Extracellular Signal-regulated Kinase Activation Research Articles

Production and characterization of basic fibroblast growth factor protein in rice suspension cultures

The basic fibroblast growth factor (bFGF) plays a major role in cell growth, survival, and other mitogenic activities. General applications of this protein involve tissue repair, morphogenesis, embryonic development, cellular growth, etc. Due to its importance and high demand, research has been done on producing recombinant bFGF in different expression platforms. We have produced bFGF in transgenic rice (Oryza sativa) cells growing in liquid suspension culture. The gene construct consists of the rice alpha-amylase (RAmy) 3D sugar-inducible promotor and rice glutelin secretion signal peptide for controlled production and release of the bFGF protein by manipulating the sugar concentration for batch or semi-continuous bioreactor runs. A thermal shift assay was performed on the purified rice cell produced bFGF (rbFGF) and the results were compared with E. coli derived bFGF. The purified rbFGF protein from rice cells was tested for propagating human-induced pluripotent stem cells (hiPSCs) up to eight passages. Furthermore, the biological activity measured in both crude lysate and purified rbFGF showed a similar pattern of the extracellular signal-regulated kinase (ERK) activity compared to E. coli derived bFGF (control), indicating that rbFGF is active before purification and the protein activity is not inhibited by other components of the lysate. From the current results, the rice-produced rbFGF protein is biologically active and could serve as a viable product for various applications.

A guide to ERK dynamics, part 1: mechanisms and models.

Extracellular signal-regulated kinase (ERK) has long been studied as a key driver of both essential cellular processes and disease. A persistent question has been how this single pathway is able to direct multiple cell behaviors, including growth, proliferation, and death. Modern biosensor studies have revealed that the temporal pattern of ERK activity is highly variable and heterogeneous, and critically, that these dynamic differences modulate cell fate. This two-part review discusses the current understanding of dynamic activity in the ERK pathway, how it regulates cellular decisions, and how these cell fates lead to tissue regulation and pathology. In part 1, we cover the optogenetic and live-cell imaging technologies that first revealed the dynamic nature of ERK, as well as current challenges in biosensor data analysis. We also discuss advances in mathematical models for the mechanisms of ERK dynamics, including receptor-level regulation, negative feedback, cooperativity, and paracrine signaling. While hurdles still remain, it is clear that higher temporal and spatial resolution provide mechanistic insights into pathway circuitry. Exciting new algorithms and advanced computational tools enable quantitative measurements of single-cell ERK activation, which in turn inform better models of pathway behavior. However, the fact that current models still cannot fully recapitulate the diversity of ERK responses calls for a deeper understanding of network structure and signal transduction in general.

A guide to ERK dynamics, part 2: downstream decoding.

Signaling by the extracellular signal-regulated kinase (ERK) pathway controls many cellular processes, including cell division, death, and differentiation. In this second installment of a two-part review, we address the question of how the ERK pathway exerts distinct and context-specific effects on multiple processes. We discuss how the dynamics of ERK activity induce selective changes in gene expression programs, with insights from both experiments and computational models. With a focus on single-cell biosensor-based studies, we summarize four major functional modes for ERK signaling in tissues: adjusting the size of cell populations, gradient-based patterning, wave propagation of morphological changes, and diversification of cellular gene expression states. These modes of operation are disrupted in cancer and other related diseases and represent potential targets for therapeutic intervention. By understanding the dynamic mechanisms involved in ERK signaling, there is potential for pharmacological strategies that not only simply inhibit ERK, but also restore functional activity patterns and improve disease outcomes.

