The p53-dependent effects of macrophage migration inhibitory factor revealed by gene targeting

Abstract

Macrophage migration inhibitory factor (MIF) is a mediator of host immunity and functions as a high, upstream activator of cells within the innate and the adaptive immunological systems. Recent studies have suggested a potentially broader role for MIF in growth regulation because of its ability to antagonize p53-mediated gene activation and apoptosis. To better understand MIF's activity in growth control, we generated and characterized a strain of MIF-knockout (MIF-KO) mice in the inbred, C57BL/6 background. Embryonic fibroblasts from MIF-KO mice exhibit p53-dependent growth alterations, increased p53 transcriptional activity, and resistance to ras-mediated transformation. Concurrent deletion of the p53 gene in vivo reversed the observed phenotype of cells deficient in MIF. In vivo studies showed that fibrosarcomas induced by the carcinogen benzo[alpha]pyrene are smaller in size and have a lower mitotic index in MIF-KO mice relative to their WT counterparts. The data provide direct genetic evidence for a functional link between MIF and the p53 tumor suppressor and indicate an important and previously unappreciated role for MIF in carcinogenesis.

Fig. 1.

Generation of MIF mutant mice. (A) A schematic representation of the MIF-WT allele (exons 1, 2, and 3, Top), the targeting vector, the allele flanked by loxP sites (floxed allele), and the KO allele (Bottom). Bg, BglII; RI, EcoRI; X, XbaI; tk, thymidine kinase; ATG, translation initiation codon. In targeted ES cells, the MIF gene and the neomycin-resistance cassette (neo^r), both flanked by the loxP sites, were subsequently deleted by Cre-mediated recombination. (B) Southern blot analysis of WT and floxed (neo^r) alleles using EcoRI-digested genomic DNA and external hybridization probes. (C) G418-sensitive ES clones, derived after neo^r deletion, were analyzed by Southern hybridization using EcoRI-digested genomic DNA and the 5′ probe. (D) PCR amplification using ES-derived genomic DNA and primers specific for the WT (544 bp), floxed (683 bp), and MIF-KO (383 bp) alleles. The position of the primers A, B, and C is indicated in A for the floxed allele.

Fig. 2.

Validation of the MIF-KO mouse. (A) EcoRI-digested genomic DNA prepared from WT (+/+), heterozygous (+/–), and MIF-KO (–/–) mice was hybridized with the external 5′ probe. (B) Northern blot analysis of RNA prepared from the spleens of WT (+/+), heterozygous (+/–), and MIF-KO mice (–/–) using MIF cDNA as probe. (C) Immunoblot analysis of protein extracts prepared from the livers of WT (+/+), heterozygous (+/–), and MIF-KO mice (–/–) using a MIF-specific polyclonal antibody (R102). The expression of mitogen-activated protein kinase ERK1/2 is shown as a loading control. rMIF, recombinant MIF.

Fig. 3.

The growth properties of primary MIF-KO fibroblasts. (A) Representative growth curves of MIF-KO and WT embryonic fibroblasts. (B) MIF-KO and WT fibroblasts were grown in 10-cm plates, and the cell density of cultures was determined 4 days after visible confluency. The data were derived from four independent experiments. (C) Immunoblot analysis of proliferating (P) and confluent (C) MIF-KO and WT fibroblasts. (D) MIF-KO and WT fibroblasts were synchronized in G0G1 by serum starvation and were then induced by serum to re-enter the cell cycle. The proportion of cells in S phase was determined by ³H-thymidine incorporation. *, P < 0.01.

Fig. 4.

Growth characterization of MIF^–/–p53^–/– DKO fibroblasts. (A) The deletion of the Trp53 gene in DKO mice was confirmed by PCR amplification, using the primers specific for the p53 WT and KO (neo) allele. (B) Representative growth curves of p53^–/–, DKO, and MIF-KO fibroblasts. (C) Immunoblot analysis of proliferating (P) and confluent (C) DKO and p53^–/– fibroblasts with the indicated antibodies. The expression of p21 by MIF-KO MEFs (Right) is shown as a control.

Fig. 5.

MIF modulates cisplatin-induced p53 transactivation. (A) Immunoblot analysis of p53, p21, and MIF expression by MIF-KO and WT MEFs incubated in the presence of 25 μM cisplatin. (B) Relative expression of p21 by MIF-KO and WT MEFs at the indicated periods of time after cisplatin treatment. Densitometric measurements were converted into fold induction relative to untreated WT MEFs. (C) Immunoblot analysis of DKO fibroblasts exposed to 25 μM cisplatin (cp) for 4 and 8 h. Untreated MIF-KO fibroblasts (C) are shown as controls.

Fig. 6.

Induction of ras-mediated senescence response in MIF-deficient fibroblasts. (A) Growth curves of MIF-KO and WT fibroblasts after infection with empty vector (V)- or H-ras^V12 (R)-expressing retroviruses. (B) Kinetics of SA-βGal expression by MIF-KO and WT fibroblasts infected with V- or R-expressing retroviruses. *, P < 0.02.

Fig. 7.

The reduced susceptibility of MIF-KO fibroblasts to ras-mediated transformation is p53-dependent (A) Focus formation by MIF-KO and WT fibroblasts coexpressing E1A and H-ras^V12. (Upper) Representative morphology of a single colony induced by E1A and H-ras^V12 in MIF-KO and WT MEFs (×10 magnification). (B) Western blot analysis of MIF-KO and WT MEFs infected with empty vector (V)- or E1A- and H-ras^V12 (R)-containing retroviruses. (C) Growth curves of MIF-KO and WT fibroblasts coexpressing E1A and H-ras^V12.(D) Focus formation by p53^–/– and MIF^–/–p53^–/– DKO fibroblasts expressing H-Ras^V12, N-Ras^D12, or K-Ras^V12 mutants.

Fig. 8.

MIF-deficient mice exhibit delayed development of B[α]P-induced fibrosarcomas. (A) Tumor development by MIF-KO and WT BALB/c mice after treatment with DMBA as a tumor initiator and TPA as a promoter. *, significant with P < 0.05. (B) Tumor formation by C57BL/6 MIF-KO and WT mice after injection of B[α]P. (Left) Tumors developed by MIF-KO, and WT mice were excised in week 16 and weighed (P = 0.02). (Right) Mitotic index in B[α]P-induced fibrosarcomas from MIF-KO and WT mice (P = 0.003). (C) Histological examination of B[α]P-induced tumors in C57BL/6 MIF-KO and WT mice. Hematoxylin/eosin stain. Magnification: Upper, ×400; Lower, ×5. Arrowheads indicate cells undergoing mitoses.