G protein coupled receptors in embryonic stem cells: a role for Gs-alpha signaling

Abstract

Background: Identification of receptor mediated signaling pathways in embryonic stem (ES) cells is needed to facilitate strategies for cell replacement using ES cells. One large receptor family, largely uninvestigated in ES cells, is G protein coupled receptors (GPCRs). An important role for these receptors in embryonic development has been described, but little is known about GPCR expression in ES cells.

Methodology/principal findings: We have examined the expression profile of 343 different GPCRs in mouse ES cells demonstrating for the first time that a large number of GPCRs are expressed in undifferentiated and differentiating ES cells, and in many cases at high levels. To begin to define a role for GPCR signaling in ES cells, the impact of activating Gs-alpha, one of the major alpha subunits that couples to GPCRs, was investigated. Gs-alpha activation resulted in larger embryoid bodies (EBs), due, in part, to increased cell proliferation and prevented the time-related decline in expression of transcription factors important for maintaining ES cell pluripotency.

Significance/conclusions: These studies suggest that Gs-alpha signaling contributes to ES cell proliferation and pluripotency and provide a framework for further investigation of GPCRs in ES cells.

Figure 1. Changes in mRNA levels for markers of pluripotency and the three germ cell layers in undifferentiated ES cells relative to EBs at day 4 and day 20.

(A) Relative changes in the level of mRNA encoding markers of pluripotency, Nanog and Oct4, during EB differentiation at day 4 (blue) and at day 20 (red) as compared to undifferentiated ES cells (green). Values are the mean ± SEM (n = 5 for Nanog and n = 4 for Oct4) of the relative level of mRNA in day 4 and day 20 EBs compared to the level in undifferentiated ES cells which was defined as 1.0. (B) Relative changes in the level of mRNA encoding markers of the three germ layers; mesoderm (Brachyury), endoderm (CXCR4), and ectoderm (Nestin) during EB differentiation, at day 4 (blue) and at day 20 (red), as compared to the level of mRNA in undifferentiated ES cells (green). Values are the mean ± SEM (n = 4 for Brachyury, n = 3 for CXCR4, and n = 3 for Nestin) of the level of mRNA in day 4 and day 20 EBs compared to the level in undifferentiated ES cells, which was defined as 1.0.

Figure 2. GPCR expression in ES cells using a real time RT-PCR microarray.

(A) The level of mRNA encoding GPCRs from day 4 (blue) and day 20 (red) EBs and undifferentiated ES cells (green) was categorized as undetectable, low, medium, and high. The cycle number (Ct) for the low, medium, and high groups were greater than 31.0, 31.0–28.0, and less than 28.0, respectively. Ct values for each GPCR were the average of the results from 2 independent experiments. (B) Receptors with the highest level of expression in day 4 EBs are indicated with their corresponding relative level of expression and gene name. The relative expression is presented as 2 ^{Ct (GPCR)–Ct (housekeeping gene)}×10⁵. (C) Receptors with the highest level of expression in day 20 EBs are indicated with their corresponding relative level of expression and gene name. The relative expression is presented as 2 ^{Ct (GPCR)–Ct (housekeeping gene)}×10⁵. (D) Receptors with the highest level of expression in undifferentiated ES cells are indicated with their corresponding relative level of expression and gene name. The relative expression is presented as 2 ^{Ct (GPCR)–Ct (housekeeping gene)}×10⁵.

Figure 3. Verification of GPCR mRNA levels using real time RT-PCR.

(A–B) Relative changes in the level of mRNA encoding MC4R and SSTR1(A) and the EDG4 and GLP-1 receptors (B) in day 20 compared to day 4 EBs, as determined by real time RT-PCR (mean ± SEM, n = 3 or 4). (C) Western blot analysis of MC4R protein expression (representative of the results from three independent experiments) in extracts of day 4 (D4) and day 20 (D20) EBs. Following hybridization with antibodies directed against the above receptors, the blots were stripped and re-probed with antibody against GAPDH.

