Nuclear Ago2/HSP60 contributes to broad spectrum of hATSCs function via Oct4 regulation

Abstract

Aims: Argonaute2 (Ago2) plays a fundamental role in microRNA-mediated gene regulation through its intrinsic endonuclease activity. In this study we demonstrate the novel functions and molecular mechanisms by which nuclear Ago2 directly regulates HSP (heat shock protein) 60 expression and stem cell self-renewal. HSP60 is a crucial regulator of ROS (reactive oxygen species), senescence, and apoptotic cell death in several tissues and cell types.

Results: HSP60 is regulated via inactivation of p38/JNK and p53 and binds directly to the regulatory regions of the TERT, c-myc, GPx3, p53, and STAT3 genes. Using HSP60 CHIP-PCR experiments, we show that HSP60 binds directly to the Oct4 and Nanog genes and directly regulates Oct4 and other stemness genes involved in human adipose tissue-derived stem cell (hATSC) differentiation. HSP60 also positively regulates ROS-scavenging factors, including GPx3 and TXNL1, which directly modulate cytosolic ROS in hATSCs. Moreover, our study shows that Oct4 regulates HSP60 expression and controls hATSC survival and self-renewal after binding to the HSP60 gene. Furthermore, HSP60-mediated regulation of Oct4 contributes to neuronal and endodermal β-cell differentiation of hATSCs in vitro and in vivo and downregulates mesoderm-specific gene expression.

Innovation and conclusion: We show that increased levels of Ago2 or HSP60 effectively induce nuclear localization of HSP60, which directly controls Oct4, c-Myc, p53, TERT, and STAT3 for transdifferentiation programs. Collectively, we suggest a novel model in which nuclear Ago2 controls HSP60 in hATSCs.

FIG. 1.

Nuclear localization and gene expression regulation of Ago2 enhances hATSC cell self-renewal along with telomere extension. (A) Immunocytochemical images show nuclear localization of Ago2 and co-localization with BrdU+ mitotic hATSCs. (B) Determination of DNA binding activity of Ago2 to the stemness genes Oct4, Sox2, and Nanog by CHIP/immunoblot analysis. (C) Luciferase activity of Oct4 and Ago2 promoters in Ago2- or Oct4-overexpressing or control hATSCs. Cells were transfected with pGL3-PSA, a phRL-SV40 plasmid. In hATSCs, Oct4 and Ago2 promoter luciferase activity was significantly increased by Ago2 or Oct4. (D) Point-mutation-induced Oct4 binding motif of Ago2 promoter significantly decreases Oct4 expression compared to scramble DNA transfected cells in Ago2-overexpressing hATSCs. (E) The proliferation activity of Ago2-overexpressing or Ago2-knockdown hATSC cells and their phase contrast images (n=5). (F) For flow cytometric analysis, cells were harvested and stained with propidium iodide to detect DNA. The percentages of cells in the G0/G1, S, and G2/M phases of the cell cycle were determined using a DNA histogram-fitting program. (G) Determination of differential expression of stemness and cell proliferation-associated genes by real-time RT-PCR and Western blotting. (H) Telomerase assay of Ago2-hATSCs compared to control hATSCs. Telomere length analysis was performed by Southern blotting. **p<0.01 (n=5), *p<0.05 with ANOVA comparison to control cells (n=3).

FIG. 2.

Ago2 controls HSP60 expression to enhance hATSC self-renewal. (A, B) Ago2 directly binds the promoter regions of HSP60 as shown by CHIP/PCR/sequencing. Red marks show potential Ago2 binding regions. (C) Immunocytochemical image showing coexpression of Ago2 and HSP60 in the nuclei of hATSCs. Arrows indicate co-localized Ago2 and HSP60 in the nucleus. (D) Regulation of HSP60 transcript expression before and after interference of Ago2 expression and Ago2 overexpression. (E) Ago2 controls HSP60, HSP70, and HSP90 expression and also affects P38/JNK phosphorylation in cultured hATSCs. (F) Luciferase activity of HSP60 promoter in Ago2 overexpressed or interfered Ago2 expression in hATSCs. Cells were transfected with PGL3-PSA, a phRL-SV40 plasmid. In hATSCs, HSP60 promoter luciferase activity was significantly increased by Ago2. (G) Ago2 expression was positively regulated by HSP60. (H) Luciferase activity of Ago2 promoter in HSP60 overexpression in hATSCs. Ago2 promoter luciferase activity was significantly increased by HSP60 in hATSCs. **p<0.01 (n=5), *p<0.05 with ANOVA comparison to control cells (n=3).

FIG. 3.

