Subcellular Localization of Mitf in Monocytic Cells is Attachment-Dependent

Objectives: Mitf is a bHLH-Zip transcription factor important for the development of melanocytes, retinal pigmented epithelial cells and osteoclasts, with minor effects on the cell numbers of NK cells, mast cells, basophils, macrophages and B cells. The localization of Mitf protein is widely accepted to be nuclear. In preosteoclasts, it was reported that without M-CSF or RANKL, Mitf is retained in the cytoplasms. Addition of either cytokines abolishes the retention and Mitf becomes nuclear. We investigated whether the presence of M-CSF or M-CSF plus RANKL is sufficient to provide nuclear translocation signals for Mitf in human monocytes. Methods: Monocytes were purified by plate adhesion in chamber slides from Ficoll-fractionated human PBMC. Cells were subsequently cultured in MEMá supplemented with M-CSF or M-CSF plus RANKL. Cells were fed every 3 days for 3 weeks when foci formation is evident. Some foci cells were removed by inoculating rings and cytospined onto glass slides. Both cells on chamber slides and cytospined slides were stained with anti-Mitf antibody C5 to determine the subcellular localization of Mitf. Results: In long term monocytic cultures supplemented with M-CSF or M-CSF plus RANKL, cells form foci composed of small round cells surrounded by epithelioid/spindle cells when the cell density is low. These cells attach to the surface loosely and are detached and floating in the media upon light shear force. Upon immunofluorescent stainings, the foci-surrounding cells exhibit a nuclear signal of Mitf with moderate expression levels. In contrast, the foci cells exhibit a strong cytoplasmic Mitf signal that is resistant to M-CSF or MCSF plus RANKL. Mitf becomes nuclear again when the foci cells are forced to attach to a surface by centrifugation. Conclusions: In addition to cytokines, the Mitf subcellular localization in monocytic cells is also attachment-dependent.