Effects Of Tetradecanoyl Phorbol Acetate Research Articles

12-O-tetradecanoylphorbol-13-acetate (TPA) increases murine intestinal crypt stem cell survival following radiation injury.

Radiation enteropathy is a common complication in cancer patients following radiation therapy. Thus, there is a need for agents that can protect the intestinal epithelium against radiation. 12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown to induce differentiation and/or apoptosis in multiple cell lines and primary cells. In the current report, we studied the function of TPA in radiation induced enteropathy in cultured rat intestinal epithelial cell line IEC-6 after ionizing radiation (IR) and in mice after high dose total-body gamma-IR (TBI). In IEC-6 cells, there were reduced apoptosis and cell cycle arrest in TPA treated cells after IR. We detected a four-fold increase in crypt cell survival and a two-fold increase in animal survival post TBI in TPA treated mice. The beneficial effects of TPA were accompanied by upregulation of stem cells markers and higher level of proteins that are involved in PKC signaling pathway. In addition, TPA also decreased the TBI-augmented levels of the DNA damage indicators. The effects were only observed when TPA was given before irradiation. These results suggest that TPA has the ability to modulate intestinal crypt stem cells survival and this may represent a promising countermeasure against radiation induced enteropathy.

Melatonin inhibits aromatase promoter expression by regulating cyclooxygenases expression and activity in breast cancer cells

Background:Melatonin reduces the development of breast cancer interfering with oestrogen-signalling pathways, and also inhibits aromatase activity and expression. Our objective was to study the promoters through which melatonin modifies aromatase expression, evaluate the ability of melatonin to regulate cyclooxygenases and assess whether the effects of melatonin are related to its effects on intracellular cAMP, in MCF-7 cells.Methods:Total aromatase mRNA, aromatase mRNA promoter regions and cyclooxygenases mRNA expression were determined by real-time RT–PCR. PGE2 and cAMP were measured by kits.Results:Melatonin downregulated the gene expression of the two major specific aromatase promoter regions, pII and pI.3, and also that of the aromatase promoter region pI.4. Melatonin 1 nM was able to counteract the stimulatory effect of tetradecanoyl phorbol acetate on PGE2 production and inhibit COX-2 and COX-1 mRNA expression. Melatonin 1 nM elicited a parallel time-dependent decrease in both cyclic AMP formation and aromatase mRNA expression.Conclusions:This study shows that melatonin inhibits aromatase activity and expression by regulating the gene expression of specific aromatase promoter regions. A possible mechanism for these effects would be the regulation by melatonin of intracellular cAMP levels, mediated by an inhibition of cyclooxygenase activity and expression.

Histone Hyperacetylation Occurs on Promoters of Lytic Cycle Regulatory Genes in Epstein-Barr Virus-Infected Cell Lines Which Are Refractory to Disruption of Latency by Histone Deacetylase Inhibitors

Activation of the Epstein-Barr virus (EBV) lytic cycle is mediated through the combined actions of ZEBRA and Rta, the products of the viral BZLF1 and BRLF1 genes. During latency, these two genes are tightly repressed. Histone deacetylase inhibitors (HDACi) can activate viral lytic gene expression. Therefore, a widely held hypothesis is that Zp and Rp, the promoters for BZLF1 and BRLF1, are repressed by chromatin and that hyperacetylation of histone tails, by allowing the access of positively acting factors, leads to transcription of BZLF1 and BRLF1. To investigate this hypothesis, we used chromatin immunoprecipitation (ChIP) to examine the acetylation and phosphorylation states of histones H3 and H4 on Zp and Rp in three cell lines, Raji, B95-8, and HH514-16, which differ in their response to EBV lytic induction by HDACi. We studied the effects of three HDACi, sodium butyrate (NaB), trichostatin A (TSA), and valproic acid (VPA). We also examined the effects of tetradecanoyl phorbol acetate (TPA) and 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor, on histone modification. In Raji cells, TPA and NaB act synergistically to activate the EBV lytic cycle and promote an increase in histone H3 and H4 acetylation and phosphorylation at Zp and Rp. Surprisingly, however, when Raji cells were treated with NaB or TSA, neither of which is sufficient to activate the lytic cycle, an increase of comparable magnitude of hyperacetylated and phosphorylated histone H3 at Zp and Rp was observed. In B95-8 cells, NaB inhibited lytic induction by TPA, yet NaB promoted hyperacetylation of H3 and H4. In HH514-16 cells, NaB and TSA strongly activated the EBV lytic cycle and caused hyperacetylation of histone H3 on Zp and Rp. However, when HH514-16 cells were treated with VPA, lytic cycle mRNAs or proteins were not induced, although histone H3 was hyperacetylated as measured by immunoblotting or by ChIP on Zp and Rp. Taken together, our data suggest that open chromatin at EBV BZLF1 and BRLF1 promoters is not sufficient to activate EBV lytic cycle gene expression.

Pituitary Adenylyl Cyclase-activating Polypeptide 38 Reduces Astroglial Proliferation by Inhibiting the GTPase RhoA

Pituitary adenylyl cyclase-activating polypeptide 38 (PACAP38) plays an important role in the proliferation and differentiation of neural cells. In the present study, we have investigated how PACAP38 inhibits the proliferation of cultured neocortical astroglial cells. When applied to synchronized cells during the G(1) phase of the cell cycle, PACAP38 diminished the subsequent nuclear uptake of bromodeoxyuridine. When applied for 2 days, it reduced the cell number. PACAP38 did not exert its antiproliferative effect by activating protein kinase A. It also did not reduce the activity of mitogen-activated protein kinases essential for G(1) phase progression. Instead, PACAP38 acted on a member of the Rho family of small GTPases. It reduced the activity of RhoA as was shown with a Rhotekin pull-down assay. The decrease in endogenous RhoA activity induced by treatment of the cells with C3 exotoxin or by expression of dominant negative RhoA also reduced the nuclear uptake of bromodeoxyuridine. In contrast, expression of constitutively active RhoA prevented the effect of PACAP38. Our data show a novel signal transduction pathway by which the neuropeptide influences cell proliferation.

Early activation of the Kaposi's sarcoma-associated herpesvirus RTA, RAP, and MTA promoters by the tetradecanoyl phorbol acetate-induced AP1 pathway.

Kaposi's sarcoma-associated herpesvirus (KSHV) maintains a latent infection in primary effusion lymphoma (PEL) cells, but treatment with tetradecanoyl phorbol acetate (TPA) can trigger the full lytic-cycle replication in some of these cells. During lytic-cycle replication, the KSHV-encoded replication and transcription activator (RTA or ORF50), the mRNA transport and accumulation protein (MTA), and the replication-associated protein (RAP) all play crucial roles in expression of downstream viral genes as well as in mediation of viral DNA replication. The cellular CCAAT/enhancer-binding protein alpha (C/EBP alpha) is induced in TPA-treated PEL cells and contributes to transactivation of the promoters for all of these genes through both direct binding and cooperative interactions with RTA and RAP targeted to upstream C/EBP sites. However, little is known about how RTA expression is triggered initially at the earliest stages after TPA induction when the C/EBP alpha levels are still limited. Treatment with TPA proved to significantly induce both AP1 DNA-binding activity and levels of activated phosphorylated cJUN in PEL cells and ectopic expression of cJUN-plus-cFOS-induced RTA protein expression in PEL cells. Cotransfected cJUN plus cFOS or TPA treatment transactivated the KSHV RTA, RAP, and MTA promoters in an AP1-binding site-dependent manner in all three promoters. Chromatin immunoprecipitation assays confirmed that cJUN associates with these KSHV target promoters in PEL cells as early as 4 h after TPA treatment. Furthermore, the KSHV RTA and RAP proteins both interact with cJUN or both cJUN and cFOS in vitro or by coimmunoprecipitation from induced PEL cells and enhance cJUN-plus-cFOS-mediated transactivation of these viral promoters. Both increased phosphorylated cJUN and AP1 DNA-binding activity was detected as early as 1 h after TPA treatment in PEL cells, suggesting that AP1 activity may be crucial for very early activation of the RAP, MTA, and RTA promoters during the KSHV lytic cycle. Finally, expression of RTA alone increased cJUN protein levels severalfold in DG75 cells but did not induce cJUN phosphorylation. Therefore, we suggest that the initiating effects of TPA via the AP1 pathway in PEL cells need to be amplified by RTA for full lytic-cycle induction.

12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits osteoclastogenesis by suppressing RANKL-induced NF-kappaB activation.

The mechanism by which TPA-induced PKC activity modulates osteoclastogenesis is not clear. Using a RAW(264.7) cell culture system and assays for NF-kappaB nuclear translocation, NF-kappaB reporter gene activity, and MAPK assays, we demonstrated that TPA inhibits osteoclastogenesis through the suppression of RANKL-induced NF-kappaB activation. The protein kinase C (PKC) pathway has been suggested to be an important regulator of osteoclastic bone resorption. The role of PKC in RANKL-induced osteoclastogenesis, however, is not clear. In this study, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, on osteoclastogenesis and studied its role in RANKL-induced signaling. RANKL-induced RAW(264.7) cell differentiation into osteoclast-like cells was used to assess the effect of TPA on osteoclastogenesis. Assays for NF-kappaB nuclear translocation, NF-kappaB reporter gene activity, protein kinase activity, and Western blotting were used to examine the effects of TPA on RANKL-induced NF-kappaB, c-Jun N-terminal kinase (JNK), and MEK/ERK and p38 signal transduction pathways. We found that TPA inhibited RANKL-induced RAW(264.7) cell differentiation into osteoclasts in a dose-dependent manner. Time course analysis showed that the inhibitory effect of TPA on RANKL-induced osteoclastogenesis occurs predominantly at an early stage of osteoclast differentiation. TPA alone had little effect on NF-kappaB activation in RAW(264.7) cells, but it suppresses the RANKL-induced NF-kappaB activation in a dose-dependent fashion. Interestingly, the suppressive effect of TPA on RANKL-induced NF-kappaB activation was prevented by a conventional PKC inhibitor, Go6976. Supershift studies revealed that the RANKL-induced DNA binding of NF-kappaB complexes consisted of C-Rel, NF-kappaB1 (p50), and RelA (p65). In addition, TPA induced the activation of JNK in RAW(264.7) cells but had little effect on RANKL-induced activation of JNK. TPA also inhibited RANKL-induced activation of ERK but had little effect on p38 activation. Given that NF-kappaB activation is obligatory for osteoclast differentiation, our studies imply that inhibition of osteoclastogenesis by TPA is, at least in part, caused by the suppression of RANKL-induced activation of NF-kappaB during an early stage of osteoclastogenesis. Selective modulation of RANKL signaling pathways by PKC activators may have important therapeutic implications for the treatment of bone diseases associated with enhanced bone resorption.

Stimulation of melanogenesis by tetradecanoylphorbol 13-acetate (TPA) in mouse melanocytes and neural crest cells.

In vitro studies have shown that the phorbol ester, 12-tetradecanoylphorbol 13-acetate (TPA) induces neural crest cell differentiation into melanocytes, and stimulates proliferation and differentiation of normal melanocytes. As TPA is not a physiological agent, its action is clearly mimicking some in vivo pathway involved in these processes. An understanding of the effect of TPA on the expression of melanogenic genes will therefore provide valuable insight into the molecular mechanisms regulating melanocyte differentiation. In this study, we utilized primary cultures of neural crest cells and an immortalized melanocyte cell line (DMEL-2) which proliferates in the absence of TPA, to explore the effects of TPA on key melanogenic effectors. In neural crest cells, TPA was found to be necessary for both microphthalmia associated transcription factor (Mitf) up-regulation and for melanin synthesis. Using northern blots, we show that in DMEL-2 cells, TPA significantly increases the messenger ribonucleic acid (mRNA) levels of the tyrosinase gene family (tyrosinase, Tyrp1 and Dct) and the expression of Mitf. Western blots demonstrate that in these TPA-treated cells there is a concomitant increase in Tyr, Tyrp1 and glycosylated Dct protein levels. Pax3, a known Mitf regulator, is unaltered by TPA treatment. This study demonstrates the utility of a novel cell line for investigating the long-term effects of TPA on melanogenesis and provides an understanding of how TPA enhances mouse melanocyte differentiation.

Nitroglycerin: a NO donor inhibits TPA-mediated tumor promotion in murine skin.

Nitroglycerin (GTN), a nitric oxide (NO) generating vasodilator has been used in the present study to assess the role of NO during tumor promotion in murine skin. Administration of GTN to 12-O tetradecanoyl phorbol 13-acetate (TPA)-treated mice resulted in a dose-dependent inhibition in the level of glutathione and the activity of antioxidant enzymes by approximately 16-40% of acetone-treated control. We also observed that GTN application led to a significant reduction in the ornithine decarboxylase (ODC) activity and decreased the rate of [3H]thymidine incorporation into epidermal DNA when compared with the acetone-treated control (P < 0.001). Treatment of DMBA-initiated TPA-promoted mice with GTN increased the latency period, decreased the tumor incidence by 32% and there was a 2-fold decrease in tumor yield (tumor/mouse) as compared with the TPA (alone)-treated group by 20 weeks. From these data, it can be concluded that NO can abrogate the toxic and tumor promoting effects of TPA and GTN can be used as a chemopreventive agent to inhibit tumorigenesis in murine skin.