Ago2 controls HSP60 expression to enhance hATSC survival against P38MAPK-mediated cell death. (A) HSP60 significantly induces cell self-renewal and knockdown of HSP60 results in attenuation of cell growth in cultured hATSCs. Viable cell counting and (B) immunofluorescence staining with an anti-BrdU antibody showed that cell populations were significantly increased after HSP60 overexpression (C) along with an increase in the S-phase subpopulation. For flow cytometric analysis, cells were harvested and stained with propidium iodide to detect DNA. The percentage of cells in the G0/G1, S, and G2/M phases of the cell cycle were determined using a DNA histogram-fitting program. Evaluation of cells' self-renewal activity via colony forming unit (CFU) formation assay after HSP60+, siHSP60, Ago2+, or siAgo2 treatment in cultured hATSCs. (D) Telomerase assay of HSP60+ ATSCs compared to control hATSCs. Telomerase activity was assessed with a modified TRAP (telomere repeat amplification protocol) assay. (E) Telomere length analysis was performed by Southern blotting before and after HSP60 overexpression in cultured hATSCs. (F) Expression of the cell cycle regulating genes c-Myc, P53, p21, CDK2,4, and RUNX3 before and after HSP60 overexpression or knockdown induction in P6 or P15 cultured ATSCs. (G) Western blot analysis of differential phosphorylation of JAK/STAT and ERK1/2 and (H) the effect of HSP60 on Ago2 and HSP70 and HSP90 expression in P6 and P16 hATSCs by Western blot analysis. **p<0.01 (n=5), *p<0.05. ANOVA was used for comparisons with control cells. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 4.

Involvement of HSP60 in senescence and cell aging via cytosolic ROS modulation in cultured hATSCs. (A) The effect of HSP60 expression on ROS generation or scavenging in cultured hATSCs. For detection of cytosolic ROS, DCFDA staining was performed for quantified positive cell counting (B) and FACS analysis. (C) The effect of HSP60 expression on hATSC senescence. We determined cell senescence by β-galactosidase staining and positive cell counting. (D, E) The crucial role of HSP60 for P38/JNK mediated apoptotic cell death in hATSCs. We detected apoptotic cell death by TUNEL staining. Disruption of HSP60 expression induces apoptotic cell death and expression of the proapoptotic signal mediators Bax, cytochrome C, and caspase 3. Overexpression of HSP60 or siHSP60/SB203580 treatment in hATSCs finally resulted in significantly downregulated apoptotic cell death signal (P15) along with decreased numbers of TUNEL+ cells. (F) Real time RT-PCR and quantification results showed that knockdown of HSP60 expression effectively induces GPx1 and GPx3 downregulation in hATSCs. (G) Cell survival before and after Ago2, Oct4, or HSP60 overexpression against H₂O₂ mediating apoptotic cell death in cultured hATSCs. Compared to untreated control cells, Ago2, Oct4, or HSP60 overexpression effectively induced cell survival against H₂O₂-mediated apoptotic cell death (TUNEL+ cells). (H) The effect of exogenic Ago2, Oct4, or HSP60 overexpression on the phosphorylation of p38/JUNK and expression of apoptotic cell death involving the signal mediators Bax and caspase 3 in H₂O₂-treated hATSCs. Exogenic Ago2, Oct4, or HSP60 overexpression effectively induces inactivation of p38/JUNK and significantly downregulates Bax and caspase 3 expression in hATSCs. (I) Differential distribution of HSPs in different passages of hATSCs (P6 and P18). At an early passage (P6), the nuclear localization of HSP60 was higher than cytosolic HSP60. In contrast, late passage (P15) cells showed markedly higher cytosolic localization compared to nuclear localization. (J) Direct controlling of ROS scavenging genes by HSP60 for hATSCs self-renewal and the related increase in gene expression. We performed HSP60 CHIP/PCR and presented relative HSP60 binding frequency compared to HSP60 overexpressing and control hATSCs. To confirm the relevance of the P38/JNK signaling pathways in terms of controlling cell survival, cells were stimulated with siHSP60 for 6 hours. The siHSP60-stimulated cells were treated with the P38 MAPK inhibitor SB203580 (10 μM; Promega, Madison, WI) and incubated for 1 h. After a medium change, cells were further incubated for 12 h. Treated cells were then analyzed for relative self-renewal activity and differential gene expression via viable cell counting, western blotting. **p<0.01 (n=5), *p<0.05. ANOVA was used for comparisons with control cells.

FIG. 5.

The effect of HSP60 on Oct4, including stemness genes expression in hATSCs. (A) HSP60 expression induces HSF1 upregulation in hATSCs. (B) Oct4, Sox2, Nanog, and Rex1 expression before and after induction of HSP60 overexpression or knockdown in different passage (P6 or P15) cultured hATSCs at the protein level and (C) immunocytochemical image showing co-localization of stemness proteins and HSP60 in cultured hATSCs. *p<0.05 (n=5). ANOVA was used for comparisons with control cells.

