Amphiregulin (AR) is a recently discovered member of the EGF-receptor-binding family of ligands that also includes, among others, Epidermal Growth Factor (EGF), Transforming Growth Factor-alpha (TGF-a), Heregulin (HRG), and Heparin-binding EGF (HB-EGF). The biological explanation for the ostensible redundancy within this family is unclear; however, emerging experimental data suggest principally a proliferative function for EGF and TGF-alpha, and a differentiative one for AR, HR, and HB-EGF. Tissue macrophages derive from circulating monocytes, and are thought to play a role in the initial inflammatory phase of wound repair through phagocytosis and release of growth factors. Our prior immunohistochemical results in human venous stasis ulcers had indicated AR staining mainly in vascular cuffs and at wound margins where active epithelial proliferation was evident. However, to date, no study has yet published on AR expression during the healing process. Consequently, using freshly isolated human monocytes, we examined AR mRNA expression with the differential RT-PCR technique, and found AR to be expressed in a time-dependent manner following induction with either lipopolysaccaride or physiologic concentrations of platelet releasate. Neither EGF-receptor expression by kinase phosphorylation assays, nor unexpectedly mRNA for TGF-a by RT-PCR was detected under these two inducing conditions. To enhance mRNA quantitation of AR and TGF-a, currently a competition-based RT-PCR approach is being developed which uses an internal RNA reference template containing primer sequences identical to the target mRNAs of interest but which generate different bp-sized cDNA products. Platelet releasate derives from the alpha-granules of platelets, and contributes to the mileau of the initial phase of wound healing. Which individual components of platelet releasate are responsible for AR mRNA induction (e.g., perhaps PDGF or TGF-b), and whether or not AR protein is anchored to monocyte cell membranes or is secreted in its glycosylated form, are subjects for future investigation.
