Cell density and cell shape-related regulation of vimentin and cytokeratin synthesis : Inhibition of vimentin synthesis and appearance of a new 45 kD cytokeratin in dense epithelial cell cultures

The pattern of the intermediate type filament protein synthesis was examined in cultured bovine mammary gland epithelial (BMGE) cells under conditions of varied cell shape and cell-cell contact. In dense monolayer and suspension cultures BMGE cells expressed a new cytokeratin of 45 kD identified as a member of the acidic subfamily of cytokeratins. This polypeptide has a phosphorylated component and is dissociated from the cytokeratins complex in the presence of 6.5 M urea. The mRNA of the new cytokeratin accumulated in dense cell cultures, as revealed by in vitro translation in a cell-free system. In BMGE-H cells that express also vimentin, the synthesis of vimentin decreased dramatically in dense cell cultures, while the synthesis of the 45 kD cytokeratin was maximal under these conditions. The results suggest that the expression of certain cytokeratins and that of vimentin can be coordinately regulated by factors in the cellular environment that effect cell shape and cell surface contacts.     

Differential control of cytokeratins and vimentin synthesis by cell- cell contact and cell spreading in cultured epithelial cells

The expression of cytokeratins and vimentin was investigated in Madin- Darby bovine epithelial cells (MDBK) in culture under conditions of varied cell spreading and cell-cell contact. When extensive cell-cell contact was achieved by seeding cells at high density in monolayer, or in suspension culture in which multicellular aggregates formed, the cells synthesized high levels of cytokeratins and low levels of vimentin. In contrast, in sparse monolayer and suspension cultures where cell-cell contact was minimal, the cells synthesized very low levels of cytokeratins. The level of vimentin synthesis was high in sparse monolayer culture and was low in both sparse and dense suspension cultures. The ratio of cytokeratin to vimentin synthesis was not affected during the cell cycle, or when cell growth was inhibited by ara C and in serum-starvation-stimulation experiments. The variations in the synthesis of cytokeratins and vimentin under the various culture conditions were also reflected at the level of mRNA activity in a cell-free in vitro translation system and as determined by RNA blot hybridization with cDNA to vimentin and cytokeratins. The results suggest that control of cytokeratin synthesis involves cell- cell contact, characteristic of epithelia in vivo, while vimentin synthesis responds to alterations in cell spreading.     

Induction of cytokeratin expression in human mesenchymal cells

We studied the phenotypic features of some typical human mesenchymal cells, including decidual stromal cells and adult and fetal fibroblasts under different cell culture conditions by using antibodies to intermediate filament proteins and desmoplakins. In cell culture, the decidual stromal cells rapidly acquired typical fibroblastoid appearance with abundant arrays of vimentin filaments while the cytokeratin-positive epithelial cells, occasionally found in typical epithelioid colonies, lacked vimentin positivity and showed desmoplakin positivity. Within a few days, many of the stromal cells started to present cytokeratin positivity when cultured either in Condimed or in Chang medium. The cytokeratin positivity was first detected in small, scattered cytoplasmic dotted fibrils or in perinuclear dotlike aggregates with fibrillar projections. Later, denser cytokeratin-positive fibrillar arrays could also be seen in stromal cells, which lacked desmoplakin positivity as judged by two monoclonal antibodies. Decidual stromal cells were also cloned and in five out of ten clones some of the cells acquired a similar cytokeratin positivity when transferred into Chang or Condimed medium. Immunoblotting results indicated that cytokeratins 8, 18, and 19 can be found in these cultures. Similar cytokeratin positivity could also be seen in the same culture conditions in cultured fetal fibroblasts from skin, chorionic villi, and lung but not in young or adult skin fibroblast cultures. The present results suggest that decidual stromal cells as well as some embryonal mesenchymal cells can acquire epithelial differentiation in vitro as judged by the emergence of cytokeratin proteins. This ability appears to be lost in the corresponding adult cell. The results furthermore suggest that cytokeratin fibrils can be organized in the cytoplasm without an apparent organization center and that neither the appearance of desmoplakins nor the formation of cell-to-cell contacts are required for cytokeratin filament assembly.     

