PiggyBac transposase-mediated inducible trophoblast-specific knockdown of Mtor decreases placental nutrient transport and fetal growth

Abstract

Abnormal fetal growth is associated with perinatal complications and adult disease. The placental mechanistic target of rapamycin (mTOR) signaling activity is positively correlated with placental nutrient transport and fetal growth. However, if this association represents a mechanistic link, it remains unknown. We hypothesized that trophoblast-specific Mtor knockdown in late pregnant mice decreases trophoblast nutrient transport and inhibits fetal growth. PiggyBac transposase-enhanced pronuclear injection was performed to generate transgenic mice containing a trophoblast-specific Cyp19I.1 promoter-driven, doxycycline-inducible luciferase reporter transgene with a Mtor shRNAmir sequence in its 3' untranslated region (UTR). We induced Mtor knockdown by administration of doxycycline starting at E14.5. Dams were killed at E 17.5, and trophoblastspecific gene targeting was confirmed. Placental mTOR protein expression was reduced in these animals, which was associated with a marked inhibition of mTORC1 and mTORC2 signaling activity. Moreover, we observed a decreased expression of System A amino acid transporter isoform SNAT2 and the System L amino acid transporter isoform LAT1 in isolated trophoblast plasma membranes and lower fetal, placental weight, and fetal:placental weight ratio. We also silence the MTOR in cultured primary human trophoblast cells, which inhibited the mTORC1 and C2 signaling, System A and System L amino acid transport activity, and markedly decreased the trafficking of LAT1 and SNAT2 to the plasma membrane. Inhibition of trophoblast mTOR signaling in late pregnancy is mechanistically linked to decreased placental nutrient transport and reduced fetal growth. Modulating trophoblast mTOR signaling may represent a novel intervention in pregnancies with abnormal fetal growth.

Keywords: FGR; fetal development; maternal–fetal exchangeMaternal-Fetal Exchange; mechanistic target of rapamycin; placenta.

Figure 1. Transgene construct and experimental design for developing a trophoblast-specific inducible Mtor knockdown mouse.

(A) A transgene construct is depicted, consisting of TRE3G & rtTA3, Tet-On system; GL3, luciferase reporter gene; shRNAmir, Mtor knockdown shRNAmir; CYP19I.1, Aromatase P450 fragment as the promoter for trophoblast-specific gene expression; CAG, CMV early enhancer/chicken beta-actin promoter; pB, piggyBac transposase and TRE, terminal repeat elements. (B) A schematic of the experimental design for developing a trophoblast-specific inducible Mtor knockdown mouse. Transposase-enhanced pronuclear injection was performed using B6D2F1 (B57BL/6 x DBA/2) and CD1 mice. Genomic DNA was isolated from pups, and genotyping PCR was performed to determine the number of transgenes for the founder generation and F1 (crossed with WT B57BL/6)

Figure 2. Trophoblast-specific inducible Mtor knockdown in mice.

Mtor knockdown was induced by the administration of doxycycline at E14.5 in pregnant mice transgenic for a construct including a Tet-On system, luciferase, Mtor shRNA mir, and CYP19I.1. The vehicle was administered in the control transgenic animals (No Doxycycline). Laparotomy was performed at E17.5, and the luciferase signal was detected in isolated placentas and fetuses using an in vivo imaging system (IVIS). The luciferase signal could be detected only in the (A) placentas of dams that received doxycycline with no visible signal in the embryo proper, (B) maternal tissues, or (C-D) in transgenic animals in which Mtor knockdown had not been induced by doxycycline

Figure 3. Doxycycline treatment did not affect fetal and placental weight and placental caspase/mTOR signaling in mice.

