Parathyroid hormone-like hormone (PTHLH) represses decidualization of human uterine fibroblast cells by an autocrine/paracrine mechanism

Abstract

Context: Parathyroid hormone-like hormone (PTHLH) is abundantly expressed by human endometrial stromal cells during decidualization. However, the role for PTHLH in the decidualization process is unknown.

Objective: To examine the effects of PTHLH on the induction and maintenance of decidualization of human uterine fibroblast (HUF) cells in vitro.

Design: HUF cells were treated with a PTHLH siRNA or a PTHLH receptor antagonist (bPTH(7-34)) before or after decidualization with medroxyprogesterone acetate (MPA), estradiol (E(2)), and prostaglandin E(2) (PGE(2)). Decidualization was monitored by immunocytochemistry and the induction of decidualization-specific marker genes, including IGFBP-1, prolactin, lefty, and transcription factor FOXO1.

Results: HUF cells decidualized after pretreatment with a PTHLH siRNA showed greater morphologic changes of decidualization, greater IGFBP-1 protein, and two- to threefold more IGFBP-1, prolactin, lefty, and FOXO1 mRNAs than cells pretreated with a nonsilencing RNA. The PTHLH siRNA pretreated cells also had 31% less DNA fragmentation (TUNEL assay) and 30-35% less caspase 3 levels during decidualization than cells pretreated treated with nonsilencing RNA. Treatment of HUF cells with PTHLH siRNA or bPTH(7-34) at 9 d after the induction of decidualization also resulted in 2.1- to 3.2-fold greater IGFBP-1, prolactin, lefty, and FOXO1 mRNA levels than that noted in control cells treated with nonsilencing RNA.

Conclusions: These finding strongly suggest that PTHLH represses the induction of human decidualization, stimulates stromal cell apoptosis, and limits the extent of uterine stromal cell differentiation. Because PTHLH and its receptor are expressed by HUF cells and placental cells, the inhibitory effect of PTHLH on decidualization appears to be due, at least in part, to an autocrine/paracrine mechanism.

Fig. 1.

PTHLH mRNA levels during the decidualization of HUF cells. HUF cells were decidualized in vitro in response to MPA, E₂, and PGE₂ as described in Materials and Methods. Each point represents the mean of triplicate wells, and the bars enclose ± 1 sem. Similar patterns of PTHLH mRNA expression were observed during the decidualization of two other preparations of HUF cells from different pregnancies.

Fig. 2.

Silencing of PTHLH expression enhances the induction of decidualization. HUF cells were pretreated for 3 d with either a PTHLH siRNA (right) or a control non-silencing RNA (left) for 3 d and then exposed for 3 d to MPA, E₂, and PGE₂. At the end of this period, the cells were fixed and immunostained with a specific polyclonal antiserum to human IGFBP-1 and counterstained with hemotoxylin. No staining was observed in control slides in which the HUF cells were incubated with a nonimmune rabbit serum in place of the specific antiserum.

Fig. 3.

Silencing of PTHLH expression by a PTHLH siRNA enhances the induction of decidualization marker genes. HUF cells were exposed for 3 d to a specific PTHLH siRNA or a nonsilencing RNA. The medium in both groups of cells was then exposed to MPA, E₂, and PGE₂ for 3 d as in Fig. 2. The relative amounts of PTHLH, IGFBP-1, prolactin, and lefty mRNA levels, normalized to the amount of GAPDH mRNA in the same RNA sample, were determined by real-time PCR. Each bar represents the mean of triplicate wells, and the bracket above each bar represents + 1 sem. *, P < 0.05; **, P < 0.01; ****, P < 0.005; ****, P < 0.0001.

Fig. 4.

Silencing of PTHLH expression in decidualized HUF cells by a PTHLH siRNA enhances the expression of decidualization-specific genes. HUF cells that had been decidualized with MPA, E₂, and PGE₂ (decidualizing medium) for 9 d were exposed to a PTHLH siRNA or a nonsilencing RNA and incubated for an additional 4 d in decidualizing medium. RNA was extracted from the cells at the end of this 4-d interval, and the relative amounts of PTHLH, IGFBP-1, prolactin, and lefty mRNA levels, normalized to the amount of GAPDH mRNA in the same RNA sample, were determined by real-time PCR. Each bar represents the mean of triplicate wells, and the bracket above each bar encloses + 1 sem. *, P < 0.05; **, P < 0.01; ****, P < 0.005.

Fig. 5.

Blocking PTHLH expression in decidualized HUF cells by the PTHLH antagonist bPTH_7-34 enhances the expression of decidualization-specific genes. HUF cells that had been decidualized with MPA, E₂, and PGE₂ (decidualizing medium) for 9 d were exposed for an additional 4 d to bPTH_7-34 in decidualizing medium or decidualizing medium alone. RNA was extracted from the cells at the end of the 4-d interval, and the relative amounts of PTHLH, IGFBP-1, prolactin, and lefty mRNA levels, normalized to the amount of GAPDH mRNA in the same RNA sample, were determined by real-time PCR. Each bar represents the mean of triplicate wells, and the bracket above each bar encloses + 1 sem. *, P < 0.05; **, P < 0.01; ****, P < 0.005.

Fig. 6.

Silencing of PTHLH expression in decidualized HUF cells by a PTHLH siRNA blocks caspase 3 expression. HUF cells were cultured as described in Fig. 3. Whole cell extracts were prepared 4-d after the cells had been transfected with the PTHLH siRNA or the nonsilencing RNA. Western blot analyses of the extracts for caspase 3 were performed as described in Materials and Methods. Relative signal sum density for each caspase 3 protein band was measured by autoradiography on Hyperfilm using Kodak Digital Science ID Image Analysis software. Each bar represents the density of triplicate wells, and the bracket encloses + 1 sem. **, P < 0.01.