Protein Kinase A Signaling Pathway Research Articles

Hepatocyte Nuclear Factor‐4 Phosphorylation Mediated by Inflammatory Response

Hepatocyte nuclear factor (HNF)‐4, a liver‐enriched transcription factor, regulates a large number of liver‐specific genes and plays an important role in liver development and maintenance of liver phenotype. While HNF‐4 function is regulated by phosphorylation, only a limited number of phosphorylation sites have been identified, and its roles and dynamic changes in response to inflammatory stimulation are not explored. Utilizing the cytokine treated HNF‐4 enriched HepG2 cells, we have quantitatively analyzed HNF‐4 phosphorylation by using the combined stable isotopic variants of dithiothreitol with mass spectrometry. Twelve serine/threonine phosphorylation sites in the diverse functional domains of HNF‐4 were identified and quantified after cytokine exposure. Cytokine treatment (IL‐6, IL‐1β and TNF‐α) causes a dynamic change in HNF‐4 phosphorylation in a peptide‐and time‐dependent manner. Protein kinase A (PKA) significantly reduces HNF‐4 binding activity, which mimics the repressive effect of cytokines, and a PKA inhibitor partially restores the reduced HNF‐4 binding activity induced by cytokines. These results suggest that cytokines mediate the phosphorylation/dephosphorylation levels of HNF‐4, and a PKA signaling pathway involves in the modification of HNF‐4 function during the inflammatory response.

Melanocortin 4 Receptors Reciprocally Regulate Sympathetic and Parasympathetic Preganglionic Neurons

Melanocortin 4 receptors (MC4Rs) in the central nervous system are key regulators of energy and glucose homeostasis. Notably, obese patients with MC4R mutations are hyperinsulinemic and resistant to obesity-induced hypertension. Although these effects are probably dependent upon the activity of the autonomic nervous system, the cellular effects of MC4Rs on parasympathetic and sympathetic neurons remain undefined. Here, we show that MC4R agonists inhibit parasympathetic preganglionic neurons in the brainstem. In contrast, MC4R agonists activate sympathetic preganglionic neurons in the spinal cord. Deletion of MC4Rs in cholinergic neurons resulted in elevated levels of insulin. Furthermore, re-expression of MC4Rs specifically in cholinergic neurons (including sympathetic preganglionic neurons) restores obesity-associated hypertension in MC4R null mice. These findings provide a cellular correlate of the autonomic side effects associated with MC4R agonists and demonstrate a role for MC4Rs expressed in cholinergic neurons in the regulation of insulin levels and in the development of obesity-induced hypertension.

Cyclic AMP-dependent protein kinase A negatively regulates conidia formation by the tangerine pathotype of Alternaria alternata

The necrotrophic fungal pathogen Alternaria alternata causes brown spot diseases in many citrus cultivars. The FUS3 and SLT2 mitogen-activated protein kinases (MAPK)-mediated signaling pathways have been shown to be required for conidiation. Exogenous application of cAMP to this fungal pathogen decreased conidia formation considerably. This study determined whether a cAMP-activated protein kinase A (PKA) is required for conidiation. Using loss-of-function mutations in PKA catalytic and regulatory subunit-coding genes, we demonstrated that PKA negatively regulates conidiation. Fungal mutants lacking PKA catalytic subunit gene (PKA ( cat )) reduced growth, lacked detectable PKA activity, and produced higher amounts of conidia compared to wild-type. Introduction of a functional copy of PKA ( cat ) into a null mutant partially restored PKA activity and produced wild-type level of conidia. In contrast, fungi lacking PKA regulatory subunit gene (PKA ( reg )) produced detectable PKA activity, exhibited severe growth reduction, formed swelling hyphal segments, and produced no mature conidia. Introduction of the PKA ( reg ) gene to a regulatory subunit mutant restored all phenotypes to wild type. PKA ( reg )-null mutants induced fewer necrotic lesions on citrus compared to wild-type, whereas PKA ( cat ) mutant displayed wild-type virulence. Overall, our studies indicate that PKA and FUS3-mediated signaling pathways apparently have very different roles in the regulation of conidia production and A. alternata pathogenesis in citrus.

