Introduction: Urine of female patients with overactive bladder syndrome (OAB) is characterized by low levels of the neurotrophin NGF with stable levels of its precursor proNGF. The resulting imbalance between NGF and proNGF may originate from an elevated activity of the proteolytic enzyme matrix metalloproteinase-9 (MMP-9) observed in the same samples. On the other hand, an inhibitor of the proinflammatory p75NTR receptor, THX-B, restored NGF levels to normal in a murine model of diabetic voiding dysfunction (Mossa et al 2020). The aim of the present work is to: 1) determine if NGF and MMP-9 are synthesized by cells of the bladder; 2) assess the role of MMP-9 in the synthesis and release of NGF by the same cells; and 3) examine how NGF secretion and MMP-9 might be affected by THX-B.
Methods: Rat bladders were digested by collagenase type IV to generate primary cultures of urothelial and smooth muscle cells. The expression of NGF, proNGF and MMP-9 were assessed by RT-qPCR and by immunoblotting. Cellular localisation of proteins was obtained by immunohistochemistry. Knock-down of MMP-9 was achieved using Crispr-Cas9. Levels of NGF and proNGF were measured by ELISA and enzyme activities by specific enzymatic kits.
Results: Urothelial and smooth muscle cells were found to contain and release significant amounts of NGF, proNGF and MMP-9 as revealed by RT-qPCR, immunoblotting and microscopy. The knock-down of MMP-9 using Crispr-Cas9 resulted in the disappearance of MMP-9 mRNA, proteins and enzymatic activity, in both cell types. In the MMP-9 knock-down cells, levels of secreted NGF were multiplied by a factor 4 to 9 while proNGF concentrations were not affected. On the other hand, incubation of urothelial cells with the p75 antagonist THX-B (5 μg/mL) for 24 hours increased NGF secretion and concomitantly decrease the activity of MMP-9. MMP-7, the enzyme converting proNGF to NGF, displayed an increased activity as well. On the other hand, THX-B had no effect on smooth muscle cells. Levels of mRNA were not affected by THX-B in both cell types. Pathways associated to p75NTR, namely erk, jnk, p38MAPK and cyclic AMP were also unchanged by treatment with THX-B.
Conclusions: Bladder cells express, synthesize and release NGF, proNGF and MMP-9. The latter is central in the control of NGF secretion. THX-B targets urothelial cell to enhance NGF secretion by downregulation of MMP-9 and increased in MMP-7 activities, which could explain the improvement of diabetic voiding dysfunction in vivo.
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