Mutual repression between JNK/AP-1 and JAK/STAT stratifies senescent and proliferative cell behaviors during tissue regeneration.

Epithelial repair relies on the activation of stress signaling pathways to coordinate tissue repair. Their deregulation is implicated in chronic wound and cancer pathologies. Using TNF-α/Eiger-mediated inflammatory damage to Drosophila imaginal discs, we investigate how spatial patterns of signaling pathways and repair behaviors arise. We find that Eiger expression, which drives JNK/AP-1 signaling, transiently arrests proliferation of cells in the wound center and is associated with activation of a senescence program. This includes production of the mitogenic ligands of the Upd family, which allows JNK/AP-1-signaling cells to act as paracrine organizers of regeneration. Surprisingly, JNK/AP-1 cell-autonomously suppress activation of Upd signaling via Ptp61F and Socs36E, both negative regulators of JAK/STAT signaling. As mitogenic JAK/STAT signaling is suppressed in JNK/AP-1-signaling cells at the center of tissue damage, compensatory proliferation occurs by paracrine activation of JAK/STAT in the wound periphery. Mathematical modelling suggests that cell-autonomous mutual repression between JNK/AP-1 and JAK/STAT is at the core of a regulatory network essential to spatially separate JNK/AP-1 and JAK/STAT signaling into bistable spatial domains associated with distinct cellular tasks. Such spatial stratification is essential for proper tissue repair, as coactivation of JNK/AP-1 and JAK/STAT in the same cells creates conflicting signals for cell cycle progression, leading to excess apoptosis of senescently stalled JNK/AP-1-signaling cells that organize the spatial field. Finally, we demonstrate that bistable separation of JNK/AP-1 and JAK/STAT drives bistable separation of senescent signaling and proliferative behaviors not only upon tissue damage, but also in RasV12, scrib tumors. Revealing this previously uncharacterized regulatory network between JNK/AP-1, JAK/STAT, and associated cell behaviors has important implications for our conceptual understanding of tissue repair, chronic wound pathologies, and tumor microenvironments.

Distinct signaling signatures drive compensatory proliferation via S-phase acceleration.

Regeneration relies on cell proliferation to restore damaged tissues. Multiple signaling pathways activated by local or paracrine cues have been identified to promote regenerative proliferation. How different types of tissue damage may activate distinct signaling pathways and how these differences converge on regenerative proliferation is less well defined. To better understand how tissue damage and proliferative signals are integrated during regeneration, we investigate models of compensatory proliferation in Drosophila imaginal discs. We find that compensatory proliferation is associated with a unique cell cycle profile, which is characterized by short G1 and G2 phases and, surprisingly, by acceleration of the S-phase. S-phase acceleration can be induced by two distinct signaling signatures, aligning with inflammatory and non-inflammatory tissue damage. Specifically, non-autonomous activation of JAK/STAT and Myc in response to inflammatory damage, or local activation of Ras/ERK and Hippo/Yki in response to elevated cell death, promote accelerated nucleotide incorporation during S-phase. This previously unappreciated convergence of different damaging insults on the same regenerative cell cycle program reconciles previous conflicting observations on proliferative signaling in different tissue regeneration and tumor models.

JNK-dependent cell cycle stalling in G2 promotes survival and senescence-like phenotypes in tissue stress.

The restoration of homeostasis after tissue damage relies on proper spatial-temporal control of damage-induced apoptosis and compensatory proliferation. In Drosophila imaginal discs these processes are coordinated by the stress response pathway JNK. We demonstrate that JNK signaling induces a dose-dependent extension of G2 in tissue damage and tumors, resulting in either transient stalling or a prolonged but reversible cell cycle arrest. G2-stalling is mediated by downregulation of the G2/M-specific phosphatase String(Stg)/Cdc25. Ectopic expression of stg is sufficient to suppress G2-stalling and reveals roles for stalling in survival, proliferation and paracrine signaling. G2-stalling protects cells from JNK-induced apoptosis, but under chronic conditions, reduces proliferative potential of JNK-signaling cells while promoting non-autonomous proliferation. Thus, transient cell cycle stalling in G2 has key roles in wound healing but becomes detrimental upon chronic JNK overstimulation, with important implications for chronic wound healing pathologies or tumorigenic transformation.

JAK/STAT signalling mediates cell survival in response to tissue stress.

Tissue homeostasis relies on the ability of tissues to respond to stress. Tissue regeneration and tumour models in Drosophila have shown that c-Jun amino-terminal kinase (JNK) acts as a prominent stress-response pathway promoting injury-induced apoptosis and compensatory proliferation. A central question remaining unanswered is how both responses are balanced by activation of a single pathway. Signalling through the Janus kinase/Signal transducers and activators of transcription (JAK/STAT) pathway, which is a potential JNK target, is implicated in promoting compensatory proliferation. While we observe JAK/STAT activation in imaginal discs upon damage, our data demonstrate that JAK/STAT and its downstream effector Zfh2 promote the survival of JNK signalling cells. The JNK component fos and the pro-apoptotic gene hid are regulated in a JAK/STAT-dependent manner. This molecular pathway restrains JNK-induced apoptosis and spatial propagation of JNK signalling, thereby limiting the extent of tissue damage, as well as facilitating systemic and proliferative responses to injury. We find that the pro-survival function of JAK/STAT also drives tumour growth under conditions of chronic stress. Our study defines the function of JAK/STAT in tissue stress and illustrates how crosstalk between conserved signalling pathways establishes an intricate equilibrium between proliferation, apoptosis and survival to restore tissue homeostasis.

Recognition of a basic AP-2 binding motif within the C2B domain of synaptotagmin is dependent on multimerization.

Synaptotagmin is a multifunctional membrane protein that may regulate exo-endocytic cycling of synaptic vesicles at the presynaptic plasmalemma. Its C2B domain has been postulated to interact with a variety of effector molecules including acidic phospholipids, phosphoinositides, SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), calcium channels, and the clathrin adaptor complex AP-2. Here we report that a basic motif within the C2B domain is required and sufficient for binding to AP-2 via its mu2 subunit and that this interaction is dependent on multimerization of the AP-2 binding site. Moreover, we show that upon fusion to a plasma membrane reporter protein this sequence is sufficient to target the chimeric molecule for internalization. We hypothesize that basic motifs within multimeric membrane proteins may represent a novel type of clathrin/AP-2-dependent endocytosis signal.