Exendin-4 improved rat cortical neuron survival under oxygen/glucose deprivation through PKA pathway

Previous studies demonstrated that exendin-4 (Ex-4) may possess neurotrophic and neuroprotective functions in ischemia insults, but its mechanism remained unknown. Here, by using real-time PCR and ELISA, we identified the distribution of active GLP-1Rs in the rat primary cortical neurons. After establishment of an in vitro ischemia model by oxygen/glucose deprivation (OGD), neurons were treated with various dosages of Ex-4. The MTT assay showed that the relative survival rate increased with the dosage of Ex-4 ranging from 0.2 to 0.8μg/ml (P<0.001, vs. OGD group). The apoptosis rate was reduced from (49.47±2.70)% to (14.61±0.81)% after Ex-4 treatment (0.4μg/ml) 12h after OGD (P<0.001). Moreover, immunofluorescence staining indicated that Ex-4 increased glucose-regulated proteins 78 (GRP78) and reduced C/EBP-homologous protein (CHOP). Western blot analysis demonstrated that, after neurons were treated with Ex-4, GRP78 was up-regulated over time (P<0.01, vs. OGD group), while CHOP levels rose to a peak 8h after OGD and then decreased (P<0.05, vs. OGD group). This effect was changed by both the protein kinase A (PKA) inhibitor H89 (P<0.01, P<0.05, respectively, vs. Ex-4 group) and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (P<0.01, P<0.01, respectively, vs. Ex-4 group) but not by the mitogen-activated protein kinase (MAPK) inhibitor U0126. Our study also revealed that, compared with the Ex-4 group, inhibition of the PKA signaling pathway significantly decreased the survival rate of neurons, down-regulated the expression of B-cell lymphoma 2 (Bcl-2) and up-regulated the Bax expression 3h after ODG (P<0.05, P<0.01, respectively), while neither PI3K nor MAPK inhibition exerted such effects. Furthermore, Western blotting exhibited that PKA expression was elevated in the presence or absence of OGD insults (P<0.05). This study indicated that Ex-4 protected neurons against OGD by modulating the unfolded protein response (UPR) through the PKA pathway and may serve as a novel therapeutic agent for stroke.

PKA phosphorylation redirects ERα to promoters of a unique gene set to induce tamoxifen resistance

Protein kinase A (PKA)-induced estrogen receptor alpha (ERα) phosphorylation at serine residue 305 (ERαS305-P) can induce tamoxifen (TAM) resistance in breast cancer. How this phospho-modification affects ERα specificity and translates into TAM resistance is unclear. Here, we show that S305-P modification of ERα reprograms the receptor, redirecting it to new transcriptional start sites, thus modulating the transcriptome. By altering the chromatin-binding pattern, Ser305 phosphorylation of ERα translates into a 26-gene expression classifier that identifies breast cancer patients with a poor disease outcome after TAM treatment. MYC-target genes and networks were significantly enriched in this gene classifier that includes a number of selective targets for ERαS305-P. The enhanced expression of MYC increased cell proliferation in the presence of TAM. We demonstrate that activation of the PKA signaling pathway alters the transcriptome by redirecting ERα to new transcriptional start sites, resulting in altered transcription and TAM resistance.

Ten paths of PKA signaling pathway regulate hepatocyte proliferation in rat liver regeneration

Generally, protein kinase A (PKA) signaling pathway regulates many physiological activities such as cell proliferation and metabolism. However, little is known about how PKA signaling pathway composed of 16 paths participates in regulating hepatocyte proliferation during rat liver regeneration (LR). To explore the influence PKA signaling pathway upon hepatocytes at gene transcription level, Rat Genome 230 2.0 Array was used to detect expression changes of 225 related genes in isolated hepatocytes from rat regenerating livers, showing that 197 genes were contained in the array and 78 genes were significantly changed. Moreover, bioinformatics and systems biology methods were applied to analyze the precise role of PKA signaling pathway in regulating hepatocyte proliferation during LR, and it was found that 10 paths were related to hepatocyte proliferation during rat LR. In detail, 5 paths including SR→Gα-S→PKA, GPCR→Gα-S→PKA, GPCR→ PLC→PKA, ET-1→PLC→PKA and hypoxia→AMP→PKA launched hepatocyte proliferation in initiating phase of LR, and then 3 paths including βR→Gα-S→PKA, GPCR→PLC→ PKA and GPCR→Gβγ ┤ AcV VI→PKA enhanced hepatocyte proliferation in progressing phase, whereas 3 paths including TGFB→SMAD3/4→PKA, CaCn→ACIX ┤ PKA, SST→Gα-I ┤ PKA inhibited hepatocyte proliferation in terminating phase of LR. In conclusion, 78 genes and 10 paths of PKA signaling pathway regulate hepatocyte proliferation in rat LR.

