Staurosporine induces neurite outgrowth in neuronal hybrids (PC12EN) lacking NGF receptors.

A novel neuronal model (PC12EN cells), obtained by somatic hybridization of rat adrenal medullary pheochromocytoma (PC12) and bovine adrenal medullary endothelial (BAME) cells, was developed. PC12EN cells maintained numerous neuronal characteristics: they expressed neuronal glycolipid conjugates, synthesized and secreted catecholamines, and responded to differentiative agents with neurite outgrowth. PC12EN lacked receptors for EGF and both the p75 and trk NGF receptors, while FGF receptor expression was maintained. Staurosporine (5-50 nM), but not other members of the K252a family of protein kinase inhibitors, rapidly induced neurite outgrowth in PC12EN, as also found in the parental PC12 cells, but not in BAME cells. Similarly, both acidic and basic FGF (1-100 ng/ml) were neurotropic in PC12EN. In contrast to the mechanism by which FGF promoted neurite outgrowth in PC12EN, the neurotropic effect of staurosporine did not involve activation of established signalling pathways, such as tyrosine phosphorylation of erk (ras pathway) or SNT (a specific target of neuronal differentiation). In addition, staurosporine induced the tyrosine phosphorylation of the focal adhesion kinase p125FAK. However, since the latter effect was also observed with other protein kinase inhibitors of the K252a family, which induced PC12EN cells flattening but no neurite extension, we propose that FAK tyrosine phosphorylation may be related to ubiquitous changes in cell shape. We anticipate that PC12EN neuronal hybrids will become useful models in neuroscience research for evaluating unique cellular signalling mechanisms of novel neurotropic compounds.

K252a and staurosporine microbial alkaloid toxins as prototype of neurotropic drugs.

K252 family of alkaloid toxins-kinase inhibitors are the most widely used compounds in biological research on the role of protein kinases in cellular transduction systems, biological functions and pathophysiology of neurological disorders. The wide research interest in these toxins is due to their potency in inhibiting cellular protein kinases. However, lack of kinase specificity is one of their major drawbacks. Synthesis of new K252 derivatives can be expected to open up a new generation of kinase inhibitors. Staurosporine might be considered as a prototype neurotropic drug in view of its ability to induce neurite outgrowth and to increase tau protein levels. Because it mimics some of the neuroprotective effects of NGF and might blocks certain signals required to enhance cellular levels and/or beta amyloid processing, staurosporine might play a beneficial role in the treatment of Alzheimer's disease. The ability of staurosporine to promote neuronal regeneration and brain cholinergic neurons survival has been also demonstrated in animal studies (Nabeshima et al., 1991). The beneficial effects of K252a on the experimental autoimmune encephalomyelitis (EAE) disease mice model and it's ability to supress macrophage activation suggest an important role of protein kinases inhibitors as immunosupressive agents. These results may also point to the potential clinical relevance of K252 microbial toxins as prototypes for the development of new drugs for the management of neuronal diseases.

Short- and long-term mechanisms of tau regulation in PC12 cells.

Induction by nerve growth factor of neurite outgrowth in PC12 cells is transcription-dependent and is associated with the accumulation of tau protein. It was recently shown that short-term treatment with staurosporine, a protein kinase alkaloid inhibitor, induced an elevation of tau protein levels and outgrowth of stable neurites. In this study, we analyzed the mechanism(s) by which nerve growth factor and staurosporine exert their effects on tau levels. We demonstrate that nerve growth factor affects tau mRNA stability, thus contributing to the observed increase in tau mRNA levels. On the other hand, tau mRNA levels were not affected by the treatment with staurosporine. We also demonstrate that the phosphorylation of tau protein was reduced after treatment of PC12 cells with nerve growth factor or staurosporine, as shown by immunoblot analysis using specific antibodies and alkaline phosphatase treatment. Thus, regulation of tau levels by nerve growth factor appears to be mediated by transcriptional, post-transcriptional and posttranslational steps, whereas the effect of staurosporine on tau levels may be attributed to its effect on the state of phosphorylation of the protein.

Staurosporine induces tyrosine phosphorylation of a 145 kDa protein but does not activate gp140trk in PC12 cells.

