Extracellular Signal-regulated Kinase Mitogen-activated Protein Research Articles

Bidirectional transfer of a small membrane-impermeable molecule between the C. elegans intestine and germline

The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) is a positive regulator of cell proliferation often upregulated in cancer. Its C. elegans ortholog MPK-1 stimulates germline stem cell (GSC) proliferation non-autonomously, from the intestine or somatic gonad. How MPK-1 can perform this task from either of these two tissues however remains unclear. We reasoned that somatic MPK-1 activity could lead to the generation of pro-proliferative small molecules that could transfer from the intestine and/or somatic gonad to the germline. Here, in support of this hypothesis, we demonstrate that a significant fraction of the small membrane-impermeable fluorescent molecule, 5-carboxyfluorescein (5-CF), transfers to the germline after its microinjection in the animal’s intestine. The larger part of this transfer targets oocytes and requires the germline RME-2 yolk receptor. A minor quantity of the dye is however distributed independently from RME-2 and more widely in the animal, including the distal germline, gonadal sheath, coelomocytes and hypodermis. We further show that the intestine-to-germline transfer efficiency of this RME-2 independent fraction does not vary together with GSC proliferation rates or MPK-1 activity. Therefore, if germline proliferation was influenced by small membrane-impermeable molecules generated in the intestine, it is unlikely that proliferation would be regulated at the level of molecule transfer rate. Finally, we show that conversely, a similar fraction of germline injected 5-CF transfers to the intestine, demonstrating transfer bidirectionality. Altogether, our results establish the possibility of an intestine-to-germline signaling axis mediated by small membrane-impermeable molecules that could promote GSC proliferation cell non-autonomously downstream of MPK-1 activity.

EIF4F controls ERK MAPK signaling in melanomas with BRAF and NRAS mutations

The eIF4F translation initiation complex plays a critical role in melanoma resistance to clinical BRAF and MEK inhibitors. In this study, we uncover a function of eIF4F in the negative regulation of the rat sarcoma (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) signaling pathway. We demonstrate that eIF4F is essential for controlling ERK signaling intensity in treatment-naïve melanoma cells harboring BRAF or NRAS mutations. Specifically, the dual-specificity phosphatase DUSP6/MKP3, which acts as a negative feedback regulator of ERK activity, requires continuous production in an eIF4F-dependent manner to limit excessive ERK signaling driven by oncogenic RAF/RAS mutations. Treatment with small-molecule eIF4F inhibitors disrupts the negative feedback control of MAPK signaling, leading to ERK hyperactivation and EGR1 overexpression in melanoma cells in vitro and in vivo. Furthermore, our quantitative analyses reveal a high spare signaling capacity in the ERK pathway, suggesting that eIF4F-dependent feedback keeps the majority of ERK molecules inactive under normal conditions. Overall, our findings highlight the crucial role of eIF4F in regulating ERK signaling flux and suggest that pharmacological eIF4F inhibitors can disrupt the negative feedback control of MAPK activity in melanomas with BRAF and NRAS activating mutations.

Interdependency and differential expression of ERK1 and ERK2 in breast and melanoma cell lines

BackgroundRegulatory mechanism of ERK1 and ERK2, their mechanisms of action, and how they impact on development, growth, and homeostasis of different organisms have been given much emphasis for long. ERK1 and 2 though are isoforms of ERK mitogen-activated protein kinase but are coded by two different genes MAPK3 and MAPK1 respectively and show differential expressions and interdependency in different cancer cell lines. Our previous investigations substantially stated the effect of ERK1 and ERK2 on different extracellular molecules like MMPs and integrins, responsible for cell growth and differentiation. Here, we aim to study individual roles of ERK1 and ERK2 and their interdependency in progression and invasiveness in various cancer cell lines.MethodsDifferent cancer cell lines namely B16F10 (melanoma), MCF7, and MDAMB231 (breast cancer) for studying this particular question were used. Methodologies like gelatin zymography, immunoprecipitation, Western blotting, cell invasion assay, wound healing assay, siRNA transfection, and double transfection procedures were followed for our study.ResultsOur findings suggest compensation for ERK2 deficiency by pERK1, clear ERK2 predominance in MCF7 cell line, ERK1-ERK2 interdependency in MDAMB231 cells with regard to compensating each other, and significant role of both ERK1 and ERK2 in modulation of MMP9.ConclusionIf summarized, our results prove the contribution of ERK2 in compensating ERK1 loss and vice versa and an evident role of ERK1 in cancer cell invasiveness.

