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. 2018 Dec;233(12):9563-9574.
doi: 10.1002/jcp.26859. Epub 2018 Jun 26.

BMAL1 and CLOCK proteins in regulating UVB-induced apoptosis and DNA damage responses in human keratinocytes

Yang Sun  1 , Peiling Wang  1   2 , Hongyu Li  1 , Jun Dai  1   2
Affiliations

BMAL1 and CLOCK proteins in regulating UVB-induced apoptosis and DNA damage responses in human keratinocytes

Yang Sun et al. J Cell Physiol. 2018 Dec.

Abstract

A diverse array of biological processes are under circadian controls. In mouse skin, ultraviolet ray (UVR)-induced apoptosis and DNA damage responses are time-of-day dependent, which are controlled by core clock proteins. This study investigates the roles of clock proteins in regulating UVB responses in human keratinocytes (HKCs). We found that the messenger RNA expression of brain and muscle ARNT-like 1 (BMAL1) and circadian locomotor output cycles kaput (CLOCK) genes is altered by low doses (5 mJ/cm2 ) of UVB in the immortalized HaCat HKCs cell line. Although depletion of BMAL1 or CLOCK has no effect on the activation of Rad3-related protein kinases-checkpoint kinase 1-p53 mediated DNA damage checkpoints, it leads to suppression of UVB-stimulated apoptotic responses, and downregulation of UVB-elevated expression of DNA damage marker γ-H2AX and cell cycle inhibitor p21. Diminished apoptotic responses are also observed in primary HKCs depleted of BMAL1 or CLOCK after UVB irradiation. While CLOCK depletion shows a suppressive effect on UVB-induced p53 protein accumulation, depletion of either clock gene triggers early keratinocyte differentiation of HKCs at their steady state. These results suggest that UVB-induced apoptosis and DNA damage responses are controlled by clock proteins, but via different mechanisms in the immortalized human adult low calcium temperature and primary HKCs. Given the implication of UVB in photoaging and photocarcinogenesis, mechanistic elucidation of circadian controls on UVB effects in human skin will be critical and beneficial for prevention and treatment of skin cancers and other skin-related diseases.

Keywords: DNA damage; apoptosis; brain and muscle ARNT-like 1 (BMAL1); clock; keratinocytes; ultraviolet B (UVB).

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Conflict of interest statement

CONFLICT OF INTEREST

The authors state no conflict of interest.

