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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 9, 2001 as doi:10.1096/fj.00-0909fje. |
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Department of Basic Gerontology, National Institute for Longevity Sciences, Morioka-cho, Obu, Aichi 474-8522, Japan
2Correspondence: Department of Basic Gerontology, National Institute for Longevity Sciences, 36–3, Gengo, Morioka-cho, Obu, Aichi, 474-8522, Japan. E-mail: kenisobe{at}nils.go.jp
SPECIFIC AIMS
We hypothesized that manganese superoxide dismutase (Mn-SOD) that plays a major role in the protection of the mitochondria from oxidative damage is induced by platelet-derived growth factor (PDGF). We investigated the transcriptional regulation of the Mn-SOD gene mediated by PDGF to clarify the relation between the antioxidant enzyme and survival-signaling pathways mediated by PDGF.
PRINCIPAL FINDINGS
1. Mn-SOD promoter contains regulatory DNA elements responsive to
PDGF
NIH3T3 cells were treated with or without 50 ng/ml PDGF to
examine the effect on Mn-SOD gene expression. Mn-SOD mRNA in
PDGF-treated cells peaked at 6 h with a twofold induction relative
to the level in the untreated cells. A series of 5' end deletion murine
Mn-SOD promoter luciferase reporter plasmids was transfected into
NIH3T3 cells and then treated with 10 ng/ml PDGF for 16 h.
Constructs containing 231, 250, 538, and 1452 bp of 5' flanking DNA
exhibited a high level of relative luciferase activity in the untreated
cells, and the activity was increased significantly by treatment with
10 ng/ml PDGF. Deletion up to the position of -170 resulted in a
drastic decrease of the luciferase activity in both untreated and
PDGF-treated cells. These transfection studies indicated that the
Mn-SOD promoter region nucleotide between -250 and -170 was
responsible for the basal and PDGF-induced transcription.
2. Sp1 family proteins and early growth-responsive-1 (Egr-1) bind
to Mn-SOD proximal promoter region
Electrophoretic mobility shift assays (EMSAs) were carried
out with nuclear extracts prepared from NIH3T3 cells that were either
untreated or treated with 50 ng/ml PDGF for 1 h. We used three
different probes spanning nucleotides (-192 to -162, -219 to -185,
or -263 to -235) of the Mn-SOD gene. Nuclear extracts from
the untreated cells formed two major bands using
32P-labeled Mn-P (-192, -162) and Mn-P (-219,
-185) and three bands using 32P-labeled Mn-P
(-263, -235). Although we failed to identify a protein forming a
third band with Mn-P (-263, -235), the two major complexes also
disappeared by excess of an unlabeled Sp1 consensus oligonucleotide and
by the addition of anti-Sp1 and -Sp3 antibodies. Nuclear extracts from
the PDGF-treated cells interacted with the three probes formed an
additional complex between the Sp1- and Sp3-related complexes. This
additional complex disappeared by an excess of an Egr-1 consensus
oligonucleotide or an anti-Egr-1 antibody, suggesting a possible
involvement of Egr-1 in the assembly of the PDGF-induced complex. Sp1
binding with Mn-P (-192, -162) probe was increased by the addition of
anti-Egr-1 antibody or unlabeled Egr-1 consensus oligonucleotide. EMSAs
using oligonucleotides containing mutated sequences as a competitor
showed that the binding elements of Egr-1 and Sp1 family proteins
overlapped but were not identical.
3. PDGF-induced Mn-SOD transcription mediated by Egr-1
We performed cotransfection studies using the expression vector
for Egr-1 (pcDNA-Egr-1). Cotransfection of pcDNA-Egr-1 with Mn-pro
(-250) induced the luciferase activities in a dose-dependent manner.
To determine whether Egr-1 mediates Mn-SOD transcription induced by
PDGF, we constructed pcDNA3-Egr-ZF, which contains a DNA binding domain
but lacks the activation domain. Cotransfection of pcDNA-Egr-ZF
resulted in a gradual decrease of the PDGF-activated luciferase
activities in a dose-dependent manner and finally abolished the
PDGF-induced activities.
A 250 bp fragment of the Mn-SOD proximal promoter contained three GC clusters that were bound with Sp1 family proteins and Egr-1 protein. We performed luciferase assays with reporter plasmids containing the site-specific mutations to clarify the cis-elements for PDGF-mediated Mn-SOD transcription. The disruption of two Egr-1 binding sites, which were located from -207 to -205 and from -243 to -238 in the proximal promoter, abrogated PDGF-induced Mn-SOD transcriptional activity. Sp1 protein was indispensable factor for basal and PDGF-mediated Mn-SOD transcription, because the disruption of the major Sp1 binding site located from -182 to -176 failed to activate Mn-SOD transcription.
