Extracellular ATP drives tryptophan metabolism and aryl hydrocarbon receptor activation to promote cellular senescence.

We have shown that extracellular ATP promotes senescence through the activation of the P2Y11 receptor (P2Y11R). The underlying molecular mechanisms remain to be fully established. Synthesis of tryptophan (Trp)-derived indole metabolites is mediated mostly by the gut microbiota. Trp metabolites can activate the aryl hydrocarbon receptor (AhR). Whether eukaryotic cells can generate Trp-derived indoles and their functional significance remains to be fully established. Here, we investigated the role of Trp metabolites and AhR activation in purinergic-mediated senescence of human fibroblasts. We find that ATP activated AhR in a P2Y11R-dependent manner and that AhR activation was necessary for ATP-induced senescence. Stimulation with an AhR agonist was sufficient to induce senescence. Interestingly, depletion of Trp in the conditioned medium inhibited ATP-induced senescence. We show that ATP stimulation upregulated the expression of the L-amino acid oxidase interleukin-4-induced-1, which has been shown to metabolize Trp into indole-3-pyruvate (I3P), in a P2Y11R-dependent fashion. We find that I3P-derived Trp metabolites are upregulated in ATP-induced senescent human fibroblasts and that stimulation with I3P and I3P-derived Trp metabolites was sufficient to promote senescence in these cells. In addition, I3P stimulation activated AhR, and AhR inhibition impaired I3P-induced senescence. Downregulation of interleukin-4-induced-1 inhibited ATP-induced AhR activation and senescence. Finally, we show that conditioned medium derived from senescent lung fibroblasts, which were induced to senesce by I3P treatment, promoted the proliferation of breast cancer cells and their tumorigenic potential. Our study identifies the existence of a novel purinergic dependent signaling pathway that functionally couples Trp metabolism to the development of a premature senescent phenotype in human fibroblasts.