Search Results (21)

Oil-Gan is the fruit of the genus Phyllanthus emblica L. The fruits have excellent effects on health care and development values. There are many methods for the management of diabetic nephropathy (DN). However, there is a lack of effective drugs for treating DN throughout the disease course. The primary aim of this study was to examine the protective effects (including analyses of urine and blood, and inflammatory cytokine levels) and mechanisms of the ethyl acetate extract of P. emblica (EPE) on db/db mice, an animal model of diabetic nephropathy; the secondary aim was to examine the expression levels of p- protein kinase Cα (PKCα)/t-PKCα in the kidney and its downregulation of vascular endothelial growth factor (VEGF) and fibrosis gene transforming growth factor-β1 (TGF-β1) by Western blot analyses. Eight db/m mice were used as the control group. Forty db/db mice were randomly divided into five groups. Treatments included a vehicle, EPE1, EPE2, EPE3 (at doses of 100, 200, or 400 mg/kg EPE), or the comparative drug aminoguanidine for 8 weeks. After 8 weeks of treatment, the administration of EPE to db/db mice effectively controlled hyperglycemia and hyperinsulinemia by markedly lowering blood glucose, insulin, and glycosylated HbA1c levels. The administration of EPE to db/db mice decreased the levels of BUN and creatinine both in blood and urine and reduced urinary albumin excretion and the albumin creatine ratio (UACR) in urine. Moreover, EPE treatment decreased the blood levels of inflammatory cytokines, including kidney injury molecule-1 (KIM-1), C-reactive protein (CRP), and NLR family pyrin domain containing 3 (NLRP3). Our findings showed that EPE not only had antihyperglycemic effects but also improved renal function in db/db mice. A histological examination of the kidney by immunohistochemistry indicated that EPE can improve kidney function by ameliorating glomerular morphological damage following glomerular injury; alleviating proteinuria by upregulating the expression of nephrin, a biomarker of early glomerular damage; and inhibiting glomerular expansion and tubular fibrosis. Moreover, the administration of EPE to db/db mice increased the expression levels of p- PKCα/t-PKCα but decreased the expression levels of VEGF and renal fibrosis biomarkers (TGF-β1, collagen IV, p-Smad2, p-Smad3, and Smad4), as shown by Western blot analyses. These results implied that EPE as a supplement has a protective effect against renal dysfunction through the amelioration of insulin resistance as well as the suppression of nephritis and fibrosis in a DN model. Full article

The use of manufactured silica nanoparticles (SiNPs) has become widespread in everyday life, household products, and various industrial applications. While the harmful effects of crystalline silica on the lungs, known as silicosis or chronic pulmonary diseases, are well understood, the impact of SiNPs on the airway is not fully explored. This study aimed to investigate the potential effects of SiNPs on human tracheal smooth muscle cells (HTSMCs). Our findings revealed that SiNPs induced the expression of cyclooxygenase-2 (COX-2) mRNA/protein and the production of prostaglandin E₂ (PGE₂) without causing cytotoxicity. This induction was transcription-dependent, as confirmed by cell viability assays and COX-2 luciferase reporter assays. Further analysis, including Western blot with pharmacological inhibitors and siRNA interference, showed the involvement of receptor tyrosine kinase (RTK) EGF receptor (EGFR), non-RTK Pyk2, protein kinase Cα (PKCα), and p42/p44 MAPK in the induction process. Notably, EGFR activation initiated cellular signaling that led to NF-κB p65 phosphorylation and translocation into the cell nucleus, where it bound and stimulated COX-2 gene transcription. The resulting COX-2 protein triggered PGE₂ production and secretion into the extracellular space. Our study demonstrated that SiNPs mediate COX-2 up-regulation and PGE₂ secretion in HTSMCs through the sequential activation of the EGFR/Pyk2/PKCα/p42/p44MAPKs-dependent NF-κB signaling pathway. Since PGE₂ can have both physiological bronchodilatory and anti-inflammatory effects, as well as pathological pro-inflammatory effects, the increased PGE₂ production in the airway might act as a protective compensatory mechanism and/or a contributing factor during airway exposure to SiNPs. Full article

