Induction of p57(KIP2) expression by p73beta

Abstract

The p53-related protein p73 has many functions similar to that of p53 including the ability to induce cell-cycle arrest and apoptosis. Both p53 and p73 function as transcription factors, and p73 activates expression of many genes that also are regulated by p53. Despite their similarities, it is evident that p53 and p73 are not interchangeable functionally, with p73 playing a role in normal growth and development that is not shared by p53. In this paper we describe the ability of p73beta but not p53 to activate expression of the cyclin-dependent kinase inhibitor p57(KIP) and KvLQT1, two genes that are coregulated in an imprinted region of the genome. Our results suggest that p73 may regulate expression of genes through mechanisms that are not shared by p53, potentially explaining the different contributions of p53 and p73 to normal development.

Figure 1

Saos-2 cell lines with doxycycline-inducible expression of various p73 splice forms. Western blot analysis showing inducible expression of HA-tagged p73β, p73α, or p73γ in Saos-2 cells without (−) and after (+) treatment with 2 μg/ml doxycycline (Dox) for 40 h. p73β protein was detected by using the rabbit polyclonal antibody specific for p73β (37); p73α and p73γ were detected by using an anti-HA antibody. Induction of endogenous MDM2 and p21^CIP1 is shown also. Actin expression was examined as a loading control.

Figure 2

Activation of p57^KIP2 mRNA expression by p73β. (a) Saos-2p73 cells were untreated (0) or treated with 2 μg/ml doxycycline for the indicated times (hours Dox), and RNA was analyzed by RNase protection. Protected probes specific for p57^KIP2, p27^KIP1, p21^CIP1, and GAPDH are shown. (b) MEFs were transiently transfected with 10 μg of vector control (v), human p73β, or human p73γ expression constructs and harvested 24 h posttransfection. (Upper) Northern blot analysis demonstrating induction of endogenous p57^KIP2 is shown. GAPDH expression was examined as a loading control. (Lower) Expression of p73β and p73γ protein in the same transfected cells by Western blot using an anti-HA antibody.

Figure 3

Activation of p57^KIP2 protein expression by p73β. (a) Various Saos-2-p73 cells were untreated (−) or treated (+) with doxycycline (Dox) for 40 h as described for Fig. 1, and p57^KIP2 protein expression was analyzed by Western blotting as indicated. (b) Saos-2-p73 and Saos-2-p53 cells were untreated (0) or treated with 2 μg/ml doxycycline (hours Dox) for the indicated times, and p57^KIP2 and p21^CIP1 protein was analyzed by Western blotting. Actin expression was examined as a loading control. (c) Saos-2-p73β and Saos-2-p53 cells were untreated (−) or treated (+) with doxycycline for 40 h, and p57^KIP2 and MDM2 protein were analyzed by Western blotting. (d) U2OS cells were transfected with 15.5 μg of DNA consisting of 0, 5, 10, or 15 μg of p53 or p73β expression plasmids (as indicated) supplemented with vector control DNA, and 0.5 μg of green fluorescent protein (GFP) was added as a transfection efficiency control. Protein expression was analyzed as described above; p73β was detected by using the p73β-specific polyclonal antibody, and p53 was detected by using the p53-specific monoclonal antibody DO-1.

Figure 4

E2F1 induction of p57^KIP2 is p73-dependent. (a) Saos-E2F1 and Saos-E2F1-DDp73 cells were untreated (−) or treated (+) with 2 μg/ml doxycycline (Dox) for 12 h, and total RNA was analyzed by RNase protection. Protected probes specific for p57^KIP2 and GAPDH are shown. (b) Saos-E2F1 and Saos-E2F1-DDp73 cells were untreated (0) or treated with doxycycline for the indicated times (hours Dox). Expression of E2F1, DDp73, and p57^KIP2 proteins was analyzed by Western blotting. Actin expression was examined as a loading control. (c) Primary human retinal pigment epithelial cells without (−) or with (+) E1A were examined for p73β and p57^KIP2 mRNA expression levels by RT-PCR. GAPDH expression was included as a loading control.

Figure 5

p57^KIP2 and KvLQT1 are regulated coordinately by p73β. (a) Genomic organization of the p57^KIP2 gene. The sequences studied by luciferase reporter assays are indicated as 1 kb, 1.2 kb, and 3 kb. (b) Saos-2p73β cells were untreated (− dox) or treated (+ dox) with 2 μg/ml doxycycline for 20 h. Cells were harvested at the indicated time points after the addition of 5 μg/ml actinomycin D, and the remaining p57^KIP2 mRNA levels were assessed. (c) Regulation of p57^KIP2 and KvLQT1 by a putative shared enhancer (E?, closed circle). The drawing represents ≈500 kb of the genome located on chromosome 11p15.5. Closed, open, and hatched boxes represent the p57^KIP2, KvLQT1, and LIT1 genes, respectively, small arrows show gene activation, and large arrows show the direction of transcription. Cen and tel refer to the orientation of the centromere and telomere, respectively. (d) Saos-2p73α, p73β, and p73γ cells were untreated (−) or treated (+) with 2 μg/ml doxycycline (Dox) for 12 h, and RNA was analyzed by RT-PCR. (e) U2OS cells were transfected with 10 μg of vector control (v) or p73β expression plasmid. Twenty-four hours after transfection, total RNA was analyzed by RT-PCR.

Figure 6

p73β cannot activate expression of p57^KIP2 in uniparental AG MEFs. Retroviral infection was used to achieve efficient expression of p73β or p53 in wild-type (WT), AG, or PG MEFs. Cells were harvested 24 h postinfection, and expression of p57^KIP2 mRNA was analyzed by RT-PCR. GAPDH expression was included as a loading control.