RESUMEN
Immune-mediated inflammatory diseases (IMIDs) comprise a broad spectrum of conditions characterized by systemic inflammation affecting various organs and tissues, for which there is no known cure. The isoform-specific inhibition of phosphodiesterase-4B (PDE4B) over PDE4D constitutes an effective therapeutic strategy for the treatment of IMIDs that minimizes the adverse effects associated with non-selective PDE4 inhibitors. Thus, we report a new class of isoquinolone derivatives as next-generation PDE4 inhibitors for effective management of rheumatoid arthritis (RA) and psoriasis. Among the series, 8 compounds i.e. 1e, 1l, 1m, 1n, 1o, 2m, 2o and 3o showed promising PDE4B inhibition (>80â¯%) in vitro with IC50â¯â¼â¯1.4-6.2⯵M. The compound 1l was identified as an initial hit and was pursued for further studies. According to structure-activity relationship (SAR), an allyl group at C-4 position improved PDE4B inhibition. The correlation between in vitro activity data and binding affinities obtained via molecular docking suggested that the high-affinity binding to PDE4B is a prerequisite for the effective inhibition of PDE4B. Notably, the hit 1l showed selectivity towards PDE4B over PDE4D in vitro. Furthermore, 1l treatment (30â¯mg/kg) in the adjuvant-induced arthritis (AIA) rat model induced by complete Freund's adjuvant (CFA) demonstrated anti-arthritic potential via ameliorating paw swelling and body weight, narrowing joint space, reducing excessive immune cells infiltration and pannus formation in addition to reducing mRNA expression of pro-inflammatory cytokines such as TNF-α and IL-6 in synovial tissues of experimental rats. Additionally, 1l reduced the hyper-proliferative state and colony forming potential of IMQ-induced psoriatic keratinocytes. The treatment of these cells with 1l markedly reduced the protein levels of Ki67 and mRNA levels of pro-inflammatory cytokines e.g. IL-17A and TNF-α suggesting its potent anti-psoriatic potential. Furthermore, 1l did not show any significant adverse effects when evaluated in a systematic toxicity (e.g. teratogenicity, hepatotoxicity and cardiotoxicity) studies in zebrafish at the tested concentrations (1-100⯵M) and the NOAEL (no-observed-adverse-effect level) was found to be 100⯵M. Thus, with promising anti-inflammatory effects both in vitro and in vivo along with PDE4B selectivity with an acceptable safety margin, 1l emerged as a new and promising inhibitor for further studies.
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Pancreatic beta cell function is an important component of glucose homeostasis. Here, we investigated the function of PIMT (PRIP-interacting protein with methyl transferase domain), a transcriptional co-activator binding protein, in the pancreatic beta cells. We observed that the protein levels of PIMT, along with key beta cell markers such as PDX1 (pancreatic and duodenal homeobox 1) and MafA (MAF bZIP transcription factor A), were reduced in the beta cells exposed to hyperglycemic and hyperlipidemic conditions. Consistently, PIMT levels were reduced in the pancreatic islets isolated from high fat diet (HFD)-fed mice. The RNA sequencing analysis of PIMT knockdown beta cells identified that the expression of key genes involved in insulin secretory pathway, Ins1 (insulin 1), Ins2 (insulin 2), Kcnj11 (potassium inwardly-rectifying channel, subfamily J, member 11), Kcnn1 (potassium calcium-activated channel subfamily N member 1), Rab3a (member RAS oncogene family), Gnas (GNAS complex locus), Syt13 (synaptotagmin 13), Pax6 (paired box 6), Klf11 (Kruppel-Like Factor 11), and Nr4a1 (nuclear receptor subfamily 4, group A, member 1) was attenuated due to PIMT depletion. PIMT ablation in the pancreatic beta cells and in the rat pancreatic islets led to decreased protein levels of PDX1 and MafA, resulting in the reduction in glucose-stimulated insulin secretion (GSIS). The results from the immunoprecipitation and ChIP experiments revealed the interaction of PIMT with PDX1 and MafA, and its recruitment to the insulin promoter, respectively. Importantly, PIMT ablation in beta cells resulted in the nuclear translocation of insulin. Surprisingly, forced expression of PIMT in beta cells abrogated GSIS, while Ins1 and Ins2 transcript levels were subtly enhanced. On the other hand, the expression of genes, PRIP/Asc2/Ncoa6 (nuclear receptor coactivator 6), Pax6, Kcnj11, Syt13, Stxbp1 (syntaxin binding protein 1), and Snap25 (synaptosome associated protein 25) associated with insulin secretion, was significantly reduced, providing an explanation for the decreased GSIS upon PIMT overexpression. Our findings highlight the importance of PIMT in the regulation of insulin synthesis and secretion in beta cells.
