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.

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).

proNGF decreased the viability of UT cells when incubated at concentration of 5 and 10nM for 24 hours (Figure 1.A) while SM cells showed increased viability (Figure 1.B). Treating UT cells with proNGF for 24 and 48 hours increased the expression of the transmembrane TNF-α (Figure 1.C) and reduced NO secretion. After 48 hours of incubation with 5 and 10 nM proNGF caused a significant reduction in junctional protein occludin expression. Short incubation of UT cells with proNGF (10nM) activated RhoA. On the other hand, SM did not show a reduction in viability nor an increase in TNF-α or RhoA. Interestingly, there was a significant nuclear translocation of NF-κB in the SM cells in the presence of proNGF (Figure 1.D) which was unseen in urothelial cells. Long incubation of SM cells with proNGF (48 and 72 hours) did not cause changes in contractile protein expression (smoothelin and myosin). MTT test and cell migration assay showed a significant increase in SM cell proliferation and migration when incubated with proNGF which was dose- and time-dependent. SM cell viability increased by 15-20% when incubated with proNGF [10nM] for 24 and 48 hours compared to cells incubated without proNGF (Figure 1.B). An increase of 20-40% in migration capacity of SM incubated with 5 and 10 nM of proNGF was observed after 24 and 48 hours.

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.

These results suggest that proNGF causes degenerative changes in urothelial cells and opposing effects on SMC to promote cell response to stress. Our findings also reiterate the observation that p75NTR receptor have cell-type specific response that include a wide range of responses that can promote cell apoptosis as well as cell survival and growth. Further understanding of this axis can help to identify cell-type changes in the bladder diseases in context of metabolic stress and inflammation.