Differentiation of trophoblast giant cells and their metabolic functions are dependent on peroxisome proliferator-activated receptor beta/delta

Abstract

Mutation of the nuclear receptor peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) severely affects placenta development, leading to embryonic death at embryonic day 9.5 (E9.5) to E10.5 of most, but not all, PPARbeta/delta-null mutant embryos. While very little is known at present about the pathway governed by PPARbeta/delta in the developing placenta, this paper demonstrates that the main alteration of the placenta of PPARbeta/delta-null embryos is found in the giant cell layer. PPARbeta/delta activity is in fact essential for the differentiation of the Rcho-1 cells in giant cells, as shown by the severe inhibition of differentiation once PPARbeta/delta is silenced. Conversely, exposure of Rcho-1 cells to a PPARbeta/delta agonist triggers a massive differentiation via increased expression of 3-phosphoinositide-dependent kinase 1 and integrin-linked kinase and subsequent phosphorylation of Akt. The links between PPARbeta/delta activity in giant cells and its role on Akt activity are further strengthened by the remarkable pattern of phospho-Akt expression in vivo at E9.5, specifically in the nucleus of the giant cells. In addition to this phosphatidylinositol 3-kinase/Akt main pathway, PPARbeta/delta also induced giant cell differentiation via increased expression of I-mfa, an inhibitor of Mash-2 activity. Finally, giant cell differentiation at E9.5 is accompanied by a PPARbeta/delta-dependent accumulation of lipid droplets and an increased expression of the adipose differentiation-related protein (also called adipophilin), which may participate to lipid metabolism and/or steroidogenesis. Altogether, this important role of PPARbeta/delta in placenta development and giant cell differentiation should be considered when contemplating the potency of PPARbeta/delta agonist as therapeutic agents of broad application.

FIG. 1.

Targeted disruption of the PPARβ gene in mouse. The PPARβ gene was disrupted in ES cells by homologous recombination with a replacement-type vector, using an approach based on positive-negative selection (A). In this vector, PPARβ genomic sequences containing the exons encoding the DNA-binding domain of the receptor (exon 4 and part of exon 5) were replaced with a PGK-neo cassette. Homologous recombination at the PPARβ locus in ES cells led to the deletion of both exon 4 and part of exon 5 encoding the two zinc fingers of the DNA-binding domain. ES cells carrying the mutant allele were confirmed by Southern blot analysis (B). Two independent positive ES cell clones were injected into blastocysts to generate chimeras, and heterozygous mice were obtained from a germ line transmitter chimera. Panel A shows the structure of the wt PPARβ allele, targeting vector, and recombinant PPARβ allele. The exons as well as locations of restriction sites and probes for PCR and southern blot are indicated. B, BamHI; E, EcoRI; K, KpnI; N, NotI; X, XhoI. Panel B shows a Southern blot analysis of genomic DNA digested with BamHI and KpnI from E9.5 embryos produced by a PPARβ heterozygous intercross. (C) PCR analysis of yolk sac DNA derived from E9.5 embryos. (D) Western blot analysis performed on nuclear extracts with a specific PPARβ antibody. The nuclear protein c-Jun was used as an internal control. In order to obtain a sufficient amount of material, the control at the protein level was performed on pups obtained from homozygous matings.

FIG. 2.

Defects in extra-embryonic tissues associated with the PPARβ-null genotype. (A) Morphology of E9.5 and E10.5 embryos from wt (a and c) and PPARβ^−/− (b and d). (B) Hematoxylin-eosin-stained sections of wt (a to c and g to i) and PPARβ-null (d to f and j to l) placentas. Higher magnification views of PPARβ-null placenta at E9.5 (e and f) and E10.5 (k and l) show a severe reduction of the giant cell layer in the mutant. A, heart atrium; gi, trophoblast giant cells; L, limb; la, labyrinthine trophoblast; ma, maternal decidual tissue; V, heart ventricle; sp, spongiotrophoblast. Bars = 1 mm (A), 400 μm (B, frames a, d, g, and j), and 100 μm (B, frames b, c, e, f, h, i, k, and l).

FIG. 3.