Asunto(s)
Células Secretoras de Insulina , Insulina , Animales , Ratones , Ratas , Genes Homeobox , Glucosa/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Insulina/metabolismo , Insulina Regular Humana , Células Secretoras de Insulina/metabolismo , Potasio/metabolismo , Transactivadores/metabolismo , HistonasRESUMEN
Adaptability to intracellular or extracellular cues is essential for maintaining cellular homeostasis. Metabolic signals intricately control the morphology and functions of mitochondria by regulating bioenergetics and metabolism. Here, we describe the involvement of PHLPP1, a Ser/Thr phosphatase, in mitochondrial homeostasis. Microscopic analysis showed the enhanced globular structure of mitochondria in PHLPP1-depleted HEK 293T and C2C12 cells, while forced expression of PHLPP1 promoted mitochondrial tubularity. We show that PHLPP1 promoted pro-fusion markers MFN2 and p-DRP1Ser637 levels using over-expression and knockdown strategies. Contrastingly, PHLPP1 induced mitochondrial fragmentation by augmenting pro-fission markers, t-DRP1 and pDrp1Ser616 upon mitochondrial stress. At the molecular level, PHLPP1 interacted with and caused dephosphorylation of calcineurin, a p-DRP1Ser637 phosphatase, under basal conditions. Likewise, PHLPP1 dimerized with PINK1 under basal conditions. However, the interaction of PHLPP1 with both calcineurin and PINK1 was impaired upon CCCP and oligomycin-induced mitochondrial stress. Interestingly, upon mitochondrial membrane depolarization, PHLPP1 promoted PINK1 stabilization and parkin recruitment to mitochondria, and thereby activated the mitophagy machinery providing a molecular explanation for the dual effects of PHLPP1 on mitochondria under different conditions. Consistent with our in-vitro findings, depletion of phlp-2, ortholog of PHLPP1 in C. elegans, led to mitochondrial fission under basal conditions, extended the lifespan of the worms, and enhanced survival of worms subjected to paraquat-induced oxidative stress.
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Longevidad , Proteínas Quinasas , Animales , Caenorhabditis elegans/metabolismo , Calcineurina , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Células HEK293 , Humanos , RatonesRESUMEN
The physiological and metabolic functions of PIMT/TGS1, a third-generation transcriptional apparatus protein, in glucose homeostasis sustenance are unclear. Here, we observed that the expression of PIMT was upregulated in the livers of short-term fasted and obese mice. Lentiviruses expressing Tgs1-specific shRNA or cDNA were injected into wild-type mice. Gene expression, hepatic glucose output, glucose tolerance, and insulin sensitivity were evaluated in mice and primary hepatocytes. Genetic modulation of PIMT exerted a direct positive impact on the gluconeogenic gene expression program and hepatic glucose output. Molecular studies utilizing cultured cells, in vivo models, genetic manipulation, and PKA pharmacological inhibition establish that PKA regulates PIMT at post-transcriptional/translational and post-translational levels. PKA enhanced 3'UTR-mediated translation of TGS1 mRNA and phosphorylated PIMT at Ser656, increasing Ep300-mediated gluconeogenic transcriptional activity. The PKA-PIMT-Ep300 signaling module and associated PIMT regulation may serve as a key driver of gluconeogenesis, positioning PIMT as a critical hepatic glucose sensor.
