Phosphatase of regenerating liver 2 (PRL2) is essential for placental development by down-regulating PTEN (Phosphatase and Tensin Homologue Deleted on Chromosome 10) and activating Akt protein

Abstract

The PRL (phosphatase of regenerating liver) phosphatases are implicated in the control of cell proliferation and invasion. Aberrant PRL expression is associated with progression and metastasis of multiple cancers. However, the specific in vivo function of the PRLs remains elusive. Here we show that deletion of PRL2, the most ubiquitously expressed PRL family member, leads to impaired placental development and retarded growth at both embryonic and adult stages. Ablation of PRL2 inactivates Akt and blocks glycogen cell proliferation, resulting in reduced spongiotrophoblast and decidual layers in the placenta. These structural defects cause placental hypotrophy and insufficiency, leading to fetal growth retardation. We demonstrate that the tumor suppressor PTEN is elevated in PRL2-deficient placenta. Biochemical analyses indicate that PRL2 promotes Akt activation by down-regulating PTEN through the proteasome pathway. This study provides the first evidence that PRL2 is required for extra-embryonic development and associates the oncogenic properties of PRL2 with its ability to negatively regulate PTEN, thereby activating the PI3K-Akt pathway.

FIGURE 1.

Generation and characterization of PRL2 knock-out mice. A, structure of the mouse PRL2 locus and the mutant allele containing the pGT01 gene trap cassette. The exons and the initiation codon (ATG) of PRL2 are indicated. Lac Z and neo: expression units of the β-galactosidase and neomycin resistance genes. pA, polyadenylation signal. SA, splice acceptor. Solid arrows indicate primer sites for PCR genotyping. B, representative PCR amplification fragments using primers indicated in A for genotyping. C, quantitative RT-PCR analysis of PRL2 mRNA expression in tissues from PRL2^+/+, PRL2^+/−, and PRL2^−/− mice. n = 3 per group. D, Western blot analyses of PRL2 protein levels in skeletal muscle from PRL2^+/+, PRL2^+/−, and PRL2^−/− mice. E, quantitative RT-PCR analysis of PRL1, PRL2, and PRL3 mRNA expression in E16.5 embryos with PRL2^+/+, PRL2^+/−, or PRL2^−/− genotypes (n = 3 per genotype). The results were presented as the percentage of the wild type ± S.E.

FIGURE 2.

PRL2 mutant mice are growth-retarded. A, gross appearance of newborn PRL2 mutant and wild-type littermates. B, body weight of PRL2^+/+ (n = 6), PRL2^+/− (n = 15), and PRL2^−/− (n = 6) mutant newborn mice (mean ± S.E. *, p < 0.01). C, gross appearance of 6-week-old PRL2 mutant and wild-type littermates. D, body weight of PRL2^+/+, PRL2^+/−, and PRL2^−/− mice (males, n = 9, 12, and 10, respectively; females, n = 10, 11, and 11, respectively) from 1 to 21 weeks of age. Data represent mean for PRL2^+/− mice and mean ± S.E. for PRL2^+/+and PRL2^−/− mice (p < 0.01 for all time points except 1 week female).

FIGURE 3.

Placental hypotrophy and insufficiency in PRL2^−/− mice. A, embryos and their connected placentas of a PRL2^−/− mouse and its wild-type littermate at E16.5. B, weight of wild-type and PRL2^−/− embryos at E16.5 (n = 9 for each genotype). *, p < 0.005. C, weight of wild-type and PRL2^−/− placentas at E16.5 (n = 9 for each genotype). *, p < 0.01. D–K, histological examination of wild-type and PRL2^−/− placentas at E16.5. D and E, H&E staining at 25×. F and G, H&E staining at 100×. Arrows designate islands of glycogen cells. H and I, in situ hybridization using Tpbp probe marking spongiotrophoblast cells (dark blue) and glycogen cells (light blue). J and K, PAS staining for glycogen-containing cells (pink signal). Arrows designate islands of glycogen cells. De, deciduas; Sp, spongiotrophoblast; La, labyrinth. L, labyrinth area on the midline section (the largest section of continuous sections) from PRL2^−/− or wild-type placentas was measured using ImageJ (n = 6 for each genotype). Data represent mean ± S.E. *, p < 0.01. M, [¹⁴C]mannitol transported by PRL2^−/− or wild-type placentas. Data are presented as mean (percentage of wild type) ± S.E. n = 6 litters, *, p < 0.005.

