Signaling Cascade Research Articles

VEGF-dependent testicular vascularisation involves MEK1/2 signalling and the essential angiogenesis factors, SOX7 and SOX17

BackgroundAbnormalities of in utero testis development are strongly associated with reproductive health conditions, including male infertility and testis cancer. In mouse testes, SOX9 and FGF9 support Sertoli cell development, while VEGF signalling is essential for the establishment of vasculature. The mitogen-activated protein kinase (MAPK) pathway is a major signalling cascade, essential for cell proliferation, differentiation and activation of Sry during primary sex-determination, but little is known about its function during fetal testis morphogenesis. We explored potential functions of MAPK signalling immediately after the establishment of testis cords in embryonic day (E)12.5 Oct4-eGFP transgenic mouse testes cultured using a MEK1/2 inhibitor.ResultsRNA sequencing in isolated gonadal somatic cells identified 116 and 114 differentially expressed genes after 24 and 72 h of MEK1/2 inhibition, respectively. Ingenuity Pathway Analysis revealed an association of MEK1/2 signalling with biological functions such as angiogenesis, vasculogenesis and cell migration. This included a failure to upregulate the master transcriptional regulators of vascular development, Sox7 and Sox17, VEGF receptor genes, the cell adhesion factor gene Cd31 and a range of other endothelial cell markers such as Cdh5 (encoding VE-cadherin) and gap junction genes Gja4 and Gja5. In contrast, only a small number of Sertoli cell enriched genes were affected. Immunofluorescent analyses of control testes revealed that the MEK1/2 downstream target, ERK1/2 was phosphorylated in endothelial cells and Sertoli cells. Inhibition of MEK1/2 eliminated pERK1/2 in fetal testes, and CD31, VE-cadherin, SOX7 and SOX17 and endothelial cells were lost. Consistent with a role for VEGF in driving endothelial cell development in the testis, inhibition of VEGFR also abrogated pERK1/2 and SOX7 and SOX17 expressing endothelial cells. Moreover, while Sertoli cell proliferation and localisation to the testis cord basement membrane was disrupted by inhibition of MEK1/2, it was unaffected by VEGFR inhibition. Instead, inhibition of FGF signalling compromised Sertoli cell proliferation and localisation to the testis cord basement membrane.ConclusionsTogether, our data highlight an essential role for VEGF-dependent MEK1/2 signalling in promoting vasculature and indicate that FGF signalling through MEK1/2 regulates Sertoli cell organisation in the developing mouse testis.

Kukoamine A alleviates nephrolithiasis by inhibiting endogenous oxalate synthesis via the IL-6/JAK/STAT3/DAO signaling pathway

BackgroundThe recurrent nature and socioeconomic burden of nephrolithiasis demand effective treatments. Delineating the crosstalk between inflammatory processes and the endogenous oxalate metabolism pathway, which underpins nephrolithiasis pathogenesis, is essential for advancing treatment strategies. PurposeWe aim to screen therapeutic Chinese herbal remedies and their bioactive constituents for kidney stone treatment using a fruit fly model, followed by efficacy and mechanism validation in a rodent nephrolithiasis model as well as in vitro human cell culture model. Study design and methodsWe developed a fruit fly model to screen for efficient traditional Chinese medicine herbs and their active compounds for kidney stone treatment. Candidate active compounds from efficient herbs were separated and identified by solid-phase chromatography coupled with LC-MS analysis. Fruit fly genetic tools were employed to manipulate the expression of related genes to explore the therapeutic mechanisms of the Lycii Cortex and kukoamine A (KuA). To confirm the therapeutic effects and mechanisms of KuA for mammalian nephrolithiasis, mouse model of glyoxylate-induced kidney stone and human renal tubular cells were used. The therapeutic role of kukoamine A in nephrolithiasis was evaluated by assessing tubular injury, crystal deposition, and adhesion. The level of expression and phosphorylation in cells and mice was assessed using RT-qPCR and western blot. ResultsOur findings indicate that Lycii Cortex potently inhibits calcium oxalate stone formation via activation of the JNK/Upd3/JAK/STAT signaling cascade, resulting in diminished endogenous oxalate synthesis by downregulating D-amino acid oxidase (DAO) gene expression, predominantly in fruit fly Malpighian tube stellate cells. KuA was identified as the principal bioactive constituent mediating these effects. Both mouse models and human cell assays confirmed KuA's efficacy in preventing calcium oxalate nephrolithiasis in mammals, through hepatic JAK/STAT3 pathway activation and upregulation of IL-6, culminating in reduced urinary crystal deposition. ConclusionOur research underscores the potential of kukoamine A as a lead compound in treating nephrolithiasis and reveals the interplay between the IL-6/JAK/STAT3 inflammatory pathway and endogenous oxalate metabolism in nephrolithiasis pathogenesis. Additionally, it highlights the utility of the fruit fly model as a powerful tool for deciphering the therapeutic mechanisms of traditional Chinese herbs.

