p75NTR-dependent modulation of cellular handling of reactive oxygen species.

Our previous studies demonstrated that p75NTR confers protection against oxidative stress-induced apoptosis upon PC12 cells; however, the mechanisms responsible for this effect are not known. The present studies reveal decreased mitochondrion membrane potential and increased generation of reactive oxygen species (ROS) in p75NTR-deficient PC12 cells as well as diminution of ROS generation after transfection of a full-length p75NTR construct into these cells. They also show that p75NTR deficiency attenuates activation of the phosphatidylinositol 3-kinase --> phospho-Akt/protein kinase B pathway in PC12 cells by oxidative stress or neurotrophic ligands and inhibition of Akt phosphorylation decreases the glutathione (GSH) content in PC12 cells. In addition, decreased de novo GSH synthesis and increased GSH consumption are observed in p75NTR-deficient cells. These findings indicate that p75NTR regulates cellular handling of ROS to effect a survival response to oxidative stress.

Bcl-2 overexpression disrupts the morphology of PC12 cells through reduced ERK activation.

Bcl-2 has been hypothesized to regulate many cellular functions in addition to its well-characterized role in the prevention of programmed cell death. To understand the role of Bcl-2 in regulating cell morphology and to explore the mechanism of this effect, we examined the effects of Bcl-2 overexpression on the morphology of PC12 cells in culture. We demonstrate that the overexpression of Bcl-2 in PC12 cells results in altered cell morphology and reduced actin expression. Analysis of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation reveals that the morphological changes seen after bcl-2 transfection are associated with reduced ERK activation. Treatment of control (mock-transfected) PC12 cells with the mitogen-activated ERK-activating kinase (MEK) inhibitor PD98059 converts their flat, process-bearing morphology into the rounded, process-free morphology of bcl-2-transfected cells, further confirming the association of ERK activation with altered cell shape. In conclusion, the present study describes a novel function of Bcl-2 in regulating cell shape through reduced ERK activation.

Bcl-2-mediated potentiation of neocarzinostatin-induced apoptosis: requirement for caspase-3, sulfhydryl groups, and cleavable Bcl-2.

Overexpression of antiapoptotic Bcl-2 family members is thought to contribute to chemotherapeutic resistance of neural crest tumors. Paradoxical potentiation by Bcl-2 of apoptosis induced by the antineoplastic prodrug, neocarzinostatin (NCS), has been observed in PC12 pheochromocytoma cells. Prior studies have indicated that the cleavage of Bcl-2 to its proapoptotic counterpart mediated by caspase-3 is responsible for this potentiation of apoptosis. This has led to the hypothesis that induction of caspase-3 expression in bcl-2-transfected, caspase-3-deficient MCF-7 cells, will result in Bcl-2 cleavage and Bcl-2-dependent potentiation of NCS-induced apoptosis. These studies have further led to the hypothesis that both cleavable Bcl-2 and sulfhydryl groups are required for the activity of caspase-3 in this regard. As hypothesized, co-transfection of bcl-2-transfected MCF-7 cells with a caspase-3 expression construct results in cleavage of Bcl-2 and potentiation of dose-dependent, NCS-mediated cell death. Furthermore, PC12 cells transfected with an expression construct for cleavage-resistant Bcl-2 demonstrated attenuated potentiation of apoptosis relative to their counterparts transfected with wild-type bcl-2. Finally, irreversible oxidative titration of sulfhydryl groups resulted in concentration-dependent attenuation of apoptosis in PC12 cells, along with prevention of caspase-3 activation and Bcl-2 cleavage. These results definitively demonstrate the requirement for caspase-3, cleavable Bcl-2, and available sulfhydryl groups (separate from those required for NCS activation) in potentiation of NCS-induced apoptosis by Bcl-2.

The intracellular domain of p75NTR as a determinant of cellular reducing potential and response to oxidant stress.

The low-affinity neurotrophin receptor, p75NTR, has been found to be pro- or anti-apoptotic depending upon the cell in which it is expressed. Reactive oxygen species play a major role in apoptosis induction and enactment. Using two polyclonal PC12 populations that, respectively, do or do not express p75NTR, this paper demonstrates that p75NTR expression confers resistance to oxidant stress upon PC12 cells maintained in serum-containing medium. The effect of p75NTR on cell survival is mimicked in p75-negative cells by expression of constructs that produce the p75NTR intracellular domain (ICD) or p75NTR with the extracellular domain deleted (DeltaECD), suggesting that binding of an extracellular ligand to p75NTR is not required. Our studies further document that the differential sensitivity to oxidant stress is serum-dependent and associated with differential oxidation of glutathione between p75-positive and p75-negative cells. These results suggest that the role of p75NTR in determining the consequences and treatment of age-related disorders and conditions in which reactive oxygen species are involved may require neither the extracellular receptor domain nor, by inference, the cognate extracellular ligands of this neurotrophin receptor.

P75 neurotrophin receptor regulates expression of neural cell adhesion molecule 1.

