The rejuvenation of bone tissue remains a formidable challenge for osteoporosis (OP) patients who suffer severe bone degeneration or structural deterioration. To reverse the bone loss associated with OP, a dual-enzyme cascade system (PCF@EA) was engineered as a biomimetic engine to regulate bone metabolism imbalance. The bienzyme-driven system was constructed by integrating functionalized polymeric composite fibers with two mineralization-promoting hydrolases: recombinant human ectonucleotide pyrophosphatase/phosphodiesterase 1 (rhENPP1) and recombinant human alkaline phosphatase (rhALP). The bienzyme-driven engine efficaciously navigates mitochondria-activated mineralization through an autophagic process, thereby promoting osteogenic differentiation, increasing intracellular inorganic phosphate (Pi), and facilitating calcium influx. Concurrently, metabolic regulation mediated by the bienzyme-driven engine involves the PI3K–Akt pathway, tricarboxylic acid (TCA) cycle and glycerophosphate metabolism, significantly down-regulating the mineralization inhibitor pyrophosphate (PPi) while accelerating the formation of phosphate-related metabolites. Moreover, the enzyme-loaded substrates inhibited bone resorption by reducing expression of osteoclast activity markers, including Trap and Cath-K. This bienzymatic cascade strategy has demonstrated efficacy in restoring bone homeostasis in osteoporotic rats, significantly improving defect maturation and catalyzing the recovery of bone mineral density from severe loss back to baseline levels. The unique features of the bienzyme-driven engine provide a promising approach for the therapeutic treatment of degenerative skeletal diseases.
The cyclic GMP-AMP synthase (cGAS)–stimulator of IFN genes (STING) pathway is instrumental to antitumor immunity, yet the underlying molecular and cellular mechanisms are complex and still unfolding. A new paradigm suggests that cancer cells’ cGAS-synthesized cGAMP can be transferred to tumor-infiltrating immune cells, eliciting STING-dependent IFN-β response for antitumor immunity. Nevertheless, how the tumor microenvironment may shape this process remains unclear. In this study, we found that extracellular ATP, an immune regulatory molecule widely present in the tumor microenvironment, can potentiate cGAMP transfer, thereby boosting the STING signaling and IFN-β response in murine macrophages and fibroblasts. Notably, genetic ablation or chemical inhibition of murine volume-regulation anion channel LRRC8/volume-regulated anion channel (VRAC), a recently identified cGAMP transporter, abolished ATP-potentiated cGAMP transfer and STING-dependent IFN-β response, revealing a crucial role of LRRC8/VRAC in the cross-talk of extracellular ATP and cGAMP. Mechanistically, ATP activation of the P2X family receptors triggered Ca2+ influx and K+ efflux, promoting reactive oxygen species production. Moreover, ATP-evoked K+ efflux alleviated the phosphorylation of VRAC’s obligate subunit LRRC8A/SWELL1 on S174. Mutagenesis studies indicated that the phosphorylation of S174 on LRRC8A could act as a checkpoint for VRAC in the steady state and a rheostat of ATP responsiveness. In an MC38-transplanted tumor model, systemically blocking CD39 and ENPP1, hydroxylases of extracellular ATP and cGAMP, respectively, elevated antitumor NK, NKT, and CD8+ T cell responses and restrained tumor growth in mice. Altogether, this study establishes a crucial role of ATP in facilitating LRRC8/VRAC transport cGAMP in the tumor microenvironment and provides new insight into harnessing cGAMP transfer for antitumor immunity.
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