Molecular mechanisms of the TGFβ1 Arg25Pro polymorphism related to acute radiotherapy-induced toxicity
von Kateryna Filonenko
Datum der mündl. Prüfung:2015-03-25
Betreuer:Prof. Dr. Jürgen Brockmöller
Gutachter:Prof. Dr. Markus Bohnsack
Gutachter:Prof. Dr. Margarete Schön
EnglischIn radiochemotherapy administered for rectal cancer, all patients harboring the Pro25 allele in the signal peptide of TGFβ1 experienced a higher grade of acute treatment toxicity. Pro25 features an allelic frequency of about 10% in the Caucasian population and replaces an arginine residue. The functional role of this Arg25Pro polymorphism is still not clarified. Therefore, the aim of my work was to determine the possible molecular mechanisms driving the reported clinical association. In order to investigate allele-specific effects of the Arg25Pro polymorphism stable trasnfection to overexpress either Arg25 or Pro25 was carried out. As host cells, T-REx HEK 293 cells were used. The transfected cells were characterized on genome level by specific PCRs, for mRNA expression by quantitative real time PCR, and for protein expression by Western blotting. Transient transfection with FLAG tag-containing TGFβ1 constructs was employed to localize subcellular TGFβ1 distribution visualized by immunocytochemistry. Quantitative expression was determined by ELISA directed to either an epitope of the LAP or the mature TGFβ1 both for intracellular and secreted amounts. Clinically administered radiochemotherapy was simulated on the stably transfected cells. A major effect of the Arg25Pro polymorphism was observed on the secretion rate of the inactive LAP-TGFβ1 complex. Under baseline conditions without irradiation, the stably transfected cells carrying Pro25 secreted two times higher amounts of LAP-TGFβ1 than the cells harboring Arg25 as assesd by ELISA (p=0.0002). This increased level of secreted TGFβ1 in presence of Pro25 was confirmed by Western blotting. Radiation did not result in changes of the fraction of secreted LAP-TGFβ1 compared to non-irradiated cells. Intracellular amounts of LAP-TGFβ1 and active TGFβ1 were not affected by the Arg25Pro polymorphism. FLAG epitope staining upon transient transfection indicated perinuclear localization of TGFβ1 suggesting its association with ER or Golgi apparatus. There were no apparent distribution distinctions dependent on the Arg25Pro polymorphism. Stepwise acidification was used to test the hypothesis whether the Arg25Pro polymorphism may modulate activation of TGFβ1. Increasing proton concentrations revealed more activated TGFβ1 normalized to the inactive LAP-TGFβ1 in case of Arg25 in comparison with Pro25. This allelic effect was still evident when adjusted for pH in linear regression analysis. Though secreted TGFβ1 from Arg25-containing constructs appeared more sensitive toward activation, there was a net effect of more active TGFβ1 by pH-dependent activation for Pro25. This was due to substantially higher amounts of secreted and thus potentially activatable LAP-TGFβ1 molecules. Altered secretion rate in dependence on the Arg25Pro polymorphism might be due to distinct cleavage sites patterns. Thus, prediction of the cleavages site position was conducted in silico. For Pro25 one cleavage site position between the codons Glu29/Leu30 and for Arg25 two sites, i.e. at Glu24/Arg25 and at Glu29/Leu30, were predicted. In conclusion, the data of my thesis indicate that an increased secretion rate in case of Pro25 might contribute to the clinically reported association of this allele with acute radiotoxicity. Distinct patterns of signal peptide cleavage for the Arg25 and the Pro25 allele may lead to higher secretion rates of LAP-TGFβ1. As ionizing radiation and tissue acidification are major activators of LAP-TGFβ1, increased active TGFβ1 in irradiated tissue might result for carriers of Pro25 possibly explaining the radiotherapy-related toxicity. Pharmacological interference to target elevated TGFβ1 secretion, activation or subsequent signaling might offer strategies to prevent patients at particular risk for radiation injuries. Such approaches might also be beneficial for other conditions linked to altered TGFβ1 signaling like chronic inflammations.
Keywords: LAP-TGFβ1; TGFβ1; Arg25Pro; radiotoxicity