Processes Of PC12 Cells Research Articles

Rnd1, a Novel Rho Family GTPase, Induces the Formation of Neuritic Processes in PC12 Cells

Rho family GTPases have been shown to be involved in the regulation of neuronal cell morphology, including neurite extension and retraction. Rho activation leads to neurite retraction and cell rounding, whereas Rac and Cdc42 are implicated in the promotion of filopodia and lamellipodia formation in growth cones and, therefore, in neurite extension. In this study, we examined the morphological role of Rnd1, a new member of Rho family GTPases, in PC12 cells, and found that expression of Rnd1 by itself caused the formation of many neuritic processes from the cell body with disruption of the cortical actin filaments, the processes having microtubules but few filamentous actin and neurofilaments. Treatment with cytochalasin D, an inhibitor of actin polymerization, could mimic the effects of expression of Rnd1, in that this inhibitor disrupted the cortical actin filaments and induced the formation of many thin processes containing microtubules. The process formation induced by Rnd1 was inhibited by dominant negative Rac1. These results suggest that Rnd1 induces the Rac-dependent neuritic process formation in part by disruption of the cortical actin filaments.

Anisomycin Affects Both Pro- and Antiapoptotic Mechanisms in PC12 Cells

Survival and differentiation of PC12 cells depend on the proper balance between the activities of several mitogen-activated protein kinase (MAPK) pathways. We have previously shown that low, nontoxic doses of anisomycin stimulated these MAPKs as well as the expression of several early-response genes and inhibited NGF-induced neurite formation. In the present work we show that protein synthesis-inhibiting concentrations of anisomycin, in contrast, cause apoptosis of PC12 cells. To try to characterize the apoptosis-inducing mechanisms of anisomycin we compared the signaling effects of subinhibitory and inhibitory drug concentrations. Anisomycin in a nontoxic dosis activates the same MAPK pathways and early-response genes as in protein synthesis inhibiting concentrations. In contrast, while the subinhibitory anisomycin treatment stimulates Akt and induces Bcl-2, two antiapoptotic proteins, the translation-inhibiting concentration of the drug prevents these survival-promoting biochemical events. Anisomycin thus triggers both pro- and antiapoptotic processes in PC12 cells; stimulation of stress-responsive MAPK cascades is not sufficient to mediate apoptotic signaling: the inhibition of key antiapoptotic proteins appears to be more important for PC12 cell death by anisomycin treatment.

Mediation of NGF signaling by post-translational inhibition of HES-1, a basic helix-loop-helix repressor of neuronal differentiation.

The induction of neurite outgrowth by NGF is a transcription-dependent process in PC12 cells, but the transcription factors that mediate this process are not known. Here we show that the bHLH transcriptional repressor HES-1 is a mediator of this process. Inactivation of endogenous HES-1 by forced expression of a dominant-negative protein induces neurite outgrowth in the absence of NGF and increases response to NGF. In contrast, expression of additional wild-type HES-1 protein represses and delays response to NGF. Endogenous HES-1 DNA-binding activity is post-translationally inhibited during NGF signaling in vivo, and phosphorylation of PKC consensus sites in the HES-1 DNA-binding domain inhibits DNA binding by purified HES-1 in vitro. Mutation of these sites generates a constitutively active protein that strongly and persistently blocks response to NGF. These results suggest that post-translational inhibition of HES-1 is both essential for and partially mediates the induction of neurite outgrowth by NGF signaling.

The Novel Protein-tyrosine Phosphatase PTP20 Is a Positive Regulator of PC12 Cell Neuronal Differentiation

A novel cytoplasmic protein-tyrosine phosphatase (PTPase) designated PTP20 was isolated from a PC12 cDNA library and shown to positively regulate the differentiation process in PC12 cells. The PTP20 open reading frame of 453 amino acids contains a single tyrosine phosphatase catalytic domain and displays closest homology to members of the PTP-PEST protein-tyrosine phosphatase family. Transient expression of PTP20 in Rat-1 cells resulted in the expression of a 50-kDa protein which exhibited PTPase activity in vitro. Expression of the 2.3-kilobase PTP20 mRNA increased during differentiation of nerve growth factor (NGF)-stimulated PC12 cells. Consistent with this observation, stable overexpression of PTP20 in PC12 cells resulted in accelerated neurite formation following NGF treatment. These findings suggest a positive regulatory role of PTP20 in NGF-dependent neuronal differentiation of PC12 cells.