Syndecan-4 (SDC4) consists of transmembrane heparan sulfate proteoglycan (HSPG) belonging to the syndecan family. It is present in most cell types of Mammalia. Its structure contains a heparan-sulfate-modified extracellular domain, a single transmembrane domain, and a short C-terminal cytoplasmic domain. Regarding the overall cellular function of SDC4, other cells or ligands can bind to its ecto-domain. In addition, 4,5-bisphosphate phosphatidylinositol (PIP₂) or protein kinase Cα can bind to its cyto-domain to activate downstream signaling pathways. To understand the signal transduction mechanism of syndecan, it is important to know the interactions between their actual structure and function in vivo. Therefore, it is important to identify the structure of SDC4 to understand the ligand binding behavior of SDC4. In this study, expression and purification were performed to reveal structures of the short ecto-domain, the transmembrane domain, and the cytoplasmic domain of Syd4-eTC (SDC4). Solution-state NMR spectroscopy and solid-state NMR spectroscopy were used to study the structure of Syd4-eTC in membrane environments and to demonstrate the interaction between Syd4-eTC and PIP₂. Full article

Platelet activation during hemostasis and thrombosis is facilitated by agonist-induced inside–out and integrin α_IIbβ₃-initiated outside–in signaling via protein kinases and phosphatases. Pharmacological inhibitor studies suggest that the serine/threonine protein phosphatase 1 (PP1) promotes platelet activation. However, since phosphatase inhibitors block all the isoforms of the catalytic subunit of PP1 (PP1c), the role of specific PP1c isoform in platelet signaling remains unclear. Here, we employed a platelet-specific PP1cα^−/− mice to explore the contribution of a major PP1 isoform in platelet functions. Loss of PP1cα moderately decreased activation of integrin α_IIbβ₃, binding of soluble fibrinogen, and aggregation to low-dose thrombin, ADP, and collagen. In contrast, PP1cα^−/− platelets displayed increased adhesion to immobilized fibrinogen, fibrin clot retraction, and thrombus formation on immobilized collagen. Mechanistically, post-fibrinogen engagement potentiated p38 mitogen-activated protein kinase (MAPK) activation in PP1cα^−/− platelets and the p38 inhibitor blocked the increased integrin-mediated outside–in signaling function. Tail bleeding time and light-dye injury-induced microvascular thrombosis in the cremaster venules and arterioles were not altered in PP1cα^−/− mice. Thus, PP1cα displays pleiotropic signaling in platelets as it amplifies agonist-induced signaling and attenuates integrin-mediated signaling with no impact on hemostasis and thrombosis. Full article

The cyclic AMP-dependent protein kinase (PKA) plays an essential role in the regulation of many important cellular processes and is dysregulated in several pervasive diseases, including diabetes, cardiovascular disease, and various neurodegenerative disorders. Previous studies suggest that the alpha isoform of the catalytic subunit of PKA (PKA-Cα) is oxidized on C199, both in vitro and in situ. However, the molecular consequences of these modifications on PKA-Cα’s substrate selection remain largely unexplored. C199 is located on the P + 1 loop within PKA-Cα’s active site, suggesting that redox modification may affect its kinase activity. Given the proximity of C199 to the substrate binding pocket, we hypothesized that oxidation could differentially alter PKA-Cα’s activity toward its substrates. To this end, we examined the effects of diamide- and H₂O₂-dependent oxidation on PKA-Cα’s activity toward select peptide and protein substrates using a combination of biochemical (i.e., trans-phosphorylation assays and steady-state kinetics analysis) and biophysical (i.e., surface plasmon resonance and fluorescence polarization assays) strategies. These studies suggest that redox modification of PKA-Cα differentially affects its activity toward different substrates. For instance, we found that diamide-mediated oxidation caused a marked decrease in PKA-Cα’s activity toward some substrates (e.g., Kemptide and CREBtide) while having little effect on others (e.g., Crosstide). In contrast, H₂O₂-dependent oxidation of PKA-Cα led to an increase in its activity toward each of the substrates at relatively low H₂O₂ concentrations, with differential effects at higher peroxide concentrations. Together, these studies offer novel insights into crosstalk between redox- and phosphorylation-dependent signaling pathways mediated by PKA. Likewise, since C199 is highly conserved among AGC kinase family members, they also lay the foundation for future studies designed to elucidate the role of redox-dependent modification of kinase substrate selection in physiological and pathological states. Full article

