Retinoic acid and phorbol ester synergistically up-regulate IL-8 expression and specifically modulate protein kinase C-epsilon in human skin fibroblasts.

Phorbol ester (TPA) and retinoic acid (RA) are two potent immunomodulatory agents whose actions are mediated through distinct signal transduction pathways involving protein kinase C (PKC) and nuclear RA receptors, respectively. We have investigated the interactions between these two pathways in the regulation of expression of the inflammatory cytokine IL-8 in human skin fibroblasts. TPA (as previously reported) and RA both induced IL-8 mRNA and protein in a time- and dose-dependent manner. IL-8 mRNA induction by TPA (10 nM) was maximal (15-fold) within 6 h, and returned to baseline within 24 h of treatment, although maximal induction (10-fold) by RA (1 microM) did not occur until 24 h posttreatment. Induction of IL-8 by TPA was blocked by 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine, which inhibits PKC and cAMP-dependent protein kinases (PKA), but not by N-(2-ganidinoethyl)-5-isoquinoline sulfonamide, which preferentially inhibits PKA, consistent with the participation of PKC in the induction of IL-8 by TPA. In contrast, induction of IL-8 by RA was inhibited by both 1-(5-isoquinoline sulfonamide and N-(2-gamidinoethyl)-5-isoquinoline sulfonamide, suggesting the participation of PKA in the induction of IL-8 by RA. However, activation of PKA by addition of cAMP analogues was not sufficient to induce IL-8 expression. Induction of IL-8 by RA also did not appear to be mediated indirectly through induction of IL-1, because addition of IL-1R antagonist did not block IL-8 induction by RA. RA and TPA added in combination synergistically enhanced expression of IL-8 mRNA, measured at 6 (2-fold) and 24 h (10-fold) posttreatment. To investigate the mechanism of this synergy, the effect of TPA and RA on fibroblast PKC activation and PKC isozyme levels were determined. TPA, either alone or together with RA, but not RA alone, stimulated phosphorylation of an endogenous 80-kDa PKC substrate. Dermal fibroblasts expressed three PKC isozymes (alpha, (delta, and (epsilon). TPA, but not RA, down-regulated PKC-alpha, neither TPA or RA affected the level of PKC-delta, and both TPA and RA down-regulated PKC-epsilon. This latter effect was enhanced 2-fold by addition of RA and TPA together. These data suggest that modulation of PKC-epsilon may be a common participant in the regulation of IL-8 expression by TPA and RA.     

Effect of parathyroid hormone on rat renal cAMP-dependent protein kinase and protein kinase C activity measured using synthetic peptide substrates

The actions of parathyroid hormone (PTH) on the renal cortex are thought to be mediated primarily by cAMP-dependent protein kinase (PKA) with some suggestion of a role for protein kinase C (PKC). However, present methods for assaying PKA and PKC in subcellular fractions are insensitive and require large amounts of protein. Recently, a sensitive method for measuring the activity of protein kinases has been reported. This method uses synthetic peptides as substrates and a tandem chromatographic procedure for isolating the phosphorylated peptides. We have adapted this method to study the effect of PTH on PKA and PKC activity using thin slices of rat renal cortex. PTH (250 nM) stimulated cytosolic PKA activity four- to fivefold within 30 s, and PKA activity was sustained for at least 5 min. PTH also rapidly stimulated PKC activity in the membrane fraction and decreased PKC activity in the cytosol. These changes were maximal at 30 s, but unlike changes in PKA, they declined rapidly thereafter. PTH significantly activated PKC only at concentrations of 10 nM or greater. This study demonstrates that PTH does activate PKC in renal tissue, although the duration of activation is much less than for PKA. It also demonstrates that a combination of synthetic peptides with tandem chromatography can be used as a sensitive assay procedure for protein kinase activity in biological samples.     

Effect of perinatal thyroid hormone deficiency on expression of rat hippocampal conventional protein kinase C isozymes

Thyroid hormone (TH) is essential for the proper development of mammalian central nervous system. TH deficiency during critical period of brain development results in permanent cognitive and neurological impairments. Hippocampus is a structure involved in various memory processes that are essential for creating new memories, and lesions to hippocampus result in impaired learning and memory. Protein kinase C (PKC) isoforms play an important role in many types of learning and memory, and deletion of specific PKC genes results in deficits in learning. In the present study, we used real-time PCR and Western blot to investigate the conventional PKC expression in developing rat hippocampus with different thyroid status, trying to establish a correlation between TH deficiency and conventional PKC expression in developing rat hippocampus. We found that PKCβI and PKCγ expression decreased significantly both in mRNA and protein levels in hypothyroid group compared with the normal controls, and thyroxine replacement could restore it. As for PKCα, we did not find any difference between different thyroid status. Though the expression of PKCβII also decreased in the TH deficiency group, the change was not significant. Taken together, our data indicate TH deficiency can cause hippocampal PKCβ1 and PKCγ downregulation during rat brain development. Since there are other PKC isoforms in the rat brain, whether these change is related to impaired learning and memory of perinatal hypothyroid rats requires further researches.     

Oxidative damage and antioxidant enzyme activities in experimental hypothyroidism

Free radicals are now well known to damage cellular components. To investigate whether age and thyroid level affect peroxidation speed, we examined the levels of malondialdehyde and antioxidant enzyme activities in different age groups of hypothyroid rats. Hypothyroidism was induced in 30- and 60-day-old Wistar Albino rats by the i.p. administration of propylthiouracil (10 mg kg(-1) body weight) for 15 days. While malondialdehyde levels of 30- or 60-day-old hypothyroid rats were increased in liver, they were decreased in the tissues of the heart and thyroid. While glucose-6-phosphate dehydrogenase activity levels did not change in heart, brain and liver tissues of 30-day-old rats, they increased in brain and heart tissues of 60-day-old experimental groups, but decreased in the liver. Catalase activities decreased in the liver and heart of rats with hypothyroidism, but increased in erythrocytes. In control groups while malondialdehyde levels increased in brain, heart and thymus with regard to age, they decreased in plasma. Glucose-6-phosphate dehydrogenase and catalase activities were not affected by age in tissues of the thymus, thyroid and brain, but they were decreased in the heart tissue. The changes in the levels of lipid peroxidation and antioxidant enzyme activities which were determined in different tissues of hypothyroid rats indicate a cause for functional disorder of these tissues. Moreover, there may be changes depending on age at lipid peroxidation and antioxidant enzyme activity levels.     

Differential regulation of Na,K-ATPase alpha 1, alpha 2, and beta subunit mRNA and protein levels by thyroid hormone

The purpose of this study was to determine the effect of thyroid status on the Na,K-ATPase alpha isoforms and beta in rat heart, skeletal muscle, kidney, and brain at the levels of mRNA, protein abundance, and enzymatic activity. Northern and dot-blot analysis of RNA (euthyroid, hypothyroid, and triiodothyronine-injected hypothyroids = hyperthyroids) and immunoblot analysis of protein (euthyroid and hypothyroid) revealed isoform-specific regulation of Na,K-ATPase by thyroid status in kidney, heart, and skeletal muscle and no regulation of sodium pump subunit levels in the brain. In general, in the transition from euthyroid to hypothyroid alpha 1 mRNA and protein levels are unchanged in kidney and skeletal muscle and slightly decreased in heart, while alpha 2 mRNA and protein are decreased significantly in heart and skeletal muscle. In hypothyroid heart and skeletal muscle, the decrease in alpha 2 protein levels was much greater than the decrease in alpha 2 mRNA levels relative to euthyroid indicating translational or post-translational regulation of alpha 2 protein abundance by triiodothyronine status in these tissues. The regulation of beta subunit by thyroid status is tissue-dependent. In hypothyroid kidney beta mRNA levels do not change, but immunodetectable beta protein levels decrease relative to euthyroid, and the decrease parallels the decrease in Na,K-ATPase activity. In hypothyroid heart and skeletal muscle beta mRNA levels decrease; beta protein decreases in heart and was not detected in the skeletal muscle. These findings demonstrate that the euthyroid levels of expression of alpha 1 in heart, alpha 2 in heart and skeletal muscle, and beta in kidney, heart, and skeletal muscle are dependent on the presence of thyroid hormone.     

Effect of thyroid state on susceptibility to oxidants and swelling of mitochondria from rat tissues

The effects of the thyroid state on oxidative damage, antioxidant capacity, susceptibility to in vitro oxidative stress and Ca(2+)-induced permeabilization of mitochondria from rat tissues (liver, heart, and gastrocnemious muscle) were examined. Hypothyroidism was induced by administering methimazole in drinking water for 15 d. Hyperthyroidism was elicited by a 10 d treatment of hypothyroid rats with triiodothyronine (10 micro g/100 g body weight). Mitochondrial levels of hydroperoxides and protein-bound carbonyls significantly decreased in hypothyroid tissues and were reported above euthroid values in hypothyroid rats after T(3) treatment. Mitochondrial vitamin E levels were not affected by changes of animal thyroid state. Mitochondrial Coenzyme Q9 levels decreased in liver and heart from hypothyroid rats and increased in all hyperthyroid tissues, while Coenzyme Q10 levels decreased in hypothyroid liver and increased in all hyperthyroid tissues. The antioxidant capacity of mitochondria was not significantly different in hypothyroid and euthyroid tissues, whereas it decreased in the hyperthyroid ones. Susceptibility to in vitro oxidative challenge decreased in mitochondria from hypothyroid tissues and increased in mitochondria from hyperthyroid tissues, while susceptibility to Ca(2+)-induced swelling decreased only in hypothyroid liver mitochondria and increased in mitochondria from all hyperthyroid tissues. The tissue-dependence of the mitochondrial susceptibility to stressful conditions in altered thyroid states can be explained by different thyroid hormone-induced changes in mitochondrial ROS production and relative amounts of mitochondrial hemoproteins and antioxidants. We suggest that susceptibilities to oxidants and Ca(2+)-induced swelling may have important implications for the thyroid hormone regulation of the turnover of proteins and whole mitochondria, respectively.     

Effects of thyroid state on the expression of hepatic thyroid hormone transporters in rats

Liver uptake of thyroxine (T4) is mediated by transporters and is rate limiting for hepatic 3,3',5-triiodothyronine (T3) production. We investigated whether hepatic mRNA for T4 transporters is regulated by thyroid state using Xenopus laevis oocytes as an expression system. Because X. laevis oocytes show high endogenous uptake of T4, T4 sulfamate (T4NS) was used as an alternative ligand for the hepatic T4 transporters. Oocytes were injected with 23 ng liver mRNA from euthyroid, hypothyroid, or hyperthyroid rats, and after 3-4 days uptake was determined by incubation of injected and uninjected oocytes for 1 h at 25 degrees C or for 4 h at 18 degrees C with 10 nM [125I]T4NS. Expression of type I deiodinase (D1), which is regulated by thyroid state, was studied in the oocytes as an internal control. Uptake of T4NS showed similar approximately fourfold increases after injection of liver mRNA from euthyroid, hypothyroid, or hyperthyroid rats. A similar lack of effect of thyroid state was observed using reverse T3 as ligand. In contrast, D1 activity induced by liver mRNA from hyperthyroid and hypothyroid rats in the oocytes was 2.4-fold higher and 2.7-fold lower, respectively, compared with euthyroid rats. Studies have shown that uptake of iodothyronines in rat liver is mediated in part by several organic anion transporters, such as the Na+/taurocholate-cotransporting polypeptide (rNTCP) and the Na-independent organic anion-transporting polypeptide (rOATP1). Therefore, the effects of thyroid state on rNTCP, rOATP1, and D1 mRNA levels in rat liver were also determined. Northern analysis showed no differences in rNTCP or rOATP1 mRNA levels between hyperthyroid and hypothyroid rats, whereas D1 mRNA levels varied widely as expected. These results suggest little effect of thyroid state on the levels of mRNA coding for T4 transporters in rat liver, including rNTCP and rOATP1. However, they do not exclude regulation of hepatic T4 transporters by thyroid hormone at the translational and posttranslational level.     

Cellular concentrations of protein kinase A modulate prostaglandin and cAMP induction of alkaline phosphatase.

The relationship between the concentration of cAMP-dependent protein kinase (PKA) activity and the induction of alkaline phosphatase (AP) was examined in transfected L cell lines with altered PKA levels. C alpha 12 cells were generated by transfecting mouse L cells with an expression vector coding for the mouse C alpha catalytic subunit of PKA and were shown to contain 2.5-fold more PKA activity than L cells. RAB10 cells were generated by transfection with an expression vector for a mutant regulatory subunit and had 10-fold lower levels of PKA activity than L cells. AP induction by 8-chlorophenylthio-cAMP (CPT-cAMP) was found to be 2-fold greater in C alpha 12 cells than in L cells, while RAB10 cells lacked any induction of AP in response to CPT-cAMP. Northern blot and solution hybridization analyses of AP mRNA showed that induced AP mRNA levels were comparable in C alpha 12 and in L cells. Western blot analysis demonstrated that AP protein levels were greater in C alpha 12 cells and suggested that the increased AP protein level resulted from either increased stability of the AP protein or increased rate of translation of the AP mRNA. In contrast, Northern blot analysis of the RAB10 cells failed to detect AP mRNA after CPT-cAMP treatment and suggested that PKA is required for induction of AP mRNA. Stimulation of endogenous cAMP levels by treatment with prostaglandin E1 gave similar effects on AP activity as those seen with CPT-cAMP. These results indicate that cellular levels of PKA can determine the magnitude of cellular response to hormonal stimulation and also suggest that PKA can regulate AP gene expression at both the level of the AP mRNA and AP protein.     

Cyclic AMP-dependent protein kinase (PKA) and protein kinase C phosphorylate sites in the amino acid sequence corresponding to the M3/M4 cytoplasmic domain of alpha4 neuronal nicotinic receptor subunits

To determine whether alpha4 subunits of alpha4beta2 neuronal nicotinic receptors are phosphorylated within the M3/M4 intracellular region by cyclic AMP-dependent protein kinase A (PKA) or protein kinase C (PKC), immunoprecipitated receptors from Xenopus oocytes and a fusion protein corresponding to the M3/M4 cytoplasmic domain of alpha4 (alpha4(336-597)) were incubated with ATP and either PKA or PKC. Both alpha4 and alpha4(336-597) were phosphorylated by PKA and PKC, providing the first direct biochemical evidence that the M3/M4 cytoplasmic domain of neuronal nicotinic receptor alpha4 subunits is phosphorylated by both kinases. When the immunoprecipitated receptors and the alpha4(336-597) fusion protein were phosphorylated and the labeled proteins subjected to phosphoamino acid analysis, results indicated that alpha4 and alpha4(336-597) were phosphorylated on the same amino acid residues by each kinase. Furthermore, PKA phosphorylated serines exclusively, whereas PKC phosphorylated both serines and threonines. To determine whether Ser(368) was a substrate for both kinases, a peptide corresponding to amino acids 356-371 was synthesized (alpha4(356-371)) and incubated with ATP and the kinases. The phosphorylation of alpha4(356-371) by both PKA and PKC was saturable with K(m)s of 15.3 +/- 3.3 microM and 160.8 +/- 26.8 microM, respectively, suggesting that Ser(368) was a better substrate for PKA than PKC.     

