ACTH promotion of p27(Kip1) induction in mouse Y1 adrenocortical tumor cells is dependent on both PKA activation and Akt/PKB inactivation

Here we report antimitogenic mechanisms activated by the adrenocorticotropic hormone (ACTH) in the mouse Y1 adrenocortical tumor cell line. ACTH receptors activate the Galphas/adenylate cyclase cAMP/PKA pathway to promote dephosphorylation of Akt/PKB enzymes, leading to induction of the cyclin-dependent kinases' (CDKs) inhibitor p27(Kip1). Y1 cells display high constitutive levels of phosphorylated Akt/PKB dependent on chronically elevated c-Ki-Ras.GTP and PI3K activity. Expression of the dominant negative mutant RasN17 in Y1 cells results in strong reduction of both c-Ki-Ras.GTP and phosphorylated Akt/PKB, which are restored by FGF2 treatments. Inhibitors of PI3K lead to rapid dephosphorylation of Akt/PKB and block phosphorylation of Akt/PKB promoted by FGF2. ACTH rapidly promotes dephosphorylation of Akt/PKB in Y1 adrenal cells, while constitutively high levels of c-Ki-Ras.GTP remain unchanged. ACTH and cAMP elevating agents fail to cause Akt/PKB dephosphorylation in PKA-deficient clonal mutants of Y1 cells. In addition, cholera toxin, forskolin, and 8BrcAMP all mimic ACTH, causing dephosphorylation of Akt/PKB in wild-type Y1 cells. ACTH is unable to prevent Akt/PKB phosphorylation, promoted by FGF2 in clonal lines of RasN17-Y1 transfectants displaying negligible levels of c-Ki-Ras.GTP. ACTH promotes strong p27(Kip1) protein induction in wild-type Y1 adrenocortical cells but not in PKA-deficient Y1-clonal mutants nor in RasN17-Y1 transfectants. PI3K inhibitors induce p27(Kip1) protein in all cells studied, i.e., wild type and transfectants. The inverse correlation between levels of phosphorylated Akt/PKB and of p27(Kip1) protein caused by ACTH suggests a novel antimitogenic pathway activated by ACTH and mediated by cAMP/PKA in the mouse Y1 adrenocortical tumor cell line.     

Stimulation of Muscarinic Receptors Induces Expression of Individual fos and jun Genes Through Different Transduction Pathways

Abstract: The transduction pathways coupling muscarinic receptors to induction of fos and jun genes were investigated in neuroblastoma SH-SY5Y cells. Stimulation with carbachol induced expression of c- fos , fosB , c- jun , junB , and junD . This effect was abolished by pretreatment with atropine, indicating an involvement of muscarinic receptors. These genes were also induced by activation of protein kinase C with phorbol ester or by elevating the intracellular Ca²⁺ concentration with a Ca²⁺ ionophore. The Ca²⁺ effect was inhibited by KN-62, suggesting an induction through Ca²⁺/calmodulin-dependent kinase II. Inhibition of protein kinase C with GF109203X suppressed the carbachol-stimulated increase in mRNA levels of c- fos , fosB , and junB by ∼70% but had only minor effects on the expression of c- jun and junD . On the other hand, preincubation with KN-62 attenuated the carbachol-induced increase in c- jun and junD expression by 70% but had no effect on c- fos , fosB , and junB mRNA levels. Simultaneous inhibition of both protein kinase C and Ca²⁺/calmodulin-dependent kinase II completely abolished the carbachol-stimulated expression of c- jun and junD , but c- fos , fosB , and junB were still expressed to a certain extent under this condition. Comparison of the inhibitory effects of GF109203X and Gö 6976 suggests the involvement of classical protein kinase C isozymes in muscarinic receptor-stimulated expression of fos and jun genes. These results demonstrate that the muscarinic receptor-induced expression of individual fos and jun genes is regulated via different pathways, primarily protein kinase C or Ca²⁺/calmodulin-dependent kinase II.     

Broad-Scale Phosphoprotein Profiling of Beta Adrenergic Receptor (β-AR) Signaling Reveals Novel Phosphorylation and Dephosphorylation Events

β-adrenergic receptors (β-ARs) are model G-protein coupled receptors that mediate signal transduction in the sympathetic nervous system. Despite the widespread clinical use of agents that target β-ARs, the signaling pathways that operate downstream of β-AR stimulation have not yet been completely elucidated. Here, we utilized a lysate microarray approach to obtain a broad-scale perspective of phosphoprotein signaling downstream of β-AR. We monitored the time course of phosphorylation states of 54 proteins after β-AR activation mouse embryonic fibroblast (MEF) cells. In response to stimulation with the non-selective β-AR agonist isoproterenol, we observed previously described phosphorylation events such as ERK1/2(T202/Y204) and CREB(S133), but also novel phosphorylation events such as Cdc2(Y15) and Pyk2(Y402). All of these events were mediated through cAMP and PKA as they were reproduced by stimulation with the adenylyl cyclase activator forskolin and were blocked by treatment with H89, a PKA inhibitor. In addition, we also observed a number of novel isoproterenol-induced protein dephosphorylation events in target substrates of the PI3K/AKT pathway: GSK3β(S9), 4E-BP1(S65), and p70s6k(T389). These dephosphorylations were dependent on cAMP, but were independent of PKA and correlated with reduced PI3K/AKT activity. Isoproterenol stimulation also led to a cAMP-dependent dephosphorylation of PP1α(T320), a modification known to correlate with enhanced activity of this phosphatase. Dephosphorylation of PP1α coincided with the secondary decline in phosphorylation of some PKA-phosphorylated substrates, suggesting that PP1α may act in a feedback loop to return these phosphorylations to baseline. In summary, lysate microarrays are a powerful tool to profile phosphoprotein signaling and have provided a broad-scale perspective of how β-AR signaling can regulate key pathways involved in cell growth and metabolism.     

Stimulation of protein kinase C or protein kinase A mediated signal transduction pathways shows three modes of response among serum inducible genes

Activation of the signal transduction pathways mediated by protein kinase A (PKA) or protein kinase C (PKC) led to different responses of several serum inducible genes including the jun gene family, c-fos, c-myc, krox 20 and krox 24. Whereas all of these genes were stimulated by the phorbol ester TPA, a chemical activator of protein kinase C, they were differently regulated upon cAMP stimulation of the PKA dependent pathway. The proto-oncogenes jun B, c-fos, and to a lesser extent jun D were stimulated by increasing the intracellular concentration of cAMP, whereas the TPA stimulation of c-jun and c-myc was inhibited under these conditions. Krox 20 and krox 24 were insensitive to this second messenger. This study allowed us to classify these growth stimulated genes into three distinct groups distinguished by their sensitivity to elevated concentrations of intracellular cAMP. The inhibition of c-jun and c-myc expression in the presence of increased cAMP levels may be at least partially responsible for the growth inhibitory effect of this agent in Balb/c-3T3 cells.     

Phosphodiesterase 3A (PDE3A) deletion suppresses proliferation of cultured murine vascular smooth muscle cells (VSMCs) via inhibition of mitogen-activated protein kinase (MAPK) signaling and alterations in critical cell cycle regulatory proteins

