Limits...
Differential growth factor regulation of aspartyl-(asparaginyl)-beta-hydroxylase family genes in SH-Sy5y human neuroblastoma cells.

Lahousse SA, Carter JJ, Xu XJ, Wands JR, de la Monte SM - BMC Cell Biol. (2006)

Bottom Line: 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.Further studies demonstrated that activated Cdk-5 mediated its stimulatory effects on AAH through Erk MAPK and PI3 kinase.

View Article: PubMed Central - HTML - PubMed

Affiliation: 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>

ABSTRACT

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.

Conclusion: 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.

Show MeSH

Related in: MedlinePlus

Cdk-5 stimulation of AAH and Humbug expression is mediated by signaling through Erk MAPK and PI3 kinase: SH-Sy5y cells were transfected with recombinant plasmids to over-express Luciferase or Cdk-5+p25. 36 hours after transfection, the cells were treated with vehicle (Con), PD98059 (PD9), or LY294002 (LY2). 12 hours later, i.e. 48 hours after transfection, the cells were harvested to measure (A) AAH, (B) Humbug, or (C) Junctin mRNA levels by real time quantitative RT-PCR. The graphs depict the mean ± S.D. of results normalized to 18S rRNA measured in the same samples. Statistical comparisons were made using ANOVA with post hoc Fisher LSD tests (*P < 0.001 relative to corresponding vehicle-treated control).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1764734&req=5

Figure 8: Cdk-5 stimulation of AAH and Humbug expression is mediated by signaling through Erk MAPK and PI3 kinase: SH-Sy5y cells were transfected with recombinant plasmids to over-express Luciferase or Cdk-5+p25. 36 hours after transfection, the cells were treated with vehicle (Con), PD98059 (PD9), or LY294002 (LY2). 12 hours later, i.e. 48 hours after transfection, the cells were harvested to measure (A) AAH, (B) Humbug, or (C) Junctin mRNA levels by real time quantitative RT-PCR. The graphs depict the mean ± S.D. of results normalized to 18S rRNA measured in the same samples. Statistical comparisons were made using ANOVA with post hoc Fisher LSD tests (*P < 0.001 relative to corresponding vehicle-treated control).

Mentions: To determine if the effects of Cdk-5 on AAH and Humbug expression were mediated downstream through Erk or PI3 kinase, AAH, Humbug, and Junctin mRNA levels were measured in cells transfected with pLuc or Cdk-5+p25. 24 hours prior to harvesting the cells, parallel cultures were treated with PD98059 or LY294002 to inhibit Erk MAPK or PI3 kinase, respectively. Cells transfected with the Cdk-5+p25 had increased AAH and Humbug, but not Junctin mRNA levels by real time quantitative RT-PCR, as described above. However, pre-treatment with PD98059 or LY294002 significantly inhibited AAH, Humbug, and Junctin expression, and with regard to Humbug and Junctin, the inhibition of gene expression occurred independent of Cdk-5+p25 over-expression (Figures 8A–8C).


Differential growth factor regulation of aspartyl-(asparaginyl)-beta-hydroxylase family genes in SH-Sy5y human neuroblastoma cells.

Lahousse SA, Carter JJ, Xu XJ, Wands JR, de la Monte SM - BMC Cell Biol. (2006)

Cdk-5 stimulation of AAH and Humbug expression is mediated by signaling through Erk MAPK and PI3 kinase: SH-Sy5y cells were transfected with recombinant plasmids to over-express Luciferase or Cdk-5+p25. 36 hours after transfection, the cells were treated with vehicle (Con), PD98059 (PD9), or LY294002 (LY2). 12 hours later, i.e. 48 hours after transfection, the cells were harvested to measure (A) AAH, (B) Humbug, or (C) Junctin mRNA levels by real time quantitative RT-PCR. The graphs depict the mean ± S.D. of results normalized to 18S rRNA measured in the same samples. Statistical comparisons were made using ANOVA with post hoc Fisher LSD tests (*P < 0.001 relative to corresponding vehicle-treated control).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1764734&req=5

Figure 8: Cdk-5 stimulation of AAH and Humbug expression is mediated by signaling through Erk MAPK and PI3 kinase: SH-Sy5y cells were transfected with recombinant plasmids to over-express Luciferase or Cdk-5+p25. 36 hours after transfection, the cells were treated with vehicle (Con), PD98059 (PD9), or LY294002 (LY2). 12 hours later, i.e. 48 hours after transfection, the cells were harvested to measure (A) AAH, (B) Humbug, or (C) Junctin mRNA levels by real time quantitative RT-PCR. The graphs depict the mean ± S.D. of results normalized to 18S rRNA measured in the same samples. Statistical comparisons were made using ANOVA with post hoc Fisher LSD tests (*P < 0.001 relative to corresponding vehicle-treated control).
Mentions: To determine if the effects of Cdk-5 on AAH and Humbug expression were mediated downstream through Erk or PI3 kinase, AAH, Humbug, and Junctin mRNA levels were measured in cells transfected with pLuc or Cdk-5+p25. 24 hours prior to harvesting the cells, parallel cultures were treated with PD98059 or LY294002 to inhibit Erk MAPK or PI3 kinase, respectively. Cells transfected with the Cdk-5+p25 had increased AAH and Humbug, but not Junctin mRNA levels by real time quantitative RT-PCR, as described above. However, pre-treatment with PD98059 or LY294002 significantly inhibited AAH, Humbug, and Junctin expression, and with regard to Humbug and Junctin, the inhibition of gene expression occurred independent of Cdk-5+p25 over-expression (Figures 8A–8C).

Bottom Line: 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.Further studies demonstrated that activated Cdk-5 mediated its stimulatory effects on AAH through Erk MAPK and PI3 kinase.

View Article: PubMed Central - HTML - PubMed

Affiliation: 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>

ABSTRACT

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus