Nerve growth factor signaling in caveolae-like domains at the plasma membrane.
(25/1684)
Nerve growth factor (NGF) binding to its receptors TrkA and p75(NTR) enhances the survival, differentiation, and maintenance of neurons. Recent studies have suggested that NGF receptor activation may occur in caveolae or caveolae-like membranes (CLM). This is an intriguing possibility because caveolae have been shown to contain many of the signaling intermediates in the TrkA signaling cascade. To examine the membrane localization of TrkA and p75(NTR), we isolated caveolae from 3T3-TrkA-p75 cells and CLM from PC12 cells. Immunoblot analysis showed that TrkA and p75(NTR) were enriched about 13- and 25-fold, respectively, in caveolae and CLM. Binding and cross-linking studies demonstrated that the NGF binding to both TrkA and p75(NTR) was considerably enriched in CLM and that about 90% of high affinity binding to TrkA was present in CLM. When PC12 cells were treated with NGF, virtually all activated (i.e. tyrosine phosphorylated) TrkA was found in the CLM. Remarkably, in NGF-treated cells, it was only in CLM that activated TrkA was coimmunoprecipitated with phosphorylated Shc and PLCgamma. These results document a signaling role for TrkA in CLM and suggest that both TrkA and p75(NTR) signaling are initiated from these membranes. (+info)
Protein 4.1N binding to nuclear mitotic apparatus protein in PC12 cells mediates the antiproliferative actions of nerve growth factor.
(26/1684)
Protein 4.1N is a neuronal selective isoform of the erythrocyte membrane cytoskeleton protein 4.1R. In the present study, we demonstrate an interaction between 4.1N and nuclear mitotic apparatus protein (NuMA), a nuclear protein required for mitosis. The binding involves the C-terminal domain of 4.1N. In PC12 cells treatment with nerve growth factor (NGF) elicits translocation of 4. 1N to the nucleus and promotes its association with NuMA. Specific targeting of 4.1N to the nucleus arrests PC12 cells at the G1 phase and produces an aberrant nuclear morphology. Inhibition of 4.1N nuclear translocation prevents the NGF-mediated arrest of cell division, which can be reversed by overexpression of 4.1N. Thus, nuclear 4.1N appears to mediate the antiproliferative actions of NGF by antagonizing the role of NuMA in mitosis. (+info)
Mitochondrial impact on nerve growth factor production in vascular smooth muscle-derived cells.
(27/1684)
Ht30/=Ht5). Cells with reduced mitochondrial activity also showed abnormal responses to the stimulation of NGF output. Thrombin and phorbol ester elevated NGF production from Ht100, Ht30 and Ht10 cells, but not from Ht5 cells. Ht30 cells, despite secreting less NGF basally than Ht100 cells, reached a similar or greater NGF output upon stimulation. Mitogens increased NGF output and NGF mRNA levels with the largest effect on NGF protein in Ht30 cells. Free radical production and the ability of cells to respond to NGF-inducing agents were related. These data suggest that chronic impairment of mitochondrial function associates with disturbances in cellular production of a signaling protein. (+info)
Nerve growth factor protects human keratinocytes from ultraviolet-B-induced apoptosis.
(28/1684)
Ultraviolet radiation is a potent inducer of apoptosis, whereas autocrine nerve growth factor protects human keratinocytes from programmed cell death. To evaluate the role of nerve growth factor in the mechanisms of ultraviolet B-induced apoptosis, cultured human keratinocytes were ultraviolet B irradiated following pretreatment with K252, a specific inhibitor of the tyrosine kinase high-affinity nerve growth factor receptor. Here we report that the addition of K252 significantly enhanced keratinocyte apoptosis. We then transfected normal human keratinocytes with pNUT-hNGF. Nerve growth factor overexpressing keratinocytes secreted the highest amounts of nerve growth factor in culture supernatants, were more viable, and had a higher rate of proliferation than mock-transfected cells. Whereas ultraviolet B radiation downregulated nerve growth factor mRNA and protein as well as the tyrosine kinase high-affinity nerve growth factor receptor in normal keratinocytes, it failed to do so in nerve growth factor-transfected cells. Moreover, nerve growth factor overexpressing keratinocytes were partially resistant to apoptosis induced by increasing doses of ultraviolet B at 24 and 48 h. These results indicate that downregulation of nerve growth factor function plays an important part in the mechanisms of ultraviolet B-induced apoptosis in human keratinocytes. In addition, ultraviolet B caused a decrease in BCL-2 and BCL-xL expression in mock-transfected keratinocytes, but not in nerve growth factor overexpressing cells. Finally, nerve growth factor prevented the cleavage of the enzyme poly(ADP-ribose) polymerase induced in human keratinocytes by ultraviolet B. These results are consistent with a model whereby the autocrine nerve growth factor protects human keratinocytes from ultraviolet B-induced apoptosis by maintaining constant levels of BCL-2 and BCL-xL, which in turn might block caspase activation. (+info)
Neurotrophin binding to the p75 receptor modulates Rho activity and axonal outgrowth.