Cyclic nucleotide phosphodiesterase 3 (PDE3) is an important regulator of cyclic adenosine monophosphate (cAMP) signaling within the cardiovascular system. In this study, we examined the role of PDE3A and PDE3B isoforms in regulation of growth of cultured vascular smooth muscle cells (VSMCs) and the mechanisms by which they may affect signaling pathways that mediate mitogen-induced VSMC proliferation. Serum- and PDGF-induced DNA synthesis in VSMCs grown from aortas of PDE3A-deficient (3A-KO) mice was markedly less than that in VSMCs from PDE3A wild type (3A-WT) and PDE3B-deficient (3B-KO) mice. The reduced growth response was accompanied by significantly less phosphorylation of extracellular signal-regulated kinase (ERK) in 3A-KO VSMCs, most likely due to a combination of greater site-specific inhibitory phosphorylation of Raf-1^Ser-259 by protein kinase A (PKA) and enhanced dephosphorylation of ERKs due to elevated mitogen-activated protein kinase phosphatase 1 (MKP-1). Furthermore, 3A-KO VSMCs, compared with 3A-WT, exhibited higher basal PKA activity and cAMP response element-binding protein (CREB) phosphorylation, higher levels of p53 and p53 phosphorylation, and elevated p21 protein together with lower levels of Cyclin-D1 and retinoblastoma (Rb) protein and Rb phosphorylation. Adenoviral overexpression of inactive CREB partially restored growth effects of serum in 3A-KO VSMCs. In contrast, exposure of 3A-WT VSMCs to VP16 CREB (active CREB) was associated with inhibition of serum-induced DNA synthesis similar to that in untreated 3A-KO VSMCs. Transfection of 3A-KO VSMCs with p53 siRNA reduced p21 and MKP-1 levels and completely restored growth without affecting amounts of Cyclin-D1 and Rb phosphorylation. We conclude that PDE3A regulates VSMC growth via two complementary pathways, i.e. PKA-catalyzed inhibitory phosphorylation of Raf-1 with resulting inhibition of MAPK signaling and PKA/CREB-mediated induction of p21, leading to G₀/G₁ cell cycle arrest, as well as by increased accumulation of p53, which induces MKP-1, p21, and WIP1, leading to inhibition of G₁ to S cell cycle progression.     

In vitro mitogenic and steroidogenic effects of ACTH analogues on an adrenal tumor cell line (Y-1)

Native porcine adrenocorticotropin (ACTH_1–39) as well as synthetic adrenocorticotropin (ACTH_1–24) increase cAMP and steroid production and inhibit DNA synthesis in an adrenal cell line. The COOH terminal sequence of both peptides as well as β-endorphin have no effects, while the NH₂ terminal sequence of ACTH as well as α-MSH which have very low stimulatory effect on cAMP production, have a mitogenic effect. These results suggest that ACTH might have in vitro some mitogenic action on adrenal cell, but this effect is blunted by cAMP accumulation during hormonal stimulation. The results can also explain the in vivo and in vitro contradictory effects of the hormone on adrenal cell replication.     

Melanocortin receptor signaling in RAW264.7 macrophage cell line

Melanocortin peptides modulate cytokine release and adhesion molecule expression. Here we have investigated the early cell-signaling pathway responsible for the induction of interleukin-10 (IL-10) in RAW264.7 cells. Cell incubation with ACTH(1-39) or MTII (melanotan II) did not alter ERK1/2 and JNK phosphorylation, while p38 phosphorylation and intracellular cAMP accumulation occurred within minutes. ACTH(1-39) and MTII provoked a time-dependent accumulation of IL-10 that was abrogated by the PKA inhibitor H-89 and only partially blocked by the p38 MAPK inhibitor SB203580. Thus, in RAW264.7 cells, IL-10 induction by the melanocortins is via the PKA pathway, and this mechanism could contribute to their anti-inflammatory profile.     

Cyclic AMP inhibits JNK activation by CREB-mediated induction of c-FLIP(L) and MKP-1, thereby antagonizing UV-induced apoptosis

The cyclic AMP (cAMP) signaling pathway has been reported to either promote or suppress apoptosis, in a cell context-dependent manner. Our previous study has shown that cAMP, by protein kinase A (PKA)-cAMP response element-binding protein (CREB)-dynein light chain (DLC) pathway, negatively regulates mitogen-activated protein kinase p38 activation, thereby contributing to tumor necrosis factor (TNF)-alpha-induced apoptosis in certain types of cells. However, it remains largely unknown how cAMP suppresses apoptosis. Here we report that cAMP antagonized UV-induced apoptosis in Rat-1 and NIH 3T3 cells. Despite that cAMP significantly suppressed UV-induced p38 activation, inhibition of p38 activity showed no significant effect on UV-induced cell death in both cell lines. Further studies revealed that cAMP antagonized UV-induced apoptosis by inhibition of c-Jun N-terminal protein kinase (JNK) activation. The induction of the long form of cellular FLICE-inhibitory protein (c-FLIP(L)) and mitogen-activated protein kinase phosphatase-1 (MKP-1), but not DLC and p21(WAF1) by CREB was required for cAMP-mediated inhibition of JNK activation. The suppression by cAMP of UV-induced apoptosis was reversed by c-FLIP(L) small-interfering RNA (siRNA) or MKP-1 siRNA, which released the inhibition of JNK activation by cAMP. Thus, our results provide a molecular mechanism by which cAMP suppresses JNK activation and antagonizes apoptosis.     

