Hypothesis / aims of study
The nerve growth factor precursor (proNGF) activates p75NTR receptor and promotes cell death and degeneration in different tissues. Tissue levels of proNGF build up in conditions such as diabetes, inflammation and ischemia. Given that proNGF and p75NTR are expressed in different layers of the bladder, we aimed to identify the biological effect of proNGF/p75NTR activation on urothelial (UT) and smooth muscle (SM) cells of rodents’ bladder.
Study design, materials and methods
UT and SM cells cultured from bladder of Sprague Dawley rats (passages 2-7) were incubated with proNGF (5 or 10 nM) for different durations. Total cellular or nuclear protein extraction was performed, and the extracts were tested for TNF-α, RhoA, p-JNK and NF-κB levels by western blotting. Nitric oxide (NO) estimation was performed on cells medium. Nuclear translocation of NF-κB was also confirmed with immunocytofluorescence. Cell viability and proliferation were assessed by MTT test and migration assay (wound healing assay).
Interpretation of results
The reduced viability of UT with increased expression of TNF-α and RhoA can be related to the activation of the death domain of p75NTR receptor, a member of the TNF family. The activation of these pathways through proNGF/p75NTR impacted secretory and functional integrity, as represented with lower NO and junctional protein occludin. However, p75NTR seems to promote cell survival in detrusor smooth muscle cells mostly via NF-κB activation, with enhanced proliferation and migration.