Using affinity-purified antibodies against mouse liver gap junction protein (26 K), discrete fluorescent spots were seen by indirect immunofluorescence labelling on apposed membranes of contiguous cells in several mouse and rat tissues: pancreas (exocrine part), kidney, small intestine (epithelium and circular smooth muscle), Fallopian tube, endometrium, and myometrium of delivering rats. No reaction was seen on sections of myocardium, ovaries and lens. Specific labeling of gap junction plaques was demonstrated by immunoelectron microscopy on ultrathin frozen sections through liver and the exocrine part of pancreas after treatment with gold protein A. Weak immunoreactivity was found on the endocrine part of the pancreas (i.e., Langerhans islets) after glibenclamide treatment of mice and rats, which causes an increase of insulin secretion and of the size as well as the number of gap junction plaques in cells of Langerhans islets. Furthermore, the affinity purified anti-liver 26 K antibodies were shown by immunoblot to react with proteins of similar mol. wt. in pancreas and kidney membranes. Taken together these results suggest that gap junctions from several, morphogenetically different tissues have specific antigenic sites in common. The different extent of specific immunoreactivity of anti-liver 26 K antibodies with different tissues is likely due to differences in size and number of gap junctions although structural differences cannot be excluded.

We have analyzed the structure and the regulation of Krox-20, a mouse zinc finger-encoding gene which is transiently activated following serum stimulation of quiescent fibroblast cells in culture. The gene is localized on chromosome 10, band B5, in the mouse, and the homologous human gene also maps to chromosome 10 (region q21.1 to q22.1). Alternative splicing of the 5'-most intron of the Krox-20 gene gives rise to mRNAs encoding putative zinc finger proteins with different N termini. The first exon contains a sequence element with strong similarity to the c-fos proto-oncogene serum response element (SRE). This element can functionally substitute for the c-fos SRE, and it binds the same nuclear protein. It is probably responsible for the serum induction of Krox-20, possibly in combination with a weaker SRE located in the 5'-flanking region of the gene. Our findings suggest that c-fos, Krox-20, and a number of immediate-early serum response genes are coregulated and that the SRE and its cognate protein are essential components of this regulatory pathway.

We studied the expression of arachidonate 5-lipoxygenase (5-LO) in a cell line of human keratinocytes (HaCaT) and in normal human skin keratinocytes in tissue culture. In undifferentiated keratinocytes 5-LO gene expression was low or undetectable as determined by 5-LO mRNA, protein, cell-free enzyme activity, and leukotriene production in intact cells. However, after shift to culture conditions that promote conversion of prokeratinocytes into a more differentiated phenotype, 5-LO gene expression was markedly induced in HaCaT cells and, to a lesser extent, in normal keratinocytes. These results show that 5-LO gene expression is an intrinsic property of human skin keratinocytes.After the discovery of arachidonate 5-lipoxygenase (5-LO; arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) in polymorphonuclear neutrophils the enzyme was identified in other leukocyte lineages (1, 2). Products of the 5-LO pathway, including leukotriene (LT) B4 and the cysteinyl LTs, in addition to their roles as inflammatory agonists, may act as physiological autocrine or paracrine signaling molecules. Until recently 5-LO expression was thought to be restricted to the hematopoietic system. However, attempts to identify the enzyme in other tissues yielded results that support the hypothesis that it is expressed in distinctive extrahematopoietic epithelia. Thus, Natsui et al. (3) Expression of the 5-LO pathway in skin epithelium likewise remains debatable. Several authors using primary skin keratinocytes reported the production of 5-hydroxyeicosatetraenoic acid (5-HETE) and LTB4, but subsequent work challenged some of the earlier interpretations (refs. 6-11; for review, see ref. 12).We have used a nontransformed human keratinocyte cell line, HaCaT, and normal human skin keratinocytes (NHKs) as in vitro models to study expression of the 5-LO pathway in extrahematopoietic epithelia. HaCaT cells are derived from a normal skin biopsy and maintain a substantial differentiation potential in culture (13,14). In this report we show that the 5-LO gene is expressed in both HaCaT cells and NHKs under conditions that favor their differentiation. MATERIALS AND METHODS

We studied expression of the 5-lipoxygenase (5-LO) pathway in normal human skin. In situ hybridization revealed a 5-LO mRNA-containing epidermal cell (EC) population that was predominantly located in the midportion of the spinous layer, in outer hair root sheaths, and in the epithelial compartment of sebaceous glands. Products of the 5-lipoxygenase (5-LO; arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) pathway, including leukotriene (LT) B4 and the cysteinyl LTs, are considered potent mediators of allergic and hypersensitivity reactions and may also act as physiological autocrine͞paracrine signaling molecules (1). Several white blood cell lineages have been reported to express the 5-LO gene (1), whereas its occurrence in extrahematopoietic tissues has received less attention. We reported that the human keratinocyte cell line, HaCaT, can be induced to up-regulate the 5-LO gene when maintained for prolonged time periods in culture medium supplemented with fetal calf serum (2). Other experiments indicated that normal human epidermal keratinocytes can also be induced to express 5-LO under similar in vitro conditions (2). These data raised the possibility that skin keratinocytes in vivo express the 5-LO gene during their normal differentiation program or that it is induced in response to cytokines generated in diseased skin. To address these issues we decided to focus attention on normal human skin and to initially study epidermis, the outermost skin compartment (3). Epidermis is a highly organized stratified squamous epithelium consisting of keratinocytes at different stages of differentiation as its major cellular constituent. Moreover, epidermis harbors several numerically minor cell populations including melanocytes, Langerhans cells (LCs), Merkel cells, and occasionally white blood cells (4, 5). In the report detailed below we used in situ hybridization (ISH), immunohistochemistry, flow cytometry, and cell purification methods to identify the lineage(s) of 5-LO positive cells in the normal unperturbed epidermis. We found that the physiological keratinocyte differentiation pathway is not associated with up-regulation of the 5-LO gene but that LCs are the major, and possibly sole, 5-LO expressing cells in the normal human epidermis.