Protective Effects of Recombinant Human Angiogenin in Keratinocytes: New Insights on Oxidative Stress Response Mediated by RNases

Abstract

Human angiogenin (ANG) is a 14-kDa ribonuclease involved in different pathophysiological processes including tumorigenesis, neuroprotection, inflammation, innate immunity, reproduction, the regeneration of damaged tissues and stress cell response, depending on its intracellular localization. Under physiological conditions, ANG moves to the cell nucleus where it enhances rRNA transcription; conversely, recent reports indicate that under stress conditions, ANG accumulates in the cytoplasmic compartment and modulates the production of tiRNAs, a novel class of small RNAs that contribute to the translational inhibition and recruitment of stress granules (SGs). To date, there is still limited and controversial experimental evidence relating to a hypothetical role of ANG in the epidermis, the outermost layer of human skin, which is continually exposed to external stressors. The present study collects compelling evidence that endogenous ANG is able to modify its subcellular localization on HaCaT cells, depending on different cellular stresses. Furthermore, the use of recombinant ANG allowed to determine as this special enzyme is effectively able to counter at various levels the alterations of cellular homeostasis in HaCaT cells, actually opening a new vision on the possible functions that this special enzyme can support also in the stress response of human skin.

Keywords: oxidative stress; skin cells; stress granules; stress induced RNases; tiRNA.

Figure 1

Analysis of ANG expression level in HaCaT cells cultured in different growth conditions. (A). Expression analysis of ANG mRNA by RT-qPCR; (B). Western blot analysis of cell lysates. CTRL: cells cultured in physiological conditions; SA: HaCaT cells treated with 500 µM sodium arsenite (SA) for 1 h at 37 °C; H₂O₂: HaCaT cells treated with 1 mM of hydrogen peroxide for 2 h at 37 °C; T₄₅: HaCaT cells subjected to thermal stress at 45 °C for 1 h.

Figure 2

Immunofluorescence analyses of ANG sub-cellular localization under physiological and stresses conditions. (A) HaCaT cells cultured in physiological conditions; (B) HaCaT cells treated with SA for 1 h at 37 °C; (C) HaCaT cells treated with 1 mM of hydrogen peroxide for 2 h at 37 °C; (D) HaCaT cells subjected to thermal stress at 45 °C for 1 h. Cells were co-stained with PABP (red), ANG (green) and blue staining (DAPI) for nuclei. Yellow spots indicate the co-localization between ANG and PABP at the level of SGs.

Figure 3

CD analysis of rANG and its variants. (A) CD spectra recorded at 20 °C in 20 mM of MES/NaOH pH 6.0 and 100 mM NaCl, using a protein concentration of 0.1 mg mL⁻¹; (B) thermal denaturation profiles, as followed by monitoring changes in the molar ellipticity at 220 nm as a function of temperature. The denaturation temperature (T_d) for each protein is shown in panel B.

Figure 4

Superimposition of the CD spectra of (A) rANG, (B) rANG_6xHis and (C) H13A_rANG, registered at 20 °C before heating (plane line) and after cooling (dashed line). Spectra were recorded in 20 mM of MES/NaOH pH 6.0 and 100 mM NaCl, using a protein concentration of 0.1 mg mL⁻¹.

Figure 5

HaCaT cells viability measured after the treatment of cells for 24 h with increasing doses of rANG. Statistical analyses were carried out by using GraphPad Prism. Values are the means ± SEM of biological replicates (* p < 0.05, *** p < 0.001) compared to the respective controls (one-way ANOVA, followed by Bonferroni’s post-test).

Figure 6

HaCaT cells viability measured after the treatment of cells with increasing concentration of SA at three different incubation time (1, 6 and 24 h). Experiments were performed in triplicate and statistical analysis were carried out by using GraphPad Prism. Values are the means ± SEM of biological replicates (*** p < 0.001 or **** p < 0.0001) compared to the respective controls (one-way ANOVA, followed by Bonferroni’s post-test).

Figure 7

rANG effects on stress response of HaCaT cells. (A) RT-qPCR analysis of the expression of HSPA6 gene; (B) intracellular ROS detection by DCFH-Da assay; (C) distribution of HaCaT cells population among cell cycle G1 phase and S/G2 phase. (−) untreated cells; (+) pre-treatment of cells with 2µM rANG or 500 µM SA. Values are the means ± SEM of biological replicates (** p < 0.01, *** p < 0.001, **** p < 0.0001) compared to the respective controls (one-way ANOVA, followed by Bonferroni’s post-test).

Figure 8

Model of the variant rANG_6xHis. The linker peptide is highlighted in turquoise. Amino acids from the catalytic triad and the 6xHis tag are shown as sticks. The image was generated by the molecular visualization software PyMOL (https://pymol.org, accessed on 10 May 2022).

Figure 9

HaCaT cells viability measured after the treatment of cells for 24 h with increasing doses of rANG_6xHis. Statistical analyses were carried out by using GraphPad Prism. Values are the means ± SEM of biological replicates (* p < 0.05, **** p < 0.0001) compared to the respective controls (one-way ANOVA, followed by Bonferroni’s post-test).

Figure 10

rANG_6xHis internalization and localization in HaCaT cells. (A) Western blot analysis of 200 ng of purified rANG_6xHis used for cells treatments; (B) Western blot analysis of cell lysates obtained by IMAC chromatography (M: protein ladder; 1: untreated HaCaT cell lysate; 2: 2 µM rANG_6xHis treated HaCaT cell lysate). (C) Immunofluorescence analysis of unstressed 2 µM rANG_6xHis pre-treated cells. (D) Immunofluorescence analysis of SA-stressed 2 µM rANG_6xHis pre-treated cells. PABP (red), rANG_6xHis (green) and blue staining (DAPI) for nuclei.

Figure 11

rANG_6xHis effects on HaCaT cells stress response. (A) RT-qPCR analysis of the expression of HSPA6 gene; (B) intracellular ROS detection by DCFH-Da assay; (C) distribution of HaCaT cells population among cell cycle G1 phase and S/G2 phase. (−) untreated cells; (+) pre-treatment of cells with 2 µM rANG_6xHis or 500 µM SA. Values are the means ± SEM of biological replicates (** p < 0.01, *** p < 0.001, **** p < 0.0001) compared to the respective controls (one-way ANOVA, followed by Bonferroni’s post-test).

Figure 12

H13A_rANG effects on HaCaT cells stress response. (A) Real-time qPCR analysis of the expression of HSPA6 gene; (B) intracellular ROS detection by DCFH-Da assay; (C) distribution of HaCaT cells population among cell cycle G1 phase and S/G2 phase. (−) untreated cells; (+) pre-treatment of cells with 2 µM H13A_rANG or 500 µM SA. Values are the means ± SEM of biological replicates (* p < 0.05, ** p < 0.01, *** p < 0.001) compared to the respective controls (one-way ANOVA, followed by Bonferroni’s post-test).