Date Published: May 10, 2013
Publisher: Public Library of Science
Author(s): Zhongquan Dai, Feng Wu, Jian Chen, Hongjie Xu, Honghui Wang, Feima Guo, Yingjun Tan, Bai Ding, Jinfu Wang, Yumin Wan, Yinghui Li, Linda M. Hendershot. http://doi.org/10.1371/journal.pone.0063661
Microgravity decreases osteoblastic activity, induces actin microfilament disruption and inhibits the responsiveness of osteoblast to cytokines, but the mechanisms remains enigmatic. The F-actin cytoskeleton has previously been implicated in manifold changes of cell shape, function and signaling observed under microgravity. Here we investigate the involvement of microfilament in mediating the effects of microgravity and BMP2 induction on Cbfa1 activity. For this purpose we constructed a fluorescent reporter cell line (OSE-MG63) of Cbfa1 activity by stably transfecting MG63 cells with a reporter consisting of six tandem copies of OSE2 and a minimal mOG2 promoter upstream of enhanced green fluorescent protein (EGFP). The fluorescence intensity of OSE-MG63 showed responsiveness to bone-related cytokines (IGF-I, vitamin D3 and BMP2) and presented an accordant tendency with alkaline phosphatase (ALP) activity. Using OSE-MG63 reporter fluorescence, we performed a semi-quantitative analysis of Cbfa1 activity after treatment with simulated microgravity, microfilament-disrupting agent (cytochalasin B, CB), microfilament-stabilizing agent (Jasplakinolide, JAS) or any combination thereof. In parallel, ALP activity, DNA binding activity of Cbfa1 to OSE2 (ChIP), F-actin structure (immunofluorescence) and EGFP mRNA expression (RT-qPCR) were analyzed. Simulated microgravity inhibited Cbfa1 activity, affected the responsiveness of Cbfa1 to cytokine BMP2, and caused a thinning and dispersed distribution of microfilament. Under normal gravity, CB significantly attenuated BMP2 induction to Cbfa1 activity as well as DNA binding activity of Cbfa1 to OSE2. The addition of JAS reversed the inhibitory effects of microgravity on the responsiveness of Cbfa1 to BMP2. Our study demonstrates that disrupting the microfilament organization by CB or simulated microgravity attenuates the responsiveness of Cbfa1 to BMP2. A stabilization of the microfilament organization by JAS reverses this inhibition. Taken together, these results suggest that actin microfilament participates in BMP2’s induction to Cbfa1 activity and that their disruption might be an important contributor to microgravity’s inhibition on BMP2’s osteogenic induction.
During spaceflight, 1–2% of bone mass, particularly of weight-bearing bone, is lost each month . The reduction of bone formation is considered to be the main cause of decrease in bone density during spaceflight . Real and simulated microgravity by clinorotation inhibits the differentiation of osteoprogenitor cells into mature osteoblasts – and simulated microgravity by hindlimb unloading decreases the osteogenic potential of bone marrow mesenchymal stem cells (BMSCs) . Taken together, bone loss induced by microgravity has been attributed to osteoblasts due to their (a) reduced proliferation and activity, (b) reduced differentiation and (c) decreased responsiveness of osteoblast to bone related factors in the microenvironment. However, the mechanisms are not fully understood , .
Accumulating evidence demonstrates that microgravity inhibits the initial as well as subsequent stages of osteoblast differentiation , . Cbfa1, an osteoblast-specific transcription factor, not only initiates the differentiation of osteoblasts, but regulates the expression of osteoblast-specific genes during differentiation. Expression of Cbfa1 is decreased under real and simulated microgravity . On the other hand, we and other investigators have demonstrated that microgravity decreases the responsiveness of osteoblasts to cytokines that promote osteoblast proliferation, differentiation and bone formation, such as BMP2 and IGF-I. These cytokines regulate the expression and activity of Cbfa1 during osteogenesis , . The mechanisms by which microgravity has an effect on Cbfa1 activation or inhibits its responsiveness to cytokines are not clearly understood. Here, we investigated whether the effects of microgravity on BMP2-induced osteogenic differentiation are related to actin disruption.