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Transforming growth factor-β1 (TGF-β1) is a potent regulator of extracellular matrix production, wound healing, differentiation, and immune response, and is implicated in the progression of fibrotic diseases and cancer. Extracellular activation of TGF-β1 from its latent form provides spatiotemporal control over TGF-β1 signaling, but the current understanding of TGF-β1 activation does not emphasize cross talk between activators. Plasmin (PLS) and thrombospondin-1 (TSP1) have been studied individually as activators of TGF-β1, and in this work we used a systems-level approach with mathematical modeling and in vitro experiments to study the interplay between PLS and TSP1 in TGF-β1 activation. Simulations and steady-state analysis predicted a switch-like bistable transition between two levels of active TGF-β1, with an inverse correlation between PLS and TSP1. In particular, the model predicted that increasing PLS breaks a TSP1-TGF-β1 positive feedback loop and causes an unexpected net decrease in TGF-β1 activation. To test these predictions in vitro, we treated rat hepatocytes and hepatic stellate cells with PLS, which caused proteolytic cleavage of TSP1 and decreased activation of TGF-β1. The TGF-β1 activation levels showed a cooperative dose response, and a test of hysteresis in the cocultured cells validated that TGF-β1 activation is bistable. We conclude that switch-like behavior arises from natural competition between two distinct modes of TGF-β1 activation: a TSP1-mediated mode of high activation and a PLS-mediated mode of low activation. This switch suggests an explanation for the unexpected effects of the plasminogen activation system on TGF-β1 in fibrotic diseases in vivo, as well as novel prognostic and therapeutic approaches for diseases with TGF-β dysregulation.

Plasmin (PLS) and urokinase-type plasminogen activator (UPA) are ubiquitous proteases that regulate the extracellular environment. Although they are secreted in inactive forms, they can activate each other through proteolytic cleavage. This mutual interplay creates the potential for complex dynamics, which we investigated using mathematical modeling and in vitro experiments. We constructed ordinary differential equations to model the conversion of precursor plasminogen into active PLS, and precursor urokinase (scUPA) into active urokinase (tcUPA). Although neither PLS nor UPA exhibits allosteric cooperativity, modeling showed that cooperativity occurred at the system level because of substrate competition. Computational simulations and bifurcation analysis predicted that the system would be bistable over a range of parameters for cooperativity and positive feedback. Cell-free experiments with recombinant proteins tested key predictions of the model. PLS activation in response to scUPA stimulus was found to be cooperative in vitro. Finally, bistability was demonstrated in vitro by the presence of two significantly different steady-state levels of PLS activation for the same levels of stimulus. We conclude that ultrasensitive, bistable activation of UPA-PLS is possible in the presence of substrate competition. An ultrasensitive threshold for activation of PLS and UPA would have ramifications for normal and disease processes, including angiogenesis, metastasis, wound healing, and fibrosis.

Fibrotic diseases are often caused by chronic injuries, and are characterized by pathological over-activation of certain stages of wound healing, particularly extracellular matrix secretion. Matrix secretion is driven by the fibrotic cytokine TGF-β1 (transforming growth factorβ1), which is secreted by cells as a latent complex, and must be activated extracellularly for its biological function. Plasmin is a direct proteolytic activator of TGF-β1, but puzzling results in fibrotic animal have shown that the plasmin pathway causes decreased TGF-β1.We used computational modelling and experimental tests to understand the activation of TGF-β1, by plasmin and by the matricellular glycoprotein thrombospondin1 (TSP1). Ordinary differential equations were used to model the dynamics of activation and feedback. TGF-β1 and TSP1 create a fibrogenic feedback loop, because TGF-β1 causes transcriptional up-regulation of TSP1, while TSP1 activates TGF-β1 through conformational change. Simulations showed that, although plasmin is a direct activator of TGF-β1, increasing doses of plasmin caused a decrease in TGF-β1 activation by cleaving TSP1 and antagonizing the TSP1-TGF-β1 positive feedback loop. Furthermore, the system was capable of bistability, switching between high and low levels of active TGF-β1. Key predictions of the mathematical model were validated in vitro with co-culture models of liver fibrosis, which showed inverse correlation between active TGF-β1 and plasmin. Bistability was demonstrated by showing hysteresis, where the same system achieved two different steady states, after being given the same two input stimuli but in opposite orders.Our model explains how plasmin can decrease or increase TGF-β1 activation, depending on the presence of TSP1. The bistable transition between plasmin-mediated and TSP-mediated TGF-β1 activation pathways may be relevant to the transition between inflammatory and fibrogenic phases of wound healing, suggesting novel mechanisms for addressing TGFβ1-related diseases.