PHF10 inhibits gastric epithelium differentiation and induces gastric cancer carcinogenesis.

Gastric cancer (GC) is characterized with differentiation disorders, the precise mechanisms of which remain unknown. Our previous study showed that PHF10 exhibits oncogenic properties in GC, with its histological presentation indicating a potential role in the modulation of differentiation disorders in GC. This study reveals a significant upregulation of PHF10 in GC tissues, showing a negative correlation with differentiation level. PHF10 was found to impede the differentiation of GC cells while promoting their stemness properties. This was attributed to the formation of a positive feedback loop between PHF10 and E2F1, resulting in dysregulated expression levels in GC. Additionally, PHF10 was found to mediate the transcriptional repression of the target gene DUSP5 in GC cells through the assembly of the SWI/SNF complex, leading to an elevation in pERK1/2 levels. In GC tissues, a negative association was noted between the expression of E2F1 or PHF10 and DUSP5, whereas a positive correlation was observed between the expression of E2F1 or PHF10 and pERK1/2. Additional rescue experiments confirmed that the inhibitory effect on differentiation of GC cells by PHF10 is dependent on the DUSP5-pERK1/2 axis. The signaling cascade involving E2F1-PHF10-DUSP5-pERK1/2 was identified as an important player in regulating differentiation and stemness in GC cells. PHF10 emerges as a promising target for differentiation induction therapy in GC.

Unveiling signaling pathways inducing MHC class II expression in neutrophils.

Gram-negative bacillary bacteremia poses a significant threat, ranking among the most severe infectious diseases capable of triggering life-threatening sepsis. Despite the unambiguous involvement of neutrophils in this potentially fatal disease, there are limited data about the molecular signaling mechanisms, phenotype, and function of human neutrophils during the early phase of gram-negative bacillary bacteremia. By using an unbiased proteomics and flow cytometry approach, we identified an antigen-presenting cell (APC)-like phenotype in human peripheral blood neutrophils (PMN) with MHC class II molecule expression in the early phase of bacteremia. Using an in-vitro model of GM-CSF-mediated induction of APC-like phenotype in PMN, we investigated downstream signaling pathways leading to MHC class II expression. GM-CSF stimulation of neutrophils leads to the activation of three major signaling pathways, the JAK-STAT, the mitogen-activated protein kinase (MAPK), and the phosphoinositide 3-kinase (PI3K)-Akt-mTOR pathways, while MHC class II induction is mediated by a MAPK-p38-MSK1-CREB1 signaling cascade and the MHC class II transactivator CIITA in a strictly JAK1/2 kinase-dependent manner. This study provides new insights into the signaling pathways that induce MHC class II expression in neutrophils, highlighting the potential for therapeutic targeting of JAK1/2 signaling in the treatment of gram-negative bacteremia and sepsis. Understanding these mechanisms may open up novel approaches for managing inflammatory responses during sepsis.

Reactive Oxygen Species in Cystic Kidney Disease.

Polycystic kidney disease (PKD) is a rare but significant renal condition with major implications for global acute and chronic patient care. Oxidative stress and reactive oxygen species (ROS) can significantly alter its pathophysiology, clinical outcomes, and treatment, contributing to negative outcomes, including hypertension, chronic kidney disease, and kidney failure. Inflammation from ROS and existing cysts propagate the generation and accumulation of ROS, exacerbating kidney injury, pro-fibrotic signaling cascades, and interstitial fibrosis. Early identification and prevention of oxidative stress and ROS can contribute to reduced cystic kidney disease progression and improved longitudinal patient outcomes. Increased research regarding biomarkers, the pathophysiology of oxidative stress, and novel therapeutic interventions alongside the creation of comprehensive guidelines establishing methods of assessment, monitoring, and intervention for oxidative stress in cystic kidney disease patients is imperative to standardize clinical practice and improve patient outcomes. The integration of artificial intelligence (AI), genetic editing, and genome sequencing could further improve the early detection and management of cystic kidney disease and mitigate adverse patient outcomes. In this review, we aim to comprehensively assess the multifactorial role of ROS in cystic kidney disease, analyzing its pathophysiology, clinical outcomes, treatment interventions, clinical trials, animal models, and future directions for patient care.

