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. 2016 May 24;7(21):30018-31.
doi: 10.18632/oncotarget.9065.

Vitamin D receptor signaling improves Hutchinson-Gilford progeria syndrome cellular phenotypes

Ray Kreienkamp  1 , Monica Croke  1 , Martin A Neumann  1 , Gonzalo Bedia-Diaz  1 , Simona Graziano  1 , Adriana Dusso  2 , Dale Dorsett  1 , Carsten Carlberg  3 , Susana Gonzalo  1
Affiliations

Vitamin D receptor signaling improves Hutchinson-Gilford progeria syndrome cellular phenotypes

Ray Kreienkamp et al. Oncotarget. .

Abstract

Hutchinson-Gilford Progeria Syndrome (HGPS) is a devastating incurable premature aging disease caused by accumulation of progerin, a toxic lamin A mutant protein. HGPS patient-derived cells exhibit nuclear morphological abnormalities, altered signaling pathways, genomic instability, and premature senescence. Here we uncover new molecular mechanisms contributing to cellular decline in progeria. We demonstrate that HGPS cells reduce expression of vitamin D receptor (VDR) and DNA repair factors BRCA1 and 53BP1 with progerin accumulation, and that reconstituting VDR signaling via 1α,25-dihydroxyvitamin D3 (1,25D) treatment improves HGPS phenotypes, including nuclear morphological abnormalities, DNA repair defects, and premature senescence. Importantly, we discovered that the 1,25D/VDR axis regulates LMNA gene expression, as well as expression of DNA repair factors. 1,25D dramatically reduces progerin production in HGPS cells, while stabilizing BRCA1 and 53BP1, two key factors for genome integrity. Vitamin D/VDR axis emerges as a new target for treatment of HGPS and potentially other lamin-related diseases exhibiting VDR deficiency and genomic instability. Because progerin expression increases with age, maintaining vitamin D/VDR signaling could keep the levels of progerin in check during physiological aging.

Keywords: DNA repair; Gerotarget; genomic instability; laminopathies; progeria; vitamin D receptor.

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Conflict of interest statement

All other authors have no conflict of interest.

