Circadian clock protein BMAL1 regulates cellular senescence in vivo

Abstract

Deficiency of the circadian clock transcriptional factor BMAL1 results in the development of premature aging in mice. In agreement with the accelerated aging phenotype, we observed an increase in the number of senescent cells in different tissues (lungs, liver and spleen) of Bmal1(-/-) mice, which suggests the important role of BMAL1 in the control of senescence in vivo. However, no difference in the rate of proliferation and senescence between primary fibroblasts isolated from wild-type and Bmal1(-/-) mice has been detected, suggesting that BMAL1 does not play a significant role in replicative senescence in vitro. BMAL1 deficient fibroblasts had an increased sensitivity to hydrogen peroxide treatment, and reduced sensitivity to DNA damaging anticancer drugs etoposide and daunorubicin. Increased sensitivity of Bmal1(-/-) cells to oxidative stress was p53 independent and correlated with the disrupted regulation of reactive oxygen species (ROS) homeostasis in BMAL1 deficient cells: indeed, circadian oscillations of ROS level can be induced in wild-type but not in Bmal1(-/-) cells. We propose that BMAL1 is important for the regulation of oxidative stress and DNA damage responses, while deregulation of these processes upon BMAL1 deficiency leads to development of stress induced senescence in vivo.

Figure 1

BMAL1 deficiency leads to accumulation of senescent cells in vivo. Representative histological images of the lungs (left column), liver (middle column) and spleen (right column) isolated from 6 mo old wildtype (upper row) or Bmal1^-/- (lower row) mice were stained for senescent associated β-galactosidase.

Figure 2

BMAL1 deficiency does not affect fibroblast replicative senescence in vitro. Growth curves of primary lung fibroblasts isolated from wild type (closed circles) and Bmal1^-/- (open circles). On indicated days cells were counted and plated with equal density.

Figure 3

BMAL1-deficient cells have increased sensitivity to hydrogen peroxide. Relative survival of wild-type (black circles) and Bmal1^-/- (open circles) fibroblasts treated with indicated concentrations of hydrogen peroxide for 24 h (A), 48 h (B) and 72 h (C). Survival of untreated cells was set as 100%. *p < 0.01.

Figure 4

Increased sensitivity of BMAL1-deficient cells to hydrogen peroxide is due to increased growth inhibition. (A) Relative cell death of wildtype (black circles) and Bmal1^-/- (open circles) fibroblasts treated with indicated concentrations of hydrogen peroxide for 4 h were calculated based on fluorescent signal using kit. Signal of untreated cells was set as 0 and signal of cells lysed with SDS was set as 1.0. (B) Numbers of colonies formed by wild-type (black bars) and Bmal1^-/- (gray bars) fibroblasts. Equal amount of cells were plated, cells grew in regular growth media for 96 h (U/T), or in the presence of 250 mM of hydrogen peroxide for 96 h (250 mM H₂O₂), or treated with 250 mM of hydrogen peroxide for 24 h and then in regular growth media for additional 72 h (250 mM H₂O₂/media). (C) Representative images of colony formation experiment. *p < 0.05.

Figure 5

BMAL1 deficiency cells have differential sensitivity to anticancer drugs. Relative survival of wild-type (black circles) and Bmal1^-/- (open circles) fibroblasts treated with indicated concentrations of paclitaxel (A), 5-fluorouracil (B), daunorubicin (C) and etoposide (D). Survival of untreated cells was set as 100%. *p < 0.01.

Figure 6

Increased sensitivity of BMAL1-deficient cells to hydrogen peroxide is p53-independent. Relative survival of GSE 56 (dominant negative fragment of p53) wild-type (black circles) and Bmal1^-/- (open circles) fibroblasts treated with indicated concentrations of hydrogen peroxide for 72 h. Survival of untreated cells was set as 100%. *p < 0.01.

Figure 7

Circadian rhythms of intracellular ROS level can be induced in cell culture. Confluent wild-type (black circles) and Bmal1^-/- (open circles) fibroblasts were maintained in DMEM with 5% FBS for 7 d, at time 0 h media were replaced with media with 50% FBS (serum shock); cells were incubated for 2 h and then 50% FBS media was replaced with serum free media; cells were collected every 10 min during serum shock and every 3 h after serum replacement and ROS level was measured as described in Materials and Methods. *Statistically significant (p < 0.001) difference between peak and throw for wild-type cells.