Treatment With 12-O-tetradecanoylphorbol-13-acetate Research Articles

Regulation of Bcl-2 protein expression in human neuroblastoma SH-SY5Y cells: positive and negative effects of protein kinases C and A, respectively.

The regulatory mechanism of Bcl-2 protein expression was investigated in SH-SY5Y cells, the human neuroblastoma cell line that expresses natively Bcl-2 proteins. WHen the cells were treated with 12-O-tetradecanoylphorbol 13-acetate (TPA) or retinoic acid, the level of Bcl-2 protein was increased compared with the control. These effects were inhibited by pretreatment with a protein kinase C (PKC) inhibitor, staurosporine or calphostin C. The level of Bcl-2 protein was also increased by treatment with carbachol, a muscarinic acetylcholine receptor (mAChR) agonist, and the effect were also inhibited by pretreatment with staurosporine or calphostin C. An addition, a carbachol-induced increase in Bcl-2 protein levels and a transient elevation of [Ca2+]i were inhibited by pretreatment with 4-DAMP (4-diphenylacetoxy-N-methylpiperidine), an m3 mAChR antagonist. In contrast, the level of Bcl-2 protein was decreased by treatment with dibutyryl cAMP (diBu-cAMP), forskolin, or cholera toxin, and the effects of diBu-cAMP were inhibited by pretreatment with a protein kinase A (PKA) inhibitor, H-89. From these results, we suggest that the expression of Bcl-2 proteins is regulated by PKC and PKA in positive and negative manners, respectively, in SH-SY5Y cells. Furthermore, the nucleosomal DNA fragmentation induced by serum depletion for 4 h was observed in SH-SY5Y cells when the level of Bcl-2 protein was down-regulated by treatment with 1 mM diBu-cAMP for 3 days, although the DNA fragmentation by serum depletion for 4 h was not observed in nontreatment cells, indicating that Bcl-2 proteins whose expression is regulated by PKC and PKA play important roles in serum depletion-induced apoptosis.

Differential control of 17 alpha-hydroxylase and 3 beta-hydroxysteroid dehydrogenase expression in human adrenocortical H295R cells.

Previous studies of human adrenocortical cells have given inconsistent findings concerning the effects of angiotensin II (AII) alone or in combination with activators of the protein kinase A-signaling pathway on expression of cholesterol side-chain cleavage cytochrome P450 (P450scc), 17 alpha-hydroxylase cytochrome P450 (P450c17), and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), as well as the corresponding effects on adrenocortical cell steroid secretory products. We have used the human adrenocortical carcinoma H295R cell to evaluate further this question and determine the role of protein kinase C in each of these responses to AII. Treatment with AII alone (10 nmol/L, 48 h) resulted in a significant increase in cortisol production (1.8-fold), as well as a much greater effect on aldosterone production. This increased formation of 17 alpha-hydroxysteroids was accompanied by increased expression of P450c17 as determined at the level of messenger RNA (mRNA) and enzyme activity. Similar increases in expression of P450scc were observed at the level of mRNA. Increases in 3 beta-HSD expression were also seen at the level of mRNA and, to a lesser extent, at the level of enzyme activity. Because of the comparatively low basal 17 alpha-hydroxylase and high basal 3 beta-HSD activity of H295R cells, however, the overall effect of AII treatment was actually a rise in the 17 alpha-hydroxylase/3 beta-HSD activity ratio, resulting in increased formation of 17 alpha-hydroxysteroids such as cortisol. Whereas treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) reproduced the effect of AII on 3 beta-HSD expression, TPA failed to reproduce the effects of AII on P450c17 and P450scc and even resulted in a marked decrease in expression of P450c17. Thus, the stimulatory effect of AII alone on P450c17 expression was not mediated via protein kinase C but, like the action of K+, was probably mediated via the Ca(2+)-signaling pathway. Treatment with forskolin (10 mumol/L, 48 h) resulted in a dramatic increase in both cortisol and dehydroepiandrosterone production together with increases in expression of P450c17, P450scc, and 3 beta-HSD as measured at the level of mRNA and activity. Consistent with the increase in 17 alpha-hydroxysteroid formation, the effect on 17 alpha-hydroxylase expression was greater than that on 3 beta-HSD at the level of enzyme activity, so a larger 17 alpha-hydroxylase/3 beta-HSD activity ratio was achieved. Cotreatment with forskolin and AII, however, resulted in a dose-dependent reduction in cortisol and DHEA production concomitant with a marked attenuation of P450scc and P450c17 expression. Although forskolin-induced expression of 3 beta-HSD was not further increased at the level of mRNA by cotreatment with AII, additivity was observed at the level of changes in enzyme activity. Thus, AII cotreatment resulted in a marked reduction of the forskolin-induced increase in 17 alpha-hydroxylase/3 beta-HSD activity ratio, and so, 17 alpha-hydroxysteroid synthesis was attenuated. These effects of AII cotreatment on expression of P450c17 and P450scc were reproduced by cotreatment with TPA (10 nmol/L), suggesting the involvement of protein kinase C in these attenuative responses. Furthermore, the effect of AII cotreatment on changes in forskolin-induced 17 alpha-hydroxylase and 3 beta-HSD activities were blocked by the AII Type 1 (AT1) receptor antagonist DuP753 (Losartan), confirming the involvement of an AT1 receptor-linked phospholipase C in activating protein kinase C.