The BDNF-ERK/MAPK axis reduces phosphatase and actin regulator1, 2 and 3 (PHACTR1, 2 and 3) mRNA expressions in cortical neurons.

Actin rearrangement and phosphorylation-dephosphorylation in the nervous system contribute to plastic alteration of neuronal structure and function. Phosphatase and actin regulator (PHACTR) family members are actin- and protein phosphatase 1 (PP1)-binding proteins. Because some family members act as regulators of neuronal morphology, studying the regulatory mechanisms of PHACTR is valuable for understanding the basis of neuronal circuit formation. Although expression patterns of PHACTR family molecules (PHACTR1-4) vary across distinct brain areas, little is known about the extracellular ligands that influence their mRNA levels. In this study, we focused on an important neurotrophin, brain-derived neurotrophic factor (BDNF), and examined its effect on mRNA expression of PHACTR family member in cortical neurons. PHACTR1-3, but not PHACTR4, were affected by stimulation of primary cultured cortical neurons with BDNF; namely, sustained downregulation of their mRNA levels was observed. The observed downregulation was blocked by an inhibitor of the extracellular signal-regulated protein kinase/mitogen-activated protein kinase (ERK/MAPK) pathway, U0126, suggesting that ERK/MAPK plays an inhibitory role for gene induction of PHACTR1-3. These findings aid the elucidation of how BDNF regulates actin- and PP1-related neuronal functions.

Sodium Tungstate Promotes Neurite Outgrowth and Confers Neuroprotection in Neuro2a and SH-SY5Y Cells.

Sodium tungstate (Na2WO4) normalizes glucose metabolism in the liver and muscle, activating the Mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. Because this pathway controls neuronal survival and differentiation, we investigated the effects of Na2WO4 in mouse Neuro2a and human SH-SY5Y neuroblastoma monolayer cell cultures. Na2WO4 promotes differentiation to cholinergic neurites via an increased G1/G0 cell cycle in response to the synergic activation of the Phosphatidylinositol 3-kinase (PI3K/Akt) and ERK1/2 signaling pathways. In Neuro2a cells, Na2WO4 increases protein synthesis by activating the mechanistic target of rapamycin (mTOR) and S6K kinases and GLUT3-mediated glucose uptake, providing the energy and protein synthesis needed for neurite outgrowth. Furthermore, Na2WO4 increased the expression of myocyte enhancer factor 2D (MEF2D), a member of a family of transcription factors involved in neuronal survival and plasticity, through a post-translational mechanism that increases its half-life. Site-directed mutations of residues involved in the sumoylation of the protein abrogated the positive effects of Na2WO4 on the MEF2D-dependent transcriptional activity. In addition, the neuroprotective effects of Na2WO4 were evaluated in the presence of advanced glycation end products (AGEs). AGEs diminished neurite differentiation owing to a reduction in the G1/G0 cell cycle, concomitant with lower expression of MEF2D and the GLUT3 transporter. These negative effects were corrected in both cell lines after incubation with Na2WO4. These findings support the role of Na2WO4 in neuronal plasticity, albeit further experiments using 3D cultures, and animal models will be needed to validate the therapeutic potential of the compound.

Cinobufacini Inhibits Survival and Metastasis of Hepatocellular Carcinoma via c-Met Signaling Pathway.

To investigate the anti-tumor effects of cinobufacini (CINO) on hepatocellular carcinoma (HCC) induced by des-gamma-carboxy-prothrombin (DCP) and to uncover the underlying mechanisms. The inhibitory effect of CINO on HCC cell proliferation was evaluated using the cell counting kit-8 method, and the apoptosis rate was quantified using flow cytometry. Immunofluorescence and Western blot analyses were used to investigate the differential expression of proteins associated with cell growth, apoptosis, migration, and invasion pathways after CINO treatment. The therapeutic potential of CINO for HCC was confirmed, and the possibility of combining cinobufacini with c-Met inhibitor for the treatment of primary HCC was further validated by in vivo experiments. Under the induction of DCP, CINO inhibited the activity of HCC cells, induced apoptosis, and inhibited migration and invasion. Upon the induction of DCP, CINO regulated c-Met activation and the activation of the phosphatidylinositol-3 kinase/protein kinase B (PI3K/AKT) and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) pathways. In a mouse model of HCC, CINO exhibited significant antitumor effects by inhibiting the phosphorylation of c-Met and the downstream PI3K/AKT and MEK/ERK pathways in tumor tissues. CINO inhibited HCC cell growth, promoted apoptosis, and suppressed HCC cell invasion and migration by targeting c-Met and PI3K/AKT and MEK/ERK signaling pathways under DCP induction.

GPCR–MAPK signaling pathways underpin fitness trade-offs in whitefly

Trade-offs between evolutionary gain and loss are prevalent in nature, yet their genetic basis is not well resolved. The evolution of insect resistance to insecticide is often associated with strong fitness costs; however, how the fitness trade-offs operates remains poorly understood. Here, we show that the mitogen-activated protein kinase (MAPK) pathway and its upstream and downstream actors underlie the fitness trade-offs associated with insecticide resistance in the whitefly Bemisia tabaci. Specifically, we find a key cytochrome P450 gene CYP6CM1, that confers neonicotinoids resistance to in B. tabaci, is regulated by the MAPKs p38 and ERK through their activation of the transcription factor cAMP-response element binding protein. However, phosphorylation of p38 and ERK also leads to the activation of the transcription repressor Cap "n" collar isoform C (CncC) that negatively regulates exuperantia (Ex), vasa (Va), and benign gonial cell neoplasm (Bg), key genes involved in oogenesis, leading to abnormal ovary growth and a reduction in female fecundity. We further demonstrate that the transmembrane G protein-coupled receptor (GPCR) neuropeptide FF receptor 2 (NPFF2) triggers the p38 and ERK pathways via phosphorylation. Additionally, a positive feedback loop between p38 and NPFF2 leads to the continuous activation of the MAPK pathways, thereby constitutively promoting neonicotinoids resistance but with a significant reproductive cost. Collectively, these findings provide fundamental insights into the role of cis-trans regulatory networks incurred by GPCR-MAPK signaling pathways in evolutionary trade-offs and applied knowledge that can inform the development of strategies for the sustainable pest control.

MAPK signaling pathway participates in the regulation of intestinal phosphorus and calcium absorption in broiler chickens via 1,25-dihydroxyvitamin D3

Four experiments were performed to investigate the role of the mitogen-activated protein kinase (MAPK) signaling pathway in intestinal absorption of phosphorus (P) and calcium (Ca) in broiler chickens. Experiment 1 assessed how dietary levels of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) influence the gene expression of intestinal P and Ca transporters in broilers. Experiment 2 evaluated the effects of 1,25(OH)2D3 administered via intraperitoneal injection on the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38MAPK) signaling pathways. Experiments 3 and 4 investigated the effect of ERK and p38MAPK inhibitors on the expression of intestinal P and Ca transporters. The findings demonstrated that broilers (1-21 days old) fed a 1,25(OH)2D3-deficient diet (0.625 µg/kg) exhibited reduced body weight, tibia P and Ca levels, and mRNA levels of P transporters (NaPi-IIb, PiT-1, and PiT-2), Ca transporters (NCX1, PMCA1b, and CaBP-D28k), vitamin D receptors (VDR), ERK, and p38MAPK in the duodenum (Experiment 1) (P < 0.05). By comparison, the growth, bone quality, and mRNA levels of genes (except for duodenal NaPi-IIb) in broilers were similar to those in broilers fed the control diet when dietary 1,25(OH)2D3 was adequate (5 µg/kg) (Experiment 1) (P > 0.05). After intraperitoneal injection of 1,25(OH)2D3, the mRNA level of jejunal NaPi-IIb and the protein level of p-p38MAPK/t-p38MAPK in broilers (9-14 days old) decreased (P < 0.05), whereas the mRNA level of CaBP-D28k and the protein level of p-ERK/t-ERK increased (Experiment 2) (P < 0.05). The mRNA and protein expression of jejunal NaPi-IIb and the protein expression of CaBP-D28k in broilers (9-17 days old) treated with the ERK inhibitor PD98059 were greater than those in the control group (Experiment 3) (P < 0.05). Similarly, compared with control broilers, broilers (9-17 days old) treated with the p38MAPK inhibitor SB203580 showed elevated mRNA expression of jejunal NaPi-IIb and CaBP-D28k (Experiment 4) (P < 0.05). These results suggest that adequate supplementation with 1,25(OH)2D3 (5 µg/kg) can restore broiler growth and bone quality by upregulating the transcription of genes involved in intestinal P and Ca absorption. Additionally, the ERK and p38MAPK signaling pathways are implicated in the modulatory effect of 1,25(OH)2D3 on the absorption of P and Ca in broilers.

