Growth factor-dependent phosphorylation of Gαi shapes canonical signaling by G protein-coupled receptors.

A long-standing question in the field of signal transduction is how distinct signaling pathways interact with each other to control cell behavior. Growth factor receptors and G protein-coupled receptors (GPCRs) are the two major signaling hubs in eukaryotes. Given that the mechanisms by which they signal independently have been extensively characterized, we investigated how they may cross-talk with each other. Using linear ion trap mass spectrometry and cell-based biophysical, biochemical, and phenotypic assays, we found at least three distinct ways in which epidermal growth factor affected canonical G protein signaling by the Gi-coupled GPCR CXCR4 through the phosphorylation of Gαi. Phosphomimicking mutations in two residues in the αE helix of Gαi (tyrosine-154/tyrosine-155) suppressed agonist-induced Gαi activation while promoting constitutive Gßγ signaling. Phosphomimicking mutations in the P loop (serine-44, serine-47, and threonine-48) suppressed Gi activation entirely, thus completely segregating growth factor and GPCR pathways. As expected, most of the phosphorylation events appeared to affect intrinsic properties of Gαi proteins, including conformational stability, nucleotide binding, and the ability to associate with and to release Gßγ. However, one phosphomimicking mutation, targeting the carboxyl-terminal residue tyrosine-320, promoted mislocalization of Gαi from the plasma membrane, a previously uncharacterized mechanism of suppressing GPCR signaling through G protein subcellular compartmentalization. Together, these findings elucidate not only how growth factor and chemokine signals cross-talk through the phosphorylation-dependent modulation of Gαi but also how such cross-talk may generate signal diversity.

SAP deletion promotes malignant insulinoma progression by inducing CXCL12 secretion from CAFs via the CXCR4/p38/ERK signalling pathway.

Malignant insulinoma is an extremely rare type of functioning pancreatic neuroendocrine tumour with a high degree of malignancy and a high incidence of metastasis. However, it is still unclear how malignant insulinomas develop and metastasize. Serum amyloid P component (SAP), a member of the pentraxin protein family, is an acute-phase protein secreted by liver cells. The role of SAP in insulinoma and the related mechanism are still unknown. To determine the effect of SAP on insulinoma, we crossed Rip1-Tag2 mice, which spontaneously develop insulinoma, and SAP knockout (KO) mice to generate Rip1-Tag2;SAP-/- mice. We found that SAP deletion significantly promoted the growth, invasion and metastasis of malignant insulinoma through C-X-C motif chemokine ligand 12 (CXCL12) secreted by cancer-associated fibroblasts (CAFs). Further study showed that SAP deletion promoted CXCL12 secretion by CAFs through the CXCR4/p38/ERK signalling pathway. These findings reveal a novel role and mechanism of SAP in malignant insulinoma and provide direct evidence that SAP may be a therapeutic agent for this disease.

Construction of a prognostic model for autophagy in Wilm's tumor.

BACKGROUND: Wilm's tumor (WT) is one of the most common childhood urological tumors, ranking second in the incidence of pediatric abdominal tumors. The development of WT is associated with various factors, and the correlation with autophagy is currently unclear. PURPOSE: To develop a new prognostic model of autophagy-related genes (ATG) for WT. METHODS: Using the Therapeutically applicable research to generate effective treatments (TARGET) database to screen for differentially expressed ATGs in WT and normal tissues. ATGs were screened for prognostic relevance to WT using one-way and multifactorial Cox regression analyses and prognostic models were constructed. The risk score was calculated according to the model, and the predictive ability of the constructed model was analyzed using the ROC (receiver operating characteristic) curve to verify the significance of the model for the prognosis of WT. RESULTS: Sixty-eight differentially expressed ATGs were identified by univariate Cox regression analysis, and two critical prognostic ATGs (CXCR4 and ERBB2) were identified by multivariate Cox regression analysis. Patients were divided into high-risk and low-risk groups according to the differential expression of these two ATGs. Kaplan-Meier (KM) curves showed a significant difference in survival time between the two groups. The critical prognostic ATGs were combined with race, age, and stage in a multifactorial regression analysis, and the final prognostic model was produced as a line graph. CONCLUSION: The prognostic model of autophagy-related genes composed of the CXCR4 gene and ERBB2 gene has a specific predictive value for the prognosis of WT, and the present study provides a clear basis for future research on biomarkers and therapeutic targets.

In vitro effects and mathematical modelling of CTCE-9908 (a chemokine receptor 4 antagonist) on melanoma cell survival.