Independent regulation of matrix metalloproteinases and plasminogen activators in human fibrosarcoma cells.

Serine proteases and matrix metalloproteinases have been shown to often cooperate in multiple physiological and pathological processes associated with changes in the extracellular matrix (ECM). We have examined the interaction between the plasminogen activator (PA)-plasmin system and matrix metalloproteinases (MMPs) in HT1080 human fibrosarcoma cells treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). While TPA treatment evoked a temporary increased expression of urokinase type PA (uPA), the production of both types of human plasminogen activator inhibitors (PAI) was induced and sustained over 12 h by TPA treatment shifting the protease-protease inhibitors balance in favor of the inhibitors. TPA treatment of HT1080 cells induced the expression of interstitial collagenase (MMP-1) and increased the expression of gelatinase B (MMP-9), tissue inhibitor of metalloproteinases-1 (TIMP-1), and MT-MMP, a membrane-bound activator of progelatinase A (proMMP-2), while MMP-2 and TIMP-2 expression were decreased. Increased MT-MMP expression by TPA treatment was associated with increased activation of proMMP-2. These data show that the regulation of PA-plasmin and metalloproteinase and their specific inhibitors is uncoordinated. In addition, inhibition of the PA-plasmin system by PAI-2 or aprotinin did not prevent the activation of proMMP-2 by TPA, suggesting that plasmin is not involved in MT-MMP-mediated activation of proMMP-2.

Effect of diverse tumor promoters on the expression of gap-junctional proteins connexin (Cx) 26, Cx31.1, and Cx43 in SENCAR mouse epidermis

The inhibition of gap-junctional intercellular communication (GJIC) between initiated and surrounding normal cells by tumor promoters is believed to be important in the promotion stage of carcinogenesis. Therefore, we examined the effect of skin-tumor promoters on the expression of the gap-junctional proteins connexin (Cx) 26, Cx43, and Cx31.1 in SENCAR mouse skin. Animals were treated with 12-0-tetradecanoylphorbol-13-acetate (TPA) (8.3 nmol), okadaic acid (OA) (2.5 nmol), chrysarobin (220 nmol), or benzoyl peroxide (BzPo) (83 micromol). Northern blot and immunofluorescence analyses revealed that keratinocytes in adult mouse skin expressed Cx31.1 and Cx43 but not Cx26. All four of the skin-tumor promoters switched on the Cx26 gene, transiently increased expression of Cx43, and significantly inhibited the expression of Cx31.1. The time courses for changes in Cx26, Cx3l. 1, and Cx43 mRNA levels coincided in most cases and in general corresponded well to the time-response curves for hyperplastic changes in mouse skin. The peaks of Cx26 and Cx43 expression and Cx31.1 inhibition appeared 12 h after TPA application and 24 h after OA and chrysarobin application. BzPo elevated the levels of Cx26 and Cx43 transcripts later (peak at 2-4 d). In tumor promoter-treated skin, Cx26 and Cx43 plaques were on the plasma membrane of most keratinocytes. Cx31.1 staining was much weaker than in untreated epidermis. Thus, tumor promoters induce a large change in the expression of several Cxs, which in turn may affect both the level of GJIC and the sensitivity of GJlC to regulatory factors.