SIRT6 Inhibits the Proliferation and Collagen Synthesis of Keloid Fibroblasts through MAPK/ERK Pathway.

Keloid, a fibroproliferative disorder, significantly impacts patients' quality of life, yet effective therapies remain elusive. This study explored the role of silent information regulator 6 (SIRT6) in modulating the proliferation, invasion, and collagen synthesis of keloid fibroblasts. Keloid and normal skin specimens were collected, and fibroblasts were isolated from the keloid tissue. SIRT6 recombinant adenovirus (Ad) was constructed to infect keloid fibroblasts to overexpress SIRT6. This study entails three groups: Control group, adenovirus-Negative Control (Ad-NC) group, and Ad-SIRT6 group. SIRT6 protein and mRNA levels were measured via Western blotting and Quantitative reverse transcription polymerase chain reaction (qRT-PCR), respectively. Cell viability was determined using 5-ethynyl-2'-deoxyuridine (EdU) assay. Flow cytometry was exploited to measure cell apoptosis. To investigate cell migration, wound healing assay and Transwell assay were employed. Western blotting was also utilized to study the expression levels of apoptotic proteins, collagen deposition-related proteins, and Mitogen-Activated Protein Kinases (MAPK)/extracellular regulated protein kinases (ERK) pathway-related proteins. Compared to the control and Ad-NC groups, the Ad-SIRT6 group exhibited significantly elevated SIRT6 level; diminished cell proliferation, migration and invasion; reduced protein levels of α-smooth muscle actin (α-SMA), collagen I, collagen III, phospho SMAD Family Member 3 (p-Smad3), transforming growth factor-β 1 (TGF-β1), and MAPK/ERK pathway proteins (phospho extracellular signal-regulated protein kinase 1/2 (p-ERK1/2), phospho MAP kinase-ERK kinase (p-MEK) and phospho-c-Raf (p-c-Raf)). Treatment with epidermal growth factor (EGF), an MAPK/ERK pathway agonists, reversed the inhibitory effect of SIRT6 on cell activity and inhibited apoptosis in keloid fibroblasts. SIRT6 overexpression in keloid fibroblasts attenuates proliferation, invasion, and collagen synthesis, while fostering apoptosis, likely through the suppression of MAPK/ERK pathway activity. This suggests a potential therapeutic target for keloid treatment.

TLR3-mediated Astrocyte Responses in High and Normal Glucose Adaptation Differently Regulated by Metformin.

Toll-like receptors 3 (TLR3) are innate immune receptors expressed on a wide range of cell types, including glial cells. Inflammatory responses altered by hyperglycemia highlight the need to explore the molecular underpinnings of these changes in cellular models. Therefore, here we estimated TLR3-mediated response of astrocytes cultured at normal (NG, 5 mM) and high (HG, 22.5 mM) glucose concentrations for 48 h before stimulation with polyinosinic:polycytidylic acid Poly(I:C) (PIC) for 6 h. Seahorse Extracellular Flux Analyzer (Seahorse XFp) was used to estimate the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). Although adaptation to HG affected ECAR and OCR, the stimulation of cells with PIC had no effect on ECAR. PIC reduced maximal OCR, but this effect disappeared upon adaptation to HG. PIC-stimulated release of cytokines IL-1β, IL-10 was reduced, and that of IL-6 and iNOS was increased in the HG model. Adaptation to HG reduced PIC-stimulated synthesis of COX-derived oxylipins measured by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Adaptation to HG did not alter PIC-stimulated p38 activity, ERK mitogen-activated protein kinase, STAT3 and ROS production. Metformin exhibited anti-inflammatory activity, reducing PIC-stimulated synthesis of cytokines and oxylipins. Cell adaptation to high glucose concentration altered the sensitivity of astrocytes to TLR3 receptor activation, and the hypoglycemic drug metformin may exert anti-inflammatory effects under these conditions.

