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.

Connexin43 is involved in the effect of endothelin‐1 on astrocyte proliferation and glucose uptake

“…Thus, several authors have reported that endothelins upregulate the expression of cyclins D1 and D3, promoting the phosphorylation of pRb (Koyama et al, 2004;Tabernero et al, 2006b;Teixeira et al, 2000) required for the transition of astrocytes from the G0/G1 to the S phase of the cell cycle (Sherr, 1995). In this study, we addressed the participation of Cx43 in these effects.…”

“…The protein extract (90 lg) was applied to a 10% SDS-polyacrylamide gel under reducing conditions and then transferred to a PVDF sheet as described (Tabernero et al, 2006b). The PVDF sheet was blocked with 10% fat-free dried milk in TTBS and then exposed to primary antibody against Cx43 (1:100), type I hexokinase antibody (1:500), GLUT-1 antibody (1:500), cyclin D1 (1:250), cyclin D3 (1:250), ki-67 (1:400), pRb phosphorylated on Ser-795 (1:500), or pRb phosphorylated on Ser-780 (1:200) for at least 4 h. Peroxidase-conjugated anti-rabbit IgG and peroxidase-conjugated anti-mouse IgG were used and developed with a chemiluminiscent substrate.…”

“…ET-1, endothelin-1; CBX, carbenoxolone. expression in astrocytes (Spinella et al, 2003;Tabernero et al, 2006b). Thus, in astrocytes transfected with NTsiRNA both ET-1 and CBX reduced Cx43 expression by about 50% (Fig.…”

“…In this sense, the ability of ET-1 to inhibit gap junctional communication between astrocytes both in vitro and in vivo is well documented (Blomstrand and Giaume, 2006;Blomstrand et al, 2004;Giaume et al, 1992;Hasselblatt et al, 2003;Tabernero et al, 1996a;Tabernero et al, 2001;Venance et al, 1997). Indeed, ET-1 downregulates the expression of connexin43 (Cx43) (Tabernero et al, 2006b), the main protein forming gap junction channels in astrocytes (Giaume et al, 1991). Cx43 has been widely investigated owing to its effect in regulating cell proliferation (for recent review, see Kardami et al, 2007).…”

“…Thus, tolbutamide, which prevents the ET-1-induced inhibition of gap junctional communication and the downregulation of Cx43 (Granda et al, 1998;Tabernero et al, 2001;Tabernero et al, 2006b), also abolishes the effects caused by ET-1 on glucose uptake, astrocyte proliferation (Tabernero et al, 2001;Tabernero et al, 2006b), increases in protein content, and changes in morphology (Hasselblatt et al, 2003). To confirm the involvement of Cx43 in these effects, in this study, we investigated the effects of ET-1 in astrocytes whose Cx43 had been silenced by siRNA and in astrocytes obtained from Cx43 KO mice.…”