ABSTRACT
Besnoitia besnoiti is an obligate intracellular apicomplexan parasite and the causal agent of bovine besnoitiosis. Bovine besnoitiosis has a considerable economic impact in Africa and Asia due to reduced milk production, abortions, and bull infertility. In Europe, bovine besnoitiosis is classified as an emerging disease. Polymorphonuclear neutrophils (PMN) are one of the most abundant leukocytes in cattle blood and amongst the first immunological responders toward invading pathogens. In the case of B. besnoiti, bovine PMN produce reactive oxygen species (ROS), release neutrophil extracellular traps (NETs), and show increased autophagic activities upon exposure to tachyzoite stages. In that context, the general processes of NETosis and autophagy were previously reported as associated with AMP-activated protein kinase (AMPK) activation. Here, we study the role of AMPK in B. besnoiti tachyzoite-induced NET formation, thereby expanding the analysis to both upstream proteins, such as the calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK), and downstream signaling and effector molecules, such as the autophagy-related proteins ULK-1 and Beclin-1. Current data revealed early AMPK activation (<30 min) in both B. besnoiti-exposed and AMPK activator (AICAR)-treated bovine PMN. This finding correlated with upstream responses on the level of CAMKK activation. Moreover, these reactions were accompanied by an augmented autophagic activity, as represented by enhanced expression of ULK-1 but not of Beclin-1. Referring to neutrophil effector functions, AICAR treatments induced both AMPK phosphorylation and NET formation, without affecting cell viability. In B. besnoiti tachyzoite-exposed PMN, AICAR treatments failed to affect oxidative responses, but led to enhanced NET formation, thereby indicating that AMPK and autophagic activation synergize with B. besnoiti-driven NETosis.
Subject(s)
AMP-Activated Protein Kinases , Calcium-Calmodulin-Dependent Protein Kinase Kinase , Extracellular Traps , Neutrophils , Sarcocystidae , Signal Transduction , Animals , Cattle , Neutrophils/metabolism , Neutrophils/drug effects , Neutrophils/immunology , AMP-Activated Protein Kinases/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism , Extracellular Traps/metabolism , Sarcocystidae/metabolism , Signal Transduction/drug effects , Autophagy/drug effects , Coccidiosis/parasitology , Coccidiosis/veterinary , Coccidiosis/immunology , Cattle Diseases/parasitology , Cattle Diseases/metabolism , Cattle Diseases/immunology , Reactive Oxygen Species/metabolismABSTRACT
Calcium/calmodulin dependent protein kinase kinase (CaMKK), a highly conserved protein kinase, is involved in the downstream processes of various biological activities by phosphorylating and activating 5'-AMP-activated protein kinase (AMPK) in response to the increase of cytosolic-free calcium (Ca2+). In the present study, a CaMKKI was identified from Yesso scallop Patinopecten yessoensis. Its mRNA was ubiquitously expressed in haemocytes and all tested tissues with the highest expression level in mantle. The expression level of PyCaMKKI mRNA in adductor muscle was significantly upregulated at 1, 3 and 6 h after high temperature treatment (25 °C), which was 3.43-fold (p < 0.05), 5.25-fold (p < 0.05), and 5.70-fold (p < 0.05) of that in blank group, respectively. At 3 h after high temperature treatment (25 °C), the protein level of PyAMPKα, as well as the phosphorylation level of PyAMPKα at Thr170 in adductor muscle, and the positive co-localized fluorescence signals of PyCaMKKI and PyAMPKα in haemocyte all increased significantly (p < 0.05) compared to blank group (18 °C). The pull-down assay showed that rPyCaMKKI and rPyAMPKα could bind each other in vitro. After PyCaMKKI was silenced by siRNA, the mRNA and protein levels of PyCaMKKI and PyAMPKα, and the phosphorylation level of PyAMPKα at Thr170 in adductor muscle were significantly down-regulated (p < 0.05) compared with the negative control group receiving an injection of siRNA-NC. These results collectively suggested that PyCaMKKI was involved in the activation of PyAMPKα in response to high temperature stress and would be helpful for understanding the function of PyCaMKKI-PyAMPKα pathway in maintaining energy homeostasis under high temperature stress in scallops.
