Phosphorylation Of Transcription Factor Research Articles

Targeted ablation of Fn14 receptor improves exercise capacity and inhibits neurogenic muscle atrophy.

Skeletal muscle atrophy is a prevalent complication in multiple chronic diseases and disuse conditions. Fibroblast growth factor-inducible 14 (Fn14) is a member of the TNF receptor superfamily and a bona fide receptor of the TWEAK cytokine. Accumulating evidence suggests that Fn14 levels are increased in catabolic conditions as well as during exercise. However, the role of Fn14 in the regulation of skeletal muscle mass and function remains poorly understood. In this study, through the generation of novel skeletal muscle-specific Fn14-knockout mice, we have investigated the muscle role of Fn14 in the regulation of exercise capacity and denervation-induced muscle atrophy. Our results demonstrate that there was no difference in skeletal muscle mass between control and muscle-specific Fn14-knockout mice. Nevertheless, the deletion of Fn14 in skeletal muscle significantly improved exercise capacity and resistance to fatigue. This effect of Fn14 deletion is associated with an increased proportion of oxidative myofibers and higher capillaries number per myofiber in skeletal muscle. Furthermore, our results demonstrate that targeted deletion of Fn14 inhibits denervation-induced muscle atrophy in adult mice. Deletion of Fn14 reduced the expression of components of the ubiquitin-proteasome system and non-canonical NF-kappa B signaling in denervated skeletal muscle, as well as increased the phosphorylation of Akt kinase and FoxO3a transcription factor. Collectively, our results demonstrate that targeted inhibition of Fn14 improves exercise tolerance and inhibits denervation-induced muscle atrophy in adult mice.

Abstract 15847: Mass Cytometry Reveals Immune Cell Subtypes Responsive to Il-1b and Il-6 Stimulation in CAD Subjects and Controls

Introduction: Inflammation plays a key role in the development of atherosclerosis, yet targeted therapies are lacking. Previous trials have demonstrated that IL-1b is a potential target, albeit with adverse effects on immune suppression. IL-6 has emerged as another mediator of coronary artery disease. It clear that a strategy to identify a biomarker customized for inflammatory inhibition is needed. Mass cytometry shows promise as a technology for customized biomarker identification. Hypothesis: A mass cytometry panel including markers to identify circulating immune subtypes in humans and phosphorylation of key transcription factors driving downstream inflammation can identify cell subtypes most responsive to cytokine stimulation. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from patients undergoing coronary computed tomography angiography (CCTA). Using PBMCs from 12 pairs of matched subjects, we stimulated cells with vehicle, IL-6, and IL-1b. These samples were analyzed using a custom mass cytometry panel. Results: Mass cytometry analysis of PMBCs after 15 minute cytokine stimulation identified specific lymphocyte and monocyte subtypes that were particularly responsive to IL-1b and IL-6. Notably, there was clear heterogeneity in this response suggesting this approach may identify those most impacted by cytokine inhibition or those with most severe disease. Conclusions: High dimensional mass cytometry has the potential to better identify individuals at risk and those that may respond to cytokine inhibition. We are in the process of performing advanced bioinformatic analysis including machine learning to leverage this novel technology for precision medicine.

Calcium/calmodulin-mediated microbial symbiotic interactions in plants.

Cytoplasmic calcium (Ca2+) transients and nuclear Ca2+ oscillations act as hubs during root nodulation and arbuscular mycorrhizal symbioses. Plants perceive bacterial Nod factors or fungal signals to induce the Ca2+ oscillation in the nucleus of root hair cells, and subsequently activate calmodulin (CaM) and Ca2+/CaM-dependent protein kinase (CCaMK). Ca2+ and CaM-bound CCaMK phosphorylate transcription factors then initiate down-stream signaling events. In addition, distinct Ca2+ signatures are activated at different symbiotic stages: microbial colonization and infection; nodule formation; and mycorrhizal development. Ca2+ acts as a key signal that regulates a complex interplay of downstream responses in many biological processes. This short review focuses on advances in Ca2+ signaling-regulated symbiotic events. It is meant to be an introduction to readers in and outside the field of bacterial and fungal symbioses. We summarize the molecular mechanisms underlying Ca2+/CaM-mediated signaling in fine-tuning both local and systemic symbiotic events.

