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Actin Microfilament Mediates Osteoblast Cbfa1 Responsiveness to BMP2 under Simulated Microgravity

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 obs...

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Autores principales: Dai, Zhongquan, Wu, Feng, Chen, Jian, Xu, Hongjie, Wang, Honghui, Guo, Feima, Tan, Yingjun, Ding, Bai, Wang, Jinfu, Wan, Yumin, Li, Yinghui
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3651164/
https://www.ncbi.nlm.nih.gov/pubmed/23675497
http://dx.doi.org/10.1371/journal.pone.0063661
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author Dai, Zhongquan
Wu, Feng
Chen, Jian
Xu, Hongjie
Wang, Honghui
Guo, Feima
Tan, Yingjun
Ding, Bai
Wang, Jinfu
Wan, Yumin
Li, Yinghui
author_facet Dai, Zhongquan
Wu, Feng
Chen, Jian
Xu, Hongjie
Wang, Honghui
Guo, Feima
Tan, Yingjun
Ding, Bai
Wang, Jinfu
Wan, Yumin
Li, Yinghui
author_sort Dai, Zhongquan
collection PubMed
description 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.
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spelling pubmed-36511642013-05-14 Actin Microfilament Mediates Osteoblast Cbfa1 Responsiveness to BMP2 under Simulated Microgravity Dai, Zhongquan Wu, Feng Chen, Jian Xu, Hongjie Wang, Honghui Guo, Feima Tan, Yingjun Ding, Bai Wang, Jinfu Wan, Yumin Li, Yinghui PLoS One Research Article 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. Public Library of Science 2013-05-10 /pmc/articles/PMC3651164/ /pubmed/23675497 http://dx.doi.org/10.1371/journal.pone.0063661 Text en © 2013 Dai et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Dai, Zhongquan
Wu, Feng
Chen, Jian
Xu, Hongjie
Wang, Honghui
Guo, Feima
Tan, Yingjun
Ding, Bai
Wang, Jinfu
Wan, Yumin
Li, Yinghui
Actin Microfilament Mediates Osteoblast Cbfa1 Responsiveness to BMP2 under Simulated Microgravity
title Actin Microfilament Mediates Osteoblast Cbfa1 Responsiveness to BMP2 under Simulated Microgravity
title_full Actin Microfilament Mediates Osteoblast Cbfa1 Responsiveness to BMP2 under Simulated Microgravity
title_fullStr Actin Microfilament Mediates Osteoblast Cbfa1 Responsiveness to BMP2 under Simulated Microgravity
title_full_unstemmed Actin Microfilament Mediates Osteoblast Cbfa1 Responsiveness to BMP2 under Simulated Microgravity
title_short Actin Microfilament Mediates Osteoblast Cbfa1 Responsiveness to BMP2 under Simulated Microgravity
title_sort actin microfilament mediates osteoblast cbfa1 responsiveness to bmp2 under simulated microgravity
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3651164/
https://www.ncbi.nlm.nih.gov/pubmed/23675497
http://dx.doi.org/10.1371/journal.pone.0063661
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