Distinct P2 receptors initiate intracellular signaling involved in mechanotransduction in tenocytes

Tendons adapt to mechanical load through mechanotransduction, but the molecular mechanisms underlying this process have not been fully elucidated. In other musculoskeletal tissues, purinergic (P2) receptors-activated by extracellular nucleotides released during mechanical stress-are critical regulators of cellular responses. Here, we investigated whether similar P2 signalling pathways are present in tendons. We show that primary tenocytes express several P2 receptors, including P2X4, P2Y₂ and P2Y₆. Live-cell imaging revealed that both ATP and UTP trigger transient intracellular calcium signalling via P2Y₂. Surprisingly, only ATP significantly increased expression of mechanosensitive cytokines (<i>Il6, Ptgs2</i>) and the tendon-specific transcription factor Scx, suggesting that additional P2 receptors contribute to ATP-driven gene regulation. Using bulk RNA-sequencing (RNA-seq), we performed the first unbiased, genome-wide analysis of early transcriptional responses to extracellular nucleotide stimulation in tenocytes. Transcriptomic profiling revealed that ATP selectively activates gene programs linked to inflammation and immune responses, whereas both ATP and UTP regulate genes associated with cell differentiation, angiogenesis, metabolism, and responses to mechanical stimuli. Consistent with these findings, receptor-specific inhibition demonstrated that P2Y₂ and P2X4 both contribute to ATP-induced transcriptional changes, whereas broader P2 receptor inhibition completely abrogated ATP-driven gene expression, supporting redundant or convergent purinergic signalling in tenocytes. Together, these findings suggest that while P2Y₂ governs calcium dynamics, the broader purinergic signalling network, including P2Y₂, P2X4, and possibly others, serves as a central mediator of tenocyte mechanotransduction.