Alleviation of Copper-Induced Hepatotoxicity by Bergenin: Diminution of Oxidative Stress, Inflammation, and Apoptosis via Targeting SIRT1/FOXO3a/NF-κB Axes and p38 MAPK Signaling.

Despite its biological importance, excess copper induces organ damage, especially to the liver. Disruption of critical signaling cascades that control redox status, inflammatory responses, and cellular apoptosis significantly contributes to the copper-induced hepatotoxicity. The present work explored the hepatoprotective ability of bergenin against the copper-induced hepatotoxicity using male Wistar rats as a mammalian model. The results revealed that bergenin suppressed the copper-evoked histopathological changes and hepatocellular necrosis as indicated by decreased activity of the liver enzymes ALT and AST in the sera of the copper-intoxicated rats. It decreased hepatic copper content and the copper-induced oxidative stress as revealed by reduced lipid peroxidation and improved activity of the antioxidant enzymes thioredoxin reductase, glutathione peroxidase, catalase, and superoxide dismutase. Bergenin downregulated the inflammatory cytokines TNF-α and IL-6, and the inflammatory cell infiltration to the liver tissues. Additionally, it inhibited the copper-induced apoptosis as indicated by significant reduction in caspase-3 activity. At the molecular level, bergenin activated the antioxidant transcription factor FOXO3a, inhibited the nuclear translocation of the inflammatory transcription factor NF-κB, and suppressed the inflammatory signaling molecules p38 MAPK and c-Fos. Interestingly, bergenin improved the expression of the anti-apoptotic protein Bcl2 and reduced the pro-apoptotic protein BAX. Bergenin markedly enhanced the expression of the histone deacetylase protein SIRT1 that regulates activity of NF-κB and FOXO3a. Collectively, these findings highlight the alleviating activity of bergenin against the copper-induced hepatotoxicity via controlling oxidative stress, inflammation, and apoptosis potentially through upregulation of SIRT1, activation of FOXO3a along with suppression of NF-κB and p38 MAPK signaling.

The role of PTPN11 in MAPK/ERK signaling pathway and its impact on the occurrence and development of glioma

The protein tyrosine phosphatase, PTPN11, exerts a crucial inhibitory influence on the MAPK/ERK signaling cascade. This research endeavored to unravel the involvement of PTPN11 in the initiation and progression of glioma, as well as its underlying molecular interactions. To achieve this, Western blotting was employed to observe alterations in ERK phosphorylation upon PTPN11 depletion or amplification. Furthermore, immunoprecipitation coupled with mass spectrometry was utilized to pinpoint the targets of PTPN11's enzymatic activity. The outcomes demonstrated that the absence of PTPN11 significantly enhanced ERK phosphorylation, whereas an excess of the protein had the converse effect. In-depth explorations into the molecular mechanisms highlighted that PTPN11's inhibitory action is chiefly executed through the deactivation of Ras proteins by dephosphorylation. These revelations accentuate the critical part played by PTPN11 within the MAPK/ERK pathway in relation to glioma development and offer insights that could lead to innovative treatments for this disease.

Phlpp1 alters the murine chondrocyte phospho-proteome during endochondral bone formation