A new signalling pathway for parallel fibre presynaptic type 4 metabotropic glutamate receptors (mGluR4) in the rat cerebellar cortex

In the rodent cerebellum, pharmacological activation of mGluR4 acutely depresses excitatory synaptic transmission at parallel fibre–Purkinje cell synapses. This depression involves the inhibition of presynaptic calcium (Ca2+) influx that ultimately controls glutamate release. In this study, we investigate the molecular basis of mGluR4-mediated inhibition of presynaptic Ca2+ transients. Our results demonstrate that the mGluR4 effect does not depend on selective inhibition of a specific type of presynaptic voltage-gated Ca2+ channel, but rather involves modulation of all classes of Ca2+ channels present in the presynaptic terminals. In addition, this inhibitory effect does not involve the activation of G protein-activated inwardly rectifying potassium channels, TEA-sensitive potassium channels or two-pore-domain potassium channels. Furthermore, this inhibition does not require pertussis toxin-sensitive G proteins, and is independent of any effect on adenylyl cyclases, protein kinase A, mitogen-activated protein kinases or phosphoinositol-3 kinase activity. Interestingly we found that mGluR4 inhibition of presynaptic Ca2+ influx employs a newly defined signalling pathway, notably that involving the activation of phospholipase C and ultimately protein kinase C.

Phosphorylation of endothelial NOS contributes to simvastatin protection against myocardial no-reflow and infarction in reperfused swine hearts: partially via the PKA signaling pathway

The cholesterol-lowering drugs statins could enhance the activities of endothelial nitric oxide synthase (eNOS) and protect myocardium during ischemia and reperfusion. The aim of this study was to examine whether protein kinase A (PKA) was involved in statin-mediated eNOS phosphorylation and cardioprotection. 6-Month-old Chinese minipigs (20-30 kg) underwent a 1.5-h occlusion and 3-h reperfusion of the left anterior descending coronary artery (LAD). In the sham group, the LAD was encircled by a suture but not occluded. Hemodynamic and cardiac function was monitored using a polygraph. Plasma activity of creatine kinase and the tissue activities of PKA and NOS were measured spectrophotometrically. p-CREB, eNOS and p-eNOS levels were detected using Western blotting. Sizes of the area at risk, the area of no-reflow and the area of necrosis were measured morphologically. Pretreatment of the animals with simvastatin (SIM, 2 mg/kg, po) before reperfusion significantly decreased the plasma activity of creatine kinase, an index of myocardial necrosis, and reduced the no-reflow size (from 50.4%±2.4% to 36.1%±2.1%, P<0.01) and the infarct size (from 79.0%±2.7% to 64.1%±4.5%, P<0.01). SIM significantly increased the activities of PKA and constitutive NOS, and increased Ser(133) p-CREB protein, Ser(1179) p-eNOS, and Ser(635) p-eNOS in ischemic myocardium. Intravenous infusion of the PKA inhibitor H-89 (1 μg·kg(-1)·min(-1)) partially abrogated the SIM-induced cardioprotection and eNOS phosphorylation. In contrast, intravenous infusion of the eNOS inhibitor L-NNA (10 mg·kg(-1)) completely abrogated the SIM-induced cardioprotection and eNOS phosphorylation during ischemia and reperfusion, but did not affect the activity of PKA. Pretreatment with a single dose of SIM 2.5 h before reperfusion attenuates myocardial no-reflow and infarction through increasing eNOS phosphorylation at Ser(1179) and Ser(635) that was partially mediated via the PKA signaling pathway.

Ksp1 Kinase Regulates Autophagy via the Target of Rapamycin Complex 1 (TORC1) Pathway

