Reconstitution of cyclin D1-associated kinase activity drives terminally differentiated cells into the cell cycle

Abstract

Terminal cell differentiation entails definitive withdrawal from the cell cycle. Although most of the cells of an adult mammal are terminally differentiated, the molecular mechanisms preserving the postmitotic state are insufficiently understood. Terminally differentiated skeletal muscle cells, or myotubes, are a prototypic terminally differentiated system. We previously identified a mid-G(1) block preventing myotubes from progressing beyond this point in the cell cycle. In this work, we set out to define the molecular basis of such a block. It is shown here that overexpression of highly active cyclin E and cdk2 in myotubes induces phosphorylation of pRb but cannot reactivate DNA synthesis, underscoring the tightness of cell cycle control in postmitotic cells. In contrast, forced expression of cyclin D1 and wild-type or dominant-negative cdk4 in myotubes restores physiological levels of cdk4 kinase activity, allowing progression through the cell cycle. Such reactivation occurs in myotubes derived from primary, as well as established, C2C12 myoblasts and is accompanied by impairment of muscle-specific gene expression. Other terminally differentiated systems as diverse as adipocytes and nerve cells are similarly reactivated. Thus, the present results indicate that the suppression of cyclin D1-associated kinase activity is of crucial importance for the maintenance of the postmitotic state in widely divergent terminally differentiated cell types.

FIG. 1

Exogenous cyclin E and cdk2 expression. (A) Western blot analysis of C2C12 myotubes (Mt) infected with the Ad-Track control virus (Ctr; MOI, 300) or with the Ad-cycE and Ad-cdk2 viruses (cycE/cdk2; MOIs, 200 and 350, respectively) at 48 h p.i. Proliferating C2C12 myoblasts (Mb) and/or human 293 cells, expressing high levels of endogenous cyclin E, are shown for comparison. 293 cells are shown because human cyclin E levels in myotubes could not be compared with those physiologically expressed in mouse C2C12 myoblasts, since an antibody reacting equally with murine and human cyclin E was not available. (B) Cyclin E-associated kinase activity immunoprecipitated from myotubes infected with Ad-cycE and Ad-cdk2, the E1A-expressing virus dl520 (for reference), or a control virus. Myoblasts are shown for comparison. A nonspecific (n.s.) antibody was used as a control. The precipitates were assayed by using histone H1 (H1) as the substrate. (C) Western blot analysis of pRb in myotubes infected with the Ad-cycE and Ad-cdk2, dl520, and control viruses and myoblasts. The slow-migrating, hyperphosphorylated (ppRb) and the hypophosphorylated (pRb) forms of Rb are indicated.

FIG. 2

Endogenous and exogenous expression of cyclin D1 and cdk4 in myotubes. (A) Western blot (WB) analysis of endogenous cyclin D1 expression in TD C2C12 myotubes treated with serum for the indicated times. (B) Kinase activities precipitated by anti-cyclin D1 or anti-cdk4 antibodies from C2C12 myoblasts or TD myotubes treated with serum for 0 or 48 h. Nonspecific immunoprecipitation (IP) was done as described in the legend to Fig. 1. The kinase activities were assayed by using a glutathione S–transferase Rb (Rb) fusion protein as the substrate. (C) Western blot analysis of cyclin D1 in the immunoprecipitates whose kinase activity is shown in panel B. (D) Western blot analysis of cyclin D1 and cdk4 proteins in myotubes infected with the control virus (MOI, 830) or coinfected with the recombinant adenoviruses Ad-cycD1 and J-cdk4 (MOIs, 60 and 770, respectively) and switched to 5% FBS at 12 h p.i.; whole-cell lysates were prepared 24 h p.i.; proliferating myoblasts are included for comparison. (E) Anti-cyclin D1- and anti-cdk4-precipitated kinase activities were measured in proliferating C2C12 myoblasts and C2C12 myotubes infected as described above with the control virus (20 h p.i.) or coinfected with the Ad-cycD1 and J-cdk4 viruses, stimulated with 5% serum from 12 h p.i., and analyzed at 20 h p.i. Protein extracts (2.5 mg per sample) were immunoprecipitated by using a nonspecific antibody, a mixture of two distinct mouse MAbs to cyclin D1, or a mixture of two different anti-cdk4 antibodies (see Materials and Methods).