Effects of the Cc2d1a/Freud-1 Knockdown in the Hippocampus of BTBR Mice on the Autistic-Like Behavior, Expression of Serotonin 5-HT1A and D2 Dopamine Receptors, and CREB and NF-kB Intracellular Signaling.

The mechanisms of autism are of extreme interest due to the high prevalence of this disorder in the human population. In this regard, special attention is given to the transcription factor Freud-1 (encoded by the Cc2d1a gene), which regulates numerous intracellular signaling pathways and acts as a silencer for 5-HT1A serotonin and D2 dopamine receptors. Disruption of the Freud-1 functions leads to the development of various psychopathologies. In this study, we found an increase in the expression of the Cc2d1a/Freud-1 gene in the hippocampus of BTBR mice (model of autistic-like behavior) in comparison with C57Bl/6J mice and examined how restoration of the Cc2d1a/Freud-1 expression in the hippocampus of BTBR mice affects their behavior, expression of 5-HT1A serotonin and D2 dopamine receptors, and CREB and NF-κB intracellular signaling pathways in these animals. Five weeks after administration of the adeno-associated viral vector (AAV) carrying the pAAV_H1-2_shRNA-Freud-1_Syn_EGFP plasmid encoding a small hairpin RNA (shRNA) that suppressed expression of the Cc2d1a/Freud-1 gene, we observed an elevation in the anxiety levels, as well as the increase in the escape latency and path length to the platform in the Morris water maze test, which was probably associated with a strengthening of the active stress avoidance strategy. However, the Cc2d1a/Freud-1 knockdown did not affect the spatial memory and phosphorylation of the CREB transcription factor, although such effect was found in C57Bl/6J mice in our previous study. These results suggest the impairments in the CREB-dependent effector pathway in BTBR mice, which may play an important role in the development of the autistic-like phenotype. The knockdown of Cc2d1a/Freud-1 in the hippocampus of BTBR mice did not affect expression of the 5-HT1A serotonin and D2 dopamine receptors and key NF-κB signaling genes (Nfkb1 and Rela). Our data suggest that the transcription factor Freud-1 plays a significant role in the pathogenesis of anxiety and active stress avoidance in autism.

Endothelial β-arrestins regulate mechanotransduction by the type II bone morphogenetic protein receptor in primary cilia.

Modulation of endothelial cell behavior and phenotype by hemodynamic forces involves many signaling components, including cell surface receptors, intracellular signaling intermediaries, transcription factors, and epigenetic elements. Many of the signaling mechanisms that underlie mechanotransduction by endothelial cells are inadequately defined. Here we sought to better understand how β-arrestins, intracellular proteins that regulate agonist-mediated desensitization and integration of signaling by transmembrane receptors, may be involved in the endothelial cell response to shear stress. We performed both in vitro studies with primary endothelial cells subjected to β-arrestin knockdown, and in vivo studies using mice with endothelial specific deletion of β-arrestin 1 and β-arrestin 2. We found that β-arrestins are localized to primary cilia in endothelial cells, which are present in subpopulations of endothelial cells in relatively low shear states. Recruitment of β-arrestins to cilia involved its interaction with IFT81, a component of the flagellar transport protein complex in the cilia. β-arrestin knockdown led to marked reduction in shear stress response, including induction of NOS3 expression. Within the cilia, β-arrestins were found to associate with the type II bone morphogenetic protein receptor (BMPR-II), whose disruption similarly led to an impaired endothelial shear response. β-arrestins also regulated Smad transcription factor phosphorylation by BMPR-II. Mice with endothelial specific deletion of β-arrestin 1 and β-arrestin 2 were found to have impaired retinal angiogenesis. In conclusion, we have identified a novel role for endothelial β-arrestins as key transducers of ciliary mechanotransduction that play a central role in shear signaling by BMPR-II and contribute to vascular development.

Structural basis for FLCN RagC GAP activation in MiT-TFE substrate-selective mTORC1 regulation.