TPA-Induced Arrest of Erythroid Differentiation Is Coupled With Downregulation of GATA-1 and Upregulation of GATA-2 in an Erythroid Cell Line SAM-1

GATA-1 protein is thought to be a positive regulator of erythroid differentiation. However, ectopic expression of a conditional GATA-2/estrogen receptor chimera was shown to inhibit erythroid differentiation in a hormone-dependent manner, suggesting the negative regulation of erythroid differentiation by GATA-2 protein. Accordingly, we reasoned that the quantitative balance of GATA-1 and GATA-2 protein might affect erythroid differentiation. In this report, we performed specific and quantitative measurements of GATA-1 and GATA-2 protein in a new erythroid cell line, SAM-1, after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). On the basis of these measurements, we show that TPA-induced arrest of erythroid differentiation is coupled with the upregulation of GATA-2 protein, as well as the downregulation of GATA-1 protein. Our results suggest that it is the precise quantitative balance of GATA-1 and GATA-2 protein that regulates erythroid differentiation.

The expression of integrin α2β1 and attachment to type I collagen of melanoma cells are preferentially induced by tumour promoter, TPA (12-O-tetradecanoyl phorbol-13-acetate)

The adhesion of melanoma cells to the extracellular matrix (ECM) protein is likely to be essential in their invasive metastatic processes. Treatment with 12-O-tetradecanoyl phorbol-13-acetate (TPA), a potent protein kinase C (PKC) activator, preferentially induced the expression of alpha 2 beta 1 integrin, the receptor for collagen/laminin. The number of cells attached to type I collagen, but not laminin, was increased by treatment with TPA. Prior exposure to PKC inhibitors such as H-7 (20 mumol/l) and calphostin C (50 mumol/l) had no effect on TPA-induced alpha 2 beta 1 integrin expression and cell attachment to type I collagen, whereas prior exposure to the calmodulin antagonist W-7(50 mumol/l) inhibited these TPA-induced events. The augmented adhesion was also inhibited by anti-alpha 2 antibody. These data suggest that the increased attachment of melanoma cells to type I collagen appears to be mediated by the preferential augmentation of integrin alpha 2 beta 1, and the activation of calmodulin kinase, but not via the activation of PKC. Analysis of the expression of integrins and of cell attachment to ECMs is important in elucidating the mechanisms involved in the progression and metastasis of malignant melanoma.

Chronic TPA treatment inhibits expression of proximal tubule-specific properties by LLC-PK1 cells.

Confluent LLC-PK1 cell populations expressed progressively proximal tubule-specific properties, including gamma-glutamyl transpeptidase activity, sodium hexose symport activity, alkaline phosphatase activity, and villin protein. This was paralleled by an increase in villin protein manifested at the single cell level. Chronic treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibited expression of proximal tubule-specific properties at the levels of enzyme activity, protein content, and mRNA content. Inhibition occurred in all cells of the population. TPA treatment induced a decrease in total protein kinase C (PKC)-alpha protein content and a change in subcellular localization from predominantly soluble to predominantly particulate. PKC-epsilon protein content was unchanged by TPA treatment. PKC-epsilon was localized in both soluble and particulate fractions of control cells but was localized predominantly in particulate fractions after TPA treatment. PKC-delta was barely detectable in control cells, but content was markedly increased by TPA. These results suggest that TPA-induced inhibition of expression of proximal tubule-specific properties is mediated through modulation of content and/or subcellular localization of one or more PKC isozymes, likely PKC-alpha.

Catecholamines modulate growth and differentiation of human preosteoclastic cells.

