Introduction:
The cannabinoid receptor (CBR) subtypes 1 (CB
1
R) and 2 (CB
2
R) are key components of the endocannabinoid system (ECS), playing a central role in the control of peripheral pain, ...inflammation and the immune response, with further roles in the endocrine regulation of food intake and energy balance. So far, few medicines targeting these receptors have reached the clinic, suggesting that a better understanding of the receptor signalling properties of existing tool compounds and clinical candidates may open the door to the development of more effective and safer treatments. Both CB
1
R and CB
2
R are Gα
i
protein-coupled receptors but detecting Gα
i
protein signalling activity reliably and reproducibly is challenging. This is due to the inherent variability in live cell-based assays and restrictions around the use of radioactive
35
S-GTPγS, a favoured technology for developing higher-throughput membrane-based Gα
i
protein activity assays.
Methods:
Here, we describe the development of a membrane-based Gα
i
signalling system, produced from membrane preparations of HEK293TR cells, stably overexpressing CB
1
R or CB
2
R, and components of the Gα
i
-CASE biosensor. This BRET-based system allows direct detection of Gα
i
signalling in both cells and membranes by monitoring bioluminescence resonance energy transfer (BRET) between the α and the βγ subunits. Cells and membranes were subject to increasing concentrations of reference cannabinoid compounds, with 10 μM furimazine added to generate RET signals, which were detected on a PHERAstar FSX plate reader, then processed using MARS software and analysed in GraphPad PRISM 9.2.
Results:
In membranes expressing the Gi-CASE biosensor, the cannabinoid ligands profiled were found to show agonist and inverse agonist activity. Agonist activity elicited a decrease in the BRET signal, indicative of receptor activation and G protein dissociation. Inverse agonist activity caused an increase in BRET signal, indicative of receptor inactivation, and the accumulation of inactive G protein. Our membrane-based Gi-CASE NanoBRET system successfully characterised the potency (pEC
50
) and efficacy (E
max
) of CBR agonists and inverse agonists in a 384-well screening format. Values obtained were in-line with whole-cell Gi-CASE assays and consistent with literature values obtained in the GTPγS screening format.
Discussion:
This novel, membrane-based Gα
i
protein activation assay is applicable to other Gα
i
-coupled GPCRs, including orphan receptors, allowing real-time higher-throughput measurements of receptor activation.
Cells differentiate into specific and functional lineages to build up tissues. It has been shown in several tissues that mitochondrial morphology, levels of “mitochondria-shaping” proteins, and ...mitochondrial functions change upon differentiation. In this review, we highlight the significance of mitochondrial dynamics and functions in tissue development, cell differentiation, and reprogramming processes. Signalling cascades are critical for tissue stem cell maintenance and cell fate determination, and growing evidence demonstrates mitochondria could act as a centre of intra and extracellular signals to coordinate signalling pathways, such as Notch, Wnt, and YAP/TAZ signalling. Just an organelle, however, emerges as a master regulator of cell differentiation, and can be a target to manipulate cell fates.
•Mitochondrial fusion and fission are essential in mouse embryogenesis, and cell differentiation.•The mitochondrial shape changes in differentiation and dedifferentiation.•Mitochondrial dynamics act as a hub to consolidate intra-and-extracellular signals in cell differentiation.
Wnt signaling is a highly conserved signaling pathway that plays a critical role in controlling embryonic and organ development, as well as cancer progression. Genome-wide sequencing and gene ...expression profile analyses have demonstrated that Wnt signaling is involved mainly in the processes of breast cancer proliferation and metastasis. The most recent studies have indicated that Wnt signaling is also crucial in breast cancer immune microenvironment regulation, stemness maintenance, therapeutic resistance, phenotype shaping, etc. Wnt/β-Catenin, Wnt-planar cell polarity (PCP), and Wnt-Ca
signaling are three well-established Wnt signaling pathways that share overlapping components and play different roles in breast cancer progression. In this review, we summarize the main findings concerning the relationship between Wnt signaling and breast cancer and provide an overview of existing mechanisms, challenges, and potential opportunities for advancing the therapy and diagnosis of breast cancer.
Background and Aims
Cancer‐associated fibroblasts (CAFs) are key players in multicellular, stromal‐dependent alterations leading to HCC pathogenesis. However, the intricate crosstalk between CAFs and ...other components in the tumor microenvironment (TME) remains unclear. This study aimed to investigate the cellular crosstalk among CAFs, tumor cells, and tumor‐associated neutrophils (TANs) during different stages of HCC pathogenesis.
