ECHO: the executable chondrocyte

Introduction: We recently presented ANIMO (Analysis of Networks with Interactive Modeling), a software tool for modeling dynamic molecular networks for use by biologists [1, 2]. We used ANIMO to generate a computational model of articular cartilage. Materials and methods: Based on a largescale literature study [3] and our own experiments, we developed ECHO (Executable Chondrocyte), a computational model of the key processes that regulate expression and activity of SOX9 and RUNX2, two master transcriptional regulators of the chondrocyte phenotype. ECHO consists of 93 nodes with 274 interactions that describe the expression and activity of 52 genes and proteins. Simulations in ECHO were performed to investigate the robustness of the chondrocyte network. To validate ECHO predictions, we used FRAP to measure mobility of SOX9 and RUNX2, which we have shown to be a faithful readout of their activity. Results: In its unperturbed form, ECHO displays two stable states in which activities of SOX9 and RUNX2 are mutually exclusive. SOX9 rep resents a stable articular cartilage phenotype, while RUNX2 represents transient hypertrophic cartilage. We tested in silico the hypothesis that addition of WNT (performed with a few clicks of the mouse) will change permanent cartilage into transient cartilage by inducing hyper trophy. Indeed, when we add WNT, a known regulator of bone forma tion, the permanent or SOX9 + state changes to a transient or RUNX2 + state in the model. However, it is known that healthy articular cartilage is resistant to hypertrophic differentiation. Our group has previously shown experimentally that this was probably due to the secretion of DKK1, FRZB and GREM1[4, 5]. We therefore added nodes to ECHO representing DKK1, FRZB and GREM1 (fig. 1). GREM1 and DKK1 are able to stabilize the permanent cartilage or SOX9 + state even after addition of WNT in ECHO. We observed that in our model activation of WNT leads to a switch from a SOX9 + state to a RUNX2 + state. To prove that WNT/bcate nin signaling can directly regulate SOX9 function, we investigated the response of SOX9 mobility to WNT3A in live primary chondrocytes. Addition of WNT3A to human chondrocytes transfected with SOX9GFP resulted in a significant decrease of the immobile SOX9 frac tion from 53% to 34% within 15 minutes after addition, indicating a loss of transcriptional activity of SOX9. Discussion and conclusion: Using ECHO we predicted the stimuli that prevent hypertrophic differentiation of articular cartilage, and tested this experimentally with FRAP using SOX9 and RUNX2 mobility as a readout.