Modulation of melanotransferrin and transferrin receptor 1 (TFRC)- and CD44-based signaling for TFRC up-regulation in human melanoma cells

Abstract

Background: The human melanoma cell line IGR- 1 was used for the detection and regulation of both melanotransferrin (MTf) and transferrin receptor 1 (TFRC, CD71). While the function in iron transport of the TFRC is well documented the functional importance of MTf is not yet fully understood. Due to the up-regulation of TFRC by hyaluronan (HA) some components and aspects of CD44 signaling were investigated. Materials and Methods: The cell-surface proteins MTf, TFRC and ERBB2 receptor tyrosine kinase 2 (ERBB2) were detected by immunoluminescent technique using different polyclonal and monoclonal antibodies. Ionomycin was used to inhibit β-catenin/T-cell-specific transcription factor (TCF) association, essential in HA-CD44-ERBB2 signaling. Results: MTf, was found to be resistant to phosphatidylinositol-specific phospholipase C. However, MTf as well as TFRC were sensitive to partial proteolytic degradation by pronase E and trypsin. The expression of MTf was shown to be up-regulated by mannose-6-phosphate and that of TFRC by HA. Ionomycin at 10 μM inhibited TFRC up-regulation. However, at 50 μM it induced a 7.5-fold increase of TFRC concentration. Conclusion: Our results suggest that human melanoma cells are able to up-regulate TFRC expression using HA/CD44 signaling. The whole pathway comprises of the sequence: HA/CD44, neural Wiskott-Aldrich syndrome protein (N-WASP), ERBB2, β-catenin/TCF, c-MYC and TFRC. Since β-catenin is also known to be a component of wingless/Int-1-Frizzled signaling that also leads to transcriptional c-MYC activation, the pathway found here might be alternatively used by melanoma cells for iron supply, necessary for cell proliferation.