Protein kinase Cα (PKCα/PRKCA) is a crucial regulator of circadian rhythm and is associated with human mental illnesses such as autism spectrum disorders and schizophrenia. However, the roles of PRKCA in modulating animal social behavior and the underlying mechanisms remain to be explored. Here we report the generation and characterization of prkcaa-deficient zebrafish (Danio rerio). The results of behavioral tests indicate that a deficiency in Prkcaa led to anxiety-like behavior and impaired social preference in zebrafish. RNA-sequencing analyses revealed the significant effects of the prkcaa mutation on the expression of the morning-preferring circadian genes. The representatives are the immediate early genes, including egr2a, egr4, fosaa, fosab and npas4a. The downregulation of these genes at night was attenuated by Prkcaa dysfunction. Consistently, the mutants demonstrated reversed day–night locomotor rhythm, which are more active at night than in the morning. Our data show the roles of PRKCA in regulating animal social interactions and link the social behavior defects with a disturbed circadian rhythm. Full article

Dermal papilla cells (DPCs) are growth factor reservoirs that are specialized for hair morphogenesis and regeneration. Due to their essential role in hair growth, DPCs are commonly used as an in vitro model to investigate the effects of hair growth-regulating compounds and their molecular mechanisms of action. Cyclic adenosine monophosphate (cAMP), an intracellular second messenger, is currently employed as a growth-promoting target molecule. In a pilot test, we found that α-phellandrene, a naturally occurring phytochemical, increased cAMP levels in DPCs. Therefore, we sought to determine whether α-phellandrene increases growth factors and proliferation in human DPCs and to identify the underlying mechanisms. We demonstrated that α-phellandrene promotes cell proliferation concentration-dependently. In addition, it increases the cAMP downstream effectors, such as protein kinase A catalytic subunit (PKA Cα) and phosphorylated cAMP-responsive element-binding protein (CREB). Also, among the CREB-dependent growth factor candidates, we identified that α-phellandrene selectively upregulated vascular endothelial growth factor (VEGF) mRNA expression in DPCs. Notably, the beneficial effects of α-phellandrene were nullified by a cAMP inhibitor. This study demonstrated the cAMP-mediated growth effects in DPCs and the therapeutic potential of α-phellandrene for preventing hair loss. Full article

cAMP-dependent protein kinase (PKA) signaling plays various roles during mammalian spermatogenesis, ranging from the regulation of gene expression to the modulation of sperm motility. However, the molecular mechanisms that govern the multifaceted functions of PKA during spermatogenesis remain largely unclear. We previously found that PKA regulatory subunit I α (RIα) and catalytic subunit α (Cα) co-sediment with polyribosomal fractions of mouse testis lysate on sucrose gradient and the stimulation of PKA activity facilitates protein synthesis in post-meiotic elongating spermatids, indicating that type I PKA is intricately associated with protein translation machinery and regulates protein synthesis during mouse spermiogenesis. Since PKA activity is often regulated by interacting proteins that form complexes with its regulatory subunits, the identification of PKA-RIα interacting proteins in post-meiotic spermatogenic cells will facilitate our understanding of its regulatory roles in protein synthesis and spermiogenesis. In the present study, we applied a yeast two-hybrid screen to identify PKA-Riα-binding proteins using a cDNA library generated from mouse round and elongating spermatids. Numerous proteins were found to potentially interact with PKA-RIα, including proteostasis modulators, metabolic enzymes, cytoskeletal regulators, and mitochondrial proteins, many of which are specifically expressed in testes. Consistently, the examination of MENA (mouse ENA/VASP homolog) in developing mouse testes suggested that post-meiotic spermatogenic cells express a short isoform of MENA that interacts with PKA-RIα in yeast two-hybrid assay. The identification of PKA-RIα interacting proteins provides us solid basis to further explore how PKA signaling regulates protein synthesis and cellular morphogenesis during mouse spermatogenesis. Full article

The pyrazolyl-urea Gege3 molecule has shown interesting antiangiogenic effects in the tumor contest. Here, we have studied the role of this compound as interfering with endothelial cells activation in response to the paracrine effects of annexin A1 (ANXA1), known to be involved in promoting tumor progression. ANXA1 has been analyzed in the extracellular environment once secreted through microvesicles (EVs) by pancreatic cancer (PC) cells. Particularly, Gege3 has been able to notably prevent the effects of Ac2-26, the ANXA1 mimetic peptide, and of PC-derived EVs on endothelial cells motility, angiogenesis, and calcium release. Furthermore, this compound also inhibited the translocation of ANXA1 to the plasma membrane, otherwise induced by the same ANXA1-dependent extracellular stimuli. Moreover, these effects have been mediated by the indirect inhibition of protein kinase Cα (PKCα), which generally promotes the phosphorylation of ANXA1 on serine 27. Indeed, by the subtraction of intracellular calcium levels, the pathway triggered by PKCα underwent a strong inhibition leading to the following impediment to the ANXA1 localization at the plasma membrane, as revealed by confocal and cytofluorimetry analysis. Thus, Gege3 appeared an attractive molecule able to prevent the paracrine effects of PC cells deriving ANXA1 in the tumor microenvironment. Full article

