Protein Kinase A Phosphorylation Research Articles

Low phosphorus causes hepatic energy metabolism disorder through Dynamin-related protein 1-mediated mitochondrial fission in fish

BackgroundLow phosphorus (LP) diets perturb hepatic energy metabolism homeostasis in fish. However, the specific mechanisms in LP-induced hepatic energy metabolism disorders remain to be fully elucidated. ObjectivesThis study sought to elucidate the underlying mechanisms of mitochondria involved in LP-induced energy metabolism disorders. MethodsSpotted seabass were fed diets with 0.72% (S-AP, control) or 0.36% (S-LP) available phosphorus for 10 weeks. Drp1 was knocked down or protein kinase A (PKA) was activated using 8Br-cAMP (5 μM, a PKA activator) in spotted seabass hepatocytes under LP medium. Zebrafish were fed Z-LP diets (0.30% available phosphorus) containing Mdivi-1 (5 mg/kg, a Drp1 inhibitor) or 8Br-cAMP (0.5 mg/kg) for 6 weeks. Biochemical and molecular parameters, along with transmission electron microscopy and immunofluorescence, were used to assess hepatic glycolipid metabolism, mitochondrial function and morphology. ResultsSpotted seabass fed S-LP diets showed reduced ATP (0.52-fold) and cyclic adenosine monophosphate (cAMP) (0.52-fold) levels, along with reduced Drp1 (s582) (0.38-fold) and PKA (0.61-fold) phosphorylation levels in the liver compared with those fed S-AP diets (P < 0.05). Drp1 knockdown elevated ATP levels (1.99-fold), decreased mitochondrial DRP1 protein levels (0.45-fold), and increased mitochondrial aspect ratio (1.82-fold) in LP-treated hepatocytes (P < 0.05). Furthermore, 8Br-cAMP-treated hepatocytes exhibited higher PKA phosphorylation (2.85-fold), ATP levels (1.60-fold), and mitochondrial aspect ratio (2.00-fold), along with decreased mitochondrial DRP1 protein levels (0.29-fold) under LP medium (P < 0.05). However, mutating s582 to alanine mimic Drp1 dephosphorylation decreased ATP levels (0.63-fold) and mitochondrial aspect ratio (0.53-fold) in 8Br-cAMP-treated hepatocytes (P < 0.05). In addition, zebrafish were fed Z-LP diets containing Mdivi-1 or 8Br-cAMP had higher ATP levels (3.44-fold or 1.98-fold) than that fed Z-LP diets (P < 0.05). ConclusionsThese findings provide a potential mechanistic elucidation for LP-induced energy metabolism disorders through the cAMP/PKA/Drp1-mediated mitochondrial fission signaling pathway.

Hyperfunction of AMPA receptors in the amygdala and hippocampus contributes to enhanced fear memory in diabetic mice

Patients with diabetes mellitus show an elevated prevalence of psychiatric disorders such as anxiety. We have reported that fear memory, a model related to anxiety as reflected in the freezing response, is enhanced in diabetic mice and was ameliorated by an AMPA receptor antagonist. The present study investigated whether functions of AMPA receptors in the amygdala and hippocampus are altered in streptozotocin (STZ)-induced diabetic mice. While protein levels of the GluA1 subunit of AMPA receptors were not altered in the amygdala and hippocampus, protein levels of GluA1 phosphorylated at serine 845 in the amygdala and hippocampus and of GluA1 phosphorylated at serine 831 in the hippocampus were increased in STZ-induced diabetic mice. L-lactate, which is increased in the amygdala and hippocampus of STZ-induced diabetic mice, did not alter these protein levels in either brain area. In contrast, protein levels of phosphorylated protein kinase A (PKA) catalytic subunit and phosphorylated calcium calmodulin kinase II (CaMKII), which are known to phosphorylate serine 845 and serine 831 of GluA1, respectively, were increased in the amygdala and hippocampus of STZ-induced diabetic mice. In the fear memory test, the PKA inhibitor H-89 injected before test sessions and the CaMKII inhibitor KN-62 injected before conditioning or test sessions each reduced the increase in freezing in STZ-induced diabetic mice. These results indicate that the functions of AMPA receptors in the amygdala and hippocampus are enhanced due to increased phosphorylation by PKA and CaMKII, which enhances fear memory in diabetic mice.

Catecholamines Attenuate LPS-Induced Inflammation through β2 Adrenergic Receptor Activation- and PKA Phosphorylation-Mediated TLR4 Downregulation in Macrophages.

Inflammation is a tightly regulated process involving immune receptor recognition, immune cell migration, inflammatory mediator secretion, and pathogen elimination, all essential for combating infection and restoring damaged tissue. However, excessive inflammatory responses drive various human diseases. The autonomic nervous system (ANS) is known to regulate inflammatory responses; however, the detailed mechanisms underlying this regulation remain incompletely understood. Herein, we aimed to study the anti-inflammatory effects and mechanism of action of the ANS in RAW264.7 cells. Quantitative PCR and immunoblotting assays were used to assess lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNFα) expression. The anti-inflammatory effects of catecholamines (adrenaline, noradrenaline, and dopamine) and acetylcholine were examined in LPS-treated cells to identify the receptors involved. Catecholamines inhibited LPS-induced TNFα expression by activating the β2 adrenergic receptor (β2-AR). β2-AR activation in turn downregulated the expression of Toll-like receptor 4 (TLR4) by stimulating protein kinase A (PKA) phosphorylation, resulting in the suppression of TNFα levels. Collectively, our findings reveal a novel mechanism underlying the inhibitory effect of catecholamines on LPS-induced inflammatory responses, whereby β2-AR activation and PKA phosphorylation downregulate TLR4 expression in macrophages. These findings could provide valuable insights for the treatment of inflammatory diseases and anti-inflammatory drug development.