Figures

Figure 1
Figure 1. Low-dose UVB resets expression of BMAL1 and CLOCK genes in HaCat cells, and clock gene depletion increases the viability of normal and UVB-irradiated cells
(A, B) Human HaCat keratinocytes were exposed to 5 mJ/cm2 UVB irradiation and harvested at indicated time points for (A) real time RT-PCR; (B) western blot analysis of BMAL1 and CLOCK genes and proteins. (C) HaCat cells were reversely transfected with indicated siRNAs and exposed to UVB radiation 48 h later. Cells were harvested at 24 h post UVB for real time RT-PCR or western blot analysis of BMAL1 or CLOCK. (D) Cells transfected with different siRNAs were exposure to UVB, as described in Fig. 1C. Cells were fixed at 24 h post UVB and stained with Hoechst 33342. Images of nuclei were taken from 3 different fields per experiment for cell counting. The average cell number/field was presented as mean-percentage over control ± S.E.M. *, p < 0.05, **, p < 0.01, ns, not significant, N=3 independent experiments. The mRNA level of each gene is normalized to 36B4, and presented as mean-fold over control ± S.E.M. ***, p < 0.001, N=3. Relative protein levels were quantified by densitometry scanning with ImageJ, and normalized to a-tubulin. Data were presented as mean-fold over control ± S.E.M., N=3.
Figure 2
Figure 2. BMAL1 and CLOCK are required for UVB-induced apoptosis in HaCat keratinocytes
HaCat cells were transfected with control siRNA or siRNAs against BMAL1 or CLOCK and subjected to UVB irradiation as described in Fig. 1C. (A) At 24 h post UVB irradiation, live cells were double stained with Annexin V-FITC and propidium iodide (PI) and analyzed by flow cytometry. Cells that stained positive for Annexin V and negative for PI were categorized as in the early apoptotic stage. Cells that stained positive for both Annexin V and PI were categorized as in the late apoptosis. Data were presented as average percentage of cells ± S.E.M. *, p < 0.05, **, p < 0.01, ***, p < 0.001, N=3. (B) At 24 h post UVB, cells were harvested for western blot analysis using an antibody against the cleaved form of PARP. Relative protein levels were quantified by densitometry scanning with ImageJ, normalized to a-tubulin, and presented as mean-fold over control ± S.E.M. ***, p < 0.001, N=3.
Figure 3
Figure 3. UVB-induced expression of γ-H2AX is suppressed by BMAL1 or CLOCK depletion in HaCat keratinocytes
HaCat cells were transfected with control siRNA or siRNAs against BMAL1 or CLOCK, and subjected to UVB irradiation as described in Fig. 1C. (A) At 3 h and 24 h post UVB irradiation, cells were fixed and subjected to immunostaining with an antibody against γ–H2AX (green). DNA was counter stained with propidium iodide (PI) [red]. Bar = 50 µm. (B) At 24 h post UVB, cells were collected for western blot analysis of γ–H2AX or H2AX. Relative protein levels were quantified by densitometry scanning with ImageJ and normalized to α–tubulin. Data were presented as mean-fold over control ± S.E.M, *, p < 0.05, **, p < 0.01, ***, p < 0.001, ns, not significant, N=3.
Figure 4
Figure 4. Effects of BMAL1 or CLOCK depletion on UVB-induced DNA damage checkpoint responses in HaCat keratinocytes
HaCat cells were transfected with control siRNA or siRNAs against BMAL1 or CLOCK and subjected to UVB irradiation as described in Fig. 1C. (A) Protein lysates were collected at 2 h post UVB for western blot analysis of phospho-CHK1 (p-CHK1) or phosphor-p53 (p-p53). (B) Protein lysates were collected at 24 h post UVB for western blot analysis of p-p53 and total p53 protein. (C) Cells were harvested at 24 h post UVB for RT-PCR analysis of PUMA and p21 genes, or for western blot analysis of p21 protein. The mRNA level of each gene is normalized to 36B4, and presented as mean-fold over control ± S.E.M. *, p < 0.01, ***, p < 0.001, ns, not significant, N=3. Relative protein levels were quantified by densitometry scanning with ImageJ, normalized to α–tubulin, and presented as mean-fold over control ± S.E.M., *, p < 0.05, **, p < 0.01, ***, p < 0.001, ns, not significant, N=3.
Figure 5
Figure 5. Depletion of BMAL1 or CLOCK attenuates UVB-induced apoptosis in primary human keratinocytes (HKCs)
(A) HKCs were exposed to 5 mJ/cm2 UVB radiation and harvested at indicated time points for real time RT-PCR analysis of BMAL1 and CLOCK mRNA expression. (B–D) HKCs were transfected with control siRNA or siRNAs against BMAL1 or CLOCK. At 48 h post transfection, cells were exposed to 5 mJ/cm2 UVB radiation, as described in Fig. 1C. Cells were harvested at 24 h post UVB irradiation for (B) Western blot analysis of BMAL1 or CLOCK proteins; (C) Cell viability analysis using the Alamar Blue assay; (D) Western blot analysis of cleaved PARP. Relative cell viability was presented as mean-percentage over control ± S.E.M. *, p < 0.05, **, p < 0.01, ***, p < 0.001, ns, not significant, N=3. The relative protein level of cleaved PARP was measured by densitometry scanning with ImageJ, normalized to α–tubulin, and presented as mean-fold over control ± S.E.M. ***, p < 0.001, N=3.
Figure 6
Figure 6. Depletion of BMAL1 or CLOCK alters UVB-induced DNA damage responses and induces terminal differentiation in HKCs
(A) HKCs were transfected with control siRNA or siRNAs against BMAL1 or CLOCK and exposed to 5 mJ/cm2 UVB radiation. Cells were harvested at 6 h or 24 h post UVB irradiation for western blot analysis of γ–H2AX. (B) Protein lysates collected at 24 h post UVB irradiation were analyzed for p-p53 and total p53 protein expressions. (C) Cells were collected at 48 h or 72 h after siRNAs transfection (without UVB irradiation) and subjected to RT-PCR analysis or western blot analysis of keratin 1 (KRT1) or keratin 10 (KRT10). The mRNA level of each gene was normalized to 36B4, and presented as mean-fold over control ± S.E.M. *, p < 0.05, ***, p < 0.001, ****, p < 0.0001, N=3. Relative protein levels were quantified by densitometry scanning with ImageJ and normalized to α–tubulin. Data were presented as mean-fold over control ± S.E.M. *, p < 0.05, **, p < 0.01, ***, p < 0.001, ns, not significant, N=3.
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