Inducing cyclooxygenase-2 expression, prostaglandin E2 and prostaglandin F2α production of human dental pulp cells by activation of toll-like receptor-3, mitogen-activated protein kinase kinase/extracellular signal-regulated kinase and p38 signaling

Background/purposeBacterial infection was the major etiology for pulpal/root canal infection. This study aimed to investigate the activation of toll-like receptor-3 (TLR) on cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) and PGF2α production of human dental pulp cells (HDPCs) and associated signaling. Materials and methodsHDPCs were exposed to different concentrations of Poly (I:C) (a TLR3 activator). Cell viability was determined by 3- (4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay and alkaline phosphatase (ALP) activity was evaluated by ALP staining. Activation of extracellular signal-regulated kinase (ERK) and p38 by Poly (I:C) was determined by immunofluorescent staining. The COX-2 protein expression was analyzed by Western blot. PGE2 and PGF2α production was measured by enzyme-linked immunosorbent assay. The mRNA expression was studied by real-time polymerase-chain reaction. Moreover, HDPCs were exposed to Poly(I:C) with/without U0126 or SB203580 treatment and analysis of COX-2 expression and prostanoid production were conducted. ResultsPoly (I:C) showed little effect on ALP activity, but decreased viability of HDPCs. It stimulated COX-2 mRNA and protein expression. Poly (I:C) induced PGE2 and PGF2α production of HDPCs. Poly (I:C) activated p-ERK, and p-p38 protein expression. Treatment by U0126 (a mitogen-activated protein kinase kinase (MEK)/ERK inhibitor) and SB203580 (a p38 inhibitor) attenuated Poly (I:C)-induced COX-2 mRNA and protein expression as well as PGE2 and PGF2α production. ConclusionTLR3 activation is involved in the infection and inflammatory responses of pulp tissues, via MEK/ERK, and p38 signaling to mediate COX-2 expression as well as PGE2 and PGF2α production, contributing to the pathogenesis and progression of pulpal/periapical diseases.

Cold Atmospheric Plasma Jet Irradiation Decreases the Survival and the Expression of Oncogenic miRNAs of Oral Carcinoma Cells

Despite recent advancements, therapies against advanced oral squamous cell carcinoma (OSCC) remain ineffective, resulting in unsatisfactory therapeutic outcomes. Cold atmospheric plasma (CAP) offers a promising approach in the treatment of malignant neoplasms. Although the effects of CAP in abrogating OSCC have been explored, the exact mechanisms driving CAP-induced cancer cell death and the changes in microRNA (miRNA) expression are not fully understood. We fabricated and calibrated an argon-CAP device to explore the effects of CAP irradiation on the growth and expression of oncogenic miRNAs in OSCC. The analysis revealed that, in OSCC cell lines following CAP irradiation, there was a significant reduction in viability; a downregulation of miR-21, miR-31, miR-134, miR-146a, and miR-211 expression; and an inactivation of the v-akt murine thymoma viral oncogene homolog (AKT) and extracellular signal-regulated kinase (ERK) signals. Pretreatment with blockers of apoptosis, autophagy, and ferroptosis synergistically reduced CAP-induced cell death, indicating a combined induction of variable death pathways via CAP. Combined treatments using death inhibitors and miRNA mimics, alongside the activation of AKT and ERK following the exogenous expression, counteracted the cell mortality associated with CAP. The CAP-induced downregulation of miR-21, miR-31, miR-187, and miR-211 expression was rescued through survival signaling. Additionally, CAP irradiation notably inhibited the growth of SAS OSCC cell xenografts on nude mice. The reduced expression of oncogenic miRNAs in vivo aligned with in vitro findings. In conclusion, our study provides new lines of evidence demonstrating that CAP irradiation diminishes OSCC cell viability by abrogating survival signals and oncogenic miRNA expression.

Crocin Inhibits Orbital Fibroblasts Fibrosis in Thyroid-Associated Ophthalmopathy