The mechanistic target of rapamycin complex 1 (mTORC1) regulates cell growth and catabolism in response to nutrients through phosphorylation of key substrates. The tumor suppressor folliculin (FLCN) is a RagC/D guanosine triphosphatase (GTPase)–activating protein (GAP) that regulates mTORC1 phosphorylation of MiT-TFE transcription factors, controlling lysosome biogenesis and autophagy. We determined the cryo–electron microscopy structure of the active FLCN complex (AFC) containing FLCN, FNIP2, the N-terminal tail of SLC38A9, the RagAGDP:RagCGDP.BeFx- GTPase dimer, and the Ragulator scaffold. Relative to the inactive lysosomal FLCN complex structure, FLCN reorients by 90°, breaks contact with RagA, and makes previously unseen contacts with RagC that position its Arg164 finger for catalysis. Disruption of the AFC-specific interfaces of FLCN and FNIP2 with RagC eliminated GAP activity and led to nuclear retention of TFE3, with no effect on mTORC1 substrates S6K or 4E-BP1. The structure provides a basis for regulation of an mTORC1 substrate-specific pathway and a roadmap to discover MiT-TFE family selective mTORC1 antagonists.

Dose-dependent phosphorylation and activation of Hh pathway transcription factors.

Graded Hedgehog (Hh) signaling is mediated by graded Cubitus interruptus (Ci)/Gli transcriptional activity, but how the Hh gradient is converted into the Ci/Gli activity gradient remains poorly understood. Here, we show that graded Hh induces a progressive increase in Ci phosphorylation at multiple Fused (Fu)/CK1 sites including a cluster located in the C-terminal Sufu-binding domain. We demonstrated that Fu directly phosphorylated Ci on S1382, priming CK1 phosphorylation on adjacent sites, and that Fu/CK1-mediated phosphorylation of the C-terminal sites interfered with Sufu binding and facilitated Ci activation. Phosphorylation at the N-terminal, middle, and C-terminal Fu/CK1 sites occurred independently of one another and each increased progressively in response to increasing levels of Hh or increasing amounts of Hh exposure time. Increasing the number of phospho-mimetic mutations of Fu/CK1 sites resulted in progressively increased Ci activation by alleviating Sufu-mediated inhibition. We found that the C-terminal Fu/CK1 phosphorylation cluster is conserved in Gli2 and contributes to its dose-dependent activation. Our study suggests that the Hh signaling gradient is translated into a Ci/Gli phosphorylation gradient that activates Ci/Gli by gradually releasing Sufu-mediated inhibition.

Oleanolic acid stimulation of cell migration involves a biphasic signaling mechanism

Cell migration is a critical process for wound healing, a physiological phenomenon needed for proper skin restoration after injury. Wound healing can be compromised under pathological conditions. Natural bioactive terpenoids have shown promising therapeutic properties in wound healing. Oleanolic acid (OA), a triterpenoid, enhances in vitro and in vivo cell migration. However, the underlying signaling mechanisms and pathways triggered by OA are poorly understood. We have previously shown that OA activates epidermal growth factor receptor (EGFR) and downstream effectors such as mitogen-activated protein (MAP) kinase cascade and c-Jun N-terminal kinase (JNK), leading to c-Jun transcription factor phosphorylation, all of which are involved in migration. We performed protein expression or migration front protein subcellular localization assays, which showed that OA induces c-Jun activation and its nuclear translocation, which precisely overlaps at wound-edge cells. Furthermore, c-Jun phosphorylation was independent of EGFR activation. Additionally, OA promoted actin cytoskeleton and focal adhesion (FA) dynamization. In fact, OA induced the recruitment of regulator proteins to FAs to dynamize these structures during migration. Moreover, OA changed paxillin distribution and activated focal adhesion kinase (FAK) at focal adhesions (FAs). The molecular implications of these observations are discussed.

The Kinesin Gene KIF26B Modulates the Severity of Post-Traumatic Heterotopic Ossification.

