siRNA regulering av signalveier i den mesenkymalen stamcellelinjen hMSC-TERT

Abstract

Abstract

The intention of this study was to identify and characterize signal pathways of the mesenchymal stem cell line hMSC-TERT which are being influenced by different mediators of inflammation. The purpose was also to characterize the effect of such pathways on the wnt-pathway. The wnt-pathway seems to be central in the understanding of regulated differentiation and proliferation in stem cells. The primary goal was to investigate and establish molecular biological methods that could be used in the study of these pathways in hMSC-TERT. Transient transfection of plasmids overexpressing selected mutants and constructs was one of the considered methods. The second method investigated was targeted gene silencing of signal proteins by the use of RNAi and siRNA.

It was obtained an increased expression of the signal protein Raf-1 and the mutant Raf-1 S43A as a result of electroporation by use of a commercial kit delivered by Amaxa Biosystems (USA). An optimized siRNA-transfection method with use of the cationic lipid DOTAP was established. By use of this method it was obtained a reduced synthesis of the signal proteins Akt1/PKBá, Raf-1 and the PKA catalytic units á and â.

hMSC-TERT transfected with Raf-1 siRNA or PKA catalytic unit á and â (PKAc siRNA), where stimulated by the two inflammatory substances PGE2 and adenosin. PGE2 and adenosin affects G-protein coupled receptors (GPCR). As a result of these affections there have been obtained an increased intracellular cyclic AMP synthesis level in hMSC-TERT. Less synthesis of cAMP was obtained in cells stimulated with adenosin than cells stimulated with PGE2. cAMP is an intracellular signal molecule that activates PKA.

It was obtained a reduced phosphorylation of an inhibitory phosphorylation site of the wnt-pathway signal protein GSK3â in PKAc siRNA knockdown cells. This result insinuates that GSK3â is being negatively regulated by an active PKA. No altered phosphorylation patterns where obtained of a presumed activating phosphorylation site of the signal protein Akt/PKB in PKAc siRNA knockdown cells. This result implies that PKA is not activating Akt/PKB.

PKA is inhibiting the MAP3-kinase Raf-1 by phosphorylating an inhibitory phosphorylation site and activated Raf-1 leads to phosphorylation of the further downstream MAP-kinase ERK1/2. Both adenosin and PGE2 resulted in dephosphorylated ERK1/2 among cells not transfected with siRNA. These forthcoming results appeared when Raf-1 or PKAc siRNA transfected cells where stimulated with adenosin or PGE2. In general, adenosin seems to be mediating its effect on ERK1/2 phosphorylation through PKA. PGE2 may mediate two parallel inhibitory mechanisms that results in dephosphorylated ERK1/2, through PKA and maybe by an unknown mechanism.

During the work with the PKA catalytic unit á siRNA it was obtained reduced expression of a protein that could be a new isoform of PKAc á with the molecular weight ~37 kD. The same band was detected by Westernblotting of an isolated, purified and concentrated sample.