Phosphorylation of membrane-bound acetylcholine receptor by protein kinase C: characterization and subunit specificity

Acetylcholine receptor (AChR) from Torpedo electric organ in its membrane-bound or solubilized form is phosphorylated by the Ca2+/phospholipid-dependent protein kinase (PKC). The subunit specificity for PKC is different from that observed for cAMP-dependent protein kinase (PKA). Whereas PKC phosphorylates predominantly the delta subunit and the phosphorylation of the gamma subunit by this enzyme is very low, PKA phosphorylates both subunits to a similar high extent. We have extended our phosphorylation studies to a synthetic peptide from the gamma subunit, corresponding to residues 346-359, which contains a consensus PKA phosphorylation site. This synthetic peptide is phosphorylated by both PKA and PKC, suggesting that in the intact receptor both kinases may phosphorylate the gamma subunit at a similar site, as has been previously demonstrated by us for the delta subunit [Safran, A., et al. (1987) J. Biol. Chem. 262, 10506-10510]. The diverse pattern of phosphorylation of AChR by PKA and PKC may play a role in the regulation of its function.     

Neuromodulation of Na+ channel slow inactivation via cAMP-dependent protein kinase and protein kinase C

Neurotransmitters modulate sodium channel availability through activation of G protein-coupled receptors, cAMP-dependent protein kinase (PKA), and protein kinase C (PKC). Voltage-dependent slow inactivation also controls sodium channel availability, synaptic integration, and neuronal firing. Here we show by analysis of sodium channel mutants that neuromodulation via PKA and PKC enhances intrinsic slow inactivation of sodium channels, making them unavailable for activation. Mutations in the S6 segment in domain III (N1466A,D) either enhance or block slow inactivation, implicating S6 segments in the molecular pathway for slow inactivation. Modulation of N1466A channels by PKC or PKA is increased, whereas modulation of N1466D is nearly completely blocked. These results demonstrate that neuromodulation by PKA and PKC is caused by their enhancement of intrinsic slow inactivation gating. Modulation of slow inactivation by neurotransmitters acting through G protein-coupled receptors, PKA, and PKC is a flexible mechanism of cellular plasticity controlling the firing behavior of central neurons.     

Selectivity of protein kinase inhibitors in human intact platelets

The specificity of commonly used protein kinase inhibitors has been evaluated in the intact human platelet. Protein kinase C (PKC) and cyclic AMP-dependent protein kinase (PKA) were activated selectively by treating platelets with phorbol dibutyrate (PDBu) or prostacyclin (PGl2). PKC activity was quantitated by measuring PDBu-specific phosphorylation of a 47,000 molecular weight protein, and PKA activity monitored by measuring prostacyclin-dependent phosphorylation of a 22,000 molecular weight protein. Staurosporine and 1-(5-isoquinolinylsulphonyl)-2-methyl-piperazine (H-7) were found to be non-specific inhibitors in the intact platelet, consistent with their effects on the isolated enzymes. Tamoxifen inhibited PKC activity (IC50 = 80 microM) but increased PKA-dependent protein phosphorylation. These results support the use of human platelets for measuring the specificity of protein kinase inhibitors and indicate that tamoxifen might have value for experimental purposes as a relatively selective PKC inhibitor.     

The role of protein kinase C-delta in PTH stimulation of IGF-binding protein-5 mRNA in UMR-106-01 cells

