Ceramide-induced inhibition of Akt is mediated through protein kinase Czeta: implications for growth arrest.

We recently demonstrated that ceramide-coated balloon catheters limit vascular smooth muscle cell (VSMC) growth after stretch injury in vivo. In that study, inhibition of VSMC growth was correlated with a decrease in phosphorylation of the cell survival kinase Akt (protein kinase B). Utilizing cultured A7r5 VSMCs, we have now examined the mechanism by which ceramide inhibits Akt phosphorylation/activation. Our initial studies showed that ceramide-induced inhibition of Akt phosphorylation was not mediated through diminution in phosphoinositide 3-kinase activity. As we have previously demonstrated that protein kinase Czeta (PKCzeta) is a target of ceramide, we proposed an alternative signaling mechanism by which ceramide induces inhibition of Akt through activation of PKCzeta. We demonstrate that C(6)-ceramide (but not the inactive analog dihydro-C(6)-ceramide) induced PKCzeta activity and also caused a selective increase in the association between Akt and PKCzeta, without affecting PKCepsilon, in A7r5 cells. In addition, the ability of ceramide to significantly decrease platelet-derived growth factor-induced Akt phosphorylation or cell proliferation was abrogated in A7r5 cells overexpressing a dominant-negative mutant of PKCzeta. Taken together, these data suggest that ceramide-mediated activation of PKCzeta leads to diminished Akt activation and consequent growth arrest in VSMCs. The therapeutic potential for ceramide to limit dysregulated VSMC growth has direct applicability to vascular diseases such as restenosis and atherosclerosis.     

Possible involvement of ceramide in the regulation of rat Leydig cell function

In the present study, a possible role of a ceramide-dependent pathway in the regulation of Leydig cell function was investigated. Intracellular ceramide levels were increased by: (a) adding ceramide analogs; (b) inhibiting ceramidase activity; and (c) adding sphingomyelinase (SMase). The cell-permeable ceramide analogs N-acetyl-, N-hexanoyl- and N-octanoylsphingosine (C2, C6 and C8) were used. As inhibitor of ceramidase activity 1S,2R-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (MAPP) was used. Sphingomyelinase from S. aureus origin was utilized. Leydig cells were cultured for 3 or 24 h with or without the different drugs (10 microM) and SMase (0.3 U/ml) in the presence or absence of hCG (10 ng/ml). Basal testosterone production was not modified under any of the experimental conditions. A decrease in hCG-stimulated testosterone production was observed at 3 and 24 h in all cases. The inactive analog (N-hexanoyl dihydrosphingosine) did not produce inhibition in hCG-stimulated testosterone production. TNFalpha and IL1beta, two possible inducers of sphingomyelin hydrolysis, produced similar effects on hCG-stimulated testosterone production. In experiments performed in the presence of C6, inhibition in hCG-stimulated cAMP production was observed. The inhibitory effect of ceramide was also observed in dbcAMP-stimulated cultures indicating that this pathway inhibits post-cAMP formation events. To study possible loci for the action of ceramide on the steroidogenic pathway, cells were incubated with C6 and MAPP in the presence of different testosterone precursors. The drugs inhibited testosterone produced from 22(R)-hydroxycholesterol (22R-OHChol), pregnenolone and 17alpha-hydroxyprogesterone (17OHP4) but not from androstenedione (Delta4). These results suggest that a ceramide-dependent pathway regulates hCG-stimulated Leydig cell steroidogenesis at the level of cAMP production and at post-cAMP events.     