(A-D) Doxycycline (2.5 mg/kg (IP)) was administered at E14.5 in pregnant wild type (WT) mice. The vehicle (PBS) was administered to pregnant WT mice (No Doxycycline, Control). At E18.5, animals were sacrificed, and fetal and placentas from each litter were weighed individually. Fetal weight (A), placental weight (B), fetal: placental weight ratio (C) and litter size (D) in control and doxycycline administered conceptuses (PBS group: n = 7 dams, 49 conceptuses; Doxycycline group: n = 11 dams, 78 conceptuses). (E-J) Doxycycline treatment did not affect placental PARP (Poly (ADP-ribose) polymerase), caspase 3/cleaved caspase 3, mTORC1 and mTORC2 signaling in mice. Placentas from each litter were pooled and homogenized. Western blot determined the (E,F) PARP (analyzing 89 and 116 kDa bands together)/caspase 3/cleaved caspase 3, total expression and phosphorylation of key intermediates in the (G,H) mTORC1 (S6^Ser-235/236, total S6, 4E-BP1 ^Thr-70, and 4E-BP1) and (I,J) mTORC2 (Akt^ser-473 and total Akt) signaling pathways. (E,G,I) Representative western blots of PARP/caspase 3/cleaved caspase 3, S6^Ser-235/236, total S6, 4E-BP1 ^Thr-70, 4E-BP1, Akt^ser-473 and total akt expression in placental homogenates of vehicle (PBS) and doxycycline administered mice. Equal loading was performed. (F,H,I) Summary of the western blot data. n = 7–10 dams in each group. Values are expressed as means ± SEM

Figure 4. Trophoblast-specific inducible Mtor knockdown in mice decreases placental mTOR protein expression and fetal weight.

Trophoblast-specific Mtor knockdown was induced by the administration of doxycycline starting at E14.5 (Mtor ^kD). The vehicle was administered at E14.5 in control animals, resulting in mice without Mtor knockdown (Mtor). At E17.5, animals were sacrificed, and placentas from each litter were collected, pooled and homogenized. Western blot was used to determine the total mTOR protein expression. (A) Representative western blot of total mTOR expression in placental homogenates of WT, Mtor and Mtor ^kD placentas. Equal loading was performed. (B) Summary of the western blot data. n = 6–7 dams in each group. (C-G) Fetal and placental weight, fetal: placental (F:P) weight ratio, litter size and fetal weight distribution curves following induction of trophoblast-specific Mtor knockdown in mice. at E17.5, animals were sacrificed, and placentas from each litter were weighed. Compared with control and wildtype mice, trophoblast-specific Mtor knockdown (induced by doxycycline at E14.5) mice exhibit reduced (C) fetal weights and (D) placental weights at embryonic day 17.5. Trophoblast-specific Mtor knockdown in mice reduced (E) fetal:placental weight ratio but not (F) litter size. (C-F) Symbols show the individual fetal/ placental weight, and F:P ratio in WT, Mtor, and Mtor ^kD dams. Values are expressed as means ± SEM. (B,C,E) Means without a common letter are statistically different by one-way ANOVA with Tukey–Kramer multiple comparisons post hoc test (P<0.05). Each value represents individual fetal weight, placental weight, and F:P ratio in WT (n = 46 fetuses from 7 dams), Mtor (n = 48 fetuses from 7 dams), and Mtor ^kD (n = 40 fetuses from 6 dams) dams. (D) Means without a common letter are statistically different by Kruskal–Wallis test with Dunn’s multiple comparisons test (P<0.05). (F) Frequency distribution of individual fetal weights in WT (n = 46), Mtor (n = 48), and Mtor ^kD (n = 40) dams. Mean fetal weight of trophoblast-specific Mtor knockdown mice (solid line, r ² = 0.67; n = 40 fetuses, 6 dams) was significantly lower than in wildtype (WT, dotted line, r ² = 0.99; n = 46 fetuses, 7 dams) and control type (Mtor, dashed line, r ² = 0.84; N = 48 fetuses, 7 dams) mice. The vertical dashed line represents the 5th centile on the WT curve (0.647 gm), revealing 77.5% of Mtor ^kD fetuses fall below this. Values are expressed as means ± SEM

Figure 5. Placental mTORC1 and mTORC2 signaling expression following induction of trophoblast-specific Mtor knockdown in mice.

