ASTROCYTE PROGRESSION FROM G(1) TO S-PHASE OF THE CELL-CYCLE DEPENDS UPON MULTIPLE PROTEIN-INTERACTION

The proliferation of cultured astrocytes is positively and negatively regulated, respectively, by the endogenous neuropeptides, endothelin-3 (ET-3) and atrial natriuretic peptide (ANP), Here, we determined the important steps for the modulation by ET and ANP of G(1) to S phase ce ll cycle progression. ET-3 stimulated an increased number of fetal rat diencephalic astrocytes to progress through G(1)/S, and this was bloc ked significantly by ANP, ET augmented the gene expression and/or prot ein production of D-type, A and E cyclins, whereas ANP inhibited these events significantly, ET also stimulated the activation of the cyclin -dependent kinases Cdk2, Cdk4, and Cdk6, directed against the retinobl astoma protein pRb, and this was inhibited by as much as 80% by ANP, A s an additional mechanism of cell cycle restraint, ANP stimulated the production of multiple cyclin-dependent kinase inhibitory (CKI) protei ns, including p16, p27, and p57, This was critical because antisense o ligonucleotides to each CKI reversed ANP-induced inhibition of ET-stim ulated DNA synthesis by as much as 85%, CKI antisense oligonucleotides also reversed the ANP inhibition of Cdk phosphorylation of pRb, In tu rn, ET inhibited ANP-stimulated production of the CKIs, thereby promot ing cell cycle progression. Specific and changing associations of the CKI with Cdk2 and Cdk4 were stimulated by ANP and inhibited by ET, Our findings identify several mechanisms by which endogenous modulators o f astrocyte proliferation can control the G(1)-S progression and indic ate that multiple CKIs are necessary to restrain cell cycle progressio n in these cells.