Differentiation of neuroblastoma cells by phorbol esters and insulin-like growth factor 1 is associated with induction of retinoic acid receptor β gene expression

The retinoic acid (RA) receptor beta isoform (RARbeta) plays an important role in RA-induced differentiation of human neuroblastoma. In this study we show that insulin-like growth factor 1 (IGF-1) and tetradecanoyl phorbol acetate (TPA) induce RARbeta gene expression in neuroblastoma SH-SY5Y cells. IGF-1 and TPA caused a marked induction of RARbeta2 promoter activity and had a synergistic effect with RA that also upregulates transcription. The effect of RA is mediated by two RA responsive elements (RAREs), whereas the IGF-1 and TPA actions are independent of the RAREs and map to sequences that overlap the TATA box. These results suggest that the signaling pathways stimulated by TPA and IGF-1 could modify the components assembled at the core RARbeta2 promoter and activate transcription. Expression of RasVal12 mimics the effect of IGF-1 and TPA on the promoter, and a dominant negative Ras mutant abrogates activation. A dominant negative Raf also blocks activation showing that the Ras-Raf pathway mediates stimulation of the RARbeta2 promoter. Our results show that neuronal differentiation induced by non-retinoid agents that activate Ras is accompanied by increased transcription of the RARbeta gene.

The effect of cytokines on parathyroid hormone-related protein (PTH-rP) production in human amnion cells.

In the present study, the effects of various cytokines on parathyroid hormone-related protein (PTH-rP) production and PTH-rP mRNA expression in human amnion cells were studied. Immunoreactive (ir) PTH-rP was measured by immunoradiometric assay and the expression of PTH-rP mRNA was determined by Northern blot analysis. The addition of interleukin-1beta (IL-1beta, 10 ng/ml) and IL-6 (10 ng/ml) in culture medium for 24 hours resulted in a significant increase in ir-PTH-rP levels by 1.5 and 1.6 fold, respectively. The effects of these agents were dose dependent. In contrast, IL-2 (10 ng/ml) and IL-8 (10 ng/ml) showed no effect on the production of ir-PTH-rP from amnion cells. Treatment with IL-1beta or IL-6 for 6 hours increased the expression of PTH-rP mRNA in amnion cells. The stimulatory effect of IL-1beta was reduced by IL-1 receptor antagonist (IL-1Ra) in a dose dependent manner. Both tetradecanoyl phorbol acetate (TPA), and forskolin increased PTH-rP mRNA levels and the PTH-rP production in amnion cells, and the effect of TPA was much greater than that of forskolin. The findings of the present study suggest of the participation of inflammatory cytokines for the regulation of PTH-rP production in human amnion cells.

AP-1 Activity Affects the Levels of Induced Erythroid and Megakaryocytic Differentiation of K562 Cells

The effect of modulating AP-1 activity on the levels of induced erythroid and megakaryocytic differentiation of the erythroleukemia cell line K562 was examined. Cells were stably transfected with expression vectors encoding either a tetracycline-responsive dominant negative c-Jun (JunDN) or a hybrid Raf protein inducible by estrogen. Down-regulation of AP-1 activity by induction of JunDNenhanced erythroid differentiation by two agents, cytosine arabinoside and activin A. Induction of AP-1 activity by elevated Raf activity inhibited erythroid differention, thus mimicking the well-known effect of tetradecanoyl phorbol acetate (TPA) on this process. Induced Raf activity also brought about partial megakaryocytic differentiation of the line. However, inhibition of TPA-induced AP-1 activity by induction of JunDNgave mixed results. While the cytological effects of TPA treatment observed on cytochemical staining were inhibited by JunDN, two protein markers for megakaryocytic differentiation were increased. These results, while supportive of current models of hematopoietic lineage-specific gene expression, suggest a complex and temporal mechanism of lineage commitment.

Transforming growth factor-beta1 and protein kinase C synergistically activate the c-fos serum response element in myocardial cells.