Purpose To investigate the role of Crocin on proliferation, fibrosis, and migration of orbital fibroblasts, as well as the possible signaling pathway. Methods Immunofluorescence assay was performed to detect the expression of fibroblast marker proteins vimentin cytokeratin, desmin, and S-100. The quantity of 5‑ethynyl‑2′‑deoxyuridine-positive cells in orbital fibroblast was analyzed. Quantitative real-time PCR and western blots were performed to evaluate the expression level of fibrosis-related marker including alpha-smooth muscle actin, connective-tissue growth factor, collagen 1A1, and fibronectin. Scratch wound assays were performed to assess wound widths of orbital fibroblast. The expression and phosphorylation of extracellular signal-regulated kinase/signal transducer and activator of transcription 3 were evaluated using western blots. The phosphorylation of smad2 and smad3 was evaluated using immunofluorescence assay. Results Crocin treatment reduced 5‑ethynyl‑2′‑deoxyuridine-positive cells, indicating inhibitory effect on orbital fibroblast proliferation. The expression levels of alpha-smooth muscle actin, connective-tissue growth factor, collagen 1A1 and fibronectin were declined in Crocin treatment. Delayed wound closures were observed in Crocin treatment. Furthermore, Crocin did not affect the expression of extracellular signal-regulated kinase and signal transducer and activator of transcription 3, but weakened extracellular signal-regulated kinase and signal transducer and activator of transcription 3 phosphorylation in orbital fibroblast. The phosphorylation of smad2 and smad3 was attenuated by Crocin as well. Conclusion In conclusion, Crocin inhibits the phosphorylation of extracellular signal-regulated kinase and signal transducer and activator of transcription 3, contributing to the inhibitory effect on proliferation, fibrosis, and migration of orbital fibroblast, suggesting that Crocin has potential to be a novel therapeutic candidate for thyroid-associated ophthalmopathy treatment.

Time-resolved live-cell spectroscopy reveals EphA2 multimeric assembly.

Ephrin type-A receptor 2 (EphA2) is a receptor tyrosine kinase that initiates both ligand-dependent tumor-suppressive and ligand-independent oncogenic signaling. We used time-resolved, live-cell fluorescence spectroscopy to show that the ligand-free EphA2 assembles into multimers driven by two types of intermolecular interactions in the ectodomain. The first type entails extended symmetric interactions required for ligand-induced receptor clustering and tumor-suppressive signaling that inhibits activity of the oncogenic extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) protein kinases and suppresses cell migration. The second type is an asymmetric interaction between the amino terminus and the membrane proximal domain of the neighboring receptors, which supports oncogenic signaling and promotes migration in vitro and tumor invasiveness in vivo. Our results identify the molecular interactions that drive the formation of the EphA2 multimeric signaling clusters and reveal the pivotal role of EphA2 assembly in dictating its opposing functions in oncogenesis.

The CD2v protein of African swine fever virus inhibits macrophage migration and inflammatory cytokines expression by downregulating EGR1 expression through dampening ERK1/2 activity

African swine fever virus (ASFV) is a highly contagious and deadly virus that leads to high mortality rates in domestic swine populations. Although the envelope protein CD2v of ASFV has been implicated in immunomodulation, the molecular mechanisms underlying CD2v-mediated immunoregulation remain unclear. In this study, we generated a stable CD2v-expressing porcine macrophage (PAM-CD2v) line and investigated the CD2v-dependent transcriptomic landscape using RNA-seq. GO terms enrichment analysis and gene set enrichment analysis revealed that CD2v predominantly affected the organization and assembly process of the extracellular matrix. Wound healing and Transwell assays showed that CD2v inhibited swine macrophage migration. Further investigation revealed a significant decrease in the expression of transcription factor early growth response 1 (EGR1) through inhibiting the activity of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Notably, EGR1 knockout in swine macrophages restricted cell migration, whereas EGR1 overexpression in PAM-CD2v restored the ability of macrophage migration, suggesting that CD2v inhibits swine macrophage motility by downregulating EGR1 expression. Furthermore, we performed chromatin immunoprecipitation and sequencing for EGR1 and the histone mark H3K27 acetylation (H3K27ac), and we found that EGR1 co-localized with the activated histone modification H3K27ac neighboring the transcriptional start sites. Further analysis indicated that EGR1 and H3K27ac co-occupy the promoter regions of cell locomotion-related genes. Finally, by treating various derivatives of swine macrophages with lipopolysaccharides, we showed that depletion of EGR1 decreased the expression of inflammatory cytokines including TNFα, IL1α, IL1β, IL6, and IL8, which play essential roles in inflammation and host immune response. Collectively, our results provide new insights into the immunomodulatory mechanism of ASFV CD2v.

