Abstract
Since heparin-binding epidermal growth factor (HB-EGF) is a member of the EGF family and binds to EGF receptor, we tested recombinant HB-EGF for its ability to stimulate human keratinocyte proliferation. The effect of HB-EGF on human keratinocytes was dependent on the cell density. HB-EGF optimally increased the cell number by 1.8-fold at 1.0 ng/ml for a 4-day incubation period under subconfluent culture. In contrast, under confluent culture, 10 ng/ml HB-EGF optimally increased the DNA synthesis 2.1-fold. To examine the production of HB-EGF by human keratinocytes, the analysis of human keratinocyte-conditioned medium was undertaken by a combination of heparin affinity column chromatography, EGF receptor-stimulating assay, immunoblotting, and neutralization. Heparin column chromatography fractionated three activities, peaks 1, 2, and 3, which contained immunoreactive 30- and 27-, 19-, and 14.5-kDa bands, respectively. The anti-HB-EGF-blocking antibody neutralized the activities of peaks 2 and 3 by 38 and 22%, respectively, but did not neutralize the activity of peak 1 at all. The antibody reduced the cell growth by 37% for a 4-day incubation period. Northern blot analysis detected a 2.5-kilobase transcript of HB-EGF. The addition of 1 ng/ml HB-EGF optimally increased the levels of HB-EGF mRNA 5.4-fold at 1 h and TGF-alpha mRNA 3.1-fold at 3 h. Interestingly, the addition of TGF-alpha at 1 ng/ml to keratinocyte cultures enhanced the level of HB-EGF mRNA 10.2-fold at 6 h. 1 ng/ml EGF also increased HB-EGF mRNA levels 10.9-fold at 1 h. These results suggest that HB-EGF is an autocrine growth factor for human keratinocytes, and HB-EGF and TGF-alpha act not only by an autoinductive mechanism but also by mutual amplification.
Highlights
EGF) is a member of the EGF family and binds to EGF of 208 amino acidscomposed of a signal peptide, a hydrophilic receptor, we tested recombinant Heparin-binding EGF-like growth factor (HB-EGF) for its ability domain, an EGF-like domain, and a transmembrane domain
Tected a 2.5-kilobase transcript of HB-EGF.The addition In the presentwork, we demonstrate for the first time that of 1ng/ml HB-EGFoptimally increased the levels of H B - human keratinocytes produce HB-EGF, which stimulates the EGF mRNA SA-foldat 1h and transforming growth factor-a (TGF-a) mRNA 3.1-foldat 3 growth of these cells in culture
The effective doserange of HB-EGF for keratinocytes is almost the same as that of BalbIc-3T3 cells and bovine aortic smooth muscle cells [1].Since keratinocytes are at confluency i n vivo, we studied the effect of HB-EGF on the growth of human keratinocytes under confluent conditions
Summary
EGF) is a member of the EGF family and binds to EGF of 208 amino acidscomposed of a signal peptide, a hydrophilic receptor, we tested recombinant HB-EGF for its ability domain, an EGF-like domain, and a transmembrane domain. Heparin column chromatography fractionated three activities, peaks 1, 2, and 3, which contained immunoreactive30-and 27-, 19-a, nd 14.5-kDa protein containing at least 86 amino acids and comprising an EGF-like domain and a basic region responsible for heparin binding [2]. Tected a 2.5-kilobase transcript of HB-EGF.The addition In the presentwork, we demonstrate for the first time that of 1ng/ml HB-EGFoptimally increased the levels of H B - human keratinocytes produce HB-EGF, which stimulates the EGF mRNA SA-foldat 1h and TGF-a mRNA 3.1-foldat 3 growth of these cells in culture. 1ng/ml EGF increased H B - EGF mRNA levels 10.9-foldat 1h These results suggest that HB-EGF is an autocrine growth factor for human keratinocytes, and HB-EGF and TGF-a act by an autoinductive mechanism and by mutual amplification
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
