Cross talk among tyrosine kinase receptors in PC12 cells: desensitization of mitogenic epidermal growth factor receptors by the neurotrophic factors, nerve growth factor and basic fibroblast growth factor.

PC12 cells, derived from a rat pheochromocytoma, have been extensively used as a model of neuronal differentiation (1), because the cells acquire the phenotype of sympathetic neurons and stop dividing in response to nerve growth factor (NGF) 1 (2).In PC12 cells, NGF interacts with two distinct plasma membrane receptor proteins: p75 NGFR , a cysteine-rich glycoprotein having a relatively low affinity for NGF (3), and p140 trk , a receptor tyrosine kinase activated by NGF, which binds NGF with high affinity and mediates many of the biological activities of this neurotrophin (4). The binding of NGF to the p140 trk receptor stimulates rapid tyrosine autophosphorylation of the receptor and activation of several signal-transducing proteins (4). Activated p140 trk receptors bind to and tyrosine phosphorylate the signaling substrates phospholipase C␥1, phosphatidylinositol-3 kinase, and Src homologous collagen protein (4), resulting in their activation. The latter stimulates Ras activity which subsequently activates a series of serine/threonine kinases including B-Raf, myelin basic protein kinase kinase, the Erks, and p90 rsk (4). This Ras-Erk pathway plays a major role in the activation of transcriptional events by NGF and in NGFinduced neuronal differentiation (5), as illustrated by inhibition of NGF actions upon expression of dominant-negative HaRas (Asn-17) proteins (6, 7). In addition a role for pp60 c-src in NGF actions has been suggested (5) by experiments involving the expression of a dominant-interfering kinase-inactive Src (8).PC12 cells also express plasma membrane receptors for epidermal growth factor (EGF) (9, 10), a mild mitogen for these cells (9). PC12 cells treated with NGF exhibit an attenuated response to EGF (9) because EGF receptors are down-regulated (9, 11). The cellular mechanisms that mediate NGF-induced EGF receptor down-regulation are unknown and represent an interesting question in neuronal development. The existence of functional receptors for both NGF and EGF on PC12 cells and the recent advances in understanding the signaling pathways activated by these receptors make these cells a useful model for the study of cross-regulation between NGF and EGF receptors during differentiation.In this study, we have used a number of PC12 variant cell lines with different levels of p140 trk (12, 13) and EGF receptor expression and dominant-negative Ras (7,14,15) or Src (5,6,8) PC12 transfectants in an attempt to elucidate the molecular mechanisms of the receptor cross-talk required by NGF to down-regulate EGF receptors. Our findings implicate the involvement of p140 trk -, Ras-, and Src-dependent signaling pathways in NGF-induced down-regulation of EGF receptors while * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ Permanent address:

Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

Down-regulation of Epidermal Growth Factor Receptors by Nerve Growth Factor in PC12 Cells Is p140 -, Ras-, and Src-dependent

Lazarovici

Oshima

Shavit

et al. 1997

“…It is known that treatment of PC12 cells with NGF leads to a rapid down-regulation of the EGF receptor (20,21). However, it is unlikely that this is the reason for the prevention of the ROS increase by NGF because the down-regulation is only partial and a comparable action of NGF is seen when 3T3 cells transfected with p140 trk are treated with PDGF (data not shown), and there is no reason to believe that NGF has any effect on the levels of PDGF receptors in these cells.…”

Section: Resultsmentioning

confidence: 99%

Nerve Growth Factor Treatment Prevents the Increase in Superoxide Produced by Epidermal Growth Factor in PC12 Cells