(29/1684)
While the neurotrophin receptor p75NTR is expressed by many developing neurons, its function in cells escaping elimination by programmed cell death remains unclear. The lack of intrinsic enzymatic activity of p75NTR prompted a search for protein interactors expressed in the developing retina, which resulted in the identification of the GTPase RhoA. In transfected cells, p75NTR activated RhoA, and neurotrophin binding abolished RhoA activation. In cultured neurons, inactivation of Rho proteins mimicked the effect of neurotrophins by increasing the rate of neurite elongation. In vivo, axonal outgrowth was retarded in mice carrying a mutation in the p75NTR gene. These results indicate that p75NTR modulates in a ligand-dependent fashion the activity of intracellular proteins known to regulate actin assembly. (+info)
Mediation by a CREB family transcription factor of NGF-dependent survival of sympathetic neurons.
(30/1684)
Nerve growth factor (NGF) and other neurotrophins support survival of neurons through processes that are incompletely understood. The transcription factor CREB is a critical mediator of NGF-dependent gene expression, but whether CREB family transcription factors regulate expression of genes that contribute to NGF-dependent survival of sympathetic neurons is unknown. CREB-mediated gene expression was both necessary for NGF-dependent survival and sufficient on its own to promote survival of sympathetic neurons. Moreover, expression of Bcl-2 was activated by NGF and other neurotrophins by a CREB-dependent transcriptional mechanism. Overexpression of Bcl-2 reduced the death-promoting effects of CREB inhibition. Together, these data support a model in which neurotrophins promote survival of neurons, in part through a mechanism involving CREB family transcription factor-dependent expression of genes encoding prosurvival factors. (+info)
Gab1 mediates neurite outgrowth, DNA synthesis, and survival in PC12 cells.
(31/1684)
The Gab1-docking protein has been shown to regulate phosphatidylinositol 3-kinase PI3K activity and potentiate nerve growth factor (NGF)-induced survival in PC12 cells. Here, we investigated the potential of Gab1 to induce neurite outgrowth and DNA synthesis, two other important aspects of NGF-induced neuronal differentiation of PC12 cells and NGF-independent survival. We generated a recombinant adenovirus encoding hemagglutinin (HA)-epitope-tagged Gab1 and expressed this protein in PC12 cells. HA-Gab1 was constitutively tyrosine-phosphorylated in PC12 cells and induced the phosphorylation of Akt/protein kinase B and p44/42 mitogen-activated protein kinase. HA-Gab1-stimulated a 10-fold increase in neurite outgrowth in the absence of NGF and a 5-fold increase in NGF-induced neurite outgrowth. HA-Gab1 also stimulated DNA synthesis and caused NGF-independent survival in PC12 cells. Finally, we found that HA-Gab1-induced neuritogenesis was completely suppressed by pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) activity and 50% suppressed by inhibition of PI3K activity. In contrast, HA-Gab1-stimulated cell survival was efficiently suppressed only by inhibition of both PI3K and MEK activities. These results indicate that Gab1 is capable of mediating differentiation, DNA synthesis, and cell survival and uses both PI3K and MEK signaling pathways to achieve its effects. (+info)
Activation of c-Ha-Ras by nitric oxide modulates survival responsiveness in neuronal PC12 cells.
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p21(c-Ha-Ras) (Ras) can be activated by the guanine nucleotide exchange factor mSOS1 or by S-nitrosylation of cysteine 118 via nitric oxide (NO). To determine whether these two Ras-activating mechanisms modulate distinct biological effects, a NO-nonresponsive Ras mutant (Ras(C118S)) was stably expressed in the PC12 cells, a cell line that generates NO upon nerve growth factor treatment. We report here that Ras(C118S) functions indistinguishably from wild type Ras in activating and maintaining the mSOS1- and Raf-1-dependent mitogen-activated protein kinase cascade necessary for neuronal differentiation. However, continuous (>5 days) exposure to nerve growth factor reveals that, in contrast to parental or wild-type Ras-overexpressing PC12 cells, Ras(C118S)-expressing PC12 cells cannot sustain the basal interaction between Ras and phosphatidylinositol 3-kinase. This results in spontaneous apoptosis of these cells despite the presence of nerve growth factor and serum. Thus unique downstream effector interactions and biological outcomes can be differentially modulated by distinct modes of Ras activation. (+info)