Faculty Bibliography

Nerve growth factor (NGF) initiates its biological effects by promoting the dimerization and activation of the tyrosine kinase receptor TrkA. The requirements for NGF signaling through the TrkA receptor have been defined extensively from studies in immortalized cells, involving transfection of NIH 3T3, COS, and PC12 cells. In the present study, we tested the effects of extracellular and intracellular mutations of TrkA after DNA-mediated transfection in primary cultures of embryonic day 17 hippocampal neurons. We found that the action of the TrkA receptor on neuronal differentiation depends on specific motifs in the extracellular domain and on tyrosine 490 (Y490), the site for SHC protein binding. In contrast with previous observations in a PC12 background, a mutation in the SHC Y490 binding site in TrkA resulted in a loss of NGF-dependent process formation. These results indicate that tyrosine 490 is necessary for neurite outgrowth in hippocampal neurons. Moreover, a constitutively active form of TrkA did not give enhanced responsiveness in hippocampal neurons, indicating that the behavior of TrkA receptors in primary neuronal cells is distinct from that of other cell types

Reduced expression of the low-affinity p75 neurotrophin receptor (p75(NTR)) occurs in prostate epithelial cells during malignant transformation. Recent studies indicating that the p75(NTR) can transduce signals that induce apoptosis suggest that diminished p75(NTR) in transformed prostate cells may contribute to immortalization. Mutations in the transmembrane domain of the p75(NTR) gene have been associated with decreased p75(NTR) protein expression and may block the ability of the p75(NTR) to induce apoptosis. Therefore, we used Western blot to analyze prostate cancer (PC) cell lines for p75(NTR) protein expression and gene single strand conformation polymorphism (SSCP) analysis and direct DNA sequencing to analyze mutations in the transmembrane domain of the p75(NTR). p75(NTR) Protein was present in all cell lines, and mutations in the p75(NTR) gene were not detected in cDNA derived from any cell line. To define the expression pattern of p75(NTR) in PCs in vivo, we used immunohistochemical techniques to examine tissue specimens from 20 benign, 19 malignant primary, and 14 metastatic prostate specimens. In benign prostate tissues, expression of p75(NTR) was universally detected in basal cells but not in secretory epithelial or stromal cells. In both primary and metastatic PC tissues, p75(NTR) immunoreactivity could not be detected in malignant prostate epithelial cells. However, in contrast to the benign prostate, p75(NTR) protein was expressed in stromal cells surrounding malignant epithelial cells. Stromal p75(NTR) expression was present in 84% (16 of 19) primary and in 86% (12 of 14) metastatic specimens. These data show that in the benign prostate p75(NTR) protein is expressed by basal cells and not stromal cells whereas in malignant prostate p75(NTR) protein is expressed by stromal cells but not prostatic carcinoma cells. Reversal of the p75(NTR) stromal-epithelial pattern of expression between benign and malignant prostate suggests that p75(NTR) may contribute to the development and maintenance of prostate cancer

Oligodendrocyte differentiation is a complex process believed to be controlled by an intrinsic mechanism associated with cell-cycle arrest. Recently, the cell-cycle inhibitor protein p27 Kip1 has been proposed as a key element in causing growth arrest of oligodendrocyte precursor cells. To investigate the effects of p27 upon oligodendrocyte cell development, we have introduced the p27 cDNA in oligodendrocyte progenitor cells using an adenovirus vector. Progenitor cells normally express low levels of p27. After adenoviral infection and p27 overexpression, progenitor cells were able to undergo cell-cycle arrest, even in the presence of strong mitogens. The effects of p27 were shown to be directly upon cyclin-dependent kinase-2 (CDK2), the protein kinase complex responsible for G1/S transition, as immunodepletion of oligodendrocyte extracts of p27 protein resulted in the activation of CDK2 activity. However, cells that became growth arrested owing to infection with p27 adenovirus did not display conventional oligodendrocyte differentiation markers, such as O4 or O1. Taken together, these data provide mechanistic evidence indicating that p27 is primarily involved in oligodendroglial progenitor proliferation by inhibiting CDK2 activity and inducing oligodendrocyte cell-cycle arrest

Multicellular organisms have the challenging task of coordinating the activities of many distinct cell types. This coordination is accomplished largely by cell-associated and soluble signalling molecules that act locally or distantly to alter target-cell physiology. The tumour necrosis factor family of cytokines are type II transmembrane proteins that are important regulators of homeostasis and have been implicated as mediators of disease. These molecules serve as ligands for a family of cell-surface receptors termed the tumour necrosis factor/nerve growth factor (TNF/NGF) receptor family. The receptors are type I transmembrane proteins capable of mediating a wide range of responses in vitro and in vivo. Signal transduction is mediated by several newly discovered cytoplasmic proteins that couple these receptors to downstream signalling events. The elucidation and use of spontaneously occurring mutants in TNF-related ligands and receptors in addition to gene-targeting experiments have begun to clarify the diverse biological effects mediated by this superfamily of cytokines

A stress-responsive gene highly expressed in brain and reproductive organs (BRE) is down-regulated after UV irradiation, DNA damaging agents, or retinoic acid treatment. The human BRE gene encodes a mRNA of 1.9 kb, which gives rise to a protein of 383 amino acids with a molecular size of 44 kilodaltons. BRE is not homologous to any known gene and its function has not been defined. Here we report that BRE was identified multiple times in a yeast two-hybrid screen of a murine cerebellar cDNA library, using the juxtamembrane domain of the p55 tumor necrosis factor alpha (TNF) receptor. The interaction between the p55 receptor and BRE was verified by an in vitro biochemical assay by using recombinant fusion proteins and by co-immunoprecipitation of transfected mammalian cells. In the yeast two-hybrid assay, BRE specifically interacted with p55 TNF receptor but not with other TNF family members such as the Fas receptor, the p75 TNF receptor, and p75 neurotrophin receptor. Overexpression of BRE inhibited TNF-induced NFkappaB activation, indicating that the interaction of BRE protein with the cytoplasmic region of p55 TNF receptor may modulate signal transduction by TNF-alpha