5-Lipoxygenase expression in Langerhans cells of normal human epidermis.

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. Examination of skin specimens by immunohistochemistry and of primary ECs by flow cytometry mapped the 5-LO protein exclusively to Langerhans cells (LCs). The LC 5-LO protein was largely found in the nuclear matrix, in nuclear envelopes, and perinuclear regions as indicated by in situ confocal laser scan microscopy. Reverse transcription-PCR and immunoblot analyses of purified primary EC populations further indicated that LCs are major 5-LO expressing cells. Enriched primary LCs were also found to contain 5-LO activating protein (FLAP), leukotriene (LT) C4 synthase, and LTA4 hydrolase. By contrast, 5-LO, FLAP, and LTC4 synthase were undetectable or largely reduced, but LTA4 hydrolase transcripts and protein were identified in ECs depleted of LCs. These data show that naive LCs are major, and possibly the sole, 5-LO pathway expressing cells in the normal human epidermis.

Expression of 5-lipoxygenase in differentiating human skin keratinocytes.

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.

The arachidonic acid cascade, eicosanoids, and signal transduction.

Eicosanoid biosynthesis in animal cells either results from agonist-stimulated phospholipase activation (endogenous pathway) or from lipoprotein receptor-mediated uptake and lysosomal lipid hydrolase-dependent release of AA (exogenous pathway) (see Fig. 1 for schematic representation). LDL stimulates eicosanoid formation through delivery of substrate AA to enzymes of oxidative AA metabolism. The classical LDL receptor is a control point of the effects of LDL AA on eicosanoid formation in different tissues: LDL AA metabolism occurs in several cell types of mesenchymal and epithelial origin and generates the formation of distinct eicosanoid patterns in each case. The LDL AA pathway does appear to couple directly to the PGH synthase reaction, whereas it does not couple directly to the 5-lipoxygenase reaction. We expect that a more complete characterization of the LDL unsaturated fatty acid pathway in different tissue will yield additional information on the biochemistry of lipoproteins, AA, and eicosanoids.

Lipoprotein-dependent unsaturated fatty acid transport and metabolism in cultured cells.

Our results can be summarized as follows. LDL stimulates eicosanoid formation through delivery of substrate AA to enzymes of oxidative AA metabolism. The classical LDL receptor controls the effect of LDL AA on eicosanoid formation. LDL AA metabolism occurs in several cell types of mesenchymal and epithelial origin and generates the formation of distinct eicosanoid patterns in a tissue-specific way. LDL inhibits the PGH synthase, and the LDL-dependent inhibition of the enzyme resembles the inhibition by unesterified AA. The LDL AA pathway does appear to couple directly to the PGH synthase reaction, but it does not appear to couple directly to the 5-lipoxygenase reaction. We expect that a more complete characterization of the LDL unsaturated fatty acid pathway in different tissues will yield additional information on the biochemistry of both lipoproteins and AA metabolism.

Differential low density lipoprotein receptor-dependent formation of eicosanoids in human blood-derived monocytes.

We studied the ability of low density lipoproteins (LDLs) to provide arachidonic acid (AA) for eicosanoid biosynthesis in human blood-derived monocytes. When incubated in the presence of reconstituted LDL that contained cholesteryl [1-14C]arachidonate (recLDL-[14C]AA-CE), resting monocytes formed three labeled products of the prostaglandin (PG) H synthase pathway: 6-keto-PGF1 alpha, thromboxane B2, and PGE2. The amounts of these eicosanoids in response to recLDL-[14C]AA-CE were comparable to or exceeded those that were produced in response to the addition of 10 microM unesterified [1-14C]AA. By contrast, resting monocytes formed only small amounts of products of the 5-lipoxygenase pathway, leukotriene (LT) B4 and LTC4 from either recLDL-[14C]AA-CE or [14C]AA, indicating preferential utilization of AA in the PGH synthase reaction. However, they converted LDL-derived [14C]AA efficiently into LTB4 and LTC4, when they were first incubated with recLDL-[14C]AA-CE and subsequently stimulated with the chemotactic peptide N-formylmethionylleucylphenylalanine or the Ca2+ ionophore A23187. The classical LDL receptor pathway mediated the synthesis of all of the above eicosanoids from LDL but not from unesterified AA. These results demonstrate that the LDL receptor pathway preferentially promotes the synthesis of PGH synthase products in resting human blood-derived monocytes and that an additional mechanism is required to promote effective synthesis of 5-lipoxygenase pathway products from AA that originates in LDL cholesteryl esters.

