Blockage of hemichannels alters gene expression in osteocytes in a high magneto-gravitational environment

Abstract

Osteocytes, the most abundant cells in bone, are highly responsive to external environmental changes. We tested how Cx43 hemichannels which mediate the exchange of small molecules between cells and extracellular environment impact genome wide gene expression under conditions of abnormal gravity and magnetic field. To this end, we subjected osteocytic MLO-Y4 cells to a high magneto-gravitational environment and used microarray to examine global gene expression and a specific blocking antibody was used to assess the role of Cx43 hemichannels. While 3 hr exposure to abnormal gravity and magnetic field had relatively minor effects on global gene expression, blocking hemichannels significantly impacted the expression of a number of genes which are involved in cell viability, apoptosis, mineral absorption, protein absorption and digestion, and focal adhesion. Also, blocking of hemichannels enriched genes in multiple signaling pathways which are enaged by TGF-beta, Jak-STAT and VEGF. These results show the role of connexin hemichannels in bone cells in high magneto-gravitational environments.

Figure 1

Diagram illustrates three positions of different magnetic force and gravity in the center of superconducting magnet. From top to bottom, μg position, where the direction of magnet force and gravity are opposite and the magnitude is equal, and the magnetic field strength is 12T; 1g, where the magnetic force is zero with only the existence of gravity, and the magnetic field strength is 16T; 2g, where the direction and magnitude of magnet force and gravity are the same, and the magnetic field strength is 12T.

Figure 2

Gene ontology analysis of differentially expressed genes. The subsets of genes that were 1.5-fold upregulated or downregulated were analyzed after exposure of HMGE for 3 hrs and incubating with or without Cx43(E2) antibody in MLO-Y4 cells. (A) μg group vs 2g group (gravity effects); (B) 1g group vs control group (magnetic effects); (C) E2 groups vs control groups (effects of hemichannel inibition).

Figure 3

Hierarchical clustering shown by heatmap analysis. (A) the whole gene expression profile; (B) bone metabolism related genes differentially expressed. Red represents up-regulated genes while green represents down-regulated genes.

Figure 4

Relationship of differentially expressed genes related with bone metabolism and their responses to hemichannel inhibition by Gene-Act-Net analysis. In the network, red arrowheads are upregulated genes and green arrowheads are downregulated genes. a: activation; b: binding; c: compound; dep: dephosphorylation; dis: dissociation; e: expression; ind: indirect effect; inh: inhibition; p: phosphorylation; u: ubiquination.

Figure 5

Relationship of changed pathways after hemichannel inbition by Path-Act-Net analysis. Genes in MAPK signaling pathway, apoptosis, cell cycle and ubiquitin-mediated proteolysis were affected by the inhibition of Cx43 hemichannels.

Figure 6

Validation of relative expression levels of representative genes by Real time PCR. Total RNA was extracted and real time PCR assay was used to further identify for 15 selected central genes, including Pdgfra, Kras, Jak2, Figf, Arhgap5, Csf2rb, IL6, Xiap, Col3a1, Rock1, Csf3, Tgfbr1, IL8, Ccl3 and IL12rb1. The expression levels of corresponding genes were normalized by GAPDH. The results were means ± SD. The differences between μg vs control, μg vs 2g, 1g vs control and antibody-treated groups vs control groups were statistically analyzed by one-way ANOVA. ***, P< 0.001; **, P < 0.01; *, P < 0.1.