The formation of pathological bone deposits within soft tissues, termed heterotopic ossification (HO), is common after trauma. However, the severity of HO formation varies substantially between individuals, from relatively isolated small bone islands through to extensive soft tissue replacement by bone giving rise to debilitating symptoms. The aim of this study was to identify novel candidate therapeutic molecular targets for severe HO. We conducted a genome-wide scan in men and women with HO of varying severity following hip replacement for osteoarthritis. HO severity was dichotomized as mild or severe, and association analysis was performed with adjustment for age and sex. We next confirmed expression of the gene encoded by the lead signal in human bone and in primary human mesenchymal stem cells. We then examined the effect of gene knockout in a murine model of osseous trans-differentiation, and finally we explored transcription factor phosphorylation in key pathways perturbed by the gene. Ten independent signals were suggestively associated with HO severity, with KIF26B as the lead. We subsequently confirmed KIF26B expression in human bone and upregulation upon BMP2-induced osteogenic differentiation in primary human mesenchymal stem cells, and also in a rat tendo-Achilles model of post-traumatic HO. CRISPR-Cas9 mediated knockout of Kif26b inhibited BMP2-induced Runx2, Sp7/Osterix, Col1A1, Alp, and Bglap/Osteocalcin expression and mineralized nodule formation in a murine myocyte model of osteogenic trans-differentiation. Finally, KIF26B deficiency inhibited ERK MAP kinase activation during osteogenesis, whilst augmenting p38 and SMAD 1/5/8 phosphorylation. Taken together, these data suggest a role for KIF26B in modulating the severity of post-traumatic HO and provide a potential novel avenue for therapeutic translation.

Brassinosteroids promote thermotolerance through releasing BIN2-mediated phosphorylation and suppression of HsfA1 transcription factors in Arabidopsis

High temperature adversely affects plant growth and development. The steroid phytohormones brassinosteroids (BRs) are recognized to play important roles in plant heat stress responses and thermotolerance, but the underlying mechanisms remain obscure. Here, we demonstrate that the glycogen synthase kinase 3 (GSK3)-like kinase BRASSINOSTEROID INSENSITIVE2 (BIN2), a negative component in the BR signaling pathway, interacts with the master heat-responsive transcription factors CLASS A1 HEAT SHOCK TRANSCRIPTION FACTORS (HsfA1s). Furthermore, BIN2 phosphorylates HsfA1d on T263 and S56 to suppress its nuclear localization and inhibit its DNA-binding ability, respectively. BR signaling promotes plant thermotolerance by releasing the BIN2 suppression of HsfA1d to facilitate its nuclear localization and DNA binding. Our study provides insights into the molecular mechanisms by which BRs promote plant thermotolerance by strongly regulating HsfA1d through BIN2 and suggests potential ways to improve crop yield under extreme high temperatures.

Asprosin inhibits macrophage lipid accumulation and reduces atherosclerotic burden by up-regulating ABCA1 and ABCG1 expression via the p38/Elk-1 pathway

BackgroundAsprosin, a newly discovered adipokine, is a C-terminal cleavage product of profibrillin. Asprosin has been reported to participate in lipid metabolism and cardiovascular disease, but its role in atherogenesis remains elusive.MethodsAsprosin was overexpressed in THP-1 macrophage-derived foam cells and apoE−/− mice using the lentiviral vector. The expression of relevant molecules was determined by qRT-PCR and/or western blot. The intracellular lipid accumulation was evaluated by high-performance liquid chromatography and Oil red O staining. HE and Oil red O staining was employed to assess plaque burden in vivo. Reverse cholesterol transport (RCT) efficiency was measured using [3H]-labeled cholesterol.ResultsExposure of THP-1 macrophages to oxidized low-density lipoprotein down-regulated asprosin expression. Lentivirus-mediated overexpression of asprosin promoted cholesterol efflux and inhibited lipid accumulation in THP-1 macrophage-derived foam cells. Mechanistic analysis revealed that asprosin overexpression activated p38 and stimulated the phosphorylation of ETS-like transcription factor (Elk-1) at Ser383, leading to Elk-1 nuclear translocation and the transcriptional activation of ATP binding cassette transporters A1 (ABCA1) and ABCG1. Injection of lentiviral vector expressing asprosin diminished atherosclerotic lesion area, increased plaque stability, improved plasma lipid profiles and facilitated RCT in apoE−/− mice. Asprosin overexpression also increased the phosphorylation of p38 and Elk-1 as well as up-regulated the expression of ABCA1 and ABCG1 in the aortas.ConclusionAsprosin inhibits lipid accumulation in macrophages and decreases atherosclerotic burden in apoE−/− mice by up-regulating ABCA1 and ABCG1 expression via activation of the p38/Elk-1 signaling pathway.