Effects of Magnoliae Flos on Atopic Dermatitis-Like Inflammation Evaluated via Extracellular Signal-regulated Kinase or Signal Transducers and Activators of Transcription 1/3 Signalling Pathways

Atopic dermatitis is a chronic inflammatory skin disease. Skin is the largest organ and plays a pivotal role in protecting the body. Not only does the skin act as a physical barrier against the external environment, but it also has its own immune system. Atopic dermatitis is caused by prolonged excessive inflammatory responses that worsen under imbalanced cutaneous immune system skin conditions. Although the prevalence and burden of atopic dermatitis is increasing, the standard therapeutic agents remain unclear due to the complicated pathophysiology of the condition. The objective of this study is to examine the use of Magnoliae flos, the dried flower bud of Magnolia biondii or related plants. The effects and underlying mechanism of action of aqueous extract of the buds of Magnoliae flos (MF) were evaluated. Immortalized human keratinocytes (HaCaT) stimulated with tumour necrosis factor-α and interferon-γ mixture and NC/Nga mice stimulated with 2,4-dinitrochlorobenzene were used as atopic dermatitis models, in vitro and in vivo, respectively. The effects of MF were determined by measuring the suppression of pro-inflammatory signalling pathways, such as extracellular signal-regulated kinase or signal transducers and activators of transcription 1/3 and restoring skin barrier molecules. In conclusion, MF is a potential therapeutic alternative for the treatment of atopic dermatitis through repressing inflammatory pathways.

Disruption of phosphofructokinase activity and aerobic glycolysis in human bronchial epithelial cells by atmospheric ultrafine particulate matter

Exposure to ambient ultrafine particulate matter (UPM) causes respiratory disorders; however, the underlying molecular mechanisms remain unclear. In this study, we synthesized simulated UPM (sUPM) with controlled physicochemical properties using the spark-discharge method. Subsequently, we investigated the biological effects of sUPM using BEAS-2B human bronchial epithelial cells (HBECs) and a mouse intratracheal instillation model. High throughput RNA-sequencing and bioinformatics analyses revealed that dysregulation of the glycolytic metabolism is involved in the inhibited proliferation and survival of HBECs by sUPM treatment. Furthermore, signaling pathway and enzymatic analyses showed that the treatment of BEAS-2B cells with sUPM induces the inactivation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB, also known as AKT), resulting in the downregulation of phosphofructokinase 2 (PFK2) S483 phosphorylation, PFK enzyme activity, and aerobic glycolysis in HBECs in an oxidative stress-independent manner. Additionally, intratracheal instillation of sUPM reduced the phosphorylation of ERK, AKT, and PFK2, decreased proliferation, and increased the apoptosis of bronchial epithelial cells in mice. The findings of this study imply that UPM induces pulmonary toxicity by disrupting aerobic glycolytic metabolism in lung epithelial cells, which can provide novel insights into the toxicity mechanisms of UPM and strategies to prevent their toxic effects.

Increased intracellular diffusivity of macromolecules within a mammalian cell by low-intensity pulsed ultrasound