Regulation of myeloperoxidase gene expression by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate in human leukemia HL-60 cells

Myeloperoxidase synthesis during induction of differentiation of human promyelocytic leukemia HL-60 cells by 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied. Differentiation was characterized by morphological changes, arrest of cell proliferation, development of cell adherence, and increased secretion of lysozyme. The cellular myeloperoxidase activity decreased early during induction of differentiation by TPA. Pulse-labeling experiments indicated that the rate of myeloperoxidase synthesis decreased to an undetectable level in cells exposed to TPA for 22 h. The relative amounts of myeloperoxidase mRNA in TPA-treated and untreated cells were determined by measuring translatable mRNA activity in a reticulocyte lysate system. Reduction in the myeloperoxidase mRNA level was observed as early as after 3 h treatment with TPA, and no myeloperoxidase mRNA was detected after 24 h. Time course experiments indicated that the time required for 50% reduction of myeloperoxidase mRNA in TPA-treated cells was approximately 5 h. These results suggest that TPA induces decrease of myeloperoxidase activity in HL-60 cells at a pretranslational level.     

Evidence of protein kinase C involvement in phorbol diester-stimulated arachidonic acid release and prostaglandin synthesis

Many stimulators of prostaglandin production are thought to activate the Ca2+- and phospholipid-dependent protein kinase first described by Nishizuka and his colleagues (Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T., and Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695. In this paper we report evidence that the activation of protein kinase C caused by 12-O-tetradecanoylphorbol-13-acetate (TPA) is involved in the increased prostaglandin production induced by 12-O-tetradecanoylphorbol-13-acetate in Madin-Darby canine kidney (MDCK) cells. We have shown that TPA activates protein kinase C in MDCK cells with similar dose response curve as observed for TPA induction of arachidonic acid release in MDCK cells. Activation of protein kinase C was associated with increased phosphorylation of proteins of 40,000 and 48,000 daltons. We used two compounds (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OMe) and 1-(5-isoquinolinesulfonyl)piperazine) known to inhibit protein kinase C by different mechanisms to further examine if activation of protein kinase C was involved in the increased synthesis of prostaglandins in TPA-treated MDCK cells. We found that both compounds inhibited protein kinase C partially purified from MDCK cells and that ET-18-OMe inhibited the phosphorylation of proteins by protein kinase C in the intact cells. Addition of either compound during or after TPA treatment decreased both release of arachidonic acid from phospholipids and prostaglandin synthesis. Release of [3H]arachidonic acid from phosphatidylethanolamine in TPA-treated cells was blocked by ET-18-OMe or 1-(5-isoquinolinesulfonyl)piperazine addition. However, arachidonic acid release stimulated by A23187 is not blocked by Et-18-OMe. When assayed in vitro, treatment of cells with Et-18-OMe did not prevent the enhanced conversion of arachidonic acid into prostaglandins induced by pretreatment of cells with TPA. Our results suggest that the stimulation of phospholipase A2 activity by TPA occurs via activation of protein kinase C by TPA.     

K562 erythroleukemia cells express cytokeratins 8, 18, and 19 and epithelial membrane antigen that disappear after induced differentiation.

