REDD1 Protects Osteoblast Cells from Gamma Radiation-Induced Premature Senescence

Radiotherapy is commonly used for cancer treatment. However, it often results in side effects due to radiation damage in normal tissue, such as bone marrow (BM) failure. Adult hematopoietic stem and progenitor cells (HSPC) reside in BM next to the endosteal bone surface, which is lined primarily by hematopoietic niche osteoblastic cells. Osteoblasts are relatively more radiation-resistant than HSPCs, but the mechanisms are not well understood. In the present study, we demonstrated that the stress response gene REDD1 (regulated in development and DNA damage responses 1) was highly expressed in human osteoblast cell line (hFOB) cells after γ irradiation. Knockdown of REDD1 with siRNA resulted in a decrease in hFOB cell numbers, whereas transfection of PCMV6-AC-GFP-REDD1 plasmid DNA into hFOB cells inhibited mammalian target of rapamycin (mTOR) and p21 expression and protected these cells from radiation-induced premature senescence (PS). The PS in irradiated hFOB cells were characterized by significant inhibition of clonogenicity, activation of senescence biomarker SA-β-gal, and the senescence-associated cytokine secretory phenotype (SASP) after 4 or 8 Gy irradiation. Immunoprecipitation assays demonstrated that the stress response proteins p53 and nuclear factor κ B (NFkB) interacted with REDD1 in hFOB cells. Knockdown of NFkB or p53 gene dramatically suppressed REDD1 protein expression in these cells, indicating that REDD1 was regulated by both factors. Our data demonstrated that REDD1 is a protective factor in radiation-induced osteoblast cell premature senescence

Gamma-radiation induced senescence in hFOB cells.

<p>Following radiation (0, 4, or 8 Gy), senescent hFOB cells were determined by (A) clonogenicity assays, (B) MTS-proliferation assay and (C) SA-β-gal staining. SA-β-gal-positive cells were increased 72 h after 8 Gy γ irradiation. Representative data was from one of a total of 3 independent experiments. (D) Conditioned medium (CM) from hFOB cells was collected and pooled from three experiments 48 h after irradiation and the concentration of cytokines was analyzed by Luminex in triplicate. Gamma radiation-induced IL-6, IL-8, G-CSF and GM-CSF were significantly increased. Means ± SD. *: p<0.05, **: p<0.01, IR vs. non-irradiated cells.</p

REDD1 expression in human hematopoietic cells.

<p>Purified human CD34+ cells were cultured for 4 and 14 days before being exposed to 2 Gy radiation. (A) mRNA levels for <i>REDD1</i> in 14-day cultured CD34+ cells using 18S rRNA as a control to calculate the relative quantity (RQ) of gene expression at different times after IR. Means ± SD. *, p <0.05, 2 Gy vs. 0 Gy sham-irradiated control. (B) Western blot shows REDD1 protein levels in cultured CD34+ cells at different times after 2 Gy irradiation. Representative immunoblots and ratio of REDD1/β-actin expression from 3 independent experiments are shown. Flow cytometric analysis for the apoptotic cell death marker Annexin-V/7AAD and CD34 surface marker was performed. Radiation resulted in more apoptotic cells in 4-day (C-1) cultured cells than in 14-day (D-1) culture. (C-2) and (D-2) CD34-positive (progenitor) and CD34-negative (mature) populations were separately gated for Annexin-V/7AAD analysis. FS, forward angle light scatter.</p

Effects of miR-30c on radiation injury of hFOB and CD34+ cells.

<p>(A) Cell death and proliferation potential were measured using the CellTiter 96R AQueous non-radioactive cell proliferation assay (MTS-assay). The quantity of formazan product as measured by the amount of 490 nm absorbance (OD) is directly proportional to the number of living cells in culture. Levels of OD were dramatically lower in pre-miR30-transfected hFOB cells in both irradiated and non-irradiated samples, compared with controls and miR30 inhibitor-transfected samples (p<0.01). Representative expression from 3 independent experiments is shown. (B) Clonogenicity of human hematopoietic progenitor CD34+ cells was quantified in standard semisolid cultures in triplicate. Colonies were counted 14 days later. Results from two experiments showed that transfection of miR30 inhibitor resulted in significant colony number increases in irradiated cells. Means ± SD. **, p <0.01, miR30c inhibitor vs. controls. Non-TD: miRNA-untransfected control.</p

REDD1 protects hFOB cells from γ radiation-induced senescence.