Whilst a number of studies have demonstrated that low-intensity pulsed ultrasound (LIPUS) is a promising therapeutic ultrasound technique that can be used for delivering mild mechanical stimuli to target tissue non-invasively, the underlying biophysical mechanisms still remain unclear. Most mechanism studies have focused explicitly on the effects of LIPUS on the cell membrane and mechanosensitive receptors. In the present study, we propose an additional mechanism by which LIPUS propagation through living cells may directly impact intracellular dynamics, particularly the diffusion transport of biomolecules. To support our hypothesis, human epithelial-like cells (SaOS-2 and HeLa) seeded on a confocal dish placed on a microscope stage were exposed to LIPUS with various exposure conditions (ultrasound frequencies of 0.5, 1 and 3 MHz, peak acoustic pressure of 200 and 400 kPa, a pulse repetition frequency of 1 kHz and a 20 % duty cycle), and the diffusivities of various sizes of biomolecules in the cytoplasm area were measured using fluorescence recovery after photobleaching (FRAP). Furthermore, giant unilamellar vesicles (GUVs) filled with macromolecules were used to examine the physical causal relationship between LIPUS and molecular diffusion changes. Nucleocytoplasmic transport coefficients were also measured by modified FRAP that bleaches the whole cell nuclear region. Extracellular signal-regulated kinases (ERK) activity (the phosphorylation dynamics) was monitored using fluorescence resonance energy transfer (FRET) microscopy. All the measurements were taken during, before and after the LIPUS exposure. Our experimental results clearly showed that the diffusion coefficients of macromolecules within the cell increased with acoustic pressure by 12.1 to 33.5 % during the sonication, and the increments were proportional to their molecular sizes regardless of the ultrasound frequency used. This observation in living cells was consistent with the GUVs exposed to the LIPUS, which indicated that the diffusivity increase was a passive physical response to the acoustic energy of LIPUS. Under the 1 MHz LIPUS exposure with 400 kPa, the passive nucleocytoplasmic transport of enhanced green fluorescent protein (EGFP) was accelerated by 21.4 %. With the same LIPUS exposure condition, both the diffusivity and phosphorylation of ERK induced by EGF treatment were significantly elevated simultaneously, which implied that LIPUS could also modify the kinase kinetics in the signal transduction process. Taken together, this study is the first attempt to uncover the physical link between LIPUS and the dynamics of intracellular macromolecules and related biological processes that LIPUS can possibly increase the diffusivity of intracellular macromolecules, leading to the changes in the basic cellular processes: passive nucleocytoplasmic transport and ERK. Our findings can provide a novel perspective that the mechanotransduction process that the intracellular region, in addition to the cell membrane, can convert the acoustic stimuli of LIPUS to biochemical signals.

SAT027 Novel Obesity-Associated Melanocortin 4 Receptor Variants Modulate Signaling Differently

Abstract Disclosure: A.V. Rodriguez Rondon: None. M.S. Welling: None. E.L. van den Akker: None. E.F. van Rossum: None. P.J. Delhanty: None. J.A. Visser: None. Melanocortin-4 receptor (MC4R) is a G-protein-coupled receptor expressed in the hypothalamus regulating appetite and energy expenditure. Extensive evidence from genetic and biological studies show that MC4R is a key player in the homeostatic regulation of body weight. MC4R loss of function (LoF) variants are the most common cause of monogenic obesity. We have identified 1 homozygous and 16 heterozygous MC4R variants in patients with obesity at our academic Obesity Center CGG, Erasmus MC. The impact of these variants on MC4R signaling and the association with obesity are unknown. Here, we functionally characterized these variants by analyzing four different aspects of MC4R signaling. HEK293 cells were transiently transfected with expression plasmids encoding wild type (WT) or variant MC4R and stimulated with melanocyte stimulating hormone. Cyclic AMP response (cAMP) was measured using GloSensor cAMP bioluminescence assay, and β-arrestin-2 recruitment (βarr2) was measured using NanoBiT complementation luminescence assay. Furthermore, HiBiT Detection System was used to measure cell surface expression, and serum response element (SRE) reporter assay was used to measure extracellular signal-regulated kinase (ERK) activation. Variants were classified according to the percent of maximum response (Emax) relative to WT: complete LoF (cLoF): 0-20%; partial LoF (pLoF): 20-80%; WT: 80-120%; gain of function (GoF): >120%. The MC4R variants were identified by sequencing the MC4R gene of adult and pediatric patients with obesity using an obesity gene panel. Eleven of these variants have not been functionally studied previously. The median age of onset obesity for all patients is 3 years (range 0 – 6). Furthermore, the median BMI of the adult patients was 48.8 kg/m2 (range 36 - 58.8) and the median BMI-SDS of the pediatric patients was +4.2 SDS (range +3 – 7). Twelve out of 17 patients presented with hyperphagia. MC4R variants had differential effects on signaling. In 7 out of 17 variants, cLoF for cAMP was observed. This could be explained in 6 out of 7 of these variants due to lack of cell surface expression. Intriguingly, five out of 17 variants had a normal or GoF cAMP response and normal ERK activation. However, three out of these 5 variants showed cLoF or pLoF for βarr2, indicating a disrupted desensitization mechanism. In addition, the remaining five variants displayed pLoF for most studied pathways. We show that these MC4R variants identified in our patients with obesity affect signaling differently, through modulation of cAMP, βarr2, cell surface expression, and ERK pathways. This demonstrates the value of examining different aspects of MC4R signaling to understand possible biased effects of variants on these pathways. Overall, our results show the clinical importance of assessing the effect of MC4R variants on a range of molecular signaling mechanisms to determine their association with obesity. Presentation: Saturday, June 17, 2023