The effects of differentiation-modulating drugs were studied on the expression of intermediate filaments (IFs) in the human K562 erythroleukemic cell line. The untreated cells contained typical cytoplasmic coiling bundles, positive for both vimentin and cytokeratin as judged by indirect immunofluorescence microscopy with monoclonal antibodies (Mabs). Some of the cells also showed bright immunoreactivity for epithelial membrane antigen (EMA), as revealed with a Mab and polyclonal antiserum. When exposed to hemin or to sodium butyrate, most of the cells became cytokeratin negative within 3 days and showed dispersion of vimentin fibrils. Upon exposure to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), the amount of both vimentin and cytokeratin appeared to be greatly increased within 3 days and was found both in dispersed cytoplasmic fibrils, in large spherical, eccentric aggregates, as well as in cytoplasmic fibrils in cells spreading on fibronectin. TPA induced a complete loss of proliferation, as judged by immunostaining with the Mab Ki-67. The effects of TPA were found to be irreversible and could be induced by only a short exposure to the drug. Western blotting analysis and monoclonal antibodies to individual cytokeratins revealed that untreated K562 cells expressed Mr 52,000 (No. 8), 46,000 (No. 18), and 40,000 (No. 19) cytokeratin polypeptides, which disappeared when the cells were exposed to hemin or to sodium butyrate to induce erythroid differentiation but were greatly enhanced when exposed to TPA. The monoclonal anti EMA antibody reacted in K562 cells with a single Mr 320,000 polypeptide that was also revealed in MCF-7 breast carcinoma cells. Human bone marrow cells or other leukemic cell lines with erythroid differentiation capacity (HEL and KG-1) did not contain cytokeratin- or EMA-immunoreactive cells, suggesting that in K562 cells these properties may rather represent abnormal cytodifferentiation or retrodifferentiation toward early embryonic mesenchymal cells, than a more general expression of epithelial features in human leukemic cells.     

Tissue type-specific expression of intermediate filament proteins in a cultured epithelial cell line from bovine mammary gland

Different clonal cell lines have been isolated from cultures of mammary gland epithelium of lactating cow’s udder and have been grown in culture media containing high concentrations of hydrocortisone, insulin, and prolactin. These cell (BMGE+H), which grow in monolayers of typical epithelial appearance, are not tightly packed, but leave intercellular spaces spanned by desmosomal bridges. The cells contain extended arrays of cytokeratin fibrils, arranged in bundles attached to desmosomes. Gel electophoresis show that they synthesize cytokeratins similar, if not identical, to those found in bovine epidermis and udder, including two large (mol wt 58,500 and 59,000) and basic (pH range: 7-8) and two small (mol wt 45,500 and 50,000) and acidic (pH 5.32 and 5.36) components that also occur in phosphorylated forms. Two further cytokeratins of mol wts 44,000 (approximately pH 5.7) and 53,000 (pH 6.3) are detected as minor cytokeratins in some cell clones. BMGE+H cells do not produce vimentin filaments as determined by immunofluorescence microscopy and gel electrophoresis. By contrast, BMGE-H cells, which have emerged from the same original culture but have been grown without hormones added, are not only morphologically different, but also contain vimentin filaments and a different set of cytokeratins, the most striking difference being the absence of the two acidic cytokeratins of mol wt 50,000 and 45,500. Cells of the BMGE+H line are characterized by an unusual epithelial morphology and represent the first example of a nonmalignant permanent cell line in vitro that produces cytokeratin but not vimentin filaments. The results show that (a) tissue-specific patterns of intermediate filament expression can be maintained in permanent epithelial cell lines in culture, at least under certain growth conditions; (b) loss of expression of relatively large, basic cytokeratins is not an inevitable consequence of growth of epithelial cells in vitro. Our results further show that, during culturing, different cell clones with different cytoskeletal composition can emerge from the same cell population and suggest that the presence of certain hormones may have an influence on the expression of intermediate filament proteins.     

Differences in phorbol-ester-induced down-regulation of protein kinase C between cell lines.

Down-regulation of protein kinase C induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) was examined in Swiss 3T3, V79, MDBK and C6 cells by Western blotting. Variations in the rate of down-regulation caused by treatment with 100 nM-TPA were observed; TPA treatment for 5 h caused maximal down-regulation in V79 cells, whereas TPA treatment for 10 h or 30 h was needed for maximal down-regulation of protein kinase C in MDBK or Swiss 3T3 cells respectively. The decrease in amount of immunologically detectable protein kinase C was 30% in MDBK cells and 100% in V79 and Swiss 3T3 cells. MDBK and C6 cells could be completely depleted of protein kinase C by treatment with 250 nM-TPA. In C6 cells, after treatment with 500 nM-TPA, an 80% loss of protein kinase C was seen over 10 h. Measurement of the numbers of phorbol-ester-binding sites remaining in each cell line when protein kinase C was maximally down-regulated indicated that in MDBK and Swiss 3T3 cells loss of phorbol-ester-binding sites paralleled loss of protein kinase C, whereas in V79 and C6 cells no such correlation was observed.     

