A mammalian monothiol glutaredoxin, Grx3, is critical for cell cycle progression during embryogenesis

Abstract

Glutaredoxins (Grxs) have been shown to be critical in maintaining redox homeostasis in living cells. Recently, an emerging subgroup of Grxs with one cysteine residue in the putative active motif (monothiol Grxs) has been identified. However, the biological and physiological functions of this group of proteins have not been well characterized. Here, we characterize a mammalian monothiol Grx (Grx3, also termed TXNL2/PICOT) with high similarity to yeast ScGrx3/ScGrx4. In yeast expression assays, mammalian Grx3s were localized to the nuclei and able to rescue growth defects of grx3grx4 cells. Furthermore, Grx3 inhibited iron accumulation in yeast grx3gxr4 cells and suppressed the sensitivity of mutant cells to exogenous oxidants. In mice, Grx3 mRNA was ubiquitously expressed in developing embryos, adult tissues and organs, and was induced during oxidative stress. Mouse embryos absent of Grx3 grew smaller with morphological defects and eventually died at 12.5 days of gestation. Analysis in mouse embryonic fibroblasts revealed that Grx3(-/-) cells had impaired growth and cell cycle progression at the G(2) /M phase, whereas the DNA replication during the S phase was not affected by Grx3 deletion. Furthermore, Grx3-knockdown HeLa cells displayed a significant delay in mitotic exit and had a higher percentage of binucleated cells. Therefore, our findings suggest that the mammalian Grx3 has conserved functions in protecting cells against oxidative stress and deletion of Grx3 in mice causes early embryonic lethality which could be due to defective cell cycle progression during late mitosis.

Fig. 1

Mammalian Grx3s can rescue the growth defects of yeast grx3grx4 cells. (A) Vector-expressing wild-type cells, and vector-, ScGrx3-, HsGrx3- and MmGrx3-expressing grx3grx4 cells were grown on nutrient rich YPD and SC-Ura media for 48 h at 30 °C. (B) Subcellular localization of ScGrx3–RFP (upper) and MmGrx3–GFP (lower) in yeast cells. Scale bars = 10 μm.

Fig. 2

Mammalian Grx3s are able to suppress the sensitivity of grx3grx4 cells to oxidants and iron accumulation. (A) Yeast grx3grx4 cells expressing plasmids as indicated were grown in SC-Ura liquid media and the same media supplemented with 1.0 mM H₂O₂, 1.5 mM diamide, 0.3 mM tBHP, respectively. Cell density was measured at A₆₀₀ after growth for 24 h at 30 °C. Shown is one representative experiment from four independent experiments conducted. The bars indicate the standard deviation (n = 3). (B) Whole-cell iron contents were measured using inductively coupled plasma mass spectrometry (ICP-MS). All results shown here are the means of three independent experiments, and the bars indicate the standard deviation. (C) Intracellular iron levels were measured by a QuantiChron™ Iron Assay Kit. Shown is one representative experiment of four independent experiments. The bars represent standard deviations (n = 3). Student’s t-test, *P < 0.01; **P < 0.001; ***P < 0.0001.

Fig. 3

MmGrx3 expression is in tissues and embryos and induced by oxidative stress. (A) Ten micrograms of total RNA isolated from brain, heart, spleen, liver, kidney, stomach, muscle, testis, lung and fat tissues were prepared, blotted and probed for MmGrx3. Ethidium bromide-stained rRNAs are shown as a loading control. (B) In situ hybridization. Embryo specimens at E10.5 were hybridized with digoxygenin-labeled antisense RNA probe (left) and sense RNA probe (right). Magnification of images is 7.5×. a, heart atrium; ce, cerebellum; h, hypothalamus; ic, infecrior colliculus; l, liver; m, medulla; n, neocortex; s, spinal cord; sc, superior colliculus; t, tongue; ta, tail; th, thalamus; v, heart ventricle. (C–E) Quantitative real-time PCR analysis of MmGrx3 mRNA levels in C2C12 cells treated with various concentrations of H₂O₂ (C), diamide (D) and tBHP (E) for different time points as indicated. The data shown are relative mRNA levels (fold change) as compared with C2C12 cells without treatments. The housekeeping gene cyclophilin was used to normalize Grx3 expression. All values are means ± SD. Student’s t-test, *P < 0.05; **P < 0.01.