RESUMEN
Site-directed mutagenesis is an invaluable technique that enables the elucidation of the contribution of specific residues to protein structure and function. The simultaneous introduction of mutations at a large number of sites (>10), singly and in multiple combinations, is often necessary to fully understand the functional contributions. We report a simple, efficient, time and cost-effective method to achieve this using commonly available molecular biology reagents and protocols, as an alternative to gene synthesis. We demonstrate this method using the Omicron Spike DNA construct as an example, and create a construct bearing 37 mutations (as compared to wild-type Spike DNA), as well as 4 other constructs bearing subsets of the full spectrum of mutations. We believe that this method can be an excellent alternative to gene synthesis, especially when three or more variants are required.
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In this report, we describe an approach to generate a zebrafish larval model of lipid accumulation that can be used as an in vivo system to study hyperlipidemic conditions such as atherosclerosis. Furthermore, we detail steps on staining techniques, lipid estimation assays, RNA isolation, and utilization of ImageJ to evaluate larval dimensions and to explore the model in the context of hyperlipidemia. Researchers should be aware of context specificity of the proposed protocols and interpret results accordingly. For complete details on the use and execution of this protocol, please refer to Balamurugan et al., (2022).
Asunto(s)
Hipercolesterolemia , Hiperlipidemias , Animales , Pez Cebra , Larva , Hiperlipidemias/diagnóstico , Hipercolesterolemia/genética , LípidosRESUMEN
That reversible protein phosphorylation by kinases and phosphatases occurs in metabolic disorders is well known. Various studies have revealed that a multi-faceted and tightly regulated phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP)-1/2 displays robust effects in cardioprotection, ischaemia/reperfusion (I/R), and vascular remodelling. PHLPP1 promotes foamy macrophage development through ChREBP/AMPK-dependent pathways. Adipocyte-specific loss of PHLPP2 reduces adiposity, improves glucose tolerance,and attenuates fatty liver via the PHLPP2-HSL-PPARα axis. Discoveries of PHLPP1-mediated insulin resistance and pancreatic ß cell death via the PHLPP1/2-Mst1-mTORC1 triangular loop have shed light on its significance in diabetology. PHLPP1 downregulation attenuates diabetic cardiomyopathy (DCM) by restoring PI3K-Akt-mTOR signalling. In this review, we summarise the functional role of, and cellular signalling mediated by, PHLPPs in metabolic tissues and discuss their potential as therapeutic targets.
Asunto(s)
Resistencia a la Insulina , Fosfoproteínas Fosfatasas , Proteínas Quinasas Activadas por AMP , Glucosa , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Proteínas Nucleares/metabolismo , PPAR alfa , Fosfatidilinositol 3-Quinasas , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TORRESUMEN
Infiltration of arterial intima by foamy macrophages is a hallmark of early atherosclerotic lesions. Here, we investigated the potential role of Ser/Thr phosphatase PHLPP1 in foam cell development. PHLPP1 levels were elevated in OxLDL-exposed macrophages and high-fat diet (HFD)-fed zebrafish larvae. Using overexpression and knockdown approaches, we show that PHLPP1 promotes the accumulation of neutral lipids, and augments cellular total cholesterol and free fatty acid (FFA) levels. RNA-Seq analysis uncovered PHLPP1 role in lipid metabolism pathways. PHLPP1 interacted with and modestly increased ChREBP recruitment to Fasn promoter. PHLPP1-mediated lipid accumulation was attenuated by AMPK activation. Pharmacological inhibition or CRISPR/Cas9-mediated disruption of PHLPP1 resulted in lower lipid accumulation in the intersegmental vessels of HFD-fed zebrafish larvae along with a reduction in total cholesterol and triglyceride levels. Deficiency of phlp-2, C. elegans PHLPP1/2 ortholog, abolished lipid accumulation in high cholesterol-fed worms. We conclude that PHLPP1 exerts a significant effect on lipid buildup.