Regulation by sphingolipids of the fate of FRTL-5 cells

Sphingolipids, including ceramide (Cer), sphingosine (Sph), and sphingosine 1-phosphate (Sph-1-P) have recently emerged as signal-transducing molecules. Functionally, a distinguishing characteristic of these lipids is their apparent participation in pro- or anti-proliferative cell regulation pathways. In this study, we examined the involvement of sphingolipids in the fate of FRTL-5 thyroid follicular cells. We first examined the effects of sphingolipids on FRTL-5 cell viability. Sph and Cer induced apoptosis, as revealed by fluorescence microscopy of TUNEL-positive fragmented nuclei and 180-300 bp DNA fragmentation on agarose gel electrophoresis while Sph-1-P was confirmed to prevent FRTL-5 cell apoptosis induced by deprivation of serum and TSH, possibly via cell surface receptors. We then analysed the metabolism of radiolabelled Sph and C(6)-Cer (a synthetic cell-permeable Cer) in FRTL-5 cells by thin layer chromatography, followed by autoradiography. Sph was mainly metabolized to Cer, and then to sphingomyelin, while Sph conversion into Sph-1-P was hardly detected. These changes were not affected by stimulation of the cells with TSH. Our results indicate the involvement of sphingolipid mediators in the fate of FRTL-5 thyroid cells.     

Direct binding to ceramide activates protein kinase Czeta before the formation of a pro-apoptotic complex with PAR-4 in differentiating stem cells

We have reported that ceramide mediates binding of atypical protein kinase C (PKC) zeta to its inhibitor protein, PAR-4 (prostate apoptosis response-4), thereby inducing apoptosis in differentiating embryonic stem cells. Using a novel method of lipid vesicle-mediated affinity chromatography, we showed here that endogenous ceramide binds directly to the PKCzeta.PAR-4 complex. Ceramide and its analogs activated PKCzeta prior to binding to PAR-4, as determined by increased levels of phosphorylated PKCzeta and glycogen synthase kinase-3beta and emergence of a PAR-4-to-phosphorylated PKCzeta fluorescence resonance energy transfer signal that co-localizes with ceramide. Elevated expression and activation of PKCzeta increased cell survival, whereas expression of PAR-4 promoted apoptosis. This suggests that PKCzeta counteracts apoptosis, unless its ceramide-induced activation is compromised by binding to PAR-4. A luciferase reporter assay showed that ceramide analogs activate nuclear factor (NF)-kappaB unless PAR-4-dependent inhibition of PKCzeta suppresses NF-kappaB activation. Taken together, our results show that direct physical association with ceramide and PAR-4 regulates the activity of PKCzeta. They also indicate that this interaction regulates the activity of glycogen synthase kinase-3beta and NF-kappaB.     

Ceramide disables 3-phosphoinositide binding to the pleckstrin homology domain of protein kinase B (PKB)/Akt by a PKCzeta-dependent mechanism

Ceramide is generated in response to numerous stress-inducing stimuli and has been implicated in the regulation of diverse cellular responses, including cell death, differentiation, and insulin sensitivity. Recent evidence indicates that ceramide may regulate these responses by inhibiting the stimulus-mediated activation of protein kinase B (PKB), a key determinant of cell fate and insulin action. Here we show that inhibition of this kinase involves atypical PKCzeta, which physically interacts with PKB in unstimulated cells. Insulin reduces the PKB-PKCzeta interaction and stimulates PKB. However, dissociation of the kinase complex and the attendant hormonal activation of PKB were prevented by ceramide. Under these circumstances, ceramide activated PKCzeta, leading to phosphorylation of the PKB-PH domain on Thr(34). This phosphorylation inhibited phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) binding to PKB, thereby preventing activation of the kinase by insulin. In contrast, a PKB-PH domain with a T34A mutation retained the ability to bind PIP(3) even in the presence of a ceramide-activated PKCzeta and, as such, expression of PKB T34A mutant in L6 cells was resistant to inhibition by ceramide treatment. Inhibitors of PKCzeta and a kinase-dead PKCzeta both antagonized the inhibitory effect of ceramide on PKB. Since PKB confers a prosurvival signal and regulates numerous pathways in response to insulin, suppressing its activation by a PKCzeta-dependent process may be one mechanism by which ceramide promotes cell death and induces insulin resistance.     