Targeting the IKs Channel PKA Phosphorylation Axis to Restore Its Function in High-Risk LQT1 Variants.

The KCNQ1+KCNE1 (IKs) potassium channel plays a crucial role in cardiac adaptation to stress, in which β-adrenergic stimulation phosphorylates the IKs channel through the cyclic adenosine monophosphate (cAMP)/PKA (protein kinase A) pathway. Phosphorylation increases the channel current and accelerates repolarization to adapt to an increased heart rate. Variants in KCNQ1 can cause long-QT syndrome type 1 (LQT1), and those with defective cAMP effects predispose patients to the highest risk of cardiac arrest and sudden death. However, the molecular connection between IKs channel phosphorylation and channel function, as well as why high-risk LQT1 mutations lose cAMP sensitivity, remain unclear. Regular patch clamp and voltage clamp fluorometry techniques were utilized to record pore opening and voltage sensor movement of wild-type and mutant KCNQ1/IKs channels. The clinical phenotypic penetrance of each LQT1 mutation was analyzed as a metric for assessing their clinical risk. The patient-specific-induced pluripotent stem-cell model was used to test mechanistic findings in physiological conditions. By systematically elucidating mechanisms of a series of LQT1 variants that lack cAMP sensitivity, we identified molecular determinants of IKs channel regulation by phosphorylation. These key residues are distributed across the N-terminus of KCNQ1 extending to the central pore region of IKs. We refer to this pattern as the IKs channel PKA phosphorylation axis. Next, by examining LQT1 variants from clinical databases containing 10 579 LQT1 carriers, we found that the distribution of the most high-penetrance LQT1 variants extends across the IKs channel PKA phosphorylation axis, demonstrating its clinical relevance. Furthermore, we found that a small molecule, ML277, which binds at the center of the phosphorylation axis, rescues the defective cAMP effects of multiple high-risk LQT1 variants. This finding was then tested in high-risk patient-specific induced pluripotent stem cell-derived cardiomyocytes, where ML277 remarkably alleviates the beating abnormalities. Our findings not only elucidate the molecular mechanism of PKA-dependent IKs channel phosphorylation but also provide an effective antiarrhythmic strategy for patients with high-risk LQT1 variants.

Albiflorin Decreases Glutamate Release from Rat Cerebral Cortex Nerve Terminals (Synaptosomes) through Depressing P/Q-Type Calcium Channels and Protein Kinase A Activity.

The purpose of this study was to investigate whether and how albiflorin, a natural monoterpene glycoside, affects the release of glutamate, one of the most important neurotransmitters involved in neurotoxicity, from cerebrocortical nerve terminals (synaptosomes) in rats. The results showed that albiflorin reduced 4-aminopyridine (4-AP)-elicited glutamate release from synaptosomes, which was abrogated in the absence of extracellular Ca2+ or in the presence of the vesicular glutamate transporter inhibitor or a P/Q-type Ca2+ channel inhibitor, indicating a mechanism of action involving Ca2+-dependent depression of vesicular exocytotic glutamate release. Albiflorin failed to alter the increase in the fluorescence intensity of 3,3-diethylthiacarbocyanine iodide (DiSC3(5)), a membrane-potential-sensitive dye. In addition, the suppression of protein kinase A (PKA) abolished the effect of albiflorin on glutamate release. Albiflorin also reduced the phosphorylation of PKA and synaptosomal-associated protein of 25 kDa (SNAP-25) and synapsin I at PKA-specific residues, which correlated with decreased available synaptic vesicles. The results of transmission electron microscopy (TEM) also observed that albiflorin reduces the release competence of synaptic vesicles evoked by 4-AP in synaptosomes. In conclusion, by studying synaptosomally released glutamate, we suggested that albiflorin reduces vesicular exocytotic glutamate release by decreasing extracellular Ca2+ entry via P/Q-type Ca2+ channels and reducing PKA-mediated synapsin I and SNAP-25 phosphorylation.

The Extract of Humulus japonicus Inhibits Lipogenesis and Promotes Lipolysis via PKA/p38 Signaling