Figure 4. Gs-alpha is expressed and functional in mouse ES cells forming EBs.

(A) Representative immunoblot of Gs-alpha expression in extracts of day 4 (D4) and day 20 (D20) EBs. The findings are representative of the results of three independent experiments. After hybridization, the blots were stripped and re-probed with antibody against GAPDH. (B–C) Immunohistochemical localization of Gs-alpha in EBs. Day 4 (B) and day 20 (C) EBs were immunostained with an antibody to Gs-alpha (red), and nuclei were stained with DAPI (blue), magnification was 40×. (D) Immunoblot of phospo-CREB in day 4 EBs treated with 1 µg/ml CTX for 0, 5, 15, 30 and 60 mins, as well as 4 days. Immunoblots for total CREB and GAPDH using the same samples are also shown. The results are representative of the results from 3 independent experiments.

Figure 5. The effect of Gs-alpha activation by CTX on EB size.

(A–D) EB formation in the absence (A,C) or presence (B,D) of 1 µg/ml CTX at day 4 (A,B) and day 12 (C,D). (E) A comparison of the diameter (mean ± SEM) of EBs incubated in the absence (blue bars) and presence (red bars) of 1 µg/ml CTX over a 20 day period. One random image was taken of each sample of EBs, and the greatest horizontal diameter for each EB in the horizontal plane was measured. A total of 4 to 9 (independently prepared) EB preparations were studied at each time point. *, P<0.05 compared to control, untreated EBs. Total number of EBs counted for each condition was for day 4 (control, CTX; 417, 395), day 8 (346, 195), day 12 (255, 129), day 16 (58, 50) and day 20 (36, 25). (F) A comparison of the diameter (mean ± SEM) of EBs produced by the hanging drop method and incubated in the absence (n = 10 at each time point, blue bars) and presence (n = 11 at each time point, red bars) of 1 µg/ml CTX over a 20 day period. An image was taken on each individually grown EB, and the greatest horizontal diameter for each EB in the horizontal plane was measured. *, P<0.05 compared to control, untreated EBs.

Figure 6. Effect of Gs-alpha activation on cell proliferation in EBs.

(A) Cell metabolic activity in EBs over a 20 day period of CTX treatment was evaluated, and the percentage change in the CTX-treated compared to control cells was determined in three independent samples at each time point. The values represent the relative percentage change in absorbance A450 in the CTX-treated EBs as compared to the control EBs, and the percentage change is shown. *, P<0.05 compared to control, untreated EBs. (B) The percentage of Ki67⁺ cells in CTX-treated and control EBs at day 20 relative to DAPI⁺ positive cells was determined as described in the Methods . *, P<0.05 compared to control cells. Representative image of immunohistochemical localization of Ki67 (red) in control (C) and CTX-treated (D) day 20 EBs is shown. EBs were immunostained with an antibody to Ki67, and nuclei were stained with DAPI (blue).

Figure 7. Effect of Gs-alpha activation on Nanog and Oct4 expression.

(A–B) Fold changes in the level of Nanog (A) and Oct4 (B) mRNA in CTX-treated compared to control EBs at day 12, 16, and 20 as determined by real time RT-PCR. Values are the mean ± SEM (n = 3 for Nanog and n = 4 for Oct4). (C) Western blot analysis of Nanog expression in CTX-treated (X) compared to control cells (C) at different time points (D4, D8, D12, D16, D20). The blots were stripped and re-probed for GAPDH. The results are representative of the results of three independent experiments. (D–E) Immunohistochemical localization of Nanog (red) in control (D) and CTX-treated (E) day 20 EBs. EBs were immunostained with an antibody to Nanog (red), and nuclei were stained with DAPI (blue). The images are representative of the results of three independent experiments. (F) The percentage of Nanog⁺ cells in CTX-treated and control EBs at day 20 relative to DAPI⁺ positive cells was determined as described in the Methods . *, P<0.05 compared to control cells.