PTEN/AKT signaling pathway related to hTERT downregulation and telomere shortening induced in Toxoplasma GRA16-expressing colorectal cancer cells

This study investigated whether the molecular mechanism of granule protein 16 (GRA16), a dense granule protein of Toxoplasma gondii (T. gondii) that induces cancer cell apoptosis, results in telomere shortening in cancer cells. The molecular mechanism of GRA16 responsible for regulating telomerase reverse transcriptase (hTERT) activity and telomere shortening was investigated using GRA16-transferred HCT116 human colorectal cancer cells (GRA16-stable cells). GRA16 directly decreased hTERT expression by downregulating the expression and phosphorylation of hTERT transcriptional factors accompanied by decreased expression of shelterin complex molecules. Moreover, GRA16 resulted in cancer cell death through reduction of telomerase activity which leads to telomere shortening (decreased relative ratio of telomeric repeat-amplified sequence to that of a single-copy gene) (T/S ratio)), and at the same time gamma-H2A histone family member X (γ-H2A.X) stained nucleus was increased in the cells. The molecular mechanism between GRA16 and hTERT inactivation was revealed using inhibitors for phosphatase and tensin homolog (PTEN) and protein phosphatase 2A (PP2A) as well as siRNAs against PTEN and PP2A. hTERT dephosphorylation was induced effectively by the signaling pathway of HAUSP/PTEN/p-AKT(S473) but not by PP2A-B55/p-AKT(T308). Inhibition of the PTEN signaling pathway increased mRNA expressions in hTERT transcriptional factors, cell cycle activating factors, and apoptosis-inhibiting factors. When HCT116 cells were infected with T. gondii, the number of γ-H2A.X-stained nuclei also increased and p-hTERT/hTERT decreased as in GRA16-stable cells. Altogether, our results emphasize that GRA16 is a novel promising telomerase inhibitor that causes telomere shortening through telomerase inactivation by inducing the activation of the tumor suppressor PTEN.

Abstract 4225: IL-2Rβ/IL-2R γsynthetic cytokines induce activation of human T and NK cells

Abstract Introduction: Heterodimerization of the intermediate affinity Interleukin-2 Receptors (IL-2R), IL-2Rβ and IL-2Rγ, initiates a signaling cascade in T and NK cells that ultimately results in proliferation and production of Interferon-gamma (IFN-γ). Binding of IL-2 in a dimeric complex with intermediate affinity or in the high affinity trimeric complex, which also includes IL-2Rα and is predominantly expressed on antigen activated T cells and Tregs leads to trans-phosphorylation of signaling motifs on the IL-2Rβ and IL-2Rγ intracellular domains by the associated JAK kinases. The function of the wild type IL-2 is to bring the receptor chains in close proximity to produce an optimal level of phosphorylation and activation of associated STAT transcription factors. Here we established a way to bring IL-2Rβ and IL-2Rγ chains together in a cytokine-independent manner that results in functional activation. Experimental Procedures: Human IL-2Rβ and IL-2Rγ specific heavy chain single domain antibodies (VHH) were generated by camel immunization and screening of VHH libraries prepared from peripheral blood cells for binding. Ten IL-2Rβ VHH segments and six IL-2Rγ VHH segments, all with low nanomolar affinity, were identified and coupled as IL-2Rβ/IL-2Rγ VHH dimers in all possible combinations in both amino-carboxy and carboxy-amino orientations yielding 120 different proteins that were tested in human T and NK cell functional assays. Results: IL-2Rβ/IL-2Rγ VHH dimers were biologically active and induced pSTAT-5 phosphorylation in the IL-2 dependent NK cell line NKL at various levels. The biological activity of these IL-2Rβ/IL-2Rγ VHH dimers was further confirmed on primary cells. IL-2Rβ/IL-2Rγ VHH dimers induced pSTAT5 phosphorylation on NK cells isolated from human peripheral blood and culture with IL-2Rβ/IL-2Rγ VHH dimers resulted in proliferation and production of high but varied levels of IFN-γ. The activity of IL-2Rβ/IL-2Rγ VHH dimers on primary T cells was also examined. Similar to NK cells, IL-2Rβ/IL-2Rγ VHH dimers induced pSTAT5 phosphorylation, proliferation and IFN-γ production by CD4 positive and CD8 positive T cell blasts generated after CD3/CD28 activation of human PBMC. Conclusions: In conclusion, we have generated a series of functional IL-2Rβ/IL-2Rγ VHH dimers representing surrogate cytokine agonists each with unique signaling strengths that will now be further analyzed for potential therapeutic application in tumor immunology and auto-immunity Citation Format: Rene de Waal Malefyt, PJ Aspuria, Sandro Vivona, Priyanka Balasubrahmanyam, Cindy Buffone, Mahalakshmi Ramadass, Romina Riener, Martin Oft, Patrick Lupardus, Robert Kastelein. IL-2Rβ/IL-2R γsynthetic cytokines induce activation of human T and NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4225.

