Regulation of heme oxygenase-1 gene expression through the phosphatidylinositol 3-kinase/PKC-zeta pathway and Sp1.
The molecular mechanisms involved in modulation of the antioxidant cell defence by survival signals remain largely unexplored. Here, we report a mechanistic connection between the survival signal elicited by nerve growth factor (NGF) and the antioxidant cell defence represented by heme oxygenase-1 (HO-1) at the level of a newly identified Sp1 site in the human ho1 proximal promoter. By using luciferase reporter constructs we identified a PI3K-responsive region containing a GC-box that resembled the response element for Sp1. Indeed, transfection of Sp1-deficient SL2 cells, electrophoretic mobility shift assays, the use of the GC-box binding drug mithramycin, and mutation of the GC-box provided evidence for a Sp1-like site in the PI3K-sensitive region. Then, we observed with the use of a Sp1-Gal4 chimera that PI3K regulates the transactivating capacity of Sp1. Cotransfection of active PI3K and PKC-zeta expression vectors resulted in substantial increase of Sp1 phosphorylation and in synergistic activation of both Sp1-Gal4 and endogenous Sp1. Moreover, these effects were mimicked by cotransfection of active MEK and ERK expression vectors and were blocked by the MEK inhibitor PD98059. Inhibition of HO-1 with Sn protoporphyrin IX and blockage of Sp-1-mediatied upregulation of HO-1 with mithramycin attenuated antioxidant and cytoprotective functions of NGF against hydrogen peroxide. This study elucidates how NGF contributes to protection of target cells against oxidative stress.
Rojo
Ana I
AI
Instituto de Investigaciones Biomédicas A. Sols UAM-CSIC and Departamento de Bioquímica, Facultad de Medicina, Universidad Autónoma de Madrid, 28029 Madrid, Spain.
Salina
Marta
M
Salazar
María
M
Takahashi
Shigeru
S
Suske
Guntram
G
Calvo
Victor
V
de Sagarra
María Rosa
MR
Cuadrado
Antonio
A
D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic; D051547:Heme Oxygenase-1 / Q000235:genetics*; D006801:Humans; D020932:Nerve Growth Factor / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000378:metabolism*; D034741:RNA, Small Interfering; D051381:Rats; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D015533:Transcriptional Activation; D015854:Up-Regulation; D000099221:Protein Kinase C zeta
D017931:DNA Primers; D051547:Heme Oxygenase-1; D020932:Nerve Growth Factor; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D034741:RNA, Small Interfering; D016329:Sp1 Transcription Factor; D000099221:Protein Kinase C zeta