Dysregulated expression of IDO may cause unexplained recurrent spontaneous abortion through suppression of trophoblast cell proliferation and migration

Shanshan Zong, Chunqing Li, Chengfeng Luo, Xin Zhao, Chunhong Liu, Kai Wang, Wenwen Jia, Mingliang Bai, Minghong Yin, Shihua Bao, Jie Guo, Jiuhong Kang, Tao Duan, Qian Zhou, Shanshan Zong, Chunqing Li, Chengfeng Luo, Xin Zhao, Chunhong Liu, Kai Wang, Wenwen Jia, Mingliang Bai, Minghong Yin, Shihua Bao, Jie Guo, Jiuhong Kang, Tao Duan, Qian Zhou

Abstract

In pregnancy, trophoblast proliferation, migration and invasion are important for the establishment and maintenance of a successful pregnancy. Impaired trophoblast function has been implicated in recurrent spontaneous abortion (RSA), a major complication of pregnancy, but the underlying mechanisms remain unclear. Indoleamine 2,3-dioxygenase (IDO), an enzyme that catabolizes tryptophan along the kynurenine pathway, is highly expressed in the placenta and serum during pregnancy. Here, we identified a novel function of IDO in regulating trophoblast cell proliferation and migration. We showed that IDO expression and activity were decreased in unexplained recurrent spontaneous abortion (URSA) compared to normal pregnancy. Furthermore, blocking IDO in human trophoblast cells led to reduced proliferation and migration, along with decreased STAT3 phosphorylation and MMP9 expression. Increased STAT3 phosphorylation reversed the IDO knockdown-suppressed trophoblast cell proliferation and migration. In addition, the overexpression of IDO promoted cell proliferation and migration, which could be abolished by the STAT3 signaling inhibitor (AG490). Finally, we observed similar reductions of STAT3 phosphorylation and MMP9 expression in URSA patients. These results indicate that the level of IDO expression may be associated with pregnancy-related complications, such as URSA, by affecting trophoblast cell proliferation and migration via the STAT3 signaling pathway.