Bladder cancer has a high recurrence rate; therefore, frequent and effective monitoring is essential for disease management. Cystoscopy is considered the gold standard for the diagnosis and continuous monitoring of bladder cancer. However, cystoscopy is invasive and relatively expensive. Thus, there is a need for non-invasive, relatively inexpensive urinary biomarker-based diagnoses of bladder cancer. This study aimed to investigate the presence of activated protein kinase Cα (PKCα) in urine samples and the possibility of PKCα as a urinary biomarker for bladder cancer diagnosis. Activated PKCα was found to be present at higher levels in bladder cancer tissues than in normal bladder tissues. Furthermore, high levels of activated PKCα were observed in urine samples collected from orthotopic xenograft mice carrying human bladder cancer cells compared to urine samples from normal mice. These results suggest that activated PKCα can be used as a urinary biomarker to diagnose bladder cancer. To the best of our knowledge, this is the first report describing the presence of activated PKCα in the urine of orthotopic xenograft mice. Full article

Cancer stem cells (CSCs) are a limited cell population inside a tumor bulk characterized by high levels of glutathione (GSH), the most important antioxidant thiol of which cysteine is the limiting amino acid for GSH biosynthesis. In fact, CSCs over-express xCT, a cystine transporter stabilized on cell membrane through interaction with CD44, a stemness marker whose expression is modulated by protein kinase Cα (PKCα). Since many chemotherapeutic drugs, such as Etoposide, exert their cytotoxic action by increasing reactive oxygen species (ROS) production, the presence of high antioxidant defenses confers to CSCs a crucial role in chemoresistance. In this study, Etoposide-sensitive and -resistant neuroblastoma CSCs were chronically treated with Etoposide, given alone or in combination with Sulfasalazine (SSZ) or with an inhibitor of PKCα (C2-4), which target xCT directly or indirectly, respectively. Both combined approaches are able to sensitize CSCs to Etoposide by decreasing intracellular GSH levels, inducing a metabolic switch from OXPHOS to aerobic glycolysis, down-regulating glutathione-peroxidase-4 activity and stimulating lipid peroxidation, thus leading to ferroptosis. Our results suggest, for the first time, that PKCα inhibition inducing ferroptosis might be a useful strategy with which to fight CSC chemoresistance. Full article

Coffea arabica pulp (CP) is a by-product of coffee processing. CP contains polyphenols that have exhibited beneficial effects, including antioxidant and lipid-lowering effects, as well as enhanced insulin sensitivity, in in vitro and in vivo models. How polyphenols, as found in CP aqueous extract (CPE), affect type 2 diabetes (T2D) has not been investigated. Thus, the present study examined the potential antidiabetic, antioxidant, and renoprotective effects of CPE-rich polyphenols, using an experimental model of T2D in rats induced by a high-fat diet and a single low dose of streptozotocin. The T2D rats received either 1000 mg/kg body weight (BW) of CPE, 30 mg/kg BW of metformin (Met), or a combination treatment (CPE + Met) for 3 months. Plasma parameters, kidney morphology and function, and renal organic transport were determined. Significant hyperglycemia, hypertriglyceridemia, insulin resistance, increased renal lipid content and lipid peroxidation, and morphological kidney changes related to T2D were restored by both CPE and CPE + Met treatments. Additionally, the renal uptake of organic cation, ³H-1-methyl-4-phenylpyridinium (MPP⁺), was reduced in T2D, while transport was restored by CPE and CPE + Met, through an up-regulation of antioxidant genes and protein kinase Cα deactivation. Thus, CPE has antidiabetic and antioxidant effects that potentially ameliorate kidney function in T2D by preserving renal organic cation transport through an oxidative stress pathway. Full article

