Purinergic P2Y14 receptor modulates stress-induced hematopoietic stem/progenitor cell senescence
Joonseok Cho, … , David T. Scadden, Byeong Chel Lee
Joonseok Cho, … , David T. Scadden, Byeong Chel Lee
Published June 17, 2014
Citation Information: J Clin Invest. 2014;124(7):3159-3171. https://doi.org/10.1172/JCI61636.
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Research Article

Purinergic P2Y14 receptor modulates stress-induced hematopoietic stem/progenitor cell senescence

Abstract

Purinergic receptors of the P2Y family are G protein–coupled surface receptors that respond to extracellular nucleotides and can mediate responses to local cell damage. P2Y-dependent signaling contributes to thrombotic and/or inflammatory consequences of tissue injury by altering platelet and endothelial activation and immune cell phagocytosis. Here, we have demonstrated that P2Y14 modifies cell senescence and cell death in response to tissue stress, thereby enabling preservation of hematopoietic stem/progenitor cell function. In mice, P2Y14 deficiency had no demonstrable effect under homeostatic conditions; however, radiation stress, aging, sequential exposure to chemotherapy, and serial bone marrow transplantation increased senescence in animals lacking P2Y14. Enhanced senescence coincided with increased ROS, elevated p16INK4a expression, and hypophosphorylated Rb and was inhibited by treatment with a ROS scavenger or inhibition of p38/MAPK and JNK. Treatment of WT cells with pertussis toxin recapitulated the P2Y14 phenotype, suggesting that P2Y14 mediates antisenescence effects through Gi/o protein–dependent pathways. Primitive hematopoietic cells lacking P2Y14 were compromised in their ability to restore hematopoiesis in irradiated mice. Together, these data indicate that P2Y14 on stem/progenitor cells of the hematopoietic system inhibits cell senescence by monitoring and responding to the extracellular manifestations of tissue stress and suggest that P2Y14-mediated responses prevent the premature decline of regenerative capacity after injury.

Authors

Joonseok Cho, Rushdia Yusuf, Sungho Kook, Eyal Attar, Dongjun Lee, Baehang Park, Tao Cheng, David T. Scadden, Byeong Chel Lee

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Figure 7

Impact of P2Y14 deficiency on senescence-associated molecules.

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Impact of P2Y14 deficiency on senescence-associated molecules. (A) WT an...
(A) WT and P2ry14–/– BM cells were transplanted as described. Eight months after transplantation, recipient mice were either left untreated (n = 4, left) or irradiated (n = 4, right) with TBI (6 Gy). LSK cells derived from WT (CD45.1+) or P2ry14–/– (CD45.2+) donors in the recipients were sorted and subjected to Q-PCR analysis, respectively. The expression level in WT cells was arbitrarily set to 1. The fold change in expression of each gene was calculated using the ΔΔCt method. The expression was normalized to GAPDH. The 2-tailed Student’s t test was used. (B) Four weeks after TBI (6 Gy), LSK and CD150+CD48– LSK cells were gated and analyzed for the expression of p16Ink4a by flow cytometry analysis. Mice were analyzed individually (n > 3 mice/group). Representative flow cytometric analysis of p16Ink4a in gated LSK and CD150+CD48–LSK cells is shown (left). The 2-tailed Student’s t-test was used. (C) Western blot analysis of WT and P2ry14–/– MEF cells: early passage WT or P2ry14–/– MEFs were prepared and serially passaged following a 3T3 protocol. Cell lysates were probed with the indicated antibodies. Autoradiographs were analyzed by densitometry. The intensity observed in passage no. 2 WT MEF cells was normalized to the β-actin and arbitrarily set to 1.0. The normalized signal intensities of ppRb and pRb proteins were expressed as a ppRb/pRb ratio. The ratio of ppRb/pRb in passage no. 2 WT MEF cells was arbitrarily set to 1.0. Number denotes passage numbers. *P < 0.05; **P < 0.01.