GSK3β activity is essential for senescence-associated heterochromatin foci (SAHF) formation induced by HMGA2 in WI38 cells

Abstract

Cellular senescence is an irreversible form of cell cycle arrest, which is often characterized by domains of facultative heterochromatin substructures also known as senescence-associated heterochromatin foci (SAHF). SAHF assembly is likely mediated through the downregulation of the Wnt pathway, which inhibits Glycogen Synthase Kinase 3 Beta (GSK3β) in cells undergoing replicative senescence. Alternatively, High Mobility Group AT-Hook 2 (HMGA2) can also induce SAHF formation in primary cells, through a process in which the involved cell signaling pathway is unknown. Accordingly, it is important to determine whether GSK3β and the Wnt pathway are necessary during HMGA2-induced SAHF formation. In this study, we developed a senescence model for SAHF assembly in WI38 cell through ectopic expression of HMGA2. In this model, typical senescent features were identified, including elevated SA-β-galactosidase staining and the downregulation of the Wnt pathway. We also showed that the GSK3β inhibitor LiCl can partly disable SAHF formation through the HMGA2 overexpression in WI38 cells. However, the disabled SAHF formation resulting from the inactivity of GSK3β in our senescence model cannot be restored through ectopic overexpression of Catenin Beta 1 (CTNNB1), a downstream transcription factor of the Wnt pathway. This indicates that the GSK3β activity alone, and not those of downstream target genes, is crucial for the HMGA2-induced SAHF formation following the downregulation of the Wnt pathway.

Keywords: GSK3β; HMGA2; SAHF; Wnt2.

Figure 1

HMGA2 induced the senescence phenotype and formation of SAHF in WI38 cell line. A. β-gal staining shown the increased SA-β-galactosidase activity in WI38 cells 5 days after HMGA2-GFP overexpression indicating that HMGA2 induced senescence in these cells. Scale bar: 20 μm. B. Fluorescent images of WI38 cells displaying SAHF-like foci 5 days after HMGA2-GFP overexpression. Scale bar: 20 μm. C. Confocal immunofluorescent images of co-localization of HMGA2 in chromatin foci with the SAHF markers HP1γ in WI38 cells 5 d after over-expression of HMGA2-GFP, which indicates that the foci induced by HMGA2 are indeed senescence-associated heterochromatin foci (SAHF). Scale bar: 10 μm.

Figure 2

Canonical Wnt Signaling is Down-regulated in HMGA2 Induced SAHF Formation. (A) RT-PCR analysis showing the down-regulation of Wnt2 & Western-blot analysis showing the downregulation of β-catenin and the phosphorylated GSK3β, but not the unphosphorylated GSK3β, in WI38 cells after HMGA2 (H2) overexpression at the indicated time points in days (d). β-actin, a housekeeping gene, was used for normalization here. These results show that HMGA2 downregulated the Wnt pathway in WI38 cell line. (B) The increased GSK3β activity was measured at the indicated time points in days (d) after HMGA2 (H2) overexpression and the GSK3β activity analyzed by the overall quantitation of the GSK3β and phosphorylated GSK3β in (A), the details are described in the Methods section. Statistical analysis was performed using the Student’s t-test and the statistical significance is indicated by “*” and “**” which denote a P value of < 0.05 and < 0.01, respectively. (C) Reporter assay showing the downregulation of the activity of downstream transcription factor of the Wnt pathway indicated by POT/POF. Statistical analysis was performed using the Student’s t-test and the statistical significance is indicated by “*” which denotes a P value of < 0.05.

Figure 3

HMGA2-induced SAHF formation does not involve β-catenin regulated downstream factors in WI38 cells. A. POT/POF reporter assay in WI38 cells transfected with either GFP, GSK3β or HMGA2 (1, 2, 3) and co-transfected with each of GFP, GSK-3β, HMGA2 and β-catenin (4, 5, 6). Overexpression of β-catenin increased the activity of a downstream gene promoter (compare 3 with 6), a process independent from HMGA2 (compare 1 with 4), whereas GSK3β inhibited such a process (compare 4 with 5). Statistical analysis was performed using the Student’s t-test and the statistical significance is indicated by “*” and “**”, which denote P value of < 0.05 and < 0.01, respectively. B. Western-blotting showing upregulation of Cyclin D1, a downstream target gene of the Wnt pathway, induced by ectopic overexpression of β-catenin in WI38 cells. Overexpression of β-catenin increased the expression level of the Wnt downstream genes. C. The Fluorescent images of WI38 cells displaying the SAHF formation 5 days (d) after transfection with Mock+HMGA2-GFP or β-catenin+HMGA2-GFP (Scale bar: 20 μm), indicating that in WI38 cells the HMGA2-induced SAHF formation is independent from β-catenin-regulated downstream factors.

Figure 4

The activity of GSK3β is required for the SAHF formation induced by HMGA2 in WI38 cells. (A) Western-blotting showing changes in phosphorylated GSK3β and unphosphorylated GSK3β after adding a specific inhibitor (LiCl) or mock inhibitor (NaCl) in WI38 cells with or without HMGA2-GFP. (B) Activity of GSK3β analyzed by the quantitation of (A). LiCl effectively inhibited the GSK3β activity regardless of whether HMGA2 existed or not. Statistical analysis was performed using the Student’s t-test and the statistical significance is defined by “*” which denotes a P value of < 0.05. (C) Fluorescent images showing changes in SAHF formation in WI38 cells expressing HMGA2 incubated with NaCl or LiCl, as well as the absence of SAHF formation in MEF cells expressing only HMGA2-GFP. Scale bar: 20 μm. GSK3β is essential for SAHF formation induced by HMGA2 in WI38 cells.