NP-inducible NFκB/UPS activation
We have observed that at baseline level there is an inverse expression pattern between the NEP/NPs and NFκB/UPS pathways in AD and AI states
in vitro[
40]. We have also shown that NPs may act as inducers of NFκB activation in PC-3 cells [
45]. In the current work we have further investigated whether this mirror phenotype of the two pathways at steady state also translates to a dynamic relationship following dose- and time-dependent stimulation and blocking of the NEP/NPs pathway.
Our evidence shows that increasing concentrations of ET-1 and BBS stimulate translocation of NFκB to the cell nucleus in PC-3 cells (lacking NEP), with resultant activation of NFκB as a transcription factor, evident by increased binding on DNA. Our results concur with these of Levine et al. [
23]. They showed that in PC-3 cells BBS induced a time-dependent increase in DNA binding of NFκB peaking at 1 hour which returned to near baseline levels after that. Our results supplemented the above by showing that BBS-induced nuclear translocation is also time-dependent and peaks at the same time as the reported increase in NFκB binding (1 hour), the latter also confirmed in our EMSA results. Furthermore, we have shown that ET-1 has the exact same effect on NFκB activation, evident both at ICC and EMSA analysis.
Our results strongly support that this effect on NFκB activation is due to the NPs themselves and not a chance association. No perceivable nuclear translocation occurred at the same incubations in NEP-expressing LnCaP cells with either ET-1 or BBS. EMSA analysis, being a more sensitive technique did indeed detect a level of NFκB binding activity in these cells, but this was significantly lower compared to PC-3 cells. Similarly, rhNEP incubation successfully prevented nuclear translocation and activation in PC-3 cells.
Further evidence was provided by the fact that this upregulation in NFκB nuclear activity was prevented by the use of the respective receptor inhibitors, and this was shown both via ICC and EMSA. This indicates that this NP-induced effect is receptor-mediated. ET
AR blockade was predictably able to completely prevent nuclear translocation, as it is established that the main receptor responsible for mediating the mitogenic effects of ET-1 in PC cells is indeed the ET
A receptor. BB
2 receptor blockade was selected for our experiments based on the fact that prostate carcinomas and PC-3 cells in specific are known to abundantly express GRP-R [
23,
27,
30] and that the mitogenic/proliferative effects of BBS in prostate and other types of cancer are predominantly mediated via BB
2 receptor [
28,
46]. However, BBS acts on two other receptors, neuromedin B receptor (NMB-R) and BBS receptor subtype 3 (BRS-3), shown to be expressed in 14% and 9% of prostate carcinomas respectively [
30]. Levine et al. assumed that BBS-induced NFκB activation is due to activation of GRP-R based on the ability of BB
2 receptor antagonist to block the BBS-induced increase in intracellular Ca
++[
23]. However, our previously published concentration-series results [
45] suggest that the actual NFκB translocation and preceding proteasomal activation is mainly but not completely due to activation of this receptor. Whether blocking of the other two receptors as well would completely prevent nuclear translocation of NFκB remains to be elucidated.
We did not perform a separate analysis of the effect of ET-1 and BBS receptor inhibitors on NFκB at baseline (without NP stimulation). Firstly, a constitutive activation of NFκB has been consistently reported in androgen-independent PC-3 cells, at least partially mediated through epidermal growth factor receptors (EGFR) tyrosine kinases [
47], the extracellular signal regulated kinase-1/2 (ERK) [
48], NF-κB-inducing kinase (NIK), and IKK activation [
38,
49]. Therefore, it might seem unlikely to detect a significant effect of NP receptor inhibitors on protein levels and intracellular localization of NFκB at baseline conditions, as NFκB is regulated by multiple signaling pathways which do not necessarily involve upstream NP receptor binding. On the other hand, the use of a specific NP receptor inhibitor in the NEP-expressing LnCaP cells might not offer a significant additional blockage of the mitogenic effects of NPs, including NFκB activation, given that cleavage of NPs by NEP effectively prevents NP receptor binding. In a previous study using BQ-123 at a 10-fold higher dose compared to ours, no effect was observed on baseline secreted levels of IL-6, which is a known NFκB-target gene [
50]. In another work, prolonged exposure (72 h) to high doses (25 μM) of another endothelin receptor inhibitor (ABT-627) was needed to produce a discernible effect on NFκB activity [
51]. Based on the results of our study examining the stimulated activation of NFκB by ET-1 and BBS, it may be suggested that this is at least partially a receptor-mediated effect as it was reversed by their specific inhibitors.
This effect is associated with increase in proteasomal activity with resultant decrease in IκBα, suggesting that the NP-induced nuclear translocation is IκB-dependent, also prevented by use of NFκB/UPS inhibitors, NP receptor inhibitors and NEP. This suggests that the NP-stimulated NFκB is indeed activated via the canonical pathway.
NP-induced early proteasomal upregulation model
Our results specifically indicate that NPs are able to upregulate 20 S proteasomal activity at lower concentrations [
45] and at shorter incubations than these necessary to achieve NFκB activation. It can therefore be deduced that the increase in proteasomal activity occurs early during NP stimulation and precedes the NFκB nuclear translocation. So it might be that NPs induce proteasomal activity, and when this reaches a critical level it results in NFκB activation via decrease of total IκBα status.
The NP-associated upregulation of proteasomal activity could also explain our finding that ET
AR antagonist, blocking the action of not only the exogenous but even autocrine- and paracrine-acting ET-1 in PC-3 cells, results in a 50% reduction of baseline proteasomal activity even if it is followed by ET-1 stimulation [
45]. The fact that BB
2 receptor antagonist pre-incubation could not reduce proteasomal activity to lower than baseline [
45] could be attributed to the fact that BBS might exert its effect via other receptors as well, as discussed above. It should however be noted that, as the regulation of the proteasome complex activity is a very complicated process, it might not be possible to draw unequivocal conclusions or deduct linear relationships.
LnCaP cells have intrinsic NEP production so paracrine-secreted ET-1 is cleaved. Furthermore, there’s evidence that they have decreased expression of endothelin-converting enzyme 1 (ECE-1), with resultant decrease in production of ET-1 [
52]. It is not, therefore, surprising that the effect of exogenous NPs on proteasomal activity is comparably less intense in LnCaP cells [
45] or following rhNEP incubation in PC-3 cells. Even at high concentrations, the NP-induced proteasomal activity upregulation [
45] does not seem to be strong enough to result in critically low levels of IκBα, thereby not being able to stimulate any visible nuclear NFκB translocation, as we have demonstrated.
The underlying mechanism of NP-induced proteasomal activity upregulation is not known. It could be that NP-mediated increase in IκBα levels results in substrate induction of the proteasome. On the other hand, a direct NP-proteasome interaction or an NFκB-dependent induction of expression of regulatory components of the UPS pathway cannot be excluded and need to be further elucidated. Also, it should be acknowledged as a limitation of this study that our findings were generated only with two different cell lines (LnCaP, PC-3), although it is generally accepted that they do represent preclinical models of AD and AI states, respectively. It is therefore not possible to draw a final conclusion without further studies on other PC cell lines.