Abstract 3163: PR/STAT3 nuclear transcriptional complexes mediate aurora-A kinase-induced stemness plasticity in ER+ breast cancer

Abstract Purpose: Emergence of de novo or acquired resistance to standard of care endocrine therapies represents a major challenge in the clinical management of estrogen receptor alpha positive (ER+) breast tumors. This process is characterized by high tumor plasticity that confers to cancer cells the ability to shift from a differentiated state, with limited tumorigenic potential, to an undifferentiated state which is responsible for drug resistance and tumor relapse. In ER+ breast tumors, this plasticity is manifest by the enrichment of breast tumor initiating cells (BTICs) with stemness features, such as self-renewal capacity, CD44 expression and high ALDH activity, and ER down-regulation. Development of clinically relevant ER+ breast tumor models to test novel therapeutic strategies that inhibit the enhanced tumor cell plasticity and restore or extend endocrine sensitivity are urgently needed. Experimental Design: Unique endocrine resistant and metastatic breast cancer models were utilized to define in vitro and in vivo the pivotal role of Aurora-A mitotic kinase (AURKA) in the induction of stemness plasticity, endocrine therapy resistance and tumor progression. Unbiased phospho-proteomic analysis was performed in the absence or presence of alisertib, a clinically relevant AURKA inhibitor, that was used to identify novel down-stream targets of AURKA signaling pathway. Results: Phospho-proteomic analysis showed that AURKA targeting reduced the phosphorylation of STAT3 transcription factor at the serine-727 site. Significantly, herein we define a novel mechanism by which AURKA-induced self-renewal capacity is mediated through the activation of a phosphorylated serine727-STAT3/serine294-progesterone receptor (PR) transcriptional complex that regulates the expression of KLF4 stemness reprogramming gene. Remarkable, pharmacologic and genetic targeting of AURKA/STAT3/PR oncogenic axis impairs self-renewal capacity of endocrine therapy resistant breast cancer cells. Conclusions: In this study we demonstrate the pivotal role of a novel AURKA/STAT3/PR oncogenic axis in promoting stemness plasticity and endocrine therapy resistance. These findings provide the strong preclinical rationale for the development of novel combinatorial therapeutic strategies targeting cooperative AURKA/serine727-STAT3 and serine294-PR pathways predicted to result in the selective eradication of CD44High/ALDHHigh BTICs from the bulk tumor with consequent benefits to the clinical outcomes of patients with advanced ER+ breast cancer refractory to standard of care endocrine therapy. Citation Format: Antonino B. D'Assoro. PR/STAT3 nuclear transcriptional complexes mediate aurora-A kinase-induced stemness plasticity in ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3163.

Epigallocatechine-3-gallate Inhibits the Adipogenesis of Human Mesenchymal Stem Cells via the Regulation of Protein Phosphatase-2A and Myosin Phosphatase.

Epigallocatechin-3-gallate (EGCG) has widespread effects on adipocyte development. However, the molecular mechanisms of EGCG are not fully understood. We investigate the adipogenic differentiation of human-derived mesenchymal stem cells, including lipid deposition and changes in the expression and phosphorylation of key transcription factors, myosin, protein phosphatase-2A (PP2A), and myosin phosphatase (MP). On day 6 of adipogenic differentiation, EGCG (1–20 µM) suppressed lipid droplet formation, which was counteracted by an EGCG-binding peptide for the 67 kDa laminin receptor (67LR), suggesting that EGCG acts via 67LR. EGCG decreased the phosphorylation of CCAAT-enhancer-binding protein beta via the activation of PP2A in a protein kinase A (PKA)-dependent manner, leading to the partial suppression of peroxisome proliferator-activated receptor gamma (PPARγ) and adiponectin expression. Differentiated cells exhibited a rounded shape, cortical actin filaments, and lipid accumulation. The EGCG treatment induced cell elongation, stress fiber formation, and less lipid accumulation. These effects were accompanied by the degradation of the MP target subunit-1 and increased the phosphorylation of the 20 kDa myosin light chain. Our results suggest that EGCG acts as an agonist of 67LR to inhibit adipogenesis via the activation of PP2A and suppression of MP. These events are coupled with the decreased phosphorylation and expression levels of adipogenic transcription factors and changes in cell shape, culminating in curtailed adipogenesis.