Using a clonal cell line of human osteoclast precursors (FLG 29.1 cells), that after treatment with 12-O-tetradecanoyl phorbol 13-acetate (TPA) show many functional characteristics of osteoclasts, we demonstrated that catecholamines act as inducers of osteoclast maturation in vitro and as stimulators of osteoclast activity via the binding to beta 2 adrenergic receptors. Scatchard analysis of 125I-labelled iodocyanopindolol to untreated (undifferentiated) or TPA-treated (differentiated) FLG 29.1 cells revealed the presence of a single high-affinity site with a Kd value around 24 pM and 8 pM respectively and with superimposable binding capacity (1.18 fmol/mg protein). Catecholamines increased in a dose-dependent fashion the intracellular cyclic AMP (cAMP) accumulation in both undifferentiated and TPA-treated FLG 29.1 cells. Pretreatment of untreated and TPA-treated FLG 29.1 cells with propranolol inhibited the catecholamine effect on cAMP accumulation, while pretreatment with clonidine had no effect. Catecholamines also reduced cell proliferation, increased tartrate-resistant acid phosphatase (TRAcP) activity, interleukin 6 (IL-6) production, multi-nuclearity and response to salmon calcitonin (sCT) in undifferentiated FLG 29.1 cells. In differentiated FLG 29.1 cells only IL-6 release was induced by catecholamine treatment. These findings support a potential role for catecholamines in modulating osteoclast differentiation and mature osteoclast activity.

A novel factor, TA20, involved in neuronal differentiation: cDNA cloning and expression

Combined treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) and dibutyryl cyclic AMP (diBu-cAMP) induced significantly longer neurites than treatment with each alone in NG108-15 cells. We performed differential screening to identify genes expressed only by treatment with TPA plus diBu-cAMP but not by that with diBu-cAMP for 72 h alone in NG108-15 cells, and isolated a novel gene, TA20. Over-expression of the gene in NG108-15 and neuroblastoma N18TG-2 cells caused intense neurite elongation and suppressed cell growth. TA20 did not cause, however, any morphological changes in glioma C6Bu-1 cells. These results suggest that TA20 is a novel neuronal differentiation factor.

Epstein-Barr virus (EBV) hypersensitivity of peripheral B lymphocytes in patients with EBV genome-positive Burkitt's lymphoma

Peripheral blood mononuclear cells (PBMC) from four Japanese patients with Epstein-Barr virus (EBV) genome-positive Burkitt's lymphoma (BL) during remission were exposed to the B95-8 strain of EBV. Maximum concentrations of the EBV-determined nuclear antigen (EBNA) before cellular DNA synthesis were similar to those of healthy counterparts. Subsequently, EBV-immortalised cell lines were established. These immortalised lymphoblastoid cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) and superinfected with the P3HR-1 strain of EBV. EBV early antigens (EA) and viral capsid antigen (VCA) were expressed in approximately 3-10 fold higher concentrations by these lymphoblastoid cells than by those from patients with other types of malignant neoplasia including EBV genome-negative BL and from healthy counterparts. Moderate to extremely high IgG antibody titres to EBV VCA as well as IgG antibodies to EA were demonstrated in these patients during the study. These results suggest that defective underlying cellular mechanisms for regulating the replication of EBV may be present in patients with EBV genome-positive BL.

Differential Regulation of a Multipromoter Gene.: SELECTIVE 12-O-TETRADECANOYLPHORBOL-13-ACETATE INDUCTION OF A SINGLE TRANSCRIPTION START SITE IN THE HMG-I/Y GENE

The human HMG-I/Y gene, encoding the non-histone "high mobility group" proteins HMG-I and HMG-Y, is transcriptionally activated in human K562 erythroleukemia cells by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA treatment induces differentiation of K562 cells within 2-4 days after treatment. In this report, we show that transcriptional activation of the HMG-I/Y gene is dependent on protein synthesis and is an early event (2 h after induction) in the TPA-mediated differentiation process. Of the four functional transcription start sites present in the gene, only one (start site 2) is preferentially induced upon TPA treatment. This is the first report, to our knowledge, of the preferential utilization of a specific transcription start site in response to a particular stimulus in a gene that contains multiple promoters. This indicates that each start site in the gene has the potential to be independently regulated instead of being coordinately controlled as shown in a number of other genes. In addition, sequences upstream of the inducible start site, which contains a TPA-responsive element, mediates TPA inducibility through AP1 (or an AP1-like) transcription factor. The HMG-I/Y proteins function as key regulators of gene expression and play a significant role in chromatin structural changes as well. The cloning and sequence analyses previously reported indicated the structure of the HMG-I/Y gene to be highly complex and predicted its expression to be tightly regulated. The results presented here confirm and extend these earlier findings.

