Antagonism of the p75NTR receptor decreases the extracellular activity of matrix metalloproteinase-9 and increases secretion of nerve growth factor by mouse urothelial cells in culture
Aalya Hamouda1,2, Aya Hajj1,2, Stephanie Sirmakesyan1,2, Philippe Cammisotto1, Uri Saragovi1, Lysanne Campeau1,2,3.
1Molecular and Regenerative Medicine, Lady Davis Research Institute, Montreal, QC, Canada; 2Experimental Surgery, McGill University, Montreal, QC, Canada; 3Surgery, Jewish General Hospital, Montreal, QC, Canada
Introduction: Overactive bladder (OAB) syndrome was reported to be characterized by low levels of nerve growth factor (NGF) in the urine of aging female patients, with stable levels of proNGF and a decreased NGF/proNGF ratio.1 This was linked to high activity of metalloproteinase-9 (MMP-9) that digests NGF.1 P75 neurotrophin receptor (p75NTR) antagonism with THX-B restored normal NGF levels in bladders of type 1 diabetic mice with voiding dysfunction.2 Here, we examine the in vitro effect of THX-B on MMP-9 activity in bladder cells and the consequences on secreted NGF.
Methods: Primary culture of urothelial cells (UCs) and smooth muscle cells (SMCs) were grown from rat bladders. RT-qPCR assessed NGF and MMP-9 gene expression. Immunohistochemistry and immunoblotting assessed protein expression. NGF and proNGF secretion were measured by ELISA and MMP-9 activity by enzymatic assays.
Results: NGF and MMP-9 mRNAs were expressed in UCs and SMCs at similar levels. At the intracellular protein level, NGF and proNGF were abundant in UCs, while SMCs produced a limited amount. Intracellular MMP-9 was seven times higher in SMCs than in UCs. The opposite patterns were observed extracellularly; secretion of active MMP-9 was 40 times higher in UC medium and was paralleled with lower extracellular NGF and proNGF compared to SMCs. THX-B treatment decreased the synthesis and secretion of MMP-9 and doubled the NGF concentration in the UC medium. ProNGF secretion was unaffected. Reduced MMP-9 was paralleled, with decreased intracellular α2-macroglobulin (α2m), a protein that prevents MMP-9 degradation. THX-B had little effect on SMCs both at the level of NGF and MMP-9.
Conclusions: UCs secrete most of the active MMP-9 and appear to be the primary target of p75NTR antagonism. The reduction MMP-9 expression and secretion by THX-B may be explained by reduced α2m in the same cells. Our results suggest that THX-B could be a therapeutic tool to improve OAB by targeting the urothelium to increase NGF.
[1] Mossa, A. H. et al. Imbalance of nerve growth factor metabolism in aging women with overactive bladder syndrome. World J Urol 2021;39(6):2055-2063
[2] Mossa, A. H. et al. Antagonism of proNGF or its receptor p75(NTR) reverses remodelling and improves bladder function in a mouse model of diabetic voiding dysfunction. Diabetologia 2020;63(9):1932-1946
