Expression of Reg family proteins in embryonic stem cells and its modulation by Wnt/beta-catenin signaling

Abstract

Regenerating islet (Reg) proteins are involved in the proliferation and differentiation of diverse cell types. However, whether embryonic stem cells (ESCs) express Reg genes and their corresponding proteins remains unknown. In this study, we probed the expression of Reg family members by mouse ESCs (mESCs). Mouse Reg1 and Reg3gamma were detected in undifferentiated stem cells. Furthermore, we tested if gastrin--an inducer of Reg1 expression in committed cells--up-regulates the Reg1 gene in mESCs. Gastrin did not affect the expression of Reg1 either in self-renewing mESCs or under conditions permitting their differentiation. Moreover, overexpression of Reg genes found in various forms of cancer has been linked to dysregulated activation of the canonical Wnt/beta-catenin cascade. Given the important roles of Wnt signaling in stem cells, we investigated if activation of Wnt alters the expression of Reg genes in mESCs. Wnt activation led to an increase in Reg1 gene expression with a concomitant increase in the amount of secreted Reg1 protein. Finally, the expression pattern of genes indicative of differentiation was examined in mESCs that were either exposed to soluble Reg1 or overexpressed the Reg1 gene. This is the first account of expression of Reg family members by ESCs. Our results show that the canonical Wnt cascade affects Reg expression and warrants further studies into the potential roles of Reg proteins in stem cell physiology.

FIG. 1.

Expression of Reg family members in embryonic stem cells (ESCs). (A) Mouse ESC lines E14Tg2a and R1, MIN6 (insulinoma) β-cells, and 3T3 fibroblasts were probed by RT-PCR for the expression of Reg genes. The expression of β-actin is also shown. (B) Western blot analysis of lysates from mESCs, MIN6, and 3T3 cells for their content of Reg1 protein. (C) The amount of Reg1 protein secreted in the medium by mESCs and MIN6 cells was quantified by enzyme-linked immunosorbent assay (ELISA). No Reg1 protein was detectable in supernatants from cultured 3T3 cells. Values are shown as mean ± standard deviation (SD) from 3 experiments. (D) Expression of the putative Reg1 receptor Extl3 in mESCs and MIN6 cells.

FIG. 2.

Induction of Reg1 expression in mouse embryonic stem cells (mESCs) by gastrin. (A) Relative expression of the Reg1 gene in MIN6 cells and mESCs (E14Tg2a) after incubation with 1 nM gastrin for 24 h. Reg1 gene expression is shown as fold-increase compared to untreated cells. Values are shown as mean ± SD. (B) The CCK2R is not detected in undifferentiated mESCs cultured in the presence of leukemia inhibitor factor (LIF) (mESCs). In contrast, MIN6 cells and mESCs grown without LIF (mESCs–LIF) for 4 days express the CCK2R. The expression of the housekeeping gene β-actin is also shown. (C) Mouse ESCs cultivated in the absence of LIF for 4 days were incubated without (−) or with 1 nM gastrin (+) for 24 h. Results are shown compared to the Reg1 expression of E14Tg2a mESCs incubated without gastrin.

FIG. 3.

Activation of the canonical Wnt pathway in mESCs. (A) The activation of Wnt in mESCs transfected with a Wnt/β-catenin activatable promoter driving the expression of luciferase (SuperTOP) was examined under different conditions as shown. The luciferase signal was normalized to that due to Renilla luciferase activity and was expressed in relative luciferase units (RLUs). Cells transfected with SuperFOP (carrying mutated TCF/LEF-binding sites) did not yield signal above the background. *P < 0.005 compared to untreated mESCs. **P < 0.005 compared to mESCs exposed in 0.25% dimethyl sulfoxide (DMSO). # P < 0.005 compared to mESCs treated with Wnt3a only. (B–E) Immunostaining of mESCs for β-catenin and Oct3/4A. Unlike (B) untreated mESCs, cells incubated with Wnt3a (D) displayed nuclear accumulation of β-catenin as indicated by the arrowheads. Co-staining of the cells depicted in (B and D) for Oct3/4A is also shown (C and E). Bars in (B–E): 20 μm. (F) Relative expression of Oct3/4A and Nanog in mESCs treated with 100 ng/mL Wnt3a (white bars) or 30 mM LiCl (dark bars). Results are shown as mean ± SD (n = 3) relative to the expression of the corresponding genes in samples of untreated mESCs.

FIG. 4.

Reg gene expression is enhanced by activation of the canonical Wnt cascade. (A) The expression of the Reg1 gene was increased in E14Tg2a mESCs treated with 100 ng/mL Wnt3a or 30 mM LiCl compared to untreated (control) cells (their Reg1 gene expression was set to 1). The increase in Reg1 gene expression due to incubation with Wnt3a was curtailed in mESCs transfected with a plasmid encoding ΔnTCF4 (Wnt3a + ΔnTCF4). Cells transfected with an empty vector and treated with Wnt3a (Wnt3a + control vector) were also included in this series of experiments. Results are shown as mean values ± SD from a representative experiment (triplicate samples). *P < 0.005 and **P < 0.001 compared to control (untreated) cells. # P < 0.005 compared to Wnt3a + control vector cells. (B) Wnt activation in mESCs by LiCl or Wnt3a did not lead to statistically significant differences in the expression of the Reg3γ gene in comparison with untreated (control) cells. (C) Other Reg genes were not detected in mESCs even after activation of the Wnt/β-catenin cascade. (D) Expression of the Reg1 receptor Extl3 in mESCs treated with Wnt3a or LiCl. *P < 0.05 compared to untreated (control) mESCs.

FIG. 5.

Reg1 protein secretion by mESCs. When incubated with 100 ng/mL Wnt3a, E14Tg2a, and R1 mESCs secreted higher amounts of Reg1 protein than control mESCs as enzyme-linked immunosorbent assay (ELISA) analysis revealed. *P < 0.05.

FIG. 6.

Gene expression in mESCs exposed to exogenous Reg1 or transduced with AdReg1GFP. (A) Quantitative PCR results for the expression of the differentiation markers brachyury (Bry) and eomes [mesoderm], neuroD1 and pax6 [ectoderm]. White bars: mESCs (−LIF) with 100 ng/mL Reg1 (control: mESCs without leukemia inhibitor factor [LIF]). Dark bars: mESCs (−LIF) infected with AdReg1GFP at MOI of 100 (control: mESCs without LIF but infected with AdGFP at a MOI of 100). The cells were probed after 4 days of incubation with Reg1 or adenoviral infection. (B) The endoderm genes foxa2 and sox17 displayed a more pronounced up-regulation in mESCs that were either treated with exogenous Reg1 (white bars) or overexpressed Reg1 after infection with AdReg1GFP (dark bars). Results are shown after 2 and 4 days of exposure to Reg1 or infection with AdReg1GFP. Gene expression from the control samples was set to unity. #P < 0.001 and *P < 0.05 compared to controls. (C) Foxa2 (red) and (D) Sox17 (red) staining (nuclear DNA counterstaining with DAPI) of mESCs exposed to 100 ng/mL Reg1. Undifferentiated E14Tg2a cells stained for (E) Foxa2 and (F) Sox17 (and DAPI) are also shown. Bars: 50 μm. (G) The fraction of Sox17⁺cells in cultures without LIF (−LIF) and containing 100 ng/mL Reg1 (−LIF + Reg1) was assessed by flow cytometry (# P < 0.001). Results are shown from 3 independent experiments. Color images available online at www.liebertonline.com/scd.