Increased levels of cyclins D1 and D3 after inhibition of gap junctional communication in astrocytes Arantxa Tabernero 1 , Rosa Sa ´nchez-Alvarez 1 and Jose ´ M. Medina Departamento de Bioquı ´mica y Biologı ´a Molecular, Instituto De Neurociencias De Castilla Y Leon, Universidad de Salamanca, Salamanca, Spain 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 prolifer- ation. In the present study we investigated the changes in the cell cycle of astrocytes promoted by the inhibition of intercel- lular communication through gap junctions. Thus, the pres- ence of endothelin-1 and carbenoxolone, two gap junction uncouplers, promoted an increase in the percentage of ast- rocytes 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 pre- vented 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. Keywords: brain, carbenoxolone, endothelin, gap junctions, glia, retinoblastoma. J. Neurochem. (2006) 96, 973–982. Several lines of evidence suggest that the regulation of gap junctional communication is required for an adequate rate of cell proliferation (for review see Loewenstein 1992; Yama- saki and Naus 1996). Thus, the neoplastic phenotype of several tumours includes not only the absence of gap junctional communication but also a lack of expression of connexins, the proteins that form gap junction channels. In fact, connexins have been proposed as tumour suppressor proteins as their overexpression in tumour cells reverses the neoplastic phenotype (for review see Yamasaki and Naus 1996). However, the expression of connexins needs fine regulation because their forced expression in tumour cells has been associated with invasion of the surrounding tissue (Lin et al. 2002; Pollmann et al. 2005). Astrocytes are extensively coupled through gap junctions (Mugnaini 1986), which allow the cell-to-cell passage of small molecules such as ions, second messengers and metabolites (Loewenstein 1987; Tabernero et al. 1996a; Giaume et al. 1997; for review see Kumar and Gilula 1996). In tumours derived from astrocytes, the expression of connexin 43 (Cx43), the main connexin in astrocytes (Giaume et al. 1991), is inversely correlated with the degree of malignancy (Shinoura et al. 1996; Huang et al. 1999; Received June 6, 2005; revised manuscript received October 11, 2005; accepted November 11, 2005. Address correspondence and reprint requests to Jose ´ M. Medina, Departamento de Bioquı ´mica y Biologı ´a Molecular, Universidad de Salamanca, Edificio Departamental, Pza Doctores de la Reina 37007 Salamanca, Spain. E-mail: medina@usal.es 1 Both authors contributed equally to this work. Abbreviations used: BrdU, bromodeoxyuridine; CBX, carbenoxolone; Cdk, cyclin-dependent kinase; Cx43, connexin 43; DMEM, Dulbecco’s modified Eagle’s medium; ET-1, endothelin-1; FCS, fetal calf serum; NCBI, National Center for Biotechnology Information; pRb, retinobla- stoma protein; SDS, sodium dodecyl sulphate; SSC, saline sodium cit- rate; tolb, tolbutamide. Journal of Neurochemistry , 2006, 96, 973–982 doi:10.1111/j.1471-4159.2005.03623.x Ó 2006 The Authors Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2006) 96, 973–982 973