Transcription factor JunD, deprived of menin, switches from growth suppressor to growth promoter

Abstract

Different components of the AP1 transcription factor complex appear to have distinct effects on cell proliferation and transformation. In contrast to other AP1 components, JunD has been shown to inhibit cell proliferation. Also, in prior studies, JunD alone bound menin, product of the MEN1 tumor suppressor gene, and JunD's transcriptional activity was inhibited by menin, suggesting that JunD might achieve all or most of its unique properties through binding to menin. Analyses of JunD and menin effects on proliferation, morphology, and cyclin D1 in stable cell lines unmasked an unexpected growth promoting activity of JunD. Whereas stable overexpression of wild-type (wt) mouse JunD in JunD-/- immortalized fibroblasts inhibited their proliferation and reverted their transformed-like phenotype, overexpression of a missense mouse JunD mutant (mJunDG42E) with disabled binding to menin showed opposite or growth promoting effects. Similarly, stable overexpression of wt mouse JunD in wt immortalized fibroblasts inhibited growth. In contrast, its overexpression in Men1-/- immortalized fibroblasts enhanced their already transformed-like characteristics. To conclude, JunD changed from growth suppressor to growth promoter when its binding to menin was prevented by a JunD mutant unable to bind menin or by Men1-null genetic background.

Fig. 1.

Menin expression in JunD^–/– immortalized fibroblasts and JunD expression in Men1^–/– immortalized fibroblasts. (A) Western blot of nuclear extracts (25 μg) from immortalized fibroblasts, JunD-WT, JunD-heterozygote, or JunD-null. Blot was probed with an anti-menin antibody (SQV) (Lower) and reprobed with anti-NUMA antibody as loading control and nuclear protein marker (Upper). Lower arrow points to the menin-specific band. Decrease or absence of JunD did not affect menin expression. (B) Western blot of nuclear extracts (25 μg) from immortalized fibroblasts, which are wt or Men1-null. Blot was probed with an anti-JunD antibody (Lower) and reprobed with the p84 antibody as loading control and nuclear protein marker (Upper). Solid black arrow below indicates the JunD-specific band, and the open arrowhead denotes a nonspecific band. Both Men1^–/– lines express far more JunD than wt. The faster migrating band below the JunD likely represents an alternate smaller form of JunD generated from an internal translation initiation site (22).

Fig. 2.

mJunD and mJunD mutant protein expression, proliferation, and morphology of JunD^–/– stable lines. Cell morphology under phase contrast was examined in 10% FCS, and proliferation was analyzed in 0.5% FCS. (A) Western blot of nuclear extracts (25 μg) from JunD-null immortalized fibroblasts stably transfected with mJunD (from JunD-NULL-2-JD-1 to -JD7). (B) Western blot of nuclear extracts (25 μg) from JunD-null immortalized fibroblasts stably transfected with mutant mJunD^G42E (from JunD-NULL-2-G42E-1 to -G42E-6). Blots in A and B were probed with an anti-JunD antibody (Lower) and reprobed with the p84 antibody as loading control (Upper). Untransfected JunD-null and wt is also shown as control. The open arrowhead shows a nonspecific band also seen in the JunD-null cells. The solid arrow shows full-length JunD. The faster migrating band below the JunD band likely represents an alternate smaller form of JunD generated from an internal translation initiation site (22). (C) Proliferation curves for three independent JunD-NULL-2 stable lines overexpressing mJunD or overexpressing mutant mJunD^G42E that express numerically increasing protein from transfection. Proliferation curves for JunD-NULL-2 and a genetically matched wt line are shown for comparison. Three independent lines of JunD-NULL-2 stably transfected with vector alone showed proliferation curves similar to JunD-NULL-2 (data not shown). Each point represents the mean of triplicate cultures from the same experiment. Standard deviations were 5–20% of the mean (data not shown). (D) Morphology of JunD^–/– immortalized fibroblasts. Cells are spindle-like and pile up at high cell density. (E) Morphology of JunD^–/– cells stably overexpressing mJunD. Compared with JunD^–/–, cells are flattened and are highly spread. (F) Morphology of JunD^–/– cells stably overexpressing mutant mJunD^G42E. Cells pile up to form foci at high cell densities.

Fig. 3.

Proliferation and morphology of stable lines based on wt immortalized fibroblasts or Men1^–/– immortalized fibroblasts. Cell morphology under phase contrast or proliferation were examined in 10% FCS. Each point in the growth curve represents the mean of triplicate cultures from the same experiment. Standard deviations were 3–15% of the mean (data not shown). (A) Men1-WT-10 transfected with mJunD and two independent lines transfected with cJun that express numerically increasing protein from transfection. Lines stably transfected with vector are shown for comparison. (B) Men1-NULL-41 vector transfected lines and three independent mJunD-transfected lines that express numerically increasing levels of mJunD. The proliferation of mJunD expressing Men1-NULL-41 was unaltered compared with vector control. Men1-NULL-17 gave similar results (data not shown). (C) Vector-transfected line. (D) mJunD-transfected line. Cells are more flattened and more highly spread than in C.(E) Vector-transfected line Men1-NULL-17-V1. Cells are spindle-shaped, compared with vector-transfected Men1-WT-10 (A). (F) mJunD-transfected line Men1-NULL-17-JD6. Cells are refractile, are elongated, and pile up. (G) Vector-transfected line Men1-NULL-41-V1. Cells are spindle-shaped, compared with vector-transfected Men1-WT-10 (C). The cells pile up at high cell densities (data not shown). (H) mJunD-transfected line Men1-NULL-41-JD4. Cells are refractile, slightly elongated, and pile up to form foci (one focus occupies the center of the image).

Fig. 4.

Consequence on growth from genotype difference and from stable overexpression of wt or missense mutant mJunD (mJunD^G42E is unable to bind menin). The JunD^–/– and Men1^–/– genotypes are variably displaced to the right from the basal point to reflect their germ-line-positive effect on growth, compared with the wt genotype. Because the JunD-null and the Men1-null immortalized fibroblasts have very different genetic backgrounds, all comparisons between these backgrounds must be qualitative.