Macroautophagy (hereafter autophagy) is a bulk degradation system conserved in all eukaryotes, which engulfs cytoplasmic components within double-membrane vesicles to allow their delivery to, and subsequent degradation within, the vacuole/lysosome. Autophagy activity is tightly regulated in response to the nutritional state of the cell and also to maintain organelle homeostasis. In nutrient-rich conditions, Tor kinase complex 1 (TORC1) is activated to inhibit autophagy, whereas inactivation of this complex in response to stress leads to autophagy induction; however, it is unclear how the activity of TORC1 is controlled to allow precise adjustments in autophagy activity. In this study, we performed genetic analyses in Saccharomyces cerevisiae to identify factors that regulate TORC1 activity. We determined that the Ksp1 kinase functions in part as a negative regulator of autophagy; deletion of KSP1 facilitated dephosphorylation of Atg13, a TORC1 substrate, which correlates with enhanced autophagy. These results suggest that Ksp1 down-regulates autophagy activity via the TORC1 pathway. The suppressive function of Ksp1 is partially activated by the Ras/cAMP-dependent protein kinase A (PKA), which is another negative regulator of autophagy. Our study therefore identifies Ksp1 as a new component that functions as part of the PKA and TORC1 signaling network to control the magnitude of autophagy.

Activation of spinal TDAG8 and its downstream PKA signaling pathway contribute to bone cancer pain in rats

Bone cancer pain is difficult to treat and has a strong impact on the quality of life of patients. Few therapies have emerged because the molecular mechanisms underlying bone cancer pain are poorly understood. Recently, T-cell death-associated gene 8 (TDAG8) has been shown to participate in complete Freund's adjuvant-induced chronic inflammatory pain. In this study, we aimed to examine whether TDAG8 and its downstream protein kinase A (PKA) pathway are involved in bone cancer pain. A bone cancer pain model was made by inoculation of Walker 256 cells into the intramedullary space of rat tibia. Spinal TDAG8 expression was increased after inoculation with tumor cells. Intrathecal TDAG8 siRNA attenuated bone cancer pain behaviors during the initiation and maintenance phases; there were also concomitant decreases in TDAG8 mRNA and protein levels in spinal cord. Moreover, we found spinal PKA and phosphorylated cAMP response element-binding (pCREB) protein levels were up-regulated in the rat model of bone cancer pain. Knockdown of TDAG8 resulted in reduced bone cancer pain-induced spinal PKA and pCREB protein expression in two procedures. Furthermore, intrathecal H-89 (a PKA inhibitor) significantly attenuated bone cancer pain behaviors in rats. Our results suggest a causal relationship between TDAG8 expression and the initiation and maintenance of bone cancer pain. Activation of spinal TDAG8 contributes to bone cancer pain through the PKA signaling pathway in rats. These findings may lead to novel strategies for the treatment of bone cancer pain.

Momilactione B inhibits protein kinase A signaling and reduces tyrosinase-related proteins 1 and 2 expression in melanocytes

Momilactone B (MB) is a terpenoid phytoalexin present in rice bran that exhibits several biological activities. MB reduced the melanin content in B16 melanocytes melanin content and inhibited tyrosinase activities. Using transcriptome analysis, the genes involved in protein kinase A (PKA) signaling were found to be markedly altered. B16 cells stimulated with MB had decreased concentrations of cAMP protein kinase A activity, and cAMP-response element-binding protein which is a key transcription factor for microphthalmia-associated transcription factor (MITF) expression. Accordingly, the expression of MITF and its target genes, which are essential for melanogenesis, were reduced. MB thus exhibits anti-melanogenic effects by repressing tyrosinase enzyme activity and inhibiting the PKA signaling pathway which, in turn, decreases melanogenic gene expression.

Serum Amyloid A Induces Lipolysis by Downregulating Perilipin Through ERK1/2 and PKA Signaling Pathways

Serum amyloid A (SAA) is not only an apolipoprotein, but also a member of the adipokine family with potential to enhance lipolysis. The purpose of this study was to explore how SAA facilitates lipolysis in porcine adipocytes. We found that SAA increased the phosphorylation of perilipin and hormone-sensitive lipase (HSL) after 12-h treatment and decreased perilipin expression after 24-h treatment, and these effects were prevented by extracellular signal-regulated kinase (ERK) or protein kinase A (PKA) inhibitors in primary adipocyte cell culture. SAA treatment decreased HSL and adipose triglyceride lipase (ATGL) expression. SAA treatment also activated ERK and PKA by increasing the phosphorylation of these kinases. Moreover, SAA significantly increased porcine adipocyte glycerol release and lipase activity, which was inhibited by either ERK (PD98059) or PKA (H89) inhibitors, suggesting that ERK and PKA were involved in mediating SAA enhanced lipolysis. SAA downregulated the expression of peroxisome proliferator-activated receptor γ (PPARγ) mRNA, which was reversed by the ERK inhibitor. We performed a porcine perilipin promoter assay in differentiated 3T3-L1 adipocytes and found that SAA reduced the porcine perilipin promoter specifically through the function of its PPAR response element (PPRE), and this effect was reversed by the ERK inhibitor. These findings demonstrate that SAA-induced lipolysis is a result of downregulation of perilipin and activation of HSL via ERK/PPARγ and PKA signaling pathways. The finding could lead to developing new strategies for reducing human obesity.