RESUMEN
In search of potent and new anti-inflammatory agents, we explored a new class of isocoumarin derivatives possessing the 3-oxoalkyl moiety at C-4 position. These compounds were synthesized via the FeCl3 catalyzed construction of isocoumarin ring. The methodology involved coupling of 2-alkynyl benzamides with alkyl vinyl ketone and proceeded via a regioselective cyclization to give the desired compound as a result of formation of CO and CC bonds. A large number of isocoumarins were synthesized and assessed against PDE4B in vitro. While isocoumarins containing an aminosulfonyl moiety attached to the C-3 aryl ring showed encouraging inhibition of PDE4B, some of the derivatives devoid of aminosulfonyl moiety also showed considerable inhibition. According to the SAR analysis the C6H4NHSO2R2-m moiety at C-3 position of the isocoumarin ring was favorable when the R2 was chosen as an aryl or 2-thienyl group whereas the presence of F or OMe substituent at C-7 of the isocoumarin ring was found to be beneficial. The compound 5f with IC50 values 0.125 ± 0.032 and 0.43 ± 0.013 µM against PDE4B and 4D, respectively was identified as an initial hit. It showed in silico interaction with the PHE678 residue in the CR3 region of PDE4B and relatively less number of interactions with PDE4D. Besides showing the PDE4 selectivity over other PDEs and TNF-α inhibition in vitro the compound 5f at an intraperitoneal dose of 30 mg/kg demonstrated the protective effects against the development of arthritis and potent immunomodulatory activity in adjuvant induced arthritic (AIA) rats. Furthermore, no significant adverse effects were observed for this compound when evaluated in a systematic toxicity (e.g. teratogenicity, hepatotoxicity and cardiotoxicity) studies in zebrafish at various concentrations. Collectively, being a new, potent, moderately selective and safe inhibitor of PDE4B the isocoumarin 5f can be progressed into further pharmacological studies.
Asunto(s)
Compuestos Férricos , Isocumarinas , Animales , Catálisis , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/química , Isocumarinas/química , Ratas , Relación Estructura-Actividad , Pez Cebra/metabolismoRESUMEN
A Pd-catalysed regioselective synthesis of 4,5-disubstituted 7-membered N/O-heterocycles was achieved via the 7-endo-dig cyclization followed by C-C bond formation of 2-(1-alkynyl)phenylacetamide. The ligand/additive free cascade reaction proceeded in the presence of PdCl2 in aqueous MeCN when the separate and individual use of methyl vinyl ketone and allyl bromide generally afforded an O- and N-heterocycle, respectively. The pharmacological assay was performed to identify the first example of a 1H-benzo[d]azepin-2(3H)-one based novel inhibitor of PDE4B.
RESUMEN
While anti-inflammatory properties of isocoumarins are known their PDE4 inhibitory potential was not explored previously. In our effort the non-PDE4 inhibitor isocoumarins were transformed into the promising inhibitors via introducing an aminosulfonyl/aminocarboxamide moiety to the C-3 benzene ring attached to the isocoumarin framework. This new class of isocoumarins were synthesized via a PdCl2-catalyzed construction of the 4-allyl substituted 3-aryl isocoumarin ring starting from the appropriate 2-alkynyl benzamide derivative. Several compounds showed good inhibition of PDE4B in vitro and the SAR indicated superiority of aminosulfonamide moiety over aminocarboxamide in terms of PDE4B inhibition. Two compounds 3q and 3u with PDE4B IC50 = 0.43 ± 0.11 and 0.54 ± 0.19 µM and ≥ 2-fold selectivity over PDE4D emerged as initial hits. The participation of aminosulfonamide moiety in PDE4B inhibition and the reason for selectivity though moderate shown by 3q and 3u was revealed by the in silico docking studies. In view of potential usefulness of moderately selective PDE4B inhibitors the compound 3u (that showed PDE4 selectivity over other PDEs) was further evaluated in adjuvant induced arthritic rats. At an intraperitoneal dose of 30 mg/kg the compound showed a significant reduction in paw swelling (in a dose dependent manner), inflammation and pannus formation (in the knee joints) as well as pro-inflammatory gene expression/mRNA levels and increase in body weight. Moreover, besides its TNF-α inhibition and no significant toxicity in an MTT assay the compound did not show any adverse effects in a thorough toxicity studies e.g. teratogenicity, hepatotoxicity, cardiotoxicity and apoptosis in zebrafish. Thus, the isocoumarin 3u emerged as a new, safe and moderately selective PDE4B inhibitor could be useful for inflammatory diseases possibly including COVID-19.
