Differential growth factor regulation of aspartyl-(asparaginyl)-beta-hydroxylase family genes in SH-Sy5y human neuroblastoma cells.
BACKGROUND
Aspartyl (asparaginyl)-beta-hydroxylase (AAH) hydroxylates Asp and Asn residues within EGF-like domains of Notch and Jagged, which mediate cell motility and differentiation. This study examines the expression, regulation and function of AAH, and its related transcripts, Humbug and Junctin, which lack catalytic domains, using SH-Sy5y neuroblastoma cells.


RESULTS
Real time quantitative RT-PCR demonstrated 8- or 9-fold higher levels of Humbug than AAH and Junctin, and lower levels of all 3 transcripts in normal human brains compared with neuroblastic tumor cells. AAH and Humbug expression were significantly increased in response to insulin and IGF-I stimulation, and these effects were associated with increased directional motility. However, over-expression of AAH and not Humbug significantly increased motility. Treatment with chemical inhibitors of Akt, Erk MAPK, or cyclin-dependent kinase 5 (Cdk-5) significantly reduced IGF-I stimulated AAH and Humbug expression and motility relative to vehicle-treated control cells. In addition, significantly increased AAH and Humbug expression and directional motility were observed in cells co-transfected with Cdk-5 plus its p35 or p25 regulatory partner. Further studies demonstrated that activated Cdk-5 mediated its stimulatory effects on AAH through Erk MAPK and PI3 kinase.


CONCLUSIONS
AAH and Humbug are over-expressed in SH-Sy5y neuroblastoma cells, and their mRNAs are regulated by insulin/IGF-1 signaling through Erk MAPK, PI3 kinase-Akt, and Cdk-5, which are known mediators of cell migration. Although AAH and Humbug share regulatory signaling pathways, AAH and not Humbug mediates directional motility in SH-Sy5y neuroblastoma cells.
Lahousse
Stephanie A
SA
Departments of Medicine and Pathology, Rhode Island Hospital, Brown Medical School, and the Pathobiology Graduate Program, Brown University, Providence, RI, USA. Lahousses@niehs.nih.gov <Lahousses@niehs.nih.gov>
Carter
Jade J
JJ
Xu
Xaolai J
XJ
Wands
Jack R
JR
de la Monte
Suzanne M
SM
D001921:Brain / Q000378:metabolism; D002135:Calcium-Binding Proteins; D045744:Cell Line, Tumor; D002465:Cell Movement; D004268:DNA-Binding Proteins / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005786:Gene Expression Regulation; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007004:Hypoglycemic Agents; D007328:Insulin / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D008565:Membrane Proteins; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D009447:Neuroblastoma / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction; D014157:Transcription Factors / Q000494:pharmacology; D014162:Transfection
D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; D007004:Hypoglycemic Agents; C489892:IGHMBP2 protein, human; D007328:Insulin; D008565:Membrane Proteins; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D014157:Transcription Factors; C089495:neuronal Cdk5 activator (p25-p35); D007334:Insulin-Like Growth Factor I; D006899:Mixed Function Oxygenases; C058291:aspartic acid 2-oxoglutarate-dependent dioxygenase; C419389:ASPH protein, human; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases