Phorbol ester regulation of the human gamma-glutamyltransferase gene promoter

In the present study the molecular mechanisms underlying tetradecanoylphorbol-13-acetate (TPA) mediated regulation of the human gamma-glutamyltransferase (GGT) gene were examined. TPA challenge of HeLa cells resulted in an increase of GGT mRNA and enzyme activity. Deletion analysis of the promoter revealed that the -348 to +60 fragment was able to mediate TPA induced expression. Gel shift and supershift analyses showed that TPA treatment increased nuclear protein binding to a putative AP-1 site (-225 to -214) and that c-Jun was part of the complex. This AP-1 element, when cloned either in its native arrangement or as tandem repeat 5' of the minimal thymidine kinase promoter, mediated a significant increase of luciferase activity after TPA treatment of transfected HeLa cells, while its mutated counterpart abolished the induction. The same AP-1 element was able to mediate TPA induced expression in HepG2 cells. Collectively these results indicate that like other GSH metabolising enzymes, GGT too is a target for AP-1 mediated regulation.     

Aromatic‐turmerone attenuates invasion and expression of MMP‐9 and COX‐2 through inhibition of NF‐κB activation in TPA‐induced breast cancer cells

Recent evidence suggests that breast cancer is one of the most common forms of malignancy in females, and metastasis from the primary cancer site is the main cause of death. Aromatic (ar)‐turmerone is present in Curcuma longa and is a common remedy and food. In the present study, we investigated the inhibitory effects of ar‐turmerone on expression and enzymatic activity levels of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced matrix metalloproteinase (MMP)‐9 and cyclooxygenaase‐2 (COX‐2) in breast cancer cells. Our data indicated that ar‐turmerone treatment significantly inhibited enzymatic activity and expression of MMP‐9 and COX‐2 at non‐cytotoxic concentrations. However, the expression of tissue inhibitor of metalloproteinase (TIMP)‐1, TIMP‐2, MMP‐2, and COX‐1 did not change upon ar‐turmerone treatment. We found that ar‐turmerone inhibited the activation of NF‐κB, whereas it did not affect AP‐1 activation. Moreover, The ChIP assay revealed that in vivo binding activities of NF‐κB to the MMP‐9 and COX‐2 promoter were significantly inhibited by ar‐turmerone. Our data showed that ar‐turmerone reduced the phosphorylation of PI3K/Akt and ERK1/2 signaling, whereas it did not affect phosphorylation of JNK or p38 MAPK. Thus, transfection of breast cancer cells with PI3K/Akt and ERK1/2 siRNAs significantly decreased TPA‐induced MMP‐9 and COX‐2 expression. These results suggest that ar‐turmerone suppressed the TPA‐induced up‐regulation of MMP‐9 and COX‐2 expression by blocking NF‐κB, PI3K/Akt, and ERK1/2 signaling in human breast cancer cells. Furthermore, ar‐turmerone significantly inhibited TPA‐induced invasion, migration, and colony formation in human breast cancer cells. J. Cell. Biochem. 113: 3653–3662, 2012. © 2012 Wiley Periodicals, Inc.     

Expressions of inhibitory Smads, Smad6 and Smad7, are differentially regulated by TPA in human lung fibroblast cells

Smad6 and Smad7 are inhibitory Smads (I-Smads) with differential inhibitory effects on the regulation of the cellular signalings induced by TGF-beta superfamily. Here, we show that phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) down-regulates Smad6 mRNA expression and up-regulates Smad7 mRNA expression in IMR-90, a human lung fibroblast cell line. These regulations of I-Smads by TPA were suppressed by one PKC inhibitor (G?6983), but not by another (G?6976). TPA treatment had little effect on the phosphorylation of novel PKCs (PKCdelta and PKCepsilon), but specifically induced PKCmu phosphorylation, and this effect was inhibited by G?6983, but not by G?6976. Additionally, G?6983 but not G?6976 inhibited ERK- and JNK-phosphorylation as well as Smad7 promoter activity induced by TPA. MEK inhibitor U0126 inhibited the down-regulation of Smad6 mRNA expression but not the up-regulation of Smad7 mRNA expression. In contrast, JNK inhibitor SP600125 had no such effects. Luciferase reporter analysis revealed that TPA did not induce NF-kappaB activation. In addition, TPA up-regulated Smad7 expression in the presence of NF-kappaB inhibitor TLCK. These findings indicate that TPA down-regulates Smad6 expression presumably via PKCmu-ERK-dependent pathway and up-regulates Smad7 expression via PKCmu-dependent mechanism(s) which need no MAPK and NF-kappaB activation.     

佛波酯对NIH3T3细胞整合蛋白α_5亚基表达的影响

１００ｎｍｏｌ／Ｌ佛波酯（１２－Ｏ－ｔｅｔｒａｄｅｃａｎｏｙｌｐｈｏｂｏｌ１３－ａｃｅｔａｔｅ,ＴＰＡ）作用于ＮＩＨ３Ｔ３细胞２４ｈ,流式细胞仪检测到细胞表面整合蛋白α５亚基含量增加５２．３％．Ｎｏｒｔｈｅｒｎ杂交方法测定结果亦表明整合蛋白α５亚基ｍＲＮＡ量增加,于２ｈ时达到高峰,为对照的４．１４倍．蛋白激酶Ｃ（ｐｒｏｔｅｉｎｋｉｎａｓｅＣ,ＰＫＣ）的活性增加趋势与之基本一致．运用ＰＫＣ的抑制剂鞘氨醇（ｓｐｈｉｎｇｏ－ｓｉｎｅ）和酷氨酸激酶（ｔｙｒｏｓｉｎｅｋｉｎａｓｅ,ＴＫ）抑制剂４,５,７－三羟基异黄酮（ｇｅｎｅｓｔｅｉｎ）进一步研究,发现两者均可抑制佛波酯对整合蛋白α５亚基表达的上调作用．提示佛波酯对ＮＩＨ３Ｔ３细胞整合蛋白α５亚基表达的调控与ＰＫＣ和ＴＫ均有关．     

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.     

Phenotypic effects of overexpression of PKC beta 1 in rat liver epithelial cells

We have used a previously described retroviral expression vector pMV7-PKC beta 1 to develop derivatives of two rat liver epithelial cell lines, K16 and K22, that stably express about tenfold-higher PKC activity than control cells. Despite these high levels of PKC, these cells did not exhibit gross morphologic changes, anchorage-independent growth, or tumorigenicity. K16PKC-4 and K22PKC-2, two lines with the highest PKC enzyme activity, were studied further in terms of several responses to the phorbol ester tumor promoter TPA. When treated with 100 ng/ml of TPA, the control K16MV7 and K22MV7 cells displayed a slight change in morphology, whereas the K16PKC-4 and K22PKC-2 cells displayed a marked change in morphology. Northern blot analyses demonstrated that TPA induced increased levels of fos, myc, phorbin, and ODC RNAs in control K16MV7 and K22MV7 cells, with maximum induction occurring at about 0.5, 1, 8, and 8 h, respectively. In K16PKC-4 and K22PKC-2 cells, TPA induction of phorbin and ODC RNAs was markedly enhanced, but this was not the case for myc and fos RNAs. In addition, the levels of myc RNA were constitutively higher in both K16PKC-4 and K22PKC-2 cells than in the control cells. Taken together, these results provide direct evidence that PKC plays a critical role in modulating the expression of myc, phorbin, and ODC RNAs. On the other hand, overexpression of PKC beta 1 is not itself sufficient to cause cell transformation.     

Epidermal growth factor (EGF) and hormones stimulate phosphoinositide hydrolysis and increase EGF receptor protein synthesis and mRNA levels in rat liver epithelial cells. Evidence for protein kinase C-dependent and -independent pathways

Epidermal growth factor (EGF) stimulated the rapid accumulation of inositol trisphosphate in WB cells, a continuous line of rat hepatic epithelial cells. Since we previously had shown that EGF stimulates EGF receptor synthesis in these cells, we tested whether hormones that stimulate PtdIns(4,5)P2 hydrolysis would increase EGF receptor protein synthesis and mRNA levels. Epinephrine, angiotensin II, and [Arg8]vasopressin activate phospholipase C in WB cells as evidenced by the accumulation of the inositol phosphates, inositol monophosphate, inositol bisphosphate, and inositol trisphosphate. A 3-4-h treatment with each hormone also increased the rate of EGF receptor protein synthesis by 3-6-fold as assessed by immunoprecipitation of EGF receptor from [35S]methionine-labeled cells. Northern blot analyses of WB cell EGF receptor mRNA levels revealed that agents linked to the phosphoinositide signaling system increased receptor mRNA content within 1-2 h. A maximal increase of 3-7-fold was observed after a 3-h exposure to EGF and hormones. The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), which activates protein kinase C also stimulated EGF receptor synthesis. Pretreatment of WB cells for 18 h with high concentrations of TPA "down-regulated" protein kinase C and blocked TPA-directed EGF receptor mRNA synthesis. In contrast, the effect of EGF on EGF receptor mRNA levels was not significantly decreased by TPA pretreatment. Epinephrine-induced increases in EGF receptor mRNA were reduced from 4- to 2-fold. Similarly, 18 h TPA pretreatment abolished the effect of TPA on EGF receptor protein synthesis but did not affect EGF-dependent EGF receptor protein synthesis. The 18-h TPA pretreatment diminished by 30-50% the induction of receptor protein synthesis by epinephrine or angiotensin II. We conclude that in WB cells EGF receptor synthesis can be regulated by EGF and other hormones that stimulate PtdIns(4,5)P2 hydrolysis. In these cells, EGF receptor synthesis appears to be regulated by several mechanism: one pathway is dependent upon EGF receptor activation and can operate independently of protein kinase C activation; another pathway is correlated with PtdIns(4,5)P2 hydrolysis and is dependent, at least in part, upon protein kinase C activation.     

Osteopontin, a transformation-associated cell adhesion phosphoprotein, is induced by 12-O-tetradecanoylphorbol 13-acetate in mouse epidermis

A murine mRNA (provisionally called 2ar) is described whose abundance is greatly increased by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate both in JB6 epidermal cells in vitro and in epidermis in vivo. We have previously shown induction of 2ar in epidermal or fibroblast cell lines by tumor promoters, growth factors, and transformation with H-ras. The 2ar mRNA appears to be derived from a single copy gene. It encodes the mouse homolog of rat osteopontin, a 41.5-kDa glycosylated bone phosphoprotein that binds to fibroblasts and osteosarcoma cells and to hydroxylapatite (bone matrix). The rat and mouse sequences are 84% identical at the amino acid level and 87% identical at the nucleotide level. Many of the primary structural features are conserved, including a run of 9-10 aspartic residues and a Gly-Arg-Gly-Asp-Ser cell adhesion sequence. Antiserum raised against portions of the predicted polypeptide immunoprecipitated proteins of apparent Mr 55,000-70,000 both from reticulocyte lysates containing the translation products of hybrid-selected mRNA and from cell culture medium containing metabolically labeled proteins secreted by JB6 cells. The results presented here demonstrate that osteopontin is identical to a transformation-associated phosphoprotein whose level of expression by cultured cells and abundance in human sera has been correlated with tumorigenicity. These results suggest a role for osteopontin in carcinogenesis. The murine version of osteopontin has been given the formal name "secreted phosphoprotein 1" and the designation spp.     

Regulation of alternative splicing of Bcl-x by IL-6, GM-CSF and TPA

The splicing of many alternative exons in the precursor messenger RNA (pre-mRNA) is regulated by extracellular factors but the underlying molecular bases remain unclear. Here we report the differential regulation of Bcl-x pre-mRNA splicing by extracellular factors and their distinct requirements for pre-mRNA elements. In K562 leukemia cells, treatment with interleukin-6 (IL-6) or granulocyte-macrophage colony stimulating factor (GM-CSF) reduced the proportion of the Bcl-xL variant mRNA while treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) had no effect. In U251 glioma cells, however, TPA efficiently increased the Bcl-xL level. These regulations were also seen for a transfected splicing reporter mini-gene. Further analyses of deletion mutants indicate that nucleotides 1-176 of the downstream intron are required for the IL-6 effect, whereas additional nucleotides 177-284 are essential for the GM-CSF effect. As for the TPA effect, only nucleotides 1-76 are required in the downstream intron. Thus, IL-6, GM-CSF and TPA differentially regulate Bcl-x splicing and require specific intronic pre-mRNA sequences for their respective effects.