Subject(s)
AMP-Activated Protein Kinases , Calcium-Calmodulin-Dependent Protein Kinase Kinase , Pectinidae , Animals , Pectinidae/immunology , Pectinidae/genetics , AMP-Activated Protein Kinases/metabolism , AMP-Activated Protein Kinases/genetics , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Kinase/genetics , Phosphorylation , Heat-Shock Response , Hemocytes/metabolism , RNA, Small Interfering/genetics , Hot Temperature , Stress, PhysiologicalABSTRACT
BACKGROUND: The increasing prevalence of heated tobacco products (HTPs) has heightened concerns regarding their potential health risks. Previous studies have demonstrated the toxicity of cigarette smoke extract (CSE) from traditional tobacco's mainstream smoke, even after the removal of nicotine and tar. Our study aimed to investigate the cytotoxicity of CSE derived from HTPs and traditional tobacco, with a particular focus on the role of reactive oxygen species (ROS) and intracellular Ca2+. METHODS: A human oral squamous cell carcinoma (OSCC) cell line, HSC-3 was utilized. To prepare CSE, aerosols from HTPs (IQOS) and traditional tobacco products (1R6F reference cigarette) were collected into cell culture media. A cell viability assay, apoptosis assay, western blotting, and Fluo-4 assay were conducted. Changes in ROS levels were measured using electron spin resonance spectroscopy and the high-sensitivity 2',7'-dichlorofluorescein diacetate assay. We performed a knockdown of calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) by shRNA lentivirus in OSCC cells. RESULTS: CSE from both HTPs and traditional tobacco exhibited cytotoxic effects in OSCC cells. Exposure to CSE from both sources led to an increase in intracellular Ca2+ concentration and induced p38 phosphorylation. Additionally, these extracts prompted cell apoptosis and heightened ROS levels. N-acetylcysteine (NAC) mitigated the cytotoxic effects and p38 phosphorylation. Furthermore, the knockdown of CaMKK2 in HSC-3 cells reduced cytotoxicity, ROS production, and p38 phosphorylation in response to CSE. CONCLUSION: Our findings suggest that the CSE from both HTPs and traditional tobacco induce cytotoxicity. This toxicity is mediated by ROS, which are regulated through Ca2+ signaling and CaMKK2 pathways.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Kinase , Carcinoma, Squamous Cell , Mouth Neoplasms , Reactive Oxygen Species , Smoke , Tobacco Products , Humans , Reactive Oxygen Species/metabolism , Mouth Neoplasms/metabolism , Mouth Neoplasms/pathology , Cell Line, Tumor , Smoke/adverse effects , Carcinoma, Squamous Cell/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism , Tobacco Products/adverse effects , Apoptosis/drug effects , Nicotiana/chemistry , Calcium/metabolism , Cell Survival/drug effectsABSTRACT
Cathepsin B (CTSB) and inflammatory cytokines are critical in initiating and developing pancreatitis. Calcineurin, a central calcium (Ca2+)-responsive signaling molecule, mediates acinar cell death and inflammatory responses leading to pancreatitis. However, the detailed mechanisms for regulating CTSB activity and inflammatory cytokine production are unknown. Myricetin (MC) exhibits various biological activities, including anti-inflammatory effects. Here, we aimed to investigate MC effects on pancreatitis and the underlying mechanisms. Prophylactic and therapeutic MC treatment ameliorated the severity of cerulein-, L-arginine-, and PDL-induced acute pancreatitis (AP). The inhibition of CTSB activity by MC was mediated via decreased calcineurin activity and macrophage infiltration, not neutrophils infiltration, into the pancreas. Additionally, calcineurin activity inhibition by MC prevented the phosphorylation of Ca2+/CaM-dependent protein kinase kinase 2 (CaMKK2) during AP, resulting in the inhibition of CaMKIV phosphorylation and adenosine monophosphate-activated protein kinase (AMPK) dephosphorylation. Furthermore, MC reduced nuclear factor-κB activation by modulating the calcineurin-CaMKIV-IKKα/ß-Iκ-Bα and calcineurin-AMPK-sirtuin1 axes, resulting in reduced production of tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6. Our results showed that MC alleviated AP severity by inhibiting acinar cell death and inflammatory responses, suggesting that MC as a calcineurin and CaMKK2 signaling modulator may be a potential treatment for AP.
Subject(s)
Calcineurin , Cathepsin B , Cytokines , Flavonoids , Mice, Inbred C57BL , Pancreatitis , Animals , Pancreatitis/drug therapy , Pancreatitis/immunology , Pancreatitis/pathology , Pancreatitis/chemically induced , Flavonoids/pharmacology , Flavonoids/therapeutic use , Cytokines/metabolism , Cathepsin B/metabolism , Mice , Male , Calcineurin/metabolism , Anti-Inflammatory Agents/therapeutic use , Anti-Inflammatory Agents/pharmacology , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism , Ceruletide , NF-kappa B/metabolism , Pancreas/pathology , Pancreas/drug effects , Pancreas/immunology , Signal Transduction/drug effects , Arginine/metabolism , Disease Models, Animal , AMP-Activated Protein Kinases/metabolismABSTRACT
OBJECTIVE: Liraglutide, a glucagon-like peptide-1 receptor agonist, has been shown to regulate blood sugar and control body weight, but its ability to treat obesity-related nephropathy has been poorly studied. Therefore, this study was designed to observe the characteristics and potential mechanism of liraglutide against obesity-related kidney disease. METHODS: Thirty-six C57BL/6J male mice were randomly divided into six groups (n = 6 per group). Obesity-related nephropathy was induced in mice by continuous feeding of high-fat diet (HFD) for 12 weeks. After 12 weeks, liraglutide (0.6 mg/kg) and AMP-activated protein kinase (AMPK) agonists bortezomib (200 µg/kg) were injected for 12 weeks, respectively. Enzyme-linked immunosorbent assay was employed to detect the levels of total cholesterol, triglycerides, low-density lipoprotein cholesterol, blood urea nitrogen, creatinine in serum, as well as urinary protein in urine. Besides, hematoxylin-eosin staining and periodic acid-Schiff staining were used to observe the pathological changes of kidney tissue; immunohistochemistry, western blot, and real-time quantitative PCR to assess the calmodulin-dependent protein kinase kinase beta (CaMKKß)/AMPK signaling pathway activation. RESULTS: Liraglutide significantly reduced serum lipid loading, improved kidney function, and relieved kidney histopathological damage and glycogen deposition in the mouse model of obesity-related kidney disease induced by HFD. In addition, liraglutide also significantly inhibited the CaMKKß/AMPK signaling pathway in kidney tissue of HFD-induced mice. However, bortezomib partially reversed the therapeutic effect of liraglutide on HDF-induced nephropathy in mice. CONCLUSIONS: Liraglutide has a therapeutic effect on obesity-related kidney disease, and such an effect may be achieved by inhibiting the CaMKKß/AMPK signaling pathway in kidney tissue.
Subject(s)
AMP-Activated Protein Kinases , Calcium-Calmodulin-Dependent Protein Kinase Kinase , Diet, High-Fat , Liraglutide , Mice, Inbred C57BL , Obesity , Signal Transduction , Animals , Liraglutide/pharmacology , Liraglutide/therapeutic use , Male , Diet, High-Fat/adverse effects , Mice , AMP-Activated Protein Kinases/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism , Signal Transduction/drug effects , Obesity/complications , Obesity/drug therapy , Kidney/pathology , Kidney/drug effects , Kidney/metabolism , Disease Models, Animal , Hypoglycemic Agents/pharmacology , Hypoglycemic Agents/therapeutic useABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Jiedu Tongluo Tiaogan Formula (JTTF), a traditional Chinese herbal decoction, exhibits the potential to treat type 2 diabetes mellitus (T2DM) by inhibiting endoplasmic reticulum stress (ERS) and excessive autophagy, which are the risk factors for the abnormal development and progression of ß cells. AIM OF THE STUDY: We aimed to assess the effect of JTTF on pancreatic glucotoxicity by inhibiting ERS and excessive autophagy, for which db/db mice and INS-1 insulinoma cells were used. MATERIALS AND METHODS: The chemical composition of the JTTF was analyzed by UPLC-Q/TOF-MS. Diabetic (db/db) mice were treated with distilled water or JTTF (2.4 and 7.2 g/kg/day) for 8 weeks. Furthermore, INS-1 cells induced by high glucose (HG) levels were treated with or without JTTF (50, 100, and 200 µg/mL) for 48 h to elucidate the protective mechanism of JTTF on glucose toxicity. The experimental methods included an oral glucose tolerance test, hematoxylin-eosin staining, immunohistochemistry, western blotting, RT-qPCR, and acridine orange staining. RESULT: 28 chemical components of JTTF were identified. Additionally, treatment with JTTF significantly decreased the severity of glycemic symptoms in the db/db mice. Moreover, the treatment partially restored glucose homeostasis in the db/db mice and protected the pancreatic ß-cell function. JTTF protected INS-1 cells from HG injury by upregulating GSIS and PDX1, MafA mRNA expression. Further, treatment with JTTF downregulated GRP78 and ATF6 expression, whereas it inhibited Beclin-1 and LC3 activation. The treatment protected the cells from HG-induced ERS and excessive autophagy by downregulating the CaMKKß/AMPK pathway. CONCLUSIONS: The present study findings show that JTTF may protects ß-cells by inhibiting the CaMKKß/AMPK pathway, which deepens our understanding of the effectiveness of JTTF as a treatment strategy against T2DM.
Subject(s)
AMP-Activated Protein Kinases , Autophagy , Calcium-Calmodulin-Dependent Protein Kinase Kinase , Drugs, Chinese Herbal , Endoplasmic Reticulum Stress , Insulin-Secreting Cells , Signal Transduction , Animals , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Endoplasmic Reticulum Stress/drug effects , Drugs, Chinese Herbal/pharmacology , Autophagy/drug effects , Mice , Male , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism , Signal Transduction/drug effects , AMP-Activated Protein Kinases/metabolism , Rats , Endoplasmic Reticulum Chaperone BiP , Mice, Inbred C57BL , Cell Line, Tumor , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Experimental/drug therapyABSTRACT
Sleep deprivation (SD) has emerged as a critical concern impacting human health, leading to significant damage to the cardiovascular system. However, the underlying mechanisms are still unclear, and the development of targeted drugs is lagging. Here, we used mice to explore the effects of prolonged SD on cardiac structure and function. Echocardiography analysis revealed that cardiac function was significantly decreased in mice after five weeks of SD. Real-time quantitative PCR (RT-q-PCR) and Masson staining analysis showed that cardiac remodeling marker gene Anp (atrial natriuretic peptide) and fibrosis were increased, Elisa assay of serum showed that the levels of creatine kinase (CK), creatine kinase-MB (CK-MB), ANP, brain natriuretic peptide (BNP) and cardiac troponin T (cTn-T) were increased after SD, suggesting that cardiac remodeling and injury occurred. Transcript sequencing analysis indicated that genes involved in the regulation of calcium signaling pathway, dilated cardiomyopathy, and cardiac muscle contraction were changed after SD. Accordingly, Western blotting analysis demonstrated that the cardiac-contraction associated CaMKK2/AMPK/cTNI pathway was inhibited. Since our preliminary research has confirmed the vital role of Casein Kinase-2 -Interacting Protein-1 (CKIP-1, also known as PLEKHO1) in cardiac remodeling regulation. Here, we found the levels of the 3' untranslated region of Ckip-1 (Ckip-1 3'UTR) decreased, while the coding sequence of Ckip-1 (Ckip-1 CDS) remained unchanged after SD. Significantly, adenovirus-mediated overexpression of Ckip-1 3'UTR alleviated SD-induced cardiac dysfunction and remodeling by activating CaMKK2/AMPK/cTNI pathway, which proposed the therapeutic potential of Ckip-1 3'UTR in treating SD-induced heart disease.
Subject(s)
3' Untranslated Regions , AMP-Activated Protein Kinases , Calcium-Calmodulin-Dependent Protein Kinase Kinase , Signal Transduction , Sleep Deprivation , Animals , Male , Mice , 3' Untranslated Regions/genetics , AMP-Activated Protein Kinases/metabolism , AMP-Activated Protein Kinases/genetics , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Kinase/genetics , Carrier Proteins/genetics , Carrier Proteins/metabolism , Mice, Inbred C57BL , Myocardium/metabolism , Myocardium/pathology , Sleep Deprivation/genetics , Sleep Deprivation/metabolism , Sleep Deprivation/complications , Troponin I/metabolism , Troponin I/geneticsABSTRACT
Cisplatin is a particularly potent antineoplastic drug. However, its usefulness is restricted due to the induction of nephrotoxicity. More recent research has indicated that ß-hydroxybutyrate (ß-HB) protects against acute or chronic organ damage as an efficient healing agent. Nonetheless, the therapeutic mechanisms of ß-HB in acute kidney damage caused by chemotherapeutic drugs remain unclear. Our study developed a model of cisplatin-induced acute kidney injury (AKI), which involved the administration of a ketogenic diet or ß-HB. We analyzed blood urea nitrogen (BUN) and creatinine (Cr) levels in serum, and used western blotting and immunohistochemical staining to assess ferroptosis and the calcium/calmodulin-dependent kinase kinase 2 (Camkk2)/AMPK pathway. The mitochondrial morphology and function were examined. Additionally, we conducted in vivo and in vitro experiments using selective Camkk2 inhibitor or activator to investigate the protective mechanism of ß-HB on cisplatin-induced AKI. Exogenous or endogenous ß-HB effectively alleviated cisplatin-induced abnormally elevated levels of BUN and Cr and renal tubular necrosis in vivo. Additionally, ß-HB reduced ferroptosis biomarkers and increased the levels of anti-ferroptosis biomarkers in the kidney. ß-HB also improved mitochondrial morphology and function. Moreover, ß-HB significantly attenuated cisplatin-induced cell ferroptosis and damage in vitro. Furthermore, western blotting and immunohistochemical staining indicated that ß-HB may prevent kidney injury by regulating the Camkk2-AMPK pathway. The use of the Camkk2 inhibitor or activator verified the involvement of Camkk2 in the renal protection by ß-HB. This study provided evidence of the protective effects of ß-HB against cisplatin-induced nephrotoxicity and identified inhibited ferroptosis and Camkk2 as potential molecular mechanisms.
ß-HB protects against cisplatin-induced renal damage both in vivo and in vitro.Moreover, ß-HB is effective in attenuating cisplatin-induced lipid peroxidation and ferroptosis.The regulation of energy metabolism, as well as the treatment involving ß-HB, is associated with Camkk2.
Subject(s)
3-Hydroxybutyric Acid , Acute Kidney Injury , Calcium-Calmodulin-Dependent Protein Kinase Kinase , Cisplatin , Ferroptosis , Cisplatin/adverse effects , Cisplatin/toxicity , Animals , Ferroptosis/drug effects , Acute Kidney Injury/chemically induced , Acute Kidney Injury/prevention & control , Acute Kidney Injury/metabolism , Acute Kidney Injury/pathology , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism , Male , Mice , 3-Hydroxybutyric Acid/pharmacology , Disease Models, Animal , Kidney/drug effects , Kidney/pathology , Kidney/metabolism , Antineoplastic Agents/toxicity , Antineoplastic Agents/adverse effects , Mice, Inbred C57BL , Signal Transduction/drug effects , AMP-Activated Protein Kinases/metabolism , Blood Urea Nitrogen , Mitochondria/drug effects , Mitochondria/metabolism , Creatinine/blood , HumansABSTRACT
The protection of the replication fork structure under stress conditions is essential for genome maintenance and cancer prevention. A key signaling pathway for fork protection involves TRPV2-mediated Ca2+ release from the ER, which is triggered after the generation of cytosolic DNA and the activation of cGAS/STING. This results in CaMKK2/AMPK activation and subsequent Exo1 phosphorylation, which prevent aberrant fork processing, thereby ensuring genome stability. However, it remains poorly understood how the TRPV2 channel is activated by the presence of cytosolic DNA. Here, through a genome-wide CRISPR-based screen, we identify TRPM8 channel-associated factor 1 (TCAF1) as a key factor promoting TRPV2-mediated Ca2+ release under replication stress or other conditions that activate cGAS/STING. Mechanistically, TCAF1 assists Ca2+ release by facilitating the dissociation of STING from TRPV2, thereby relieving TRPV2 repression. Consistent with this function, TCAF1 is required for fork protection, chromosomal stability, and cell survival after replication stress.