Fig. 4

Disruption of Grx3 in mice. (A) Shown is the schematic diagram of MmGrx3 genomic DNA structure and the gene trap vector. (B) Genotyping of embryos dissected at E10.5 from F2 sibling-crossing female by PCR using a combination of gene-specific and target vector-specific primers. The large PCR fragments indicated target alleles, whereas the smaller bands indicated wild-type alleles. (C) Western blot analysis of the lysates from the same group of embryos shown in (B). Shown are MmGrx3 protein levels in wild-type, reduced levels in heterozygous alleles and absence of MmGrx3 in homozygous alleles. (D) Shown are examples for wild-type and homozygous embryos at E10.5. Magnification is 20×. (E) Wild-type embryo showing closed neural tube (arrowhead). (F) Homozygous embryo showing open neural tube and pericardial effusion (arrowhead).

Fig. 5

Grx3 null allele MEFs display impaired cell proliferation and are defective in cell-cycle progression. (A) Cell lysates from Grx3^+/+, Grx3^+/−, and Grx3^−/− MEFs were subjected to western blot analysis of MmGrx3 (1:1000). Monoclonal antibody against Gapdh (1: 1000) was used as a loading control. (B) MEF proliferation (Passage 2) was examined during a 6-day period. (C) Cell-cycle profiles of MEFs (P2) were conducted by flow cytometry (FACScan, Coulter). Cells were grown in 10% fetal bovine serum Dulbecco’s modified Eagle’s medium media for 72 h, fixed then stained with propidium iodide. (D) DAPI staining of nuclei of Grx3^+/+ (a) and Grx3^−/− (b) MEFs showed Grx3^−/− MEFs accumulated binucleated cells (arrow). Grx3^+/+ (c) and Grx3^−/− (d) MEFs were counterstained with β-actin (1:200). Scale bars = 10 μm. (E) Quantification of binucleated cells in Grx3^+/+ and Grx3^−/− MEFs. Total 615 cells counted for Grx3^+/+ and 527 cells counted for Grx3^−/−. The bars represent means ± SD. Student’s t-test *P < 0.0001. (F,G) Grx3^+/+ and Grx3^−/− MEFs were pulse-labeled with BrdU for 5 h before being harvested. Cells were first stained with anti-BrdU-Alex 688 and then counterstained with Sytox orange. Results in (D) show the same field of cells stained with Sytox orange (a and c) or anti-BrdU-Alex 688 (b and d). (a,b) Grx3^+/+ MEFs; (c,d) Grx3^−/− MEFs. Scale bars = 30 μm. The percentage of BrdU-positive cells in (E) was calculated by counting the BrdU-positive cells in six independent fields and dividing by the total number of Sytox orange-stained cells (~ 200 cells counted in each field). The bars represent means ± SD.

Fig. 6

Grx3 is critical for cell-cycle progression at G₂/M phase. (A) Western blot analysis of Grx3 expression in Grx3-KD HeLa cells in comparison with control cells. (B) Quantification of Grx3 protein levels in Grx3-KD cells compared with control cells. The bars represent standard deviations (n = 3). Student’s t-test *P < 0.05, **P < 0.01. (C) Grx3-KD and control HeLa cells were synchronized using double thymidine block, then released, and progressed through S, G₂ and M phases to finish cell cycle back to G₁ phase. Cells were harvested at 2-h intervals. Half of cells were stained with propidium iodide, and analyzed by FACS. Another half of cells were prepared for cell lysate. (D) Analysis of key cell-cycle regulators in Grx3-KD and control cells Cell lysates from control, Grx3-KD shRNA#1, Grx3-KD shRNA#2 cells were prepared as described above and subjected to western blotting for cyclin B1, Plk1, Securin, Grx3 and Gapdh. (E) DAPI staining of nuclei of control (a) and Grx3-KD (b) cells showed Grx3-KD cells accumulated binucleated cells (arrowhead). Control (c) and Grx3-KD (d) were counterstained with β-actin (1: 200). (e,f) Mergered images of control (e) and Grx3-KD (f) staining cells. Scale bars = 200 μm. (F) Quantification of binucleated cells in control and Grx3-KD cells. Total 600 cells counted for control cells and 360 cells counted for Grx3-KD cells. The bars represent means ± SD. Student’s t-test *P < 0.0001.