ERK Signaling Pathway Research Articles

Light-Inducible Activation of TrkA for Probing Chronic Pain in Mice.

Chronic pain is a prevalent problem that plagues modern society, and better understanding its mechanisms is critical for developing effective therapeutics. Nerve growth factor (NGF) and its primary receptor, Tropomyosin receptor kinase A (TrkA), are known to be potent mediators of chronic pain, but there is a lack of established methods for precisely perturbing the NGF/TrkA signaling pathway in the study of pain and nociception. Optobiological tools that leverage light-induced protein-protein interactions allow for precise spatial and temporal control of receptor signaling. Previously, our lab reported a blue light-activated version of TrkA generated using light-induced dimerization of the intracellular TrkA domain, opto-iTrkA. In this work, we show that opto-iTrkA activation is able to activate endogenous ERK and Akt signaling pathways and causes the retrograde transduction of phospho-ERK signals in dorsal root ganglion (DRG) neurons. Opto-iTrkA activation also sensitizes the transient receptor potential vanilloid 1 (TRPV1) channel in cellular models, further corroborating the physiological relevance of the optobiological stimulus. Finally, we show that opto-iTrkA enables light-inducible potentiation of mechanical sensitization in mice. Light illumination enables nontraumatic and reversible (<2 days) sensitization of mechanical pain in mice transduced with opto-iTrkA, which provides a platform for dissecting TrkA pathways for nociception in vitro and in vivo.

From Bone Remodeling to Wound Healing: An miR-146a-5p-Loaded Nanocarrier Targets Endothelial Cells to Promote Angiogenesis.

Wound healing is a complex challenge that demands urgent attention in the clinical realm. Efficient angiogenesis is a pivotal factor in promoting wound healing. microRNA-146a (miR-146a) inhibitor has angiogenic potential in the periodontal ligament. However, free microRNAs (miRNAs) are poorly delivered into cells due to their limited tissue specificity and low intracellular delivery efficiency. To address this hurdle, we developed a nanocarrier for targeted delivery of the miR-146a inhibitor into endothelial cells. It is composed of a polyethylenimine (PEI)-modified mesoporous silica nanoparticle (MSN) core and a pentapeptide (YIGSR) layer that recognizes endothelial cells. In vitro, we defined that the miR-146a inhibitor and adiponectin (ADP) can modulate angiogenesis and the remodeling of periodontal tissues by activating the ERK and Akt signaling pathways. Then, we confirm the specificity of YIGSR to endothelial cells, and importantly, the nanocarrier effectively delivers the miR-146a inhibitor into endothelial cells, promoting angiogenesis. In a C57 mouse skin wound model, the miR-146a inhibitor is successfully delivered into endothelial cells at the wound site using the nanocarrier, resulting in the formation of new blood vessels with strong CD31 expression. Additionally, no significant differences are found in the expression levels of inflammatory markers interleukin-6 and tumor necrosis factor-α. This outcome not only brings new strategies for angiogenesis but also exhibits broader implications for bone remodeling and wound healing. The breakthrough holds significance for future research and clinical interventions.

Galectin-3 inhibition alleviated LPS-induced periodontal inflammation in gingival fibroblasts and experimental periodontitis mice.

Clinical studies have confirmed that galectin-3 (Gal-3) levels are significantly elevated in periodontitis patients. The present study aimed to explore the effects of Gal-3 inhibition on periodontal inflammation in vitro and in vivo. Human gingival fibroblasts (HGFs) with or without Gal-3 knockdown were stimulated by lipopolysaccharide (LPS), and a ligation-induced mouse periodontitis model treated with a Gal-3 inhibitor was established. Hematoxylin-eosin (H&E) and immunohistochemistry (IHC) staining were used to evaluate Gal-3 levels in gingival tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect Gal-3, interleukin (IL)-6, IL-8, and C-C motif ligand 2 (CCL2) expression. Immunofluorescence and western blotting were used to detect NF-κB and ERK signaling pathway activation. Micro-computed tomography was used to analyse the degree of bone loss. Gal-3 was significantly up-regulated in inflamed gingival tissues and LPS-induced HGFs. Gal-3 knockdown markedly decreased LPS-induced IL-6, IL-8, and CCL2 expression and blocked NF-κB and ERK signaling pathway activation in HGFs. In the mouse periodontitis model, Gal-3 inhibition significantly alleviated IL-1β and IL-6 infiltration in gingival tissue and mitigated periodontal bone loss. Gal-3 inhibition notably alleviated periodontal inflammation partly through blocking NF-κB and ERK signaling pathway activation.