(1) Background: It is known that sickle cells contain a higher amount of Ca²⁺ compared to healthy red blood cells (RBCs). The increased Ca²⁺ is associated with the most severe symptom of sickle cell disease (SCD), the vaso-occlusive crisis (VOC). The Ca²⁺ entry pathway received the name of P_sickle but its molecular identity remains only partly resolved. We aimed to map the involved Ca²⁺ signaling to provide putative pharmacological targets for treatment. (2) Methods: The main technique applied was Ca²⁺ imaging of RBCs from healthy donors, SCD patients and a number of transgenic mouse models in comparison to wild-type mice. Life-cell Ca²⁺ imaging was applied to monitor responses to pharmacological targeting of the elements of signaling cascades. Infection as a trigger of VOC was imitated by stimulation of RBCs with lysophosphatidic acid (LPA). These measurements were complemented with biochemical assays. (3) Results: Ca²⁺ entry into SCD RBCs in response to LPA stimulation exceeded that of healthy donors. LPA receptor 4 levels were increased in SCD RBCs. Their activation was followed by the activation of G_i protein, which in turn triggered opening of TRPC6 and Ca_V2.1 channels via a protein kinase Cα and a MAP kinase pathway, respectively. (4) Conclusions: We found a new Ca²⁺ signaling cascade that is increased in SCD patients and identified new pharmacological targets that might be promising in addressing the most severe symptom of SCD, the VOC. Full article

Recently, Toll-like receptors (TLRs), a family of pattern recognition receptors, are reported as potential modulators for neuropathic pain; however, the desired mechanism is still unexplained. Here, we operated on the sciatic nerve to establish a pre-clinical rodent model of chronic constriction injury (CCI) in Sprague-Dawley rats, which were assigned into CCI and Decompression groups randomly. In Decompression group, the rats were performed with nerve decompression at post-operative week 4. Mechanical hyperalgesia and mechanical allodynia were obviously attenuated after a month. Toll-like receptor 5 (TLR5)-immunoreactive (ir) expression increased in dorsal horn, particularly in the inner part of lamina II. Additionally, substance P (SP) and isolectin B4 (IB4)-ir expressions, rather than calcitonin-gene-related peptide (CGRP)-ir expression, increased in their distinct laminae. Double immunofluorescence proved that increased TLR5-ir expression was co-expressed mainly with IB4-ir expression. Through an intrathecal administration with FLA-ST Ultrapure (a TLR5 agonist, purified flagellin from Salmonella Typhimurium, only the CCI-induced mechanical hyperalgesia was attenuated dose-dependently. Moreover, we confirmed that mu-opioid receptor (MOR) and phospho-protein kinase Cα (pPKCα)-ir expressions but not phospho-protein kinase A RII (pPKA RII)-ir expression, increased in lamina II, where they mostly co-expressed with IB4-ir expression. Go 6976, a potent protein kinase C inhibitor, effectively reversed the FLA-ST Ultrapure- or DAMGO-mediated attenuated trend towards mechanical hyperalgesia by an intrathecal administration in CCI rats. In summary, our current findings suggest that nerve decompression improves CCI-induced mechanical hyperalgesia that might be through the cross-talk of TLR5 and MOR in a PKCα-dependent manner, which opens a novel opportunity for the development of analgesic therapeutics in neuropathic pain. Full article

cAMP-dependent protein kinase (PKA) is the major receptor of the second messenger cAMP and a prototype for Ser/Thr-specific protein kinases. Although PKA strongly prefers serine over threonine substrates, little is known about the molecular basis of this substrate specificity. We employ classical enzyme kinetics and a surface plasmon resonance (SPR)-based method to analyze each step of the kinase reaction. In the absence of divalent metal ions and nucleotides, PKA binds serine (PKS) and threonine (PKT) substrates, derived from the heat-stable protein kinase inhibitor (PKI), with similar affinities. However, in the presence of metal ions and adenine nucleotides, the Michaelis complex for PKT is unstable. PKA phosphorylates PKT with a higher turnover due to a faster dissociation of the product complex. Thus, threonine substrates are not necessarily poor substrates of PKA. Mutation of the DFG+1 phenylalanine to β-branched amino acids increases the catalytic efficiency of PKA for a threonine peptide substrate up to 200-fold. The PKA Cα mutant F187V forms a stable Michaelis complex with PKT and shows no preference for serine versus threonine substrates. Disease-associated mutations of the DFG+1 position in other protein kinases underline the importance of substrate specificity for keeping signaling pathways segregated and precisely regulated. Full article