Hepatocyte-specific Wtap deficiency promotes hepatocellular carcinoma by activating GRB2–ERK depending on downregulation of proteasome-related genes

Wilm's tumor 1-associating protein (WTAP), a regulatory protein of the m6A methyltransferase complex, has been found to play a role in regulating various physiological and pathological processes. However, the invivo role of WTAP in the pathogenesis of hepatocellular carcinoma (HCC) is unknown. In this study, we have elucidated the crucial role of WTAP in HCC progression and shown that hepatic deletion of Wtap promotes HCC pathogenesis through activation of multiple signaling pathways. A single dose of diethylnitrosamine injection causes more and larger HCCs in hepatocyte-specific Wtap knockout (Wtap-HKO) mice than Wtapflox/flox mice fed with either normal chow diet or a high-fat diet. Elevated CD36, IGFBP1 (insulin-like growth factor-binding protein 1), and chemokine (C-C motif) ligand 2 (CCL2) expression leads to steatosis and inflammation in the Wtap-HKO livers. The hepatocyte proliferation is dramatically increased in Wtap-HKO mice, which is due to higher activation of extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription-3 signaling pathways. Hepatic deletion of Wtap activates the ERK signaling pathway by increasing the protein stability of GRB2 and ERK1/2, which is due to the decreased expression of proteasome-related genes. Restoring PSMB4 or PSMB6 (two key components of the proteasome) leads to the downregulation of GRB2 and ERK1/2 in Wtap-HKO hepatocytes. Mechanistically, WTAP interacts with RNA polymerase II and H3K9ac to maintain expression of proteasome-related genes. These results demonstrate that hepatic deletion of Wtap promotes HCC progression through activating GRB2-ERK1/2-mediated signaling pathway depending on the downregulation of proteasome-related genes especially Psmb4 and Psmb6.

KZ02 enhances the radiosensitivity of BRAF-mutated CRC in vitro and in vivo

Colorectal cancer (CRC) is a common malignant tumor with a high incidence and mortality worldwide. Preoperative chemoradiotherapy is a common treatment for patients with metastatic colorectal cancer (mCRC) as it reduces colostomy and local recurrence. The RAS (rat sarcoma)-RAF (extracellular signal-regulated kinase)-MEK (mitogen-activated protein kinase)-ERK (extracellular signal-regulated kinase) pathway regulates important cellular processes in the CRC. Abnormal ERK activation stimulates cell growth and provides a survival advantage. Our group has previously reported that the compound KZ02 has a stronger ability to inhibit tumor growth than AZD6244 (a MEK inhibitor). In this study, we evaluated the antitumor activity of KZ02 in combination with ionizing radiation (IR) and investigated its mechanism of action in BRAF-mutated colorectal cancer. Our results showed that this combination kills tumor cells better than either radiation or drugs alone, both in vivo and in vitro. Furthermore, studies have shown that KZ02 inhibits ERK overactivation. The combination resulted in a G1 phase arrest, a reduction in the radioresistant S phase, and aggravating DNA damage. It can also inhibit Pim-1 (Moloney murine leukemia virus-1), p-BAD (Bcl-2 associated agonist of cell death), Bcl-2 (B-cell lymphoma 2) and Bcl-XL (B-cell lymphoma-extra large) levels and promote apoptosis when combined with radiation. Our results suggest that KZ02 significantly increases the radiosensitivity of BRAF-mutated CRC cells by perturbing the cell cycle, increasing DNA damage, and promoting tumor apoptosis.

