| Popis: |
In the frame of this work it was investigated which signalling pathways are involved in the regulation of MMP2 and MMP9 gens as well as how they are influenced by TIMP-1. For this purpose both certain kinases as well as their phosphorylated form (STAT 1, P-STAT 1, STAT 3, P-STAT-3, JAK 1, P-JAK 1, JAK 2, P-JAK 2, Ras GAP and JNK 1) were investigated by means of western blot analysis and western blot analysis after immunoprecipitation in wild type HepG2 cells as well as TIMP-1 overexpressing hepatoma cells. In all cases the activation of the kinases was not influenced by TIMP-1. The changed cell morphology (nest shaped growth) of TIMP-1 overexpressing cells was not due to an extended expression of cell cycle inhibitor p21. Instead an extended expression of p21 was found for hepatoma cells overexpressing the TIMP-1 antagonist MMP-9-H401A. These findings regarding p21 could affirmed via cell proliferation kits.In a second run to identify the signalling pathways leading to an increased expression of MMP-2 and MMP-9, both wild type HepG2 cells and TIMP-1 overexpressing hepatoma cells were incubated together with genistein (inhibitor of tyrosine kinase), SB 202190 (inhibitor of p38 MAP kinase) and UO126 (inhibitor of MEK-1 and -2). Afterwards the gen expression was checked by means of RT-PCR. It could be revealed that in human hepatoma cells (wild type HepG2) each of the chosen inhibitors blocks the MMP-2 and MMP-9 gen expression nearly completely. The results indicate that the regulation of gen expression is effected by activated MAP kinases. Contrary to the other inhibitors SB 202190 does not block the MMP-2 and MMP-9 expression in TIMP-1 overexpressing HepG2. Obviously in this case the expression is independent of p38 MAP kinase. Other kinases could be involved that are influenced by TIMP-1. An indirect effect of TIMP-1 cannot be excluded. From these experiments it can be concluded that the activation of MMP-2 and MMP-9 inducing signalling pathways can be controlled, perhaps via components of the extracellular matrix.Furthermore the migration of TIMP-1 overexpressing hepatoma cells was investigated in the Boyden chamber. The TIMP-1 overexpression seems to promote the migration. Presumably this is due to an increased secretion of MMP (MMP-2 and MMP-9) in TIMP-1 overexpression cells, as via MMP inhibition the migration of these cells could be reduced significantly. |