Appendicular skeletal growth and bone mass acquisition are controlled by a variety of growth factors, hormones, and mechanical forces in a dynamic process called endochondral ossification. In long bones, chondrocytes in the growth plate proliferate and undergo hypertrophy to drive bone lengthening and mineralization. Pleckstrin homology (PH) domain and leucine rich repeat phosphatase 1 and 2 (Phlpp1 and Phlpp2) are serine/threonine protein phosphatases that regulate cell proliferation, survival, and maturation via Akt, PKC, Raf1, S6k, and other intracellular signaling cascades. Germline deletion of Phlpp1 suppresses bone lengthening in growth plate chondrocytes. Here, we demonstrate that Phlpp2 does not regulate endochondral ossification, and we define the molecular differences between Phlpp1 and Phlpp2 in chondrocytes. Phlpp2−/− mice were phenotypically indistinguishable from their wildtype (WT) littermates, with similar bone length, bone mass, and growth plate dynamics. Deletion of Phlpp2 had moderate effects on the chondrocyte transcriptome and proteome compared to WT cells. By contrast, Phlpp1/2−/− (double knockout) mice resembled Phlpp1−/− mice phenotypically and molecularly, as the chondrocyte phospho-proteomes of Phlpp1−/− and Phlpp1/2−/− chondrocytes had similarities and were significantly different from WT and Phlpp2−/− chondrocyte phospho-proteomes. Data integration via multiparametric analysis showed that the transcriptome explained less variation in the data as a result of Phlpp1 or Phlpp2 deletion than proteome or phospho-proteome. Alterations in cell proliferation, collagen fibril organization, and Pdpk1 and Pak1/2 signaling pathways were identified in chondrocytes lacking Phlpp1, while cell cycle processes and Akt1 and Aurka signaling pathways were altered in chondrocytes lacking Phlpp2. These data demonstrate that Phlpp1, and to a lesser extent Phlpp2, regulate multiple and complex signaling cascades across the chondrocyte transcriptome, proteome, and phospho-proteome and that multi-omic data integration can reveal novel putative kinase targets that regulate endochondral ossification.

Myricetin-loaded SBA-15 silica nanoparticles for enhanced management of pyrexia, pain, and inflammation through modulation of MAPK/NF-κB and COX-2/PGE-2 pathways: Evidence from the biochemical, histological, and metabolomic analysis

Myricetin (MYR) is a natural flavonoid that has several biological functions. However, some of its beneficial effects are diminished due to low water solubility, stability, and bioavailability. Herein, several kinds of silica nanoparticles (MCM-41 and SBA-15) were loaded with MYR to improve its biological activity as an analgesic, antipyretic, and anti-inflammatory component, thereby overcoming its drawbacks. The nanoparticles (MYR@SBA-15) were formulated optimally, transforming MYR into an amorphous state. This transformation was confirmed via several strategies, including differential scanning calorimetry, Fourier transform infrared spectroscopy, and powder x-ray diffraction. As a result, there was a significant enhancement in the solubility and rate of dissolution in water. The anti-inflammatory benefits as an innovative strategy and the underlying mechanism of action of MYR and its SBA-15 silica nanoparticles (MYR@SBA-15) were investigated based on the biochemical, histological, immunohistochemical, and metabolomic assays alongside their antipyretic and analgesic characteristics. Compared to the usage of raw MYR, the administration of MYR@SBA-15 at doses of 25, 50, and 100 mg/kg significantly decreases pain perception by inhibiting the body’s writhing motions induced by acetic acid. Furthermore, it helps regulate increased body temperature caused by baking yeast and effectively stabilizes it. It reduces the release of NO and PGE-2 in a concentration-dependent manner by down-regulating iNOS and COX-2 expression in the inflammatory model. MYR and MYR@SBA-15 also inhibit the nuclear translocation of NF-κB, downregulate the expression of mitogen-activated protein kinases (MAPKs), such as p38, ERK1/2, and JNK protein, and reduce the generation of proinflammatory cytokines, such as TNF-α. In addition, inflammatory cardinal signs like paw edema caused by carrageenan in rats are greatly suppressed by MYR and MYR@SBA-15 treatment when compared to the untreated group. More noteworthy outcomes are shown in the MYR@SBA-15, particularly at a dose of 100 mg/kg. These results of biochemical and immuno-histochemistry suggest that MYR@SBA-15 may be a useful analgesic antipyretic and may also help reduce inflammation by altering MAPKs/NF-κB and COX-2/PGE-2 signaling cascades. Serum metabolomics study demonstrated modifications in various low molecular weight metabolites with arthritis development. These metabolite levels were restored to normal when MYR@SBA-15 was administered via modulating several metabolic pathways, i.e., pyrimidine, energy metabolism, and proteins. Overall, MYR-loaded SBA-15 silica nanoparticles have demonstrated significant promise in enhancing the disturbed metaboloic pathways and providing a substantial capacity to regulate several oxidative stress and inflammatory mediators.

Thyroid Endocrine Disruption and Mechanism of the Marine Antifouling Pollutant 4,5-Dichloro-2-n-octyl-4-isothiazolin-3-one.