Asunto(s)
Antiinflamatorios/uso terapéutico , Artritis Experimental/tratamiento farmacológico , Isocumarinas/uso terapéutico , Inhibidores de Fosfodiesterasa 4/uso terapéutico , Sulfonamidas/uso terapéutico , Animales , Antiinflamatorios/síntesis química , Antiinflamatorios/metabolismo , Antiinflamatorios/toxicidad , Artritis Experimental/patología , Catálisis , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Embrión no Mamífero/efectos de los fármacos , Femenino , Isocumarinas/síntesis química , Isocumarinas/metabolismo , Isocumarinas/toxicidad , Articulación de la Rodilla/efectos de los fármacos , Articulación de la Rodilla/patología , Masculino , Ratones , Simulación del Acoplamiento Molecular , Estructura Molecular , Paladio/química , Inhibidores de Fosfodiesterasa 4/síntesis química , Inhibidores de Fosfodiesterasa 4/metabolismo , Inhibidores de Fosfodiesterasa 4/toxicidad , Unión Proteica , Células RAW 264.7 , Ratas Wistar , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/metabolismo , Sulfonamidas/toxicidad , Pez CebraRESUMEN
In spite of their various pharmacological properties the anti-inflammatory potential of benzo[c]phenanthridines remained underexplored. Thus, for the first time PDE4 inhibitory potential of 11,12-dihydro benzo[c]phenanthridine/benzo[c]phenanthridine was assessed in vitro. Elegant synthesis of these compounds was performed via a multi-step sequence consisting of a Pd-catalyzed unusual construction of 4-allyl isocoumarin ring and FeCl3-mediated intramolecular regio- as well as site-selective arene-allyl cyclization as key steps. The overall strategy involved Sonogashira coupling followed by isocoumarin and isoquinolone synthesis, then chlorination and subsequent cyclization to afford a range of 11,12-dihydro derivatives. One of these dihydro compounds was converted to the corresponding benzo[c]phenanthridine that showed concentration dependent inhibition of PDE4B affording an initial hit molecule. The SAR study suggested that 11,12-dihydro analogs were less potent than the compound having unsaturation at the same part of the ring.