FIG. 3

Cyclin D1 and cdk4 expression induces DNA synthesis in myotubes. (A) C2C12 myotubes were coinfected with the Ad-cycD1 and J-cdk4 viruses as described in the legend to Fig. 2 and cultured, from 12 h p.i., in 5% FBS and 20 μM BrdUrd. Cells were fixed at the indicated time points p.i. and subjected to double immunofluorescence staining for MyHC and BrdUrd. The graph shows the percentages of BrdUrd⁺ myotubes (BrdUrd⁺ MyHC⁺ cells). (B) Primary murine satellite cell-derived myotubes were infected with the Ad-cycD1 and J-cdk4 viruses (top) or the control virus (bottom) as described in the legend to Fig. 2 and cultured in 5% FBS. The cells were fixed at 48 h p.i. and immunostained for MyHC (red) and BrdUrd (green). The upper image shows examples of BrdUrd⁺ MyHC⁺ cells, while none of the cells in the lower image incorporated BrdUrd.

FIG. 3

Cyclin D1 and cdk4 expression induces DNA synthesis in myotubes. (A) C2C12 myotubes were coinfected with the Ad-cycD1 and J-cdk4 viruses as described in the legend to Fig. 2 and cultured, from 12 h p.i., in 5% FBS and 20 μM BrdUrd. Cells were fixed at the indicated time points p.i. and subjected to double immunofluorescence staining for MyHC and BrdUrd. The graph shows the percentages of BrdUrd⁺ myotubes (BrdUrd⁺ MyHC⁺ cells). (B) Primary murine satellite cell-derived myotubes were infected with the Ad-cycD1 and J-cdk4 viruses (top) or the control virus (bottom) as described in the legend to Fig. 2 and cultured in 5% FBS. The cells were fixed at 48 h p.i. and immunostained for MyHC (red) and BrdUrd (green). The upper image shows examples of BrdUrd⁺ MyHC⁺ cells, while none of the cells in the lower image incorporated BrdUrd.

FIG. 4

Cyclin D1 and cdk4 expression allows myotube progression through G₁ and S phases, but reactivated muscle cells meet a block in G₂. Mononucleated myocytes derived from primary murine satellite cells were infected with the Ad-cycD1 and J-cdk4 viruses or the control Ad-Track virus as described in the legend to Fig. 2 and subjected to cytofluorimetric analysis at the indicated time points. To maximize recruitment into the cell cycle, 5% FBS was added immediately after infection.

FIG. 5

Cell cycle-related events in myotubes reactivated by cyclin D1 and cdk4. (A) TD C2C12 myotubes were infected with the Ad-cycD1 and J-cdk4 viruses or a control virus as described in the legend to Fig. 2 and treated with 5% FBS from 12 h p.i.; total protein extracts were prepared at the indicated time points (h) after infection. Proliferating myoblasts and uninfected myotubes (0 h) were included for comparison. The indicated gene products were analyzed by Western blotting. (B) Cyclin E-associated kinase activity was measured in myotubes infected as described above. Myoblasts synchronized in late G₁ (peak kinase activity) were included for comparison. A precipitation performed with a nonspecific (n.s.) antibody on a myoblast extract was also included. Total protein extracts were prepared at 24 h p.i. Precipitations were performed with an anti-cyclin E antibody from 2 mg of protein extract from control (Ctr) virus or Ad-cycD1–J-cdk4 virus-infected myotubes or 1 mg of protein extract from myoblasts (see Materials and Methods); as a positive control, a similar kinase assay was also performed on 1 mg of protein extract from myotubes infected with the 12S E1A-expressing dl520 virus. (C) Western blot analysis of the phosphorylation status of pRb. Proteins were extracted from myoblasts, uninfected (Uninf.) myotubes, or myotubes infected with the Ad-cycD1 and J-cdk4 viruses or a control virus at the indicated time points p.i. (hours).

