Ph.D Thesis


Ph.D StudentRam Elsebeth
SubjectThe Role of Transcription Factors in MMP Expression and
Trophoblast Invasion in vitro
DepartmentDepartment of Medicine
Supervisor PROFESSOR EMERITUS Eliezer Shalev
Full Thesis textFull thesis text - English Version


Abstract

The Gelatinases, MMP-2 and MMP-9, are regarded as key enzymes in trophoblast invasion. The aim of this study was to study the secretion, expression and transcriptional regulation of MMP-2 and MMP-9 in the trophoblasts, under stimulation of the major signal pathways.

Gelatinase secretion from JAR cell-line and 1st trimester trophoblasts (6-8w and 9-12w) was examined with zymography, under Protein Kinase A (PKA), Protein Kinase C (PKC) and Protein Tyrosine Kinase (PTK) stimulation. Gelatinase involvement in trophoblast invasion was assessed with the Matrigel invasion assay. MMP-2 and -9 expressions were analyzed by Northern blot and RT-PCR. Transcription factors (TFs) involved in MMP-2 and MMP-9 regulation were detected with gelshift assay, supershift assay and Western blot. Antisense inhibition of relevant TFs, followed by immuno-fluorescence staining, zymography, RT-PCR and Matrigel invasion assay explored the involvement of these TFs in MMP-2 regulation and trophoblast invasion.

Gelatinase secretion was differential during the 1st trimester, with MMP-9 being dominant from week 9. MMP-2 and MMP-9 secretion, transcription and trophoblast invasion were induced by PKA (Forskolin) and PTK (Epidermal growth factor) in the 1st trimester trophoblasts. Inhibition of MMP-2 reduced trophoblast invasion in JAR, 6-8w and 9-12w trophoblasts, whereas inhibition of MMP-9 only reduced 9-12w trophoblast invasion. SP-1, AP-1, NFkB and ETS, or SP-1 and p53 were identified as binding sites involved in Forskolin and EGF (respectively) induction of MMP-9 transcription. The expressions of C-Fos, C-Jun and JunD, SP-1 and -4 transcription factors were increased by Forskolin.

p53, AP-2 and C/EBP binding sites were involved in both Forskolin and EGF induction of MMP-2 transcription, in addition to Ets-2 (Forskolin) and SP-1 (EGF). Antibodies to AP-2a, C/EBPe and -g and phosphorylated p53 (Forskolin and EGF), Ets-2 (Forskolin) and AP-2g (EGF) displaced this binding. Forskolin and EGF increased the expression of these TFs, whereas EGF reduced SP-1 and -4. Ets-2 inhibition reduced Forskolin-induced MMP-2 secretion and trophoblast invasion. p53 inhibition reduced Forskolin-induced MMP-2 expression, secretion and trophoblast invasion, and EGF-induced MMP-2 expression. We conclude that PKA and PTK induce MMP-2 and -9 expressions, secretions and trophoblast invasion. MMP-2 is the key enzyme in early trophoblast invasion. Several, both identical and diverse transcription factors mediate PKA and PTK regulation of MMP-2 and MMP-9 expression and trophoblast invasion, including p53, AP-2a, C/EBPe and-g and Ets-2 (MMP-2) and C-Fos, C-Jun, junD, NKkB and p53 (MMP-9).