Trophoblast-specific Mtor knockdown was induced by the administration of doxycycline starting at E14.5 (Mtor ^kD). The vehicle was administered at E14.5 in control animals, resulting in mice without Mtor knockdown (Mtor). At E17.5, animals were sacrificed, and placentas from each litter were weighed, pooled and homogenized. (A-B) Inhibition of placental mTORC1 signaling following induction of trophoblast-specific Mtor knockdown. Western blot determined the total expression and phosphorylation of key intermediates in the mTORC1 signaling pathways. (A) Representative western blots of S6^{Serine-235/236}, and total S6 expression in placental homogenates of WT, Mtor and Mtor ^kD placentas. (C-D) Inhibition of placental mTORC2 signaling following induction of trophoblast-specific Mtor knockdown. (C) Representative western blots of Akt ^Serine-473, and Akt expression in placental homogenates of WT, Mtor, and Mtor ^kD placentas. Equal loading was performed. (B,D) Summary of the western blot data. n = 6–7 dams in each group. Values are expressed as means ± SEM. (B) Means without a common letter are statistically different by Kruskal-Wallis test with Dunn’s multiple comparisons test (P<0.05). (D) means without a common letter are statistically different by one-way ANOVA with Tukey–Kramer multiple comparisons post hoc test (P<0.05).

Figure 6. Placental trophoblast plasma membrane SNAT2 and LAT1 expression following induction of trophoblast-specific Mtor knockdown in mice.

Trophoblast-specific Mtor knockdown was induced by the administration of doxycycline starting at E14.5 (Mtor ^kD). The vehicle was administered at E14.5 in control animals, resulting in mice without Mtor knockdown (Mtor). At E17.5, animals were sacrificed, and placentas from each litter were weighed, pooled and homogenized. (A-B) Decreased trophoblast plasma membrane SNAT2 and LAT1 expression following induction of trophoblast-specific Mtor knockdown. trophoblast plasma membranes were isolated, and the protein expression of the amino acid transporter isoforms SNAT2 (System A) and LAT1 (System L) was determined using Western blot. (A,C) Representative western blots of SNAT2 and LAT1 expression in TPM of WT, Mtor, and Mtor ^kD placentas. Equal loading was performed. (B,D) Summary of the Western blot data. n = 6–7 dams in each group. Values are expressed as means ± SEM. means without a common letter are statistically different by one-way ANOVA with Tukey–Kramer multiple comparisons post hoc test (P<0.05). .

Figure 7. Decreased trophoblast plasma membrane System L and System A activities following induction of trophoblast-specific Mtor knockdown.

Mtor knockdown was induced by the administration of doxycycline starting at E14.5 (Mtor ^kD). In control transgenic animals (Mtor), the vehicle was administered at E14.5. At E17.5, animals were sacrificed, and placentas from each litter were pooled and homogenized. Trophoblast plasma membranes were isolated. System L (A) and System A (B) transporter activities were determined using isotope-labeled substrates and rapid filtration techniques in TPM isolated from WT, Mtor, and Mtor ^kD placenta at E 17.5. Values are expressed as means ± SEM. means without a common letter are statistically different by one-way ANOVA with Tukey–Kramer multiple comparisons post hoc test (P<0.05). .

Figure 8. The present study’s findings are consistent with the model that trophoblast-specific knockdown of Mtor mRNA in mice is mechanistically linked to decreased trophoblast mTORC1 and mTORC2 signaling.

Inhibition of placental mTORC1 and mTORC2 signaling decreased the transporter trafficking of System L amino acid transport isoform LAT1 and System A amino acid transport isoform SNAT2, which contributes to decreased fetal amino acid supply and fetal growth restriction.