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

Abstract

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 microm 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.

Estradiol, Progesterone, and Transforming Growth Factor α Regulate Insulin-Like Growth Factor Binding Protein-3 (IGFBP3) Expression in Mouse Endometrial Cells

“…Furthermore, excess expression of heparin-binding epidermal growth factor (HB-EGF) lowered IGFBP3 expression (Provenzano et al, 2005). These findings suggest that the production of IGFBP3 is partly regulated by members of the EGF family.…”

“…In the present study, we demonstrated for the first time that TGFα decreases IGFBP3 expression in mouse endometrial stromal cells. Members of the EGF family, such as EGF and HB-EGF, have been shown to reduce IGFBP3 expression in various cell types (Hembree et al, 1994;Wraight and Werther, 1995;Provenzano et al, 2005;Takaoka et al, 2006). In contrast, TGFα stimulates IGFBP3 synthesis in human mammary epithelial cells (Sivaprasad et al, 2004).…”

EGFR Signaling in Liver Diseases

“…So far, it has not been possible to model EGFR-induced liver cancer in mice, as all the attempts to generate GEMMs that overexpress EGFR have been unsuccessful [ 24 ], whereas transgenic overexpression has been successful for the EGFR ligands EGF [ 145 , 146 ], TGF-α [ 147 , 148 ], AR [ 149 ], BTC [ 150 ], HB-EGF [ 151 ] and EPGN [ 152 ]. Livers of transgenic mice that constitutively overexpress human TGF-α from the mouse metallothionein 1 promoter frequently developed multifocal, well-differentiated HCCs [ 147 , 148 , 153 ] and treatment of these mice with hepatic carcinogens further accelerated HCC development [ 153 ].…”

Cellular reprogramming into a brown adipose tissue-like phenotype by co-expression of HB-EGF and ADAM 12S

“…Membranes were blocked with Tris-buffered saline with Tween-20 (TBST) (0.1%Tween-20) plus 5% non-fat dry milk overnight at 4 C with gentle shaking, washed with TBST three times (15 min each), and probed with primary antibodies overnight at 4 C [rabbit anti C-terminus of human HB-EGF, 1:200; goat anti N-terminus of human HB-EGF, 1:200, R&D Systems (Minneapolis, MN); sheep anti-ADAM 12, 0.1mg/ml, R&D Systems], followed by incubation with HRP-conjugated secondary antibodies 1 h at room temperature (goat anti-rabbit IgG; rabbit anti-goat IgG, or rabbit anti-sheep IgG, Jackson ImmunoResearch Laboratories, West Grove, PA). Specific immunoreactivity was determined by application of SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Inc., Rockford, IL) to the membrane and normalized to actin (Provenzano et al, 2005).…”