Effects of methotrexate on human bone cell responses to mechanical stimulation.
OBJECTIVE
Methotrexate (MTX), which is prescribed in the treatment of malignancy and autoimmune disease, has detrimental effects on a number of organ systems, including bone. At present, the exact mechanism of action of MTX on bone at the cellular level is unclear. Mechanical stimuli imparted by stretch, pressure, fluid flow and shear stress result in a variety of biochemical responses that are important in bone metabolism. Cyclical mechanical stimulation at 0.33 Hz induces rapid cell membrane hyperpolarization of human bone cells (HBC) via an integrin-mediated pathway which includes an IL-1beta autocrine/paracrine loop. This study was undertaken to investigate the effect of MTX on responses of HBC to 0.33 Hz mechanical stimulation.


METHODS
Electrophysiological responses of HBC were measured before and after mechanical stimulation at 0.33 Hz in the presence or absence of MTX. Semiquantitative RT-PCR was used to investigate effects of MTX on relative levels of type-1 collagen and bone morphogenetic protein-4 (BMP-4) following 0.33 Hz mechanical stimulation.


RESULTS
MTX dose-dependently inhibited HBC hyperpolarization in response to 0.33 Hz mechanical stimulation. Production/release of IL-1beta was inhibited by MTX, whereas its effects on target cells were not. Mechanical stimulation of HBC at 0.33 Hz caused a significant decrease in relative levels of BMP-4 mRNA, whereas relative levels of type-1 collagen mRNA were consistently increased, although these increases did not reach statistical significance. These trends were unaffected by MTX.


CONCLUSIONS
These studies show that MTX affects HBC mechanotransduction by interfering with integrin-mediated signalling. The data also suggest that the mechanotransduction pathway responsible for the regulation of type-1 collagen and BMP-4 gene expression may be distinct from the IL-1beta-mediated signalling pathway.
Elliot
K J
KJ
Division of Pathology, Edinburgh University Medical School, Teviot Place, Edinburgh EH8 9AG, UK.
Millward-Sadler
S J
SJ
Wright
M O
MO
Robb
J E
JE
Wallace
W H B
WH
Salter
D M
DM
D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000235:genetics; D001842:Bone and Bones / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D024042:Collagen Type I / Q000032:analysis; D017077:Culture Media, Conditioned; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D016023:Integrins / Q000378:metabolism; D007375:Interleukin-1 / Q000032:analysis; D008564:Membrane Potentials; D008727:Methotrexate / Q000494:pharmacology*; D010812:Physical Stimulation / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D015398:Signal Transduction
C525809:BMP4 protein, human; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D024042:Collagen Type I; D017077:Culture Media, Conditioned; D007166:Immunosuppressive Agents; D016023:Integrins; D007375:Interleukin-1; D012333:RNA, Messenger; D008727:Methotrexate