Asunto(s)
Fenantridinas/síntesis química , Fenantridinas/farmacología , Inhibidores de Fosfodiesterasa 4/síntesis química , Inhibidores de Fosfodiesterasa 4/farmacología , Animales , Derivados del Benceno/síntesis química , Derivados del Benceno/química , Derivados del Benceno/farmacología , Catálisis , Línea Celular , Técnicas de Química Sintética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/química , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Ciclización , Humanos , Isocumarinas/síntesis química , Isocumarinas/química , Simulación del Acoplamiento Molecular , Paladio/química , Fenantridinas/química , Inhibidores de Fosfodiesterasa 4/químicaRESUMEN
A new class of PDE4 inhibitors were designed and synthesized via the InCl3 mediated heteroarylation of indoles and their further derivatization through the Pd(II)-catalyzed CH activation strategy. This effort allowed us to discover a series of 2-(1H-indol-3-yl)-quinoxaline based inhibitors possessing PDE4B selectivity over PDE4D and PDE4C. One of these compounds i.e. 3b (PDE4B IC50â¯=â¯0.39⯱â¯0.13⯵M with â¼27 andâ¯>â¯250 fold selectivity for PDE4B over PDE4D and C, respectively) showed effects in Zebrafish experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis when dosed at 3, 10 and 30â¯mg/kg intraperitoneally. Indeed, it halted the progression of the disease across all these doses tested. At an intraperitoneal dose of 30â¯mg/kg the compound 3b showed promising effects in adjuvant induced arthritic rats. The compound reduced paw volume, inflammation and pannus formation (in the knee joints) as well as pro-inflammatory gene expression/mRNA levels significantly in arthritic rats. Moreover, this compound was found to be selective towards PDE4 over other families of PDEs in vitro and safe when tested for its probable toxicity (e.g. teratogenicity, hepatotoxicity and cardiotoxicity) in Zebrafish.
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Artritis/tratamiento farmacológico , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Indoles/uso terapéutico , Esclerosis Múltiple/tratamiento farmacológico , Inhibidores de Fosfodiesterasa 4/uso terapéutico , Quinoxalinas/uso terapéutico , Animales , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Adyuvante de Freund , Indio , Indoles/síntesis química , Indoles/química , Indoles/toxicidad , Estructura Molecular , Esclerosis Múltiple/inducido químicamente , Glicoproteína Oligodendrócito-Mielina , Inhibidores de Fosfodiesterasa 4/síntesis química , Inhibidores de Fosfodiesterasa 4/química , Inhibidores de Fosfodiesterasa 4/toxicidad , Quinoxalinas/síntesis química , Quinoxalinas/química , Quinoxalinas/toxicidad , Ratas , Relación Estructura-Actividad , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
AMPK is considered as a potential high value target for metabolic disorders. Here, we present the molecular modeling, in vitro and in vivo characterization of Activator-3, 2-[2-(4-(trifluoromethyl)phenylamino)thiazol-4-yl]acetic acid, an AMP mimetic and a potent pan-AMPK activator. Activator-3 and AMP likely share common activation mode for AMPK activation. Activator-3 enhanced AMPK phosphorylation by upstream kinase LKB1 and protected AMPK complex against dephosphorylation by PP2C. Molecular modeling analyses followed by in vitro mutant AMPK enzyme assays demonstrate that Activator-3 interacts with R70 and R152 of the CBS1 domain on AMPK γ subunit near AMP binding site. Activator-3 and C2, a recently described AMPK mimetic, bind differently in the γ subunit of AMPK. Activator-3 unlike C2 does not show cooperativity of AMPK activity in the presence of physiological concentration of ATP (2 mM). Activator-3 displays good pharmacokinetic profile in rat blood plasma with minimal brain penetration property. Oral treatment of High Sucrose Diet (HSD) fed diabetic rats with 10 mg/kg dose of Activator-3 once in a day for 30 days significantly enhanced glucose utilization, improved lipid profiles and reduced body weight, demonstrating that Activator-3 is a potent AMPK activator that can alleviate the negative metabolic impact of high sucrose diet in rat model.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Acetatos/farmacología , Tiazoles/farmacología , Proteínas Quinasas Activadas por AMP/química , Acetatos/metabolismo , Acetatos/farmacocinética , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Activación Enzimática/efectos de los fármacos , Células Hep G2 , Humanos , Simulación del Acoplamiento Molecular , Dominios Proteicos , Ratas , Tiazoles/metabolismo , Tiazoles/farmacocinéticaRESUMEN