4. MEK1-ERK1/2 pathway involves Mn-SOD transcription mediated by
PDGF
PDGF induced phosphorylations of both extracellular
signal-regulated kinases 1 and 2 (ERK1/2) and Akt within 5 min in the
cells. To determine the more prominent pathway by which PDGF activates
Mn-SOD transcription, we examined the effect of PDGF on Mn-SOD
transcriptional activity in the existence of a specific
mitogen-activated protein kinase kinase 1 (MEK1) inhibitor U0126 or a
specific phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin.
PDGF-induced Mn-SOD transcription was suppressed in the cells
pretreated with 50 µM U0126, whereas wortmannin did not.
CONCLUSIONS
We have demonstrated that PDGF-induced Mn-SOD transcription was
mediated by Egr-1, a zinc finger DNA binding protein that interacts
with a consensus GC-rich region (Fig. 1
). PDGF-induced Egr-1 expression preceded the induction of Mn-SOD
transcription. Sp1, Sp3, and Egr-1 bound to three distinct regions in
the Mn-SOD promoter. The binding elements of Egr-1 and Sp1 family
proteins overlapped but were not identical. The binding affinity of
Egr-1 for the three distinct regions in Mn-SOD gene was
different from that of Sp1 family proteins in vitro. Transfection
studies showed that the Mn-SOD promoter region nucleotide between -250
and -170 was responsive to both basal and PDGF-induced promoter
activities. Egr-1 could serve as a positive regulator of the Mn-SOD
transcription through the two Egr-1 binding sites located from -207 to
-205 and from -243 to -238 in the proximal promoter. However, the
rest Egr-1 binding site located from -182 to -174 in the promoter did
not contribute to activate Mn-SOD transcription in response to PDGF.
These observations indicated that elevated amounts of Egr-1 in response
to PDGF might block Sp1 binding to the essential cis element
required for the basal and PDGF-mediated transcription of Mn-SOD.
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PDGF led to simultaneous phosphorylation of both ERK1/2 and Akt within
5 min in NIH3T3 cells. PDGF-induced Egr-1 expression was dependent on
MEK1-ERK1/2 signaling pathway. In addition to phosphorylation of ERK1/2
and Akt, activation of nuclear factor 
B (NF-B) has been
implicated in various antiapoptotic signaling pathways. Since the
Mn-SOD gene contains an NF-B binding site responsive to
cytokines and bacterial endotoxins within the second intron, we
attempted to examine the possible involvement of NF-B on
PDGF-mediated Mn-SOD transcription through the intronic enhancer region
by transfection studies. However, PDGF-induced transcriptional activity
was not detected in the cells transfected with the plasmid containing
the intronic enhancer region of Mn-SOD (K. Maehara and K. Isobe,
unpublished data), suggesting that NF-B has a little effect on
PDGF-mediated Mn-SOD transcription.
Accumulation of oxidative cellular damage is a major cause of aging in invertebrates and mammals. Enhanced resistance to environmental stress contributes to extend individual life span in invertebrates. With its short life span and complete genome sequence, Caenorhabditis elegans is becoming an important system for genetic studies on aging. Mutations of daf-2, a homologue of a member of the insulin receptor family, and age-1, a homologue of the mammalian PI3K, confer not only constitutive dauer formation phenotype, but also the life extension phenotype. The defects in the insulin-like signaling pathway up-regulate sod-3 mRNA by activating daf-16 gene. In contrast to C. elegans, the biological significance of the relationship between cell survival signaling and Mn-SOD gene expression to extend life span in mammals is not clearly understood. Here we demonstrated that stimulation by a growth factor (PDGF) could induce the expression of the Mn-SOD gene by activating cell survival signaling MEK1-ERK1/2 cascade in cultured cell system. Although we did not show direct evidence that PDGF-induced Mn-SOD transcription could contribute to extend individual life span in mammals, our findings help explain the mechanism by which PDGF activates transcription of the Mn-SOD gene underlying cell survival processes.
FOOTNOTES
1 To read the full text of this article, go
to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0909fje ; to
cite this article, use FASEB J. (July 9, 2001)
10.1096/fj.00-0909fje 
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