Inflammation Promotes the Processing of Glial Cell-Line Derived Neurotrophic Factor by Matrix Metalloproteinase-9 in Intestinal Smooth Muscle Cells
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The control and regulation of all major intestinal functions are coordinated by neurons within the enteric nervous system (ENS). While intestinal inflammation initially causes a profound loss of enteric neurons, the ENS demonstrates remarkable changes in neuronal structure and function to retain control of gastrointestinal functions. Expression of the neurotrophin glial cell line-derived neurotrophic factor (GDNF) has been implicated in promoting postnatal regenerative processes in the ENS. It has been proposed that intestinal smooth muscle cells (ISMC) express GDNF as a precursor molecule that requires proteolytic processing to achieve the mature form (15 kDa). The expression of mature GDNF is increased during intestinal inflammation, however, little is known about how inflammatory conditions affect the synthesis, processing, and biological activity of GDNF. Analysis of a neonatal rat co-culture model confirmed that the pro-inflammatory cytokine TNF-α caused a 2-fold increase in both the expression of 15 kDa GDNF and in the axonal outgrowth of myenteric neurons, compared to control. Evaluation of broad-spectrum protease inhibitors or inhibitors of metalloproteinases (MMP) showed that only inhibition of MMP-9 specifically blocked TNFα-induced axonal proliferation. The inhibition of MMP-9 in co-cultures also blocked the increase in 15 kDa GDNF expression, while the overall expression of GDNF was not affected. Assessment of MMP-9 expression in ISMC showed that TNF-α caused a greater than 2-fold increase in intracellular expression, compared to control. Significantly, the addition of exogenous 15 kDa GDNF rescued axonal proliferation during MMP-9 inhibition, while transfection with full-length GDNF did not. Finally, a neuron survival bioassay showed that 15 kDa GDNF in conditioned media from a GDNF-expression system doubled enteric neuron survival over serum-free control, while addition of the MMP-9 inhibitor blocked the increase in survival. These findings suggest that both the expression and subsequent intracellular processing of GDNF in the intestine are influenced by inflammatory factors, through the induction of MMP-9 activity. Furthermore, this evidence indicates that GDNF processing to the mature form is required for GDNF-mediated neurotrophic outcomes in the co-culture model.