LDL receptor-dependent polyunsaturated fatty acid transport and metabolism.

It is widely assumed that eicosanoid biosynthesis is initiated by an increase in the intracellular concentration of unesterified arachidonic acid (AA) as a consequence of the activation of cellular phospholipases and/or inhibition of AA reacylation reactions. Here, we describe a mechanism of eicosanoid formation that is entirely dependent on low density lipoprotein (LDL) receptor-mediated delivery of AA to eicosanoid producing target cells. This LDL AA pathway introduces a new regulatory component into the provision of unsaturated fatty acids to mammalian cells.

A new role for the low density lipoprotein receptor.

It is well established that the low density lipoprotein (LDL) pathway functions to maintain a constant concentration of cellular cholesterol, but LDL effects that are unrelated to cholesterol metabolism have not been studied in great detail. In the present investigation we demonstrate that the LDL receptor pathway regulates cellular levels of free arachidonic acid (AA) and hence prostaglandin (PG) synthesis. We used platelet-derived growth factor (PDGF)-stimulated fibroblasts as a model system to investigate mechanism of LDL-dependent PG synthesis. PDGF-stimulated but not quiescent cells formed radiolabelled prostacyclin (PGI2) and PGE2 upon incubation with LDL that had been reconstituted with cholesteryl-(1-14C)-arachidonate (rec-LDL), while fibroblasts from patients that are afflicted with the LDL receptor negative phenotype of familial hypercholesterolaemia (FH) failed to synthesize significant amounts of PGs. Furthermore cells that had been preincubated with chloroquine or an anti LDL receptor antibody, that prevents binding of LDL to its receptor, did not produce significant amounts of PGs upon incubation with rec-LDL. Moreover incubation of PDGF-stimulated cells with LDL or AA led to a time and concentration-dependent inactivation of PGH synthase, the rate limiting enzyme of PG synthesis. When taken together our results establish a new role of the classical LDL receptor pathway of Brown and Goldstein by demonstrating that LDL provides AA to fibroblasts for eicosanoid formation and that LDL has a profound inhibitory effect on the key enzyme of PG synthesis, the PGH synthase.

The LDL receptor pathway delivers arachidonic acid for eicosanoid formation in cells stimulated by platelet-derived growth factor.

Animal cells can convert 20-carbon polyunsaturated fatty acids into prostaglandins (PGs) and leukotrienes. These locally produced mediators of inflammatory and immunological reactions act in an autocrine or paracrine fashion. Arachidonic acid (AA), the precursor of most PGs and leukotrienes, is present in the form of lipid esters within plasma lipoproteins and cannot be synthesised de novo by animal cells. Therefore, AA or its plant-derived precursor, linoleic acid, must be provided to cells if PGs or leukotrienes are to be formed. Because several classes of lipoproteins, including low-density lipoproteins (LDL), very-low-density lipoproteins, and chylomicron remnants, are taken up by means of the LDL receptor, and because LDL and very-low-density lipoproteins, but not high-density lipoproteins, stimulate PG synthesis, we have suggested previously that PG formation is directly linked to the LDL pathway. Using fibroblasts with the receptor-negative phenotype of familial hypercholesterolaemia and anti-LDL receptor antibodies, we show here that LDL deliver AA for PG production and that an LDL receptor-dependent feedback mechanism inhibits the activity of PGH synthase, the rate-limiting enzyme of PG synthesis. These results indicate that the LDL pathway has a regulatory role in PG synthesis, in addition to its well-known role in the maintenance of cellular cholesterol homeostasis.

Platelet-derived growth factor--a growth factor with an expanding role in health and disease.

Platelet-derived growth factor (PDGF) is the principal mitogen for connective tissue-derived cells such as fibroblasts, smooth muscle cells, and glial cells. It is synthesized by a variety of cell types and the synthesis of PDGF and its receptors is tightly controlled. Accumulating evidence obtained in vitro and in vivo suggests that PDGF plays important roles in the pathogenesis of clinically important diseases such as atherogenesis and cancer. Moreover, PDGF is an important research tool to study the signal transmission pathway of growth factors and other hormones.

Structure, chromosome mapping and regulation of the mouse zinc-finger gene Krox-24; evidence for a common regulatory pathway for immediate-early serum-response genes.

