טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
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).