We previously reported that transforming growth factor-beta1 (TGF-beta1) potentiated alpha1-adrenergic and stretch-induced c-fos mRNA expression and norepinephrine (NE)-induced amino acid incorporation in rat cultured myocardial cells (MCs). In the present study, we attempted to explore the mode of TGF-beta1 action for c-fos gene expression in MCs. In the transient transfection assay, TGF-beta1 potentiated NE- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-activated c-fos promoter/enhancer, but not forskolin-activated c-fos promoter/enhancer. The c-fos serum response element (SRE) and the TPA response element (TRE) were responsible for TGF-beta1-induced potentiation of the NE or TPA action. Although TGF-beta1 activated not only the wild-type c-fos SRE, but also the mutated c-fos SRE, which contains an intact binding site for the serum response factor (SRF) but lacks the ternary complex factor (TCF) binding site, TPA activated the wild-type c-fos SRE but not the mutated c-fos SRE. TGF-beta1 did not potentiate the effects of TPA on the activation of mitogen-activated protein kinase (MAPK) and the phosphorylation of Elk-1 and SAP-1a, which belong to TCF at the c-fos SRE. These results indicate that TGF-betaf potentiates the c-fos SRE activated by PKC through the SRF binding site. TGF-beta1 is involved in the regulation of c-fos gene expression through the c-fos SRE and is subsequently involved in the regulation of the gene which has the TRE in the promoter/enhancer region.

Evidence for involvement of protein kinase C (PKC)-zeta and noninvolvement of diacylglycerol-sensitive PKCs in insulin-stimulated glucose transport in L6 myotubes.

We examined the question of whether insulin activates protein kinase C (PKC)-zeta in L6 myotubes, and the dependence of this activation on phosphatidylinositol (PI) 3-kinase. We also evaluated a number of issues that are relevant to the question of whether diacylglycerol (DAG)-dependent PKCs or DAG-insensitive PKCs, such as PKC-zeta, are more likely to play a role in insulin-stimulated glucose transport in L6 myotubes and other insulin-sensitive cell types. We found that insulin increased the enzyme activity of immunoprecipitable PKC-zeta in L6 myotubes, and this effect was blocked by PI 3-kinase inhibitors, wortmannin and LY294002; this suggested that PKC-zeta operates downstream of PI 3-kinase during insulin action. We also found that treatment of L6 myotubes with 5 microM tetradecanoyl phorbol-13-acetate (TPA) for 24 h led to 80-100% losses of all DAG-dependent PKCs (alpha, beta1, beta2, delta, epsilon) and TPA-stimulated glucose transport (2-deoxyglucose uptake); in contrast, there was full retention of PKC-zeta, as well as insulin-stimulated glucose transport and translocation of GLUT4 and GLUT1 to the plasma membrane. Unlike what has been reported in BC3H-1 myocytes, TPA treatment did not elicit increases in PKCbeta2 messenger RNA or protein in L6 myotubes, and selective retention of this PKC isoform could not explain the retention of insulin effects on glucose transport after prolonged TPA treatment. Of further interest, TPA acutely activated membrane-associated PI 3-kinase in L6 myotubes, and acute effects of TPA on glucose transport were inhibited, not only by the PKC inhibitor, LY379196, but also by both wortmannin and LY294002; this suggested that DAG-sensitive PKCs activate glucose transport through cross-talk with phosphatidylinositol (PI) 3-kinase, rather than directly through PKC. Also, the cell-permeable, myristoylated PKC-zeta pseudosubstrate inhibited insulin-stimulated glucose transport both in non-down-regulated and PKC-depleted (TPA-treated) L6 myotubes; thus, the PKC-zeta pseudosubstrate appeared to inhibit a protein kinase that is required for insulin-stimulated glucose transport but is distinct from DAG-sensitive PKCs. In keeping with the latter dissociation of DAG-sensitive PKCs and insulin-stimulated glucose transport, LY379196, which inhibits PKC-beta (preferentially) and other DAG-sensitive PKCs at relatively low concentrations, inhibited insulin-stimulated glucose transport only at much higher concentrations, not only in L6 myotubes, but also in rat adipocytes, BC3H-1 myocytes, 3T3/L1 adipocytes and rat soleus muscles. Finally, stable and transient expression of a kinase-inactive PKC-zeta inhibited basal and insulin-stimulated glucose transport in L6 myotubes. Collectively, our findings suggest that, whereas PKC-zeta is a reasonable candidate to participate in insulin stimulation of glucose transport, DAG-sensitive PKCs are unlikely participants.

