Activated N-ras gene induces neuronal differentiation of PC12 rat pheochromocytoma cells

Activated mouse N-ras gene transfected into PC12 rat pheochromocytoma cells suppressed proliferation and promoted neuronal differentiation. Normal mouse N-ras in a LTR-containing vector caused differentiation with a reduced efficiency, but normal N-ras in a vector lacking LTR sequences failed to alter the PC12 phenotype. Cultures of NGF-resistant PC12 variant subline U7 also showed outgrowth of neurites and cessation of cell division following transfection with the mutated ras gene. The present findings suggest that ras genes can, in certain cells, play a role in promoting differentiation and suppressing proliferation, in contrast to their established oncogenic neoplasia-promoting activity in other cells.     

Subtype-specific neuronal differentiation of PC12 cells transfected with alpha2-adrenergic receptors

Cells of the PC12 rat pheochromocytoma cell line acquire characteristics of sympathetic neurons under appropriate treatment. Stably transfected PC12 cells expressing individual alpha2-adrenergic receptor (alpha2-AR) subtypes were used to assess the role of alpha2-ARs in neuronal differentiation and to characterise the signalling pathways activated by the alpha2-AR agonist epinephrine in these cells. The effects of alpha2-AR activation were compared with the differentiating action and the signalling mechanisms of nerve growth factor (NGF). Epinephrine induced neuronal differentiation of PC12alpha2 cells through alpha2-AR activation in a subtype-dependent manner, internalization of all human alpha2-AR subtypes, and activation of mitogen-activated protein kinase (MAPK) and the serine-threonine protein kinase Akt. Epinephrine and NGF showed synergism in their differentiating effects. The MAPK kinase (MEK-1) inhibitor PD 98059 abolished the differentiating effect of epinephrine indicating that the differentiation is dependent on MAPK activation. Activating protein-1 (AP-1) DNA-binding activity was increased after epinephrine treatment in all three PC12alpha2 subtype clones. Evaluation of the potential physiological consequences of these findings requires further studies on endogenously expressed alpha2-ARs in neuronal cells.     

Regulation of protein kinase C activity in neuronal differentiation induced by the N-ras oncogene in PC-12 cells.

Expression of the N-ras oncogene under the control of the glucocorticoid-responsive promoter in the pheochromocytoma cell line UR61, a subline of PC-12 cells, has been used to investigate the differentiation process to neuronal cells triggered by ras oncogenes (I. Guerrero, A. Pellicer, and D. E. Burstein, Biochem. Biophys. Res. Commun. 150:1185-1192, 1988). Using ras-inducible cell lines, we observed that expression of the oncogenic N-ras p21 protein interferes with the ability of phorbol esters to induce downregulation of protein kinase C. This effect was associated with the appearance of immunologically detectable protein kinase C as well as the activity of the enzyme as analyzed either by binding of [3H]phorbol-12,13-dibutyrate in intact cells or by in vitro kinase activity. These results indicate a relationship between ras p21 and protein kinase C in neuronal differentiation in this model system. Comparison to the murine fibroblast system suggests that this relationship may be functional.     

alpha-Synuclein expression levels do not significantly affect proteasome function and expression in mice and stably transfected PC12 cell lines

alpha-Synuclein (alpha-syn) is a small protein of unknown function that is found aggregated in Lewy bodies, the histopathological hallmark of sporadic Parkinson disease and other synucleinopathies. Mutations in the alpha-syn gene and a triplication of its gene locus have been identified in early onset familial Parkinson disease. alpha-Syn turnover can be mediated by the proteasome pathway. A survey of published data may lead to the suggestion that overexpression of alpha-syn wild type, and/or their variants (A53T and A30P), may produce a decrease in proteasome activity and function, contributing to alpha-syn aggregation. To investigate the relationship between synuclein expression and proteasome function we have studied proteasome peptidase activities and proteasome subunit expression (alpha, beta-constitutive, and inducible) in mice either lacking alpha-syn (knock-out mice) or transgenic for human alpha-syn A30P (under control of PrP promoter, at a time when no clear gliosis can be observed). Similar studies are presented in PC12 cells overexpressing enhanced yellow fluorescent protein fusion constructs of human wild type, A30P, and A53T alpha-syn. In these cell lines we have also analyzed the assembly of 20 S proteasome complex and the degradation rate of a well known substrate of the proteasome pathway, Ikappabalpha. Overall the data obtained led us to the conclusion that alpha-synuclein expression levels by themselves have no significant effect on proteasome peptidase activity, subunit expression, and proteasome complex assembly and function. These results strengthen the suggestion that other mechanisms resulting in synuclein aggregation (not simply expression levels) may be the key to understand the possible effect of aggregated synuclein on proteasome function.     