Vasoactive intestinal peptide gene expression in the rat pheochromocytoma cell line PC12

Vasoactive intestinal peptide (VIP) gene expression was analyzed in PC12 cells. VIP mRNA was detected in PC12 cells treated with VIP or forskolin whereas no VIP mRNA was detected in the untreated cells. The induction of the VIP mRNA was enhanced by the simultaneous treatment with 12- O-tetradecanoylphorbol-13-acetate (TPA). PC12 cells stimulated with forskolin plus TPA released immunoreactive VIP. Sephadex G-50 column chromatography revealed that the immunoreactive VIP secreted from PC12 cells is comprised of multiple forms, one of which was indistinguishable from the authentic VIP. PC12 cells supported an efficient transcription from the human VIP gene promoter in a cell-specific as well as cAMP-dependent manner. These results definitely demonstrated the expression of the VIP gene in PC12 cells. VIP biosynthesis may be positively regulated by VIP in an autocrine fashion in PC12 cells.

On the reaction kinetics of the radioadaptive response in cultured mouse cells.

The reaction kinetics of radioadaptive response of low doses of X-rays have been studied in quiescent cultured mouse cells. Mouse m5S cells pre-exposed in G1 to low doses of X-rays became insensitive to the induction of chromosome aberrations, mutation toward 6-thioguanine resistance, and cell killing. Adapted cells were, however, more susceptible to morphological transformation by subsequent high challenging doses of X-rays. The cytogenetic adaptation, which lasted about 20 h pertained to a narrow dose range. X-ray doses below and above 0.1 Gy appeared to be recognized as different signals; higher doses of X-rays were incapable of inducing adaptation and rapidly extinguished the adapted condition. Treatment with 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and hydrogen peroxide, but not the xanthine/xanthine oxidase superoxide-generating system, mimicked X-rays in inducing adaptation when applied at low doses. Over-exposure to TPA or inhibitors of protein kinase C (PKC) abrogated the adaptive response to X-rays, providing evidence for the involvement of a PKC-mediated signalling pathway. The lack of radioadaptive response in a tumorigenic variant, clone 6110, and its restoration in the morphological revertant obtained by introducing human chromosome 11 further suggested that interference of signalling pathways may alter radioadaptive responses in malignant cells.

Expression of neurotrophin genes in human fibroblasts: Differential regulation of the brain-derived neurotrophic factor gene

Brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3) are structurally related survival and differentiation factors for distinct sets of peripheral and central neurons. We previously reported that BDNF and NGF gene expression are differentially regulated in mouse L929 fibroblasts. Here we examine expression of these three neurotrophins in human fibroblasts. Northern blots detected BDNF and NT-3 mRNAs in fibroblasts derived from lung (WI-38), calvarium and foreskin. WI-38 cells and foreskin fibroblasts expressed 1.6 kb as well as 4 kb BDNF mRNAs whereas only the smaller BDNF mRNA was detected in calvarium fibroblasts. NGF mRNA was present in foreskin and calvarium but not lung fibroblasts. In WI-38 cells serum treatment increased levels of BDNF mRNA within 2 hr. Cycloheximide did not inhibit the increase. Treatment with 12-O-tetradecanoyl phorbol-13-acetate (TPA) transiently suppressed BDNF mRNA. Treatment with both serum and TPA first stimulated and then transiently suppressed BDNF mRNA. TPA and/or serum did not significantly affect BDNF mRNA in calvarium fibroblasts. These results show that human fibroblasts derived from different tissues express and regulate neurotrophin genes differentially.

High resolution analysis of cell cycle‐correlated vimentin expression in asynchronously grown, TPA‐treated MPC‐11 cells by the novel flow cytometric multiparameter BrdU‐hoechst/PI and immunolabeling technique

High resolution, multiparameter analysis using the flow cytometric BrdU/Hoechst quenching technique has been applied to study cell cycle kinetics and vimentin expression in individual cells of asynchronously grown MPC-11 mouse plasmacytoma cell cultures treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) to induce in vitro differentiation. BrdU treatment up to 16 h in the absence or presence of TPA did not affect either cell cycle progression or the kinetics or quantity of vimentin expression. TPA-treated cells became arrested in G1 phase of the second cell cycle; however, this G1 phase arrest was transient only. In addition, G1 phase cells located prior to a putative transition point at the beginning of TPA treatment were completely blocked in cell cycle progression. There is also evidence that cells located in G1 or G2/M phase at the beginning of TPA treatment finally expressed low levels of vimentin. On the contrary, cells located in S phase at TPA exposure showed high vimentin levels after treatment. The results presented here show that, with the flow cytometric BrdU/Hoechst quenching technique, one can correlate time-dependent protein expression at the single cell level in asynchronously grown cultures not only with the actual cell cycle state, but also with the history of cell replication.