The antifoulant 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT) is an emerging pollutant in the marine environment, which may disrupt the thyroid endocrine system. However, DCOIT toxicity in relation to thyroid endocrine disruption and the underlying mechanisms remains largely unclear. In this study, in vivo, in silico, in vitro, and ex vivo assays were performed to clarify DCOIT's thyroid toxicity. First, marine medaka (Oryzias melastigma) were exposed to environmentally realistic concentrations of DCOIT for an entire life cycle. The results demonstrated that DCOIT exposure potently stimulated the hypothalamic-pituitary-thyroid axis, characterized by hyperthyroidism symptom induction and prevalent key gene and protein upregulation in the brain. Moreover, the in silico and in vitro results evidenced that DCOIT could bind to thyroid hormone receptor β (TRβ) and interact synergistically with triiodothyronine, thus promoting GH3 cell proliferation. The CUT&Tag experiment found that DCOIT interfered with the affinity fingerprint of TRβ to target genes implicated in thyroid hormone signaling cascade regulation. Furthermore, ex vivo, Chem-seq revealed that DCOIT directly bound to the genomic sequences of thyrotropin-releasing hormone receptor b and thyroid-stimulating hormone receptor in marine medaka brain tissues. In conclusion, the current multifaceted evidence confirmed that DCOIT has a strong potency for thyroid endocrine system disruption and provided comprehensive insights into its toxicity mechanisms.

Development of an Ultrasensitive Competitive Double-Enzyme Cascade Fluorescence Signal Amplification Method for Factor XIa Inhibitor Screening.

Coagulation factor XIa (FXIa) is associated with a low risk of bleeding and has been identified as an effective and safe target for the development of novel anticoagulant drugs. In this study, we established an ultrasensitive competitive dual-enzyme cascade signal amplification method for the quantitative analysis and screening of FXIa inhibitors. Due to the specific recognition of FXIa's active site by the aptamer AptE40, the AptE40-QDs-EK recognition probe modified with enterokinase (EK) and the aptamer AptE40, was attached to the MNPs-FXIa capture probe. When FXIa inhibitor was present, it competed with AptE40 for binding to FXIa, resulting in the detachment of AptE40-QDs-EK from MNPs-FXIa. After magnetic separation, the enterokinase of AptE40-QDs-EK in the supernatant hydrolyzed N-terminal hexapeptide of trypsinogen, leading to the production of a large amount of trypsin as part of the first-stage signal cascade amplification. Next, trypsin could hydrolyze the hexameric arginine peptide (RRRRRR, R6), leading to the dissociation of RQDs from the R6-RQDs signal probe; this resulted in a dramatic increase in the fluorescence intensity of the supernatant as the second-stage signal cascade was amplified. The feasibility of the method was investigated using the FXIa inhibitor aptamer FELIAP as a positive model drug. Furthermore, the method was applied to screen the FXIa inhibitors in Eupolyphaga sinensis Walker. Two fractions with more active anticoagulated ingredients were successfully identified and validated via the conventional method, and the results were consistent. The established method provides a key technique for the sensitive detection, high-throughput analysis, and screening of the FXIa inhibitors.

Neuronal LAMP2A-mediated reduction of adenylyl cyclases induces acute neurodegenerative responses and neuroinflammation after ischemic stroke.

Lysosomes regulate cellular metabolism to maintain cell survival, but the mechanisms whereby they determine neuronal cell fate after acute metabolic stress are unknown. Neuron-enriched lysosomal membrane protein LAMP2A is involved in selective chaperone-mediated autophagy and exosome loading. This study demonstrates that abnormalities in the neuronal LAMP2A-lysosomal pathway cause neurological deficits following ischemic stroke and that this is an early inducer of the PANoptosis-like molecular pathway and neuroinflammation, simultaneously inducing upregulation of FADD, RIPK3, and MLKL after ischemia. Quantitative proteomic and pharmacological analysis showed that after acute metabolic stress, the neuronal LAMP2A pathway induced acute synaptic degeneration and PANoptosis-like responses involving downregulation of protein kinase A (PKA) signaling. LAMP2A directed post-stroke lysosomal degradation of adenylyl cyclases (ADCY), including ADCY1 and ADCY3 in cortical neurons. Post-stroke treatment with cAMP mimetic or ADCY activator salvaged cortical neurons from PANoptosis-like responses and neuroinflammation, suggesting that the neuronal ADCY-cAMP-PKA axis is an upstream arrester of the pathophysiological process following an ischemic stroke. This study demonstrates that the neuronal LAMP2A-lysosmal pathway drives intricate acute neurodegenerative and neuroinflammatory responses after brain metabolic stress by downregulating the ADCY-PKA signaling cascade, and highlights the therapeutic potential of PKA signal inducers for improving stroke outcomes.

