, S. Harada*, G.A. Rodan*, S.A. Goldstein

*Merck Research Laboratories, West Point, PA

Keywords: mechanotransduction, bone remodeling, gene regulation

Introduction

The remarkable potential for the repair, regeneration, and adaptation of bone is well known, however the influence of physical forces on these processes remains incompletely characterized. We hypothesized that the cascade of changes involved in the transduction of an effective mechanical stimulus into a biological response involves the temporal induction of early genes, growth and differentiation factors, which have autocrine and paracrine effects and transcription factors which direct expression of phenotypic-specific genes. We investigated the effect of mechanical loading on the expression of three early response genes/transcription factors, zif-268 (egr-1), c-fos, and msx-2, which have been proposed to play a role in the regulation of bone formation.

Materials and Methods

Bone chambers equipped with hydraulic actuators were aseptically inserted into each proximal tibial metaphysis of seven adult, male dogs, utilizing protocols approved by the Institutional Animal Care and Use Committee. After 8 weeks of bone infiltration the dogs were returned to the operating room, and the servohydraulic loading mechanism was activated to apply a maximum compressive load of 17.8 N at 1 Hz, for 1800 cycles to the woven trabecular bone that had formed in one chamber. The contralateral chamber served as the unloaded control. Six mm cylindrical bone specimens were then harvested 30 minutes, 12 or 24 hours following the single load episode. The empty chambers were sealed with a hemispherical cap, and the incisions were closed routinely. The dogs were allowed normal cage activity while a second volume of trabecular bone infiltrated the chamber for another 8 week period. The in situ loading procedure was repeated until specimens were harvested from each dog at each of the three time points. Harvested specimens were immediately rinsed in cold saline, then snap frozen in liquid nitrogen for Northern blot analysis.

Results

Application of a single load stimulus (17.8 N @ 89 N/sec, 1 Hz for 1800 cycles) followed by a temporal Northern blot analysis demonstrated load responsiveness of the two immediate early genes, zif-268 and c-fos. There was on average, a 2.2 fold induction of zif-268 mRNA expression in loaded versus unloaded specimens in 6 out of 7 dogs. Six out of 7 dogs demonstrated increased zif-268 mRNA expression at 24 hours in loaded versus unloaded specimens. Similarly, c-fos mRNA expression was upregulated in 5 out of 7 dogs within 30 minutes of a single load episode, and in all 7 dogs by 24 hours. A 12 hour specimen for 1 dog was inadvertently damaged during surgical extraction, and thus was not included in the analysis. At 12 hours both early genes were downregulated in 4 of 6 dogs.

These data demonstrate a biphasic pattern of mRNA expression of both zif-268 and c-fos mRNA, with induction at 30 minutes, downregulation at 12 hours, and upregulation again 24 hours following a single load stimulus. The temporal pattern of induction may reflect either the developmental stage of the bone cell responding (ie. proliferative versus differentiated), or simply the type of cell responding to the mechanical stimulus (ie. endothelial cell, fibroblast or bone cell). The absence of msx-2 expression suggests that in contrast to its critical role in the differentiation of calvarial osteoblasts, msx-2 may not play a role in the regulation of bone cells in long bone and consequently may not be involved in long bone signaling responses to mechanical load. Additional candidate growth factors, early genes and matrix proteins are being investigated by ribonuclease protection assay or Northern analysis for load-responsiveness.