Nutritional abundance associated with chronic inflammation and dyslipidemia impairs the functioning of endoplasmic reticulum (ER) thereby hampering cellular responses to insulin. PHLPP1 was identified as a phosphatase which inactivates Akt, the master regulator of insulin mediated glucose homeostasis. Given the suggestive role of PHLPP1 phosphatase in terminating insulin signalling pathways, deeper insights into its functional role in inducing insulin resistance are warranted. Here, we show that PHLPP1 expression is enhanced in skeletal muscle of insulin resistant rodents which also displayed ER stress, an important mediator of insulin resistance. Using cultured cells and PHLPP1 knockdown mice, we demonstrate that PHLPP1 facilitates the development of ER stress. Importantly, shRNA mediated ablation of PHLPP1 significantly improved glucose clearance from systemic circulation with enhanced expression of glucose transporter 4 (GLUT-4) in skeletal muscle. Mechanistically, we show that endogenous PHLPP1 but not PP2Cα interacts with and directly dephosphorylates AMPK Thr172 in myoblasts without influencing its upstream kinase, LKB1. While the association between endogenous PHLPP1 and AMPK was enhanced in ER stressed cultured cells and soleus muscle of high fat diet fed mice, the basal interaction between PP2Ac and AMPK was minimally altered. Further, we show that PHLPP1α is phosphorylated by ERK1/2 at Ser932 under ER stress which is required for its ability to interact with and dephosphorylate AMPK and thereby induce ER stress. Taken together, our data position PHLPP1 as a key regulator of ER stress.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Estrés del Retículo Endoplásmico , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Músculo Esquelético/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Animales , Transportador de Glucosa de Tipo 4/genética , Transportador de Glucosa de Tipo 4/metabolismo , Células HEK293 , Humanos , Ratones , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteínas Nucleares/genética , Fosfoproteínas Fosfatasas/genética , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2C/genética , Proteína Fosfatasa 2C/metabolismo , Ratas , Ratas WistarRESUMEN
Electroporation is the most widely employed method of gene transfer into macrophages which are hard to transfect. RAW 264.7 is a widely used cell line for studying macrophage responses. Electroporation of RAW 264.7 cells with commercial reagents although very efficient is expensive necessitating the development of cost effective alternatives. In this study, we have formulated an economical electroporation buffer for electroporation of RAW 264.7 cells compatible with commercial nucleofector apparatus. We observed that supplementation of membrane fusogenic agents such as Ficoll, PEG and membrane resealing agent, poloxamer P188, enhanced the transfection efficiency of macrophages to a level comparable to the commercially available solutions thereby providing us a cost effective solution for genetic manipulation of macrophages especially in large numbers.
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Medios de Cultivo Condicionados/economía , Ingeniería de Proteínas/economía , Células RAW 264.7/fisiología , Proteínas Recombinantes/economía , Transfección/economía , Transfección/métodos , Animales , Técnicas de Cultivo Celular por Lotes/economía , Técnicas de Cultivo Celular por Lotes/métodos , Tampones (Química) , Análisis Costo-Beneficio/economía , Análisis Costo-Beneficio/métodos , Medios de Cultivo Condicionados/metabolismo , India , Ratones , Ingeniería de Proteínas/métodos , Células RAW 264.7/citología , Proteínas Recombinantes/biosíntesisRESUMEN
We have previously reported that bacterial endotoxin LPS attenuates expression of PHLPP, a ser/thr phosphatase, at both transcript and protein levels in different immune cells, however the underlying molecular mechanism is unknown and is of significant interest. Here, in line with the decreased transcript levels upon LPS treatment, we observed that LPS caused significant reduction in PHLPP promoter activity. We observed that SP1, a transcription factor frequently associated with inflammation, was recruited to the PHLPP promoter region. Ectopic expression of SP1 enhanced both transcript and protein levels of PHLPP while knockdown of SP1 or pharmacological inhibition of SP1 DNA binding by mithramycin reduced PHLPP expression. Moreover, over-expression of SP1 co-activators CBP/p300 augmented SP1 driven PHLPP promoter activity. Of note, LPS treatment depleted SP1 and CBP protein levels due to which recruitment of SP1 to PHLPP promoter was reduced. Further, we found that re-introduction of SP1 restored promoter activity and transcript levels of PHLPP in LPS stimulated cells. Collectively, our data revealed the molecular mechanism underlying the regulation of PHLPP expression during LPS induced macrophage inflammatory response.