Epac, not PKA, mediate the antiproliferative effects of cyclic AMP on human arteriolar smooth muscle cells

Peroxisome proliferator-activated receptor-γ (PPAR-γ) is required for the conversion of pre-adipocytes. However, the mechanism underlying activation of PPAR-γ is unclear. Here we showed that cAMP-induced activation of protein kinase A (PKA) and Akt is essential for the transcriptional activation of PPAR-γ. Hormonal induction of adipogenesis was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), by a protein kinase A (PKA) inhibitor (H89), and by a Rap1 inhibitor (GGTI-298). Transcriptional activity of PPAR-γ was markedly enhanced by 3-isobutyl-1-methylxanthine (IBMX), but not insulin and dexamethasone. In addition, IBMX-induced PPAR-γ transcriptional activity was blocked by PI3K/Akt, PKA, or Rap1 inhibitors. 8-(4-Chlorophenylthio)-2′-O-methyl-cAMP (8-pCPT-2′-O-Me-cAMP) which is a specific agonist for exchanger protein directly activated by cAMP (Epac) significantly induced the activation of Akt. Furthermore, knock-down of Akt1 markedly attenuated PPAR-γ transcriptional activity. These results indicate that both PKA and Akt signaling pathways are required for transcriptional activation of PPAR-γ, suggesting post-translational activation of PPAR-γ might be critical step for adipogenic gene expression.

Cryptococcal Titan Cell Formation Is Regulated by G-Protein Signaling in Response to Multiple Stimuli

The titan cell is a recently described morphological form of the pathogenic fungus Cryptococcus neoformans. Occurring during the earliest stages of lung infection, titan cells are 5 to 10 times larger than the normal yeast-like cells, thereby resisting engulfment by lung phagocytes and favoring the persistence of infection. These enlarged cells exhibit an altered capsule structure, a thickened cell wall, increased ploidy, and resistance to nitrosative and oxidative stresses. We demonstrate that two G-protein-coupled receptors are important for induction of the titan cell phenotype: the Ste3a pheromone receptor (in mating type a cells) and the Gpr5 protein. Both receptors control titan cell formation through elements of the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. This conserved signaling pathway, in turn, mediates its effect on titan cells through the PKA-regulated Rim101 transcription factor. Additional downstream effectors required for titan cell formation include the G(1) cyclin Pcl103, the Rho104 GTPase, and two GTPase-activating proteins, Gap1 and Cnc1560. These observations support developing models in which the PKA signaling pathway coordinately regulates many virulence-associated phenotypes in diverse human pathogens.

Mitochondria breathe for autophagy

Proper mitochondrial structure and function are established by continuous cycles of fusion and fission, and a unidirectional pathway of autophagy‐mediated degradation. In this issue of The EMBO Journal , Graef and Nunnari (2011) demonstrate that autophagy requires healthy mitochondria in cells under amino‐acid starvation. Defects in mitochondrial respiration cause activation of cAMP‐dependent protein kinase A (PKA), an evolutionarily conserved regulator that inhibits autophagic flux. This study reveals a crucial interplay between mitochondrial function and autophagy, and raises the possibility that deregulation in one of these processes simultaneously compromises the other to potentiate risks for various human disorders.

Quantitative Analysis of Cytokine-Induced Hepatocyte Nuclear Factor-4α Phosphorylation by Mass Spectrometry