PLCgamma participates in insulin stimulation of glucose uptake through activation of PKCzeta in brown adipocytes

Based on recent studies showing that PLCgamma associates to insulin receptor, we investigated its role in insulin stimulation of glucose transport in brown adipocytes. Insulin stimulation induced rapid PLCgamma association to phosphorylated insulin receptor, and activation of PLCgamma, as assessed by the mobilization of Ca(2+) from intracellular stores and by the production of the second messenger DAG. Both events are dependent on activation of PI3-kinase. Inhibition of PLCgamma activity either with the chemical compound U73122 or with an inhibitor peptide precluded insulin stimulation of glucose uptake, GLUT4 translocation, and actin reorganization, as wortmannin did. In contrast, the inactive analog U73343 did not have an inhibitory effect. Furthermore, translocation of GLUT4-GFP in response to insulin was completely abolished by cotransfection with a PLCgamma-inactive mutant in HeLa cells, a cell model sensitive to insulin that express PLCgamma. U73122 did not affect PI3-kinase nor Akt activation, but impaired PKCzeta activation by insulin, as wortmannin did. PLC activity renders two products, IP(3) and DAG, and DAG can be metabolized to PA by the action of DAG-kinase. Using the compound R54494, a DAG-kinase inhibitor, insulin-induced PKCzeta activation was also suppressed, this activity being restored by addition of PA. In summary, these data indicate that PLCgamma, activated at least partially by PI3-kinase, is a link between insulin receptor and PKCzeta through the production of PA and could mediate insulin-induced glucose uptake and GLUT4 translocation.     

Tumor necrosis factor-alpha, sphingomyelinase, and ceramide inhibit store-operated calcium entry in thyroid FRTL-5 cells

Tumor necrosis factor alpha (TNF-alpha) is a potent inhibitor of proliferation in several cell types, including thyroid FRTL-5 cells. As intracellular free calcium ([Ca2+]i) is a major signal in activating proliferation, we investigated the effect of TNF-alpha on calcium fluxes in FRTL-5 cells. TNF-alpha per se did not modulate resting [Ca2+]i. However, preincubation (10 min) of the cells with 1-100 ng/ml TNF-alpha decreased the thapsigargin (Tg)-evoked store-operated calcium entry in a concentration-dependent manner. TNF-alpha did not inhibit the mobilization of sequestered calcium. To investigate whether the effect of TNF-alpha on calcium entry was mediated via the sphingomyelinase pathway, the cells were pretreated with sphingomyelinase (SMase) prior to stimulation with Tg. SMase inhibited the Tg-evoked calcium entry in a concentration-dependent manner. Furthermore, an inhibition of calcium entry was obtained after preincubation of the cells with the membrane-permeable C2-ceramide and C6-ceramide analogues. The inactive ceramides dihydro-C2 and dihydro-C6 showed only marginal effects. Neither SMase, C2-ceramide, nor C6-ceramide affected the release of sequestered calcium. C2- and C6-ceramide also decreased the ATP-evoked calcium entry, without affecting the release of sequestered calcium. The effect of TNF-alpha and SMase was inhibited by the kinase inhibitor staurosporin and by the protein kinase C (PKC) inhibitor calphostin C but not by down-regulation of PKC. However, we were unable to measure a significant activation of PKC using TNF-alpha or C6-ceramide. The effect of TNF-alpha was not mediated via activation of either c-Jun N-terminal kinase or p38 kinase. We were unable to detect an increase in the ceramide (or sphingosine) content of the cells after stimulation with TNF-alpha for up to 30 min. Thus, one mechanism of action of TNF-alpha, SMase, and ceramide on thyroid FRTL-5 cells is to inhibit calcium entry.     