The dopamine transporter constitutively internalizes and recycles in a protein kinase C-regulated manner in stably transfected PC12 cell lines

The dopamine transporter (DAT) removes dopamine from the extracellular milieu and is potently inhibited by number of psychoactive drugs, including cocaine, amphetamines, and methylphenidate (Ritalin). Multiple lines of evidence demonstrate that protein kinase C (PKC) down-regulates dopamine transport, primarily by redistributing DAT from the plasma membrane to endosomal compartments, although the mechanisms facilitating transporter sequestration are not defined. Here, we demonstrate that DAT constitutively internalizes and recycles in rat pheochromocytoma (PC12) cells. Temperature blockades demonstrated basal internalization and reliance on recycling to maintain DAT cell surface levels. In contrast, recycling blockade with bafilomycin A1 significantly decreased transferrin receptor (TfR) surface expression but had no effect on DAT surface levels, suggesting that DAT and TfR traffic via distinct endosomal mechanisms. Kinetic analyses reveal robust constitutive DAT cycling to and from the plasma membrane, independent of transporter expression levels. In contrast, phorbol ester-mediated PKC activation accelerated DAT endocytosis and attenuated transporter recycling in a manner sensitive to DAT expression levels. These data demonstrate constitutive DAT trafficking and that PKC-mediated DAT sequestration is achieved by a combination of accelerated internalization and reduced recycling. Additionally, the differential sensitivity to expression level exhibited by constitutive and regulated DAT trafficking suggests that these two processes are mediated by independent cellular mechanisms.     

MicroRNA expression profiling of NGF-treated PC12 cells revealed a critical role for miR-221 in neuronal differentiation

MicroRNAs (miRNAs) are small non-coding RNAs that control protein expression through translational inhibition or mRNA degradation. MiRNAs have been implicated in diverse biological processes such as development, proliferation, apoptosis and differentiation. Upon treatment with nerve growth factor (NGF), rat pheochromocytoma PC12 cells elicit neurite outgrowth and differentiate into neuron-like cells. NGF plays a critical role not only in neuronal differentiation but also in protection against apoptosis. In an attempt to identify NGF-regulated miRNAs in PC12 cells, we performed miRNA microarray analysis using total RNA harvested from cells treated with NGF. In response to NGF treatment, expression of 8 and 12 miRNAs were up- and down-regulated, respectively. Quantitative RT-PCR analysis of 11 out of 20 miRNAs verified increased expression of miR-181a^∗, miR-221 and miR-326, and decreased expression of miR-106b^∗, miR-126, miR-139-3p, miR-143, miR-210 and miR-532-3p after NGF treatment, among which miR-221 was drastically up-regulated. Functional annotation analysis of potential target genes of 7 out of 9 miRNAs excluding the passenger strands (*) revealed that NGF may regulate expression of various genes by controlling miRNA expression, including those whose functions and processes are known to be related to NGF. Overexpression of miR-221 induced neuronal differentiation of PC12 cells in the absence of NGF treatment, and also enhanced neuronal differentiation caused by low-dose NGF. Furthermore, miR-221 potentiated formation of neurite network, which was associated with increased expression of synapsin I, a marker for synapse formation. More importantly, knockdown of miR-221 expression by antagomir attenuated NGF-mediated neuronal differentiation. Finally, miR-221 decreased expression of Foxo3a and Apaf-1, both of which are known to be involved in apoptosis in PC12 cells. Our results suggest that miR-221 plays a critical role in neuronal differentiation as well as protection against apoptosis in PC12 cells.     

The induction of serine/threonine protein phosphorylations by a PDGFR/TrkA chimera in stably transfected PC12 cells

Stably transfected PC12 cells expressing a chimeric receptor composed of the extracellular domain of the platelet-derived growth factor receptor BB and the transmembrane and intracellular domains of TrkA, the nerve growth factor receptor, were stimulated for 20 min with platelet-derived growth factor and the resulting phosphoproteome was determined from affinity purified tryptic peptides identified by tandem MS (MS/MS) analyses. The changes in the levels of individual phosphorylation sites in stimulated cells versus control were ascertained by the stable isotope labeling of amino acids in cell culture technique. A total of 2035 peptides (806 proteins) were indentified and quantified in both data sets. Of these, 424 phosphopeptides on 259 proteins were found to be up-regulated and 392 sites on 206 proteins were down-regulated (1.8-fold or more). Protein kinases and phosphatases, as well as sites in many proteins involved in G-protein signaling, were prominently represented in the up-regulated group and more than half of the kinase up-regulated phosphosites could be clustered into three sequence motifs; a similar distribution was also found for the down-regulated sites. A comparison of the up-regulated motif profile observed to that calculated from a previous study of the EGFR-induced phosphoproteome in human HeLa cells at the same time point showed a considerable amount of similarity, supporting the view that RTK signal transduction pathways and downstream modifications are likely to be extensively overlapping.     

Oncogene N-ras mediates selective inhibition of c-fos induction by nerve growth factor and basic fibroblast growth factor in a PC12 cell line.

