Functional antagonism between high temperature requirement protein A (HtrA) family members regulates trophoblast invasion

Abstract

Human trophoblast invasion of decidualized endometrium is essential for placentation and is tightly regulated and involves trophoblast-decidual cell interaction. High temperature requirement A4 (HtrA4) is a secreted serine protease highly expressed in the invasive extravillous trophoblasts that invade decidua. In contrast, both HtrA1 and HtrA3 have been shown to inhibit trophoblast invasion. Here we provide evidence that decidua-secreted HtrA1 and HtrA3 antagonize HtrA4-mediated trophoblast invasion. We demonstrated that HtrA1 and HtrA3 interact with and degrade HtrA4 and thereby inhibit trophoblast-like JAR cell invasion. Specifically, HtrA1 and HtrA3 expression is up-regulated under decidualization conditions in endometrial stromal and epithelial cells, T-HESCs and Ishikawa cells, respectively. Conditioned media from these two cell lines after decidualization treatment suppress HtrA4-expressing JAR cell invasion in an HtrA1- or HtrA3-dependent manner. Co-culture of the HtrA4-expressing JAR cells with decidualization stimuli-treated T-HESC or Ishikawa monolayer also impairs JAR cell invasion, which can be reversed by HtrA1 or HtrA3 knockdown, supporting that HtrA1 and HtrA3 are crucial for trophoblast-decidual cell interaction in the control of trophoblast invasion. Our study reveals a novel regulatory mechanism of trophoblast invasion through physical and functional interaction between HtrA family members.

Keywords: Cell Invasion; Gene Expression; Placenta; Pregnancy; Protease; Trophoblast.

FIGURE 1.

Expression of HtrA proteins in human placenta and decidua. A, characterization of HtrA antibodies. The whole cell lysates of 293T cells transiently expressing HtrA1-FLAG, HtrA3-FLAG, and HtrA4-FLAG were subjected to immunoblotting (IB) with FLAG antibody (Ab), guinea pig (GP) anti-HtrA1 Ab, rabbit anti-HtrA3 Ab, and mouse anti-HtrA4 Ab, respectively. B, immunohistochemistry of HtrA proteins. Term human placental sections were co-stained with Abs to HtrA1 and CK7 (a–c) or HtrA3 and CK7 (d–f). Because both HtrA4 and CK7 Abs are derived from mice, consecutive sections were stained with HtrA4 (g and h) and CK7 (i and j) Abs, respectively. Sections were then stained with secondary Abs labeled with Alexa Fluor 568 (for HtrA3), RRX (for HtrA1), and Cy2 (for HtrA4 and CK7). Nuclei were stained by DAPI. The green fluorescent signals of HtrA4 were converted into a red color for better contrast. In a separate experiment, a consecutive section was stained with hematoxylin and eosin (H&E). D, decidua; V, villi; bar, 100 μm.

FIGURE 2.

Physical and functional interaction between HtrA4, HtrA1, and HtrA3. A, HtrA4 interacts with HtrA1 and HtrA3. 293T cells were transfected with the indicated combinations of expression plasmids for mutant HtrA-FLAG proteins for 48 h. The culture supernatants were collected for co-immunoprecipitation (Co-IP) with FLAG and HA Abs. As an input control, a portion of the supernatants was also analyzed by immunoblotting with FLAG and HA Abs. B, HtrA1 and HtrA3 decrease HtrA4 protein level. 293T cells were transfected with the indicated combinations of expression plasmids for wild-type and mutant HtrA-FLAG proteins for 48 h. The culture supernatants were collected for immunoblotting (IB) with FLAG and HA Abs.

FIGURE 3.

Regulation of HtrA4 proteolysis by HtrA1 and HtrA3. A, proteolysis of HtrA4 by HtrA1 and HtrA3. Recombinant HtrA4MT-FLAG was incubated with recombinant wild-type or mutant HtrA1-FLAG and HtrA-3-FLAG for the indicated periods of time followed by immunoblotting (IB) with HtrA4 Ab. B, HtrA1 and HtrA3 are less susceptible to proteolysis by HtrA4. Recombinant HtrA1MT-FLAG or HtrA3MT-FLAG was incubated with recombinant HtrA4-FLAG for the indicated periods of time followed by immunoblotting with HtrA1, HtrA3, and HtrA4 Abs. C, HtrA1 and HtrA3 Kazal domains suppress HtrA4 autocatalytic cleavage. Recombinant HtrA4-FLAG was incubated with GST, GST-Kazal1, GST-Kazal3, or GST-Kazal4 for 6 h and then subjected to immunoblotting with FLAG and GST Abs. The arrows and arrowheads in A–C indicate the positions of unprocessed and proteolytically processed forms of HtrA protein, respectively.