MAP kinases are involved in RNA polymerase III regulation upon LPS treatment in macrophages

Macrophages are transcriptionally highly dynamic cell type, rapidly adapting to a changing environment to execute innate immune functions. Activation of macrophages with lipopolysaccharides (LPS), a major component of the outer membrane of most Gram-negative bacteria, induces rapid transcriptional changes and within a few hours transcription of several hundred genes is altered. Within these genes are tRNAs, which are synthesised by RNA Polymerase (Pol) III, and whose expression is rapidly upregulated in response to LPS. However, the mechanisms that govern Pol III activation are not fully elucidated.LPS engage the Toll-like receptor (TLR) 4 and induce various signalling pathways, including mitogen-activated protein kinases (MAPK). MAPKs are serine/threonine kinases that catalyse the phosphorylation of transcription factors, protein kinases, and many other substrates including functional proteins, play a central role in mediating cellular responses to extracellular signals, including inflammatory cues.Here we show that ERK and p38 MAP kinases contribute to the activation of Pol III in macrophages stimulated with LPS. We also demonstrate that MAP kinases effector MSK1/2 kinases are involved in tRNA upregulation. Our data show that ERK, p38, and MSK kinases are required for upregulation of Pol III activity in macrophages stimulated by LPS. The possible modes of their action are discussed.

Knockdown and Inhibition of Spleen Tyrosine Kinase Decreases mTORC1 Activity and Enhances Lysosomal Biogenesis and Autolysosomal Maturation in Pancreatic Cancer Cell Lines

In cancer, oncogene dependency is a phenomenon where a dominant driver oncogene promotes tumor cell proliferation and survival, and loss of this oncogene results in tumor cell death and, eventually, tumor regression. KRAS, a GTPase that regulates cell growth and proliferation, is an oncogene constitutively activated in over 90% of pancreatic cancer. However, clinically effective inhibitors of KRAS have been unsuccessful so efforts have been focused on identifying other potential targets associated with the KRAS signaling network. Spleen tyrosine kinase (SYK), which is expressed at high levels in KRAS‐dependent pancreatic cancer cell lines, may be one of these targets. Our data indicate that SYK activates the mechanistic target of rapamycin kinase complex 1 (mTORC1), which promotes protein translation and cell growth. SYK activation also leads to decreased autolysosome count. In connecting SYK activation with increased mTORC1 activity and decreased autolysosome count, we hypothesis that the MiT/TFE transcription factors are involved. We propose that SYK inhibition in pancreatic cancer cells leads to reduced mTORC1 activity, which reduces phosphorylation of MiT/TFE transcription factors. The unphosphorylated MiT/TFE transcription factors may then enter the nucleus to activate genes for lysosomal biogenesis and autophagy. Autophagy is a process that recycles cellular macromolecules during nutrient deprivation by fusing autophagosomes and lysosomes, produced from lysosomal biogenesis, to generate autolysosomes. From our experiments, we were able to show that SYK inhibition blocks mTORC1‐dependent phosphorylation of MITF and TFEB transcription factors, members of the MiT/TFE family. MITF and TFEB activation leads to increased autophagy due to autolysosomal biogenesis and accumulation. In summary, our studies of the SYK‐mTORC1‐autophagy pathway provide support to investigate SYK as a candidate therapeutic target for pancreatic cancer treatment.

