Human transcriptional coactivator with PDZ-binding motif (TAZ) is downregulated during decidualization

Abstract

Transcriptional coactivator with PDZ-binding motif (TAZ) is known to bind to a variety of transcription factors to control cell differentiation and organ development. However, its role in uterine physiology has not yet been described. To study its regulation during the unique process of differentiation of fibroblasts into decidual cells (decidualization), we utilized the human uterine fibroblast (HuF) in vitro cell model. Immunocytochemistry data demonstrated that the majority of the TAZ protein is localized in the nucleus. Treatment of HuF cells with the embryonic stimulus cytokine interleukin 1 beta in the presence of steroid hormones (estradiol-17 beta and medroxyprogesterone acetate) for 13 days did not cause any apparent TAZ mRNA changes but resulted in a significant TAZ protein decline (approximately 62%) in total cell lysates. Analysis of cytosolic and nuclear extracts revealed that the decline of total TAZ was caused primarily by a drop of TAZ protein levels in the nucleus. TAZ was localized on the peroxisome proliferator-activated receptor response element site (located at position -1200 bp relative to the transcription start site) of the genomic region of decidualization marker insulin-like growth factor-binding protein 1 (IGFBP1) in HuF cells as detected by chromatin immunoprecipitation. TAZ is also present in human endometrium tissue as confirmed by immunohistochemistry. During the secretory phase of the menstrual cycle, specific TAZ staining particularly diminishes in the stroma, suggesting its participation during the decidualization process, as well as implantation. During early baboon pregnancy, TAZ protein expression remains minimal in the endometrium close to the implantation site. In summary, the presented evidence shows for the first time to date TAZ protein in the human uterine tract, its downregulation during in vitro decidualization, and its localization on the IGFBP1 promoter region, all of which indicate its presence in the uterine differentiation program during pregnancy.

FIG. 1.

TAZ detection in HuF cells and its downregulation during in vitro decidualization. A) Confocal microscopy of immunofluorescent staining of TAZ in HuF cells (a). Nuclear staining is visualized with DAPI (b). Merge image (c). Bar = 20 μm. Nonspecific antibody control staining is in upper corner insets. B) Distribution of TAZ in total cell lysates (35 μg of protein) of HuF following decidualization for 6 days and 13 days induced by SHs (E+P), IL1B and SHs (IL-1+E+P), cAMP and SHs (cAMP+E+P), and untreated controls (control) as detected by Western blot with TAZ antibody. Membrane was reprobed with beta-actin (ACTB) antibody (blot below). Densitometric evaluation of TAZ:ACTB ratios is illustrated in graph below blots. The mean value from three experiments for the control at Day 6 was set as 100%. Note the significant difference between control and IL1B- and SH-treated cells on Day 13 (*P ≤ 0.05). C) TAZ mRNA expression during decidualization. The PCR using primers specific for TAZ, IGFBP1, PRL (prolactin), and H3F3A (histone H3.3) was performed on RNA isolated from HuF cells treated for 13 days as indicated in the figure (left). The qPCR for relative TAZ mRNA levels confirmed no change during decidualization (upper graph), although IGFBP1 mRNA was significantly different between the control and other treatments (^#P ≤ 0.05, bottom graph). M.w., molecular weight; E+P, estradiol-17β and medroxyprogesterone acetate.

FIG. 2.

TAZ decline in nucleus during IL1B-induced decidualization. A) Distribution of TAZ in the cytosolic (c) and nuclear (n) fractions of HuF cells treated for 13 days with SHs (H), IL1B and SHs (IL1B+H), cAMP and SHs (cAMP+H), and untreated controls (Ctr) as detected by Western blot with TAZ antibody. Membrane was reprobed with MYC (c-myc) and beta-actin (ACTB) antibodies (blots below). B) Quantification of TAZ distribution in cytosol and nuclear extract fractions of HuF cells after 13 days of decidualization treatments expressed as the cytosol:nuclear extract ratio (labeled as cyt.:nucl. extr.) calculated from densitometric evaluation of TAZ bands from three experiments. The mean ratio in control cells (Ctr) from three experiments was set as 1, and other treatments were compared with the control (mean ± SD). Note the significant increase (*P < 0.05) in the cytosol:nucleus ratio for TAZ in HuF cells treated with IL1B and SHs compared with the control.

FIG. 3.

Association of TAZ and PPARG with the PPRE-containing site of the human IGFBP1 in vivo. A) PPARG mRNA in HuF cells detected by RT-PCR in total RNA. B) PPARG protein detected in the nuclear (n) fraction of HuF cells by Western blot with specific antibody. PPARG was not detected in the cytosolic extract (c). C) Chromatin was extracted from HuF cells. ChIP experiments were performed with anti-TAZ and anti-PPARG antibodies. An association of TAZ and PPARG with the PPRE-containing genomic region (−1197 to −1002 bp from the TSS) of the human IGFBP1 gene was detected. The control region of the IGFBP1 gene (−8869 to −8377 bp from the TSS), which contains no PPRE, did not show this association. The PCR on the input chromatin template (Input, 1:3100) served as a positive control and that of nonspecific rabbit IgG-precipitated template as a specificity control. Ab, antibody; negative c., negative control.

FIG. 4.

Localization of TAZ in human and baboon endometrium and at the implantation site in the pregnant baboon. Immunohistochemical detection of TAZ in the human endometrium in proliferative (a) and secretory (b) phases of menstrual cycle, baboon endometrium in proliferative (c) and secretory (d) phases of menstrual cycle, and pregnant baboon endometrium (Day 30 of pregnancy) at the implantation site (e and f). Insets show staining with nonspecific control rabbit IgG. Specific TAZ staining is brown. Blue nuclear costaining is with hematoxylin. Note the decline in TAZ staining in the stroma during the secretory phase (b and d) compared with the proliferative phase (a and c). Endometrium at the implantation region shows minimal TAZ staining (e and f). All images are taken at same magnification. Bar = 100 μm.