No abstract

Smad1, Smad5 and Smad9 (also known as Smad8) are activated by phosphorylation by bone morphogenetic protein (BMP)-bound type I receptor kinases. We examined the role of Smad1, Smad5, and Smad9 by creating constitutively active forms (SmadDVD). Transcriptional activity of Smad9DVD was lower than that of Smad1DVD or Smad5DVD, even though all three SmadDVDs associated with Smad4 and bound to the target DNA. The linker region of Smad9 was sufficient to reduce transcriptional activity. Smad9 expression was increased by the activation of BMP signaling, similar to that of inhibitory Smads (I-Smads), and Smad9 reduced BMP activity. In contrast to I-Smads, however, Smad9 did not inhibit the type I receptor kinase and suppressed the constitutively active Smad1DVD. Smad9 formed complexes with Smad1 and bound to DNA but suppressed the transcription of the target gene. Taken together, our findings suggest that Smad9 is a new type of transcriptional regulator in BMP signaling.

Leukemia inhibitory factor (LIF), a cytokine that induces macrophage differentiation in the murine M1 myeloid leukemia cell line, is essential for blastocyst implantation in mice. However, its expression and the role it plays in the human uterus are unknown. To clarify these issues, we examined LIF gene expression in the human uterus by Northern blot hybridization and by a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. Analysis of LIF mRNA showed two hybridization bands, with estimated mRNA sizes of about 4.0-kb pairs and 1.8-kb pairs. LIF mRNA was detected at high levels in endometrial tissue and decidua, but at low levels in the chorionic villus in first trimester and term placenta. In the secretory phase, the endometrial tissue showed higher LIF expression than in the proliferative phase (9.5-fold; p < 0.01). The endometrial tissues were separated into a stroma-enriched fraction (SF) and an epithelium-enriched fraction (EF), and the LIF mRNA levels in each fraction were examined by quantitative RT-PCR. These levels were higher in the EF than in the SF (3.3-fold; p < 0.05). These findings suggest that, in humans, LIF plays a role in uterine function during the menstrual cycle, as well as during pregnancy.

Bacterial Hfq is a protein that plays an important role in the regulation of genes in cooperation with sRNAs. Escherichia coli Hfq (EcHfq) has two or more sites that bind RNA(s) including U-rich and/or the poly(A) tail of mRNA. However, functional and structural information about Bacillus subtilis Hfq (BsHfq) including the RNA sequences that specifically bind to it remain unknown. Here, we describe RNA aptamers including fragment (AG)3A that are recognized by BsHfq and crystal structures of the BsHfq–(AG)3A complex at 2.2 Å resolution. Mutational and structural studies revealed that the RNA fragment binds to the distal site, one of the two binding sites on Hfq, and identified amino acid residues that are critical for sequence-specific interactions between BsHfq and (AG)3A. In particular, R32 appears to interact with G bases in (AG)3A. Poly(A) also binds to the distal site of EcHfq, but the overall RNA structure and protein–RNA interaction patterns engaged in the R32 residues of BsHfq–(AG)3A differ from those of EcHfq–poly(A). These findings provide novel insight into how the Hfq homologue recognizes RNA.

Endometrial stromal differentiation (decidualization) is essential for implantation of the developing blastocyst. To investigate the process of progesterone (P)-induced decidualization of human endometrial stromal cells (ESC), a complementary DNA library enriched with P-induced genes was constructed from cultured human ESC by subtractive hybridization and the polymerase chain reaction. One of the isolated clones was the complementary DNA for the tissue inhibitor of metalloproteinase-3 (TIMP-3), a recently identified member of the human TIMP family. When human ESC were cultured in the presence of P for 6 days, the induction of TIMP-3 messenger RNA (mRNA) expression was observed by Northern blotting. In contrast, the marked induction of PRL mRNA expression and morphological changes were observed after 9 days of culture. P-induced TIMP-3 mRNA expression was dose dependent, and this induction was inhibited by the antiprogestin RU486. Estrogen did not induce TIMP-3 mRNA expression under similar conditions. In situ hybridization analysis of endometria from nonpregnant women revealed that the TIMP-3 mRNA expression was restricted to predecidualized stromal cells. At the feto-maternal interface, TIMP-3 expression was observed in fetal extravillous trophoblasts that had invaded the maternal decidual tissues as well as in the maternal decidual cells. These findings suggest that TIMP-3 is a sensitive indicator of ESC decidualization, and that the induction of TIMP-3 expression in decidual cells and trophoblasts may be important in the regulation of trophoblast invasion.