FIGURE 4.

PRL2 mutant placenta has significantly lower amount of spongiotrophoblast and glycogen cells due to reduced proliferation. A–D, histological examination of wild-type and PRL2^−/− placentas at E12.5. A and B, PAS glycogen staining. Arrows designate islands of glycogen cells. C and D, immunohistochemistry using antibody against Ki67 to monitor proliferating cells. The blue line shows the boundary between labyrinth and spongiotrophoblast layers. De, deciduas; Sp, spongiotrophoblast; La, labyrinth. E, in situ hybridization using PRL2 probe showing PRL2 mRNA expression on frozen section of E12.5 wild-type placenta. Arrows show PRL2-positive cells in the decidual layer. F, PAS glycogen staining on the section adjacent to E. Arrows show glycogen-containing cells. Note the overlap of PRL2-positive cells and glycogen cells.

FIGURE 5.

Activation of Akt was reduced in PRL2-deficient placenta. A, analyses of phospho (p) and total protein levels of signaling molecules by Western blot using tissue lysates isolated from E12.5 PRL2^−/− or wild-type placentas. B–E, immunohistochemical analyses of E12.5 PRL2^−/− or wild-type placentas for phospho-Akt473 (B and C) or phospho-GSK3β (D and E) levels. De, deciduas; Sp, spongiotrophoblast; La, labyrinth.

FIGURE 6.

PRL2 overexpression enhanced EGF- and IGF2-induced Akt activation in HEK293 cells. A, proliferation rate of HEK293-PRL2 stable cell lines (PRL #1 and #2) and vector control cells. Cell numbers were determined after a 72-h culture by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay measured as optical density at 540 nm (OD 540). Data represent mean of three experiments ± S.E. *, p < 0.01. B, migration rate of HEK293-PRL2 and vector control cells determined by the Transwell migration assay. Cells that migrated to the lower chamber were collected and counted with a hemocytometer. Data represent mean of three experiments ± S.E. *, p < 0.001. C, Western blot showing FLAG-tagged PRL2 expression and Akt phosphorylation in HEK293-PRL2 cells. pAkt473, phospho-Akt473. D, responsiveness of HEK293-PRL2 and vector control cells to EGF and IGF2. Cells were serum-starved overnight, stimulated with 2 ng/ml EGF or 5 ng/ml IGF2 for the indicated times, lysed, and subjected to Western blot for phosphorylated Akt and total Akt signals. pAkt473, phospho-Akt473.

FIGURE 7.

PRL2 destabilized PTEN protein by reducing vinculin expression. A, lysates from E12.5 PRL2^−/− or wild-type placentas as well as HEK293-PRL2 stable cell lines (PRL2 #1 and #2) and their vector controls were analyzed by Western blot for PTEN, phospho-PTEN (pPTEN), and vinculin expression. B, PTEN mRNA levels in PRL2^−/− or wild-type placentas at E12.5, E14.5, and E16.5 were determined by quantitative RT-PCR (n = 5 for each genotype at each stage). Data represent mean ± S.E. C, HEK293-PRL2 stable cell line and its vector control were treated with cycloheximide (CHX) at 100 μg/ml for the indicated times, and cell lysates were analyzed for PTEN and actin protein levels by Western blot. Data represent three independent experiments. D, quantification of the results in C. PTEN and actin signals were measured using ImageJ. The ratio of PTEN/actin was determined for each sample and plotted as -folds of time 0 for each cell line. E, HEK293-PRL2 cell line and its vector control were treated with 20 μm MG132 for 6 h. Cells were lysed, and PTEN and actin protein levels were analyzed by Western blot. PTEN and actin signals were measured using ImageJ, and the ratio of PTEN/actin was determined. Data are representative of three independent experiments.