We have investigated the role of protein kinase C (PKC) signal transduction pathways in parathyroid hormone (PTH) regulation of insulin-like growth factor-binding protein-5 (IGFBP-5) gene expression in the rat osteoblast-like cell line UMR-106-01. Involvement of the PKC pathway was determined by the findings that bisindolylmaleimide I inhibited 40% of the PTH effect, and 1 microM bovine PTH-(3-34) stimulated a 10-fold induction of IGFBP-5 mRNA. PTH-(1-34) and PTH-(3-34) (100 nM) both stimulated PKC-delta translocation from the membrane to the nuclear fraction. Rottlerin, a PKC-delta-specific inhibitor, and a dominant negative mutant of PKC-delta were both able to significantly inhibit PTH-(1-34) and PTH-(3-34) induction of IGFBP-5 mRNA, suggesting a stimulatory role for PKC-delta in the effects of PTH. Phorbol 12-myristate 13-acetate (PMA) stimulated PKC-alpha translocation from the cytosol to the membrane and inhibited approximately 50% of the PTH-(1-34), forskolin, and 8-bromoadenosine 3',5'-cyclic monophosphate-stimulated IGFBP-5 mRNA levels, suggesting that PKC-alpha negatively regulates protein kinase A (PKA)-mediated induction of IGFBP-5 mRNA. These results suggest that the induction of IGFBP-5 by PTH is both PKA and PKC dependent and PKC-delta is the primary mediator of the effects of PTH via the PKC pathway.     

化学诱发大鼠肝癌的亚细胞组分中两类丝氨酸蛋白激酶的动态变化

用二乙基亚硝胺（ＤＥＮ）诱发大鼠肝癌,隔周测定肝脏胞液、膜性和胞核中的蛋白激酶Ａ（ＰＫＡ）和蛋白激酶Ｃ（ＰＫＣ）的活力。发现胞液ＰＫＡ在诱癌过程中活力改变不大,胞液ＰＫＣ则逐步增高,在第１３周和２０周形成两个活力高峰。膜性ＰＫＡ和ＰＫＣ都呈双相变化,即在癌前期（１０－１４周）增加,癌形成期（１７－２０周）反而降至正常以下,胞核ＰＫＡ和ＰＫＣ也都在癌前期升至高峰,而癌形成期则低于癌前期,但仍高于正常（ＰＫＡ）或接近正常（ＰＫＣ）。因只有膜性ＰＫＣ在大鼠老化时降低,故这些变化不是鼠龄变化的结果,而是ＤＥＮ诱癌所引起,其变化机理可能与下降调节、细胞内转位或两型同工酶相反的升降变化有关。     

Regulation of matrix Gla protein by parathyroid hormone in MC3T3-E1 osteoblast-like cells involves protein kinase A and extracellular signal-regulated kinase pathways

Inhibition of osteoblast-mediated mineralization is one of the major catabolic effects of parathyroid hormone (PTH) on bone. Previously, we showed that PTH induces matrix gamma-carboxyglutamic acid (Gla) protein (MGP) expression and established that this induction is critical for PTH-mediated inhibition of osteoblast mineralization. In the present study, we focus on the mechanism through which PTH regulates MGP expression in osteoblastic MC3T3-E1 cells. Following transient transfection of these cells with a -748 bp murine MGP promoter-luciferase construct (pMGP-luc), PTH (10 (-7) M) induced promoter activity in a time-dependent manner with a maximal four- to six fold induction seen 6 h after PTH treatment. Both H-89 (PKA inhibitor) and U0126 (MEK inhibitor), suppressed PTH induction of MGP promoter activity as well as the MGP mRNA level. In addition, forskolin (PKA activator) stimulated MGP promoter activity and mRNA levels confirming that PKA is one of the signaling molecules required for regulation of MGP by PTH. Co-transfection of MC3T3-E1 cells with pMGP-luc and MEK(SP), a plasmid encoding the constitutively active form of MEK, led to a dose-dependent increase in MGP promoter activity. Both MGP promoter activity and MGP mRNA level were not affected by the protein kinase C (PKC) inhibitor, GF109203X. However, phorbol 12-myristate 13-acetate (PMA), a selective PKC activator induced MGP mRNA expression through activation of extracellular signal-regulated kinase (ERK). Taken together, these results indicate that PTH regulates MGP via both PKA- and ERK-dependent pathways.