Subcellular localization of tissue polypeptide antigen and cytokeratins in epithelial cells (salivary ad mammary glands). Combined use of the cryoultramicrotomy and the protein A-gold technique

Epithelial cells from various sites and at various stages of differentiation reveal distinct cytokeratin polypeptide patterns. WE have localized these heterogeneous elements at the subcellular level in human salivary glands and in a solid tumor of the breast using a monoclonal and a polyclonal antibody against cytokeratin, and an antibody against tissue polypeptide antigen (TPA) which seems to be related to some cytokeratins. Labeling by the cytokeratin antibodies was more intense in squamous and duct cells than in acinar cells. The TPA:B1 antibody reacted predominantly with duct cells and to a lesser extent with acinar and squamous cells. A precise evaluation of the labeling pattern and a well-preserved cell structure appeared to be important factors in obtaining more detailed information about intermediate filament proteins. The cryoultramicrotomy and the protein A-gold technique are suitable for these studies.     

Phorbol ester-induced regulation of transferrin receptors in human leukemia K562 cells

The treatment of human leukemia K562 cells with 12-0-tetradecanoyl phorbol-13-acetate (TPA) caused a decrease of transferrin receptors. The mechanism of the decrease of the receptors with TPA has been investigated. In cells incubated with TPA, the rate of biosynthesis of transferrin receptors was reduced to 10-20% of that in untreated cells. Pulse-chase experiments showed that turnover of the receptors in TPA-treated cells was accelerated over that in untreated cells. These results indicated that the decrease of transferrin receptors in TPA-treated cells was caused by reduced biosynthesis and accelerated degradation of the receptors.     

What determines differentiation of urothelial umbrella cells?

Cytokeratins, uroplakins and the asymmetric unit membrane are biochemical and morphological markers of urothelial differentiation. The aim of our study was to follow the synthesis, subcellular distribution and supramolecular organization of differentiation markers, cytokeratins and uroplakins, during differentiation of umbrella cells of mouse bladder urothelium. Regenerating urothelium after destruction with cyclophosphamide was used to simulate de-novo differentiation of cells, which was followed from day 1 to day 14 after cyclophosphamide injection. Cytokeratin 7 and uroplakins co-localized in the subapical cytoplasm of superficial cells from the early stage of differentiation on. At early stages of superficial cell differentiation cytokeratin 7 was filamentary organized, and rare uroplakins were found on the membranes of relatively small cytoplasmic vesicles, which were grouped in clusters under the apical membrane. Later, cytokeratin 7 gradually reorganized into a continuous trajectorial network, and uroplakins became organized into plaques of asymmetric unit membrane, which formed fusiform vesicles. After insertion of fusiform vesicles into the apical plasma membrane, the surface acquired microridged appearance of umbrella cells. Cytokeratin 20 appeared as the last differentiation marker of umbrella cells. Cytokeratin 20 was incorporated into the pre-existing trajectorial cytokeratin network. These results indicate that differentiation of urothelial cells starts with the synthesis of differentiation-related proteins i.e., cytokeratins and uroplakins, and later with their specific organization. We consider that the umbrella cell has reached its final stage of differentiation when uroplakins form plaques of asymmetric unit membrane that are inserted into the apical plasma membrane and when cytokeratin 20 becomes included in a trajectorial cytokeratin network in the subapical area of cytoplasm.     