FIG. 6.

Molecular behavior of HSP60 in the control of self-renewal and differentiation of hATSCs. (A) CHIP/immunoblot analysis to determine DNA binding activity of HSP60 before and after HSP60 knockdown or overexpression in hATSCs. (Con; control ATSCs, HSP60−; knocked down HSP60 in hATSCs, HSP60+; HSP60 overexpressed in ATSCs). (B) HSP60 CHIP/PCR analysis also showed regulation of Ago2, Oct4, Nanog, c-Myc, P53, TERT, and STAT3 expression via direct binding to regulatory regions of functional genes involved in cell self-renewal and differentiation of hATSCs. CHIP/PCR results also showed that high doses of HSP60 induce active binding to regulatory regions of functional genes compared to the low levels of HSP60. (C) Differential expression of epigenetic reprogramming regulatory genes before and after HSP60 overexpression or knockdown in P6 or P15 hATSCs. All epigenetic reprogramming regulatory genes were downregulated or upregulated in the case of HSP60 knockdown or upregulation, respectively, at each passage. (D) Epigenetic reprogramming of the Oct4 promoter region in HSP60-overexpressing hATSCs. Compared to control cells, HSP60-overexpressing cells showed significant demethylation. (E) Oct4 also controls HSP60 expression after binding to regulatory regions of the HSP60 gene. The red character in upper figure represents the promoter region of HSP60, and the red character in the lower figure shows the detailed Oct4-binding nucleotide sequence on the promoter region of the HSP60 gene. (F) Luciferase activity of the HSP60 promoter in Oct4-overexpression hATSCs. In hATSCs, HSP60 promoter luciferase activity was significantly increased by Oct4. (G) Point mutation in the Oct4-binding motif in the HSP60 promoter significantly decreases HSP60 expression compared to scrambled-DNA-transfected cells in Oct4-overexpressing hATSCs. A HSP60 promoter with a single base pair point mutation led to significantly reduced HSP60 expression compared to scrambled DNA transfected and Oct4-overexpressing control hATSCs. (H) Luciferase activity of the Oct4 promoter in HSP60-overexpression hATSCs. In hATSCs Oct4 promoter luciferase activity was significantly increased by HSP60. (I) The effect of Oct4 on Ago2, HSP60, stemness, and self-renewal gene expression. **p<0.01, *p<0.05 (n=5). ANOVA was used for comparisons with control cells. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 7.

Exogenic expression of HSP60 effectively attenuates differentiation into mesodermal linage, but beta cell and neural differentiation ability was significantly increased in vitro and in vivo brain trauma. (A) Mesodermal differentiation potential of HSP60+ hATSCs and siOct4 hATSCs compared to control hATSCs. We specifically stained differentiated cartilage, fat, and bone from ectopic HSP60-treated hATSCs. Real time RT-PCR analysis of lineage-specific gene expression after bone, fat, and cartilage differentiation of HSP60+ hATSCs and siOct4 hATSCs compared to control hATSCs. **p<0.01, *p<0.05 (n=5). ANOVA was used for comparisons with control cells. (B) The effect of HSP60 and Oct4 expression on β-cell differentiation. After induction of β-cell differentiation, we evaluated differentiation potency by Western blot analysis and immunocytochemistry using anti-β insulin and c-peptide antibodies. (C) HSP60 effectively enhances neurosphere formation after neural induction in hATSCs culture. HSP60 expression induces Oct4 overexpression during neurosphere culture. (D) Expression of neural development-related transcription factors Pax6, Foxg1, and HOXB4 before or after HSP60 expression in ATSCs. **p<0.01 (n=3), *p<0.05. ANOVA was used for comparisons with control cells. (E) After neural differentiation induction in HSP60+ hATSCs, neurospheres effectively differentiated into neuronal cells and astrocytes expressing TuJ, MAP2ab, and GFAP. These data were confirmed by Western blot analysis. **p<0.01 (n=3), *p<0.05. ANOVA was used for comparisons with control cells. (F) Immunofluoresence analysis of differentiated hATSCs before or after HSP60 or siOct4 treatment using anti-Tuj and GFAP antibodies. (G) In vivo evaluation of neurogenic activity of HSP60+ hATSCs or control hATSCs. We evaluated regenerative potency of engrafted HSP60+ hATSCs by immunohistochemical analysis of traumatic brains using anti-TuJ or anti-GFAP antibodies. To trace engrafted cells in the brain, we marked engrafting cells using CM-Dil.

FIG. 8.

Global gene expression regulation of Ago2/HSP60 for hATSCs self-renewal and differentiation. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).