The TSPO ligand 3,17,19‐androsten‐5‐triol attenuates NASH by increasing FXR expression and autophagy

Translocator protein (TSPO; 18 kDa) is a ubiquitous high‐affinity cholesterol‐ and drug‐binding protein located in the outer mitochondrial membrane. 3,17,19‐androsten‐5‐triol (19‐Atriol) is a ligand binding at the cholesterol recognition amino acid consensus domain of TSPO. Our previous study suggested that TSPO deficiency ameliorated methionine‐choline‐deficient induced non‐alcoholic steatohepatitis (NASH) through downregulation of bile acid synthesis (Li et al., iScience 24: 102457, 2021). We examined the role of cholesterol binding to TSPO in NASH using the 19‐Atriol on high‐fat‐fructose‐cholesterol diet (HFFC)‐induced NASH in rats. After HFFC‐feeding for 13weeks, the rats were subcutaneously administered daily 19‐Atriol (20mg/kg) for 2 weeks (weeks 13‐15). The ratio of liver/body weight and serum triacylglycerol content were significantly reduced in 19‐Atriol‐ compared to vehicle‐treated rats. Immunoblot analyses indicated that liver inflammation (TNFα) and fibrosis markers (COL1A1, ACTA2) were reduced following 19‐Atriol treatment. Farnesoid X receptor (FXR), a bile acid receptor, was upregulated in response to 19‐Atriol treatment in HFFC‐fed rats. In search of the mechanism mediating the effects of 19‐Atriol we observed an increase in autophagy flux and the levels of phosphorylated transcription factor EB (TFEB), a major regulator of autophagy and lysosomal biogenesis. Taken together these results suggest that blocking cholesterol binding to TSPO by 19‐Atriol leads to the attenuation of NASH by increasing FXR expression levels and activating of autophagic degradation as was the case in TSPO‐deficient rats.

Curcumin selectively induces colon cancer cell apoptosis and S cell cycle arrest by regulates Rb/E2F/p53 pathway

ObjectiveCurcumin is a natural active product extracted from turmeric, has been reported to have anticancer properties against various tumors. However, the effect of curcumin on colon cancer remains still unclear. Our aimed to investigate the anti-colon cancer properties of curcumin in vitro, and determine the molecular mechanisms underlying these effects. MethodsViability assays including CCK8 and LDH were used to measure cell proliferation. Flow cytometry was performed to detect cell apoptosis and cell cycle distribution. qRT-PCR and Western blotting was used for measuring genes/protein expression/activation in apoptotic and pro-carcinogenic cellular signaling pathways. Finally, the results were confirmed using a xenograft tumor model that comprised of mice. ResultsIn vitro, Our data showed that curcumin preferentially and, in a dose, dependent way inhibited colon cancer cells proliferation, but was not toxic to normal colon mucosa epithelial cells. Curcumin induced apoptosis through a p53-mediated mechanism, pro-apoptotic proteins Bax was upregulated, and cell cycle arrest at S phase by inhibited the expression of cell cycle related proteins, phosphorylation of Rb signaling pathway proteins and E2F family transcription factors. Finally, the mouse xenograft model confirmed the suppressive effect of curcumin on tumor growth in vivo. ConclusionCurcumin may be a potential strategy for the treatment of colon cancer.

Molecular Mechanism of Cinnamomum cassia against Gastric Damage and Identification of Active Compounds.

Cinnamomum cassia is a natural product found in plants that has been used as a folk remedy for inflammation. In this study, we investigated the mechanism underlying the anti-inflammatory and antioxidant properties of C. cassia extract (ECC) in lipopolysaccharide (LPS)-induced murine RAW 264.7 cells, in comparison with 4-hydroxycinnamaldehyde, a C. cassia extract component. ECC and 4-hydroxycinnamaldehyde inhibited the production of nitrite oxide in a dose-dependent manner and did not show any change in cellular toxicity when treated with the same dose as that used in the nitrite assay. Moreover, they attenuated ROS accumulation after lipopolysaccharide (LPS) stimulation. ECC and 4-hydroxycinnamaldehyde decreased the mRNA and protein expression levels of inflammatory mediators (iNOS and COX-2) and cytokines such as TNF and IL-6. We also found that ECC and 4-hydroxycinnamaldehyde mitigated the phosphorylation of ERK, JNK, and transcription factors, such as NF-κB and STAT3, suppressing NF-κB nuclear translocation in LPS-activated macrophages. In addition, administration of ECC in a Sprague Dawley rat model of acute gastric injury caused by indomethacin significantly increased the gastric mucus volume. Analysis of serum and tissue levels of inflammatory mediators revealed a significant decrease in serum PGE2 and myeloperoxidase levels and a reduction in gastric iNOS, COX-2, and p65 protein levels. Collectively, these results suggest that ECC has antioxidant and anti-inflammatory effects and is a potential candidate for curing gastritis.