<p>The effects of REDD1 were evaluated using gene silencing (si-RNA) and overexpression (plasmid DNA transfection) approaches. (A) Western blot shows REDD1 and β-actin (loading control) expression in control (non-transfection CT), REDD1 siRNA-transfected (si-REDD1), and maxGFP siRNA-transfected control (si-CT) samples after 0 or 8 Gy irradiation. Transfection of si-REDD1 decreased the radiation-induced REDD1 expression. (B) Survival cell number (trypan blue–negative) was decreased in siREDD1-transfected hFOB cells 24 hours after IR. Means ± SD for 3 independent experiments. *: p<0.05, si-REDD1 vs. CT and si-CT. (C) Quantitative RT-PCR determined <i>REDD1</i> gene expression in non-gene transfected control (CT), vector-transfected(GFP) and REDD-gene containing construct transfected hFOB cells at 4 and 48 h after irradiation. The relative quantity of gene expression was calculated using 18 S rRNA as a control. (D) Overexpression of REDD1 inhibited SA-β-gal activation after irradiation. Representative image of SA-β-gal staining and statistical data from three experiments are shown. Means ± SD. **: p<0.01, REDD1 plasmid DNA-transfected vs. CT or vector-transfected samples.</p

hsa-miR-30 expression after γ-irradiation (Quantitative Real Time-PCR).

<p>CD34+and hFOB cells were collected at 0.5, 1, 2 and 24 h after exposure to 2 or 8 Gy radiation, respectively. miR-30b and miR30c expression were evaluated by RT-PCR. U6 was used as a loading control. miR-30b and miR-30c levels were increased at 0.5 or 1 h after γ-irradiation in (A) CD34+ cells, whereas they were decreased or not altered in (B) hFOB cells in response to radiation. Results represent one of 3 independent experiments. Means ± SD. *, p<0.05. (C) miR-30b and miR-30c binding sites in REDD1 3′UTR are shown. RQ: relative quantification.</p

REDD1inhibits senescence-associated cytokine secretory phenotype (SASP) in irradiated hFOB cells.

<p>Conditioned medium (CM) from hFOB cells were pooled from three independent experiments, the concentration of cytokines was analyzed by Luminex in triplicates. (A) Effects of REDD1 on IL-6 secretion were further evaluated in hFOB cells with si-REDD1 gene or REDD1 plasmid DNA transfection and irradiation. Means ± SD. **: p<0.01, REDD1siRNA or REDD1 plasmid DNA-transfected vs. CT or si-CT or vector-transfected samples. (B) Overexpression of REDD1 decreased levels of IL-8, G-CSF and GM-CSF in irradiated hFOB cell CM. Means ± SD. *: p<0.05, **: p<0.01, REDD1 plasmid DNA-transfected vs. vector-transfected samples.</p

Radiation regulated miRNAs expression in both CD34+ and hFOB cells.

<p>Data from miRNA Microarray (LC Sciences Co. Houston, Texas).</p

hsa-miR30c regulation by pre-miR30c or miR30c-inhibitor in human CD34+ and hFOB Cells.

<p>The pre-miR30c, miR30c inhibitor, or control miR (CT-miR) molecules were transfected into CD34+ cells and hFOB cells. miR30c expression was examined 24 h post-transfection by quantitative RT-PCR. U6 was used as a loading control. RQ: relative quantification. Non-TD: miRNA-untransfected control.</p

hsa-miR30c regulates REDD1 gene and protein expression in hFOB and CD34+ cells after γ-irradiation.

<p>Endogenous REDD1 gene (A) and protein (B) levels were measured after pre-miR30c, miR30c-inhibitor, or control-miR (CT-miR) transfection and γ-irradiation in hFOB cells. Transfection of miR30c-inhibitor upregulated REDD1 gene and protein levels in irradiated samples, whereas radiation-induced REDD1 expression was inhibited by pre-miR30c compared with control-miR transfected sample determined by western blot assays. pre-miR30c, miR30c-inhibitor, or control-miR was co-transfected with either PCMV6-AC-GFP-REDD1 plasmid DNA (1 µg or 2 µg /dish) or vector control. REDD1 gene (C) and protein (D) expression were determined at 24 h post-transfection. Effects of miR30c on REDD1-overexpressing cells are shown. (E) REDD1 protein levels were measured by western blot assays after pre-miR30c, miR30c inhibitor, or control miR (CT-miR) transfection and γ-irradiation in 14 day cultured CD34+ cells (differentiated cells). Radiation-induced REDD1 expression was inhibited by pre-miR30 transfection. Representative expression from 3 independent experiments is shown. Non-TD: miRNA-untransfected control.</p