Transcriptional coactivator Cited2 induces Bmi1 and Mel18 and controls fibroblast proliferation via Ink4a/ARF - PubMed (original) (raw)

Transcriptional coactivator Cited2 induces Bmi1 and Mel18 and controls fibroblast proliferation via Ink4a/ARF

Kamil R Kranc et al. Mol Cell Biol. 2003 Nov.

Abstract

Cited2 (CBP/p300 interacting transactivator with ED-rich tail 2) is required for embryonic development, coactivation of transcription factor AP-2, and inhibition of hypoxia-inducible factor 1 transactivation. Cited2 is induced by multiple growth factors and cytokines and oncogenically transforms cells. Here, we show that the proliferation of Cited2(-/-) mouse embryonic fibroblasts ceases prematurely. This is associated with a reduction in growth fraction, senescent cellular morphology, and increased expression of the cell proliferation inhibitors p16(INK4a), p19(ARF), and p15(INK4b). Deletion of INK4a/ARF (encoding p16(INK4a) and p19(ARF)) completely rescued the defective proliferation of Cited2(-/-) fibroblasts. However, the deletion of INK4a/ARF did not rescue the embryonic malformations observed in Cited2(-/-) mice, indicating that INK4a/ARF-independent pathways are likely to be involved here. We found that Cited2(-/-) fibroblasts had reduced expression of the polycomb-group genes Bmi1 and Mel18, which function as INK4a/ARF and Hox repressors. Complementation with CITED2-expressing retrovirus enhanced proliferation, induced Bmi1/Mel18 expression, and decreased INK4a/ARF expression. Bmi1- and Mel18-expressing retroviruses enhanced the proliferation of Cited2(-/-) fibroblasts, indicating that they function downstream of Cited2. Our results provide genetic evidence that Cited2 controls the expression of INK4a/ARF and fibroblast proliferation, at least in part via the polycomb-group genes Bmi1 and Mel18.

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Figures

FIG. 1.

FIG. 1.

Proliferation properties and morphology of _Cited2_−/− mouse embryonic fibroblasts. (A) Cumulative growth of Cited2+/+ (w) and _Cited2_−/− (n) fibroblast cultures prepared from 15.5-dpc embryos on a 129Ola/C57BL/6J mixed background shown as a plot of CPD versus passage number. (B) These results are also reproduced in Cited2+/+ (w) and _Cited2_−/− (n) fibroblast cultures isolated from 13.5-dpc embryos on a coisogenic C57BL/6J background. (C) Proliferative capacity of fibroblasts at passage 3. Fibroblasts prepared from embryos on a 129Ola/C57BL/6J background were plated in quadruplicate into 12-well plates (6 × 103 cells per well) and fixed at the indicated time points, and relative cell numbers were determined by using crystal violet. Data (means ± standard errors of the means [SEMs]) were normalized for cell numbers at day 1. (D) Morphology of Cited2+/+ (w) and _Cited2_−/− (n) fibroblasts at passage 4. _Cited2_−/− cultures rapidly accumulate cells that appear flat with cytoplasmic enlargement. (E) Cited2+/+ (w) and _Cited2_−/− (n) fibroblasts were stained for senescence-associated β-galactosidase at passage 1 (P1) or passage 4 (P4).

FIG. 2.

FIG. 2.

Growth fraction of _Cited2_−/− mouse embryonic fibroblasts. (A) Scattergrams of nuclear DNA synthesis (BrdU) vs. DNA content (PI) of Cited2+/+ (w) and _Cited2_−/− (n) fibroblast cultures at passages 0 and 4. BrdU-labeled cells are indicated in green. Unlabeled cells are indicated in blue (2n), red (4n), or black (8n DNA content). (B) Growth fraction of _Cited2_−/− and Cited2+/+ fibroblast cultures plotted against passage number.

FIG. 3.

FIG. 3.

INK4a/ARF expression in _Cited2_−/− fibroblasts. (A) Representation of the INK4a/ARF and INK4b locus showing exon structure and alternative splicing (dashed lines) that generates the three different cell cycle inhibitors p15INK4b, p16INK4a, and p19ARF. (B through F) Northern blots of total RNA obtained from independent pairs of Cited2+/+ (w) and _Cited2_−/− (n) fibroblasts derived from littermate embryos. The passage number (P) is indicated in each panel. (B) A full-length p19ARF cDNA was used as a probe. This probe detects both p19ARF and p16INK4a isoforms, which comigrate. (C) p16INK4a was detected by using a specific exon 1α probe. (D) p19ARF was detected by using a specific exon 1β probe. (E and F) p15INK4b and p19INK4d transcripts were detected by using full-length cDNA probes. The bottom section in each panel shows 28S and 18S RNA species, confirming equal loading. (G and H) Western blots of total cell lysates from Cited2+/+ and _Cited2_−/− fibroblasts at passage 1 were probed with anti-p16INK4a and anti-p19ARF antibodies (top panels) and with an antitubulin monoclonal antibody (bottom panels) to demonstrate equal loading.

FIG. 4.

FIG. 4.