Gonadotropin-releasing hormone and pituitary adenylate cyclase-activating polypeptide affect levels of cyclic adenosine 3',5'-monophosphate-dependent protein kinase A (PKA) subunits in the clonal gonadotrope alphaT3-1 cells: evidence for cross-talk between PKA and protein kinase C pathways.

We have shown previously that protein kinase A (PKA) subunit levels are regulated by activation of PKA or protein kinase C (PKC) in anterior pituitary cells. GnRH also influenced PKA subunit levels, suggesting that hormonal regulation occurs in gonadotrophs, and therefore, we have reexamined this question using the clonal gonadotrope-derived cell line (alphaT3-1 cells). Western blot analysis, using specific immunoaffinity purified immunoglobulins, revealed expression of catalytic (Cat) and regulatory type I (RI) and type II (RII) subunits of PKA in these cells. Activation of adenylyl cyclase (AC) with forskolin, or of PKC with tetradecanoyl phorbol acetate (TPA), caused a rapid (detectable at 0.5-1 h) and concentration-dependent loss of all PKA subunits. Forskolin (10-100 microM) reduced Cat and RI by 60% and RII by 30%, whereas TPA (0.1-1 microM) reduced Cat and RII by 50% and RI by 40%. Simultaneous activation of PKA and PKC caused the expected dose-dependent reductions in Cat, and the effects of forskolin or TPA were nearly additive. RI and RII were reduced similarly by 10 nM TPA, whereas 100 nM TPA tended to prevent the reduction of RI or RII caused by forskolin. GnRH, which activates phosphoinositidase C and not AC in these cells, caused a clear loss of Cat or RII at all concentrations tested and of RI at 0.1 nM. Pituitary adenylate cyclase-activating polypeptide 38, which acts via PVR-1 receptors to stimulate both phosphoinositidase C and AC in these cells, also caused a clear dose-dependent decrease in Cat, RI, and RII, although higher concentrations were needed for the latter effects. Together, the data demonstrate that catalytic and regulatory subunits of PKA are subject to both hormonal and receptor-independent regulation in alphaT3-1 cells, reinforcing the possibility that such effects occur in nonimmortalized gonadotropes. Whereas the effects of PKA activators very likely involve proteolytic degradation of the dissociated PKA holoenzyme, the effects of TPA and GnRH occur in the absence of cAMP elevation by unknown mechanisms. Whatever the mechanisms involved, the data reveal a mechanism for cross-talk between phosphoinositidase C and AC-mediated hormonal signals, in which PKC activation seems to play a pivotal role.

Phorbol Ester-Stimulated Phosphorylation of PU.l: Association With Leukemic Cell Growth Inhibition

PU.1, a member of the ets transcription factor family, has been previously shown to be necessary for tetradecanoylphorbol-13 acetate (TPA)-induced U937 leukemic cell maturation. We examined the effects of TPA on PU.1 content and PU.1 DNA binding activity in U937 cells. Unstimulated cells expressed PU.1 mRNA transcripts and TPA did not increase these levels. However, TPA treatment induced phosphorylation of PU.1. Gel-shift analysis using a labeled PU.1 oligomer showed that TPA induced a unique PU.1 binding activity. This binding activity was phosphorylation-dependent, as indicated by the ability of phosphatase treatment to abolish its detection. The PU.1 binding activity was generated at TPA-13 concentrations stimulating growth arrest and was blocked by the PKC inhibitor GF109203X, which antagonized TPA-induced growth inhibition. Bryostatin 1, another protein kinase C activator, induced only a modest degree of U937 growth inhibition and antagonized TPA-stimulated growth arrest. Bryostatin 1 was unable to induce this TPA-generated PU.1 binding activity. High bryostatin 1 concentrations inhibited generation of this TPA-induced band shift. These data suggest that TPA-induced growth inhibition is associated with phosphorylation of PU.1 and generation of a unique PU.1 binding activity.