The structure of Krox-24, a mouse zinc-finger-encoding gene that is transiently activated during G0/G1 transition, has been established. Krox-24 is located on mouse chromosome 18, bands C-D. The gene product, as anticipated for a putative DNA-binding protein, is localized within the cell nucleus. The Krox-24 5'-flanking region contains a series of serum response elements (SREs) similar to the SRE observed upstream of the c-fos proto-oncogene. These elements can substitute for the c-fos SRE, their effect is cumulative and they bind the same cellular factor, the serum response factor (SRF), as the c-fos SRE. This suggests that the SRE and its cognate protein are likely to be involved in the regulation of Krox-24 and presumably of other immediate-early serum response genes. SRE and SRF therefore constitute key components in the regulatory pathway leading from mitogenic stimulation to cellular proliferation.

Structure, chromosome location, and expression of the mouse zinc finger gene Krox-20: multiple gene products and coregulation with the proto-oncogene c-fos.

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.

Simultaneous light and electron microscopic observation of immunolabeled liver 27 KD gap junction protein on ultra-thin cryosections.

We report on immunolabeling of gap junction protein in rat liver. Simultaneous light and electron microscopic immunolabeling of ultra-thin frozen sections was performed to confirm that the antigenic targets of polyclonal antibodies and a monoclonal 27 KD antibody (12/1 C5) are the gap junctions. Our results clearly demonstrate that the immunoreactive sites determined by indirect immunofluorescence correspond to immunogold-labeled gap junctions identified in the same section according to electron microscopic criteria. Our results also support the concept that the 27 KD protein is a major constituent of gap junctions.

Reduced number of gap junctions in rat hepatocarcinomas detected by monoclonal antibody.

A new rat monoclonal antibody was characterized which recognized the 26K protein in gap junctions of mouse, rat and human liver as shown by immunoblot, indirect immunofluorescence, and immunogold electron microscopy. This monoclonal antibody was used to investigate the abundance of gap junctions in chemically induced rat hepatocarcinomas. In comparison with livers of control animals we found in hepatocarcinomas an average decrease of 71% in the number of gap junctional immunofluorescent spots. A corresponding decrease of the total amount of the 26 K protein was detected by quantitative immunoblot. Changes in the proliferative state as well as in intercellular adhesion of hepatocarcinoma cells in contrast to normal hepatocytes might have contributed to cause this decrease of gap junctions in tumor tissue. Possibly the partial loss of gap junctions provided a selective advantage for those preneoplastic liver cells which developed into rapidly proliferating tumor cells.

Gap junctions in several tissues share antigenic determinants with liver gap junctions.

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 labelling 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.

Cytoplasmic and cell surface structure of purified liver gap junctions revealed by freeze-drying.

After freeze-drying of purified liver gap junction plaques and vesicles the structural features of the inner and outer aspects of purified gap junctions were investigated. No structural details were seen on the cytoplasmic side of the connexions whereas on the cell surface side the connexions were organized in a paracrystalline pattern and exhibited a central depression or pore. We conclude that the central pore through each connexion varies in diameter along its length and that the closing site is located near the cytoplasmic face.

Immunocytochemical localization of the gap junction 26 K protein in mouse liver plasma membranes.

Specific binding sites for anti-26 K antibodies directed against the liver gap junction protein (26 K) were localized by immunoelectron microscopy in gap junction plaques purified from hepatic plasma membranes. Using immunofluorescence microscopy we found discrete fluorescent spots on plasma membranes in cross sections of liver tissues after incubation with anti-26 K antibodies. This is consistent with the notion of specific binding to gap junction plaques. Quantitative binding of anti-26 K antibodies was indirectly measured by the protein A-gold technique. We found that urea/detergent-treated, purified gap junction plaques bind 30-fold more anti-26 K antibodies than preimmune serum. Anti-26 K antibodies also bind specifically to native gap junction plaques within hepatic plasma membranes although only about one fifth as efficiently as to purified plaques. Possibly the anti-26 K antibodies raised after injection of SDS-denatured 26 K protein into rabbits recognize the cytoplasmic face of urea/detergent-treated plaques better than that of native plaques. Some, if not most, of the vesicular structures in preparations of purified plaques appear to be derived from split gap junction plaques and are probably sheets of gap junction hemichannels. In some vesicles the former cytoplasmic face of the hemichannels is turned outside, other vesicles have the former cell surface turned outside. The anti-26 K antibodies do not recognize any 26 K protein on the sheets of partially split gap junction plaques, on the heterogeneous vesicular structures, or on non-junctional areas of hepatic plasma membranes. These results suggest that the conformation of the 26 K protein in plaques must be different from that of the 26 K protein in earlier biosynthetic steps of plaque assembly.