Rocaglamide-A mitigates LPS-induced hepatic inflammation by modulating JNK/AP-1 signaling cascade and ROS production in hepatocytes

Rocaglamide-A mitigates LPS-induced hepatic inflammation by modulating JNK/AP-1 signaling cascade and ROS production in hepatocytes

Demethylzeylasteral alleviates inflammation and colitis via dual suppression of NF-κB and STAT3/5 by targeting IKKα/β and JAK2

BackgroundUlcerative colitis (UC) is a common inflammatory bowel disease and a risk factor of colorectal cancer. Demethylzeylasteral (DZT), a bioactive component mainly isolated from Tripterygium wilfordii, has been shown to inhibit inflammation and cancer. However, its anti-UC function and molecular mechanisms have not been well characterized. This study aims to explore the therapeutic effect and functional targets of demethylzeylasteral against UC. MethodsRT-qPCR, Western blot and ELISA were used to detect the generation of pro-inflammatory cytokines and chemokines in murine macrophage cells. Luciferase reporter gene, Western blot, pull-down, CETSA, DARTS, and virtual docking were employed to detect the anti-inflammatory targets and molecular mechanisms of demethylzeylasteral. The anti-inflammatory and anti-colitis effects of demethylzeylasteral were further determined in DSS-challenged mice. ResultsIn vitro, demethylzeylasteral inhibited NO and PGE2 production by suppressing the mRNA and protein expression of iNOS and COX-2, and suppressed the mRNA expression of TNF-α, IL-1β, IL-6, MCP-1, CXCL9, and CXCL10 in RAW264.7 macrophages stimulated by LPS/IFNγ. Furthermore, demethylzeylasteral was not only capable of inhibiting IKKα/β-NF-κB activation, but also able to block JAKs-STAT3/5 activation in LPS/INFγ-incubated RAW264.7 cells or DSS-exposed colon tissues of mice. Mechanistically, demethylzeylasteral was found to directly bind to IKKα/β and JAK2 kinases, leading to inactivation of pro-inflammatory signaling cascades and reduced generation of cytokines and chemokines. In vivo, oral administration of demethylzeylasteral significantly attenuated DSS-induced colitis, which was mainly manifested as mitigated symptoms of colitis, colonic mucosal barrier damage, and colonic inflammation. ConclusionWe demonstrated that demethylzeylasteral alleviated UC pathology by blocking NF-κB and STAT3/5 pathways via targeting IKKα/β and JAK2 kinases, raising the possibility that demethylzeylasteral could act as a candidate for the treatment of UC.

0JAK Inhibitor Withdrawal Causes a Transient Proinflammatory Cascade: A Potential Mechanism for Major Adverse Cardiac Events.

Our objective was to define the effect of JAK1/2 inhibitor (JAKinib) withdrawal on JAK/STAT biochemical response in the context of systemic rheumatic diseases. We tested Type I (bind kinase active conformation) and Type II (bind kinase inactive conformation) JAKinibs in vitro using mesenchymal stromal cells and endothelial cells. We translated our findings in vivo studying NK cells from rheumatoid arthritis (RA) patients treated with Type I JAKinibs or methotrexate. Type I JAKinibs (ruxolitinib and baricitinib) increased phosphoJAK1 (pJAK1) and pJAK2 of IFNγ-stimulated MSCs and HUVECs in a time- and dose- dependent manner, with effect peaking after 24 hours. As expected, pSTAT1 was completely suppressed by JAKinibs. We found a marked and rapid increase of pSTATs upon discontinuation of Type I JAKinibs, that occurred to a lesser extent after Type II JAKinib withdrawal. Type I JAKinib withdrawal increased interferon and urokinase expression when compared to Type II JAKinib withdrawal. We found NK cells from RA patients taking Type I JAKinibs had a pro-inflammatory profile after JAKinib withdrawal compared to patients on methotrexate. Type I JAKinibs paradoxically accumulate functionally defective pJAK. Upon withdrawal, the primed pJAKs are de-repressed and initiate a pSTAT signaling cascade, resulting in high interferon and urokinase. Type II JAKinibs do not cause pJAK accumulation, pSTAT cascade, and subsequent pro-inflammatory transcripts. The resultant cytokines and proteins produced from this cascade might be associated with adverse cardiac outcomes. Thus, JAKinib withdrawal is a possible mechanism contributing to the major adverse cardiac events described with JAKinib therapy.