FIG. 6

Expression and activity of dncdk4. (A) Western blot comparing expression of exogenous wild-type (wtK4) and dominant-negative cdk4 (dnK4) in myotubes infected at the indicated MOIs with the corresponding viruses along with Ad-cycD1 (MOI, 60). (B) Percentages of reactivated myotubes infected as described above. (C) Percentages of reactivated myotubes infected with Ad-cycD1 (MOI, 60) and either J-cdk4 (wt cdk4; MOI, 770) or Ad-dncdk4 (dn cdk4; MOI, 238). Three independent experiments are shown. (D) Anti-cdk4-immunoprecipitated kinase activity from myotubes infected as described for panel C. Myoblasts were included for comparison. Kinase assays were done as described in the legend to Fig. 2. (E) Cyclin E-associated kinase activity immunoprecipitated from myotubes infected with the control virus, Ad-cycE plus Ad-cdk2, Ad-cycD1 plus J-cdk4, or Ad-cycD1 plus-Ad-dncdk4. n.s., nonspecific antibody.

FIG. 7

Cyclin D1-cdk4-dependent cell cycle reactivation in myotubes impairs tissue-specific gene expression. C2C12 myotubes infected with a control virus or the Ad-cycD1 and J-cdk4 viruses as described in the legend to Fig. 2 and immediately treated with 5% FBS were lysed at the indicated times p.i. (hours), total RNA was hybridized with labeled probes to MCK, MyHC, and MLC-1. The hybridizations were performed onto two different blots. For technical reasons, normalization was carried out by hybridizing one filter with a GAPDH probe and the other with a 28S rRNA probe. Each blot should be compared with its respective normalizing hybridization, indicated by the letters in parentheses that follow the names of the genes (G, GAPDH; r, 28S rRNA). Ctr, control.

FIG. 8

Cyclin D1 and cdk4 reactivate TD adipocytes and neurons. TD 3T3-L1 adipocytes were infected with the Ad-cycD1 and J-cdk4 viruses and, 48 h later, subjected to BrdUrd immunofluorescence. Two microscopic fields (a, b and c, d) are shown. Two adipocytes (arrowheads in panel a) are BrdUrd⁺ (arrows in panel b) and show condensed chromatin, suggestive of a late telophase. The adipocyte photographed in phase contrast (arrowhead in panel c) shows a metaphase plate by Hoechst 33258 staining (arrow in panel d). P19-derived neurons were infected with the Ad-cycD1 and J-cdk4 viruses and doubly stained for the neuron-specific marker Tau and BrdUrd. Two examples of reactivated neurons (arrows) are shown in phase contrast (e and h); the same two fields are shown stained with anti-Tau (f and i) and anti-BrdUrd (g and j). Bar, 100 μm.

FIG. 9

Working model of mitotic cycle regulation in TD cells. In this schematic, the first half of the cell cycle is represented in a linear fashion (thick arrow); boundaries between cell cycle phases are marked by thin vertical lines. The approximate points where transcription of some cell cycle regulatory genes begins are marked. Growth factor or retroviral-oncogene stimulation induces G₀-arrested TD cells to enter G₁ and progress to the mid-G₁ block (thick vertical line) but not beyond. The adenovirus oncogene E1A bypasses the mid-G₁ block by acting directly at the G₁/S boundary, in part by freeing E2F from the control of pRb family proteins. In turn, E2F promotes the transcription of genes directly involved in S phase transition and DNA replication, among which are those for cyclin E and cyclin A. In contrast, simultaneous expression of cyclin D1 and cdk4 reconstitutes cdk4 activity and allows progression beyond the G₁ cell cycle block initiated by serum stimulation. A second block, represented by a thin double line close to the G₁/S boundary prevents cyclin E (this work)- or E2F (references and 31)-mediated reactivation of myotubes.