Insulin-like Growth Factor I-mediated Activation of the Transcription Factor cAMP Response Element-binding Protein in PC12 Cells

doi:10.1074/jbc.274.5.2829

Insulin-like Growth Factor I-mediated Activation of the Transcription Factor cAMP Response Element-binding Protein in PC12 Cells

INVOLVEMENT OF p38 MITOGEN-ACTIVATED PROTEIN KINASE-MEDIATED PATHWAY*

From the Section of Endocrinology, Veterans Affairs Medical Center, Denver, Colorado 80220, the Departments of ^‡Endocrinology and ^§Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262, the Department of Medicine and Center for Molecular Genetics, University of California, La Jolla, California 92093, and the Department of Veterans Affairs Medical Center, San Diego, California 92161

Abstract

IGF-I is known to support growth and to prevent apoptosis in neuronal cells. Activation of the nuclear transcription factor cAMP response element-binding protein (CREB) has emerged as a central determinant in neuronal functions. In the present investigation, we examined the IGF-I-mediated phosphorylation and transcriptional activation of CREB in rat pheochromocytoma (PC12) cells, a cellular model for neuronal differentiation, and defined three distinct postreceptor signaling pathways important for this effect including the p38 mitogen-activated protein kinase (MAPK) pathway. CREB phosphorylation at serine 133 and its transcriptional activation as measured by a CREB-specific Gal4-CREB reporter and the neuroendocrine-specific gene chromogranin A was induced 2–3.3-fold by insulin-like growth factor (IGF)-I. This activation was significantly blocked (p < 0.001) by the dominant negative K-CREB or by mutation of the CRE site. IGF-I stimulated chromogranin A gene expression by Northern blot analysis 3.7-fold. Inhibition of MAPK kinase with PD98059, PI 3-kinase with wortmannin, and p38 MAPK with SB203580 blocked IGF-I-mediated phosphorylation and transcriptional activation of CREB by 30–50% (p < 0.001). Constitutively active and dominant negative regulators of the Ras and PI 3-kinase pathways confirmed the contribution of these pathways for CREB regulation by IGF-I. Cotransfection of PC12 cells with p38β and constitutively active MAPK kinase 6 resulted in enhanced basal as well as IGF-I-stimulated chromogranin A promoter. IGF-I activated p38 MAPK, which was blocked by the inhibitor SB203580. This is the first description of a p38 MAPK-mediated nuclear signaling pathway for IGF-I leading to CREB-dependent neuronal specific gene expression.

Footnotes

↵* This work was supported by a Veterans Affairs Merit Review and Research Associate Career Development Award and NIDDK, National Institutes of Health, Grant KO8 DK02351 (to J.E.-B.R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ To whom correspondence should be addressed: Section of Endocrinology (111H), Veterans Affairs Medical Center, 1055 Clermont St., Denver, CO 80220. Tel.: 303-399-8020 (ext. 2775); Fax: 303-393-5271; E-mail: jreusch{at}sembilan.uchsc.edu.
Abbreviations:

IGF

insulin-like growth factor

CRE

cAMP response element

CREB

cAMP response element-binding protein

ERK

extracellular regulated kinase

MAPK

mitogen-activated protein kinase

MEK

MAPK kinase

PBS

phosphate-buffered saline

PI 3-kinase

phosphatidylinositol 3-kinase

NGF

nerve growth factor

TK

thymidine kinase

CgA

chromogranin A.
- Received February 20, 1998.
- Revision received October 8, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.