Role of Ca2+ and protein kinase C in shear stress-induced actin depolymerization and endothelin 1 gene expression.

Vascular endothelial cells adapt to changes in blood flow by altering the cell architecture and by producing various substances. We have previously reported that low shear stress induces endothelin 1 (ET-1) expression in endothelial cells and that this induction is mediated by depolymerization of actin fiber. In the present study, we examined the role of Ca2+ and protein kinase C (PKC) in shear stress-induced actin depolymerization and subsequent ET-1 gene expression. Exposure of cultured porcine aortic endothelial cells to low shear stress (5 dyne/cm2) for 3 hours increased the ratio of G-actin to total actin from 54 +/- 0.8% to 80 +/- 1.0%. This shear stress-induced actin depolymerization was completely blocked by chelation of extracellular Ca2+ with EGTA and partially inhibited by intracellular Ca2+ chelation with the tetraacetoxymethyl ester of BAPTA (BAPTA/AM). Pretreatment with staurosporine, a PKC inhibitor, or desensitization of PKC by treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) for 24 hours also resulted in partial inhibition of shear stress-induced actin depolymerization. Although PKC activation by TPA mildly increased G-actin content, the effect of TPA and shear stress on actin depolymerization was not additive. Moreover, shear stress-induced ET-1 gene expression was inhibited by EGTA, BAPTA/AM, and staurosporine to a degree similar to the inhibition of actin depolymerization. In contrast, ET-1 gene expression induced by cytochalasin B, an actin-disrupting agent, was not affected by staurosporine.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in the regulation of phosphatidylinositol‐specific phospholipase C in normal and neoplastic keratinocytes

The induction of epidermal differentiation by Ca2+ in vitro is associated with enhanced activity of phosphatidylinositol-specific phospholipase C (PLC). Neoplastic keratinocyte cell lines expressing a mutant c-Ha-ras gene and normal keratinocytes transformed to the neoplastic phenotype by transduction with the v-Ha-ras gene (v-Ha-ras keratinocytes) have elevated constitutive activity of PLC that increases further in response to Ca2+, but the cells do not differentiate normally. PLC-gamma 1 (145 kDa) is the major isoform detected by immunoblotting of extracts from control, v-Ha-ras, and neoplastic keratinocyte cell lines cultured in 0.05 mM Ca2+ medium. The amount of PLC-gamma 1 protein was higher in neoplastic cell lines than in normal and v-Ha-ras keratinocytes that had similar PLC-gamma 1 protein levels. Thus, higher PLC-gamma 1 protein levels cannot account for the elevated constitutive activity PLC in v-Ha-ras keratinocytes. After induction of differentiation by Ca2+, the amount of PLC-gamma 1 protein increased in all cell types, and PLC-delta 1 (85 kDa), barely detectable in 0.05 mM Ca2+, increased. PLC-beta 1 was not detected at any Ca2+ concentration. PLC-gamma 1 and PLC-delta 1 mRNA did not increase after elevation of extracellular Ca2+, suggesting that posttranscriptional mechanisms can regulate PLC-gamma 1 and PLC-delta 1 protein levels in normal and neoplastic keratinocytes. Activation of protein kinase C by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited the stimulation of inositol phosphate (InsP) formation by Ca2+ but did not alter basal InsP levels in normal keratinocytes. In contrast, TPA treatment reduced both Ca(2+)-stimulated and basal InsP formation in neoplastic cells lines and v-Ha-ras keratinocytes. In both normal and v-Ha-ras keratinocytes labeled with [32P]orthophosphate, antibodies against PLC-gamma 1 immunoprecipitated a complex of 32P-labeled proteins. The relative labeling of the PLC-gamma 1 band was greater in normal than in v-Ha-ras keratinocytes. Furthermore, treatment with TPA specifically increased the relative phosphorylation of PLC-gamma 1 in v-Ha-ras keratinocytes but not in normal keratinocytes. These results suggest that the negative regulation of constitutive activity of PLC by protein kinase C differs in normal and neoplastic keratinocytes and that this could be the mechanism of increased PLC activity produced by an oncogenic ras gene in keratinocytes.