Activation of human endometrial phospholipase D by bradykinin

Bradykinin may act as a promoter of endometrial regeneration. In [ 3H]myristate-labelled endometrial stromal cells, bradykinin and tetradecanoylphorbol acetate (TPA) mediated activation of phospholipase D (PLD) as measured by the accumulation of [ 3H]phosphatidylbutanol ([ 3H]PtdBut). Kinetics of bradykininevoked PLD activation was rapid and transient, whereas the TPA response was relatively slow in onset. Bradykinin induced a dose-dependent (EC 50 0.11 nM) [ 3H]PtdBut accumulation at concentrations at which it stimulated DNA synthesis. In [ 3H]inositol-labelled cells, bradykinin evoked a rapid increase in inositol phosphates which preceded the increase in [ 3H]PtdBut formation. Chronic pretreatment with 400 nM TPA abolished PLD activation to subsequent treatment with either TPA and bradykinin. Staurosporine, an inhibitor of protein kinase C, strongly inhibited (IC 50 96 nM) TPA-induced [ 3H]PtdBut formation, but bradykininstimulated [ 3H]PtdBut accumulation was only partially inhibited (IC 50 65 μM). The effect of bradykinin and TPA on PLD activity was synergistic, suggesting that the two agents may act via different mechanisms. These results suggest PKC-dependent and independent pathways are involved in bradykinin-induced PLD activation and that the mitogenic activity of this vasoactive peptide on endometrial stromal cells may in part be mediated via the PLD pathway. This may have significance both to implantation and endometrial cancer.

Regulation of Glial-Derived Dopaminergic Growth Factors by Glucocorticoids and Protein Kinase C

Mesencephalic glia secrete factors that support the survival and differentiation of cultured dopaminergic neurons. Crucial to the understanding of the role of glial-derived growth factors in normal and pathophysiological conditions is knowledge about the physiological regulation of their synthesis and secretion. To address this issue, several substances have been tested for effects on the secretion of dopaminergic growth factors from the mesencephalic glial cell line, Mes42. Regulatory influences were assessed by comparing the effects of conditioned medium (CM) obtained from pretreated and untreated Mes42 cells on the survival of tyrosine hydroxylase-immunoreactive (TH-IR) neurons in serum-free low density cultures of the dissociated Embryonic Day 15 rat mesencephalon. This screening demonstrated that corticosterone and dexamethasone decreased the neurotrophic activity of Mes42-CM on TH-IR neurons by 40-60% in a dose-dependent manner. In contrast, the neurotrophic activity of Mes42-CM on TH-IR neurons was enhanced with tetradecanoylphorbol acetate (TPA). Moreover, regulatory effects of glucocorticoids and TPA on secretion of dopaminergic growth factors were not restricted to mesencephalic glial cell lines but also were present in primary mesencephalic glia. Pretreatment of Mes42 cells with 17β-estradiol, testosterone, progesterone, basic fibroblast growth factor, transforming growth factor α, insulin-like growth factor-I, or activation of cAMP-dependent protein kinases was without effect on the survival promoting activity of Mes42-CM on dopaminergic neurons. These findings suggest that the secretion of dopaminergic growth factors from mesencephalic glia is regulated by glucocorticoids and protein kinase C-dependent second messenger systems.

Turnover of Glutathione S-Transferase alpha mRNAs is Accelerated by 12-O-Tetradecanoyl Phorbol-13-Acetate in Human Hepatoma and Colon Carcinoma Cell Lines

The phorbol ester, 12-O -tetradecanoyl phorbol-13-acetate (TPA), known to induce murine glutathione S -transferase (GST) Ya, was examined for its effect on the expression of human GST α. Unexpectedly, 24-h treatment of the human hepatoma cell line HepG2 with 100 nmol/1 TPA caused a decrease of the GST α mRNA level to below 5% of controls, i.e. opposite to the known response in the mouse. The level of mRNA for GST Mu was also decreased, but the mRNAs of c-jun and jun-B were elevated after 2 h. The decrease of GST α mRNAs was inhibited by staurosporine, suggesting an involvement of protein kinase C. Inhibition of transcription and translation by actinomycin D and cycloheximide also partially inhibited the effect of TPA on the expression of GST α. In the presence of Actinomycin D, GST α mRNA halflife was 14.5 h, compared to 3.5 h in the presence of TPA. The calcium ionophore A23187 caused a loss of GST α mRNAs to levels almost as low as those obtained with TPA. The effects of TPA and the calcium ionophore were also observed in CaCo2 colon carcinoma cells. As a consequence of the decrease of mRNA levels, GST α protein levels and total GST enzyme activity were also diminished. Also, the morphology of the cells was changed after 3 h exposure to TPA. These data suggest that human GST α expression can be regulated at the level of mRNA stability by a pathway involving protein kinase C.