Yeast β-glucan-based nanoparticles loading methotrexate promotes osteogenesis of hDPSCs and periodontal bone regeneration under the inflammatory microenvironment

The regeneration of absorbed alveolar bone and reconstruction of periodontal support tissue are huge challenges in the clinical treatment of periodontitis due to the limited regenerative capacity of alveolar bone. It is essential to regulate inflammatory reaction and periodontal cell differentiation. Based on the anti-inflammatory effect of baker's yeast β-glucan (BYG) with biosafety by targeting macrophages, the BYG-based nanoparticles loading methotrexate (cBPM) were fabricated from polyethylene glycol-grafted BYG through chemical crosslinking for treatment of periodontitis. In our findings, cBPM promoted osteogenesis of human dental pulp stem cells (hDPSCs) under inflammatory microenvironment, characterized by the enhanced expression of osteogenesis-related Runx2 and activation of mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/Erk) pathway in vitro. Animal experiments further demonstrate that cBPM effectively promoted periodontal bone regeneration and achieved in a better effect of recovery indicated by 19.2 % increase in tissue volume, 7.1 % decrease in trabecular separation, and a significant increase in percent bone volume and trabecular thickness, compared with the model group. Additionally, cBPM inhibited inflammation and repaired alveolar bone by transforming macrophage phenotype from inflammatory M1 to anti-inflammatory M2. This work provides an alternative strategy for the clinical treatment of periodontitis through BYG-based delivery nanoplatform of anti-inflammatory drugs.

Determining the ERK-regulated phosphoproteome driving KRAS-mutant cancer.

To delineate the mechanisms by which the ERK1 and ERK2 mitogen-activated protein kinases support mutant KRAS-driven cancer growth, we determined the ERK-dependent phosphoproteome in KRAS-mutant pancreatic cancer. We determined that ERK1 and ERK2 share near-identical signaling and transforming outputs and that the KRAS-regulated phosphoproteome is driven nearly completely by ERK. We identified 4666 ERK-dependent phosphosites on 2123 proteins, of which 79 and 66%, respectively, were not previously associated with ERK, substantially expanding the depth and breadth of ERK-dependent phosphorylation events and revealing a considerably more complex function for ERK in cancer. We established that ERK controls a highly dynamic and complex phosphoproteome that converges on cyclin-dependent kinase regulation and RAS homolog guanosine triphosphatase function (RHO GTPase). Our findings establish the most comprehensive molecular portrait and mechanisms by which ERK drives KRAS-dependent pancreatic cancer growth.

MMP-2/GSH-responsive nanobomb for tumor chemo-immunotherapy with hierarchical delivery and TAMs re-education by regulating hbegf-activated ERK/MAPK signal pathway

The low targeting of immunity regulators & chemotherapy drugs and immune tolerance in the tumor microenvironment significantly hindered the chemo-immunotherapy effect. In this work, an MMP-2/GSH-responsive nanobomb loading Resiquimod (R848) and prodrug Cisplatin (Pt) with hierarchical drug delivery and tumor associated macrophages (TAMs) polarization traits was designed for improved chemo-immunotherapy. The Pt-carrying dendrimers penetrate the depth of tumor tissues under MMP-2 and activate the cytotoxicity of Pt prodrug in response to GSH, causing effective tumor damage and enhancing immunogenicity. Meanwhile, R848 is effectively uptake by TAMs, which were re-educated and then greatly relieved immune resistance. In vitro and in vivo results collectively revealed that the nanosystem could not only overcome the barrier of tumor penetration and the heterogeneity action sites of drugs but also re-educate TAMs and suppress immune evasion via activating extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signal pathway regulated by heparin binding EGF like growth factor (Hbegf) gene. The gene silencing or knockout in vitro and in vivo demonstrated that Hbegf could regulate the re-education of TAMs and the progression of tumors.

