Genotype to phenotype: Modelling the impact of natural TGFbR2 expression variation on fibrosis initiation in vivo

Introduction: Complex diseases in man are caused by combinations of subtle variations in the expression of genes rather than total loss of function. Model systems are required to analyse genotype-to-phenotype correlation for natural expression variation in vivo. For this study we availed of BXD lines, which are recombinant inbred F2 offspring of inbred mouse strains C57BL/6J (B) and DBA/2J (D). Due to the inheritance of different combinations of regulatory molecules, BXD lines show a normal distribution of expression for all genes analysed.

Aims: We hypothesised that the natural variation of TGF-β receptor type 2 (TGFbR2) expression in livers of BXD lines represents a better model to study the consequences of expression differences on fibrogenesis than monogenic knock-out or transgenic models. TGFbR2 is the rate-limiting receptor in the transmission of TGF-β signal to the nucleus.

Methods: To test this hypothesis, we analysed the impact of variable hepatic TGFbR2 expression on the response to acute, short-term liver damage. Basal TGFbR2 protein expression was assessed by Western blot analysis of native livers from BXD lines 13 and 24a. These lines were chosen because they represent both the low and high end of TGFbR2 expression among BXD animals. Animals were challenged by a short course of CCl₄ administration (3 intraperitoneal injections at 48-hour intervals). Liver sections were stained using Ki-67 antibody, TUNEL, and Sirius red, and results quantified.

Results: Western blot analysis identified significant differences in TGFBR2 protein expression in the livers, with male mice demonstrating more pronounced effects than females. Livers challenged with CCl₄ showed significant differences in Ki-67 staining. Sirius red staining revealed less significant differences, interestingly with a reverse trend to Ki-67. TUNEL staining did not differ between lines.

Conclusions: These data indicate that the panel of BXD lines represent a basic experimental framework to study the impact of natural expression variation: Differences in TGFbR2 expression observed in native liver of BXD lines are correlated with a phenotypic difference following acute damage. Higher TGFbR2 expression is correlated with increased proliferation, but lower collagen deposition.