FIGURE 4.

Regulation of HtrA4-mediated cell invasion by HtrA1 and HtrA3. A, stable expression of HtrA4-FLAG in JAR cells. Culture supernatants and whole cell lysates (WCL) were harvested from JAR cells stably expressing the empty pCDH vector or HtrA4-FLAG and subjected to immunoblotting with the indicated Abs. On the other hand, the culture supernatants of 293T cells transiently expressing wild-type or mutant HtrA1-FLAG and HtrA3-FLAG were collected for immunoblotting (IB) with FLAG Ab. B and C, the mock and HtrA4-FLAG-expressing JAR cells were plated in Matrigel-coated chambers and incubated with the conditioned medium (CM) collected from 293T cells expressing HtrA1-FLAG or HtrA3-FLAG mentioned in A. After 24 h, invasive JAR cells in the lower surface of the filters were fixed, stained, and counted. Representative images at 100× magnification from one of three independent experiments are shown. The mean and S.D. from three independent experiments are presented. *, p < 0.05; **, p < 0.01; ns, not significant.

FIGURE 5.

Regulation of HtrA4-mediated cell invasion by decidual HtrA1 and HtrA3. A, establishment of tetracycline-inducible (Tet-On) HtrA4-FLAG-expressing JAR cells (HtrA4-FLAG-aON cells). The HtrA4-FLAG-aON cells were treated with or without Dox and then subjected to immunoblotting (IB) with HtrA4, FLAG, and β-actin Abs. In a separate experiment, the mock- and Dox-induced HtrA4-FLAG-aON cells were plated into Matrigel-coated chambers for cell invasion analysis. B, expression of HtrA3 is induced in Ishikawa endometrial cells under decidualization conditions. The scramble control or HtrA3-knockdown Ishikawa cells were treated with 17-β-estradiol (E2), 6α-methyl-17α-hydroxyl-progesterone acetate (MPA) and cAMP analog for 5 days. The effects of decidualization treatment were assessed by morphology and immunoblotting with IGFBP-1 Ab and ELISA analysis of prolactin and IL-11. In addition, the whole cell lysate and culture supernatant of the mock- and decidualization stimuli-treated scramble (Scram.) control and HtrA3-knockdown Ishikawa cells were analyzed by immunoblotting with HtrA3 and HtrA4 Abs. Note that the decidualization treatment also increases HtrA3 expression in Ishikawa cells. C, HtrA3 suppresses HtrA4-mediated trophoblast invasion. The mock- and Dox-induced HtrA4-FLAG-aON cells were plated into Matrigel-coated chambers and incubated with the conditioned medium collected from the mock- and decidualization stimuli-treated scramble control and HtrA3-knockdown Ishikawa cells for 24 h for cell invasion analysis. ***, p < 0.001; ns, not significant. D and E, HtrA1 suppresses HtrA4-mediated trophoblast invasion. The scramble control or HtrA1-knockdown T-HESCs were treated with 17-β-estradiol, 6α-methyl-17α-hydroxyl-progesterone acetate, and cAMP analog for 5 days. Note that HtrA1 gene expression is up-regulated in T-HESCs under decidualization conditions. The mock- and Dox-induced HtrA4-FLAG-aON cells were plated into Matrigel-coated chambers and incubated with the conditioned medium collected from the mock- and decidualization stimuli-treated scramble control and HtrA1-knockdown T-HESCs for 24 h for cell invasion analysis. The mean and S.D. from three independent experiments are presented in B–E. *, p < 0.05; ***, p < 0.001.

FIGURE 6.

Regulation of trophoblast invasion by trophoblast-decidual cell interaction. A and B, regulation of JAR cell invasion by Ishikawa cells. The mock- and Dox-induced HtrA4-FLAG-aON cells were pretreated with 10 μg/ml DilC₁₂(3) fluorescent dye and then plated onto mock- and decidualization stimuli-treated scramble control or HtrA3-knockdown Ishikawa monolayers for 24 h. Invasive cells in the lower surface of the filters were fixed and visualized by fluorescence microscopy analysis. Four microscopic fields per sample were randomly selected for quantification in each of three independent experiments. Representative images at 100× magnification from one of three independent experiments are shown. Insets are images at 200× magnification. ***, p < 0.001; ns, not significant. C, regulation of JAR cell invasion by T-HESCs. A similar experiment to A was performed in mock- and decidualization stimuli-treated scramble control or HtrA1-knockdown T-HESC monolayers. The mean and S.D. from three independent experiments are presented in B and C. *, p < 0.05; ***, p < 0.001.