Quercetin conjugated PSC-derived exosomes to inhibit intimal hyperplasia via modulating the ERK, Akt, and NF-κB signaling pathways in the rat carotid artery post balloon injury

The primary challenge in percutaneous coronary interventions for vascular restenosis is the occurrence of restenosis, which is defined by the excessive proliferation of neointimal tissue. Herein, our research team suggests that exosomes obtained from PSC, when paired with quercetin (Q@PSC-E), successfully reduce neointimal hyperplasia in a Sprague-Dawley rat model. Furthermore, the physical properties of the synthesized Q@PSC-E were examined using UV–vis, DLS, and FT-IR characterization techniques. The rats were subjected to balloon injury (BI) utilizing a 2-Fr Fogarty arterial embolectomy balloon catheter. Intimal hyperplasia and the degree of VSMC proliferation were evaluated using histological analysis in the rat groups that received a dosage of Q@PSC-E at 30 mg/kg/d. Significantly, Q@PSC-E inhibited cell proliferation through a pathway that does not include lipoxygenase, as demonstrated by [3H] thymidine incorporation, MTT, and flow cytometry studies. Additionally, the data indicate that Q@PSC-E hinders cell proliferation by targeting particular events that promote cell growth, including the activation of Akt and NF-κB, disruption of cell-cycle progression and also obstructs the ERK signaling pathway.

Abstract IA001: Exploiting pathway activation as a new form of synthetic lethality

Abstract Targeting activated oncogenes is an effective treatment strategy across many cancers now including therapeutics targeting KRAS. Genetic events including DNA damage deficiencies and tumor suppressor mutations require alternative strategies and the concept of synthetic lethality has been applied to these alterations. PARPi inhibitors were a founding member of this class of therapeutics demonstrating enhanced activity in the setting of BRCA-deficiency. By applying large-scale loss-of-function approaches1 we and other discovered the vulnerability to PRMT5 and WRN inhibition imposed by co-deletions of MTAP and CDKN2A, and the MSI+ genotype respectively2,3. Such inhibitors are now in clinical trials. To explore the potential for paralogous genes to act as synthetic lethal gene pairs we enacted dual knockout screens. These early efforts led to the discovery of DUSP4/6 paralog dependence in the setting of BRAF and NRAS mutations in melanoma4. Intriguingly the loss of DUSP4/6 impaired cancer cell viability through the increased activation of ERK highlighting the susceptibility of cancers to pathway activation in addition to the more common sensitivity to pathway inhibition. This inappropriate activation of the ERK signaling pathway, the conflict between EGFR and KRAS activation, the synthetic lethality enacted by TRIM8 knockouts, and the effects of AR agonists on prostate cancer viability, points to a wider than expected vulnerability of cancer to inappropriate gene activation. To systematically identify context-specific gene activation induced lethalities in cancer, we developed methods for enacting gain-of-function perturbations across ∼500 barcoded cancer cell lines. With this approach, we queried the pan-cancer vulnerability landscape upon activating 10 key cancer pathway revealing activation dependencies in MAPK and PI3K pathways. Notably, we discovered novel pathway hyperactivation dependencies in subsets of APC-mutant colorectal cancers where further activation of the WNT pathway by APC knockdown or direct β-catenin overexpression led to robust antitumor effects in xenograft and patient-derived organoid models. These latter discoveries paradoxically point to the residual activity of the APC ubiquitin-ligase complex as a target in APC-mutant CRC5. 1. McDonald, E. R., 3rd et al. Project DRIVE: A Compendium of Cancer Dependencies and Synthetic Lethal Relationships Uncovered by Large-Scale, Deep RNAi Screening. Cell 170, 577-592.e10 (2017). 2. Mavrakis, K. J. et al. Disordered methionine metabolism in MTAP/CDKN2A-deleted cancers leads to dependence on PRMT5. Science 351, 1208–1213 (2016). 3. Chan, E. M. et al. WRN helicase is a synthetic lethal target in microsatellite unstable cancers. Nature 568, 551–556 (2019). 4. Ito, T. et al. Paralog knockout profiling identifies DUSP4 and DUSP6 as a digenic dependence in MAPK pathway-driven cancers. Nat. Genet. 53, 1664–1672 (2021). 5. Chang, L. et al. Systematic profiling of conditional pathway activation identifies contextdependent synthetic lethalities. Nat. Genet. 55, 1709–1720 (2023). Citation Format: William R. Sellers. Exploiting pathway activation as a new form of synthetic lethality [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA001.