Introduction: Previous research has shown that an aqueous extract of Humulus japonicus (EH) can ameliorate hypertension, nonalcoholic fatty liver disease, and oxidative stress in adipocytes by activating the thermogenic pathway. However, the effects of an ethanol (30%) extract of EH on obesity are unknown. Methods: Various protein expression levels in fully differentiated 3T3-L1 adipocytes were assessed by Western blotting. Lipid deposition in 3T3-L1 adipocytes was examined by oil red O staining. The MTT assay was used to evaluate adipocyte viability. Caspase 3 activity and glycerol release were determined using commercial assay kits. Results: In this study, we discovered that EH treatment inhibited lipogenesis and promoted lipolysis in both differentiated 3T3-L1 adipocytes and adipose tissue of mice fed a high-fat diet. EH treatment also increased phosphorylated protein kinase A (PKA) levels while reducing p38 phosphorylation. When H89, a PKA inhibitor, was used, the effects of EH on lipogenic lipid accumulation and lipolysis in 3T3-L1 adipocytes were eliminated. Treatment with luteolin 7-O-β-d-glucoside (LU), the major active compound in EH, also suppressed lipid deposition and p38 phosphorylation but enhanced lipolysis in 3T3-L1 adipocytes. These changes were abrogated by H89. Conclusion: These findings indicate that EH containing LU reduces lipogenesis and stimulates lipolysis via the PKA/p38 signaling pathway, leading to an improvement in obesity in mice. Therefore, our study suggested that EH could be a promising therapeutic agent for treating obesity.

The PDE4 inhibitor apremilast modulates ethanol responses in Gabrb1-S409A knock-in mice via PKA-dependent and independent mechanisms

We previously showed that the PDE4 inhibitor apremilast reduces ethanol consumption in mice by protein kinase A (PKA) and GABAergic mechanisms. Preventing PKA phosphorylation of GABAA β3 subunits partially blocked apremilast-mediated decreases in drinking. Here, we produced Gabrb1-S409A mice to render GABAA β1 subunits resistant to PKA-mediated phosphorylation. Mass spectrometry confirmed the presence of the S409A mutation and lack of changes in β1 subunit expression or phosphorylation at other residues. β1-S409A male and female mice did not differ from wild-type C57BL/6J mice in expression of Gabrb1, Gabrb2, or Gabrb3 subunits or in behavioral characteristics. Apremilast prolonged recovery from ethanol ataxia to a greater extent in Gabrb1-S409A mice but prolonged recovery from zolpidem and propofol to a similar extent in both genotypes. Apremilast shortened recovery from diazepam ataxia in wild-type but prolonged recovery in Gabrb1-S409A mice. In wild-type mice, the PKA inhibitor H89 prevented apremilast modulation of ataxia by ethanol and diazepam, but not by zolpidem. In Gabrb1-S409A mice, inhibiting PKA or EPAC2 (exchange protein directly activated by cAMP) partially reversed apremilast potentiation of ethanol, diazepam, and zolpidem ataxia. Apremilast prevented acute tolerance to ethanol ataxia in both genotypes, but there were no genotype differences in ethanol consumption before or after apremilast. In contrast to results in Gabrb3-S408A/S409A mice, PKA phosphorylation of β1-containing GABAA receptors is not required for apremilast's effects on acute tolerance or on ethanol consumption but is required for its ability to decrease diazepam intoxication. Besides PKA we identified EPAC2 as an additional cAMP-dependent mechanism by which apremilast regulates responses to GABAergic drugs.

Gypenoside XVII Reduces Synaptic Glutamate Release and Protects against Excitotoxic Injury in Rats.

Excitotoxicity is a common pathological process in neurological diseases caused by excess glutamate. The purpose of this study was to evaluate the effect of gypenoside XVII (GP-17), a gypenoside monomer, on the glutamatergic system. In vitro, in rat cortical nerve terminals (synaptosomes), GP-17 dose-dependently decreased glutamate release with an IC50 value of 16 μM. The removal of extracellular Ca2+ or blockade of N-and P/Q-type Ca2+ channels and protein kinase A (PKA) abolished the inhibitory effect of GP-17 on glutamate release from cortical synaptosomes. GP-17 also significantly reduced the phosphorylation of PKA, SNAP-25, and synapsin I in cortical synaptosomes. In an in vivo rat model of glutamate excitotoxicity induced by kainic acid (KA), GP-17 pretreatment significantly prevented seizures and rescued neuronal cell injury and glutamate elevation in the cortex. GP-17 pretreatment decreased the expression levels of sodium-coupled neutral amino acid transporter 1, glutamate synthesis enzyme glutaminase and vesicular glutamate transporter 1 but increased the expression level of glutamate metabolism enzyme glutamate dehydrogenase in the cortex of KA-treated rats. In addition, the KA-induced alterations in the N-methyl-D-aspartate receptor subunits GluN2A and GluN2B in the cortex were prevented by GP-17 pretreatment. GP-17 also prevented the KA-induced decrease in cerebral blood flow and arginase II expression. These results suggest that (i) GP-17, through the suppression of N- and P/Q-type Ca2+ channels and consequent PKA-mediated SNAP-25 and synapsin I phosphorylation, reduces glutamate exocytosis from cortical synaptosomes; and (ii) GP-17 has a neuroprotective effect on KA-induced glutamate excitotoxicity in rats through regulating synaptic glutamate release and cerebral blood flow.

Role of ERK and NF-KB pathway in neurally mediated enhanced expression and traffcking of SGLT2 in congestive heart failure

Congestive heart failure (CHF) is characterized by the activation of neurohumoral systems concomitant with avid fluid retention. Recently we showed enhanced expression of sodium-glucose cotransporter 2 (SGLT2) in rats with CHF. However, the exact molecular mechanism linking enhanced sympathetic tone and enhanced expression and translocation of SGLT2 in CHF is not clear. The objective of this study was to investigate the role of enhanced sympathetic tone on the expression and traffcking of SGLT2 via activation of protein kinase A (PKA), extracellular signal-regulated kinases (ERK), and nuclear factor kappa B (NF-KB) in the renal cortex of CHF rats. Male Sprague Dawley rats were subjected to either the left coronary artery ligation for the CHF group or Sham surgery. Four weeks after surgery, expression of protein levels (PKA, ERK and NF-KB) were assessed by Western blotting, whereas the location of SGLT2 expression was determined by immunohistological and immunofluorescent staining in the cortical region of kidneys from Sham and CHF rats. Effect of exogenous norepinephrine (NE) (10μM for 24hrs) on expression and traffcking of SGLT2, phosphorylation of ERK and NF-KB in HK2 cells were examined in an in vitro model. Expression of SGLT2 was significantly enhanced in the renal cortex of rats with CHF versus Sham (0.83±0.06 vs. 0.58±0.02). Also, the translocation of SGLT2 was significantly enhanced towards the membrane in CHF rats (0.92±0.11 vs. 0.33±0.07). Phosphorylation of PKA (0.05±0.01 vs. 0.01±0.01), ERK (0.32±0.02 vs. 0.23±0.02) and NF-KB (0.71±0.14 vs. 0.08±0.06) were significantly enhanced in CHF compared to Sham. NE treatment significantly increased expression of SGLT2 (0.26±0.03 vs. 0.15±0.02), traffcking of SGLT2 (0.45 ± 0.09 vs. 0.15 ± 0.03) towards membrane, phosphorylation of ERK (0.38±0.05 vs. 0.14±0.02) and NF-KB (0.61±0.08 vs. 0.17±0.07) in HK2 cells. Thus, NE treatment enhanced the phosphorylation of ERK, NF-kB as well as traffcking of SGLT2. Immunohistological and immunofluorescent staining demonstrated increased expression of SGLT2 in the kidneys of rats with CHF. Similar results were observed in immunofluorescent staining of human kidney cells treated with NE. In conclusion, these findings indicate that NE activates ERK and NF-KB pathways which leads to enhanced traffcking and overexpression of SGLT2 in CHF. These results provide a molecular link between enhanced sympathetic tone, ERK and NF-KB pathway and the traffcking of SGLT2 to the luminal membrane of renal proximal tubular cells and consequent increase in sodium retention in rats with CHF. This study was funded by NIH R01-DK-129311. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Conservation of Protein Kinase A Substrates in the Cnidarian Coral Spermatozoa Among Animals and Their Molecular Evolution.

The coral Acropora spp., known for its reef-building abilities, is a simultaneous hermaphroditic broadcast spawning species. Acropora spp. release gametes into seawater, activating sperm motility. This activation is mediated by adenylyl cyclase (AC) and protein kinase A (PKA). Notably, membrane-permeable cAMP (8-bromo-cAMP) promotes sperm motility activation of Acropora florida. While the signal transduction for PKA-dependent motility activation is highly conserved among animals, the downstream signaling of PKA remains unclear. In this study, we used mass spectrometry (MS) analyses to identify sperm proteins in the coral Acropora digitifera, as well as the serine/threonine residues of potential PKA substrates, and then, we investigated the conservation of these proteins from corals to vertebrates. We identified 148 sperm proteins of A. digitifera with typical PKA recognition motifs, namely RRXT and RRXS. We subsequently used ORTHOSCOPE to screen for orthologs encoding these 148 proteins from corals to vertebrates. Among the isolated orthologs, we identified positive selection in 48 protein-encoding genes from 18 Acropora spp. Subsequently, we compared the conservation rates of the PKA phosphorylation motif residues between the orthologs under positive and purifying selections. Notably, the serine residues of the orthologs under positive selection were more conserved. Therefore, adaptive evolution might have occurred in the orthologs of PKA substrate candidates from corals to vertebrates, accompanied by phosphorylation residue conservation. Collectively, our findings suggest that while PKA signal transduction, including substrates in sperm, may have been conserved, the substrates may have evolved to adapt to diverse fertilization conditions, such as synchronous broadcast spawning.

A Cre-dependent reporter mouse for quantitative real-time imaging of protein kinase A activity dynamics

Intracellular signaling dynamics play a crucial role in cell function. Protein kinase A (PKA) is a key signaling molecule that has diverse functions, from regulating metabolism and brain activity to guiding development and cancer progression. We previously developed an optical reporter, FLIM-AKAR, that allows for quantitative imaging of PKA activity via fluorescence lifetime imaging microscopy and photometry. However, using viral infection or electroporation for the delivery of FLIM-AKAR is invasive and results in variable expression. Here, we developed a reporter mouse, FL-AK, which expresses FLIM-AKAR in a Cre-dependent manner from the ROSA26 locus. FL-AK provides robust and consistent expression of FLIM-AKAR over time. Functionally, the mouse line reports an increase in PKA activity in response to activation of both Gαs and Gαq-coupled receptors in brain slices. In vivo, FL-AK reports PKA phosphorylation in response to neuromodulator receptor activation. Thus, FL-AK provides a quantitative, robust, and flexible method to reveal the dynamics of PKA activity in diverse cell types.

Effect of 4-nonylphenol (4-NP) on sperm function: Insights into the PI3K/PDK1/AKT signaling pathway during capacitation

4-Nonylphenol (4-NP) is an endocrine-disrupting chemical that impairs animal and human reproduction. However, the mechanisms underlying male reproductive dysfunction by 4-NP have not been fully understood. Herein, we demonstrated the effects of 4-NP on boar sperm functions and molecular mechanisms. Spermatozoa were treated with various concentrations of 4-NP (0, 10, 25, 50, 75, and 100 μM) during capacitation. Then, we evaluated sperm motility, capacitation status, intracellular ATP level, and cell viability. Finally, we measured the expression of phosphorylated protein kinase A (PKA), tyrosine phosphorylation, and proteins related to the phosphatidylinositol 3 kinase (PI3K)/phosphoinositide-dependent kinase-1 (PDK1)/protein kinase B (AKT) signaling pathways following exposure to 4-NP. Sperm motility and motion kinematics were reduced by 4-NP, whereas intracellular ATP levels were increased significantly in a dose-dependent manner. Furthermore, the expression levels of p-PI3K, PTEN, p-PDK1, AKT, and p-AKT exhibited a significant dose-dependent increase. Moreover, abnormal activation of PKA and tyrosine phosphorylation were observed. Specifically, the ∼24 kDa p-PKA substrate demonstrated a significant reduction following exposure to 4-Np. In addition, the ∼18 kDa p-PKA substrate and tyrosine-phosphorylated proteins displayed a significant dose-dependent increase after exposure to 4-NP. Our results suggest that 4-NP may induce detrimental effects on sperm functions through abnormal changes in PKA activity and tyrosine phosphorylation during capacitation, possibly through unusual alteration of the PI3K/PDK1/AKT signaling pathway. Therefore, 4-NP must be cautiously used considering its reproductive toxicity.

Arginine vasopressin regulates the renal Na+-Cl- and Na+-K+-Cl- cotransporters through with-no-lysine kinase 4 and inhibitor 1 phosphorylation.

Vasopressin regulates water homeostasis via the V2 receptor in the kidney at least in part through protein kinase A (PKA) activation. Vasopressin, through an unknown pathway, upregulates the activity and phosphorylation of Na+-Cl- cotransporter (NCC) and Na+-K+-2Cl- cotransporter 2 (NKCC2) by Ste20-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1), which are regulated by the with-no-lysine kinase (WNK) family. Phosphorylation of WNK4 at PKA consensus motifs may be involved. Inhibitor 1 (I1), a protein phosphatase 1 (PP1) inhibitor, may also play a role. In human embryonic kidney (HEK)-293 cells, we assessed the phosphorylation of WNK4, SPAK, NCC, or NKCC2 in response to forskolin or desmopressin. WNK4 and cotransporter phosphorylation were studied in desmopressin-infused WNK4-/- mice and in tubule suspensions. In HEK-293 cells, only wild-type WNK4 but not WNK1, WNK3, or a WNK4 mutant lacking PKA phosphorylation motifs could upregulate SPAK or cotransporter phosphorylation in response to forskolin or desmopressin. I1 transfection maximized SPAK phosphorylation in response to forskolin in the presence of WNK4 but not of mutant WNK4 lacking PP1 regulation. We observed direct PP1 regulation of NKCC2 dephosphorylation but not of NCC or SPAK in the absence of WNK4. WNK4-/- mice with desmopressin treatment did not increase SPAK/OSR1, NCC, or NKCC2 phosphorylation. In stimulated tubule suspensions from WNK4-/- mice, upregulation of pNKCC2 was reduced, whereas upregulation of SPAK phosphorylation was absent. These findings suggest that WNK4 is a central node in which kinase and phosphatase signaling converge to connect cAMP signaling to the SPAK/OSR1-NCC/NKCC2 pathway.NEW & NOTEWORTHY With-no-lysine kinases regulate the phosphorylation and activity of the Na+-Cl- and Na+-K+-2Cl- cotransporters. This pathway is modulated by arginine vasopressin (AVP). However, the link between AVP and WNK signaling remains unknown. Here, we show that AVP activates WNK4 through increased phosphorylation at putative protein kinase A-regulated sites and decreases its dephosphorylation by protein phosphatase 1. This work increases our understanding of the signaling pathways mediating AVP actions in the kidney.

Abstract C030: Gene expression profiling tumors of patients with clear cell renal carcinoma: May identify race-specific therapeutic targets