Subject(s)
Calcium , Cytosol , DNA Replication , Membrane Proteins , TRPV Cation Channels , Humans , TRPV Cation Channels/metabolism , TRPV Cation Channels/genetics , Calcium/metabolism , Cytosol/metabolism , Membrane Proteins/metabolism , Membrane Proteins/genetics , HEK293 Cells , DNA/metabolism , HeLa Cells , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Kinase/genetics , Phosphorylation , Genomic Instability , DNA Damage , AnimalsABSTRACT
Lymph node metastasis, primarily caused by the migration of oral squamous cell carcinoma (OSCC) cells, stands as a crucial prognostic marker. We have previously demonstrated that EP4, a subtype of the prostaglandin E2 (PGE2) receptor, orchestrates OSCC cell migration via Ca2+ signaling. The exact mechanisms by which EP4 influences cell migration through Ca2+ signaling, however, is unclear. Our study aims to clarify how EP4 controls OSCC cell migration through this pathway. We find that activating EP4 with an agonist (ONO-AE1-473) increased intracellular Ca2+ levels and the migration of human oral cancer cells (HSC-3), but not human gingival fibroblasts (HGnF). Further RNA sequencing linked EP4 to calmodulin-like protein 6 (CALML6), whose role remains undefined in OSCC. Through protein-protein interaction network analysis, a strong connection is identified between CALML6 and calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), with EP4 activation also boosting mitochondrial function. Overexpressing EP4 in HSC-3 cells increases experimental lung metastasis in mice, whereas inhibiting CaMKK2 with STO-609 markedly lowers these metastases. This positions CaMKK2 as a potential new target for treating OSCC metastasis. Our findings highlight CALML6 as a pivotal regulator in EP4-driven mitochondrial respiration, affecting cell migration and metastasis via the CaMKK2 pathway.
Subject(s)
Calcium-Binding Proteins , Carcinoma, Squamous Cell , Cell Movement , Mitochondria , Mouth Neoplasms , Receptors, Prostaglandin E, EP4 Subtype , Animals , Humans , Mice , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Kinase/genetics , Calmodulin/metabolism , Calmodulin/genetics , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/genetics , Cell Line, Tumor , Mitochondria/metabolism , Mouth Neoplasms/pathology , Mouth Neoplasms/metabolism , Mouth Neoplasms/genetics , Receptors, Prostaglandin E, EP4 Subtype/metabolism , Receptors, Prostaglandin E, EP4 Subtype/genetics , Squamous Cell Carcinoma of Head and Neck/metabolism , Squamous Cell Carcinoma of Head and Neck/genetics , Squamous Cell Carcinoma of Head and Neck/pathology , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolismABSTRACT
Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a multi-functional, serine/threonine protein kinase with predominant roles in inflammation, systemic energy metabolism, and bone remodeling. We previously reported that global ablation of CaMKK2 or its systemic pharmacological inhibition led to bone mass accrual in mice by stimulating osteoblasts and inhibiting osteoclasts. However, a direct, cell-intrinsic role for the kinase in the osteoblast lineage has not been established. Here we report that conditional deletion of CaMKK2 from osteoprogenitors, using the Osterix 1 (Osx1) - GFP::Cre (tetracycline-off) mouse line, resulted in increased trabecular bone mass due to an acute stimulation of osteoblast function in male and female mice. The acute simulation of osteoblasts and bone formation following conditional ablation of osteoprogenitor-derived CaMKK2 was sustained only in female mice. Periosteal bone formation at the cortical bone was enhanced only in male conditional knockout mice without altering cortical bone mass or strength. Prolonged deletion of CaMKK2 in early osteoblasts was accompanied by a stimulation of osteoclasts in both sexes, indicating a coupling effect. Notably, alterations in trabecular and cortical bone mass were absent in the doxycycline-removed "Cre-only" Osx1-GFP::Cre mice. Thus, the increase in osteoblast function at the trabecular and cortical bone surfaces following the conditional deletion of CaMKK2 in osteoprogenitors is indicative of a direct but sex-divergent role for the kinase in osteoblasts.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Kinase , Osteoblasts , Sp7 Transcription Factor , Animals , Osteoblasts/metabolism , Female , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism , Male , Sp7 Transcription Factor/metabolism , Sp7 Transcription Factor/genetics , Osteogenesis/physiology , Sex Characteristics , Mice , Mice, Knockout , Osteoclasts/metabolism , Stem Cells/metabolism , Gene DeletionABSTRACT
Disturbance in mitochondrial homeostasis within proximal tubules is a critical characteristic associated with diabetic kidney disease (DKD). CaMKKß/AMPK signaling plays an important role in regulating mitochondrial homeostasis. Despite the downregulation of CaMKKß in DKD pathology, the underlying mechanism remains elusive. The expression of NEDD4L, which is primarily localized to renal proximal tubules, is significantly upregulated in the renal tubules of mice with DKD. Coimmunoprecipitation (Co-IP) assays revealed a physical interaction between NEDD4L and CaMKKß. Moreover, deletion of NEDD4L under high glucose conditions prevented rapid CaMKKß protein degradation. In vitro studies revealed that the aberrant expression of NEDD4L negatively influences the protein stability of CaMKKß. This study also explored the role of NEDD4L in DKD by using AAV-shNedd4L in db/db mice. These findings confirmed that NEDD4L inhibition leads to a decrease in urine protein excretion, tubulointerstitial fibrosis, and oxidative stress, and mitochondrial dysfunction. Further in vitro studies demonstrated that si-Nedd4L suppressed mitochondrial fission and reactive oxygen species (ROS) production, effects antagonized by si-CaMKKß. In summary, the findings provided herein provide strong evidence that dysregulated NEDD4L disturbs mitochondrial homeostasis by negatively modulating CaMKKß in the context of DKD. This evidence underscores the potential of therapeutic interventions targeting NEDD4L and CaMKKß to safeguard renal tubular function in the management of DKD.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Kinase , Diabetic Nephropathies , Down-Regulation , Homeostasis , Mitochondria , Nedd4 Ubiquitin Protein Ligases , Animals , Humans , Male , Mice , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism , Diabetic Nephropathies/metabolism , Diabetic Nephropathies/pathology , Mice, Inbred C57BL , Mitochondria/metabolism , Mitochondrial Dynamics , Nedd4 Ubiquitin Protein Ligases/metabolism , Nedd4 Ubiquitin Protein Ligases/genetics , Oxidative Stress , Protein Stability , Proteolysis , Reactive Oxygen Species/metabolismABSTRACT
Early-onset obesity is a rising health concern influenced by heredity. However, many monogenic obesity variants (MOVs) remain to be discovered due to differences in ethnicity and culture. Additionally, patients with known MOVs have shown limited weight loss after bariatric surgery, suggesting it can be used as a screening tool for new candidates. In this study, we performed whole-exome sequencing (WES) combined with postoperative data to detect candidate MOVs in a cohort of 62 early-onset obesity and 9 late-onset obesity patients. Our findings demonstrated that patients with early-onset obesity preferred a higher BMI and waist circumference (WC). We confirmed the efficacy of the method by identifying a mutation in known monogenic obesity gene, PCSK1, which resulted in less weight loss after surgery. 5 genes were selected for further verification, and a frameshift variant in CAMKK2 gene: NM_001270486.1, c.1614dup, (p. Gly539Argfs*3) was identified as a novel candidate MOV. This mutation influenced the improvement of metabolism after bariatric surgery. In conclusion, our data confirm the efficacy of WES combined with postoperative data in detecting novel candidate MOVs and c.1614dup (CAMKK2) might be a promising MOV, which needs further confirmation. This study enriches the human monogenic obesity mutation database and provides a scientific basis for clinically accurate diagnosis and treatment.
Subject(s)
Frameshift Mutation , Obesity , Humans , Exome Sequencing , Obesity/genetics , Obesity/surgery , Mutation , Weight Loss , Calcium-Calmodulin-Dependent Protein Kinase Kinase/geneticsABSTRACT
A chemical proteomics approach using Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor-immobilized sepharose (TIM-063-Kinobeads) identified main targets such as CaMKKα/1 and ß/2, and potential off-target kinases, including AP2-associated protein kinase 1 (AAK1), as TIM-063 interactants. Because TIM-063 interacted with the AAK1 catalytic domain and inhibited its enzymatic activity moderately (IC50 = 8.51 µM), we attempted to identify potential AAK1 inhibitors from TIM-063-derivatives and found a novel AAK1 inhibitor, TIM-098a (11-amino-2-hydroxy-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) which is more potent (IC50 = 0.24 µM) than TIM-063 without any inhibitory activity against CaMKK isoforms and a relative AAK1-selectivity among the Numb-associated kinases family. TIM-098a could inhibit AAK1 activity in transfected cultured cells (IC50 = 0.87 µM), indicating cell-membrane permeability of the compound. Overexpression of AAK1 in HeLa cells significantly reduced the number of early endosomes, which was blocked by treatment with 10 µM TIM-098a. These results indicate TIM-063-Kinobeads-based chemical proteomics is efficient for identifying off-target kinases and re-evaluating the kinase inhibitor (TIM-063), leading to the successful development of a novel inhibitory compound (TIM-098a) for AAK1, which could be a molecular probe for AAK1. TIM-098a may be a promising lead compound for a more potent, selective and therapeutically useful AAK1 inhibitor.
Subject(s)
Protein Kinase Inhibitors , Protein Serine-Threonine Kinases , Humans , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/metabolism , HeLa Cells , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism , PhosphorylationABSTRACT
Insect neuronal nicotinic acetylcholine receptors (nAChRs) are transmembrane receptors that play a key role in the development and synaptic plasticity of both vertebrates and invertebrates and are considered to be major targets of neonicotinoid insecticides. We used dorsal unpaired median (DUM) neurons, which are insect neurosecretory cells, in order to explore the intracellular mechanisms leading to the regulation of insect neuronal nAChRs in more detail. Using whole-cell patch-clamp and fura-2AM calcium imaging techniques, we found that a novel CaMKK/AMPK pathway could be involved in the intracellular regulation of DUM neuron nAChRs. The CaMKK selective inhibitor, STO, reduced nicotinic current amplitudes, and strongly when co-applied with α-Bgt. Interestingly, intracellular application of the AMPK activator, A-76, prevented the reduction in nicotine-induced currents observed in the presence of the AMPK inhibitor, dorsomorphin. STO prevented the increase in intracellular calcium induced by nicotine, which was not dependent on α-Bgt. Currents induced by 1 mM LMA, a selective activator of nAChR2, were reduced under bath application of STO, and mecamylamine, which blocked nAChR2 subtype, inhibited the increase in intracellular calcium induced by LMA. These findings provide insight into potential complex mechanisms linked to the modulation of the DUM neuron nAChRs and CaMKK pathway.
