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Exposure to microgravity causes bone loss in humans, and the underlying mechanism is thought to be at least partially due to a decrease in bone formation by osteoblasts. In the present study, we examined the hypothesis that microgravity changes osteoblast gene expression profiles, resulting in bone loss. For this study, we developed an in vitro system that simulates microgravity using the Random Positioning Machine (RPM) to study the effects of microgravity on 2T3 preosteoblast cells grown in gas-permeable culture disks. Exposure of 2T3 cells to simulated microgravity using the RPM for up to 9 days significantly inhibited alkaline phosphatase activity, recapitulating a bone loss response that occurs in real microgravity conditions without altering cell proliferation and shape. Next, we performed DNA microarray analysis to determine the gene expression profile of 2T3 cells exposed to 3 days of simulated microgravity. Among 10,000 genes examined using the microarray, 88 were downregulated and 52 were upregulated significantly more than twofold using simulated microgravity compared with the static 1-g condition. We then verified the microarray data for some of the genes relevant in bone biology using real-time PCR assays and immunoblotting. We confirmed that microgravity downregulated levels of alkaline phosphatase, runt-related transcription factor 2, osteomodulin, and parathyroid hormone receptor 1 mRNA; upregulated cathepsin K mRNA; and did not significantly affect bone morphogenic protein 4 and cystatin C protein levels. The identification of gravisensitive genes provides useful insight that may lead to further hypotheses regarding their roles in not only microgravity-induced bone loss but also the general patient population with similar pathological conditions, such as osteoporosis.

We have investigated the role of inhibitor kappaBalpha (IkappaBalpha) in the activation of nuclear factor kappaB (NF-kappaB) observed in human aortic endothelial cells (HAEC) undergoing a low shear stress of 2 dynes/cm(2). Low shear for 6 h resulted in a reduction of IkappaBalpha levels, an activation of NF-kappaB, and an increase in kappaB-dependent vascular cell adhesion molecule 1 (VCAM-1) mRNA expression and endothelial-monocyte adhesion. Overexpression of IkappaBalpha in HAEC attenuated all of these shear-induced responses. These results suggest that downregulation of IkappaBalpha is the major factor in the low shear-induced activation of NF-kappaB in HAEC. We then investigated the role of nitric oxide (NO) in the regulation of IkappaBalpha/NF-kappaB. Overexpression of endothelial nitric oxide synthase (eNOS) inhibited NF-kappaB activation in HAEC exposed to 6 h of low shear stress. Addition of the structurally unrelated NO donors S-nitrosoglutathione (300 microM) or sodium nitroprusside (1 mM) before low shear stress significantly increased cytoplasmic IkappaBalpha and concomitantly reduced NF-kappaB binding activity and kappaB-dependent VCAM-1 promoter activity. Together, these data suggest that NO may play a major role in the regulation of IkappaBalpha levels in HAEC and that the application of low shear flow increases NF-kappaB activity by attenuating NO generation and thus IkappaBalpha levels.