Abstract Black/African Americans are second only to Native Americans in having the highest incidence of renal cancer. Despite this statistic, very few studies have examined whether patients’ tumors exhibit racial/ethnic differences in both gene and/or protein expression. Using KIRC (clear cell renal carcinoma) TCGA dataset (n=542), we identified several genes that were significantly overexpressed in ccRCC derived from Blacks. Genes which showed significantly higher expression in tumors from Blacks (n=56) relative to Whites (n=463) included ADCK5 (adj p&amp;lt;0.001) ADRM1 (adj p=0.013), ANKRD13D (adj p=0.003), ARFGAP1 (adj p=.003), LZTS2 (adj p&amp;lt;0.001), FAM160B2 (adj p&amp;lt;0.001), and NCKAP5L (adj p=0.029). In addition, these genes were also significantly overexpressed (p&amp;lt;0.05) in advanced stages, higher grades as well as in metastatic tissues. Multivariate cox regression analysis of gene expression, ethnicity, racial demographics and clinicopathological features relative to overall survival time revealed that ARFGAP1 (HRadj=2.40 [95% CI: 1.37 to 4.22]), ANKRD13D (HRadj=2.56 [95% CI:1.43 to 4.59]), FAM160B2 (HRadj= 1.74 [95%CI: 1.01 to 2.99]), and NCKAP5L (HRadj= 1.88 [95% CI:1.04 to 3.41]) were independent prognostic markers for survival. Higher expression of ADCK5 (HRadj= 5.98[CI:1.26-28.35], p=0.012), ADMR1 (HRadj= 1.74 [95%CI: 1.05 to 11.38], p=0.03), ANKRD13D (HRadj= 9.05 [95%CI: 1.13 to 72.66], p=0.014), ARFGAP1 (HRadj=3.79 [95% CI: 0.097-14.54], p=0.041), FAM160B2 (HRadj=5.09 [95%CI: 1.01-25.75], p=0.033), LZTS2 (HRadj=3.32 [95%CI: 1.01-10.99], p=0.037) was associated with lower ccRCC survival in Blacks. ROC analysis indicated that expression of FAM160B2 and LZTS2 are strong predictors of ccRCC survival among Blacks (AUC= 0.737, p&amp;lt;0.001, AUC= 0.731, p&amp;lt;0.001, respectively), whereas ADCK5 and ADRM1, (AUC=0.692, p&amp;lt;0.001, AUC=0.625, p=0.001, respectively) were moderate predictors of ccRCC survival among Blacks. FAM160B2 (family with sequence similarity 160 member B2) is a protein kinase A anchoring protein (AKAP) and participates in PKA (protein kinase A) phosphorylation of HSP70 (heat shock protein 70). FAM160B2 is also involved in hepatocellular carcinoma and promotes tumorigenesis. LZTS2, which encodes leucine zipper tumor suppressor 2, was identified as a tumor susceptibility gene within the 10q24.3 chromosomal region and is approximately 15Mb from the PTEN locus. This region containing both loci is frequently deleted in a variety of human malignancies, including prostate cancer. Taken collectively our data suggest that the aforementioned genes, FAM160B2 and LZTS2 may be useful both as clinical biomarkers and should be explored as potential therapeutic targets for ccRCC, with possible greater impact among Black patients. Citation Format: Romonia Renee Reams, Simone O. Heyliger, Marilyn Saulsbury, Emanuela Taioli, Diana Wilkie, John Carpten. Gene expression profiling tumors of patients with clear cell renal carcinoma: May identify race-specific therapeutic targets [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C030.

β-asarone improves cognitive impairment and alleviates autophagy in mice with vascular dementia via the cAMP/PKA/CREB pathway

BackgroundVascular dementia (VD) is the second most common type of dementia after Alzheimer's disease. β-asarone, a major component of Acorus tatarinowii Schott, is important in neurodegenerative and neurovascular diseases. Studies have confirmed that β-asarone can mitigate autophagy and reduce damage in hypoxic cells. We also reported that β-asarone improves learning and memory. This study further clarifies whether β-asarone attenuates cerebral ischaemic injury by acting through the cAMP/PKA/CREB pathway in VD model mice. MethodsHere, genes and potential pathways that may be targeted by β-asarone for the treatment of transient cerebral ischaemia (TCI) and cognitive impairment (CI) were obtained using network pharmacology. The two-vessel occlusion method was used to establish the VD model. The Morris water maze test was used to evaluate the effects on memory. Then, the protein levels of mitofusin-2 (Mfn2), brain-derived neurotrophic factor (BDNF), optic atrophy 1 (OPA1), cyclic adenosine monophosphate (cAMP), myelin basic protein (MBP), matrix metalloproteinase-9 (MMP9) and neuron specific enolase (NSE) were determined by ELISA. The levels of superoxide dismutase (SOD) and malonaldehyde (MDA) were measured using commercial kits. Then, qRT-PCR was employed to investigate the expression of the candidate genes screened from the protein-protein interaction (PPI) network. Furthermore, the expression of the autophagy-related proteins Beclin-1, (microtubule-associated protein light chain 3) LC3, p62, postsynaptic density protein 95 (PSD95), protein kinase A (PKA), pPKA, cyclic-AMP response binding protein (CREB), and pCREB was determined by western blotting. The expression of autophagy-related proteins, PSD95 and translocase of outer mitochondrial membrane 20 (TOM20) was determined by immunofluorescence analyses. ResultsThe network pharmacological analysis showed 234 targets related to β-asarone, 1,118 genes related to TCI and 2,039 genes associated with CI. Our results confirm that β-asarone treatment not only alleviated brain damage in the VD model by improving mitochondrial and synaptic function, reducing neuronal injury and upregulating the expression of antioxidants but also effectively improved the cognitive behaviour of VD model mice. Moreover, β-asarone downregulated VD-induced RELA and CCND1 mRNA expression. In addition, we validated that β-asarone increased the phosphorylation of PKA and CREB and upregulated cAMP protein expression. The results showed that the cAMP/PKA/CREB signalling pathway was upregulated. Moreover, β-asarone administration decreased the protein expression levels of Beclin-1 and LC3 and increased the expression levels of p62 in VD model mice. Conclusionsβ-asarone inhibits Beclin-1-dependent autophagy and upregulates the cAMP/PKA/CREB signalling pathway to attenuate mitochondrial and synaptic damage from cerebral ischaemia and improve learning and cognitive abilities in VD model mice.

