Heparin-binding EGF-like growth factor (HB-EGF) overexpression in transgenic mice downregulates insulin-like growth factor binding protein (IGFBP)-3 and-4mRNA

An in vivo approach was taken to assess the biological significance of heparin-binding EGF-like growth factor (HB-EGF) using transgenic mice. Transgenic mice were generated using the pIRES-EGFP vector expressing a bicistronic mRNA containing both human HB-EGF (hHB-EGF) and enhanced green fluorescent protein (EGFP) coding sequences under the regulation of the cytomegalovirus immediate-early (CMV-IE) promoter. As a marker for transgene expression, EGFP fluorescence in 5 mu m tissue sections was evaluated. To confirm HB-EGF expression in EGFP-containing tissues, HB-EGF mRNA was analyzed by RT-PCR and Northern blot analysis. Protein levels of HB-EGF and insulin-like growth factor binding protein-3 (IGFBP-3), a molecule that stabilizes IGFs, which in turn helps to promote growth, were analyzed by Western blot. Also, the weights of transgenic mice were compared with the weights of wild type non-transgenic littermates over a 10-week period. EGFP fluorescence, RT-PCR and Northern analysis of a variety of tissues from hHB-EGF transgenic mice indicate recombinant EGFP/hHB-EGF mRNA expression in kidney, liver, lung and stomach. Western blot analysis confirmed that HB-EGF protein levels were greater in these tissues from hHB-EGF transgenic mice compared to wild type non-transgenic littermates. IGFBP-3 protein was absent in serum of transgenic mice prior to the onset of puberty, but indistinguishable from wild type non-transgenic mice after puberty. Furthermore, IGFBP-3 and IGFBP-4 mRNA were downregulated in the kidney, but not liver or lung of the transgenic mice. In accordance with reduced IGFBP-3 and -4 levels, hHB-EGF transgenic mice exhibited a 20% decrease in weight prior to 6 weeks of age compared to wild type non-transgenic littermates. Our laboratory has generated a biologically functional transgenic mouse model exhibiting increased expression of hHB-EGF in kidney, liver, lung and stomach. Overexpression of hHB-EGF affected the growth rate of these transgenic mice possibly through a pathway involving IGFBP-3 and IGFBP-4.