Regulation of arachidonic acid metabolism in Madin-Darby canine kidney cells. Comparison of A23187 and 12-O-tetradecanoyl-phorbol-13-acetate

Challenge of Madin-Darby canine kidney (MDCK) cells with the divalent cation ionophore A23187 caused a marked increase in the deacylation of [3H]arachidonic acid but not of [14C]palmitic acid. When the cells were treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and A23187, there was an additional increase in the deacylation of [3H]arachidonic acid compared to that observed with either agent alone. In contrast to deacylation, the stimulation of prostaglandin production by A23187 was small compared to the stimulation by TPA. Cycloheximide inhibited synthesis of prostaglandins in TPA-treated cells, but did not block the stimulated deacylation caused by either TPA or A23187. These data indicate that, while both TPA and A23187 stimulated the deacylation of [3H]arachidonic acid, TPA had an additional, cycloheximide-sensitive effect that was required for efficient conversion of the release fatty acids to prostaglandins. Thus, although required, deacylation appeared to be independent of and insufficient to stimulate maximum prostaglandin synthesis in these cells.     

K562 cells induced to differentiate by phorbol ester tumor promotors resist NK lysis

The effect of various phorbols and phorbol diesters on the NK sensitivity of the human leukemic K562 cells was studied. A marked decrease in K562 cell susceptibility was achieved by culture in the presence of either 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or beta-phorbol-dibutyrate. The maximum protection against NK lysis was achieved when K562 cells were cultured in the presence of 160 nM TPA for 48 hr (mean percentage inhibition: 61% of specific lysis). As for untreated targets, the residual killing of K562 cells after TPA treatment was mediated through large granular lymphocytes (LGL). The experimental procedures required to achieve maximal NK protection with TPA resulted simultaneously in marked phenotypical changes in K562 cells: erythroid and early myeloid markers decreased, whereas the expression of megakaryocytic markers was increased as shown by staining with antiplatelet monoclonal antibodies and assessment of platelet peroxidase activity. Chemical phorbol analogs which were unable to induce K562 cell differentiation did not affect K562 cell sensitivity to NK lysis. De novo protein synthesis is involved in the TPA-induced NK resistance, since this effect was abolished by pretreatment of K562 cells with actinomycin D or cycloheximide. TPA has been previously demonstrated to reduce NK effector activity. In our data however, the observed TPA effects were not due to release of TPA acting on effector cells during the NK assay since TPA-treated K562 cell supernatants were unable to inhibit NK activity in control assays. Thus, TPA appears to decrease NK killing of malignant cells, both by depressing NK effector cells functions and by reducing the susceptibility to NK lysis of the target cells. In single-cell agarose assays, TPA-treated K562 cells demonstrated reduced NK-binding capacity and reduced sensitivity to lysis after binding. These defects could not be reversed by activation of the NK effector cells with interferon. The results here reported extend the previously suggested relations between the expression of NK-target structures and the differentiation stage of malignant cells.     

Up-regulation of ceramide glucosyltransferase during the differentiation of U937 cells

The phorbol ester tetradecanoylphorbol acetate (TPA) induces promyelocytic leukaemia cells to differentiate to macrophage-like cells in vitro. During the course of this differentiation, the cells adhere to the bottom of the culture dish, a process that requires an increase in cell surface glycosphingolipids (GSLs). We examined the cellular content of glucosylceramide (GlcCer), the simplest of the GSLs, in a TPA-treated leukaemia cell line, U937. Following TPA treatment, we observed a 3.5-fold increase in GlcCer levels that was caused by enhanced activity of ceramide glucosyltransferase (GlcT-1), which catalyses ceramide glycosylation. Furthermore, in TPA-treated cell GlcT-1 amounts were increased at both the mRNA and protein levels. We also found decreased activity of lactosylceramide synthase in TPA-treated cells, which could also contribute to the increase in cellular GlcCer content.     

Enhancer elements directing cell-type-specific expression of cytokeratin genes and changes of the epithelial cytoskeleton by transfections of hybrid cytokeratin genes.

