Effects of hydrostatic pressure, biaxial strain, and fluid shear on osteoblastic cells: Mechanotransduction via NF-kappaB, MAP kinase, and AP-1 pathways.

Abstract

Bone adaptation, maintenance, and repair are dependent on local and systemic biochemical regulation as well as cellular responses emanating from external physical cues. Many studies of this mechanotransduction phenomenon in vitro have focused on only one of the components of the physical environment, making it difficult to compare effects across varying loading modalities. This dissertation represents a cross-sectional study to examine how variations in the character of the mechanical environment within bone may initiate signaling in osteoblasts via different pathways. Hydrostatic pressure, biaxial strain, and fluid shear stresses were applied to cells in culture using custom designed, fabricated and calibrated devices. MC3T3 preosteoblasts were used to examine the effects of load on mediators of the NF-kappaB, AP-1, and MAP kinase pathways, using transfection, immunocytochemistry, and Western blotting. The NF-kappaB pathway was activated by fluid shear and IL-1beta, but not hydrostatic pressure or biaxial strain. IL-1beta mediated NF-kappaB activation was differentially regulated by hydrostatic pressure and biaxial strain. Pressure enhanced IL-1beta mediated NF-kappaB activation, while biaxial strain repressed this pathway; both effects were amplitude dependent. A model was proposed based on distinct load regulation of MAP kinase members, specifically TGF-beta activated kinase 1 (TAK-1), which is involved in the IL-1beta activation of IkappaBalpha degradation and NF-kappaB translocation. C-fos was activated by biaxial strain and both modes of fluid shear, but not hydrostatic pressure. The biaxial strain c-fos response was transient, while c-fos induced by shear remained elevated for 3.5h. These distinct c-fos responses were related to transient and sustained phosphorylation of ERK, in strained and sheared cells respectively. A model to explain the differences between strain and shear induction of c-fos was proposed based on the duration of ERK phosphorylation, and its influence on c-fos induction and stability. Results from this dissertation outline the conditions under which specific TF pathways are activated, thereby leading to future applications in gene therapy, bioreactor design, and treatment of chronic inflammation. Mechanical loading may prove to be an important component of novel therapeutics in many of these fields.