HOXB2 promotes cisplatin resistance by upregulating lncRNA DANCR in ovarian cancer

Ovarian cancer (OV) is a highly fatal malignant disease that commonly manifests at an advanced stage. Drug resistance, particularly platinum resistance, is a leading cause of treatment failure because first-line systemic chemotherapy primarily relies on platinum-based regimens. By analyzing the gene expression levels in the Cancer Genome Atlas database, Genotype-Tissue Expression database, and Gene Expression Omnibus datasets, we discerned that HOXB2 was highly expressed in OV and was associated with poor prognosis and cisplatin resistance. Immunohistochemistry and loss-of-function experiments on HOXB2 were conducted to explore its role in OV. We observed that suppressing HOXB2 could impair the growth and cisplatin resistance of OV in vivo and in vitro. Mechanical investigation and experimental validation based on RNA-Seq revealed that HOXB2 regulated ATP-binding cassette transporter members and the ERK signaling pathway. We further demonstrated that HOXB2 modulated the expression of long non-coding RNA DANCR, a differentiation antagonizing non-protein coding RNA, and thus influenced its downstream effectors ABCA1, ABCG1, and ERK signaling to boost drug resistance and cancer proliferation. These results verified that high expression of HOXB2 correlated with platinum resistance and poor prognosis of OV. Therefore, targeting HOXB2 may be a promising strategy for OV therapy.

Canthin-6-one analogs block Newcastle disease virus proliferation via suppressing the Akt and ERK pathways

Newcastle disease virus, a member of the Paramyxoviridae family, causes significant economic losses in poultry worldwide. To identify novel antiviral agents against NDV, 36 canthin-6-one analogs were evaluated in this study. Our data showed that 8 compounds exhibited excellent inhibitory effects on NDV replication with IC50 values in the range of 5.26 to 11.76 μM. Besides, these analogs inhibited multiple NDV strains with IC50 values within 12 μM and exerted antiviral activity against peste des petits ruminants virus (PPRV) and canine distemper virus (CDV). Among these analogs, 16 presented the strongest anti-NDV activity (IC50 = 5.26 μM) and minimum cytotoxicity (CC50 > 200 μM) in DF-1 cells. Furthermore, 16 displayed antiviral activity in different cell lines. Our results showed that 16 did not affect the viral adsorption while it can inhibit the entry of NDV by suppressing the Akt pathway. Further study found that 16-treatment inhibited the NDV-activated ERK pathway, thereby promoting the expression of interferon-related genes. Our findings reveal an antiviral mechanism of canthin-6-one analogs through inhibition of the Akt and ERK signaling pathways. These results point to the potential value of canthin-6-one analogs to serve as candidate antiviral agents for NDV.

