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Amphiregulin (AR) and heparin-binding EGF-like growth factor (HB-EGF) are two recently identified members of the EGF family. Both AR and HB-EGF share with EGF the ability to interact with the type-1 EGF receptor; however, AR and HB-EGF differ from EGF in that both of these mitogens bind to heparin while EGF does not. To determine whether interactions with heparin-like molecules on the cell surface influence binding of AR and HB-EGF with EGF receptors and the subsequent mitogenic activity exerted by these growth factors, murine AKR-2B and Balb/MK-2 cells were treated with either an inhibitor of proteoglycan sulfation (chlorate) or a heparin antagonist (hexadimethrine). As expected, neither treatment significantly altered the specific binding of 125I-EGF on AKR-2B cells. Interestingly, treatment with either chlorate or hexadimethrine inhibited the ability of AR to compete with 125I-EGF for cell surface binding and also attenuated AR-mediated DNA synthesis. Thus, as has been suggested for other heparin-binding growth factors such as basic fibroblast growth factor (bFGF), the interaction of AR with an EGF-binding receptor appears to be facilitated by interaction with cell-associated sulfated glycosaminoglycans or proteoglycans. Unexpectedly, however, neither chlorate nor hexadimethrine treatment caused an inhibition of HB-EGF-induced mitogenic activity. Chlorate treatment did not significantly alter the ability of HB-EGF to compete with 125I-EGF for cell surface binding sites, however, heparin and hexadimethrine reduced the ability of HB-EGF to compete for 125I-EGF binding. These results suggest that, in AKR-2B cells, HB-EGF may mediate its mitogenic response at least in part through a receptor which appears to be selective for HB-EGF and permits HB-EGF-mediated mitogenic responses in the presence of hexadimethrine or heparin. Finally, hexadimethrine inhibited the specific binding and mitogenic activity of bFGF, suggesting that this cationic polymer can function as an antagonist of heparin-binding mitogens other than AR.

Previous investigations have shown disparate effects of adenine nucleotides on epidermal cell proliferation. Our present study demonstrates that adenosine and its related nucleotides (ATP, ADP, AMP) are antiproliferative for normal human epidermal keratinocytes cultured in the absence or presence of exogenous epidermal growth factor. Furthermore, the inhibitory effects of these compounds occur at concentrations less than 100 μM, are reversible, and do not affect the viability of the keratinocyte cultures. Our current investigation also demonstrates that both selective and nonselective adenosine receptor agonists are themselves approximately as potent as keratinocyte proliferation inhibitors, but are all less potent inhibitors than adenosine. These observations are consistent with the theory that adenosine mediates its antiproliferative response via a novel or more poorly characterized adenosine purinoreceptor subclass. Moreover, our present study demonstrates that ATP and ATP-γ-S are significantly more potent antiproliferative agents than either α,β-methylene ATP or β,γ-methylene ATP. Based on previous studies that have demonstrated that P 2y purinoreceptors possess this type of ligand specificity and that the purinoreceptor may be expressed by keratinocyte cultures, we propose that ATP may mediate its antiproliferative effects via this purinoreceptor. Collectively our results indicate that adenosine and adenine nucleotides abrogate exogenous epidermal growth factor-dependent and -independent keratinocyte proliferation at submillimolar concentrations and may be important physiologic regulators of keratinocyte growth in vivo . Further, these results suggest that these or related compounds may have application as treatments for epidermal proliferative pathologies in which the epidermal growth factor receptor-signaling pathway has been activated.