The cytokeratins, which form the intermediate filaments (IFs) characteristic of epithelial cells, are encoded by a large family of genes whose members are differentially expressed in patterns different in the various kinds of epithelia. To identify possible cis-regulatory DNA elements involved in the cell-type-specific expression of these genes, we examined, in transfection assays, 5' upstream sequence intercepts of a certain cytokeratin gene, i.e. that for bovine cytokeratin IV* (CKIV*), in combination with the coding portions of either the chloramphenicol acetyltransferase (CAT) gene or other cytokeratin genes. A 5' upstream region located between the cap-site and nucleotide -605 was found to enhance the specific expression of these reporter genes in bovine mammary gland-derived BMGE + H cells, which express the endogenous gene, but not in bovine kidney epithelium-derived MDBK cells which synthesize cytokeratins other than IV*. This epithelium-type-specific expression was also observed in heterologous combinations, e.g. in murine keratinocytes, but not in other murine cell lines such as 3T3 fibroblasts. When a fragment located between -180 and -605 was coupled to the HSV-TK promoter it stimulated the expression of the reporter gene in a cell-type-specific manner. The enhancer character of this 425 nucleotide long region is also demonstrated. Moreover, the CKIV* promoter/enhancer complex was able to direct the expression of epidermal cytokeratins characteristic for suprabasal differentiation, i.e. bovine cytokeratins Ia and VIb, in cells that normally do not express these genes. We show that the newly synthesized cytokeratins integrate into the pre-existing cytokeratin IF system of the transfected cells and that the forced expression of one of these cytokeratins does not induce the endogenous gene encoding its normal pair partner.     

Role of protein kinase C in the translational regulation of lipoprotein lipase in adipocytes

The hypertriglyceridemia of diabetes is accompanied by decreased lipoprotein lipase (LPL) activity in adipocytes. Although the mechanism for decreased LPL is not known, elevated glucose is known to increase diacylglycerol, which activates protein kinase C (PKC). To determine whether PKC is involved in the regulation of LPL, we studied the effect of 12-O-tetradecanoyl phorbol 13-acetate (TPA) on adipocytes. LPL activity was inhibited when TPA was added to cultures of 3T3-F442A and rat primary adipocytes. The inhibitory effect of TPA on LPL activity was observed after 6 h of treatment, and was observed at a concentration of 6 nM. 100 nM TPA yielded maximal (80%) inhibition of LPL. No stimulation of LPL occurred after short term addition of TPA to cultures. To determine whether TPA treatment of adipocytes decreased LPL synthesis, cells were labeled with [35S]methionine and LPL protein was immunoprecipitated. LPL synthetic rate decreased after 6 h of TPA treatment. Western blot analysis of cell lysates indicated a decrease in LPL mass after TPA treatment. Despite this decrease in LPL synthesis, there was no change in LPL mRNA in the TPA-treated cells. Long term treatment of cells with TPA is known to down-regulate PKC. To assess the involvement of the different PKC isoforms, Western blotting was performed. TPA treatment of 3T3-F442A adipocytes decreased PKC alpha, beta, delta, and epsilon isoforms, whereas PKC lambda, theta, zeta, micro, iota, and gamma remained unchanged or decreased minimally. To directly assess the effect of PKC inhibition, PKC inhibitors (calphostin C and staurosporine) were added to cultures. The PKC inhibitors inhibited LPL activity rapidly (within 60 min). Thus, activation of PKC did not increase LPL, but inhibition of PKC resulted in decreased LPL synthesis by inhibition of translation, indicating a constitutive role of PKC in LPL gene expression.     

The pattern of cytokeratin synthesis is a marker of type 2 cell differentiation in adult and maturing fetal lung alveolar cells

During the last stages of fetal life, the immature epithelial cells of the rat lung alveolus develop the properties of mature type 2 cells. Adult type 2 cells rapidly lose these same properties when isolated and maintained in cell culture. We have examined the synthesis of cytokeratin proteins by adult type 2 cells as they lose their differentiated characteristics during 1 week in culture, and of immature fetal alveolar epithelial cells as they differentiate either in utero or when cultured on an extracellular matrix. Freshly isolated adult type 2 cells synthesize four cytokeratins which by electrophoretic mobilities and Western blot analysis correspond to human cytokeratins Nos. 7, 8, 18, and 19. During 7 days in culture synthesis of cytokeratin No. 19 is dramatically decreased and cytokeratin No. 18 becomes the predominant acidic cytokeratin produced. Fetal lung epithelial cells at 18 days gestation lack most characteristics of mature type 2 cells. When freshly isolated, these cells synthesize cytokeratins Nos. 7, 8, and 18 but make only minimal amounts of cytokeratin No. 19. When these cells are allowed to mature either in utero or in culture on a whole basement membrane extract, they develop both the morphological characteristics and the pattern of cytokeratin synthesis of fully developed type 2 cells, with cytokeratins No. 19 being the major acidic cytokeratin produced.     