Isoflurane-induced neuroinflammation and NKCC1/KCC2 dysregulation result in long-term cognitive disorder in neonatal mice

BackgroundThe inhalational anesthetic isoflurane is commonly utilized in clinical practice, particularly in the field of pediatric anesthesia. Research has demonstrated its capacity to induce neuroinflammation and long-term behavioral disorders; however, the underlying mechanism remains unclear [1]. The cation-chloride cotransporters Na+–K+–2Cl−–1 (NKCC1) and K+–2Cl−–2 (KCC2) play a pivotal role in regulating neuronal responses to gamma-aminobutyric acid (GABA) [2]. Imbalances in NKCC1/KCC2 can disrupt GABA neurotransmission, potentially leading to neural circuit hyperexcitability and reduced inhibition following neonatal exposure to anesthesia [3]. Therefore, this study postulates that anesthetics have the potential to dysregulate NKCC1 and/or KCC2 during brain development.MethodsWe administered 1.5% isoflurane anesthesia to neonatal rats for a duration of 4 h at postnatal day 7 (PND7). Anxiety levels were assessed using the open field test at PND28, while cognitive function was evaluated using the Morris water maze test between PND31 and PND34. Protein levels of NKCC1, KCC2, BDNF, and phosphorylated ERK (P-ERK) in the hippocampus were measured through Western blotting analysis. Pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were quantified using ELISA.ResultsWe observed a decrease in locomotion trajectories within the central region and a significantly shorter total distance in the ISO group compared to CON pups, indicating that isoflurane induces anxiety-like behavior. In the Morris water maze (MWM) test, rats exposed to isoflurane exhibited prolonged escape latency onto the platform. Additionally, isoflurane administration resulted in reduced time spent crossing in the MWM experiment at PND34, suggesting long-term impairment of memory function. Furthermore, we found that isoflurane triggered activation of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α; downregulated KCC2/BDNF/P-ERK expression; and increased the NKCC1/KCC2 ratio in the hippocampus of PND7 rats. Bumetadine (NKCC1 specific inhibitors) reversed cognitive damage and effective disorder induced by isoflurane in neonatal rats by inhibiting TNF-α activation, normalizing IL-6 and IL-1β levels, restoring KCC2 expression levels as well as BDNF and ERK signaling pathways. Based on these findings, it can be speculated that BDNF, P-ERK, IL-1β, IL-6 and TNF - α may act downstream of the NKCC1/KCC2 pathway.ConclusionsOur findings provide evidence that isoflurane administration in neonatal rats leads to persistent cognitive deficits through dysregulation of the Cation-Chloride Cotransporters NKCC1 and KCC2, BDNF, p-ERK proteins, as well as neuroinflammatory processes.

Focal cortical dysplasia II caused by brain somatic mutation of IRS-1 is associated with ERK signaling pathway activation.

Somatic mutations have been identified in 10% to 63% of focal cortical dysplasia type II samples, primarily linked to the mTOR pathway. When the causative genetic mutations are not identified, this opens the possibility of discovering new pathogenic genes or pathways that could be contributing to the condition. In our previous study, we identified a novel candidate pathogenic somatic variant of IRS-1 c.1791dupG in the brain tissue of a child with focal cortical dysplasia type II. This study further explored the variant's role in causing type II focal cortical dysplasia through in vitro overexpression in 293T and SH-SY5Y cells and in vivo evaluation via in utero electroporation in fetal brains, assessing effects on neuronal migration, morphology, and network integrity. It was found that the mutant IRS-1 variant led to hyperactivity of p-ERK, increased cell volume, and was predominantly associated with the MAPK signaling pathway. In vivo, the IRS-1 c.1791dupG variant induced abnormal neuron migration, cytomegaly, and network hyperexcitability. Notably, the ERK inhibitor GDC-0994, rather than the mTOR inhibitor rapamycin, effectively rescued the neuronal defects. This study directly highlighted the ERK signaling pathway's role in the pathogenesis of focal cortical dysplasia II and provided a new therapeutic target for cases of focal cortical dysplasia II that are not treatable by rapamycin analogs.

