The role of proNGF is controversial, and both neurotrophic and apoptotic activities have been reported for recombinant proNGF. It has been shown that proNGF can also be neurotrophic, regardless of mutations or tags, and no matter how it is purified or in which system it is expressed. However, although proNGF is neurotrophic for primary sympathetic neurons and for PC12 cells, it is reported to be apoptotic for unprimed PC12 cells . Furthermore, proNGF has been reported to promote apoptosis via its interaction with p75NTR and sortilin receptor [7, 28]. Both proNGF and mature NGF exhibited neurotrophic activity on PC12 cells, while the pro-domain itself promotes cell death . NGF and proNGF co-exist in vivo. One might therefore expect that the proNGF/NGF mixture modulates the downstream effect of either neurotrophin form, in ways depending on their ratio, and distinct from NGF and proNGF alone. However, it is not known whether and how the concomitant presence of proNGF affects cell responses to NGF.
Our data refer to the PC12 cells system and to mRNAs expression read-out as a response signature. This experimental system was chosen, together with the gene expression read-out, as a well defined system, for investigating the complex question of the functional consequences of signalling by mixtures of NGF/proNGF, in comparison to signalling by pure proNGF or pure NGF. We expect that proNGF/NGF mixtures might modulate the downstream responses, depending on their ratio, in ways distinct from those affected by NGF or proNGF alone, also in different systems than PC12 cells. It remains however to be ascertained whether and how the distinct signalling behaviours of the NGF/proNGF mixture, here demonstrated, also apply to neuronal cells. Thus, we have analysed by qReal-Time PCR the expression of a set of genes, in PC12 cells treated for short times with proNGF, NGF or with a mixture of the two proteins. The emphasis on short times was to minimize the chance of processing of proNGF by cellular proteases in the culture medium, which is known to occur at times longer than a few hours .
From our results, we conclude that there are a significant number of cases where proNGF has a prevalent effect over NGF. This effect is time dependent, as well as dependent on the relative concentration of the two proteins. Distinct proNGF-specific effects on NGF-induced gene expressions were identified. It should be pointed out that we do not imply to put forward a functional interpretation of the gene expression changes observed. Rather, we take the gene expression patterns as a fingerprinting read-out of possible actions of proNGF/NGF mixtures, as compared to those regulated by NGF or proNGF alone. Most of the selected genes are essentially functionally unrelated (Figure 1) and any correlated behaviour, observed in the PC12 cellular model, could be attributed to the specific treatment.
Taking into account single additions, among the selected panel of genes, about 50% of Group I shows an opposite trend in the single NGF or proNGF treatments at 30 minutes. Higher doses of proNGF anticipate at 15 minutes the proNGF up-regulated response, observed at 30 minutes for that group of genes. This suggests there may be a proNGF-induced dose-dependent effect on early signalling. Hierarchical clustering of samples highlights this low degree of similarity between the NGF and proNGF responses at 30 minutes. When NGF and proNGF were added simultaneously, the effect of the mixture on gene expression was often different from the effect of the single ligands added separately. Although some NGF-like responses were observed for the mixture (indicating proNGF being neutral), in many cases the mixture behaved differently. In particular, in the case of an equimolar mixture of proNGF and NGF, synergic, additive or mutually antagonizing effects were observed for different mRNAs. We can therefore identify a non-linear “proNGF effect” interfering with the NGF-induced gene expression.
Increased proNGF levels are observed during human ageing and pathological conditions [11, 12]. This prompted us to analyse the effect of increasing proNGF concentration in the proNGF/NGF mixture. Different patterns of expression responses were obtained. At 5 minutes, the responses to NGF or proNGF alone are often different; in those cases, the response to mixtures shows a variable pattern, either similar to proNGF-response or to NGF-response or peculiar to the mixture, whereas at 15 minutes, the most represented group of genes shows a mixture-specific response. Moreover, the response is not always linear with the increasing amount of proNGF, being often reverted at the highest concentration. We conclude that the proNGF/NGF mixture shows a mixture-specific signature, that at these early time points (5 and 15 minutes) is not necessarily only transcriptional, but may involve translational regulation . This aspect warrants to be further investigated. Our data support the hypothesis that the relative concentration of NGF and proNGF might be of importance for the biological outcome of the two proteins, in physiological and/or pathological conditions and suggests that the proNGF/NGF balance is a sensitive point of regulation in the homeostasis of the system. We have recently described a transgenic mouse model expressing a form of proNGF resistant to furin cleavage , showing that different expression levels of NGF and proNGF produce neurodegeneration phenotypes of different strength in transgenic lines derived from different founders . The relative NGF and proNGF availability has been reported to regulate innervation density in postnatal Superior Cervical Ganglion (SCG) neurons: sub-saturating concentrations of proNGF and NGF act synergically to promote neurite growth, whereas the additive effect is not observed at saturating concentration. Conversely, lack of synergic effect was observed in trigeminal neurons, highlighting that subsets of NGF-responsive neurons have distinctive responses to NGF and proNGF . Our data show that the mixture of proNGF and NGF acts differently, depending on the proNGF/NGF ratio, highlighting that conflict, synergism and/or cancellation occur when both NGF and proNGF downstream pathways are similarly activated through different receptors. The biochemical site of this conflict or synergism is currently unknown, and most likely involves interactions at the level of the signalling pathways, starting from the receptor(s) level and determining distinct downstream functional consequences. In any case, the interactions between the NGF and proNGF signalling pathways, when concomitantly activated, can be described as if the proNGF/NGF mixture functionally behaves as a “new ligand”, distinct from either NGF or proNGF alone.