Schistosoma mansoni excretory-secretory products induce protein kinase signalling, hyperkinesia, and stem cell proliferation in the opposite sex

Adult male and female schistosomes in copula dwell within human blood vessels and lay eggs that cause the major Neglected Tropical Disease human schistosomiasis. How males and females communicate to each other is poorly understood; however, male-female physical interaction is known to be important. Here, we investigate whether excretory-secretory products (ESPs), released into the external milieu by mature Schistosoma mansoni, might induce responses in the opposite sex. We demonstrate that ESPs adhere to the surface of opposite sex worms inducing the activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK) pathways, particularly in the parasite tegument. Furthermore, we show that mature worms stimulated signalling in juvenile worms. Strikingly, we demonstrate that ESPs from the opposite sex promote stem cell proliferation, in an ERK- and p38 MAPK-dependent manner, in the tegument and within the testes of males, and the ovaries and vitellaria of females. Hyperkinesia also occurs following opposite sex ESP exposure. Our findings support the hypothesis that male and female schistosomes may communicate over distance to modulate key processes underlying worm development and disease progression, opening unique avenues for schistosomiasis control.

SIRPA enhances osteosarcoma metastasis by stabilizing SP1 and promoting SLC7A3-mediated arginine uptake

The function of signal regulatory protein alpha (SIRPA) has been well studied in macrophages and dendritic cells, but relatively less in tumors. Notably, SIRPA is upregulated in osteosarcoma tissues, particularly in metastatic tissues, and is associated with unfavorable clinical outcomes. Knockdown of SIRPA impaired OS cell migration by decreasing specificity protein 1 (SP1) stability and arginine uptake. Importantly, SIRPA phosphorylated SP1 at threonine 278 (Thr278) through extracellular signal-regulated kinase (ERK) activation to protect SP1 from proteasomal degradation. In addition, SP1 increased solute carrier family 7 member 3 (SLC7A3) expression by binding to the SLC7A3 promoter and increased the capability of arginine uptake, thereby facilitating OS cell migration. More interestingly, arginine promoted the stability of SP1 in an ERK-independent manner and thus formed the “SP1 stabilization circle”. Combined treatment with the anti-SIRPA antibody and arginase, which blocked the circle, impaired tumor metastasis in mice bearing xenografts formed from SIRPA-overexpressing cells. In summary, our study demonstrates that the upregulation of SIRPA promotes OS metastasis via the “SP1 stabilization circle” and SLC7A3-mediated arginine uptake, which might serve as a target for OS treatment.

Phase plane dynamics of ERK phosphorylation

The extracellular signal-regulated kinase (ERK) controls multiple critical processes in the cell and is deregulated in human cancers, congenital abnormalities, immune diseases, and neurodevelopmental syndromes. Catalytic activity of ERK requires dual phosphorylation by an upstream kinase, in a mechanism that can be described by two sequential Michaelis-Menten steps. The estimation of individual reaction rate constants from kinetic data in the full mechanism has proved challenging. Here, we present an analytically tractable approach to parameter estimation that is based on the phase plane representation of ERK activation and yields two combinations of six reaction rate constants in the detailed mechanism. These combinations correspond to the ratio of the specificities of two consecutive phosphorylations and the probability that monophosphorylated substrate does not dissociate from the enzyme before the second phosphorylation. The presented approach offers a language for comparing the effects of mutations that disrupt ERK activation and function invivo. As an illustration, we use phase plane representation to analyze dual phosphorylation under heterozygous conditions, when two enzyme variants compete for the same substrate.

Renal Fibrosis Is Alleviated through Targeted Inhibition of IL-11–Induced Renal Tubular Epithelial-to-Mesenchymal Transition

Renal fibrosis is a pathologic process that leads to irreversible renal failure without effective treatment. Epithelial-to-mesenchymal transition (EMT) plays a key role in this process. The current study found that aberrant expression of IL-11 is critically involved in tubular EMT. IL-11 and its receptorsubunitalpha-1 (IL-11Rα1) were significantly induced in renal tubular epithelial cells (RTECs) in unilateral ureteral obstruction (UUO) kidneys, co-localized with transforming growth factor-β1. IL-11 knockdown ameliorated UUO-induced renal fibrosis invivo and transforming growth factor-β1-induced EMT invitro. IL-11 intervention directly induced the transdifferentiation of RTECs to the mesenchymal phenotype and increased the synthesis of profibrotic mediators. The EMT response induced by IL-11 was dependent on the sequential activation of STAT3 and extracellular signal-regulated kinase 1/2 signaling pathways and the up-regulation of metadherin in RTECs. Micheliolide (MCL) competitively inhibited the binding of IL-11 with IL-11Rα1, suppressing the activation of STAT3 and extracellular signal-regulated kinase 1/2-metadherin pathways, ultimately inhibiting renal tubular EMT and interstitial fibrosis induced by IL-11. In addition, treatment with dimethylaminomicheliolide, a pro-drug of MCL for invivo use, significantly ameliorated renal fibrosis exacerbated by IL-11 in the UUO model. These findings suggest that IL-11 is a promising target in renal fibrosis and that MCL/dimethylaminomicheliolide exerts its antifibrotic effect by suppressing IL-11/IL-11Rα1 interaction and blocking its downstream effects.