Ceramide directly activates protein kinase C zeta to regulate a stress-activated protein kinase signaling complex

We have previously shown that interleukin 1 (IL-1)-receptor-generated ceramide induces growth arrest in smooth muscle pericytes by activating an upstream kinase in the stress-activated protein kinase (SAPK) cascade. We now report the mechanism by which ceramide activates the SAPK signaling pathway in human embryonic kidney cells (HEK-293). We demonstrate that ceramide activation of protein kinase C zeta (PKCzeta) mediates SAPK signal complex formation and subsequent growth suppression. Ceramide directly activates both immunoprecipitated and recombinant human PKCzeta in vitro. Additionally, ceramide activates SAPK activity, which is blocked with a dominant-negative mutant of PKCzeta. Co-immunoprecipitation studies reveal that ceramide induces the association of SAPK with PKCzeta, but not with PKCepsilon. In addition, ceramide treatment induces PKCzeta association with phosphorylated SEK and MEKK1, elements of the SAPK signaling complex. The biological role of ceramide to induce cell cycle arrest is mimicked by overexpression of a constitutively active PKCzeta. Together, these studies demonstrate that ceramide induces cell cycle arrest by enhancing the ability of PKCzeta to form a signaling complex with MEKK1, SEK, and SAPK.     

Interleukin 1 beta (IL-1 beta) action in porcine thyroid cells involves the ceramide signalling pathway

Interleukin 1 beta (IL-1beta) is often associated with thyroidal autoimmune diseases. This cytokine has been largely described to trigger an important biological signalling pathway: the sphingomyelin/ceramide pathway. In this report we show that IL-1beta induces ceramide formation and sphingomyelin degradation in porcine thyroid cells via the activation of a neutral sphingomyelinase. Among the potential targets of IL-1beta and ceramides action, we have investigated the role of an atypical protein kinase C (PKC), the PKC zeta. We show that both IL-1beta and ceramides lead to an increase of PKCzeta activity. All these results suggest an important role for ceramides and IL-1beta in regulation of thyroid function, leading to cell survival or to apoptosis.     

TGF-beta 1 modulation of IGF-I signaling pathway in rat thyroid epithelial cells

Transforming growth factor beta 1 (TGF-beta 1) and insulin-like growth factor I (IGF-I) have contrasting effects on cell cycle regulation in thyroid cells and TGF-beta 1 induces a dramatic decrease in IGF-I-induced cell proliferation. The aim of the present study was to investigate the molecular mechanism of cross-talk between TGF-beta 1 and IGF-I in FRTL-5 cells. TGF-beta 1 affected IGF-I-stimulated insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation and its association with Grb2 protein. Moreover, TGF-beta 1 decreased the IGF-I-induced tyrosine phosphorylation of the adaptor protein CrkII and its association with the IGF-I receptor. These results were accompanied by TGF-beta 1 inhibition of IGF-I-stimulated mitogen-activated protein kinase phosphorylation and activation. Conversely, TGF-beta 1 did not alter IGF-I-stimulated IRS-1-associated phosphatidylinositol 3-kinase activity, IGF-I-induced tyrosine phosphorylation of Shc, and its binding to Grb2. Taken together, these findings provide a molecular basis for the growth-inhibitory action of TGF-beta 1 on the IGF-I-induced mitogenic effect.     

Ghrelin potentiates TSH-induced expression of the thyroid tissue-specific genes thyroglobulin, thyroperoxidase and sodium-iodine symporter, in rat PC-Cl3 Cells

Ghrelin is a 28-amino-acid peptide that stimulates pituitary growth-hormone secretion and modulates food-intake and energy metabolism in mammals. It is mainly secreted by the stomach, but it is also expressed in many other tissues such as cartilage or the thyroid gland. In the present study we have analyzed by RT-PCR and using immunohistochemistry and immunofluorescence the expression and tissue distribution of ghrelin and its functional receptor (GHS-R type 1α) in thyroid cell-lines and in normal and pathological rat thyroid tissue. Additionally, by measuring the incorporation of BrdU, we have investigated if, as previously noted for FRTL-5 cells, ghrelin enhances the proliferation rate in the PC-Cl3 rat-thyrocyte cell-line. Finally, we have determined the stimulatory effect of ghrelin on TSH-induced expression of the tissue-specific key genes involved in the synthesis of thyroid hormone: thyroglobulin, thyroperoxidase and sodium-iodine symporter. Our data provide direct evidence that C-cell secreted ghrelin may be involved in the paracrine regulation of the thyroid follicular cell function.     

