TSPCs were isolated from 2-month-old and 20-month-old male C57BL/6 mice. The expression of Wnt5a was determined by RNA sequencing, qRT-PCR and western blotting. TSPCs were then treated with Wnt5a shRNA or recombinant Wnt5a or AG490 or IFN-γ or Ror2-siRNA. Western blotting, β-gal staining, qRT-PCR, immunofluorescence staining and cell cycle analysis were used for confirming the role of Wnt5a in TSPCs senescence.
We found a canonical to noncanonical Wnt signaling shift due to enhanced expression of Wnt5a in aged TSPCs. Functionally, we demonstrated that inhibition of Wnt5a attenuated TSPCs senescence, age-related cell polarity and the senescence-associated secretory phenotype (SASP) expression in aged TSPCs. Mechanistically, the JAK-STAT signaling pathway was activated in aged TSPCs, while Wnt5a knockdown inhibited the JAK-STAT signaling pathway, suggesting that Wnt5a modulates TSPCs senescence via JAK-STAT signaling pathway. Moreover, knockdown of Ror2 inhibited Wnt5a-induced activation of the JAK-STAT signaling pathway, which indicates that Wnt5a potentiates JAK-STAT signaling pathway through Ror2, and Ror2 acts as the functional receptor of Wnt5a in TSPCs senescence.
Our results demonstrate a critical role of noncanonical Wnt5a signaling in TSPCs senescence, and Wnt5a could be an attractive therapeutic target for antagonizing tendon aging.