GoProtein Does Not Regulate ATP-Stimulated [Ca2+]iElevation or Noradrenaline Release in PC12 Cells

The roles of Go, a heterotrimeric GTP binding (G) protein with a 40-kDa α subunit and which is localized predominantly in neuronal cells, in exocytosis have been discussed recently. PC12 pheochromocytoma cell line is a convenient model in which to study the Ca2+-dependent mechanisms of the neurosecretory process. The stimulation of ATP receptors or addition of KCl stimulated an elevation of cytosolic free Ca2+concentration ([Ca2+]i) and [3H]noradrenaline (NA) release in PC12 cells. In this study, we investigated the roles of Goin ATP- and KCl-stimulated reactions. Nerve growth factor treatment for 2 days and transfection of PC12 cells with cDNA of subunit of Go(Goα) had no effect on ATP-stimulated [3H]NA release, although both treatments increased levels of the Goαand its trimeric form by about twofold over those in unstimulated cells. The [Ca2+]irise induced by ATP in NGF-treated cells was similar to that in control cells, although the maximal response was slightly smaller. Cholera toxin treatment for 2 days inhibited ATP- and KCl-stimulated NA release, although this treatment caused an approximately twofold increase in the level of Go. Pertussis toxin treatment, which ADP ribosylated over 90% of endogenous G proteins such as Go, had no effect on ATP-stimulated reactions. These findings show that Godoes not directly regulate ATP-stimulated Ca2+channels or the NA release process in PC12 cells. Cholera toxin-sensitive protein(s) may regulate exocytosis.

Nerve Growth Factor Stimulates the Tyrosine Phosphorylation of Endogenous Crk-II and Augments Its Association with p130Cas in PC-12 Cells

The cellular homologs of the v-Crk oncogene product consist primarily of Src homology region 2 (SH2) and 3 (SH3) domains. v-Crk overexpression causes cell transformation and elevation of tyrosine phosphorylation in fibroblasts and accelerates differentiation of PC-12 cells in response to nerve growth factor (NGF). To further explore the role of Crk in NGF-induced PC-12 cell differentiation, we found that both NGF and epidermal growth factor stimulate the tyrosine phosphorylation of endogenous Crk II. Moreover, hormone stimulation enhanced the specific association of Crk proteins with the tyrosine-phosphorylated p130Cas, the major phosphotyrosine-containing protein in cells transformed with v-Crk. This interaction is mediated by the SH2 domain of Crk and can be inhibited with a phosphopeptide containing the Crk-SH2 binding motif. Furthermore, the Crk-SH2 domain binds tyrosine-phosphorylated paxillin, a cytoskeletal protein, following treatment of PC-12 cells with NGF or epidermal growth factor. These data suggest that Crk functions in a number of signaling processes in PC-12 cells.

Regulation of depolarization-dependent release of neurotransmitters by adenosine: cyclic AMP-dependent enhancement of release from PC12 cells.

We have used pheochromocytoma cells, clone PC12, as a model system for studying the effects of adenosine on neurosecretion. Exposure of the cells to adenosine or 2-chloroadenosine caused immediate activation of adenylate cyclase, increases in cellular cyclic AMP content, and inhibition of SAM-dependent phospholipid N-methylation and protein carboxymethylation. However, the effects on methylation were only observed with concentrations of adenosine 100 times greater than those that elevated cyclic AMP. Exposure of the cells to adenosine and 2-chloroadenosine did not alter the release of [3H]norepinephrine [(3H]NE) in the absence of depolarization. However, depolarization-dependent release of [3H]NE was markedly elevated by short (1-20 min) pretreatments with adenosine or 2-chloroadenosine. The enhancement of release was observed irrespective of the nature of the depolarizing stimulus (elevated K+, carbamylcholine, or veratridine). Release of [3H]acetylcholine in response to elevated K+ also was increased by adenosine pretreatment. These effects of adenosine and 2-chloroadenosine on neurotransmitter release closely paralleled elevation of cellular cyclic AMP but not inhibition of methylation. Taken together, the results show that adenosine, probably acting through adenosine receptors coupled to stimulation of adenylate cyclase, is able to modulate the neurosecretory process in PC12 cells. Furthermore, the enhancement of release occurred even though the extent of depolarization (measured as 86Rb+ flux through the acetylcholine receptor channel) and the amount of 45Ca2+ which entered upon depolarization were unchanged. Therefore, the enhancement of release produced by elevated cyclic AMP appeared to reflect increased efficiency of the stimulus-secretion coupling process.

Fine structure of initial outgrowth of processes induced in a pheochromocytoma cell line (PC12) by nerve growth factor.

Cells of the PC12 line (which is derived froma rat pheochromocytoma) develop neuron-like processes upon exposure to nerve growth factor (NGF), and thus provide an opportunity to study this phenomenon de novo. We have used the transmission electron microscope to analyse the early stages of process outgrowth (1, 2, 3 and 7 days) to determine what organelles are involved and in what sequence they appear during development. Despite the non-synchronous response to NGF, we can derive three main stages in early process formation. (1) NGF-treated cells develop conical extensions similar to, but larger and more numerous than those of controls. Extensions terminate in bulbous expansions that contain large number of chromaffin-like granules and bear microspikes filled with microfilaments. (2) The extensions of NGF-treated cells then acquire membranous organelles indicative of transmitter packaging and/or recycling of cytoplasmic membranes, for example, tubular reticulum, clear and dense-cored vesicles, multivesicular bodies, and lysosomes. (3) As processes elongate, they develop a shaft that contains an array of microtubules and fine tubular reticulum dispersed in a filamentous matrix, and varicosities that exhibit the same organelles seen in stage 2. The discussion stresses the similarities in the outgrowth of processes in PC12 cells and neurons, and speculates that NGF causes a change in organization and/or quantity or organelles that already exist in non-treated control cells.