Endothelin-1 stimulates cyclin D1 expression in rat cultured astrocytes via activation of Sp1

• Published in: Neurochemistry international Vol. 63; no. 1; pp. 25 - 34
• Main Authors: Michinaga, Shotaro, Ishida, Ayaka, Takeuchi, Risa, Koyama, Yutaka
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.07.2013
• Subjects: Animals; Astrocytes; Astrocytes - drug effects; Astrocytes - metabolism; Astrogliosis; Base Sequence; Cells, Cultured; Cyclin D1; Cyclin D1 - metabolism; DNA Primers; Endothelin; Endothelin-1 - pharmacology; ETB receptor; Mitogen-activated protein kinase; Mitogen-Activated Protein Kinases - drug effects; Phosphorylation; Plicamycin - pharmacology; Protein Kinase Inhibitors - pharmacology; Rats; Real-Time Polymerase Chain Reaction; Sp1; Sp1 Transcription Factor - metabolism; Endothelin; JNK; PDTC; G3PDH; Mitogen-activated protein kinase; MAPK; Cyclin D1; ET; GFAP; Astrogliosis; NF-κB; ETB receptor; MEM; Sp1; FBS; PI; BrdU; ELISA; ERK
• ISSN: 0197-0186; 1872-9754
• DOI: 10.1016/j.neuint.2013.04.004

•We examined the intracellular signaling of astrocytic proliferation via endothelin B (ETB) receptor.•Stimulations of ETB receptors induced activation of Sp1 and expression of cyclin D1.•ERK and JNK were involved in activation of Sp1 and increase of cyclin D1 by stimulation of ETB receptors.•Activation of Sp1 via ETB receptors was responsible for astrocytic proliferation. Endothelins (ETs), a family of vasoconstrictor peptides, are up-regulated in several pathological conditions in the brain, and induce astrocytic proliferation. We previously observed that ET-1 increased the expression of cyclin D1 protein. Thus, we confirmed the intracellular up-regulation of cyclin D1 by ET-1 in rat cultured astrocytes. Real-time PCR analysis indicated that ET-1 (100nM) and Ala1,3,11,15-ET-1 (100nM), a selective agonist of the ETB receptor, induced a time-dependent and transient increase in cyclin D1 mRNA. The effect of ET-1 was diminished by an ETB antagonist (1μM BQ788) or inhibitors of Sp1 (500nM mithramycin), ERK (50μM PD98059), p38 (20μM SB203580) and JNK (1μM SP600125), but not inhibitors of NF-κB (10μM SN50 and 100μM pyrrolidine dithiocarbamate). The binding assay for Sp1 indicated that ET-1 increased the binding activity of Sp1 to consensus sequences, and two oligonucleotides of the cyclin D1 promoter including the Sp1-binding sites diminished the effect of ET-1. Western blot analysis showed that ET-1 induced time-dependent and transient phosphorylation of Sp1 on Thr453 and Thr739 via the ETB receptor. ET-1-induced phosphorylation of Sp1 was attenuated by PD98059 and SP600125. Additionally, ET-1 increased the incorporation of bromodeoxyuridine (BrdU) in cultured astrocytes and the number of BrdU-positive cells decreased in the presence of PD98059, SP600125 and mithramycin. These results suggest that ET-1 increases the expression of cyclin D1 via activation of Sp1 and induces astrocytic proliferation.