Enhanced stability of Mcl1, a prosurvival Bcl2 relative, blunts stress-induced apoptosis, causes male sterility, and promotes tumorigenesis

Abstract

The B-cell CLL/lymphoma 2 (Bcl2) relative Myeloid cell leukemia sequence 1 (Mcl1) is essential for cell survival during development and for tissue homeostasis throughout life. Unlike Bcl2, Mcl1 turns over rapidly, but the physiological significance of its turnover has been unclear. We have gained insight into the roles of Mcl1 turnover in vivo by analyzing mice harboring a modified allele of Mcl1 that serendipitously proved to encode an abnormally stabilized form of Mcl1 due to a 13-aa N-terminal extension. Although the mice developed normally and appeared unremarkable, the homozygous males unexpectedly proved infertile due to defective spermatogenesis, which was evoked by enhanced Mcl1 prosurvival activity. Under unstressed conditions, the modified Mcl1 is present at levels comparable to the native protein, but it is markedly stabilized in cells subjected to stresses, such as protein synthesis inhibition or UV irradiation. Strikingly, the modified Mcl1 allele could genetically complement the loss of Bcl2, because introduction of even a single allele significantly ameliorated the severe polycystic kidney disease and consequent runting caused by Bcl2 loss. Significantly, the development of c-MYC-induced acute myeloid leukemia was also accelerated in mice harboring that Mcl1 allele. Our collective findings reveal that, under certain circumstances, the N terminus of Mcl1 regulates its degradation; that some cell types require degradation of Mcl1 to induce apoptosis; and, most importantly, that rapid turnover of Mcl1 can serve as a tumor-suppressive mechanism.

Keywords: programmed cell death; protein turnover.

Fig. 1.

Male mice homozygous for the Mcl1^fl/fl are unexpectedly infertile. (A) Absence of mature and elongating spermatids in the testes of adult male Mcl1^fl/fl mice. Representative histological sections (H&E stained) show epididymis (Top) and seminiferous tubules at medium (Middle) or high magnification (Bottom) from testes of Mcl1^fl/+ and Mcl1^fl/fl males and, as a control for the complete loss of spermatogenesis, Bax^−/− males (Right). (Scale bars: 100 μm.) (B) Flow cytometric analysis (fluorescence-activated cell sorting) of germ cell development shows that spermatogenesis arrests later in Mcl1^fl/fl males than Bax^−/− males. (Left) Schematic representation of normal germ cell development. The arrow depicts the order of maturation from 2N spermatogonia (R3/4), to 4N spermatocytes (R1/2), to 1N spermatids (R5), to mature sperm (R6/7). (Center) Representative forward scatter (FSC)/side scatter (SSC) profiles of germ cells from Mcl1^fl/+, Mcl1^fl/fl, and Bax^−/− males. (Right) Representative DNA content profiles of germ cells from Mcl1^fl/+, Mcl1^fl/fl, and Bax^−/− males. (Right and Center) Unlike Bax^−/− males, Mcl1^fl/fl males can produce immature 1N spermatids, but they fail to mature further.

Fig. 2.

Floxed Mcl1 allele encodes a stabilized form of Mcl1, N⁺Mcl1, that counters apoptosis more effectively than WT Mcl1 in some settings. (A) Floxed Mcl1 allele encodes an abnormally stabilized form of Mcl1. (Upper) Lysates prepared from WT (Mcl1^+/+) or Mcl1^flN/flN MEFs that were incubated for up to 6 h with the protein synthesis inhibitor cycloheximide (CHX; 50 μg/mL), with or without the proteasome inhibitor (10 μM MG132) and the broad-spectrum caspase inhibitor N-(2-Quinolyl)-L-valyl-L-aspartyl-(2,6-difluorophenoxy) methylketone (qVD-OPh) (to block degradation of Mcl1 by caspases). (Lower) Lysates prepared from the cells that were UV-irradiated (100 J/m²). The lysates were probed by immunoblotting for Mcl1 and HSP70 (loading control). WB, Western blot. (B) Under certain circumstances, N⁺Mcl1 inhibits cell death more effectively than WT Mcl1. MEFs derived from WT (Mcl1^+/+), Mcl1^flN/flN, or Bax^−/−Bak^−/− mice were treated with etoposide (2.5 μM), UV irradiation (100 J/m²), CHX (50 μg/mL), or Fas ligand (FasL, CD95L; 100 ng/mL). Cell viability was determined at the indicated times by propidium iodide staining and flow cytometric analysis. Data represent the mean ± SD of two experiments done with a representative cell line of each genotype.

Fig. 3.

N⁺Mcl1 ameliorates some of the physiological defects caused by Bcl2 deficiency. (A) N⁺Mcl1 can partially rescue some of the defects caused by Bcl2 deficiency. (Left) As previously reported (3, 4), Bcl2-deficent mice (Bcl2^−/−Mcl1^+/+) were runted and turned prematurely gray. (Right) Age-matched (52-d-old) Bcl2^−/−Mcl1^flN/flN mice grew to a normal size but still turned prematurely gray like the Bcl2^−/− mice. (B) N⁺Mcl1 prevents the lymphoid hypoplasia and developmental kidney defects caused by Bcl2 loss. Gross appearance of the thymus, spleen, and kidneys from representative Bcl2^−/−Mcl1^+/+, Bcl2^−/−Mcl1^flN/+, and Bcl2^−/−Mcl1^flN/flN mice (shown in A). Note that the kidney from a Bcl2^−/−Mcl1^flN/flN mouse was larger and well-aerated (pink, not pale) compared with that isolated from a Bcl2^−/−Mcl1^+/+ mouse. (C) N⁺Mcl1 attenuates the polycystic kidney disease caused by Bcl2 loss. Representative low-power, H&E-stained kidney sections are shown from representative mice of the indicated genotypes. Note the significant reduction in cyst formation in the kidney from a Bcl2^−/−Mcl1^flN/flN mouse compared with that from a Bcl2^−/−Mcl1^+/+ mouse. (Scale bars: 10 μm.) (D) Mcl1 stabilization extends the life span of Bcl2-deficient mice. Kaplan–Meier survival curves of cohorts of mice of the indicated genotypes are shown. Notably, even a single Mcl1^flN allele significantly prolonged the survival of Bcl2^−/− mice. The P value determined by log rank analysis of Bcl2^−/−Mcl1^+/+ mice compared with Bcl2^−/−Mcl1^flN/+ mice is 0.0032 (*), that compared with Bcl2^−/−Mcl1^flN/flN mice is <0.0001 (^#), and that compared with Bcl2^+/+Mcl1^flN/flN mice is <0.0001 (^†).

Fig. 4.

N⁺Mcl1 accelerates development of MYC-driven AML in vivo. Abnormal Mcl1 stabilization promotes c-MYC–driven development of AML. Shown is a Kaplan–Meier plot of the survival of lethally irradiated (2 × 5.5 Gy 2 h apart) mice transplanted with embryonic day 13.5 fetal liver cells (a rich source of hematopoietic stem/progenitor cells) derived from WT (Mcl1^+/+), Mcl1^flN/+, or Mcl1^flN/flN embryos and infected with a c-MYC–expressing retrovirus. Note the dose-dependent impact of the Mcl1^flN allele on AML-free survival. The P value (log rank analysis) of WT (Mcl1^+/+) mice compared with Mcl1^flN/+ mice is 0.0014 (*) and that compared with Mcl1^flN/flN mice is <0.0001(^).