Mills¹,

Takeda²,

Yu³

et al. 1998

Stimulation of pheochromocytoma (PC12) cells with the mitogen epidermal growth factor (EGF) produced a rapid and robust accumulation of intracellular reactive oxygen species (ROS), an accumulation which, in other systems, has been shown to be essential for mitogenesis. Brief pretreatment of the cells with nerve growth factor (NGF) suppressed the EGF-mediated ROS increase. EGF failed to produce elevations in ROS in a PC12 variant stably expressing a dominant-negative p21ras construct (PC12-N17) or in cells pretreated with the MEK inhibitor PD098059. NGF failed to suppress the increase in ROS in the PC12 variant nnr5, which lacks p140 trk receptors. The suppression of the increase in ROS by NGF was restored in nnr5 cells stably expressing p140 trk (nnr5-trk), but NGF failed to prevent the increase in ROS in nnr cells expressing mutant p140 trk receptors that lack binding sites for Shc and phospholipase C␥. Among several inhibitors of superoxide-generating enzymes, only the lipoxygenase inhibitor, nordihydroguaiaretic acid reduced EGF-mediated ROS accumulation. The inhibitory action of NGF on ROS production was mimicked by the nitric oxide donor, sodium nitroprusside, and was blocked by an inhibitor of nitric-oxide synthetase, Lnitroarginine methyl ester. These results suggest a novel mechanism for the rapid interruption of mitogenic signaling by the neurotrophin NGF.The neurotrophins are a family of polypeptides that are involved in the survival and differentiation of several different classes of neurons in both the central and peripheral nervous systems. Although some aspects of the intracellular mechanisms by which they act are now clear (1, 2), the way in which they produce global changes in phenotype, such as differentiation or survival, is not known. One general effect of the neurotrophins on neurons is to inhibit cell division. The mechanisms by which this occurs are a matter of substantial research interest because they may have relevance to the overall control of cell division.It was recently reported that one of the early requirements for the promotion of cell division by mitogenic growth factors involves a rapid intracellular production of reactive oxygen species (ROS), 1 such as hydrogen peroxide and superoxide (3, 4). In related experiments exogenously added ROS stimulated mitogenesis in various cell types and was associated with increased expression of cellular markers of proliferation (5). Neurotrophin-stimulated production of reactive nitrogen species, such as nitric oxide, is reportedly required for neurotrophininduced cytostasis and differentiation (6). Together these reports suggest that the signal transduction mechanisms leading to either mitogenesis or differentiation may be controlled by the relative balance between distinct forms of oxidant and nitroxide levels within cells. The PC12 cell model (7, 8) has been quite informative in providing an understanding of neurotrophin-induced signal transduction. PC12 cells stimulated with the neurotrophin nerve growth factor (NGF) undergo many of the gl...

“…Induction of antagonistic effects, by a single agent, has already been described in the literature. For instance, EGF activates MAP kinases which phosphorylate the EGF receptor, leading thereby to a decrease in the EGF binding (37). Further, tumor necrosis factor-␣ induces apoptosis and the activation of NFB that plays a key protective role against apoptosis (38).…”

Section: Discussionmentioning

confidence: 99%

Inhibition of the Mitogen-activated Protein Kinase Pathway Triggers B16 Melanoma Cell Differentiation

Englaro¹,

Bertolotto²,

Buscà³

et al. 1998

181

138

In B16 melanoma cells, mitogen-activated protein (MAP) kinases are activated during cAMP-induced melanogenesis (Englaro, W., Rezzonico, R., Durand-Clé ment, M., Lallemand, D., Ortonne, J. P., and Ballotti, R. (1995) J. Biol. Chem. 270, 24315-24320). To establish the role of the MAP kinases in melanogenesis, we studied the effects of a specific MAP kinase kinase (MEK) inhibitor PD 98059 on different melanogenic parameters. We showed that PD 98059 inhibits the activation of MAP kinase extracellular signal-regulated kinase 1 by cAMP, but does not impair the effects of cAMP either on the morphological differentiation, characterized by an increase in dendrite outgrowth, or on the up-regulation of tyrosinase that is the key enzyme in melanogenesis. On the contrary, PD 98059 promotes by itself cell dendricity and increases the tyrosinase amount and activity. Moreover, down-regulation of the MAP kinase pathway by PD 98059, or with dominant negative mutants of p21 ras and MEK, triggers a stimulation of the tyrosinase promoter activity and enhances the effect of cAMP on this parameter. Conversely, activation of the MAP kinase pathway, using constitutive active mutants of p21 ras and MEK, leads to an inhibition of basal and cAMP-induced tyrosinase gene transcription. These results demonstrate that the MAP kinase pathway activation is not required for cAMP-induced melanogenesis. Furthermore, the inhibition of this pathway induces B16 melanoma cell differentiation, while a sustained activation impairs the melanogenic effect of cAMP-elevating agents.Melanocytes are specialized cells located at the basal layer of the epidermis that synthesize and transfer melanin pigments to surrounding keratinocytes leading thereby to a uniform skin pigmentation. In vivo, melanin pigments play a key photoprotective role against the carcinogenic effects of solar ultraviolet light, which is in other respects the physiologic stimulus of melanogenesis (1, 2). UV radiation can act directly on melanocytes or indirectly through the release of keratinocyte-derived factors that regulate melanogenesis (3, 4). Among the agents secreted by keratinocytes upon UV-B treatment, ␣-melanocytestimulating hormone (␣-MSH) 1 is one of the most potent activators of melanogenesis. Indeed, addition of ␣-MSH in cultured human melanocytes (5) or in melanoma cells (6) stimulates melanization. Further, subcutaneous injection of this hormone causes a strong stimulation of the local pigmentation in humans (7). ␣-MSH binds to a G protein-coupled heptahelical receptor leading to the activation of G␣ s protein and to an increase in intracellular cAMP content. In cultured melanoma cells, the melanogenic effect of ␣-MSH can be mimicked by other cAMP-elevating agents such as cholera toxin, forskolin, and isobutylmethylxanthine (8 -10). These observations emphasize the pivotal role of cAMP in the regulation of melanogenesis, but the cellular signaling events connecting the rise in cAMP to the stimulation of melanin synthesis are still incompletely clarified. Melanin biosynthesis ...