Latent-Transforming Growth Factor β-Binding Protein 1/Transforming Growth Factor β1 Complex Drives Antitumoral Effects upon ERK5 Targeting in Melanoma

Melanoma is the deadliest skin cancer, with a poor prognosis in advanced stages. Available treatments have improved the survival, although long-term benefits are still unsatisfactory. The mitogen-activated protein kinase ERK5 promotes melanoma growth, and ERK5 inhibition determines cellular senescence and the senescence-associated secretory phenotype. Here, latent-transforming growth factor β-binding protein 1 (LTBP1) mRNA was found to be upregulated in A375 and SK-Mel-5 BRAFV600E melanoma cells after ERK5 inhibition. In keeping with a key role of LTBP1 in regulating transforming growth factor β (TGF-β), TGF-β1 protein levels were increased in lysates and conditioned media of ERK5-knock down (KD) cells, and were reduced upon LTBP1 KD. Both LTBP1 and TGF-β1 proteins were increased in melanoma xenografts in mice treated with the ERK5 inhibitor XMD8-92. Moreover, treatment with conditioned media from ERK5-KD melanoma cells reduced cell proliferation and invasiveness, and TGF-β1-neutralizing antibodies impaired these effects. In silico datasets revealed that higher expression levels of both LTBP1 and TGFB1 mRNA are associated with better overall survival of melanoma patients, and that increased LTBP1 or TGF-β1 expression proved a beneficial role in patients treated with anti-PD1 immunotherapy, making unlikely a possible immunosuppressive role of LTBP1/TGF-β1 upon ERK5 inhibition. This study, therefore, identifies additional desirable effects of ERK5 targeting, providing evidence of an ERK5-dependent tumor suppressive role of TGF-β in melanoma.

Anti-inflammatory activities of novel heat shock protein 90 isoform selective inhibitors in BV-2 microglial cells.

Heat shock protein 90 (Hsp90) is a family of chaperone proteins that consists of four isoforms: Hsp90α, Hsp90β, glucose-regulated protein 94 (Grp94), and tumor necrosis factor type 1 receptor-associated protein (TRAP1). They are involved in modulating the folding, maturation, and activation of their client proteins to regulate numerous intracellular signaling pathways. Previous studies demonstrated that pan-Hsp90 inhibitors reduce inflammatory signaling pathways resulting in a reduction of inflammation and pain but show toxicities in cancer-related clinical trials. Further, the role of Hsp90 isoforms in inflammation remains poorly understood. This study aimed to determine anti-inflammatory activities of Hsp90 isoforms selective inhibitors on the lipopolysaccharide (LPS)-induced inflammation in BV-2 cells, a murine microglial cell line. The production of inflammatory mediators such as nitric oxide (NO), interleukin 1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) was measured. We also investigated the impact of Hsp90 isoform inhibitors on the activation of nuclear factor kappa B (NF-κB), nuclear factor erythroid 2-related factor 2 (Nrf2), and mitogen-activated protein kinases (MAPKs). We found that selective inhibitors of Hsp90β reduced the LPS-induced production of NO, IL-1β, and TNF-α via diminishing the activation of NF-κB and Extracellular signal-regulated kinases (ERK) MAPK. The Hsp90α, Grp94, TRAP1 inhibitors had limited effect on the production of inflammatory mediators. These findings suggest that Hsp90β is the key player in LPS-induced neuroinflammation. Thereby providing a more selective drug target for development of medications involved in pain management that can potentially contribute to the reduction of adverse side effects associated with Hsp90 pan inhibitors.

