Involvement of protein kinase-C in the mitogenic effect of insulin-like growth factor-I on rat astrocytes.
Insulin-like growth factor-I (IGF-I) stimulates the proliferation of many cell types, including astrocytes. Astrocytes are a population of brain cells highly enriched in IGF-I receptors, which unlike neurons, retain the ability to proliferate in the adult brain. Although astrocyte proliferation in response to IGF-I is well documented, the intracellular mechanisms that mediate this phenomenon are poorly defined. Interestingly, activation of protein kinase-C (PKC) by IGF-I has been observed in several cell types. In this report we first characterized the mitogenic effects of IGF-I on highly purified type I rat astrocyte cultures. Next, we determined whether IGF-I activates PKC in our cultures. Finally, since astrocyte proliferation is stimulated by both IGF-I and the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA), we decided to test the possible involvement of PKC in the mitogenic activity of IGF-I on astrocytes. IGF-I stimulated the DNA synthesis rate in rat astrocytes. Analysis of the time course revealed that IGF-I (10 nM) induces maximal stimulation of [3H]thymidine incorporation (a 4-fold increase) 16-18 h after exposure. TPA also stimulated mitogenesis in our cultures. The dose-response of [3H]thymidine incorporation induced by IGF-I and TPA indicated that 10 nM was the lowest concentration producing a maximal effect for both agents. Analysis of proteins by Western blot revealed that 10 nM IGF-I translocates PKC(alpha), the predominant PKC isoform in astrocyte cultures, from the cytosol to the membrane fraction within 20 min. A similar activation of PKC was achieved with 100 nM TPA. When astrocytes were exposed to IGF-I (10 nM) and TPA (10 nM) in combination, [3H]thymidine uptake was significantly higher than the uptake induced by either IGF-I (10 nM) or TPA (10 nM) alone. However, the effect of IGF-I plus TPA was not fully additive. In a second experiment, the mitogenic effect of IGF-I was partially abolished in cells depleted of PKC by preincubation with high concentrations of TPA (300 nM). Finally, incubation of astrocytes with the PKC inhibitor H-7 at 20 microM, a concentration that completely blocked the mitogenic action of TPA, only reduced the ability of IGF-I to stimulate DNA synthesis by 50%. In summary, our results demonstrate that IGF-I can rapidly activate PKC in astrocytes, and that PKC activation is involved in the mitogenic effect of IGF-I on these cells. However, we conclude that IGF-I also stimulates astrocyte proliferation through PKC-independent pathways.
Tranque
P A
PA
Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Conneticut 06510.
Calle
R
R
Naftolin
F
F
Robbins
R
R
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000818:Animals; D001253:Astrocytes / Q000187:drug effects*; D002478:Cells, Cultured; D004338:Drug Combinations; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007546:Isoquinolines / Q000494:pharmacology; D008934:Mitogens / Q000494:pharmacology*; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D047428:Protein Kinase Inhibitors; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013936:Thymidine / Q000378:metabolism
D004338:Drug Combinations; D007546:Isoquinolines; D008934:Mitogens; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D007334:Insulin-Like Growth Factor I; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D013936:Thymidine