Regulation Effects Of Bone-related Cytokines On Preosteoblast Under Simulated Weightlessness Condition

Space medicine problem induced by long-term spaceflight, especially bone loss, has been one of restrictfactors of manexploring to the deeper, further and higher outer space. Exercise, drug and nutritional supplements taken by astronaut at present spaceflight, can’t effectively prevent bone loss occurrence. To assurance the health and effective performance of astronaut during future long-term spaceflight mission, it was required to clarify the mechanism of bone loss in space and develop effective countermeasure against bone loss based on the cellular and molecular intrinsic understanding of bone loss, which are also the urgent and necessary requirement of the developing manned space.Bone loss in space is primary risk factor of the susceptible disease for astronaut during spaceflight and mainly due to the decreased bone formation. Weightlessness not only affects the differentiation process of osteoblast, also influences the osteogenic initiation from osteogentior cell. Cbfa1 is a key transcription factor for the initiation and regulation of osteoblast differentiation, and a target for mechanic signaling. Bone-related cytokines participate the regulation of osteoblast proliferation and differentiation, including Cbfa1 activity. Moreover, weightlessness affects the expression and signal transduction of bone related cytokine. It is important to clarify the function of bone related cytokines to bone loss in space under weightlessness condition which also have significance for the mechanism clarification and countermeasure development against bone loss in space.Previous studies suggest that the decreased proliferation and differentiation responsiveness of BMSC and preosteoblast to bone-related cytokines stimulation attenuate the number and activity of osteoblast, which induce bone formation decrease and bone loss ultimately under weightlessness condition. In present study, we constructed osteoblast modeling in which Cbfa1 activity can be reflected by reporter and IGF-I alternative splicing model;Cell clinorotation, rat hindlimb suspension and flow shear stress were used to investigate the effects of bone-related cytokines on BMSC proliferation, Cbfa1 activity of preosteoblast and their mechanism, study the expression and regulation changes of rat bone IGF-I gene after hindlimb suspension. We aim to explore the changed of bone-related cytokine in expression, regulation and function under weightlessness condition. These studies results will provide new science evidence for clarifying the cellular and molecular mechanism of bone loss in space.Methods: (1) BMSCs were isolated using whole bone marrow culture method based on red cell lysis. The proliferation, cell cycle and promotive effects of cytokines under clinorotation condition were investigated by methylene blue staining and flow cytometry examination. Effects of clinorotation on microfilament cytoskeleton, ERK1/2 activity, osteogenic differentiation and expression profile were studied using immunofluorescence staining, RT-PCR, Western blot, gene chip etc. (2) Osteoblast models were developed by vector construction and stable transfection, in which Cbfa1 activity can be reflected by reporter activity. The effects of different gravity, VD3 and BMP2 on Cbfa1 activity and the responsiveness character of Cbfa1 to VD3 and BMP2 under clinorotation were investigated using these models by semi-quantitative analysis of EGFP fluorescence intensity or luciferase activity. Using bidirectional CO-IP, the interaction change between VDR and Cbfa1 was studied under clinorotation or hypergravity. F-actin inhibitor (CB) or stabilizer (Jas) was used to investigate the function of cytoskeleton on promotive effect of BMP2 to Cbfa1 activity. (3) Real time RT-PCR was used to examined the expression alternation of IGF-I alternative splicing isoforms under clinorotation for osteoblast or hindlimb suspension for bone tissue. We constructed 4 IGF-I alternative splicing vectors by PCR amplification and subclone, which were stably transfected into MC3T3-E1 respectively and IGF-I alternative splicing models were developed. Experiment system of Flow shear were developed to investigate the effects of FSS with 1Pa for 1h on IGF-I alternative splicing.Results:(1) Clinorotation time-dependently disrupted the microfilament cytoskeleton, made more cells arrest in G1/G0 phase of cell cycle from 86.6% to 91.4%; and inhibited proliferation of BMSC. Clinorotation also decreased the activity of ERK1/2 and the promotive effects of IGF-I, EGF and bFGF to BMSC proliferation, from 15.5%, 16.3%, 26.9% to 10.5%, 10.3%, 19.6% respectively; inhibited the osteogenic differentiation potency of BMSC. Microarray and cluster analysis suggested that the expression of some genes related to the terms of cell cycle, microfilament, osteogenic differentiation were changed, especially more upregulated negative regulation gene. (2) Osteoblast models were developed in which Cbfa1 activity can be reflected by fluorescence intensity of EGFP or Luciferase activity. Clinorotation significantly inhibited the Cbfa1 activity, but hypergravity promoted it. Both VD3 and BMP2 increased the activity of Cbfa1, but clinorotation inhibited the stimulation effects of VD3 and BMP2 to Cbfa1 activity. The increased degree of Cbfa1 activity decreased from 79%, 18% to 43%, 14% respectively. Clinorotation attenuated the interaction between VDR, VD3 receptor, and Cbfa1, also disrupted the F-actin cytoskeleton. Low concentration of cytochalasin B (0.5nmol/L), an inhibitor of F-actin, abolished the promotive effect of BMP2 to Cbfa1 activity. F-actin stabilizer (Jas) can protect, at some extent, the promotive effect of BMP2 to Cbfa1 activity under clinorotation condition. (3) Hindlimb suspension time-dependently decreased IGF-IEa and MGF mRNA level in bone tissue and IGF-I level in serum. Four cell strains which was stably transfected with alternative splicing vectors including different length intron locusing at exon 5 flank were selected and the expression of MGF-EGFP significantly increase in 5341 strain after subjected to 1Pa FSS treatment for 1h, but no changes were observed in other stable transfected cells.Conclusion: clinorotation significantly inhibits the proliferation and osteogenic differentiation of BMSC, decreases the proliferation responsiveness to cytokine IGF-I, EGF and bFGF. Clinorotation also attenuates the interaction between VDR and Cbfa1 which maybe contribute the decreased responsiveness of Cbfa1 activity to VD3. Microfilament cytoskeleton participates the promotive effect of BMP2 to Cbfa1 activity and disruption of F-actin maybe contributes to the attenuated responsiveness of Cbfa1 activity to BMP2 under clinorotation. Hindlimb suspension affects the IGF-IEa and MGF expression in bone and IGF-I level in serum. Weightlessness can influence the alternative splicing of IGF-I. FSS can regulated the IGF-I alternative splicing via the intron elements. These results provide new scientific evidences for cellular molecular mechanisms of bone loss in space.