Mechanism of miR-126 Loaded in Albumin Nanoparticles for Reversing the Multidrug Resistance in Breast Carcinoma Cells

This study discussed the mechanism of miR-126 loaded in albumin nanoparticles in reversing the multi drug resistance (MDR) in breast carcinoma cells through EGFR-MEK-ERK signal pathway. MCF-7/ADM cells were divided into blank set, empty vector set, miR-126 set and set of vector and miR-126 randomly. The change of drug susceptibility, protein expression of P-gp, BCRP, EGFR, p-EGFR, MEK, p-MEK, ERK and p-ERK, correlation between miR-126 and EGFR-MEK- ERK signal pathway were observed. miR-126 expression in set of vector was the highest. The second was in miR-126 set. IC50 of ADM in miR-126 set was 4.6 µg/mL. The reversion times were two times. The reversion times in set of vector and miR-126 set was 2.8 times. The presentation of BCRP and P-gp in miR-126 set and set of vector and miR-126 was reduced notably. The activity of EGFR-MEK-ERK signal pathway was restrained by miR-126. The content of p-EGFR, p-MEK and p-ERK in miR-126 set and set of vector and miR-126 was reduced notably compared with blank set. EGFR-MEK-ERK signal activity was targeting regulated by miR-126 loaded in albumin nanoparticles. The level of phosphoric acid activators was reduced abnormally. The expression of BCRP and P-gp was reduced notably. The MDR in breast carcinoma cells was reversed and the drug susceptibility was elevated notably.

Epigallocatechin-3-gallate inhibits osteogenic differentiation of vascular smooth muscle cells through the transcription factor JunB.

Medial arterial calcification (MAC) accompanying chronic kidney disease (CKD) leads to increased vessel wall stiffness, myocardial ischemia, heart failure, and increased cardiovascular morbidity and mortality. Unfortunately, there are currently no drugs available to treat MAC. The natural polyphenol epigallocatechin-3-gallate (EGCG) has been demonstrated to protect against cardiovascular disease; however, whether EGCG supplementation inhibits MAC in CKD remains unclear. In this study, we utilize a CKD-associated MAC model to investigate the effects of EGCG on vascular calcification and elucidate the underlying mechanisms involved. Our findings demonstrate that EGCG treatment significantly reduces calcium phosphate deposition and osteogenic differentiation of VSMCs in vivo and in vitro in a dose-dependent manner. In addition, through RNA sequencing (RNA-seq) analysis, we show a significant activation of the transcription factor JunB both in CKD mouse arteries and in osteoblast-like VSMCs. Notably, EGCG effectively suppresses CKD-associated MAC by inhibiting the activity of JunB. In addition, overexpression of JunB can abolish while knockdown of JunB can enhance the inhibitory effect of EGCG on the osteogenic differentiation of VSMCs. Furthermore, EGCG supplementation inhibits MAC in CKD via modulation of the JunB-dependent Ras/Raf/MEK/ERK signaling pathway. In conclusion, our study highlights the potential therapeutic value of EGCG for managing CKD-associated MAC, as it mitigates this pathological process through targeted inactivation of JunB.

MiR-107 Targets NSG1 to Regulate Hypopharyngeal Squamous Cell Carcinoma Progression through ERK Pathway.

Hypopharyngeal squamous cell carcinoma (HSCC) is a kind of malignant tumor with a poor prognosis and low quality of life in the otolaryngology department. It has been found that microRNA (miRNA) plays an important role in the occurrence and development of various tumors. This study found that the expression level of miRNA-107 (miR-107) in HSCC was significantly reduced. Subsequently, we screened out the downstream direct target gene Neuronal Vesicle Trafficking Associated 1 (NSG1) related to miR-107 through bioinformatics analysis and found that the expression of NSG1 was increased in HSCC tissues. Following the overexpression of miR-107 in HSCC cells, it was observed that miR-107 directly suppressed NSG1 expression, leading to increased apoptosis, decreased proliferation, and reduced invasion capabilities of HSCC cells. Subsequent experiments involving the overexpression and knockdown of NSG1 in HSCC cells demonstrated that elevated NSG1 levels enhanced cell proliferation, migration, and invasion, while the opposite effect was observed upon NSG1 knockdown. Further investigations revealed that changes in NSG1 levels in the HSCC cells were accompanied by alterations in ERK signaling pathway proteins, suggesting a potential regulatory role of NSG1 in HSCC cell proliferation, migration, and invasion through the ERK pathway. These findings highlight the significance of miR-107 and NSG1 in hypopharyngeal cancer metastasis, offering promising targets for therapeutic interventions and prognostic evaluations for HSCC.