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Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Proteínas Nucleares/genética , Fosfoproteínas Fosfatasas/genética , ARN Mensajero/genética , Factor de Transcripción Sp1/genética , Animales , Línea Celular , Regulación de la Expresión Génica , Genes Reporteros , Células HEK293 , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Macrófagos/citología , Macrófagos/inmunología , Ratones , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/inmunología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/inmunología , Plicamicina/farmacología , Regiones Promotoras Genéticas , ARN Mensajero/inmunología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Factor de Transcripción Sp1/inmunología , Transcripción Genética , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/inmunologíaRESUMEN
Post translational modifications, ubiquitination and its reversal by deubiquitination play an important role in regulating innate immune system. USP12 is a poorly studied deubiquitinase reported to regulate T-cell receptor signalling however the functional role of USP12 in macrophages, the principal architects of inflammation, is unknown. Thus, in this study we probed the involvement of USP12 in macrophage mediated inflammatory responses using bacterial endotoxin, LPS, as the model system. Here, we observed that the expression of USP12 was altered in time dependent manner in LPS stimulated RAW 264.7 macrophages at both mRNA and protein levels as revealed by qPCR and western blot analysis, respectively. Further analysis showed that LPS reduced the levels of Sp1 which enhanced the transcriptional levels of USP12. We observed that siRNA mediated ablation of USP12 expression in mouse macrophages suppressed the induction of LPS-induced iNOS and IL-6 expression but failed to alter IFN-ß synthesis, oxidative stress and phagocytic ability of macrophages. Mechanistic analysis suggest that USP12 may be required for the activation of NFκB pathway as knockdown of USP12 reduced the inhibitory phosphorylation of IκBα, a well characterized inhibitor of NFκB nuclear translocation. Further, USP12 was observed to be required for LPS elicited phosphorylation of ERK1/2 and p38. Collectively, our data suggest that USP12 may be a key mediator of LPS stimulated macrophage responses.
Asunto(s)
Endopeptidasas/metabolismo , Lipopolisacáridos/toxicidad , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Inhibidor NF-kappaB alfa/antagonistas & inhibidores , Animales , Endopeptidasas/deficiencia , Endopeptidasas/genética , Expresión Génica , Técnicas de Silenciamiento del Gen , Inflamación/metabolismo , Interferón gamma/farmacología , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/inmunología , Activación de Macrófagos/fisiología , Macrófagos/inmunología , Ratones , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fosforilación , Células RAW 264.7 , ARN Interferente Pequeño/genética , Ubiquitina TiolesterasaRESUMEN
Chronic inflammatory diseases such as insulin resistance, Type 2 diabetes, neurodegenerative diseases etc., are shown to be caused due to imbalanced activation states of macrophages. MicroRNAs which are transcriptional/post-transcriptional regulators of gene expression drive several pathophysiological processes including macrophage polarization. However the functional role of microRNAs in regulating inflammation induced insulin resistance is ill defined. In our current study we observed that the expression of miR-712 was reduced in macrophages exposed to LPS and IFN-γ. Ectopic expression of miR-712 in RAW 264.7 mouse macrophages impaired the expression of iNOS protein and secretion of pro-inflammatory cytokines such as TNF-α, IL-6 and IFN-ß which in turn led to improved insulin stimulated glucose uptake in co-cultured L6 myoblasts. Mechanistically, we identified that miR-712 targets the 3'UTR of a potent inflammatory gene LRRK2 and dampens the phosphorylation of p38 and ERK1/2 kinases. Taken together, our data underscore the regulatory role of miR-712 in restoring insulin stimulated glucose uptake by myoblasts through down-regulating macrophage mediated inflammatory responses.