Our recent transcriptome profiling studies suggest that presenilin 1 (PS1) regulates expression of neural cell adhesion molecule (Ncam1) through p75 neurotrophin receptor. To better understand regulation of Ncam1 transcript and protein levels by p75, we performed a series of in vitro and in vivo experiments. The combined results suggest that p75 receptor is required for both resting and NGF-induced Ncam1 expression. Activation of TrkA receptors alone does not upregulate Ncam1. The normal Ncam1 expression depends on the relative ratio of TrkA and p75 receptors, and p75 extracellular domain is necessary for baseline Ncam1 expression. NGF-induced Ncam1 expression is dependent on the presence of an intact palmitoylation site within p75 receptor. Finally, we show that the expression of Ncam1 is altered in brains of two transgenic mouse lines that express familial Alzheimer's disease (FAD)-linked PS1 variants, suggesting that expression of dominantly inherited mutant PS1 genes interferes with the normal Ncam1 expression via the p75 signaling pathway.

Cholesterol biosynthesis and the pro-apoptotic effects of the p75 nerve growth factor receptor in PC12 pheochromocytoma cells.

Neocarzinostatin (NCS), an enediyne antimitotic agent, induces cell death in both p75NTR neurotrophin receptor (NTR)-positive and p75NTR-negative PC12 cells in a concentration-dependent fashion. However, p75NTR-positive cells demonstrate a higher susceptibility to NCS-induced cell damage. Furthermore, treatment of p75NTR-positive cells with the p75NTR-specific ligand, MC192, resulted in apoptosis, while treatment of these cells with the TrkA-specific ligand, NGF-mAbNGF30, protected them from NCS-induced death, implying that both the naked and liganded p75NTR receptors have a pro-apoptotic effect on PC12 cells. Microarray studies aimed at examining differential gene expression between p75NTR-positive and p75NTR-negative cells suggested that enzymes of the cholesterol biosynthetic pathway are differentially expressed. We therefore tested the hypothesis that altered cholesterol biosynthesis contributes directly to the pro-apoptotic effects of p75NTR in this PC12 cell-NCS model. Subsequent Northern blotting studies confirmed that the expression of p75NTR is associated with the upregulation of cholesterol biosynthetic enzymes including 3-hydroxy-3-methylglutaryl CoA reductase (HMG CoA reductase), farnesyl-diphosphate synthase, and 7-dehydro-cholesterol reductase. Mevastatin, an HMG CoA reductase inhibitor, converts the apoptosis susceptibility of p75NTR-positive cells to that of p75NTR-negative cells. It does so at concentrations that do not themselves alter cell survival. These studies provide evidence that the pro-apoptotic effects of p75NTR in PC12 cells are related to the upregulation of cholesterol biosynthetic enzymes and consequent increased cholesterol biosynthesis.

Microarray analysis of Lyn-deficient B cells reveals germinal center-associated nuclear protein and other genes associated with the lymphoid germinal center.

Lyn is the only member of the Src family expressed in DT40 B cells, which provide a unique model to study the singular contribution of this protein tyrosine kinase (PTK) family to cell signaling. In these cells, gene ablation of Lyn leads to defective B cell receptor signaling. Complementary DNA array analysis of Lyn-deficient DT40 cells shows that the absence of Lyn leads to down-regulation of numerous genes encoding proteins involved in B cell receptor signaling, proliferation, control of transcription, immunity/inflammation response, and cytoskeletal organization. Most of these expression changes have not been previously associated with Lyn PTK signaling. They include alterations in mRNA levels of germinal center-associated nuclear protein (germinal center-associated DNA primase) (GANP), CD74, CD22, NF-kappaB, elongation factor 1alpha, CD79b, octamer binding factor 1, Ig H chain, stathmin, and gamma-actin. Changes in GANP expression were also confirmed in Lyn-deficient mice, suggesting that Lyn PTK has a unique function not compensated for by other Src kinases. Because Lyn-deficient mice have impaired development of germinal centers in spleen, the decreased expression of GANP in the Lyn-deficient DT40 cell line and Lyn-deficient mice suggests that Lyn controls the formation and proliferation of germinal centers via GANP. GANP promoter activity was higher in wild-type vs Lyn-deficient cells. Mutation of the PU.1 binding site reduced activity in wild-type cells and had no effect in Lyn-deficient cells. The presence of Lyn enhanced PU.1 expression in a Northern blot. Thus, the following new signaling pathway has been described: Lyn-->PU.1-->GANP.

Bcl-2 mediates induction of neural differentiation.

Bcl-2 is an antiapoptotic protein expressed in a wide variety of cell types. We have found that overexpression of bcl-2 in PC12 neural crest tumor cells leads to increased expression of neural differentiation-associated genes and decreased expression of proliferation-related genes. Culture growth rate decreases as well. Overexpression of bcl-2 also leads to increased expression of TrkA and increased phosphorylation of signal transductants in, albeit not specific for, the TrkA-MEK-ERK pathway. Blocking of NGF-mediated signaling through TrkA prevents Bcl-2-associated expression changes in differentiation-associated genes, raising the possibility that Bcl-2 mediates induction of neural differentiation through TrkA/NGF signaling.