Benzyl isothiocyanate inhibits TNFα-driven lipolysis via suppression of the ERK/PKA/HSL signaling pathway in 3T3-L1 adipocytes

Tumor necrosis factor α (TNFα), an inflammatory cytokine, induces lipolysis and increases circulating concentrations of free fatty acids. In addition, TNFα is the first adipokine produced by adipose tissue in obesity, contributing to obesity-associated metabolic disease. Given that benzyl isothiocyanate (BITC) is a well-known anti-inflammatory agent, we hypothesized that BITC can ameliorate TNFα-induced lipolysis and investigated the working mechanisms involved. We first challenged 3T3-L1 adipocytes with TNFα to induce lipolysis, which was confirmed by increased glycerol release, decreased protein expression of peroxisome proliferator-activated receptor γ (PPARγ) and perilipin 1 (PLIN1), and increased phosphorylation of ERK, protein kinase A (PKA), and hormone-sensitive lipase (HSL). However, inhibition of ERK or PKA significantly attenuated the lipolytic activity of TNFα. Meanwhile, pretreatment with BITC significantly ameliorated the lipolytic activity of TNFα; the TNFα-induced phosphorylation of ERK, PKA, and HSL; the TNFα-induced ubiquitination of PPARγ; the TNFα-induced decrease in PPARγ nuclear protein binding to PPAR response element; and the TNFα-induced decrease in PLIN1 protein expression. Our results indicate that BITC ameliorates TNFα-induced lipolysis by inhibiting the ERK/PKA/HSL signaling pathway, preventing PPARγ proteasomal degradation, and maintaining PLIN1 protein expression.

Glucagon-like peptide-1 receptor activation stimulates PKA-mediated phosphorylation of Raptor and this contributes to the weight loss effect of liraglutide.

The canonical target of the glucagon-like peptide-1 receptor (GLP-1R), Protein Kinase A (PKA), has been shown to stimulate mechanistic Target of Rapamycin Complex 1 (mTORC1) by phosphorylating the mTOR-regulating protein Raptor at Ser791 following β-adrenergic stimulation. The objective of these studies is to test whether GLP-1R agonists similarly stimulate mTORC1 via PKA phosphorylation of Raptor at Ser791 and whether this contributes to the weight loss effect of the therapeutic GLP-1R agonist liraglutide. We measured phosphorylation of the mTORC1 signaling target ribosomal protein S6 in Chinese Hamster Ovary cells expressing GLP-1R (CHO-Glp1r) treated with liraglutide in combination with PKA inhibitors. We also assessed liraglutide-mediated phosphorylation of the PKA substrate RRXS*/T* motif in CHO-Glp1r cells expressing Myc-tagged wild-type (WT) Raptor or a PKA-resistant (Ser791Ala) Raptor mutant. Finally, we measured the body weight response to liraglutide in WT mice and mice with a targeted knock-in of PKA-resistant Ser791Ala Raptor. Liraglutide increased phosphorylation of S6 and the PKA motif in WT Raptor in a PKA-dependent manner but failed to stimulate phosphorylation of the PKA motif in Ser791Ala Raptor in CHO-Glp1r cells. Lean Ser791Ala Raptor knock-in mice were resistant to liraglutide-induced weight loss but not setmelanotide-induced (melanocortin-4 receptor-dependent) weight loss. Diet-induced obese Ser791Ala Raptor knock-in mice were not resistant to liraglutide-induced weight loss; however, there was weight-dependent variation such that there was a tendency for obese Ser791Ala Raptor knock-in mice of lower relative body weight to be resistant to liraglutide-induced weight loss compared to weight-matched controls. Together, these findings suggest that PKA-mediated phosphorylation of Raptor at Ser791 contributes to liraglutide-induced weight loss.

Targeting ryanodine receptor type 2 to mitigate chemotherapy-induced neurocognitive impairments in mice.

Chemotherapy-induced cognitive dysfunction (chemobrain) is an important adverse sequela of chemotherapy. Chemobrain has been identified by the National Cancer Institute as a poorly understood problem for which current management or treatment strategies are limited or ineffective. Here, we show that chemotherapy treatment with doxorubicin (DOX) in a breast cancer mouse model induced protein kinase A (PKA) phosphorylation of the neuronal ryanodine receptor/calcium (Ca2+) channel type 2 (RyR2), RyR2 oxidation, RyR2 nitrosylation, RyR2 calstabin2 depletion, and subsequent RyR2 Ca2+ leakiness. Chemotherapy was furthermore associated with abnormalities in brain glucose metabolism and neurocognitive dysfunction in breast cancer mice. RyR2 leakiness and cognitive dysfunction could be ameliorated by treatment with a small molecule Rycal drug (S107). Chemobrain was also found in noncancer mice treated with DOX or methotrexate and 5-fluorouracil and could be prevented by treatment with S107. Genetic ablation of the RyR2 PKA phosphorylation site (RyR2-S2808A) also prevented the development of chemobrain. Chemotherapy increased brain concentrations of the tumor necrosis factor-α and transforming growth factor-β signaling, suggesting that increased inflammatory signaling might contribute to oxidation-driven biochemical remodeling of RyR2. Proteomics and Gene Ontology analysis indicated that the signaling downstream of chemotherapy-induced leaky RyR2 was linked to the dysregulation of synaptic structure-associated proteins that are involved in neurotransmission. Together, our study points to neuronal Ca2+ dyshomeostasis via leaky RyR2 channels as a potential mechanism contributing to chemobrain, warranting further translational studies.