CTCE-9908, a CXC chemokine receptor 4 (CXCR4) antagonist, prevents CXCR4 phosphorylation and inhibits the interaction with chemokine ligand 12 (CXCL12) and downstream signalling pathways associated with metastasis. This study evaluated the in vitro effects of CTCE-9908 on B16 F10 melanoma cells with the use of mathematical modelling. Crystal violet staining was used to construct a mathematical model of CTCE-9908 B16 F10 (melanoma) and RAW 264.7 (non-cancerous macrophage) cell lines on cell viability to predict the half-maximal inhibitory concentration (IC50). Morphological changes were assessed using transmission electron microscopy. Flow cytometry was used to assess changes in cell cycle distribution, apoptosis via caspase-3, cell survival via extracellular signal-regulated kinase1/2 activation, CXCR4 activation and CXCL12 expression. Mathematical modelling predicted IC50 values from 0 to 100 h. At IC50, similar cytotoxicity between the two cell lines and ultrastructural morphological changes indicative of cell death were observed. At a concentration 10 times lower than IC50, CTCE-9908 induced inhibition of cell survival (p = 0.0133) in B16 F10 cells but did not affect caspase-3 or cell cycle distribution in either cell line. This study predicts CTCE-9908 IC50 values at various time points using mathematical modelling, revealing cytotoxicity in melanoma and non-cancerous cells. CTCE-9908 significantly inhibited melanoma cell survival at a concentration 10 times lower than the IC50 in B16 F10 cells but not RAW 264.7 cells. However, CTCE-9908 did not affect CXCR4 phosphorylation, apoptosis,\ or cell cycle distribution in either cell line.

Bone marrow mesenchymal stromal cells support regeneration of intestinal damage in a colitis mouse model, independent of their CXCR4 expression.

Inflammatory bowel disease (IBD) is characterized by a chronically dysregulated immune response in the gastrointestinal tract. Bone marrow multipotent mesenchymal stromal cells have an important immunomodulatory function and support regeneration of inflamed tissue by secretion of soluble factors as well as through direct local differentiation. CXCR4 is the receptor for CXCL12 (SDF-1, stromal-derived factor-1) and has been shown to be the main chemokine receptor, required for homing of MSCs. Increased expression of CXCL12 by inflamed intestinal tissue causes constitutive inflammation by attracting lymphocytes but can also be used to direct MSCs to sites of injury/inflammation. Trypsin is typically used to dissociate MSCs into single-cell suspensions but has also been shown to digest surface CXCR4. Here, we assessed the regenerative effects of CXCR4high and CXCR4low MSCs in an immune-deficient mouse model of DSS-induced colitis. We found that transplantation of MSCs resulted in clinical improvement and histological recovery of intestinal epithelium. In contrary to our expectations, the levels of CXCR4 on transplanted MSCs did not affect their regenerative supporting potential, indicating that paracrine effects of MSCs may be largely responsible for their regenerative/protective effects.

Chemokine receptor hetero-oligomers regulate monocyte chemotaxis.

It is known that stress influences immune cell function. The underlying molecular mechanisms are unclear. We recently reported that many chemokine receptors (CRs) heteromerize with α1-adrenoceptors (α1-ARs) through which CRs are regulated. Here, we show that arginine vasopressin receptor 1A (AVPR1A) heteromerizes with all human CRs, except chemokine (C-X-C motif) receptor (CXCR)1, in recombinant systems and that such heteromers are detectable in THP-1 cells and human monocytes. We demonstrate that ligand-free AVPR1A differentially regulates the efficacy of CR partners to mediate chemotaxis and that AVPR1A ligands disrupt AVPR1A:CR heteromers, which enhances chemokine (C-C motif) receptor (CCR)1-mediated chemotaxis and inhibits CCR2-, CCR8-, and CXCR4-mediated chemotaxis. Using bioluminescence resonance energy transfer to monitor G protein activation and CRISPR/Cas9 gene-edited THP-1 cells lacking AVPR1A or α1B-AR, we show that CRs that share the propensity to heteromerize with α1B/D-ARs and AVPR1A exist and function within interdependent hetero-oligomeric complexes through which the efficacy of CRs to mediate chemotaxis is controlled. Our findings suggest that hetero-oligomers composed of CRs, α1B/D-ARs, and AVPR1A may enable stress hormones to regulate immune cell trafficking.

Astragalus polysaccharides augment BMSC homing via SDF-1/CXCR4 modulation: a novel approach to counteract peritoneal mesenchymal transformation and fibrosis.