Hepatocyte nuclear factor-4α (HNF-4α), a liver-enriched transcription factor, is essential for liver development and function. HNF-4α regulates a large number of liver-specific genes, many of which are modulated by injury. While HNF-4α function is regulated by phosphorylation, only a limited number of phosphorylation sites in HNF-4α have been identified, and the roles of HNF-4α phosphorylation after injury are unexplored. To address these issues, we have carried out an extensive quantitative mass spectrometry (MS)-based analysis of HNF-4α serine and threonine phosphorylation in response to cytokine stimulation. Studies were performed in HNF-4α-enriched HepG2 cells treated with cytokines for 3 h or left untreated, followed by chemical derivatization of the phosphoserine and phosphothreonine residues using stable isotopic variants of dithiothreitol (DTT) and MS analysis. This has allowed the identification and relative quantification of 12 serine/threonine phosphorylation sites in HNF-4α. Eight of these phosphorylation sites and their sensitivity to cytokine stimulation have not been previously reported. We found that cytokine treatment leads to an increase of HNF-4α phosphorylation in several phosphopeptides. The phosphorylation of HNF-4α mediated by protein kinase A (PKA) significantly reduces HNF-4α binding activity, which mimics the repressive effect of cytokines on HNF-4α binding, and the inhibition of PKA activity by PKA inhibitor can partially recover the reduced HNF-4α binding activity induced by cytokines. These results suggest that the mechanism that alters HNF-4α binding after cytokine stimulation involves modulation of specific HNF-4α phosphorylation dependent, in part, on a PKA signaling pathway.

A Role for ETO-2 In Megakaryocytic Inhibition by Cyclic AMP Agonists

AbstractAbstract 289The regulation of megakaryopoiesis by cyclic AMP (cAMP) signaling forms the basis for treatment approaches to thrombocytosis in patients with myeloproliferative neoplasms. Anagrelide, the longstanding treatment of choice, is an inhibitor of the cAMP catabolizing enzyme phosphodiesterase III, and acts to lower platelet counts by inhibiting early stages of megakaryocytic differentiation. Essential for programming megakaryocytic differentiation is the transcriptionally activating pentameric complex consisting of GATA-1, SCL/TAL-1, an E-protein (either E2A or the similar HEB), and the bridging factors Lmo2 and Ldb1. This complex becomes repressive upon the recruitment of the co-repressor ETO-2 (also called MTG16 and CBFA2T3) by either SCL or E-protein components. ETO-2 has also been described as an A-kinase floating protein (AKAP). AKAPs bind the regulatory subunits of Protein kinase A (PKA) and tether the kinase within a specific compartment of the cell, ensuring proximity to appropriate substrates following kinase activation. The current study addresses the hypothesis that elevations in cAMP, such as those induced by anagrelide, inhibit megakaryocytic differentiation through the activation of a novel PKA signaling pathway involving ETO-2 and other components of the pentameric complex. Using peripheral blood mobilized human CD34+ progenitors cultured under conditions promoting megakaryopoiesis, we have confirmed that 500 nM anagrelide or 10 μ M forskolin, a direct adenylyl cyclase agonist, suppress upregulation of CD41 by two to three-fold as well as polyploidization, as determined by FACS on day 6 cultures. Pre-treatment of cultures with 10 μ M H89, a specific inhibitor of PKA, prevented the inhibitory effect of forskolin on megakaryopoiesis, as reflected by CD41 upregulation at day 6. Analysis of the kinetics of PKA activation, by immunoblotting for phospho-CREB and for phospho-PKA substrates (Cell Signaling), surprisingly showed only transient PKA induction by forskolin, with signals returning to baseline level after 4 hours. Additionally, washout experiments in which cells were treated with forskolin for only the first 4 hours of megakaryocytic culture showed severely blunted CD41 upregulation on day 6. These findings argue that elevations in cAMP inhibit megakaryocytic differentiation via activation of PKA and that a brief window of PKA activation during early megakaryopoiesis suffices for a stable blockade of differentiation. To determine a potential role for ETO-2, expression levels were analyzed at various times during megakaryocytic culture. In contrast to the rapid downregulation described in fetal murine megakaryocyte culture (Hamlett I. et al. Blood 112:2738-2749, 2008), human CD34+ progenitors showed no downregulation of ETO-2 mRNA or protein during megakaryocytic differentiation, nor were levels significantly affected by forskolin treatment. To study the functional role of ETO-2, we utilized shRNA knockdown in CD34+ hematopoietic progenitor cells undergoing megakaryocytic culture. In contrast to cells transduced with control vector, cells expressing shRNA targeting ETO-2 showed resistance to the inhibitory effects of elevated cAMP (n = 3). In examining potential targets within the pentameric complex, we found that elevations in cAMP reproducibly lowered the protein and mRNA levels of E2A by 50% while not affecting mRNA levels of another E protein, HEB. Altogether, these data suggest that cAMP mediates inhibition of megakaryopoiesis via activation of PKA, which utilizes ETO-2, possibly as an AKAP, to modulate the composition of an essential activating complex by downregulating E2A. Interestingly, E2A+/− mice are impaired in their ability to generate megakaryocytic progenitors from bipotent megakaryocyte-erythroid precursors (Semerad CL et al. PNAS 106:1930-1935, 2009). This novel signaling pathway contributes to an understanding of the mechanism of action of anagrelide and also further elucidates the basic regulatory circuitry governing determination of the megakaryocytic lineage. Disclosures:No relevant conflicts of interest to declare.