Expression of trophoblast markers in wt and in PPARβ mutant placentas. (A) Spatial expression of PPARβ mRNA in E9.5 wt placenta. Left, hematoxylin-eosin (HE) staining; middle, in situ hybridization with digoxigenin-labeled antisense riboprobe for PPARβ; right, control with sense riboprobe. Scale bar, 200 μm. (B) Representative E9.5 placenta sections from one wt (a, d, g, and j), and two PPARβ mutant KO1 (b, e, h, and k) and KO2 (c, f, i, and l) littermates. HE corresponds to standard staining with hematoxylin-eosin. Mash-2 is a marker for spongiotrophoblast and labyrinthine trophoblast cells; Tpbpa is a specific marker of spongiotrophoblast cells; PL-I specifically labels trophoblast giant cells. Scale bars, 200 μm (a to i) and 400 μm (j to l). (C) Relative mRNA expression of PPARβ, PL-I, and Id-2 in mouse placental tissues from E8.5, E9.5, E10.5, and E16.5. L27 ribosomal protein mRNA was used for normalization. Three independent samples are shown per condition. Numbers represent the relative increase (n-fold) with respect to the basal level evaluated at E8.5 time points. gi, trophoblast giant cells; la, labyrinthine trophoblast; ma, maternal decidual tissue; sp, spongiotrophoblast.

FIG. 4.

PPARβ-dependent differentiation of Rcho-1 cells in trophoblast giant cells. Rcho-1 cells were grown to 60% confluence. The change to 1% horse serum-containing medium defines the time zero of the differentiation process. (A) Expression of PL-I in Rcho-1 cells after 4 days of differentiation in the presence of dimethyl sulfoxide (DMSO) (0.05%) used as a vehicle, the PPARβ agonist L-165041 (5 μM), the PPARβ agonist and the PI3K inhibitor LY294002 (10 μM). Scale bar, 80 μm. (B) Temporal expression of PL-I and Id-2 in differentiating Rcho-1 cells cultured in the presence of L-165041 or of its vehicle dimethyl sulfoxide (DMSO), as indicated. Numbers represent relative increase (n-fold) with respect to basal level in the presence of dimethyl sulfoxide (0.05%) at time zero. (C) Effect of RSG (1 μM) on PL-I expression after 4 days of treatment. Two independent duplicates per condition are shown. (D) Silencing of PPARβ in Rcho-1 cells. The Rcho-1 cells were noninfected or transduced with a control lentivirus vector (LV-TH), or with a vector producing a PPARβ-specific siRNA (LV-THsiPPARβ). Silencing efficiency was established by measuring the mRNA levels of PPARβ after 15 days of the differentiation protocol. L27 ribosomal protein mRNA was used as an internal control. (E) Differentiation of Rcho-1 cells either noninfected, transduced with LV-TH, or transduced with LV-THsiPPARβ. In situ hybridization with PL-I probe was used as a marker of giant cell differentiation at day 4 of differentiation in the presence of L-165041 (5 μM) or its vehicle (dimethyl sulfoxide). Red arrowheads show some of the differentiated cells that express PL-I. The nuclei were stained with a methyl-green solution. (F) Bright-field images of Rcho-1 cells in culture after 15 days of differentiation. Ni, noninfected; Ci, LV-TH; Siβ, LV-THsiPPARβ/δ.

FIG. 5.

PPARβ promoting giant cell differentiation requires an intact PI3K/Akt pathway. (A) mRNA expression levels of PPARβ, PPARγ, PL-I, Id-2, Hand1 and I-mfa after 4 days of indicated treatments with L-165041 (5 μM) and/or PI3K inhibitor (LY294002) (10 μM). Two independent duplicates per condition are shown. (B) Western blot analysis of PL-I production in Rcho-1 cells treated by PPARβ ligand and/or by LY294002. Two independent duplicates per condition are shown. The number of cells seeded in each plate was accurately quantified at the beginning of the experiment, and all culture conditions were strictly comparable. (C) mRNA expression levels of PPARβ, PL-I, and I-mfa in Rcho-1 cells either noninfected (Ni), transduced with the LV-TH (Ci), or transduced with the LV-THsiPPARβ (Siβ), after 4 days of differentiation in the presence of either dimethyl sulfoxide, used as a vehicle, or 5 μM PPARβ ligand. L27 ribosomal protein mRNA was used as an internal control. (D) Relative expression of phosphorylated Akt from Rcho-I trophoblast cells treated for 3 h with either vehicle or 5 μM PPARβ ligand. Total cellular proteins from Rcho-1 cells were used for Western blot analysis. Two independent duplicates per condition are shown. (E) Relative expression of PDK1, ILK, and PTEN in Rcho-1 cells either noninfected, transduced with the LV-TH, or transduced with the LV-THsiPPARβ, after 4 days of differentiation in the presence of either dimethyl sulfoxide, used as a vehicle, or 5 μM PPARβ ligand. Tubulin was used as an internal control. The apparent molecular mass is indicated for each protein. Numbers represent relative increase (n-fold) with respect to the basal level in the presence of dimethyl sulfoxide (0.05%) (A and B), to basal level to (Ni) in the presence of dimethyl sulfoxide (0.05%) (C and E) or to the level at time zero in the presence of dimethyl sulfoxide (0.05%) (D). DMSO, dimethyl sulfoxide; Ni, noninfected; Ci, LV-TH; Siβ, LV-THsiPPARβ.

