A proline rich acidic protein PRAP identified from uterine luminal fluid of estrous mice is able to enhance the estrogen responsiveness of Ishikawa cells

Abstract

Using mice as experimental animals, proteins in the uterine luminal fluid (ULF) from both adults and diethylstilbestrol dipropionate (DES)-treated immature animals were resolved by 2D gel electrophoresis. Two of the protein spots, (a) and (b) around the positions of 18-20 kDa, in the adult ULF were not found in the DES-treated ULF. Automated Edman degradation established the same N-terminal sequences of AHQVPVKTKGKHVFP for the two protein spots. Two trypsin digests of spot (a) were analyzed using CID MS/MS to establish the peptide sequences DNQLGPLLPEPK and RPDAMTWVETEDILSHLR. These partial sequences were confirmed in the cDNA-deduced mouse proline rich acidic protein (PRAP). Using human Ishikawa cell line as a surrogate endometrial model, we demonstrated rapid entrance of exogenous PRAP into the cells and its ability to enhance alkaline phosphatase activity of the E(2) -stimulated cells. Further, the transcripts of five estrogen-responsive genes, including ALPP (Placental alkaline phosphtase), ALPPL (placental alkaline phosphatase-like 2), TGF (transforming growth factor), PR (progesterone receptor), and Wnt7a, were measured after the cell incubation in modified Eagle medium containing 0.1 nM E(2) , or 0-25 µM PRAP, or both together at 37°C for 48 h. As compared with the control, E(2) alone increased the transcripts of ALPP, ALPPL, TGF-α, and PR, and reduced the transcript of Wnt7a, whereas PRAP alone had a slight impact on their expression. E(2) together with PRAP greatly increased the E(2) -stimulated transcriptions of ALPP, ALPPL, TGF-α, and PR, and markedly reduced the E(2) -suppressed transcription of Wnt7a.