Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, United States of America.
Orthofix, Inc., Lewisville, TX, United States of America.
PLoS One. 2021 Feb 25;16(2):e0247659. doi: 10.1371/journal.pone.0247659. eCollection 2021.
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.
脉冲电磁场（PEMF）治疗被发现可增强小鼠前成骨细胞 MC3T3-E1 的细胞分化为更成骨细胞表型。分化基因，如 Alp、BSPI、cFos、Ibsp、骨钙素、Pthr1 和 Runx2 的表达在受到 PEMF 刺激时增加。将 PEMF 与这些基因的激活联系起来的详细分子机制有限。已知两种对 PEMF 有反应的腺苷受体，Adora2A 和 Adora3，通过 CRISPR-Cas9 介导的基因破坏而功能受损，并且在 PEMF 介导的成骨细胞分化的背景下研究了这些基因破坏的后果。Adora2A 的破坏导致 Alp mRNA 表达延迟，但碱性磷酸酶蛋白表达不受影响，这与野生型细胞相似。然而，Adora3 的破坏导致在整个 PEMF 刺激期间碱性磷酸酶 mRNA 和蛋白水平的反应明显降低。在用化学定义的生长和分化诱导培养基 (DM) 进行的响应中观察到缺陷。此外，在 Adora2A 破坏的细胞中，基因表达谱显示 cFos 和 Pthr1 对 PEMF 处理的反应减弱；而 Adora3 破坏的细胞中，AlpI、BSPI、Ibsp、骨钙素和 Sp7 基因激活的反应大多减弱。为了证明 Adora3 功能的特异性，将 Adora3 开放阅读框插入到 Adora3 破坏细胞的 ROSA26 基因座中，最终导致 PEMF 反应性得到挽救，从而消除了脱靶效应的可能性。这些结果使我们提出，腺苷受体 A2A 和 A3 在 PEMF 介导的成骨细胞分化中具有互补和并行的积极作用。
