Differentiation of PC12 cells by nerve growth factor (NGF) is characterized by changes in signal transduction pathways leading to growth arrest and neurite extension. The transcription factor p53, involved in regulating cell cycle and apoptosis, is also activated during PC12 differentiation and contributes to each of these processes but the mechanisms are incompletely understood. NGF signaling stabilizes p53 protein expression which enables its transcriptional regulation of target genes, including the newly identified target, wnt7b, a member of the wnt family of secreted morphogens. We tested the hypothesis that wnt7b expression is a factor in NGF-dependent neurite outgrowth of differentiating PC12 cells. Wnt7b transcript and protein levels are increased following NGF treatment in a p53-dependent manner, as demonstrated by a reduction in wnt7b protein levels following stable shRNA-mediated silencing of p53. In addition, overexpressed human tp53 was capable of inducing marked wnt7b expression in neuronal PC12 cells but tp53 overexpression did not elevate wnt7b levels in several tested human tumor cell lines. Ectopic wnt7b overexpression was sufficient to rescue neurite outgrowth in NGF-treated p53-silenced PC12 cells which could be blocked by JNK inhibition with SP600125 and did not involve β-catenin nuclear translocation. Addition of sFRP1 to differentiation medium inhibited wnt7b-dependent phosphorylation of JNK, demonstrating that wnt7b is secreted and signals through a JNK-dependent mechanism in PC12 cells. We further identify an NGF-inducible subset of wnt receptors that likely supports wnt7b-mediated neurite extension in PC12 cells. In conclusion, wnt7b is a novel p53-regulated neuritogenic factor in PC12 cells that in conjunction with NGF-regulated Fzd expression is involved in p53-dependent neurite outgrowth through noncanonical JNK signaling.
wnt7b; wnt; p53; PC12; NGF; neurite; JNK; Fzd
Changes in the serum proteome were identified during early, fulminant and recovery phases of liver injury from acetaminophen in the rat. Male F344 rats received a single, non-injury dose or a high, injury-producing dose of acetaminophen for evaluation at 6 hr to 120 hr. Two-dimensional gel electrophoresis of immunodepleted serum separated about 800 stained proteins per sample from which differentially expressed proteins were identified by mass spectrometry. Serum ALT/AST levels and histopathology revealed the greatest liver damage at 24 and 48 hr after high dose acetaminophen corresponding to the time of greatest serum protein alterations. After 24 hr, 68 serum proteins were significantly altered of which 23 proteins were increased by >5 fold and 20 proteins were newly present compared to controls. Only minimal changes in serum proteins were noted at the low dose without any histopathology. Of the 54 total protein isoforms identified by mass spectrometry, gene ontology processes for 38 unique serum proteins revealed involvement of acute phase response, coagulation, protein degradation, intermediary metabolism and various carrier proteins. Elevated serum TNFα from 24 to 48 hr suggested a mild inflammatory response accompanied by increased antioxidant capability demonstrated by increased serum catalase activity. Antibody array and ELISA analyses also showed elevation in the chemokine, MCP-1, and the metalloprotease inhibitor, TIMP-1, during this same period of liver injury. This study demonstrates that serum proteome alterations likely reflect both liver damage and a concerted, complex response of the body for organ repair and recovery during acute hepatic injury.
ACTH18-39, adrenocorticotropic hormone fragment 18-39; ALT, alanine aminotransferase; ANIT, α-naphthylisothiocyanate; ARP3, actin related protein-3; AST, aspartate aminotransferase; BSA, bovine serum albumin; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate; CINC-2 and -3, Cytokine induced neutrophil chemoattractant-2 and -3; EDTA, ethylenediamine-tetraacetic acid; EGTA, ethylene glycol-bis(ß-aminoethylether)-N,N,N’,N’-tetra-acetic acid; ECL, enhanced chemiluminescence; ECM, extracellular matrix; EGF-1, epidermal growth factor-1; ELISA, enzyme-linked immunosorbent assay; EST, expressed sequence tag; GST, glutathione S-transferase; GM-CSF, granulocyte-macrophage colony stimulating factor; GRP, glucose responsive protein; HGF, hepatic growth factor; HEPES, N-2-Hydroxyethyl-piperazine-N'-2-ethane-sulfonic acid; 4-HPPD, 4- hydroxyphenylpyruvate dioxygenase; HRP, horseradish peroxidase; ICAT, isotope coded affinity tags; IPG, immobilized pH gradients; IEF, isoelectric focusing; LC, liquid chromatography; LIX, LPS-induced C-X-C chemokine; MALDI, matrix assisted laser desorption ionization; MCP1, monocyte chemoattractant protein-1; MS/MS, tandem mass spectrometry; Mr, relative molecular mass; MS, mass spectrometry; MSN, master spot number; PAGE, polyacrylamide gel electrophoresis; pI, isoelectric point; qRT-PCR, quantitative reverse transcriptase-polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SUMO1, small ubiquitin-like modifier-1; TIMP-1, tissue inhibitor of metalloproteinases-1; TNFα, tumor necrosis factor-alpha