Senescent cells are characterized by multiple features such as increased expression of senescence-associated β-galactosidase activity (SA β-gal) and cell cycle inhibitors such as p21 or p16. They accumulate with tissue damage and dysregulate tissue homeostasis. In the context of skeletal muscle, it is known that agents used for chemotherapy such as Doxorubicin (Doxo) cause buildup of senescent cells, leading to the inhibition of tissue regeneration. Senescent cells influence the neighboring cells via numerous secreted factors which form the senescence-associated secreted phenotype (SASP). Lipids are emerging as a key component of SASP that can control tissue homeostasis. Arachidonic acid-derived lipids have been shown to accumulate within senescent cells, specifically 15d-PGJ₂, which is an electrophilic lipid produced by the non-enzymatic dehydration of the prostaglandin PGD₂. This study shows that 15d-PGJ₂ is also released by Doxo-induced senescent cells as an SASP factor. Treatment of skeletal muscle myoblasts with the conditioned medium from these senescent cells inhibits myoblast fusion during differentiation. Inhibition of L-PTGDS, the enzyme that synthesizes PGD₂, diminishes the release of 15d-PGJ₂ by senescent cells and restores muscle differentiation. We further show that this lipid post-translationally modifies Cys184 of HRas in C2C12 mouse skeletal myoblasts, causing a reduction in the localization of HRas to the Golgi, increased HRas binding to Ras Binding Domain (RBD) of RAF Kinase (RAF-RBD), and activation of cellular Mitogen Activated Protein (MAP) kinase–Extracellular Signal Regulated Kinase (Erk) signaling (but not the Akt signaling). Mutating C184 of HRas prevents the ability of 15d-PGJ₂ to inhibit the differentiation of muscle cells and control the activity of HRas. This work shows that 15d-PGJ₂ released from senescent cells could be targeted to restore muscle homeostasis after chemotherapy.

Ligands such as insulin, epidermal growth factor, platelet-derived growth factor, and nerve growth factor (NGF) initiate signals at the cell membrane by binding to receptor tyrosine kinases (RTKs). Along with G-protein-coupled receptors, RTKs are the main platforms for transducing extracellular signals into intracellular signals. Studying RTK signaling has been a challenge, however, due to the multiple signaling pathways to which RTKs typically are coupled, including MAP/ERK, PLCγ, and Class 1A phosphoinositide 3-kinases (PI3K). The multi-pronged RTK signaling has been a barrier to isolating the effects of any one downstream pathway. Here, we used optogenetic activation of PI3K to decouple its activation from other RTK signaling pathways. In this context, we used genetic code expansion to introduce a click chemistry noncanonical amino acid into the extracellular side of membrane proteins. Applying a cell-impermeant click chemistry fluorophore allowed us to visualize delivery of membrane proteins to the plasma membrane in real time. Using these approaches, we demonstrate that activation of PI3K, without activating other pathways downstream of RTK signaling, is sufficient to traffic the TRPV1 ion channels and insulin receptors to the plasma membrane.