Experimental Cd Research 180 ~~9$9~ 314-325 reduction and Utilization of ~xtr~c~i~u~ar ti-iX ts BRIAN K. McCLENIC, RAJ S. MITRA, UCE L. R RIAN J. NICKOLOFF, VISHVA M. DIXIT, and JAMES Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109 Normal human melanocytes were separated from keratinocytes and maintained in culture using KGM medium supplemented with 120tetradecanoylphorbol acetate and cholera toxin. The melanocytes were examined for the production of extracellular matrix molecules including fibronectin, laminin, and thrombospondin and for the utilization of these molecules in adhesion and motility assays. Meianocytes produced significant amounts of fibronectin as indicated by biosynthetic labeling/immunoprecipitation and by enzyme-linked immunosorbent assay (ELBA). Fibronectin was expressed on the surface of these cells. Laminin was also produced by melanocytes and expressed on the cell surface. The amount of laminin produced was significantly less than the amount of fibronectin. In contrast, melanocytes did not produce measurable thrombospondin as indicated by biosynthetic labelingiimmunoprecipitation. Only traces of thrombospondin were detected by ELISA and no surface fluorescence was observed. When examined in adhesion and motility assays, melanocytes were found to utilize fibronectin for both processes. Laminin also stimulated adhesion but it was much less effective than fibronec- tin. Thrombospondin did not stimulate either attachment and spreading or motility. The pattern of extracellular matrix molecule production and utilization by melanocytes is significantly different from that shown previously for human epidermal keratinocytes (J. Varani et al., 1988, J. Clin. Znuest. 81, 1537). These differences may underlie the differ- ences with which the two cell types interact with basement membranes in uiw. 0 1989 Academic Press, Inc. Previous studies in our laboratory have shown that tbrombospo~~i~ is a major extracellular matrix protein produced by normal keratinocytes as well as by various squamous carcinoma cells [l-3]. Thrombospondin serves as an ad factor for normal keratinocytes and squamous carcinoma cells and o suggest that it is more effective at promoting attachment and spreading either fibronectin or laminin [l, 41. 1n Go, thrombospondin is localized to the dermal-epidermal junction and the basement membrane zone aroun glands [5]. Both are sites at which actively proliferating keratinocyte contact with the basement membrane. Melanocytes comprise a second important intcg~mentary cell ~~~~~a~i~~. There is approximately one melanocyte for every IO-15 keratinocyt melanocytes are closely associated with basal keratinocytes, they desmosomes with neighboring cells. At the basal surface of melan is in close apposition to the basement membrane, structures resembling the hemi- desmosomes of keratinocytes can be seen Anchoring filaments extend fro ’ To whom reprint requests should be addressed. 314 CopyrIght @ 1X39 by Academic Press, inc. All rights of reproductmn in any form reservrd 0014-4827!89 PjOJ 00