Insulin-like Growth Factor 1 Inhibits Apoptosis Using the Phosphatidylinositol 3′-Kinase and Mitogen-activated Protein Kinase Pathways

doi:10.1074/jbc.272.1.154

Insulin-like Growth Factor 1 Inhibits Apoptosis Using the Phosphatidylinositol 3′-Kinase and Mitogen-activated Protein Kinase Pathways*

From the Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892 and
the ^§ Department of Physiology, University of Michigan, School of Medicine and the Department of Signal Transduction, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Co., Ann Arbor, Michigan 48105

^¶ To whom correspondence should be addressed:
Diabetes Branch, NIDDK, National Institutes of Health, Bldg. 10, Rm. 8S235A, 10 Center Dr., MSC-1770, Bethesda, MD 20892-1770.
Tel.: 301-496-8090; Fax: 301-480-4386; E-mail: Derek{at}helix.nih.gov

Abstract

The role of insulin-like growth factor 1 (IGF-1) in preventing apoptosis was examined in differentiated PC12 cells. Induction of differentiation was achieved using nerve growth factor, and apoptosis was provoked by serum withdrawal. After 4-6 h of serum deprivation, apoptosis was initiated, concomitant with a 30% decrease in cell number and a 75% decrease in MTT activity. IGF-1 was capable of preventing apoptosis at concentrations as low as 10⁻⁹ M and as early as 4 h. The phosphatidylinositol 3′ (PI3′)-kinase inhibitors wortmannin (at concentrations of 10⁻⁸ M) and LY294002 (10⁻⁶ M) blocked the effect of IGF-1. The pp70 S6 kinase (pp70^S6K) inhibitor rapamycin (10⁻⁸ M) was, however, less effective in blocking IGF-1 action. Moreover, stable transfection of a dominant-negative p85 (subunit of PI3′-kinase) construct in PC12 cells enhanced apoptosis provoked by serum deprivation. Interestingly, in the cells overexpressing the dominant-negative p85 protein, IGF-1 was still capable of inhibiting apoptosis, suggesting the existence of a second pathway involved in the IGF-1 effect. Blocking the mitogen-activated protein kinase pathway with the specific mitogen-activated protein kinase/extracellular-response kinase kinase inhibitor PD098059 (10⁻⁵ M) inhibited the IGF-1 effect. When wortmannin and PD098059 were given together, the effect was synergistic. The results presented here suggest that IGF-1 is capable of preventing apoptosis by activation of multiple signal transduction pathways.

Footnotes

↵^‡ Recipient of Postdoctoral Fellowship EX-94 from the Ministerio de Educación y Ciencia (Spain).
↵* 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.
↵¹ The abbreviations used are:

NGF

nerve growth factor

IGF-1

insulin-like growth factor 1

PI3′-kinase

phosphatidylinositol 3′-kinase

IRS-1

insulin receptor substrate 1

SH2

Src homology region 2

ERK

extracellular-response kinase

MAP

mitogen-activated protein

MAPK

MAP kinase

MEK

MAPK/ERK kinase

pp70^S6K

pp70 S6 kinase

DMEM

Dulbecco's modified Eagle's medium

SFM

serum-free DMEM

PBS

phosphate-buffered saline

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
- Received August 20, 1996.
- Revision received October 18, 1996.