Antagonizing Effects of Aspartic Acid against Ultraviolet A-Induced Downregulation of the Stemness of Human Adipose Tissue-Derived Mesenchymal Stem Cells.

Ultraviolet A (UVA) irradiation is responsible for a variety of changes in cell biology. The purpose of this study was to investigate effects of aspartic acid on UVA irradiation-induced damages in the stemness properties of human adipose tissue-derived mesenchymal stem cells (hAMSCs). Furthermore, we elucidated the UVA-antagonizing mechanisms of aspartic acid. The results of this study showed that aspartic acid attenuated the UVA-induced reduction of the proliferative potential and stemness of hAMSCs, as evidenced by increased proliferative activity in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and upregulation of stemness-related genes OCT4, NANOG, and SOX2 in response to the aspartic acid treatment. UVA-induced reduction in the mRNA level of hypoxia-inducible factor (HIF)-1α was also significantly recovered by aspartic acid. In addition, the antagonizing effects of aspartic acid against the UVA effects were found to be mediated by reduced production of PGE2 through the inhibition of JNK and p42/44 MAPK. Taken together, these findings show that aspartic acid improves reduced stemness of hAMSCs induced by UVA and its effects are mediated by upregulation of HIF-1α via the inhibition of PGE2-cAMP signaling. In addition, aspartic acid may be used as an antagonizing agent to mitigate the effects of UVA

Antagonizing effects of aspartic acid against UVA-induced downregulation of stemness genes are mediated by downregulating PGE₂-cAMP-HIF-1α signaling through inhibition of AP-1.

<p>hAMSCs were irradiated with 5 J/cm² UVA or transfected with the siRNA for HIF-1α and then incubated for three days with aspartic acid (100 μM) in the presence of the indicated concentration of PGE_<b>2</b> or cAMP under serum-free conditions. After three days of incubation, total RNA was isolated and the mRNA levels of the HIF-1α gene (A) and OCT4, NANOG, SOX2 genes (B) were measured by real-time quantitative RT-PCR. The results are expressed relative to untreated cells after normalization against GAPDH. Data are expressed as the means ± S.D. *, <i>p</i><0.05 vs. UVA (5J/cm²)-treated controls. ^o, <i>p</i><0.05 vs. UVA (5J/cm²) and aspartic acid (100 μM)-treated controls. The results were verified by repeating the experiments four times, each of which was conducted in duplicate. AA: aspartic acid, dBcAMP: dibutyryl cAMP</p

Mechanisms of aspartic acid effects against UVA-induced attenuation of stemness of stem cells.

<p>UVA irradiation induces production of PGE_<b>2</b> and its downstream molecule, cAMP through activation of AP-1 and NF-κB. cAMP molecule sequentially reduces expression of HIF-1α gene through CREB activation, consequently downregulating expression of stemness genes such as NANOG, SOX2, and OCT4. In the UVA irradiation-induced signaling pathway, aspartic acid attenuates UVA-induced effects on expression of stemness genes by inhibiting AP-1, which is upstream of PGE_<b>2</b> production. AA: aspartic acid</p

Aspartic acid increases downregulated expression of HIF-1α by UVA irradiation.

<p><b>(A)</b> hAMSCs were transfected with HRE-Luc reporter along with a Renilla luciferase expression vector driven by a thymidine kinase promoter using DharmFECT Duo transfection reagent according to the manufacturer’s protocols. After incubation for 24 h, the cells were irradiated with 5J UVA and then further incubated in the presence of the indicated concentrations of aspartic acid under serum-free conditions for 14 h. These cells were then harvested, lysed, and assayed. The results were confirmed by three independent transfections. Data are expressed as the means ± S.D. *<i>P</i><0.05 compared to the UVA (5J)-treated control. <b>(B)</b> hAMSCs were irradiated with 5 J/cm² UVA or transfected with the siRNA for HIF-1α or 2α and then incubated for three days in the presence of the indicated concentrations of aspartic acid under serum-free conditions. After three days of incubation, total RNA was isolated and the mRNA levels of the HIF-1α and HIF-2α gene were measured by real-time quantitative RT-PCR. The results are expressed relative to untreated cells after normalization against GAPDH. Data are expressed as the means ± S.D. *, <i>p</i><0.05 vs. UVA (5 J/cm²)-treated controls. The results were verified by repeating the experiments four times, each of which was conducted in duplicate. <b>(C)</b> Total lysates were analyzed by Western blot using the HIF-1α and HIF-2α antibodies. The results were verified by repeating the experiments three times. AA: aspartic acid</p

Aspartic acid reduces UVA-induced production of PGE₂ and cAMP.

<p>hAMSCs were irradiated with 5 J/cm² UVA and then incubated for three days in the presence of the indicated concentrations of aspartic acid under serum-free conditions. <b>(A and B)</b> After three days of incubation, the supernatants were harvested for PGE₂ (A) and cAMP (B) measurement. Data are expressed as the means ± S.D. *, <i>p</i><0.05 vs. UVA (5J)-treated controls. The results were verified by repeating the experiments three times, each of which was conducted in duplicate. AA: aspartic acid, Fk: forskolin.</p

Aspartic acid attenuates the effects of UVA irradiation on self-renewal of hAMSCs.

<p>hAMSCs were irradiated with 5 J/cm² UVA and then incubated for three days in the presence of the indicated concentrations of aspartic acid under serum-free conditions. <b>(A)</b> After three days of incubation under serum-free conditions, total RNA was isolated and the mRNA levels of the OCT4, NANOG, SOX2, and REX1 genes were measured by real-time quantitative RT-PCR. The results are expressed relative to untreated cells after normalization against the GAPDH. Data are expressed as the means ± S.D. *, <i>p</i><0.05 vs. UVA (5J)-treated controls. The results were verified by repeating the experiments four times, each of which was conducted in duplicate. <b>(B)</b> Effects of UVA irradiation and aspartic acid treatment on expression pattern of cell surface markers were determined. AA: aspartic acid</p