Cell-penetrating superoxide dismutase attenuates oxidative stress-induced senescence by regulating the p53-p21^Cip1 pathway and restores osteoblastic differentiation in human dental pulp stem cells

Yoon Jung Choi1 Craniomaxillofacial Reconstructive Sciences, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic of Korea

Jue Yeon Lee2 Research Institute, Nano Intelligent Biomedical Engineering, Seoul, Republic of Korea

Chong Pyoung Chung2 Research Institute, Nano Intelligent Biomedical Engineering, Seoul, Republic of Korea

Yoon Jeong Park1 Craniomaxillofacial Reconstructive Sciences, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic of Korea;2 Research Institute, Nano Intelligent Biomedical Engineering, Seoul, Republic of KoreaCorrespondence[email protected]

Abstract

Background

Human dental pulp stem cells (DPSCs) have potential applications in tissue regeneration because of their convenient cell harvesting procedures and multipotent capacity. However, the tissue regenerative potential of DPSCs is known to be negatively regulated by aging in long-term culture and under oxidative stress. With an aim of reducing cellular senescence and oxidative stress in DPSCs, an intracellular delivery system for superoxide dismutase 1 (SOD1) was developed. We conjugated SOD1 with a cell-penetrating peptide known as low-molecular weight protamine (LMWP), and investigated the effect of LMWP-SOD1 conjugates on hydrogen peroxide-induced cellular senescence and osteoblastic differentiation.

Results

LMWP-SOD1 significantly attenuated enlarged and flattened cell morphology and increased senescence-associated β-galactosidase activity. Under the same conditions, LMWP-SOD1 abolished activation of the cell cycle regulator proteins, p53 and p21^Cip1, induced by hydrogen peroxide. In addition, LMWP-SOD1 reversed the inhibition of osteoblastic differentiation and downregulation of osteogenic gene markers induced by hydrogen peroxide. However, LMWP-SOD1 could not reverse the decrease in odontogenesis caused by hydrogen peroxide.

Conclusion

Overall, cell-penetrating LMWP-SOD1 conjugates are effective for attenuation of cellular senescence and reversal of osteoblastic differentiation of DPSCs caused by oxidative stress inhibition. This result suggests potential application in the field of antiaging and tissue engineering to overcome the limitations of senescent stem cells.

Keywords:

Acknowledgment

This study was supported by the Korean Research Foundation Nanobiotechnology Development Regenomics Program (2008-00889) and the Ministry of Health, Welfare, and Family Affairs (A085056) in Korea.

Disclosure

The authors report no conflicts of interest in this work.

Supplementary figures

Figure S1 The viability of DPSCs treated with SOD1 or LMWP-SOD1 was elucidated by cell cycle analysis.

Notes: SOD1 and LMWP-SOD1 (2 μM) were incubated with cells for 30 minutes, the cells were washed twice with phosphate-buffered solution, and then incubated with 1% trypsin-ethylenediamine tetra-acetic acid for 10 minutes. After incubation, the cells were washed twice with phosphate-buffered solution and fixed with 70% ethyl alcohol for one hour. The cells were then washed twice with phosphate-buffered solution and incubated with propidium iodide and RNase. Cells stained with propidium iodide were observed by flow cytometry.

Abbreviations: LMWP, low molecular weight protamine; SOD1, superoxide dismutase; DPSCs, human dental pulp stem cells, NT, no treatment.

Figure S1 The viability of DPSCs treated with SOD1 or LMWP-SOD1 was elucidated by cell cycle analysis.Notes: SOD1 and LMWP-SOD1 (2 μM) were incubated with cells for 30 minutes, the cells were washed twice with phosphate-buffered solution, and then incubated with 1% trypsin-ethylenediamine tetra-acetic acid for 10 minutes. After incubation, the cells were washed twice with phosphate-buffered solution and fixed with 70% ethyl alcohol for one hour. The cells were then washed twice with phosphate-buffered solution and incubated with propidium iodide and RNase. Cells stained with propidium iodide were observed by flow cytometry.Abbreviations: LMWP, low molecular weight protamine; SOD1, superoxide dismutase; DPSCs, human dental pulp stem cells, NT, no treatment.

Figure S2 Transduction ability of LMWP into DPSCs. FITC-labeled LMWP (2 μM) was incubated for various time intervals. After incubation of the cells with FITC-labeled LMWP, the cells were washed twice with phosphate-buffered solution and incubated with 1% trypsin-ethylenediamine tetra-acetic acid for 10 minutes. After incubation, the cells were washed twice with phosphate-buffered solution and immediately observed by flow cytometry. (A) FACS analysis of the LMWP which had penetrated into the cells. (B) Penetration level of LMWP.

Notes: Each bar represents the mean ± standard error of the mean obtained from four experiments. Three independent experiments were performed in duplicate.

Abbreviations: FITC, fluorescein isothiocyanate; LMWP, low molecular weight protamine; SOD1, superoxide dismutase; DPSCs, human dental pulp stem cells.

Figure S2 Transduction ability of LMWP into DPSCs. FITC-labeled LMWP (2 μM) was incubated for various time intervals. After incubation of the cells with FITC-labeled LMWP, the cells were washed twice with phosphate-buffered solution and incubated with 1% trypsin-ethylenediamine tetra-acetic acid for 10 minutes. After incubation, the cells were washed twice with phosphate-buffered solution and immediately observed by flow cytometry. (A) FACS analysis of the LMWP which had penetrated into the cells. (B) Penetration level of LMWP.Notes: Each bar represents the mean ± standard error of the mean obtained from four experiments. Three independent experiments were performed in duplicate.Abbreviations: FITC, fluorescein isothiocyanate; LMWP, low molecular weight protamine; SOD1, superoxide dismutase; DPSCs, human dental pulp stem cells.

Figure S3 Effect of LWMP on human DPSCs. DPSCs were pretreated with LMWP (2 μM) for various time intervals. (A) Growth curves were monitored by MTT assay. (B) FACS analysis of cell cycle distribution was determined at 0, 24, 48, and 72 hours after incubation with LMWP.

Note: Four independent experiments were performed in duplicate.

Abbreviations: LMWP, low molecular weight protamine; DPSCs, human dental pulp stem cells; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure S3 Effect of LWMP on human DPSCs. DPSCs were pretreated with LMWP (2 μM) for various time intervals. (A) Growth curves were monitored by MTT assay. (B) FACS analysis of cell cycle distribution was determined at 0, 24, 48, and 72 hours after incubation with LMWP.Note: Four independent experiments were performed in duplicate.Abbreviations: LMWP, low molecular weight protamine; DPSCs, human dental pulp stem cells; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure S4 Transduction ability of SOD1 into DPSCs. SOD1 protein (2 μM) was incubated for various time intervals. (A) Transduction of SOD1 was analyzed by Western blotting with an anti-SOD1 antibody. β-actin was detected as a loading control. (B) The activity of the transduced SOD1 protein was analyzed in cultures of human DPSCs.

Notes: Each bar represents the mean ± standard error of the mean obtained from four experiments. Four independent experiments were performed in duplicate.