Staurosporine, a protein kinase C inhibitor, induces neurite outgrowth in PC12 cells similarly to nerve growth factor (NGF). Since NGF neurotropic effects are transduced by the 'trk' gene product 140 kDa tyrosine kinase receptor, gp140trk, we investigated the role of gp140trk and tyrosine phosphorylations in staurosporine neurotropic effects. A direct correlation between staurosporine neurotropic effects and a novel stimulation of tyrosine phosphorylation of a 145 kDa protein (p145) with the following characteristics has been discovered: (1) Staurosporine specifically induced, among indolcarbazoles-K252a derivatives, in a dose-dependent manner (5-100 nM), p145 tyrosine phosphorylation and neurite outgrowth. (2) Staurosporine-induced p145 tyrosine phosphorylation was selective compared to other neurotropic compounds such as 8-Br-cAMP, acidic and basic fibroblast growth factors and NGF. (3) Staurosporine stimulation of p145 tyrosine phosphorylation gradually increased during the first 8 h of staurosporine treatment coinciding with the initiation of neurotropic effects. (4) K252a, a selective inhibitor of NGF actions, and several tyrphostins did not block staurosporine-induced p145 tyrosine phosphorylation and neurotropic effects. (5) Staurosporine stimulation of p145 tyrosine phosphorylation and neurotropic effects are independent of PKC. (6) Staurosporine did not activate gp140trk-NGF receptor in PC12 cells. The present study proposes staurosporine as a pharmacological tool to study the role of tyrosine phosphorylation pathway(s), such as p145 phosphorylation, in the action of neurotropic agents.

Neurites induced by staurosporine in PC12 cells are resistant to colchicine and express high levels of tau proteins.

Staurosporine, a protein kinase inhibitor, induces neurite outgrowth in pheochromocytoma cells and, therefore, may serve as a potential prototype for neurotropic drugs. The principal aim of the present study was to characterize the cytoskeletal properties of neurites induced in pheochromocytoma cells by staurosporine, in comparison to those induced by nerve growth factor, with emphasis on tubulin and tau proteins. Two major findings are described: a) staurosporine rapidly induces outgrowth of neurites that are resistant to colchicine treatment; and b) staurosporine treatment causes a rapid increase in tau protein levels, with a time course similar to the initiation of its neurotropic effects. The following observations exclude tubulin as the cellular target for staurosporine action: a) the level, cellular distribution, and assembly properties of tubulin are not affected by staurosporine treatment; and b) colchicine uptake, its binding to tubulin, and its interference with tubulin polymerization are not changed by staurosporine. On the other hand, staurosporine treatment causes a transient, dose-dependent increase in tau protein levels. This increase, which is already evident after 1 hr, reaches a maximum of 2 to 3 fold after 5 hr of treatment and declines to basal level within the next 10 to 15 hr. The rapid, transient increase of tau protein levels induced by staurosporine is reminiscent of its neurotropic properties. Here we characterize and compare the cytoskeletal properties of neurites induced by treatment with staurosporine and with nerve growth factor, and we offer a mechanistic explanation for the rapid stabilization of staurosporine induced neurites.

Staurosporine-induced neurite outgrowth in PC12 cells is independent of protein kinase C inhibition.

The protein kinase C (PKC) inhibitor staurosporine, a member of the K252a family of fungal alkaloids that are known as protein kinase inhibitors, induces neurite outgrowth in pheochromocytoma PC12 cells. The progressive staurosporine-induced neurotropic effect (EC50 = 50 nM) has the following characteristics: it is evident after 4 hr of incubation, requires the continuous presence of staurosporine, occurs at 37 degrees but not at 4 degrees, and is not blocked by K252a derivatives. Scanning electron micrographs showed long neurites, ruffling, and dense networks in nerve growth factor (NGF)-treated cells and short neurites, flattening, and smooth cell surface in staurosporine-treated cells. [3H]Staurosporine binding, which was time, temperature, and dose dependent, saturated at 5-10 nM. Other kinase inhibitors were poor competitors. The [3H]staurosporine bound over 20 hr at 37 degrees was poorly dissociated by acetic acid wash or unlabeled staurosporine. These results suggest an uptake process occurring at 37 degrees that is required for the neurotropic effect of staurosporine. NGF did not interfere with staurosporine binding, and staurosporine did not affect NGF receptor binding. At neurotropic concentrations of staurosporine, PKC in PC12 cells was completely inhibited. When PKC activity was down-regulated by prolonged exposure to phorbol myristate acetate, PC12 cells responded to staurosporine with neurite outgrowth similar to that of untreated cells. Although the target and mechanism of the neurotropic effects of staurosporine remain to be determined, the observed effects on PKC-deficient cells indicate that PKC may not be required for the neurotropic effect of this compound in PC12 cells. These results suggest that caution should be taken in the interpretation of staurosporine action in vivo, and they provide a pharmacological tool for the development of potential neurotropic drugs.