Brain Derived Neurotrophic Factor (BDNF) Immunolabeling in C4-C5 Spinal Cord of Adult Female Rats Following Acute Intermittent Hypoxia (AIH) Exposure Composed of 5,3-min Hypoxic Episodes (AIH5x3min) at 4-weeks After mid-thoracic SCI

Acute intermittent hypoxia (AIH) induces spinal neural plasticity in both respiratory and non-respiratory somatic motor systems. AIH-induced neural plasticity has been best studied in the respiratory neural control system, where exposure to moderate AIH (9-15% inspired O2), typically composed of 3,5-min hypoxic episodes (AIH3x5min), has been shown to consistently elicit a robust increase in phrenic motor activity due to a serotonin-dependent mechanism that promotes de novo synthesis of BDNF and activation of high affnity BDNF-TrkB receptors to initiate ERK MAP kinase signaling in phrenic motoneurons. Whether other protocols that use a total duration of 15 min of hypoxic exposure, but with an increased number of shorter duration hypoxic episodes ( e.g, AIH5x3min) similarly elicit serotonin-dependent BDNF pathway-mediated neural plasticity mechanisms in this region remains to be determined. To begin to assess this possibility, we evaluated BDNF expression in C4-C5 spinal cord segments containing the phrenic motor nucleus following single exposure to AIH5x3min (12% O2) in urethane-anesthetized spontaneously breathing adult female rats at 4-weeks after mid-thoracic SCI. Experiments using AIH3x5min (12% O2) exposure and intermittent normoxia (Nx, 21% O2) exposure served as controls. At ~60-90 min after the AIH or Nx exposure protocol, the rats were transcardially perfused with saline followed by 4% PFA, and various regions of the CNS, including the C3-C5 spinal cord were removed, post-fixed, and cryoprotected. 20 μm thick transverse sections were then cut to obtain sets of slides for single- or double-label fluorescence IHC staining to localize BDNF protein expression in the C4-C5 spinal cord, including the region containing the phrenic motor nucleus. Preliminary analyses show that both AIH5x3min and AIH3x5min exposures elicit increased BDNF expression when compared to Nx exposure, with both diffuse cytosolic and punctate immunofluorescence labeling in and around neurons within the phrenic motor nucleus region, including labeling of presumptive phrenic motoneurons. No labeling was seen in adjacent tissue sections that were incubated without the primary antibody. Immunofluorescence labeling was also seen in the more medial regions of lamina VIII in the ventral horn, the medial regions of laminae VI and VII, and throughout lamina X; these regions have been reported to contain the V0-V3 classes of spinal interneurons (SpINs) albeit we did not verify BDNF labeling of specific classes of presumptive SpINs. These preliminary observations suggest that AIH5x3min exposure likely recruits a serotonin-dependent BDNF pathway-mediated mechanism for neural plasticity in a manner similar to that seen for AIH3x5min exposure. APS Summer Undergraduate Research Fellowship; DOD CDMRP W81XWH-17-1-0260; NYS DOH SCIRB C37711GG. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

IGF-1 Induces Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells by Promoting SOX4 via the MAPK/ERK Pathway.

Tissue engineering envisions functional substitute creation for damaged tissues. Insulin-like growth factor-1 (IGF-1) plays roles in bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation (OD), and we investigated its specific mechanism. BMSCs were cultured and OD was induced. Surface antigens (CD105, CD90, CD44, CD45, CD34) were identified by flow cytometry. Adipogenic, chondrogenic, and osteogenic differentiation abilities of BMSCs were observed. BMSCs were cultured in osteogenic medium containing 80 ng/mL IGF-1 for 3 weeks. Alkaline phosphatase activity, calcification level, osteogenic factor (runt related protein 2 [RUNX2], osteocalcin [OCN], osterix [OSX]), total (t-) ERK1/2 and phosphorylated- (p-) ERK1/2 levels, and SRY-related high-mobility-group box 4 (SOX4) levels were assessed by alkaline phosphatase staining and Alizarin Red staining, Western blot, and reverse transcription-quantitative polymerase chain reaction. The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway inhibitor (PD98059) was used to inhibit the MAPK/ERK pathway in IGF-1-treated BMSCs. Small interfering-SOX4 was transfected into BMSCs to down-regulate SOX4. IGF-1 increased alkaline phosphatase activity, cell calcification, and osteogenic factor (RUNX2, OCN, OSX) levels in BMSCs, indicating that IGF-1 induced rat BMSC OD. SOX4, and p-ERK1/2 and t-ERK1/2 levels were elevated in IGF-1-induced BMSCs, which were annulled by PD98059. PD98059 partly averted IGF-1-induced rat BMSC OD. SOX4 levels, alkaline phosphatase activity, cell calcification, and osteogenic factor (RUNX2, OCN, OSX) levels were reduced after SOX4 down-regulation, showing that downregulation of SOX4 averted the effect of IGF-1 on inducing rat BMSC OD. IGF-1 induced rat BMSC OD by stimulating SOX4 via the MAPK/ERK pathway.

