Overexpression of human arginine decarboxylase rescues human mesenchymal stem cells against H₂O₂ toxicity through cell survival protein activation

Abstract

In this study, we explored the potentiality of human arginine decarboxylase (ADC) to enhance the survival of mesenchymal stem cells (MSCs) against unfavorable milieu of host tissues as the low survival of MSCs is the issue in cell transplantation therapy. To address this, human MSCs overexpressing human ADC were treated with H2O2 and the resultant intracellular events were examined. First, we examined whether human ADC is overexpressed in human MSCs. Then, we investigated cell survival or death related events. We found that the overexpression of human ADC increases formazan production and reduces caspase 3 activation and the numbers of FITC, hoechst, or propidium iodide positive cells in human MSCs exposed to H2O2. To elucidate the factors underlying these phenomena, AKT, CREB, and BDNF were examined. We found that the overexpression of human ADC phosphorylates AKT and CREB and increases BDNF level in human MSCs exposed to H2O2. The changes of these proteins are possibly relevant to the elevation of agmatine. Collectively, our data demonstrate that the overexpression of human ADC stimulates pro-survival factors to protect human MSCs against H2O2 toxicity. In conclusion, the present findings support that ADC can enhance the survival of MSCs against hostile environment of host tissues.

Keywords: Arginine Decarboxylase; Cell Survival; Hydrogen Peroxide; Mesenchymal Stem Cells; Retroviral Vector.

Fig. 1

The retroviral overexpression of human ADC in human MSCs and the measurement of agmatine with HPLC. (A) Bands of human ADC protein in control MSCs, LXSN-MSCs, and ADC-MSCs with or without H₂O₂ treatment. The photograph is a representative from 4 blots with the similar results. (B) Quantification of ADC protein bands demonstrated in Fig. 1A. Asterisks indicate the significant difference at P value less than 0.05 when compared to control and LXSN-MSCs. Data represent the mean ± standard deviation (SD) for 4 independent experiments. (C) Quantification of agmatine in control MSCs, LXSN-MSCs, and ADC-MSCs with or without H₂O₂ treatment. Asterisk indicates the significant difference at P value less than 0.05 when compared to control MSCs exposed to H₂O₂. Data represent the mean ± standard deviation (SD) for 4 independent experiments.

Fig. 2

Cell viability in control MSCs, LXSN-MSCs, and ADC-MSCs following H₂O₂ treatment. (A) Formazan formation. (B) Cell membrane integrity. Each photograph is a representative from 4 fields of microscope. The arrow indicates cells, where plasma membrane is damaged. Scale bar represents 200 µm. (a) Control MSCs, (b) LXSN-MSCs, (c) ADC-MSCs. (d) The quantitative graph showing the total number of propidium iodide (PI) positive cells in all experimental groups. Asterisk indicates the significant difference at P value less than 0.05 when compared to LXSN-MSCs exposed to H₂O₂. (C) Flow cytometric analysis. The data show that the distribution of cells undergoing apoptosis is reduced in ADC-MSCs compared to control MSCs and LXSN-MSCs under H₂O₂ exposure. Each panel is a representative from 4 independent experiments with the similar results.

Fig. 3

Apoptotic events in control MSCs, LXSN-MSCs, and ADC-MSCs following H₂O₂ treatment. (A) Caspase 3 activation. Western blotting data show that H₂O₂ treatment increases active form of caspase 3 in control MSCs and LXSN-MSCs. However, such an activation of caspase 3 was alleviated in ADC-MSCs exposed to H₂O₂. The less or no activation of caspase 3 was observed in control MSCs, LXSN-MSCs, and ADC-MSCs without H₂O₂ treatment. Each photograph is a representative from 4 blots with the similar results. (B) Nuclear morphology. Each photograph is a representative from 4 fields of microscope. Scale bar represents 200 µm. The data indicate that nuclear condensation (arrow) is lessened in ADC-MSCs compared to control MSCs and LXSN-MSCs following H₂O₂ treatment.

Fig. 4

AKT, CREB, and BDNF in control MSCs, LXSN-MSCs, and ADC-MSCs following H₂O₂ treatment. Western blotting was performed to examine AKT p-AKT, CREB, p-CREB, and BDNF in control MSCs, LXSN-MSCs, and ADC-MSCs before and after H₂O₂ treatment. Results show that the levels of p-AKT, p-CREB and BDNF were greatly increased in ADC-MSCs following H₂O₂ treatment, compared to those of control MSCs and LXSN-MSCs. Each photograph is a representative from 4 blots with the similar results.