Calpain-induced downregulation of activated protein kinase C-alpha affects lung epithelial cell morphology.

A few minutes after mouse lung epithelial cell lines were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), the cells rounded up and pulled away from their neighbors. Several hours later, the cells flattened out to resume their original morphology. To begin to characterize the enzymology underlying these changes, the subcellular distribution and intracellular content of the TPA receptor, protein kinase C (PKC), and its putative endogenous regulator, the Ca(2+)-dependent protease, calpain, were investigated. Of eight PKC isozymes examined in several tumorigenic and nontumorigenic cell lines, all cells contained PKC-alpha, PKC-delta, and PKC-zeta. TPA rapidly (5 min) translocated PKC-alpha from the cytosol to the particulate fraction; PKC-alpha concentrations then decreased with continued TPA exposure. PKC-zeta levels and intracellular location were not affected. An inhibitor of PKC activity, GF 109203X, prevented the initial morphological change. The calpain II isozyme was also found in all cell lines, and its cellular content increased as a result of TPA treatment. Calpain inhibitor I did not affect the initial shape change but prevented subsequent flattening of the cells. We therefore conclude that PKC activation is required for the TPA-induced alterations in lung cell morphology and that calpain mediates the return to a flattened epithelial appearance.

Involvement of c-Jun in the control of glucocorticoid receptor transcriptional activity during development of chicken retinal tissue.

The ability of the glucocorticoid receptor (GR) to induce gene expression in embryonic chicken retinal tissue increases dramatically during development, although the quantity of the receptor molecules does not change greatly with age. This study examines the possible involvement of c-Jun in the developmental control of GR activity. Expression of c-Jun in retinal tissue was high at early embryonic ages and declined during development. Elevation of c-Jun expression in retina of mid-developmental ages by treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), or by introduction of a c-Jun expression vector, caused a pronounced decline in the inducibility of the endogenous glutamine synthetase gene and the transiently transfected CAT constructs p delta G46TCO and pGS2.1CAT, that are controlled by a minimal consensus glucocorticoid response element (GRE) promoter and the glutamine synthetase promoter, respectively. The effect of c-Jun was dose dependent and could be reversed by overexpression of GR. C-Jun-evoked repression of GR activity could be relieved by overexpression of Jun D. Overexpression of Jun D could also elevate the responsiveness of early embryonic retina to glucocorticoids and cause a 5-fold increase in p delta G46TCO induction. The effect of Jun D could be reversed by overexpression of c-Jun. Expression of c-Jun might therefore be important for repression of GR activity at early embryonic ages.

Modulation of contractile proteins in embryonic and fetal chick cardiac cells by phorbol ester, gamma-interferon, 5-azacytidine and diacylglycerols

We studied changes in the concentration of tropomyosin, actin, desmin and vimentin in cultured myocardiocytes from Hamburger and Hamilton's stages 29 and 39 chick embryos (HH29 and HH39) (1), treated with 12-o-tetradecanoyl-phorbol-13-acetate (TPA), 5-azacytidine (AZA), gamma interferon (INF) and dyacylglycerols (DAG). In embryonic myocardiocytes at HH29, the first three agents modified the intracellular distribution of the thin filament proteins tropomyosin and actin, increasing their cytoplasmic concentration and decreasing their cytoskeletal concentration. The concentration of the intermediate filament proteins desmin and vimentin increased in both subcellular fractions after treatment with these drugs. In fetal myocardiocytes at HH39, total protein content decreased after treatment with these drugs. Cytoplasmic and cytoskeletal concentrations of actin and tropomyosin decreased to different degrees after treatment with TPA, AZA or DAG in HH39 myocardiocytes. TPA, AZA and DAG decreased desmin in the cytoplasmic and cytoskeletal fractions. These findings suggest that the drugs tested alter the normal protein composition in cultured myocardiocytes, and have different effects depending on the developmental stage in which the embryo is treated.