Liraglutide attenuates palmitate-induced apoptosis via PKA/β-catenin/Bcl-2/Bax pathway in MC3T3-E1 cells.

Liraglutide (LRG), one agonist of glucagon-like peptide-1 receptor (GLP1R), has multiple lipid-lowering effects in type 2 diabetes mellitus, however, studies on the role of LRG in saturated fatty acid-induced bone loss are limited. Therefore, our aim was to investigate whether LRG reduces palmitate (PA)-induced apoptosis and whether the mechanism involves PKA/β-catenin/Bcl-2/Bax in osteoblastic MC3T3-E1 cells. MC3T3-E1 cells were treated with different concentrations of PA, LRG, or pretreated with Exendin 9-39 and H89, cell viability, intracellular reactive oxygen species (ROS), cAMP levels, apoptosis and the expression of protein kinase A (PKA) and phosphorylation of PKA (p-PKA), β-catenin and phosphorylation of β-catenin (Ser675)(p-β-catenin), GLP1R, cleaved-capase 3, Bcl2-Associated X Protein (Bax) and B-cell lymphoma-2 (Bcl-2) along with expression of Osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) were evaluated. PA treatment inhibited cell proliferation and cAMP levels, elevated intracellular ROS levels and promoted apoptosis, increased protein expressions of RANKL, Bax and cleaved-caspase3, meanwhile decreased protein expression of OPG and Bcl-2 in a dose-dependent manner. LRG inverted PA-induced apoptosis, increased cAMP levels, promoted expression of p-PKA, p-β-catenin (Ser675) and reversed these gene expressions via increasing GLP1R expression. Pretreatment of the cells with Exendin 9-39 and H89 partially eradicated the protective effect of LRG on PA-induced apoptosis and gene expressions. Therefore, these findings indicated that LRG attenuates PA-induced apoptosis possibly by GLP1R-mediated PKA/β-catenin/Bcl-2/Bax pathway in MC3T3-E1 cells. Our results point to LRG as a new strategy to attenuate bone loss associated with high fat diet beyond its lipid-lowering actions. LRG inhibits PA-mediated apoptosis via GLP1R-mediated PKA/β-catenin/Bcl-2/ Bax pathway, while possibly enhances PA-inhibited differentiation by regulating the expression of OPG and RANKL.

Salt-inducible kinase inhibition promotes the adipocyte thermogenic program and adipose tissue browning

ObjectiveNorepinephrine stimulates the adipose tissue thermogenic program through a β-adrenergic receptor (βAR)–cyclic adenosine monophosphate (cAMP)–protein kinase A (PKA) signaling cascade. We discovered that a noncanonical activation of the mechanistic target of rapamycin complex 1 (mTORC1) by PKA is required for the βAR-stimulation of adipose tissue browning. However, the downstream events triggered by PKA-phosphorylated mTORC1 activation that drive this thermogenic response are not well understood. MethodsWe used a proteomic approach of Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC) to characterize the global protein phosphorylation profile in brown adipocytes treated with the βAR agonist. We identified salt-inducible kinase 3 (SIK3) as a candidate mTORC1 substrate and further tested the effect of SIK3 deficiency or SIK inhibition on the thermogenic gene expression program in brown adipocytes and in mouse adipose tissue. ResultsSIK3 interacts with RAPTOR, the defining component of the mTORC1 complex, and is phosphorylated at Ser884 in a rapamycin-sensitive manner. Pharmacological SIK inhibition by a pan-SIK inhibitor (HG-9-91-01) in brown adipocytes increases basal Ucp1 gene expression and restores its expression upon blockade of either mTORC1 or PKA. Short-hairpin RNA (shRNA) knockdown of Sik3 augments, while overexpression of SIK3 suppresses, Ucp1 gene expression in brown adipocytes. The regulatory PKA phosphorylation domain of SIK3 is essential for its inhibition. CRISPR-mediated Sik3 deletion in brown adipocytes increases type IIa histone deacetylase (HDAC) activity and enhances the expression of genes involved in thermogenesis such as Ucp1, Pgc1α, and mitochondrial OXPHOS complex protein. We further show that HDAC4 interacts with PGC1α after βAR stimulation and reduces lysine acetylation in PGC1α. Finally, a SIK inhibitor well-tolerated in vivo (YKL-05-099) can stimulate the expression of thermogenesis-related genes and browning of mouse subcutaneous adipose tissue. ConclusionsTaken together, our data reveal that SIK3, with the possible contribution of other SIKs, functions as a phosphorylation switch for β-adrenergic activation to drive the adipose tissue thermogenic program and indicates that more work to understand the role of the SIKs is warranted. Our findings also suggest that maneuvers targeting SIKs could be beneficial for obesity and related cardiometabolic disease.