Benjamin Friedman1 and Bruce Cronstein2, 1NYU Langone, New York, NY, 2NYU School of Medicine, New York, NY
Background/Purpose: Our lab has demonstrated the ability of an intra-articular liposomal formulation of an adenosine A2A receptor (A2AR), CGS21680, to regenerate cartilage in multiple rodent OA models. At the cellular level in chondrocytes, we have shown that activation of A2AR enhances chondrocyte homeostasis, increases mitophagy, and reduces chondrocyte senescence. Moreover, we identified an A2AR-mediated increase in an anti-senescent, anti-aging truncated p53 variant D133p53a in human chondrocytes. These anti-senescent effects have been recapitulated in transgenic mice overexpressing D122p53. Interestingly, a 2014 study reported an endogenous N-terminal mouse p53 variant in HeLa cells (2 separate deletions with loss of amino acids 42-89 and in-frame shift of aa 90-120) with no p53 transactivation potential and inability to upregulate p21 expression despite retention of the first p53 transactivation domain. Hence, we first hypothesized that this mouse p53 variant could be present in mesenchymal stem cells (MSCs), which have chondrogenic capabilities in vitro. Secondly, we hypothesized that A2AR ligation in mouse MSCs would increase expression of this variant.
Methods: Bone marrow MSCs were isolated from 12-week-old mice, treated with or without 1µM CGS21680, and RT-qPCR was performed to assess expression of total and variant p53 using variant specific primers. Primary and secondary gene sequence analysis was employed to evaluate the deleted/shifted region for CpG sites and G-quadruplex structures using the Sequence Manipulation Suite and the GQRS Mapper, respectively.
Results: We did identify this variant in mouse bone marrow MSCs. Furthermore, its expression relative to total p53 was significantly increased in the A2AR agonist treated MSCs compared to control (1.6±0.3 vs. 1.0±0.06, p=0.029, n=3). CpG site analysis revealed that the variant corresponded to a reduction in 4 CpG sites and 3 potential G-quadruplex structures if analyzed in the antisense configuration.
Conclusion: To our knowledge, this is the first report of this variant of mouse p53 noted in cell types aside from the initial report noted above. While more work needs to be done in differentiated cells, A2AR agonism seems to enhance formation of this mouse p53 variant has functional similarities to the D133p53a. Importantly, the deleted regions in this variant contain the key senescence residues in the protein. Further, It is possible that DNA modifications such as CpG methylation and/or alteration in G-quadruplex stability may play a role in such findings.
Disclosures: B. Friedman, None; B. Cronstein, Regenosine.