Mutual antagonistic interactions between the thyrotropin (adenosine 3',5'-monophosphate) and protein kinase C/epidermal growth factor (tyrosine kinase) pathways in cell proliferation and differentiation of cultured human thyroid follicles.

Our aim has been to delineate the role of the major signal transduction pathways believed implicated in the control of thyroid function and growth: the cAMP-, epidermal growth factor (EGF) (tyrosine kinase)-, and protein kinase C (PKC)-mediated mechanisms. The experimental model used was our system of thyroid follicles of human origin cultured in suspension under serum-free conditions in which the follicular three-dimensional structure is retained. The phorbol ester 12-O tetradecanoylphorbol 13-acetate (TPA) time and dose dependently (10(-11)-10(-7) M) inhibited TSH-stimulated thyroid functions (cAMP formation, iodide uptake and organification, and T3 secretion). TPA also inhibited such forskolin- and 8-BrcAMP-stimulated effects, suggesting that the attenuation of the cAMP-dependent pathway occurs at steps both pre- and post-cAMP formation. The effects of TPA were mimicked by another PKC activator, phorbol 12,13-dibutyrate, but not by a phorbol ester that fails to activate PKC, 4 alpha-phorbol 12,13-didecanoate, and were reversed by staurosporine, a PKC inhibitor. The TPA actions seem, therefore, to be PKC-mediated. EGF exhibited a time- and dose-dependent (0.02-8 nM) restraining influence on the above TSH-stimulated differentiated functions, except for cAMP, which was enhanced. EGF also blunted such forskolin- and 8-BrcAMP-induced response parameters, suggesting inhibition at a post-cAMP locus. Regarding cell proliferation, during the initial stages of culture (day 2), TPA dose dependently (10(-11)-10(-7) M) attenuated cell proliferation, but subsequently (day 7 of culture) the same doses of TPA stimulated cell multiplication. The TPA-mitogenic and antimitogenic effects could not be mimicked by 4 alpha-phorbol-12,13-didecanoate and were reversed by staurosporine, thus indicating a PKC-mediated pathway for such TPA actions. EGF, on the other hand, only enhanced cell proliferation at a late stage (coincident with the TPA-mitogenic effect). TSH (0.5 U/liter) inhibited both the mitogenic and antimitogenic actions of TPA as well as the cell-proliferative influence of EGF. In conclusion, the data demonstrate mutual antagonistic interactions between the signal transduction pathways: the PKC and EGF (tyrosine kinase) pathways seem to inhibit TSH (cAMP)-mediated human thyroid cell differentiation, whereas TSH attenuates PKC-mediated thyroid cell mitogenesis and antimitogenesis as well as EGF-mediated cell proliferation.

Transcriptional regulation of the lactate dehydrogenase A subunit gene by the phorbol ester 12- O-tetradecanoylphorbol-13-acetate

Regulation of lactate dehydrogenase (LDH) (EC 1.1.1.27) isozymes occurs through a multitude of physiological signals. Here, we show that modulation of LDH A subunit occurs via the protein kinase C pathway. Activators of protein kinase C, such as tetradecanoylphorbol acetate (TPA) and dioctanoylglycerol (DG), caused a 3–4-fold accumulation of LDH A subunit mRNA in rat C6 glioma cells. The specific protein kinase C inhibitor bisindolylmaleimide GF 109203X prevented the TPA-induced increase of LDH A subunit mRNA. To analyze the molecular basis of these effects in more detail, the transcription-modulatory effects of TPA and DG were evaluated in transient transfection assays using plasmids which contain LDH A subunit promoter fragments fused to a chloramphenicol acetyltransferase reporter gene. Both effector agents caused a marked increase of the transcriptional activity of an LDH −830 +25 bp promoter/CAT construct. In contrast, a phorbol ester which fails to activate protein kinase C, phorbol 12β,13α-didecanoate, had no effect on the LDH promoter activity. Transient transfection analysis of LDH promoter deletion/CAT constructs, DNA/protein binding assays, including footprint and gel shift analyses, identified a TRE AP-1 enhancer module at position −294 bp which was the target for the protein kinase C-mediated signal transduction pathway. Thus, our data demonstrate an active role of the protein kinase C signal pathway in regulating LDH A subunit gene expression which may be significant in regulating LDH isozyme patterns under various physiologic conditions.