FIG. 6.

Reduced Akt activity in PPARβ^−/− embryos. (A) Expression profiles of PL-I, Hand1, I-mfa, and Id-2 at E9.5 in PPAR wt and mutants. L27 ribosomal protein mRNA was used as an internal control. Three independent samples are shown per genotype. (B) Relative expression of PDK1, Akt phosphorylated in Thr308 or in Ser473, and total Akt in PPARβ wt and mutant placenta at E9.5. Tubulin was used as an internal control. Total cellular proteins from E9.5 placentas were used for these Western blot analyses. The apparent molecular mass is indicated for each protein. Two independent samples are shown per genotype. (C) wt E9.5 placenta. Hematoxylin-eosin staining is shown in frame a. Immunohistochemistry (b and c) reveals the Thr308-phosphorylated Akt (Akt-Thr308-P) in the nuclei of giant cells (red arrowheads). The red square in frame b indicates the region seen in frame c at higher magnification. Numbers in panels A and B represent the relative increase (n-fold) with respect to basal level to wt.

FIG. 7.

Lipid storage droplets accumulate in the mouse placenta. (A) Oil red O staining of sagittal sections of mouse placenta at E9.5 (a to d) and E16.5 (e to h). Abundant small lipid droplets were observed inside the cytoplasm of trophoblast giant cells. As seen in frame d, the droplets are particularly localized near the nucleus. Black arrowheads indicate some lipid droplets. Bars: 200 μm (a and e), 40 μm (b, c, and f), 20 μm (g and h), and 8 μm (d). (B) Distribution of ADRP in mouse placenta at E9.5 (a to d) and E16.5 (g to j). Red arrowheads indicate some specific labeling corresponding to ADRP protein. (C) Expression profile of ADRP mRNA in E9.5 placentas from PPARβ wt and mutant concepti (left) and from PPARγ wt and mutant concepti (right). (D) Relative mRNA expression profiles of ADRP and PPARγ in mouse placental tissues from E8.5, E9.5, E10.5, and E16.5. L27 ribosomal protein mRNA was used for normalization. Numbers represent the relative increase (n-fold) with respect to the basal level at E8.5 (D) or to wt (C). gi, trophoblast giant cells; la, labyrinthine trophoblast; ma, maternal decidual tissue; sp, spongiotrophoblast.

FIG. 8.

ADRP expression is associated to giant cell differentiation. (A) Expression profile of ADRP in Rcho-1 cells after 4 days of treatment with L-165041 (5 μM), RSG (1 μM), or PI3K inhibitor (LY294002) (10 μM) (left and bottom panels) and in cells either noninfected (Ni), transduced with the LV-TH (Ci), or transduced with the LV-THsiPPARβ (Siβ) (right panel). Dimethyl sulfoxide (DMSO) was used as a vehicle. (B) Relative mRNA expression profiles of ADRP and aP2 in mouse placental tissues at E16.5, in white adipose tissue (WAT), and in heart. L27 ribosomal protein mRNA was used for normalization. Numbers represent the relative increase (n-fold) with respect to basal level in the presence of dimethyl sulfoxide (0.05%) (A) or to level in white adipose tissue (B).

FIG. 9.

PPARβ plays a key role in trophoblast giant cell differentiation via two converging mechanisms. Hand1 and Mash-2 are two crucial transcription factors involved in promoting and inhibiting giant cell differentiation, respectively. In addition, Id-2 acts as a dominant negative factor by dimerizing and sequestering the heterodimerization partner of bHLH transcription factors and must be down-regulated to allow trophoblast giant cell differentiation. PPARβ first acts by increasing Akt activity, which leads to Id-2 down-regulation. The bHLH transcription factor targeted by Id-2 in the trophoblast cells is not ascertained but could include Hand1 (dotted line) or the heterodimerization partner of Hand1, which in the giant cells is not yet identified (56). The role of PPARβ in directly regulating Hand1 is unclear since Hand1 expression is not modified in the Rcho-1 cell model. Secondly, PPARβ also increases the expression of I-mfa, which acts as an inhibitor of Mash-2 activity, possibly by impairing its nuclear import. Finally, the subsequent differentiation is associated with ADRP expression and lipid accumulation, which also depends on PPARβ and its activity on Akt-1.