Regulation of prostaglandin H synthase activity in dog urothelial cells.

TPA regulation of prostaglandin H synthase activity in primary and subcultured dog urothelial cells was investigated. Previous studies have demonstrated an early (0-2 hr) increase in PGE2 synthesis mediated by TPA which is dependent upon release of endogenous arachidonic acid by a phospholipase-mediated pathway. In this study, prostaglandin H synthase activity was assessed directly with microsomes and indirectly after addition of exogenous arachidonic acid at a maximum effective concentration (100 microM) to media. PGE2 synthesis, measured by radioimmunoassay, served as an index of prostaglandin H synthase activity. After a 24-hr incubation with 0.1 microM TPA or 1.0 microM A23187, arachidonic acid elicited significantly more PGE2 synthesis in agonist-treated cells than it did in control cells in primary culture. Microsomes from 24-hr TPA-treated cells exhibited significantly more prostaglandin H synthase activity than did those from control cells. In addition, the PGE2 content of overnight media was approximately 10-fold greater in TPA-treated cells than in control cells. The late (24 hr) response was more sensitive to lower concentrations of TPA than was the earlier (0-2 hr) response. TPA at 0.1 microM was a maximum effective dose for both responses. The 24-hr response was blocked by cycloheximide and staurosporine, inhibitors of protein synthesis and protein kinase C, respectively. Pretreatment of cells with aspirin, an irreversible inhibitor of prostaglandin H synthase, prior to addition of TPA did not prevent the late TPA-mediated increase in PGE2 synthesis. Subcultured cells exhibited both an early and a late TPA response. Only the early response was inhibited by aspirin pretreatment. Results suggest that the late response with TPA is caused by de novo synthesis of prostaglandin H synthase. Thus, primary and subcultured dog urothelial cells possess two distinct mechanisms for regulating signal transduction by arachidonic acid metabolism. This study provides a basis for assessing these mechanisms of signal transduction in urothelial cell lines and transformed cells.     

Involvement of desmoplakin phosphorylation in the regulation of desmosomes by protein kinase C, in HeLa cells.

In the present study, we have examined how modulation of protein kinase C (PKC) activity affected desmosome organization in HeLa cells. Immunofluorescence and electron microscopy showed that PKC activation upon short exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in a reduction of intercellular contacts, splitting of desmosomes and dislocation of desmosomal components from the cell periphery towards the cytoplasm. As determined by immunoblot analysis of Triton X-100-soluble and -insoluble pools of proteins, these morphological changes were not correlated with modifications in the extractability of both desmoglein and plakoglobin, but involved almost complete solubilization of the desmosomal plaque protein, desmoplakin. Immunoprecipitation experiments and immunoblotting with anti-phosphoserine, antiphosphothreonine and anti-phosphotyrosine antibodies revealed that desmoplakin was mainly phosphorylated on serine and tyrosine residues in both treated and untreated cells. While phosphotyrosine content was not affected by PKC activation, phosphorylation on serine residues was increased by about two-fold. This enhanced serine phosphorylation coincided with the increase in the protein solubility, suggesting that phosphorylation of desmoplakin may be a mechanism by which PKC mediates desmosome disassembly. Consistent with the loss of PKC activity, we also showed that down-modulation of the kinase (in response to prolonged TPA treatment) or its specific inhibition (by GF 109203X) had opposite effects and increased desmosome formation. Taken together, these results clearly demonstrate an important role for PKC in the regulation ofdesmosomal junctions in HeLa cells, and identify serine phosphorylation of desmoplakin as a crucial event in this pathway.