Baseline and stimulated turnover of cell surface c-Kit expression in different types of human mast cells Introduction The c-Kit receptor (Kit, CD117) represents a receptor tyrosine kinase, expressed by a limited spectrum of cellular lineages such as germ cells, melanoctyes, haematopoietic stem cells and mast cells (MCs) [summarized in Refs (1–3)]. Its major importance in the regulation of prolif- eration and differentiation can be inferred from several aspects. First, in both mouse and man, it is one of the genetic sites most frequently affected by mutations, many of which eliminate or reduce particular lineages (4,5). Secondly, c-kit gene expression is reactivated, and c-kit in part mutated, in various haematopoietic as well as non-haematopoietic neoplasms, such as gas- trointestinal and breast tumors, part of which responding to newly developed c-Kit tyrosine kinase inhibitors such as imatinib mesylate (Gleevec) (6–8). Thirdly, null mutations in the mouse lead to early embryonic death, whereas function-reducing mutations have a more select- ive impact on distinct lineages (2,4). Within the haematopoietic compartment, MCs are the only lineage to maintain a high-level c-Kit expres- sion, whereas virtually all other progeny rapidly downregulate c-Kit levels, as they differentiate along the distinct pathways (1,4). In MC, c-Kit is indispensable and the most important Babina M, Rex C, Guhl S, Thienemann F, Artuc M, Henz BM, Zuberbier T. Baseline and stimulated turnover of cell surface c-Kit expression in different types of human mast cells. Exp Dermatol 2006: 15: 530–537. Ó The Authors 2006. Journal compilation Ó 2006 Blackwell Munksgaard. Abstract: The receptor tyrosine kinase c-Kit is fundamental to mast cell (MC) development and maintenance. Its regulation can occur at various levels, but nothing is known about how this is accomplished in normal human tissue MC. Likewise, the baseline turnover of c-Kit has not been addressed yet. We used mature MC from human skin, along with the MC lines LAD-2 and HMC-1 and treated them with stem cell factor (SCF), cycloheximide, actinomycin D (AD) and com- binations thereof, and determined expression levels of c-Kit and other surface receptors by flow cytometry. Ligand-induced internalization of c-Kit was found to be a universal mechanism and detectable in all MC subtypes. By Western blot analysis of LAD-2 cells, c-Kit was found to nearly disappear 3 h after the addition of SCF to slowly recover thereafter. Investigations into the baseline turnover of c-Kit expression revealed that c-Kit is strongly affected by the inhibition of de novo translation in all MC subsets, while a suppression of transcription had a weaker effect and displayed greater cell-to-cell variation. Only a minor impact on other cell surface receptors (CD29, CD50 and CD54) was noted. On combined treatment, cycloheximide, AD and SCF displayed additive effects, resulting in a complete disap- pearance of c-Kit from the cell surface. In conclusion, c-Kit represents a rapidly cycling cell surface receptor. It is not only immediately inter- nalized upon binding of its ligand, but it is also heavily affected by the inhibition of translation or transcription when viewed against an aver- age background. Interestingly, c-Kit regulation seems largely inde- pendent of the MC subtype. Magda Babina, Claudia Rex, Sven Guhl, Friedrich Thienemann, Metin Artuc, Beate M. Henz and Torsten Zuberbier Department of Dermatology and Allergy, Universita ¨tsmedizin Berlin, Berlin, Germany Key words: c-Kit – differentiation – mast cells – mastocytosis – protein turnover Magda Babina Department of Dermatology and Allergy Universita ¨tsmedizin Berlin Charite ´ Campus Mitte Schumannstrasse 20/21 D-10117 Berlin Germany Tel.: +49 30 450518408 Fax: +49 30 450518900 e-mail: magda.babina@charite.de Accepted for publication 10 April 2006 530 Experimental Dermatology 2006: 15: 530–537 Blackwell Munksgaard . Printed in Singapore Copyright Ó The Authors 2006. Journal compilation Ó 2006 Blackwell Munksgaard Experimental Dermatology ISSN 0906-6705