CircUSP13 facilitates the fast-to-slow myofiber shift via the MAPK/ERK signaling pathway in goat skeletal muscles.

Understanding how skeletal muscle fiber proportions are regulated is essential for understanding muscle function and improving the quality of mutton. While circular RNA (circRNA) has a critical function in myofiber type transformation, the specific mechanisms are not yet fully understood. Prior evidence indicates that circular ubiquitin-specific peptidase 13 (circUSP13) can promote myoblast differentiation by acting as a ceRNA, but its potential role in myofiber switching is still unknown. Herein, we found that circUSP13 enhanced slow myosin heavy chain (MyHC-slow) and suppressed MyHC-fast expression in goat primary myoblasts (GPMs). Meanwhile, circUSP13 evidently enhanced the remodeling of the mitochondrial network while inhibiting the autophagy of GPMs. We obtained fast-dominated myofibers, via treatment with rotenone, and further demonstrated the positive role of circUSP13 in the fast-to-slow transition. Mechanistically, activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway significantly impaired the slow-to-fast shift in fully differentiated myotubes, which was restored by circUSP13 or IGF1 overexpression. In conclusion, circUSP13 promoted the fast-to-slow myofiber type transition through MAPK/ERK signaling in goat skeletal muscle. These findings provide novel insights into the role of circUSP13 in myofiber type transition and contribute to a better understanding of the genetic mechanisms underlying meat quality.

Chlorovirus Glycoproteins and SOD1G93A Significantly Enhance, While Cellular Proteins IRF3 and ERK MAP-kinase Significantly Dampen, Production of ALS-associated Inflammatory Factors from Murine Macrophages (P6-11.017)

Chlorovirus Glycoproteins and SOD1G93A Significantly Enhance, While Cellular Proteins IRF3 and ERK MAP-kinase Significantly Dampen, Production of ALS-associated Inflammatory Factors from Murine Macrophages (P6-11.017)

AESIS-1, a Rheumatoid Arthritis Therapeutic Peptide, Accelerates Wound Healing by Promoting Fibroblast Migration in a CXCR2-Dependent Manner.

In patients with autoimmune disorders such as rheumatoid arthritis (RA), delayed wound healing is often observed. Timely and effective wound healing is a crucial determinant of a patient's quality of life, and novel materials for skin wound repair, such as bioactive peptides, are continuously being studied and developed. One such bioactive peptide, AESIS-1, has been studied for its well-established anti-rheumatoid arthritis properties. In this study, we attempted to use the anti-RA material AESIS-1 as a therapeutic wound-healing agent based on disease-modifying antirheumatic drugs (DMARDs), which can help restore prompt wound healing. The efficacy of AESIS-1 in wound healing was assessed using a full-thickness excision model in diabetic mice; this is a well-established model for studying chronic wound repair. Initial observations revealed that mice treated with AESIS-1 exhibited significantly advanced wound repair compared with the control group. In vitro studies revealed that AESIS-1 increased the migration activity of human dermal fibroblasts (HDFs) without affecting proliferative activity. Moreover, increased HDF cell migration is mediated by upregulating chemokine receptor expression, such as that of CXC chemokine receptor 2 (CXCR2). The upregulation of CXCR2 through AESIS-1 treatment enhanced the chemotactic reactivity to CXCR2 ligands, including CXC motif ligand 8 (CXCL8). AESIS-1 directly activates the ERK and p38 mitogen-activated protein kinase (MAPK) signaling cascades, which regulate the migration and expression of CXCR2 in fibroblasts. Our results suggest that the AESIS-1 peptide is a strong wound-healing substance that increases the movement of fibroblasts and the expression of CXCR2 by turning on the ERK and p38 MAPK signaling cascades.