Lady Davis Institute for Medical Research
Antagonism of the p75NTR receptor decreases MMP-9 activity and increases NGF in urothelial cells
Aalya Hamouda1, Philippe Cammisotto1, Lysanne Campeau1.
1Lady Davis Institute for Medical Research, McGill University, Montreal, QC, Canada
Introduction: Decreased levels of nerve growth factor (NGF) have been oberved in the urine of patients with overactive bladder syndrome (OAB) and of diabetic animal models. This unbalance appeared to result from an increased activity of the proteolytic enzyme metalloproteinase-9 (MMP-9). On the other hand, THX-B, an inhibitor of p75NTR, restored normal levels of NGF in mice with type 1 diabetes. We here examine in vitro the effect of THX-B on the activity of MMP-9 in bladder cells.
Methods: Primary culture of urothelial and smooth muscle cells (SMCs) were grown from mouse bladder and exposed to p75NTR antagonist THX. The expression of NGF and MMP-9 were assessed by RT-qPCR, by immunohistochemistry and by immunoblotting. Levels of microRNAs were measured by RT-qPCR. NGF and proNGF secretion were measured by ELISA kits and MMP-9 activity by enzymatic assays.
Results: The mRNAs for NGF and MMP-9 were detected and found expressed in both cell types at similar level. Microscopy confirmed the localization of both proteins in cells. Urothelial cells and SMCs were both major source of NGF and proNGF. On the other hand, MMP-9 protein content was 7 times higher in SMCs than in urothelial cells, which was confirmed by high levels of miR-491-5p in the latter. However, secretion of active MMP-9 in the medium was 40 times higher in urothelial cells medium. Incubation with THX-B (5 µg/mL) for 24 hours abolished the synthesis and secretion of MMP-9 and doubled the concentration of NGF in the medium of urothelial cells. ProNGF secretion levels were not affected. THX-B had little effects on SMCs both at the level of NGF and MMP-9.
Conclusions: Urothelial cells appear to be the primary target for THX-B to modulate secretion of NGF and MMP-9. These results are in accordance with our previous publications on OAB patients and diabetes type 1 in rodents, and suggest that THX-B could be a therapeutic tool to improve OAB by targeting primarily the urothelium.
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