Subject(s)
Calcium , Nicotine , Animals , Nicotine/pharmacology , Calcium/metabolism , Receptors, Nicotinic/metabolism , Receptors, Nicotinic/drug effects , Nicotinic Agonists/pharmacology , Patch-Clamp Techniques , Neurons/drug effects , Neurons/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Kinase/antagonists & inhibitors , Naphthalimides/pharmacology , Protein Kinase Inhibitors/pharmacology , BenzimidazolesABSTRACT
The ongoing COVID-19 pandemic has revealed the shortfalls in our understanding of how to treat coronavirus infections. With almost 7 million case fatalities of COVID-19 globally, the catalog of FDA-approved antiviral therapeutics is limited compared to other medications, such as antibiotics. All-trans retinoic acid (RA), or activated vitamin A, has been studied as a potential therapeutic against coronavirus infection because of its antiviral properties. Due to its impact on different signaling pathways, RA's mechanism of action during coronavirus infection has not been thoroughly described. To determine RA's mechanism of action, we examined its effect against a mouse coronavirus, mouse hepatitis virus strain A59 (MHV). We demonstrated that RA significantly decreased viral titers in infected mouse L929 fibroblasts and RAW 264.7 macrophages. The reduced viral titers were associated with a corresponding decrease in MHV nucleocapsid protein expression. Using interferon regulatory factor 3 (IRF3) knockout RAW 264.7 cells, we demonstrated that RA-induced suppression of MHV required IRF3 activity. RNA-seq analysis of wildtype and IRF3 knockout RAW cells showed that RA upregulated calcium/calmodulin (CaM) signaling proteins, such as CaM kinase kinase 1 (CaMKK1). When treated with a CaMKK inhibitor, RA was unable to upregulate IRF activation during MHV infection. In conclusion, our results demonstrate that RA-induced protection against coronavirus infection depends on IRF3 and CaMKK.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Kinase , Interferon Regulatory Factor-3 , Murine hepatitis virus , Tretinoin , Virus Replication , Animals , Mice , Amino Acids , Antiviral Agents/pharmacology , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism , Interferon Regulatory Factor-3/metabolism , Tretinoin/pharmacology , Virus Replication/drug effects , Murine hepatitis virus/drug effects , Murine hepatitis virus/physiology , RAW 264.7 Cells , L CellsABSTRACT
Peritoneal fibrosis is a severe clinical complication associated with peritoneal dialysis (PD) and impacts its efficacy and patient outcomes. The process of mesothelial-mesenchymal transition (MMT) in peritoneal mesothelial cells plays a pivotal role in fibrogenesis, whereas metabolic reprogramming, characterized by excessive glycolysis, is essential in MMT development. No reliable therapies are available despite substantial progress made in understanding the mechanisms underlying peritoneal fibrosis. Protective effect of omega-3 polyunsaturated fatty acids (ω3 PUFAs) has been described in PD-induced peritoneal fibrosis, although the detailed mechanisms remain unknown. It is known that ω3 PUFAs bind to and activate the free fatty acid receptor 4 (FFAR4). However, the expression and role of FFAR4 in the peritoneum have not been investigated. Thus, we hypothesized that ω3 PUFAs would alleviate peritoneal fibrosis by inhibiting hyperglycolysis and MMT through FFAR4 activation. First, we determined FFAR4 expression in peritoneal mesothelium in humans and mice. FFAR4 expression was abnormally decreased in patients on PD and mice and HMrSV5 mesothelial cells exposed to PD fluid (PDF); this change was restored by the ω3 PUFAs (EPA and DHA). ω3 PUFAs significantly inhibited peritoneal hyperglycolysis, MMT, and fibrosis in PDF-treated mice and HMrSV5 mesothelial cells; these changes induced by ω3 PUFAs were blunted by treatment with the FFAR4 antagonist AH7614 and FFAR4 siRNA. Additionally, ω3 PUFAs induced FFAR4, Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß), and AMPK and suppressed mTOR, leading to the inhibition of hyperglycolysis, demonstrating that the ω3 PUFAs-mediated FFAR4 activation ameliorated peritoneal fibrosis by inhibiting hyperglycolysis and MMT via CaMKKß/AMPK/mTOR signaling. As natural FFAR4 agonists, ω3 PUFAs may be considered for the treatment of PD-associated peritoneal fibrosis.
