Actin microfilament mediates osteoblast Cbfa1 responsiveness to BMP2 under simulated microgravity

Abstract

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.

Figure 1. Schematic diagram of analysis of cell fluorescence intensity.

Figure 2. Responsiveness of the OSE-MG63 cell line to BMP2, IGF-I and VD3.

OSE-MG63 cells were treated with different concentrations of these cytokines for 48 h, after which fluorescence intensity (A) and ALP activity (B, C) were analyzed. * P<0.05, ** P<0.01, vs. CN (untreated), n = 3.

Figure 3. Effects of microgravity and hypergravity on Cbfa1 activity and responsiveness to BMP2.

OSE-MG63 cells were cultured in a clinostat or in a cell centrifuge for 48 h with or without BMP2 (200 ng/ml), after which the fluorescence intensity was analyzed. CN: normal gravity, CR: clinorotation, HG: hypergravity ** P<0.01, vs. CN, # P<0.05, ## P<0.01 vs. BMP2−, n = 3.

Figure 4. Effects of CB on Cbfa1 activity and BMP2 induction effect.

OSE-MG63 cells were treated with different concentrations of CB with or without 200 ng/ml BMP2 for 48 h, then fluorescence intensity (A) and mRNA level of EGFP (B) and Cbfa1 (C) was analyzed. The CB concentration is 2.0 µmol/L in mRNA analysis (B, C). && p<0.01, VS. other groups n = 5.

Figure 5. CB suppresses DNA binding activity of BMP2-induced Cbfa1 and phosphorylated level of Smad1/5/8.

OSE-MG63 cells were treated with BMP2 (200 ng/ml), CB (2.0 µmol/L) or a combination thereof for 48 h, then analyzed by ChIP. The chromatin fragment of 6OSE2 (A) or osteocalcin (B) promoter immunoprecipitated by the Cbfa1 antibody was assayed by quantitative PCR and expressed as a relative value against the amount of input DNA. The values represent the averages plus standard errors (error bars) from triplicate samples. && p<0.01 vs. other groups * p<0.05, VS. CN, #p<0.05, ## p<0.01 VS. CB, n = 3. (C) Proteins were analyzed for phosphorylation of Smad1/5/8 by Western blot. Total SMAD1 and GAPDH protein level showed equal loading of protein (representative of n = 3).

Figure 6. Simulated microgravity disrupts F-actin in MG63 cell line.

Cells were cultured in clinorotation for 48 h and stained with Texas red isothiocyanate-conjugated phalloidin. CR: clinorotation, CN: control.

Figure 7. Effects of JAS and BMP2 on the changes of Cbfa1 activity induced by clinorotation.

OSE-MG63 cells were treated with JAS, BMP2 or a combination thereof, and cultured in clinostat for 48 h, after which the fluorescence intensity was analyzed $P<0.05, $$ P<0.01, VS. −/−(untreated group), * P<0.05, ** P<0.01 vs. CN. n = 4.