Approach and Results
In the HCC‐TME, CAF‐derived cardiotrophin‐like cytokine factor 1 (CLCF1) increased chemokine (C‐X‐C motif) ligand 6 (CXCL6) and TGF‐β secretion in tumor cells, which subsequently promoted tumor cell stemness in an autocrine manner and TAN infiltration and polarization in a paracrine manner. Moreover, CXCL6 and TGF‐β secreted by HCC cells activated extracellular signal‐regulated kinase (ERK) 1/2 signaling of CAFs to produce more CLCF1, thus forming a positive feedback loop to accelerate HCC progression. Inhibition of ERK1/2 or CLCF1/ciliary neurotrophic factor receptor signaling efficiently impaired CLCF1‐mediated crosstalk among CAFs, tumor cells, and TANs both in vitro and in vivo. In clinical samples, up‐regulation of the CLCF1−CXCL6/TGF‐β axis exhibited a marked correlation with increased cancer stem cells, “N2”‐polarized TANs, tumor stage, and poor prognosis.
Conclusions
This study reveals a cytokine‐mediated cellular crosstalk and clinical network involving the CLCF1−CXCL6/TGF‐β axis, which regulates the positive feedback loop among CAFs, tumor stemness, and TANs, HCC progression, and patient prognosis. These results may support the CLCF1 cascade as a potential prognostic biomarker and suggest that selective blockade of CLCF1/ciliary neurotrophic factor receptor or ERK1/2 signaling could provide an effective therapeutic target for patients with HCC.
Melatonin (N‐acetyl‐5‐methoxytryptamine) plays important roles in plant defences against a variety of biotic and abiotic stresses, including UV‐B stress. Molecular mechanisms underlying functions of ...melatonin in plant UV‐B responses are poorly understood. Here, we show that melatonin effect on molecular signalling pathways, physiological changes and UV‐B stress resistance in Arabidopsis. Both exogenous and endogenous melatonin affected expression of UV‐B signal transduction pathway genes. Experiments using UV‐B signalling component mutants cop1‐4 and hy5‐215 revealed that melatonin not only acts as an antioxidant to promote UV‐B stress resistance, but also regulates expression of several key components of UV‐B signalling pathway, including ubiquitin‐degrading enzyme (COP1), transcription factors (HY5, HYH) and RUP1/2. Our findings indicate that melatonin delays and subsequently enhances expression of COP1, HY5, HYH and RUP1/2, which act as central effectors in UV‐B signalling pathway, thus regulating their effects on antioxidant systems to protect the plant from UV‐B stress.
Several studies have demonstrated that melatonin plays a role in UV‐B responses, however, the molecular mechanism whereby melatonin affects the UV‐B pathway was not clear. This study examined the function of melatonin in molecular signaling pathways, physiological changes, and UV‐B stress resistance under UV‐B radiation in Arabidopsis. Exogenous melatonin treatment experiment indicated that melatonin could enhance the transcriptional level of genes on UV‐B signaling pathway and ameliorate ROS damage caused by UV‐B stress. This result was verified in SNAT overexpressing lines and knock‐down mutant.
The bone remodelling cycle Kenkre, J S; Bassett, Jhd
Annals of clinical biochemistry,
05/2018, Letnik:
55, Številka:
3
Journal Article
Recenzirano
Odprti dostop
The bone remodelling cycle replaces old and damaged bone and is a highly regulated, lifelong process essential for preserving bone integrity and maintaining mineral homeostasis. During the bone ...remodelling cycle, osteoclastic resorption is tightly coupled to osteoblastic bone formation. The remodelling cycle occurs within the basic multicellular unit and comprises five co-ordinated steps; activation, resorption, reversal, formation and termination. These steps occur simultaneously but asynchronously at multiple different locations within the skeleton. Study of rare human bone disease and animal models have helped to elucidate the cellular and molecular mechanisms that regulate the bone remodelling cycle. The key signalling pathways controlling osteoclastic bone resorption and osteoblastic bone formation are receptor activator of nuclear factor-κB (RANK)/RANK ligand/osteoprotegerin and canonical Wnt signalling. Cytokines, growth factors and prostaglandins act as paracrine regulators of the cycle, whereas endocrine regulators include parathyroid hormone, vitamin D, calcitonin, growth hormone, glucocorticoids, sex hormones, and thyroid hormone. Disruption of the bone remodelling cycle and any resulting imbalance between bone resorption and formation leads to metabolic bone disease, most commonly osteoporosis. The advances in understanding the cellular and molecular mechanisms underlying bone remodelling have also provided targets for pharmacological interventions which include antiresorptive and anabolic therapies. This review will describe the remodelling process and its regulation, discuss osteoporosis and summarize the commonest pharmacological interventions used in its management.