Figures

Figure 1
Figure 1. VDR deficiency in cells with disrupted nuclear lamina
A. Human NF lentivirally transduced with shRNA targeting lamin A/C (shLmna) or scrambled (shscr), and processed for immunoblotting (2 independent transductions). Vinculin is loading control. B. The same experiments as in (A) performed in human VSMC. Tubulin is loading control. C. Relative expression of LMNA and VDR transcripts by qRT-PCR in VSMC after depletion of lamin A/C or VDR. Results are mean±sem of 3 biological repeats. D. BJ fibroblasts were lentivirally transduced with 2 independent shRNAs targeting lamin A/C (left panels) or retrovirally transduced with progerin (right panels) and processed for immunoblotting. E. Different human fibroblasts (BJ, NF and HGPS) were collected at increasing passages in culture to monitor levels of VDR and lamin A/C by western blot. F. NF were lentivirally transduced with shscr, shLmna, or shRNA targeting VDR (shVDR) and processed for immunoblotting. G. Immunofluorescence (IF) with γH2AX antibody in NF depleted of VDR, lamin A/C, and control. Quantitated percentage of cells with more than 5 γH2AX foci. Graph represents mean±sem of 3 independent experiments. H. Images show how depletion of lamin A/C or VDR leads to accumulation of β-galactosidase positive cells, a marker of senescence (after 2 weeks). I. NF and HGPS cells were depleted of VDR and levels of BRCA1, VDR, and γH2AX monitored by immunoblotting. J. IF performed in HGPS depleted of VDR (shVDR) and control (shRNA) and percentage of γH2AX-positive cells quantitated. Images of IF showing accumulation of γH2AX in HGPS cells depleted of VDR. *p value of statistical significance (*p ≤ 0.05).
Figure 2
Figure 2. Phenotypes of HGPS cells are rescued by vitamin D
A. HGPS cells were grown in culture with 1,25D (10−7M) or vehicle and collected for western blot prior to entering senescence (passage 25). Levels of DNA repair factors 53BP1 and BRCA1, VDR, and progerin were compared to NFs. B. Densitometry of immunoblots comparing levels of 53BP1, BRCA1, VDR, and progerin between NF and HGPS cells (mean±sem of 4 biological repeats). C. Densitometry as in (B) comparing HGPS cells under prolonged treatment with 1,25D or vehicle as control (mean±sem of 3 biological repeats). D. DAPI and IF staining of progerin shows that accumulation of progerin in HGPS cells is reduced by prolonged 1,25D treatment (passage 27). Graph shows quantitation of progerin labeling intensity (relative fluorescence units) in NFs and in HGPS cells subjected to prolonged treatment with vehicle or 1,25D. DAPI staining was used to demarcate nuclei and intensity of progerin labeling measured using ImageJ program. A total of 200 cells were quantitated in each condition. E. HGPS fibroblasts were grown in culture for at least 90 days with 1,25D or vehicle, and qRT-PCR performed to monitor levels of total LMNA and progerin transcripts (passage 29). NF of similar passage were used as control. Results are the mean±sem of 3 independent experiments. F. Fibroblasts derived from a second HGPS patient were subjected to prolonged 1,25D treatment or vehicle control (passage 27). These two lines were treated with the cathepsin inhibitor E64 or vehicle for 24 h, and samples processed for immunoblotting. G. DAPI and IF staining of γH2AX shows that accumulation of DNA damage in HGPS cells is reduced by prolonged 1,25D treatment. H. Quantitation of percentage of cells positive for γH2AX in 3 biological repeats. I. Quantitation of γH2AX labeling intensity (relative fluorescence units) in NF and in HGPS cells under prolonged 1,25D treatment. DAPI staining was used to demarcate nuclei and intensity of γH2AX labeling measured using ImageJ program. A total of 200 cells were quantitated. *p value of statistical significance (*p ≤ 0.05).
Figure 3
Figure 3. Vitamin D rescues nuclear abnormalities and delays senescence in HGPS cells
A. DAPI staining and IF with lamin A antibody performed in NF and HGPS fibroblasts under prolonged treatment with vehicle or 1,25D (passage 25). Representative images are shown. B. Quantitation of percentage of cells showing aberrant nuclear morphology (extensive protrusions, lobulations, herniations, etc), an intermediate phenotype (slight change in nuclear morphology), and normal nuclear morphology. More than 500 cells were counted per condition in 3 independent blinded experiments. C. The nuclear volume of cells processed for IF as in (A) was calculated using Leica's microscope software. A total of 200 cells were quantitated. D. Proliferation rate monitored during culture of HGPS cells in media containing 1,25D or vehicle. Note how vehicle-treated HGPS cells growth arrested after approximately 8-10 weeks in culture while 1,25D-treated cells continued proliferating. E. β-galactosidase assay performed in NF and HGPS cells treated with 1,25D or vehicle, once vehicle-treated cells growth arrested (passage 33). F. Proliferation rate of a second long-term treatment of the same line of HGPS fibroblasts with 1,25D or vehicle control. G. Proliferation rate of fibroblasts from a different HGPS patient.
Figure 4
Figure 4. Regulation of LMNA gene expression by vitamin D/VDR
A. RNAseq analysis performed in NF after a 24-h treatment with 1,25D (10−7M) or vehicle (FBS) as control. Map shows the levels of transcripts containing exon sequences of the LMNA gene in control cells (blue) and 1,25D-treated cells (red). B. Relative expression of total transcripts from LMNA gene by qRT-PCR in NF treated with 1,25D for 4 h or 24 h. Results are the mean±sem of 3 biological repeats. C. Relative expression of LMNA transcripts by qRT-PCR in MAFs treated with 1,25D for 24 h. D. Relative expression of total transcripts from LMNA gene as determined by qRT-PCR in HGPS patient-derived treated with 1,25D for 24 h or vehicle control. E. IF for γH2AX in HGPS cells treated with 1,25D or vehicle for 4 and 7 days. Graph shows percentage of γH2AX-positive cells. A total of 400 cells were quantitated per condition. F. DAPI staining and IF with lamin A antibody in HGPS cells treated with 1,25D or vehicle for 4 and 7 days. The nuclear volume was calculated using Leica's microscope software. A total of 300 cells were quantitated. All graphs represent mean±sem. *p value of statistical significance (*p ≤ 0.05).
Figure 5
Figure 5. Model of functional relationship between nuclear lamina integrity, vitamin D/VDR axis, and expression of LMNA gene
We propose a model whereby in cells with an integral nuclear lamina, expression of DNA repair factors such as BRCA1 and 53BP1, and DNA repair capabilities are maintained by the vitamin D/VDR signaling axis. As such, depletion of VDR in normal fibroblasts results in BRCA1 loss, accumulation of DNA damage, and premature senescence. Cells with a disrupted nuclear lamina due to lamin A/C depletion or progerin accumulation (HGPS) experience a marked reduction in VDR levels, which in turn contributes to the down-regulation of BRCA1 and the activation of CTSL-mediated degradation of 53BP1. These cells accumulate DNA damage markers (γH2AX), nuclear morphological abnormalities and other cellular alterations that ultimately cause premature senescence. Importantly, treatment of HGPS cells with 1,25D to stabilize and activate VDR results in reduced progerin production, stabilization of BRCA1, inhibition of CTSL-mediated degradation of 53BP1, and amelioration of a variety of nuclear phenotypes. This suggests multiple benefits of a vitamin D based therapy for HGPS and other laminopathies.
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