Pulsed-electromagnetic-field induced osteoblast differentiation requires activation of genes downstream of adenosine receptors A2A and A3

Abstract

Pulsed-electromagnetic-field (PEMF) treatment was found to enhance cellular differentiation of the mouse preosteoblast, MC3T3-E1, to a more osteoblastic phenotype. Differentiation genes such as Alp, BSPI, cFos, Ibsp, Osteocalcin, Pthr1 and Runx2 showed increased expression in response to PEMF stimulation. Detailed molecular mechanisms linking PEMF to the activation of these genes are limited. Two adenosine receptors known to be modulated in response to PEMF, Adora2A and Adora3, were functionally impaired by CRISPR-Cas9-mediated gene disruption, and the consequences of which were studied in the context of PEMF-mediated osteoblastic differentiation. Disruption of Adora2A resulted in a delay of Alp mRNA expression, but not alkaline phosphatase protein expression, which was similar to that found in wild type cells. However, Adora3 disruption resulted in significantly reduced responses at both the alkaline phosphatase mRNA and protein levels throughout the PEMF stimulation period. Defects observed in response to PEMF were mirrored using a chemically defined growth and differentiation-inducing media (DM). Moreover, in cells with Adora2A disruption, gene expression profiles showed a blunted response in cFos and Pthr1 to PEMF treatment; whereas cells with Adora3 disruption had mostly blunted responses in AlpI, BSPI, Ibsp, Osteocalcin and Sp7 gene activation. To demonstrate specificity for Adora3 function, the Adora3 open reading frame was inserted into the ROSA26 locus in Adora3 disrupted cells culminating in rescued PEMF responsiveness and thereby eliminating the possibility of off-target effects. These results lead us to propose that there are complementary and parallel positive roles for adenosine receptor A2A and A3 in PEMF-mediated osteoblast differentiation.

Fig 1. PEMF induces osteoblast differentiation.

A. Relative alkaline phosphate enzymatic activity normalized to cell protein after 4 hours daily treatment with PEMF for 12 days or differentiation medium (DM) for 12 days. * indicates p<0.05, Student’s t-test compared to untreated control. B. Relative expression of osteoblast differentiation genes as indicated, normalized to GAPDH after 4 hours/day treatment with PEMF for 12 days or DM for 12 days. * indicates p<0.05, Student’s t-test compared to untreated control of same gene. C. Time series of Alizarin red staining for mineralization and alkaline phosphate activity staining after PEMF or DM treatment.

Fig 2. Adora 2A and 3 disruption by CRISPR-Cas9.

MC3T3-E1 cells were co-transfected with a pUC donor plasmid with RFP-PGK-BSD or GFP-PGK-Puro flanked by homolog arms (1) and pCas-Guide plasmid containing Cas9 and gRNA corresponding to cleavage site in the first exon in each gene (2). Expected insertions of the cassette after homology-dependent recombination (HDR) are shown at the bottom.

Fig 3. Adora3 disruption affects PEMF activation of alkaline phosphatase.

A. Relative alkaline phosphate enzymatic activity normalized to cell protein after 4 hours daily treatment with PEMF for 4, 8, 12 and 16 days. * indicates p<0.05, Student’s t-test compared to WT control for each time-point. B. Relative expression of alkaline phosphatase gene normalized to GAPDH after 4 hours daily treatment with PEMF up to 16 days or DM for 16 days. * indicates p<0.05, Student’s t-test compared to WT for each time-point. C. Alkaline phosphatase staining of the MC3T3-E1 cells without and with PEMF treatment. D. Alizarin Red staining of the MC3T3-E1 cells without and with PEMF treatment.

Fig 4

Adora2 and Adora3 disruption affects PEMF activation of cFos, Ibsp, Pthr1, Runx2 and Sp7 (A-E). Relative fold-induction of each gene expression as measured by qRT-PCR with Gapdh as standard, with PEMF treatment up to 16 days (4 hours daily) or DM for 16 days. * indicates p<0.05, Student’s t-test compared to untreated control at day 8 of same gene of same cell-type.

Fig 5. Adora3 disruption affects PEMF mediated overexpression of BSP, IBSP and Osteocalcin.

Wild-type (WT), Adora2 (Δ2), Adora3 (Δ3) and Double Knock-Out (DKO) MC3T3-E1 cells were treated with PEMF (4 hours daily) or differentiation medium (DM) for 12 days and Western Blots for fractionated whole-cell proteins were probed with each of the indicated antibodies.

Fig 6. Adora3 knock-in in Adora3 disrupted cells restores PEMF-mediated induction of alkaline phosphatase in PEMF-treated cells.

Expressions of alkaline phosphate, cFos and Pthr1 genes were measured by qRT-PCR with Gapdh as standard with PEMF treatment for 8 days. * indicates p<0.05, Student’s t-test compared to PEMF treatment in WT cells.

Fig 7. Schematic diagram role of Adenosine Receptors in PEMF-mediated MC3T3-E1 differentiation.

PEMF stimulated osteoblastic differentiation depends on Adenosine Receptor A_2A for cFos and Pthr1 overexpression, and A₃ for AlpI activation.