A Requirement for Cyclin D3–Cyclin-dependent Kinase (cdk)-4 Assembly in the Cyclic Adenosine Monophosphate–dependent Proliferation of Thyrocytes

Abstract

In different systems, cyclic adenosine monophosphate (cAMP) either blocks or promotes cell cycle progression in mid to late G1 phase. Dog thyroid epithelial cells in primary culture constitute a model of positive control of DNA synthesis initiation and G0-S prereplicative phase progression by cAMP as a second messenger for thyrotropin (TSH). The cAMP-dependent mitogenic pathway is unique as it is independent of mitogen-activated protein kinase activation and differs from growth factor–dependent pathways at the level of the expression of several protooncogenes/transcription factors. This study examined the involvement of D-type G1 cyclins and their associated cyclin-dependent kinase (cdk4) in the cAMP-dependent G1 phase progression of dog thyroid cells. Unlike epidermal growth factor (EGF)+serum and other cAMP-independent mitogens, TSH did not induce the accumulation of cyclins D1 and D2 and partially inhibited the basal expression of the most abundant cyclin D3. However, TSH stimulation enhanced the nuclear detection of cyclin D3. This effect correlated with G1 and S phase progression. It was found to reflect both the unmasking of an epitope of cyclin D3 close to its domain of interaction with cdk4, and the nuclear translocation of cyclin D3. TSH and EGF+serum also induced a previously undescribed nuclear translocation of cdk4, the assembly of precipitable cyclin D3–cdk4 complexes, and the Rb kinase activity of these complexes. Previously, cdk4 activity was found to be required in the cAMP-dependent mitogenic pathway of dog thyrocytes, as in growth factor pathways. Here, microinjections of a cyclin D3 antibody showed that cyclin D3 is essential in the TSH/ cAMP-dependent mitogenesis, but not in the pathway of growth factors that induce cyclins D1 and D2. The present study (a) provides the first example in a normal cell of a stimulation of G1 phase progression occurring independently of an enhanced accumulation of cyclins D, (b) identifies the activation of cyclin D3 and cdk4 through their enhanced assembly and/or nuclear translocation, as first convergence steps of the parallel cAMP-dependent and growth factor mitogenic pathways, and (c) strongly suggests that this new mechanism is essential in the cAMP-dependent mitogenesis, which provides the first direct demonstration of the requirement for cyclin D3 in a G1 phase progression.

Nature of the Critical Labile Event That Controls RB Phosphorylation in the Cyclic AMP-Dependent Cell Cycle of Thyrocytes in Primary Culture

“…In dog thyrocytes stimulated by TSH or forskolin, the entry of cells into S phase absolutely requires the activity of CDK4 [21] and critically depends on the most abundant cyclin D3 but not on cyclin D1 [22]. Cyclins D1 and D2 are weakly expressed and not induced by TSH and forskolin [22].…”

“…supplemented with bovine insulin (Sigma; 5 g/ml), ascorbic acid (40 g/ml), and antibiotics [24]; and stimulated at day 4 with forskolin (Calbiochem; 2 ϫ 10 Ϫ6 M) as detailed in the figure legends. Immunofluorescent detections of proliferating cell nuclear antigen (PCNA) [25], cyclin D3, and CDK4 [22] were performed exactly as described from cells fixed with 2% paraformaldehyde for 90 s and then with methanol for 10 min at Ϫ20°C. Percentages of cells in the different phases of cell cycle were determined from the different patterns of PCNA immunofluorescent staining [25,26] by counting at least 500 cells per dish.. Western blotting analyses of cyclin D3, CDK4, and p27 kip1 and analyses of proteins coimmunoprecipitated with cyclin D3 were performed as described [22] using horseradish peroxidase (HRP)-conjugated secondary antibodies (Amersham) and revealed using enhanced chemiluminescence (ECL kit, Amersham).…”

“…Immunofluorescent detections of proliferating cell nuclear antigen (PCNA) [25], cyclin D3, and CDK4 [22] were performed exactly as described from cells fixed with 2% paraformaldehyde for 90 s and then with methanol for 10 min at Ϫ20°C. Percentages of cells in the different phases of cell cycle were determined from the different patterns of PCNA immunofluorescent staining [25,26] by counting at least 500 cells per dish.. Western blotting analyses of cyclin D3, CDK4, and p27 kip1 and analyses of proteins coimmunoprecipitated with cyclin D3 were performed as described [22] using horseradish peroxidase (HRP)-conjugated secondary antibodies (Amersham) and revealed using enhanced chemiluminescence (ECL kit, Amersham). For quantitation of the proportion of hyperphosphorylated RB over total amount of RB protein, total cellular proteins were separated on SDS-6% polyacrylamide gels and immunodetected after Western blotting using the C-15 RB antibody from Santa Cruz Biotechnology (Santa Cruz, CA) and HRP-conjugated anti-rabbit immunoglobulin.…”

“…A total of 150 measurements of optical densities that cover the total area of the RB doublet were integrated and volumes of the peaks corresponding to underphosphorylated and hyperphosphorylated RB were computed by a deconvolution program using a Gaussian curve fitting. The following antibodies were used: PCNA, PC10 (Dakopatts); cyclin D3, DCS-22 (immunofluorescence and Western blotting) and DCS-28 (immunoprecipitation) [22,27]; CDK4, DCS-31 (immunofluorescence and Western blotting) or the C-22 polyclonal antibody from Santa Cruz Biotechnology (Western blotting); and p27 kip1 , C-19 polyclonal antibody from Santa Cruz Biotechnology.…”

“…Strikingly, unlike all the other classes of mitogenic factors in this and other systems, TSH does not enhance cyclin D accumulation, but it induces the nuclear translocation and assembly of required cyclin D3-CDK4 complexes [22], and it stimulates the accumulation of the nuclear p27 kip1 protein [23]. This clearly raises the question of the nature of the labile rate-limiting event associated with the passage through R point controlled by cAMP.…”