Subject(s)
Fatty Acids, Omega-3 , Peritoneal Fibrosis , Humans , Mice , Animals , Peritoneal Fibrosis/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism , AMP-Activated Protein Kinases/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolismABSTRACT
A series of 2'-hydroxychalcone derivatives with various substituents on B-ring were synthesized and evaluated for AMP-activated protein kinase (AMPK) activation activity in podocyte cells. The results displayed that hydroxy, methoxy and methylenedioxy groups on B-ring could enhance the activitiy better than O-saturated alkyl, O-unsaturated alkyl or other alkoxy groups. Compounds 27 and 29 possess the highest fold change of 2.48 and 2.73, respectively, which were higher than those of reference compound (8) (1.28) and metformin (1.88). Compounds 27 and 29 were then subjected to a concentration-response study to obtain the EC50 values of 2.0 and 4.8 µM, respectively and MTT assays also showed that cell viability was not influenced by the exposure of podocytes to compounds 27 and 29 at concentrations up to 50 µM. In addition, compound 27 was proved to activate AMPK via calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß)-dependent pathway without affecting intracellular calcium levels. The computational study showed that the potent compounds exhibited stronger ligand-binding strength to CaMKKß, particularly compounds 27 (-8.4 kcal/mol) and 29 (-8.0 kcal/mol), compared to compound 8 (-7.5 kcal/mol). Fragment molecular orbital (FMO) calculation demonstrated that compound 27 was superior to compound 29 due to the presence of methyl group, which amplified the binding by hydrophobic interactions. Therefore, compound 27 would represent a promising AMPK activator for further investigation of the treatment of diabetes and diabetic nephropathy.
Subject(s)
AMP-Activated Protein Kinases , Calcium-Calmodulin-Dependent Protein Kinase Kinase , Chalcones , AMP-Activated Protein Kinases/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism , Calcium/metabolism , PhosphorylationABSTRACT
Exercise is an effective non-pharmacological strategy for ameliorating nonalcoholic fatty liver disease (NAFLD), but the underlying mechanism needs further investigation. Cysteine dioxygenase type 1 (Cdo1) is a key enzyme for cysteine catabolism that is enriched in liver, whose role in NAFLD remains poorly understood. Here, we show that exercise induces the expression of hepatic Cdo1 via the cAMP/PKA/CREB signaling pathway. Hepatocyte-specific knockout of Cdo1 (Cdo1LKO) decreases basal metabolic rate of the mice and impairs the effect of exercise against NAFLD, whereas hepatocyte-specific overexpression of Cdo1 (Cdo1LTG) increases basal metabolic rate of the mice and synergizes with exercise to ameliorate NAFLD. Mechanistically, Cdo1 tethers Camkk2 to AMPK by interacting with both of them, thereby activating AMPK signaling. This promotes fatty acid oxidation and mitochondrial biogenesis in hepatocytes to attenuate hepatosteatosis. Therefore, by promoting hepatic Camkk2-AMPK signaling pathway, Cdo1 acts as an important downstream effector of exercise to combat against NAFLD.
Subject(s)
Non-alcoholic Fatty Liver Disease , Mice , Animals , Non-alcoholic Fatty Liver Disease/prevention & control , Non-alcoholic Fatty Liver Disease/metabolism , AMP-Activated Protein Kinases/metabolism , Liver/metabolism , Hepatocytes/metabolism , Lipid Metabolism , Mice, Inbred C57BL , Calcium-Calmodulin-Dependent Protein Kinase Kinase/genetics , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolismABSTRACT
Alcoholic liver disease(ALD), with its increasing morbidity and mortality, has seriously and extensively affected the health of people worldwide. Methyl ferulic acid(MFA) has been proven to significantly inhibit alcohol-induced lipid production in L02 cells through the AMP-activated protein kinase(AMPK) pathway, but its in-depth mechanism remains unclear. This study aimed to further clarify the mechanism of MFA in improving lipid accumulation in L02 cells through the microRNA-378b(miR-378b)-mediated calcium/calmodulin-dependent protein kinase kinase 2(CaMKK2)-AMPK signaling pathway based on existing researches. L02 cells were induced by 100 mmol·L~(-1) ethanol for 48 h to establish the model of ALD in vitro, and 100, 50, and 25 μmol·L~(-1) concentration of MFA was treated. MiR-378b plasmids(containing the overexpression plasmid-miR-378b mimics, silence plasmid-miR-378b inhibitor, and their respective negative control-miR-378b NCs) were transfected into L02 cells by electroporation to up-regulate or down-regulate the levels of miR-378b in L02 cells. The levels of total cholesterol(TC) and triglyceride(TG) in cells were detected by commercial diagnostic kits and automatic biochemical analyzers. The expression levels of miR-378b in L02 cells were detected by real-time quantitative polymerase chain reaction(qRT-PCR). CaMKK2 mRNA levels were detected by PCR, and protein expressions of related factors involved in lipid synthesis, decomposition, and transport in lipid metabolism were detected by Western blot. The results displayed that ethanol significantly increased TG and TC levels in L02 cells, while MFA decreased TG and TC levels. Ethanol up-regulated the miR-378b level, while MFA effectively inhibited the miR-378b level. The overexpression of miR-378b led to lipid accumulation in ethanol-induced L02 cells, while the silence of miR-378b improved the lipid deposition induced by ethanol. MFA activated the CaMKK2-AMPK signaling pathway by lowering miR-378b, thus improving lipid synthesis, decomposition, and transport, which improved lipid deposition in L02 cells. This study shows that MFA improves lipid deposition in L02 cells by regulating the CaMKK2-AMPK pathway through miR-378b.