Calcium (Ca2+) serves as an essential nutrient as well as a signaling agent in all eukaryotes. In plants, calcineurin B-like proteins (CBLs) are a unique group of Ca2+ sensors that decode Ca2+ ...signals by activating a family of plant-specific protein kinases known as CBL-interacting protein kinases (CIPKs). Interactions between CBLs and CIPKs constitute a signaling network that enables information integration and physiological coordination in response to a variety of extracellular cues such as nutrient deprivation and abiotic stresses. Studies in the past two decades have established a unified paradigm that illustrates the functions of CBL–CIPK complexes in controlling membrane transport through targeting transporters and channels in the plasma membrane and tonoplast.
A novel type of Ca2+ sensors, termed as calcineurin B-like proteins (CBLs), were identified in plant cells 20 years ago. They specifically target a family of plant-specific CBL-interacting protein kinases (CIPKs).To decode a Ca2+ signal, CBL binds Ca2+ and interacts with CIPK, leading to activation of the kinase. The CBL–CIPK complex phosphorylates downstream target proteins and changes their biological activities.Most CBL proteins are localized to the cell membranes and, as a result, CBL–CIPK complexes are largely associated with membranes. This unique feature underlies the core function of the CBL–CIPK network in regulating various membrane transport processes in the plasma membrane and the tonoplast, thereby linking Ca2+ signaling to plant nutrient sensing and homeostasis.
T cells orchestrate adaptive immunity against pathogens and other immune challenges, but their dysfunction can also mediate the pathogenesis of cancer and autoimmunity. Metabolic adaptation in ...response to immunological and microenvironmental signals contributes to T cell function and fate decision. Lipid metabolism has emerged as a key regulator of T cell responses, with selective lipid metabolites serving as metabolic rheostats to integrate environmental cues and interplay with intracellular signaling processes. Here, we discuss how extracellular, de novo synthesized and membrane lipids orchestrate T cell biology. We also describe the roles of lipids as regulators of intracellular signaling at the levels of transcriptional, epigenetic and post-translational regulation in T cells. Finally, we summarize therapeutic targeting of lipid metabolism and signaling, and conclude with a discussion of important future directions. Understanding the molecular and functional interplay between lipid metabolism and T cell biology will ultimately inform therapeutic intervention for human disease.
Pathogen-associated molecular patterns (PAMPs) activate innate immunity in both animals and plants. Although calcium has long been recognized as an essential signal for PAMP-triggered immunity in ...plants, the mechanism of PAMP-induced calcium signalling remains unknown
. Here we report that calcium nutrient status is critical for calcium-dependent PAMP-triggered immunity in plants. When calcium supply is sufficient, two genes that encode cyclic nucleotide-gated channel (CNGC) proteins, CNGC2 and CNGC4, are essential for PAMP-induced calcium signalling in Arabidopsis
. In a reconstitution system, we find that the CNGC2 and CNGC4 proteins together-but neither alone-assemble into a functional calcium channel that is blocked by calmodulin in the resting state. Upon pathogen attack, the channel is phosphorylated and activated by the effector kinase BOTRYTIS-INDUCED KINASE1 (BIK1) of the pattern-recognition receptor complex, and this triggers an increase in the concentration of cytosolic calcium
. The CNGC-mediated calcium entry thus provides a critical link between the pattern-recognition receptor complex and calcium-dependent immunity programs in the PAMP-triggered immunity signalling pathway in plants.
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To date, cancer is the second leading cause of death worldwide after cardiac arrest. A large number of synthetic drugs are available for the treatment of different types of cancer; ...however, a major problem associated with these drugs is its toxicity towards the normal cells. To overcome these problems, researchers explore plants derived phytochemicals because of their pleiotropic action and least toxicity towards the normal cells. Tangeretin is a polymethoxylated flavone found extensively in citrus fruits and has shown potent anti-cancer activity in different types of cancer cells. Hence, this review examines the anti-cancer activity of tangeretin via different molecular targets/pathways. Tangeretin induces apoptosis via intrinsic as well as extrinsic pathways and arrest the cell cycle. It also suppresses cell proliferation by modulating PI3K/AKT/mTOR, Notch, and MAPK signalling pathways. Besides, it induces autophagic cell death, suppresses migration, invasion, and angiogenesis. Further, the role of tangeretin in multi-drug resistance and combination therapy, different biological sources of tangeretin, its derivatives, and pharmacokinetics profile and toxicity studies are also discussed. Towards the end, the challenges associated with tangeretin usage as potential anti-cancer phytochemicals have also been discussed. Tangeretin, like a pandora's box, needs to be explored further, and more research is warranted to improve its usefulness for better human health.
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