Genistein enhances NAD+ biosynthesis by upregulating nicotinamide phosphoribosyltransferase in adipocytes

A decrease in the NAD+ level in adipocytes causes adipose-tissue dysfunction, leading to systemic glucose, and lipid metabolism failure. Therefore, it is necessary to develop small molecules and nutraceuticals that can increase NAD+ levels in adipocytes. Genistein, a nutraceutical derived from soybeans, has various physiological activities and improves glucose and lipid metabolism. In this study, we aimed to unravel the effects of genistein on the NAD+ level in adipocytes and the underlying molecular mechanisms. Genistein enhanced NAD+ biosynthesis by increasing the expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in NAD+ biosynthesis. A pull-down assay using genistein-immobilized beads revealed prohibitin 1 (PHB1) as a target protein of genistein. The knockdown of Phb1 suppressed the genistein-induced increase in NAMPT expression and NAD+ level in adipocytes. Genistein-bound PHB1 contributed to the stabilization of the transcription factor CCAAT/enhancer-binding protein β through the activation of extracellular signal-regulated kinase, resulting in increased NAMPT expression at the transcriptional level. Genistein induced the dephosphorylation of peroxisome proliferator-activated receptor at serine 273 and increased the level of the insulin-sensitizing adipokine adiponectin in adipocytes, whereas the knockdown of Nampt and Phb1 abolished these genistein-mediated effects. Our results proved the potential efficacy of genistein in increasing the NAD+ level and restoring metabolic function in adipocytes. Furthermore, we identified PHB1, localized to the plasma membrane, as a novel candidate target protein for increased expression of NAMPT in adipocytes. Overall, these findings will assist in developing NAD+-boosting nutraceuticals to alleviate metabolic dysfunctions in adipose tissues.

Novel therapeutic for multiple sclerosis protects white matter function in EAE mouse model.

Multiple sclerosis (MS) is a chronic demyelinating disease with prominent axon dysfunction. Our previous studies in an MS mouse model, experimental autoimmune encephalomyelitis (EAE), demonstrated that major histocompatibility complex Class II constructs can reverse clinical signs of EAE. These constructs block binding and downstream signaling of macrophage migration inhibitory factors (MIF-1/2) through CD74, thereby inhibiting phosphorylation of extracellular signal-regulated kinase (ERK) activation and tissue inflammation and promoting remyelination. To directly assess the effects of a novel third generation construct, DRhQ, on axon integrity in EAE, we compared axon conduction properties using electrophysiology on corpus callosum slices and optic nerves. By using two distinct white matter (WM) tracts, we aimed to assess the impact of the EAE and the benefit of DRhQ on myelinated and unmyelinated axons as well as to test the clinical value of DRhQ on demyelinating lesions in CC and optic myelitis. Our study found that EAE altered axon excitability, delayed axon conduction and slowed spatiotemporal summation correlated with diffuse astrocyte and microglia activation. Because MS predisposes patients to stroke, we also investigated and showed that vulnerability to WM ischemia is increased in the EAE MS mouse model. Treatment with DRhQ after the onset of EAE drastically inhibited microglial and astrocyte activation, improved functional integrity of the myelinated axons and enhanced recovery after ischemia. These results demonstrate that DRhQ administered after the onset of EAE promotes WM integrity and function, and reduces subsequent vulnerability to ischemic injury, suggesting important therapeutic potential for treatment of progressive MS.