Recombinant human growth hormone promotes wound angiogenesis in burned mice through the ERK signaling pathway.

Burns are the most severe type of trauma, and the resulting ischemia and hypoxia damage can promote the dysfunction and even failure of tissues and organs throughout the body, endangering patients' life safety. Recombinant human growth hormone (rhGH) has the functions of promoting protein synthesis to reverse negative nitrogen balance, accelerating wound healing, and improving immune function, which is widely used in the treatment of burns. However, the exact mechanism and pathway of rhGH's action is not yet fully understood. In this study, we observed the wound repair effect of recombinant human growth hormone (rhGH) on burned mice and further analyzed the mechanism of action, which can provide more comprehensive reference opinions for clinical practice. First, by establishing a burn mouse model and and intervening with different doses of rhGH, we found that the wound healing capacity of mice was significantly enhanced and the inflammatory and oxidative stress responses were obviously alleviated, confirming the excellent promotion of wound repair and anti-inflammatory and antioxidant effects of rhGH. Subsequently, we found that the expression of p-ERK1/2/ERK1/2, EGF, TGF-β, and VEGF proteins was elevated in the traumatic tissues of mice after rhGH intervention, suggesting that the pathway of action of rhGH might be related to the activation of ERK pathway to promote the regeneration of traumatic capillaries.

Providing biomimetic microenvironment for pulp regeneration via hydrogel-mediated sustained delivery of tissue-specific developmental signals

Regenerative endodontic therapy is a promising approach to restore the vitality of necrotic teeth, however, pulp regeneration in mature permanent teeth remains a substantial challenge due to insufficient developmental signals. The dentin is embryologically and histologically similar to the pulp, which contains a cocktail of pulp-specific structural proteins and growth factors, thus we proposed an optimizing strategy to obtain dentin matrix extracted proteins (DMEP) and engineered a DMEP functionalized double network hydrogel, whose physicochemical property was tunable by adjusting polymer concentrations to synchronize with regenerated tissues. In vitro models showed that the biomimetic hydrogel with sustained release of DMEP provided a beneficial microenvironment for the encapsulation, propagation and migration of human dental pulp stem cells (hDPSCs). The odontogenic and angiogenic differentiation of hDPSCs were enhanced as well. To elicit the mechanism hidden in the microenvironment to guide cell fate, RNA sequencing was performed and 109 differential expression of genes were identified, the majority of which enriched in cell metabolism, cell differentiation and intercellular communications. The involvement of ERK, p38 and JNK MAPK signaling pathways in the process was confirmed. Of note, in vivo models showed that the injectable and in situ photo-crosslinkable hydrogel was user-friendly for root canal systems and was capable of inducing the regeneration of highly organized and vascularized pulp-like tissues in root segments that subcutaneously implanted into nude mice. Taken together, this study reported a facile and efficient way to fabricate a cell delivery hydrogel with pulp-specific developmental cues, which exhibited promising application and translation potential in future regenerative endodontic fields.

Oxytocin attenuates hypothalamic injury-induced cognitive dysfunction by inhibiting hippocampal ERK signaling and Aβ deposition

In clinical settings, tumor compression, trauma, surgical injury, and other types of injury can cause hypothalamic damage, resulting in various types of hypothalamic dysfunction. Impaired release of oxytocin can lead to cognitive impairment and affect prognosis and long-term quality of life after hypothalamic injury. Hypothalamic injury-induced cognitive dysfunction was detected in male animals. Behavioral parameters were measured to assess the characteristics of cognitive dysfunction induced by hypothalamic–pituitary stalk lesions. Brains were collected for high-throughput RNA sequencing and immunostaining to identify pathophysiological changes in hippocampal regions highly associated with cognitive function after injury to corresponding hypothalamic areas. Through transcriptomic analysis, we confirmed the loss of oxytocin neurons after hypothalamic injury and the reversal of hypothalamic-induced cognitive dysfunction after oxytocin supplementation. Furthermore, overactivation of the ERK signaling pathway and β-amyloid deposition in the hippocampal region after hypothalamic injury were observed, and cognitive function was restored after inhibition of ERK signaling pathway overactivation. Our findings suggest that cognitive dysfunction after hypothalamic injury may be caused by ERK hyperphosphorylation in the hippocampal region resulting from a decrease in the number of oxytocin neurons, which in turn causes β-amyloid deposition.