The mycotoxin fumonisin B1 transiently activates nuclear factor-κB, tumor necrosis factor α and caspase 3 via protein kinase Cα-dependent pathway in porcine renal epithelial cells

Fumonisin B1 (FB1) is a toxic mycotoxin produced by Fusarium verticillioides, predominantly present in corn. The principal biochemical responses of FB1 involve disruption of sphingolipid metabolism from the inhibition of ceramide synthesis leading to accumulation of free sphingoid bases, particularly sphinganine. The ability of FB1 to modulate signal transduction pathways plays a role in its toxicity. We recently reported that FB1 selectively and transiently activates protein kinase Calpha (PKCalpha) in porcine renal epithelial cells (LLC-PK1). The aim of current study was to investigate the effect of PKCalpha activation by FB1 on NF-kappaB activation and subsequently on TNFalpha gene expression and caspase 3 induction in LLC-PK1 cells. FB1 (1 micromol/L for 5 min) transiently activated PKCalpha and increased nuclear translocation of NF-kappaB, followed by their down-regulation at later time points. Preincubating the cells with the PKC inhibitor, calphostin C, prevented the activation of NF-kappaB by FB1. TNFalpha mRNA expression was increased after 15 min exposure to FB1 or the PKC activator, phorbol 12-myristate 13-acetate. In addition, an increase in caspase 3 activity was observed after addition of FB1 for 1 h. Calphostin C prevented both the FB1-induced increase in TNFalpha expression and caspase 3 activation. In summary, we hereby demonstrate that the FB1 activation of NF-kappaB and sequential induction of TNFalpha expression resulting in the subsequent increase in caspase 3 activity are all dependent on PKCalpha stimulation in LLC-PK1 cells.     

Analysis of linkage between lymphotoxin alpha haplotype and polymorphisms in 5'-flanking region of tumor necrosis factor alpha gene associated with efficacy of infliximab for Crohn's disease patients

Tumor necrosis factor (TNF)alpha is increased in patients with Crohn's disease (CD) and considered to play an important role in the inflammation. Infliximab (IFX) is used as a therapeutic agent for CD. Recently, it was reported that homozygosity for a lymphotoxin alpha (LTA) haplotype (LTA 1-1-1-1) may identify subgroups with a poor response to IFX. In the present study, we characterized the linkage of the LTA haplotype with SNPs in the 5'-flanking region of the TNFalpha gene. In subjects who had homozygosity for each LTA haplotype, 6 nucleotide variations, -857C > T, -522C > G, -357A > C, -261C > G, -159G > T and -96G > T, were found in the 5'-flanking region of the TNFalpha gene. As for linking with the allele, only -857T met the LTA haplotype 1-1-1-1. We concluded that the differences in therapeutic effects of IFX among patients with CD may be explained in part by the induction ability of TNFalpha via the -857C > T polymorphism.     

Inhibitory effect of IL-1 on the TSH dependent growth of rat thyroid cells (FRTL-5)

Using a functioning rat thyroid cell line (FRTL-5), we examined the effects of some cytokines, particularly interleukin-1 (IL-1) on the growth of thyroid cells. In 5H medium, namely Coon's modified Ham's F-12 medium supplemented with 5% calf serum and a five-hormone preparation consisting of insulin, hydrocortisone, transferrin, glycyl-L-histidyl-L-lysine acetate and somatostatin, IL-1 enhanced the growth of FRTL-5 cells detected by [3H]TdR incorporation. However, in 6H medium (5H medium plus bovine TSH), IL-1 inhibited the growth of FRTL-5 cells. Both effects were neutralized by the addition of anti-IL-1 antibody. Furthermore, IL-1 inhibited the growth of FRTL-5 cells induced by forskolin which is known as an adenylate cyclase activator. FRTL-5 cells have specific IL-1 receptors detected by the binding of 125I-labeled IL-1 alpha. By Scatchard plot analysis, the numbers and the dissociation constants of IL-1 receptors on FRTL-5 cells were shown to be 5225/cell and 8.69 x 10(-10) M. Interleukin-2, interleukin-6 and interferon-gamma (IFN-gamma) had no significant effects on the cell growth in 6H medium, while IFN-gamma and insulin-like growth factor I stimulated cell growth somewhat in 5H medium. These results suggest that IL-1 plays a regulatory role in the growth of thyroid cells through binding to the IL-1 receptors.     