Figures

Figure 1. IDO expression and activity are…
Figure 1. IDO expression and activity are decreased in villi from URSA patients compared to those from normal pregnant women (CTL).
(A) Representative images of IDO expression in villous tissue. Immunostaining verifies IDO expression in tissue sections. A Brownish color represents positive staining. Magnification: 200×. (B) HPLC analysis of IDO activity, expressed as the ratio of Kyn to Trp. (C) Western blot analysis of IDO protein using GAPDH as an internal control. (D) Statistical analysis of western blot results in C (n = 20; Student’s t-test; *p < 0.05, ***p < 0.001).
Figure 2. Inhibition of IDO with l-1mT…
Figure 2. Inhibition of IDO with l-1mT or IDO-specific shRNA suppressed proliferation of HTR-8/SVneo cells.
(A) Real-time PCR analysis of IDO mRNA in HTR-8/SVneo cells infected with IDO shRNA or control (CTL) shRNA (n = 3; Student’s t-test; *p < 0.05, **p < 0.01). (B) Western blot analysis of IDO protein in HTR-8/SVneo cells infected with IDO-specific or control shRNA. GAPDH was used as an internal control. (C) Statistical analysis of protein expression in B (n = 3; Student’s t-test; **p < 0.01). (D) Proliferation of HTR-8/SVneo cells treated with vehicle or l-1mT (250−4000 μM; n = 3; Student’s t-test; ***p < 0.001). (E) Proliferation of HTR-8/SVneo cells infected with control or IDO-specific shRNA (n = 3; Student’s t-test; **p < 0.01, ***p < 0.001).
Figure 3. Inhibition of IDO with l-1mT…
Figure 3. Inhibition of IDO with l-1mT or IDO-specific shRNA suppressed the migration of HTR-8/SVneo cells.
(A) Representative images from Transwell migration assay. Cells were treated with vehicle or l-1mT (top) or with control or IDO-specific shRNA (bottom). (B) Statistical analysis of the effect of l-1mT on HTR-8/SVneo cell migration (n = 3; Student’s t-test; **p < 0.01). (C) Statistical analysis of the effect of IDO knockdown on HTR-8/SVneo cell migration (n = 3; Student’s t-test; **p < 0.01, ***p < 0.001).
Figure 4. IDO knockdown suppressed STAT3 phosphorylation…
Figure 4. IDO knockdown suppressed STAT3 phosphorylation and MMP9 expression in HTR-8/SVneo cells.
(A,B) Western blot analysis of pSTAT3 and MMP9 in HTR-8/SVneo cells infected with control or IDO-specific shRNA. GAPDH was used as an internal control. (C,D) Statistical analysis of western blotting results (n = 3; Student’s t-test; *p < 0.05, **p < 0.01).
Figure 5. LIF partially reversed the inhibitory…
Figure 5. LIF partially reversed the inhibitory effects of IDO knockdown on trophoblast cell proliferation and migration.
(A) After treatment with LIF, pSTAT3 expression in IDO knockdown HTR-8/SVneo cells was obviously increased. (B) LIF completely reversed the inhibitory effect on cell proliferation in IDO knockdown HTR-8/SVneo cells. (C,D) LIF partially reversed the inhibitory effect on migration in IDO knockdown HTR-8/SVneo cells (n = 3; Student’s t-test; **p < 0.01, ***p < 0.001).
Figure 6. STAT3 overexpression reversed the inhibitory…
Figure 6. STAT3 overexpression reversed the inhibitory effects of IDO knockdown on trophoblast cell proliferation and migration.
(A) Western blot analysis of pSTAT3 in STAT3 overexpressing shIDO2 HTR-8/SVneo cells. Total STAT3 was used as an internal control. (B) Statistical analysis of protein expression in A (n = 3; Student’s t-test; **p < 0.01). (C) Proliferation ability of STAT3-overexpressing shIDO2 HTR-8/SVneo cells was higher than the control group (n = 3; Student’s t-test; **p < 0.01). (D,E) Migration ability of STAT3-overexpressing shIDO2 HTR-8/SVneo cells was higher than the control group (n = 3; Student’s t-test; ***p < 0.001).
Figure 7. IDO promotes cell proliferation and…
Figure 7. IDO promotes cell proliferation and migration via the STAT3 signaling pathway.
(A) Western blot analysis of IDO in IDO-overexpressing HTR-8/SVneo cells. GAPDH was used as an internal control. (B) Statistical analysis of protein expression in A (n = 3; Student’s t-test; **p < 0.01). (C) Proliferation ability of IDO-overexpressing HTR-8/SVneo cells treated with vehicle or AG490 (n = 3; Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001 in comparison to the control group treated with vehicle, ##p < 0.01, ###p < 0.001 in comparison to the IDO-overexpressing group treated with vehicle) (D,E) Migration ability of IDO-overexpressing HTR-8/SVneo cells treated with vehicle or AG490 (n = 3; Student’s t-test; **p < 0.01, ***p < 0.001 in comparison to the control group treated with vehicle, ###p < 0.001 in comparison to the IDO-overexpressing group treated with vehicle).
Figure 8. pSTAT3 and MMP9 expression in…
Figure 8. pSTAT3 and MMP9 expression in IDO-overexpressing HTR-8/SVneo cells.
(A) Western blot analysis of pSTAT3 in IDO-overexpressing HTR-8/SVneo cells treated with vehicle or AG490. Total STAT3 was used as an internal control. (B) Statistical analysis of protein expression in A (n = 3; Student’s t-test; **p < 0.01 in comparison to the control group treated with vehicle, ##p < 0.01 in comparison to the IDO-overexpressing group treated with vehicle). (C) Western blot analysis of MMP9 in IDO-overexpressing HTR-8/SVneo cells treated with vehicle or AG490. GAPDH was used as an internal control (D) Statistical analysis of protein expression in A (n = 3; Student’s t-test; *p < 0.05 in comparison to the control group treated with vehicle, ##p < 0.01 in comparison to the IDO-overexpressing group treated with vehicle).
Figure 9. STAT3 phosphorylation and MMP9 expression…
Figure 9. STAT3 phosphorylation and MMP9 expression were decreased in villi from URSA patients compared to those of normal pregnant women.
(A,B) Western blot analysis of pSTAT3 and MMP9 expression in villi from normal pregnant women and URSA patients. GAPDH was used as an internal control. (C,D) Statistical analysis of western blotting results A and B (n = 20; Student’s t-test; **p < 0.01, ***p < 0.001).

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