A cell line was generated from U7 cells (a subline of PC12 rat pheochromocytoma cells) that contains a stably integrated transforming mouse N-ras (Lys-61) gene under the control of the long terminal repeat from mouse mammary tumor virus. Such cells, designated UR61, undergo neuronal differentiation upon exposure to nanomolar concentrations of dexamethasone, as a consequence of expression of the activated N-ras gene (I. Guerrero, A. Pellicer, and D.E. Burstein, Biochem, Biophys. Res. Commun. 150:1185-1192, 1988). Exposure of UR61 cells to either nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) results in a marked induction of c-fos RNA, with kinetics paralleling those of NGF- or bFGF-induced expression of c-fos RNA in PC12 cells. Dexamethasone-induced expression of activated N-ras p21 results in blocking of c-fos RNA induction by NGF or bFGF in a time-dependent manner. Activated N-ras p21-mediated inhibition of c-fos RNA induction in UR61 cells is selective for NGF and bFGF and is not due to selective degradation of c-fos RNA. Normal and transforming N-ras can trans activate the chloramphenicol acetyltransferase gene linked to mouse c-fos regulatory sequences when transient expression assays are performed. Our observations suggest that N-ras p21 selectively interacts with pathways involved in induction of c-fos expression which initiate at the receptors for NGF and bFGF.     

Effect of a dominant inhibitory Ha-ras mutation on neuronal differentiation of PC12 cells.

A dominant inhibitory mutation of Ha-ras which changes Ser-17 to Asn-17 in the gene product p21 [p21 (Asn-17)Ha-ras] has been used to investigate the role of ras in neuronal differentiation of PC12 cells. The growth of PC12 cells, in contrast to NIH 3T3 cells, was not inhibited by p21(Asn-17)Ha-ras expression. However, PC12 cells expressing the mutant Ha-ras protein showed a marked inhibition of morphological differentiation induced by nerve growth factor (NGF) or fibroblast growth factor (FGF). These cells, however, were still able to respond with neurite outgrowth to dibutyryl cyclic AMP and 12-O-tetradecanoylphorbol-13-acetate (TPA). Induction of early-response genes (fos, jun, and zif268) by NGF and FGF but not by TPA was also inhibited by high levels of p21(Asn-17)Ha-ras. However, lower levels of p21(Asn-17) expression were sufficient to block neuronal differentiation without inhibiting induction of these early-response genes. Induction of the secondary-response genes SCG10 and transin by NGF, like morphological differentiation, was inhibited by low levels of p21(Asn-17) whether or not induction of early-response genes was blocked. Therefore, although inhibition of ras function can inhibit early-response gene induction, this is not required to block morphological differentiation or secondary-response gene expression. These results suggest that ras proteins are involved in at least two different pathways of signal transduction from the NGF receptor, which can be distinguished by differential sensitivity to p21(Asn-17)Ha-ras. In addition, ras and protein kinase C can apparently induce early-response gene expression by independent pathways in PC12 cells.     

Ubiquitin and ubiquitin-protein conjugates in PC12h cells: Changes during neuronal differentiation

Ubiquitin and ubiquitin-protein conjugates in PC12h cells were detected with in vitro [¹²⁵I]ubiquitination, and quantified by immunoblotting. These levels were altered by nerve growth factor (NGF), which promotes neuronal differentiation. (i) Levels of high molecular weight (HMW) ubiquitin-protein conjugates ranging from 40 to 1,000kDa were increased by 2 days of NGF treatment, and remained high up to 10 days of NGF treatment. (ii) Ubiquitin and a 23-kDa conjugate tended to be decreased from days 2 to 10 of NGF treatment. 10-Day culture with 10 nM staurosporine, an protein kinase inhibitor, that blocks NGF-induced neurite outgrowth suppressed the NGF-induced increases in levels of HMW conjugates. Cyclic AMP and forskolin, both of which promote neurite outgrowth, mimicked the NGF-induced changes in ubiquitin and HMW conjugates, but phorbol ester and epidermal growth factor had little effect. These findings suggest that changes in ubiquitin-protein conjugates are closely coupled with neuronal differentiation.     

Dissociation of c-fos induction from macrophage differentiation in human myeloid leukemic cell lines.

Treatment of five human myeloid leukemic cell lines (KG1, ML3, HL-60, U-937, and HEL) with TPA was followed by macrophage differentiation and was accompanied by an early and transient increase in the mRNA level of c-fos proto-oncogene. The induction of c-fos was also observed in human cell lines K562 and K-Gla that did not respond to TPA with terminal macrophage differentiation. The treatment of HL-60 and U-937 cell lines with 1-oleoyl-2-acetylglycerol, a synthetic analog of diacylglycerol that, like TPA, stimulates protein kinase C activity, was followed by early and transient induction of c-fos mRNA in the absence of terminal macrophage differentiation. Finally, treatment of HL-60 with TPA in the presence of retinal, an inhibitor of protein kinase C, drastically reduced the induction of c-fos mRNA but had no effect on the terminal macrophage differentiation that is induced in this cell line by TPA. These results indicate that the induction of c-fos and terminal macrophage differentiation in response to TPA treatment can be dissociated in the in vitro models provided by human myeloid leukemic cell lines. Moreover, these findings suggest that the induction of c-fos is not only insufficient but may also be unnecessary for the differentiation along the monocyte-macrophage pathway.