MTOR/miR-142-3p/PRAS40 signaling cascade is critical for tuberous sclerosis complex-associated renal cystogenesis

BackgroundPatients with tuberous sclerosis complex (TSC) develop renal cysts and/or angiomyolipomas (AMLs) due to inactive mutations of either TSC1 or TSC2 and consequential mTOR hyperactivation. The molecular events between activated mTOR and renal cysts/AMLs are still largely unknown.MethodsThe mouse model of TSC-associated renal cysts were constructed by knocking out Tsc2 specifically in renal tubules (Tsc2f/f; ksp-Cre). We further globally deleted PRAS40 in these mice to investigate the role of PRAS40. Tsc2−/− cells were used as mTOR activation model cells. Inhibition of DNA methylation was used to increase miR-142-3p expression to examine the effects of miR-142-3p on PRAS40 expression and TSC-associated renal cysts.ResultsPRAS40, a component of mTOR complex 1, was overexpressed in Tsc2-deleted cell lines and mouse kidneys (Tsc2f/f; ksp-Cre), which was decreased by mTOR inhibition. mTOR stimulated PRAS40 expression through suppression of miR-142-3p expression. Unleashed PRAS40 was critical to the proliferation of Tsc2−/− cells and the renal cystogenesis of Tsc2f/f; ksp-Cre mice. In contrast, inhibition of DNA methylation increased miR-142-3p expression, decreased PRAS40 expression, and hindered cell proliferation and renal cystogenesis.ConclusionsOur data suggest that mTOR activation caused by TSC2 deletion increases PRAS40 expression through miR-142-3p repression. PRAS40 depletion or the pharmacological induction of miR-142-3p expression impaired TSC2 deficiency-associated renal cystogenesis. Therefore, harnessing mTOR/miR-142-3p/PRAS40 signaling cascade may mitigate hyperactivated mTOR-related diseases.

Inhibition of the NLRP3/caspase-1 cascade related pyroptosis relieved propofol-induced neuroinflammation and cognitive impairment in developing rats

BackgroundNumerous preclinical studies have demonstrated that prolonged exposure to propofol (A general anaesthetics) can lead to hippocampus injury in immature brains and impact long-term learning and memory functions. Neuroinflammation plays a pivotal role in the impairment of brain function associated with early exposure to anesthetic drugs. Nevertheless, the involvement of hippocampal pyroptosis and neuroinflammation mediated by the NLRP3/caspase-1 signaling cascade in propofol-induced developmental neurotoxicity remains unclear. MethodsPostnatal day (PND) 7 SD rats, PC12 cells, and HAPI cells were used to establish propofol neurotoxicity models in vivo and in vitro, respectively. We examined the potential hippocampal injury and cognitive dysfunction caused by propofol in neonatal rats through the NLRP3/caspase-1 signaling pathway using MCC950 and VX765 to inhibit the pathway. This investigation involved assessing histological changes in the hippocampus, behavioral performance in adulthood, NLRP3-related pyroptosis indicators, and neuroinflammatory cytokines. ResultsBoth in vivo and in vitro studies have demonstrated that exposure to propofol activates the NLRP3/caspase-1 signaling cascade in the hippocampus of PND7 rats, leading to pyroptosis, neuroinflammation, and subsequent hippocampal injury and behavioral changes in adulthood. However, MCC950 and VX765 inhibit the NLRP3/caspase-1 signaling cascade, reversing the developmental neurotoxicity of propofol. ConclusionOur study findings suggest that negative regulation of NLRP3/caspase-1 activation may serve as a potential therapeutic strategy for developmental neuroinflammation induced by propofol.