PGE2 induces IL-6 in orbital fibroblasts through EP2 receptors and increased gene promoter activity: implications to thyroid-associated ophthalmopathy

Background

IL-6 plays an important role in the pathogenesis of Graves'' disease and its orbital component, thyroid-associated ophthalmopathy (TAO). Orbital tissues become inflamed in TAO, a process in which prostanoids have been implicated. Orbital fibroblasts both generate and respond to PGE₂, underlying the inflammatory phenotype of these cells.

Methodology/Principal Findings

Using cultured orbital and dermal fibroblasts, we characterized the effects of PGE₂ on IL-6 expression. We found that the prostanoid provokes substantially greater cytokine synthesis in orbital fibroblasts, effects that are mediated through cell-surface EP₂ receptors and increased steady-state IL-6 mRNA levels. The pre-translational up-regulation of IL-6 results from increased gene promoter activity and can be reproduced with the PKA agonist, Sp-cAMP and blocked by interrupting the PKA pathway. PGE₂-induced production of cAMP in orbital fibroblasts was far greater than that in dermal fibroblasts, resulting from higher levels of adenylate cyclase. PGE₂ provokes CREB phosphorylation, increases the pCREB/CREB ratio, and initiates nuclear localization of the pCREB/CREB binding protein/p300 complex (CBP) preferentially in orbital fibroblasts. Transfection with siRNAs targeting either CREB or CBP blunts the induction of IL-6 gene expression. PGE₂ promotes the binding of pCREB to its target DNA sequence which is substantially greater in orbital fibroblasts.

Conclusion/Significance

These results identify the mechanism underlying the exaggerated induction of IL-6 in orbital fibroblasts and tie together two proinflammatory pathways involved in the pathogenesis of TAO. Moreover, they might therefore define an attractive therapeutic target for the treatment of TAO.     

α2A-AR antagonism by BRL-44408 maleate attenuates acute lung injury in rats with downregulation of ERK1/2, p38MAPK,and p65 pathway

Acute respiratory distress syndrome (ARDS), characterized by acute hypoxic respiratory dysfunction or failure, is a manifestation of multiple organ failure in the lung, and the most common risk factor is sepsis. We previously showed that blocking α₂-adrenoceptor (α₂-AR) could attenuate lung injury induced by endotoxin in rats. α_2A-adrenoceptor (α_2A-AR), a subtype of α₂-AR plays a key role in inflammatory diseases, but the mechanism remains unknown. Here, we explored the effect of BRL-44408 maleate (BRL), a specific α_2A-AR antagonist, on cecal ligation puncture (CLP)-induced ARDS in rats and the underlying mechanism. Preadministration of BRL-44408 maleate significantly alleviated CLP-induced histological injury, macrophage infiltration, inflammatory response, and wet/dry ratio in lung tissue. However, there was no statistical difference in survival rate between the CLP and CLP+BRL groups. Extracellular regulated protein kinase (ERK1/2), p38MAPK, and p65 were activated in the CLP group, and BRL-44408 maleate inhibited the activation of these signal molecules, c-Jun N-terminal kinase (JNK) and protein kinase A (PKA) showed no changes in activation between these two groups. BRL-44408 maleate decreased lipopolysaccharide (LPS)-induced expression of cytokines in NR8383 rat alveolar macrophages and reduced phosphorylation of ERK1/2, p38MAPK, and p65. JNK and PKA were not influenced by LPS. Together, these findings suggest that antagonism of α_2A-AR improves CLP-induced acute lung injury and involves the downregulation of ERK1/2, p38MAPK, and p65 pathway independent of the activation of JNK and PKA.