Complementation of _Cited2_−/− fibroblasts with CITED2. (A) _Cited2_−/− fibroblasts (derived from an embryo at 13.5 dpc) were infected with CITED2, CITED2Δ (lacking residues 215 to 270), or LZRS (control) retrovirus. Infected fibroblasts were replated on day 7 (passage 0) and passaged every 3 days. Cumulative growth of retrovirally complemented _Cited2_−/− fibroblasts is shown as a plot of CPD versus passage number. The results were reproduced in two further independent experiments by using independently isolated fibroblasts. (B and C) Northern blots of total RNA obtained from retrovirally complemented _Cited2_−/− fibroblasts. LZ indicates infection with LZRS (control) retrovirus, and C2 indicates infection with CITED2-expressing retrovirus. The transcripts detected are indicated in each panel. The bottom panels in each figure show 28S and 18S RNA species, confirming equal loading. (B) A full-length p19ARF cDNA was used as a probe. This probe detects both p19ARF and p16INK4a isoforms, which comigrate. (C) A p15INK4b transcript was detected by using a full-length cDNA probe.

FIG. 5.

FIG. 5.

Proliferative properties of fibroblasts lacking both Cited2 and INK4a/ARF. (A and B) Fibroblasts were harvested simultaneously from independent embryos at 13.5 dpc. Cumulative fibroblast growth is shown as a plot of CPD versus passage number. Each line represents fibroblasts isolated from an independent embryo. Fibroblast genotypes (w, wild-type; n, null) for Cited2 and INK4a/ARF alleles are indicated in panel B. (C) Proliferative capacity of fibroblasts at passage 4. Fibroblasts were plated in quadruplicate into 12-well plates (2.5 × 104 cells per well) and fixed at the indicated time points, and relative cell numbers were determined by using crystal violet as previously described (25). Data (means ± SEMs) were normalized for cell numbers at day 1. The genotypes are indicated in panel B. (D) Colony formation assay. Fibroblasts (passage 1) were plated at a density of 4,000 cells per 9-cm plate, and colonies were visualized with Giemsa stain after 7 days. Fibroblast genotypes are indicated as wild-type (w) or null (n) for Cited2 and INK4a/ARF alleles, as indicated in panel B.

FIG. 6.

FIG. 6.

Embryonic malformations in embryos lacking both Cited2 and INK4a/ARF. (A through D) Hematoxylin- and eosin-stained transverse sections from Cited2+/+:INK4a/_ARF_−/− and _Cited2_−/−:INK4a/_ARF_−/− embryos at 14.5 dpc. Scale bars, 0.1 mm. Axes: d, dorsal; v, ventral; r, right; l, left. (A) Transverse section through the thorax of a Cited2+/+:INK4a/_ARF_−/− embryo shows normal cardiac anatomy. The interatrial septum (ias) separates the right (ra) and left (la) atria. The right (rv) and left (lv) ventricles are separated by a normal interventricular septum (ivs) (B) Corresponding section of a _Cited2_−/−:INK4a/_ARF_−/− embryo shows a ventricular septal defect (VSD). (C) Transverse section through the abdomen of a Cited2+/+:INK4a/_ARF_−/− embryo showing normal right (rad) and left (lad) adrenal glands and left kidney (lk). (D) Corresponding section of a _Cited2_−/−:INK4a/_ARF_−/− embryo shows lack of right and left adrenal glands. (E and F) Cranial development in Cited2+/+:INK4a/_ARF_−/− and _Cited2_−/−:INK4a/_ARF_−/− embryos at 14.5 dpc. Scale bar, 1 mm. (E) Cited2+/+:INK4a/_ARF_−/− embryo showing normal cranial development. (F) _Cited2_−/−:INK4a/_ARF_−/− embryo showing exencephaly.

FIG. 7.

FIG. 7.

Expression of Mel18 and Bmi1 in fibroblasts lacking Cited2. (A) Northern blots of total RNA obtained from Cited2+/+ (w) and _Cited2_−/− (n) fibroblasts derived from littermate embryos. The top panels show blots probed for Mel18, Bmi1, or Mph1, as indicated. Two Mel18 isoforms are noted (52). The bottom panels show 28S and 18S RNA species to show equal loading. (B) Western blot of total cell lysate probed with an anti-Bmi1 monoclonal antibody (top) and reprobed with an antitubulin monoclonal antibody (bottom) to show equal loading. (C) Northern blot of total RNA obtained from _Cited2_−/− fibroblasts infected in parallel with retroviruses. LZ indicates infection with LZRS (control) retrovirus, and C2 indicates infection with CITED2-expressing retrovirus. The top panel shows a blot probed with a Mel18 cDNA. The bottom panel shows 28S and 18S RNA species to show equal loading. (D) Western blot of total cell lysate from _Cited2_−/− fibroblasts. LZ indicates infection with LZRS (control) retrovirus and C2 indicates infection with CITED2-expressing retrovirus. The experiment was performed in duplicate using independently isolated _Cited2_−/− fibroblast lines (nos. 1 and 2). The top panel shows a blot probed with an anti-Bmi1 antibody. The bottom panel shows a blot probed with an antitubulin antibody to show equal loading.

FIG. 8.

FIG. 8.

Growth properties of _Cited2_−/− and Cited2+/+ embryonic fibroblasts infected with Bmi1- and Mel18-expressing retroviruses. (A) Proliferation of _Cited2_−/− (n) and Cited2+/+ (w) fibroblasts (derived from littermate embryos on a C57BL/6J background at 13.5 dpc) infected with Bmi1, Mel18, or control retroviruses. Fibroblasts were harvested, infected with retroviruses the following day, replated on day 4 (referred to as P0), and passaged in parallel every 3 days at identical conditions. The proliferation of retrovirally complemented fibroblasts is shown as plots of CPD versus passage number. (B) Colony formation assay. _Cited2_−/− and Cited2+/+ fibroblasts (at passage 6) infected with the indicated retroviruses were plated at a density of 4,000 cells per 9-cm plate, and colonies were visualized with Giemsa stain after 16 days.

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