Chromatin accessibility and transcription factor binding at the PPARγ2 promoter during adipogenesis is protein kinase A‐dependent

The nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcription factor that specifies formation of the adipocyte lineage. PPARγ also serves as a primary target for the treatment of type 2 diabetes, illustrating both its medical relevance as well as the need to understand fundamental aspects of PPARγ expression and function. Here, we characterize molecular changes that occur at the PPARγ2 promoter within the first several hours of adipocyte differentiation in culture. Our results demonstrate that changes in chromatin accessibility at the PPARγ2 promoter and occupancy of the promoter by the c-Fos transcription factor occur within an hour of the onset of differentiation, followed closely by the binding of the CCAAT/enhancer binding protein beta (C/EBPβ) transcription factor. All three events show a remarkable dependency on protein kinase A (PKA) activity. These results reflect novel requirements for the PKA signaling pathway and reinforce the importance of PKA function during the onset of adipocyte differentiation.

Activation of cyclic amp/protein kinase: A signaling pathway enhances osteoblast cell adhesion on biomaterials for regenerative engineering

Osteoblast cell adhesion on biomaterials is an important goal for implants to be useful in bone regeneration technologies. The adhesion of osteoblastic cells to biomaterials has been investigated in the field of bone regenerative engineering. Previous work from our group demonstrated that osteoblastic cells adhering to biodegradable biomaterials require the expression of integrins on the cell surface. However, the underlying molecular signaling mechanism is still not fully clear. We report here that cyclic adenosine monophosphate (cAMP), a small signaling molecule, regulates osteoblast cell adhesion to biomaterial surfaces. We used an in vitro cell adhesion assay to demonstrate that at 0.1 mM, 8-Br-cAMP, a cell-permeable cAMP analog, significantly enhances osteoblast-like cells' (MC3T3-E1) adherence to biomaterials. Moreover, we demonstrate that a commonly used cAMP-elevating agent, forskolin, promotes cell adhesion similar to that of the cell permeable cAMP analog. By using different target-specific cAMP analogs: 8-CPT-2Me-cAMP which specifically activates exchange protein activated by cAMP (Epac), and 6-Bnz-cAMP which specifically activates protein kinase A (PKA), we observed that the PKA signaling pathway plays a dominant role in this process. Thus, this report suggests a new method to enhance osteoblast cell adhesion on biodegradable biomaterials for bone regenerative engineering applications.

Molecular mechanism of calcium channel regulation in the fight-or-flight response.

During the fight-or-flight response, the sympathetic nervous system stimulates L-type calcium ion (Ca2+) currents conducted by Ca(V)1 channels through activation of β-adrenergic receptors, adenylyl cyclase, and phosphorylation by adenosine 3',5'-monophosphate-dependent protein kinase [also known as protein kinase A (PKA)], increasing contractility of skeletal and cardiac muscles. We reconstituted this regulation of cardiac Ca(V)1.2 channels in non-muscle cells by forming an autoinhibitory signaling complex composed of Ca(V)1.2Δ1800 (a form of the channel truncated at the in vivo site of proteolytic processing), its noncovalently associated distal carboxyl-terminal domain, the auxiliary α₂δ₁ and β(2b) subunits, and A-kinase anchoring protein 15 (AKAP15). A factor of 3.6 range of Ca(V)1.2 channel activity was observed from a minimum in the presence of protein kinase inhibitors to a maximum upon activation of adenylyl cyclase. Basal Ca(V)1.2 channel activity in unstimulated cells was regulated by phosphorylation of serine-1700 and threonine-1704, two residues located at the interface between the distal and the proximal carboxyl-terminal regulatory domains, whereas further stimulation of channel activity through the PKA signaling pathway only required phosphorylation of serine-1700. Our results define a conceptual framework for Ca(V)1.2 channel regulation and identify sites of phosphorylation that regulate channel activity.