A SRC-slug-TGFβ2 signaling axis drives poor outcomes in triple-negative breast cancers

SummaryIn our study, we focused on a particular subtype of aggressive breast cancer called Triple-Negative Breast Cancer (TNBC). We investigated a complex series of events that contribute to poor outcomes in this disease and uncovered a crucial signaling cascade driving tumor growth and progression.At the core of this signaling cascade are three key proteins: SRC, AKT, and ERK2. Together, they form a pathway that activates a transcription factor called Slug. Transcription factors act like molecular switches, controlling the expression of genes. Once Slug is activated, it strongly suppresses genes that would normally restrict cell growth and cell spread.One of the genes downregulated by Slug is TGFB2-AS1. This product of the TGFB2-AS1 gene normally controls levels of its target protein called TGF-beta2 (TGFB2), a protein which has roles in cell growth, cell migration and differentiation. Slug downregulation of TGFB2-AS1 results in higher TGFB2 levels, and this in turn contributes to the uncontrolled growth and spread of cancer cells. TGFB2, and other proteins in this pathway (SRC, AKT, ERK2, and a Slug interactor called LSD1) all maintain the stability of Slug, meaning that Slug levels remain high and drive the aggressive features of this subtype of breast cancer.Overall, our research sheds light on the intricate molecular mechanisms driving aggressive TNBC. It also identifies potential targets for future therapies, aimed at disrupting this harmful signaling pathway and potentially improving patient outcomes for this disease.

Location-biased β-arrestin conformations direct GPCR signaling.

β-arrestins are multifunctional intracellular proteins that regulate the desensitization, internalization and signaling of over 800 different G protein-coupled receptors (GPCRs) and interact with a diverse array of cellular partners1,2. Beyond the plasma membrane, GPCRs can initiate unique signaling cascades from various subcellular locations, a phenomenon known as "location bias"3,4. Here, we investigate how β-arrestins direct location-biased signaling of the angiotensin II type I receptor (AT1R). Using novel bioluminescence resonance energy transfer (BRET) conformational biosensors and extracellular signal-regulated kinase (ERK) activity reporters, we reveal that in response to the endogenous agonist Angiotensin II and the β-arrestin-biased agonist TRV023, β-arrestin 1 and β-arrestin 2 adopt distinct conformations across different subcellular locations, which are intricately linked to differential ERK activation profiles. We also uncover a population of receptor-free catalytically activated β-arrestins in the plasma membrane that exhibits insensitivity to different agonists and promotes ERK activation on the plasma membrane independent of G proteins. These findings deepen our understanding of GPCR signaling complexity and also highlight the nuanced roles of β-arrestins beyond traditional G protein pathways.

Copper Dyshomeostasis And Diabetic Complications: Chelation Strategies For Management.

Cuproptosis, An Emerging Concept In The Field Of Diabetes Research, Presents A Novel And Promising Perspective For The Effective Management Of Diabetes Mellitus And Its Associated Complications. Diabetes, Characterized By Chronic Hyperglycemia, Poses A Substantial Global Health Burden, With An Increasing Prevalence Worldwide. Despite Significant Progress In Our Understanding Of This Complex Metabolic Disorder, Optimal Therapeutic Strategies Still Remain Elusive. The Advent Of Cuproptosis, A Term Coined To Describe Copper-Induced Cellular Cell Death And Its Pivotal Role In Diabetes Pathogenesis, Opens New Avenues For Innovative Interventions. Copper, An Indispensable Trace Element, Plays A Pivotal Role In A Myriad Of Vital Biological Processes, Encompassing Energy Production, Bolstering Antioxidant Defenses, And Altered Cellular Signaling. However, In The Context Of Diabetes, This Copper Homeostasis Is Perturbed, Driven By A Combination Of Genetic Predisposition, Dietary Patterns, And Environmental Factors. Excessive Copper Levels Act As Catalysts For Oxidative Stress, Sparking Intricate Intracellular Signaling Cascades That Further Exacerbate Metabolic Dysfunction. In This Review, We Aim To Explore The Interrelationship Between Copper And Diabetes Comprehensively, Shedding Light On The Intricate Mechanisms Underpinning Cuproptosis. By Unraveling The Roles Of Copper Transporters, Copper-Dependent Enzymes, And Cuproptotic Signaling Pathways, We Seek To Elucidate Potential Therapeutic Strategies That Harness The Power Of Copper Modulation In Diabetes Management. This Insight Sets The Stage For A Targeted Approach To Challenge The Complex Hurdles Posed By Diabetes, Potentially Transforming Our Therapeutic Strategies In The Ongoing Fight Against This Pervasive Global Health Concern.