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CLONING AND REGULATION OF THE BABOON INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-1 (IGFBP-1) PROMOTER.
Kim, J.1, Jaffe, Randal2, Unterman, Terry3, Fazleabas, Asgerally1,2, 1 2 3
ABSTRACT- IGFBP-1 is the major secretory product of decidualized stromal cells of the primate endometrium. Baboon endometrial stromal cells obtained during the mid-luteal phase secrete IGFBP-1 when incubated in vitro for 6 to 12 days with hormones (estradiol, medroxyprogesterone acetate, relaxin) and dibutyryl cAMP (dbcAMP). We have also demonstrated a possible relationship between the cytoskeleton and decidualization. In vitro disruption of the actin filaments of stromal cells using cytochalasin-D (CD) can induce IGFBP-1 in 24h in the presence of hormones and dbcAMP. To further study the factors involved in IGFBP-1 regulation, a baboon genomic clone was isolated from a lambda phage library. A 3.5kb 5′flank fragment was subcloned into a pGL3-basic luciferase reporter vector and sequenced. A 525bp 5′flank fragment was also generated by cutting at an internal Bgl II site in the 3.5kb fragment and inserted into the pGL3-basic luciferase reporter vector. This 525bp 5′flank reporter construct was transiently transfected using Lipofectamine 2000 into human fibroblast cells (HUF) isolated from decidua peritalis. Following transfection, the cells were treated with CD, hormones and dbcAMP for 24h. This treatment resulted in a 2-fold increase in luciferase activity in cell lysates compared to CD treatment alone. In the absence of CD, media alone or hormones and dbcAMP had no effect on luciferase activity. The transfected HUFs were also co-cultured with Jeg3 cells, a trophoblastic cell line. When Jeg3 cells were placed directly on top of transfected HUF cells, a 5-fold increase in luciferase activity was evident in cell lysates. When Jeg3 cells were cultured on cell inserts (8.0um pore size) placed above the HUF cells, there was no significant effect in luciferase activity. These data suggest that trophoblast cell contact and cytoskeletal disruption are important in IGFBP-1 gene regulation and supports our extensive in vivo data on primate decidualization. (NIH HD 36759).
KEY WORDS: IGFBP-1, decidualization, endometrium, trophoblast