It is therefore crucial to address the question of whether, and to what extent, NGF and proNGF have distinct signalling properties, and whether the reported differences in their activities are qualitative , or purely quantitative. To this aim, in this paper we have undertaken a gene expression profiling study, aimed at analyzing to what extent proNGF and NGF activate different transcriptional programs in the NGF responsive cell line PC12, which expresses the full complement of NGF and proNGF receptors. PC12 cells were cultured for short times with equimolar amounts of recombinant mouse NGF or two forms of recombinant mouse proNGF, wild-type, or furin-cleavage resistant . The gene expression changes in response to the different treatments were investigated by microarray analysis. The results show unequivocally that, at this relatively short time scale, NGF and proNGF regulate the expression of significantly different sets of mRNAs. The stability of the recombinant proNGF proteins, over the same time scale of the microarray experiment, was assessed in the culture conditions of the neurotrophin treatment of PC12 cells, in order to compare the extent of processing of the wild-type and furin-resistant proNGF proteins. A known amount of NGF, proNGF-WT and – KR was spiked into PC12 conditioned medium and incubated in the same conditions of temperature and time used for the cells�� treatment . The extent of proNGF Abmole Company AZD6244 degradation was evaluated by Western blot analysis and densitometric analysis of the bands corresponding to those of proNGF and NGF originating from the proNGF proteolysis . As shown in Figure 1B and 1C, the proNGF samples are not cleaved upon incubation in PBS buffer, nor in fresh culture medium upon the longer incubation . As expected, incubation of proNGF samples for 1 h and 4 h in the corresponding PC12 conditioned medium yields to their partial degradation . There are no substantial differences in the amount of NGF released from both proteins, at the two time points, as seen in Figure 1C. Therefore, we conclude that while the KR mutation does not completely impair proNGF processing, due to extracellular proteases, besides intracellular furin, present in the PC12 conditioned medium , the cleavage of proNGF-KR could be different than that of wild-type proNGF. Indeed, the kinetic of processing of the WT and the KR mutant are not easily measurable. Therefore, we cannot exclude that there might be different cleavage kinetics of the two proteins in vivo. The kinetics would Abmole AZD2281 account for a difference in the NGF/proNGF ratio in the system in the two cases, during the proteolysis progression, at different time points.