Cellular deformation reversibly depresses RT-PCR detectable levels of bone-related mRNA

Abstract

Osteoblastic cells respond to mechanical stimuli with alterations in proliferation and/or phenotypic expression. In some cases, these responses occur within only a few applications of stimuli (i.e. 'cycle-dependent trigger response') rather than in a dose-dependent manner. To explore potential mechanisms of the cycle dependent trigger response, we raised the following questions: (1) Does strain of bone cells alter gene expression; if so, how quickly does it occur and how long does it last? (2) Are alterations in message level strain magnitude dependent? (3) Are alterations in steady-state message levels cycle dependent? Cultures were evaluated for osteocalcin mRNA one week following a daily stretch application at four stretch magnitudes and four cycle numbers and compared to nonstretched controls. Steady state mRNA message was ascertained prior to and at 10, 20, 30, 60, 120, 180, and 240 min following initiation of stretch. Following mRNA isolation, first strand cDNA synthesis was performed and fluorometrically quantitated. A reverse transcriptase based PCR (RT-PCR) approach allowed assessment of osteocalcin mRNA levels from microcultures (50,000 cells per 10 microliters culture or 5000 cells mm2) of rat calvarial osteoblasts. Optimized PCR was performed using primers to the bone specific protein, osteocalcin (OC) and two 'housekeeping' genes, beta-actin and GAP-DH. PCR products were separated on 4% agarose gels and band intensities digitized with relative quantitation based on internal standards in each gel. The lowest magnitude of stretch (- 1 KPa) at 1800 cycles per day reproducibly depressed message for osteocalcin, but not beta-actin when assayed immediately following the cessation of strain application. By three hours following the initiation of stretch, message levels returned to control values. At the time of stretch cessation, the 1800 cycle stretch regimen diminished (p < 0.0001) steady-state osteocalcin message independently of the four stretch magnitudes. Stretch for 300 cycles failed to depress (p = 0.05) osteocalcin message cultures at any time, but 600 cycles depressed message by 30 min. By one and two hours, cultures stretch 600, 900, and 1800 cycles showed similar levels of message depression. Four hours following the initiation of stretch, message levels returning to nonstrained levels in all groups. We conclude that alterations in cell response to strain are in part mediated by gene expression, that alterations last 3-4 h in this system, and that the message mechanism itself exhibits a trigger-response dependency to cycle number.