SIMULATING INTRACELLULAR SIGNALLING PATHWAYS IN INTERVERTEBRAL DISC HOMEOSTASIS AND DEGENERATION

Intervertebral disc (IVD) degeneration (IDD) disrupts extracellular matrix homeostasis, through intricate biological processes. A prior protein-protein interaction network for nucleus pulposus cells (NPC) predicted cytokine responses but lacked resolution to capture intracellular signaling dynamics. Understanding these pathways is crucial for uncovering regulatory mechanisms and developing targeted IDD therapies. We reviewed 260 peer-reviewed papers to identify key signaling pathways in IVD regulation, focusing on NPC and general cell data. Supplemented by Reactome and KEGG databases, we selected key ligands (BMP, GDF5, IGF1, IL-1β, IL-1Ra, IL-4, IL-6, IL-10, IL-12, IL-17, IFN-?, TGF-β, TNF) and traced their signaling pathways from receptor binding to nuclear translocation. The knowledge-based network was translated into a dynamic model, through semi-quantitative interpolations of Boolean simulations. The model was assessed against 3D (trauma) and monolayer (degenerate IVD) human NPC cultures, under IL-1β stimulation. The network model included 67 signaling proteins and transcription factors, with 185 interactions, representing a non-degenerate state of NPC IVD (Fig. 1A), with high levels of: structural proteins (ACAN, COL2A); growth factors (TGF-β, GDF5, IGF1); transcription factor Sox9; SMAD2/3, a downstream mediator of TGF-β signaling. Simultaneously, it predicted low levels of: pro-inflammatory cytokines (IL-1β, IL-6, IL-17, IL-12, IFN-?, TNF); degrading enzymes (ADAMTS4/5, VEGF, MMPs); chemokines (CCL3/4); stress-activated kinases (p38, JNK); mediators of pro-inflammatory responses (TLR); transcription factor AP-1; p65 subunit of NF-?B. Upon IL-1β perturbation, model simulations agreed with the in vitro results that showed increased expression of c-Jun and p65 in 3D and monolayer culture. When activating TGF-β in the IL-1β stimulated network, ACAN, COL2A, GDF5, IL-1Ra, Sox9, Akt1 and SIRT1 were increased. The network model uniquely incorporated intracellular signaling dynamics and represented successfully the expected homeostatic NPC state in a non-degenerate IVD. Simulations aligned with experimental studies in human NPCs from IVD which demonstrated increased expression of c-Jun and p65 subunit of NF-?B following IL-1β stimulation, passing a key falsification test. Simulated rescue by TGF-β was, furthermore, consistent with prior findings in mouse and rat models. While experimental studies in human NPC remain necessary to explore signaling proteins for IDD treatments, this model offers a valuable tool to understand mechanistically intricate signaling mechanisms and guide IVD research.