Crosstalk between PKCzeta and the IL4/Stat6 pathway during T-cell-mediated hepatitis

PKCzeta is required for nuclear factor kappa-B (NF-kappaB) activation in several cell systems. NF-kappaB is a suppressor of liver apoptosis during development and in concanavalin A (ConA)-induced T-cell-mediated hepatitis. Here we show that PKCzeta-/- mice display inhibited ConA-induced NF-kappaB activation and reduced damage in liver. As the IL-4/Stat6 pathway is necessary for ConA-induced hepatitis, we addressed here the potential role of PKCzeta in this cascade. Interestingly, the loss of PKCzeta severely attenuated serum IL-5 and liver eotaxin-1 levels, two critical mediators of liver damage. Stat6 tyrosine phosphorylation and Jak1 activation were ablated in the liver of ConA-injected PKCzeta-/- mice and in IL-4-stimulated PKCzeta-/- fibroblasts. PKCzeta interacts with and phosphorylates Jak1 and PKCzeta activity is required for Jak1 function. In contrast, Par-4-/- mice have increased sensitivity to ConA-induced liver damage and IL-4 signaling. This unveils a novel and critical involvement of PKCzeta in the IL-4/Stat6 signaling pathway in vitro and in vivo.     

Bioactive Fragment of Human IL-1β [163-171] Modulates the Immune Response to Synthetic Peptides of HIV

The activation of T helper cells specific for viral antigens is critical for antibody production and the generation of cytotoxic T cells during retroviral infection. In this study, we examined the effect of linking HIV peptides with a bioactive fragment of human interleukin-1β (IL-1β) (163–171) on the induction of immune response to the peptides. A panel of highly purified synthetic peptides representing defined regions of gp41, Gag and gp120 were used as antigens. Mouse spleen cells primed with the peptide conjugates produced greater proliferation on in vitro stimulation than spleen cells primed with peptide alone. In addition, antibody production as assessed by ELISA was observed after immunization with conjugated peptides but not with peptide alone, indicating B-cell activation. We also found that a high level of IgG2a antibody production correlated with a high level of IFN-γ production. These findings favor the notion that IL-1β plays an important role in immune responses. These observations support the formulation and design of synthetic vaccines against HIV using synthetic HIV peptides conjugated with immunomodulators. Such an approach may provide an effective vaccination against other infectious agents.     

Ceramide-dependent regulation of p42/p44 mitogen-activated protein kinase and c-Jun N-terminal-directed protein kinase in cultured airway smooth muscle cells

Previous studies have demonstrated that a number of biochemical actions of ceramide are mediated through protein kinase signalling pathways, such as p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) and c-Jun N-terminal directed protein kinase (JNK). Ceramide-activated protein kinases, such as the kinase suppressor of Ras (KSR) and protein kinase Czeta (PKCzeta), are involved in the regulation of c-Raf, which promotes sequential activation of MEK-1 and p42/p44 MAPK in mammalian cells. However, in cultured airway smooth muscle (ASM) cells, neither KSR nor PKCzeta are involved in the C2-ceramide (C2-Cer)-dependent activation of this kinase cascade. Instead, we found that C2-Cer utilises a novel pathway involving tyrosine kinases, phosphoinositide 3-kinase (PI3K) and conventional PKC isoform(s). We also found that despite its ability to stimulate p42/p44 MAPK, C2-Cer inhibited platelet-derived growth factor (PDGF)-stimulated DNA synthesis. The possibility that growth arrest could be mediated by JNK was discounted on the basis that PDGF, as well as ceramide, stimulated JNK in these cells. Therefore, growth arrest in response to ceramide is mediated by an alternative mechanism.