Increased levels of cyclins D1 and D3 after inhibition of gap junctional communication in astrocytes

Abstract

We showed previously that the inhibition of gap junctional communication in astrocytes increased bromodeoxyuridine (BrdU) incorporation and promoted changes in the metabolic phenotype destined to fulfil the requirements of cell proliferation. In the present study we investigated the changes in the cell cycle of astrocytes promoted by the inhibition of intercellular communication through gap junctions. Thus, the presence of endothelin-1 and carbenoxolone, two gap junction uncouplers, promoted an increase in the percentage of astrocytes found in the S, G2 and M phases of the cell cycle, with a concomitant decrease in G0 and G1 phases. In addition, the levels of Ki-67, a protein present during all active phases of the cell cycle but absent from resting cells, increased after the inhibition of gap junctional communication. These effects were not observed when the inhibition of gap junctions was prevented with tolbutamide, indicating that the inhibition of gap junctional communication promotes the entry of astrocytes into the cell cycle. The passage of the cells from a quiescent state to the cell cycle is ultimately regulated by the degree of retinoblastoma phosphorylation. Inhibition of gap junctions increased the phosphorylation of retinoblastoma at Ser 780 but not at Ser 795 or Ser 807/811. In addition, the levels of cyclins D1 and D3 increased, whereas those of p21 and p27 were not significantly modified. Because D-type cyclins are key regulators of retinoblastoma protein phosphorylation, it is suggested that the phosphorylation of retinoblastoma protein at Ser 780, observed under our experimental conditions, is a consequence of the increase in the levels of cyclins D1 and D3. Our work provides evidence for the involvement of cyclins D1 and D3 as sensors of the inhibition of gap junctional communication in astrocytes.