Decreased thrombospondin-1 impairs endometrial stromal decidualization in unexplained recurrent spontaneous abortion†.

Inappropriate endometrial stromal decidualization has been implied as an important reason of many pregnancy-related complications, such as unexplained recurrent spontaneous abortion, preeclampsia, and intrauterine growth restriction. Here, we observed that thrombospondin-1, an adhesive glycoprotein, was significantly downregulated in endometrial decidual cells from patients with unexplained recurrent spontaneous abortion. The immortalized human endometrial stromal cell line was used to investigate the possible THBS1-mediated regulation of decidualization. In vitro experiments found that the expression level of THBS1 increased with the normal decidualization process. Knockdown of THBS1 could decrease the expression levels of prolactin and insulin-like growth factor binding protein-1, two acknowledged human decidualization markers, whereas THBS1 overexpression could reverse these effects. The RNA sequencing results demonstrated that the extracellular regulated protein kinases signaling pathway was potentially affected by the knockdown of THBS1. We further confirmed that the regulation of THBS1 on decidualization was achieved through the ERK signaling pathway by the treatment of inhibitors. Moreover, knockdown of THBS1 in pregnant mice could impair decidualization and result in an increased fetus resorption rate. Altogether, our study demonstrated a crucial role of THBS1 in the pathophysiological process of unexplained recurrent spontaneous abortion and provided some new insights into the research of pregnancy-related complications.

Structure-Based De Novo Design for the Discovery of Miniprotein Inhibitors Targeting Oncogenic Mutant BRAF.

Being a component of the Ras/Raf/MEK/ERK signaling pathway crucial for cellular responses, the VRAF murine sarcoma viral oncogene homologue B1 (BRAF) kinase has emerged as a promising target for anticancer drug discovery due to oncogenic mutations that lead to pathway hyperactivation. Despite the discovery of several small-molecule BRAF kinase inhibitors targeting oncogenic mutants, their clinical utility has been limited by challenges such as off-target effects and suboptimal pharmacological properties. This study focuses on identifying miniprotein inhibitors for the oncogenic V600E mutant BRAF, leveraging their potential as versatile drug candidates. Using a structure-based de novo design approach based on binding affinity to V600E mutant BRAF and hydration energy, 39 candidate miniprotein inhibitors comprising three helices and 69 amino acids were generated from the substructure of the endogenous ligand protein (14-3-3). Through in vitro binding and kinase inhibition assays, two miniproteins (63 and 76) were discovered as novel inhibitors of V600E mutant BRAF with low-micromolar activity, with miniprotein 76 demonstrating a specific impediment to MEK1 phosphorylation in mammalian cells. These findings highlight miniprotein 76 as a potential lead compound for developing new cancer therapeutics, and the structural features contributing to its biochemical potency against V600E mutant BRAF are discussed in detail.

A site-specific phosphorylation in FSTL1 determines its promigratory role in wound healing

Follistatin like-1 (FSTL-1) is a secreted glycoprotein of mesenchymal in origin. In human skin, FSTL1 is upregulated in the epidermal keratinocytes upon acute injury and is required for the migration of keratinocytes. Failure to upregulate FSTL1 leads to the lack of keratinocyte migration and the non-healing nature of diabetic foot ulcer (DFU). FSTL1 undergoes extensive post-translational modification (PTM) at specific residues. Glycosylation at N144, N175 and N180, are the only experimentally demonstrated PTM in FSTL1, wherein, N180 and N144 glycosylations have been found to be critical for its function in cardiac tissue regeneration and pre-adipocyte differentiation, respectively. However, it is not known if PTMs other than glycosylation occurs in FSTL1 and how it impacts its pro-migratory function. Using in-silico analysis of mass spectrometric datasets, we found a novel PTM, namely, Serine 165 (S165) phosphorylation in FSTL1. To address the role of S165 phosphorylation in its pro-migratory function, a phosphorylation defective mutant of FSTL1 (S165A) was constructed by converting serine 165 to alanine and over expressed in 293T cells. S165A mutation did not affect the secretion of FSTL1 in vitro. However, S165A abolished the pro-migratory effect of FSTL1 in cultured keratinocytes likely via its inability to facilitate ERK signaling pathway. Interestingly, bacterially expressed recombinant FSTL1, trans-dominantly inhibited wound closure in keratinocytes highlighting the prime role of FSTL1 phosphorylation for its pro-migratory function. Further, under high glucose conditions, which inhibited scratchwound migration of keratinocytes, we noticed a significant decrease in S165 phosphorylation. Taken together, our results reveal a hitherto unreported role of FSTL1 phosphorylation PTM with profound implications in wound healing.

Activation of the cGMP/PKG/ERK signaling pathway associated with PDE5Is inhibits fibroblast activation by downregulating autophagy in early progressive benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) is one of the most prevalent diseases affecting aging males. However, approximately, 8% of the BPH patients under 50-year-old experience remarkably early progression, for reasons that remain elusive. Among the various factors implicated in promoting BPH advancement, the activation of fibroblasts and autophagy hold particular importance. Our research endeavors to explore the mechanisms behind the accelerated progression in these patients. Immunohistochemistry and immunofluorescence were performed to detect the expression levels of LC3, p62, PDE5, and α-SMA in diverse BPH tissues and prostate stromal cells. The autophagy activator rapamycin, the autophagy suppressor chloroquine, and siRNA transfection were used to identify the impact of autophagy on fibroblast activation. Prostatic stromal fibroblasts in early progressive BPH tissues displayed activation of autophagy with an upregulation of LC3 and a concurrent downregulation of p62. After starvation or rapamycin treatment to a heightened level of autophagy, fibroblasts exhibited activation. Conversely, chloroquine treatment and ATG-7-knockdown effectively suppressed the level of autophagy and fibroblast activation. High expression of PDE5 was found in early progressive BPH stromal cells. The administration of PDE5 inhibitors (PDE5Is) hindered fibroblast activation through suppressing autophagy by inhibiting the ERK signaling pathway. Our findings suggest that autophagy plays a pivotal role in promoting BPH progression through fibroblast activation, while PDE5Is effectively suppress autophagy and fibroblast activation via the ERK signaling pathway. Nevertheless, further investigations are warranted to comprehensively elucidate the role of autophagy in BPH progression.

Sympathetic nerve signals: orchestrators of mammary development and stem cell vitality.

The mammary gland is a dynamic organ that undergoes significant changes at multiple stages of postnatal development. Although the roles of systemic hormones and microenvironmental cues in mammary homeostasis have been extensively studied, the influence of neural signals, particularly those from the sympathetic nervous system, remains poorly understood. Here, using a mouse mammary gland model, we delved into the regulatory role of sympathetic nervous signaling in the context of mammary stem cells and mammary development. Our findings revealed that depletion of sympathetic nerve signals results in defective mammary development during puberty, adulthood, and pregnancy, accompanied by a reduction in mammary stem cell number. Through in vitro three-dimensional culture and in vivo transplantation analyses, we demonstrated that the absence of sympathetic nerve signals hinders mammary stem cell self-renewal and regeneration, while activation of sympathetic nervous signaling promotes these capacities. Mechanistically, sympathetic nerve signals orchestrate mammary stem cell activity and mammary development through the ERK signaling pathway. Collectively, our study unveils the crucial roles of sympathetic nerve signals in sustaining mammary development and regulating mammary stem cell activity, offering a novel perspective on the involvement of the nervous system in modulating adult stem cell function and organ development.