PURPOSE: This study aimed to evaluate the potential of astragalus polysaccharide (APS) pretreatment in enhancing the homing and anti-peritoneal fibrosis capabilities of bone marrow mesenchymal stromal cells (BMSCs) and to elucidate the underlying mechanisms. METHODS: Forty male Sprague-Dawley rats were allocated into four groups: control, peritoneal dialysis fluid (PDF), PDF + BMSCs, and PDF + APSBMSCs (APS-pre-treated BMSCs). A peritoneal fibrosis model was induced using PDF. Dil-labeled BMSCs were administered intravenously. Post-transplantation, BMSC homing to the peritoneum and pathological alterations were assessed. Stromal cell-derived factor-1 (SDF-1) levels were quantified via enzyme-linked immunosorbent assay (ELISA), while CXCR4 expression in BMSCs was determined using PCR and immunofluorescence. Additionally, a co-culture system involving BMSCs and peritoneal mesothelial cells (PMCs) was established using a Transwell setup to examine the in vitro effects of APS on BMSC migration and therapeutic efficacy, with the CXCR4 inhibitor AMD3100 deployed to dissect the role of the SDF-1/CXCR4 axis and its downstream impacts. RESULTS: In vivo and in vitro experiments confirmed that APS pre-treatment notably facilitated the targeted homing of BMSCs to the peritoneal tissue of PDF-treated rats, thereby amplifying their therapeutic impact. PDF exposure markedly increased SDF-1 levels in peritoneal and serum samples, which encouraged the migration of CXCR4-positive BMSCs. Inhibition of the SDF-1/CXCR4 axis through AMD3100 application diminished BMSC migration, consequently attenuating their therapeutic response to peritoneal mesenchyme-to-mesothelial transition (MMT). Furthermore, APS upregulated CXCR4 expression in BMSCs, intensified the activation of the SDF-1/CXCR4 axis's downstream pathways, and partially reversed the AMD3100-induced effects. CONCLUSION: APS augments the SDF-1/CXCR4 axis's downstream pathway activation by increasing CXCR4 expression in BMSCs. This action bolsters the targeted homing of BMSCs to the peritoneal tissue and amplifies their suppressive influence on MMT, thereby improving peritoneal fibrosis.

Chemokines as Prognostic Factor in Colorectal Cancer Patients: A Systematic Review and Meta-Analysis.

Chemokines orchestrate many aspects of tumorigenic processes such as angiogenesis, apoptosis and metastatic spread, and related receptors are expressed on tumor cells as well as on inflammatory cells (e.g., tumor-infiltrating T cells, TILs) in the tumor microenvironment. Expressional changes of chemokines and their receptors in solid cancers are common and well known, especially in affecting colorectal cancer patient outcomes. Therefore, the aim of this current systematic review and meta-analysis was to classify chemokines as a prognostic biomarker in colorectal cancer patients. A systematic literature search was conducted in PubMed, CENTRAL and Web of Science. Information on the chemokine expression of 25 chemokines in colorectal cancer tissue and survival data of the patients were investigated. The hazard ratio of overall survival and disease-free survival with chemokine expression was examined. The risk of bias was analyzed using Quality in Prognosis Studies. Random effects meta-analysis was performed to determine the impact on overall respectively disease survival. For this purpose, the pooled hazard ratios (HR) and their 95% confidence intervals (CI) were used for calculation. Twenty-five chemokines were included, and the search revealed 5556 publications. A total of thirty-one publications were included in this systematic review and meta-analysis. Overexpression of chemokine receptor CXCR4 was associated with both a significantly reduced overall survival (HR = 2.70, 95%-CI: 1.57 to 4.66, p = 0.0003) as well as disease-free survival (HR = 2.68, 95%-CI: 1.41 to 5.08, p = 0.0026). All other chemokines showed either heterogeneous results or few studies were available. The overall risk of bias for CXCR4 was rated low. At the current level of evidence, this study demonstrates that CXCR4 overexpression in patients with colorectal cancer is associated with a significantly diminished overall as well as disease-free survival. Summed up, this systematic review and meta-analysis reveals CXCR4 as a promising prognostic biomarker. Nevertheless, more evidence is needed to evaluate CXCR4 and its antagonists serving as new therapeutic targets.

Reg4 deficiency aggravates pancreatitis by increasing mitochondrial cell death and fibrosis.

Regenerating gene family member 4 (Reg4) has been implicated in acute pancreatitis, but its precise functions and involved mechanisms have remained unclear. Herein, we sought to investigate the contribution of Reg4 to the pathogenesis of pancreatitis and evaluate its therapeutic effects in experimental pancreatitis. In acute pancreatitis, Reg4 deletion increases inflammatory infiltrates and mitochondrial cell death and decreases autophagy recovery, which are rescued by the administration of recombinant Reg4 (rReg4) protein. In chronic pancreatitis, Reg4 deficiency aggravates inflammation and fibrosis and inhibits compensatory cell proliferation. Moreover, C-X-C motif ligand 12 (CXCL12)/C-X-C motif receptor 4 (CXCR4) axis is sustained and activated in Reg4-deficient pancreas. The detrimental effects of Reg4 deletion are attenuated by the administration of the approved CXCR4 antagonist plerixafor (AMD3100). Mechanistically, Reg4 mediates its function in pancreatitis potentially via binding its receptor exostosin-like glycosyltransferase 3 (Extl3). In conclusion, our findings suggest that Reg4 exerts a therapeutic effect during pancreatitis by limiting inflammation and fibrosis and improving cellular regeneration.

[Mechanism of tetramethylpyrazine combined with neural stem cells transplantation on cerebral ischemia-reperfusion rats].

This study aimed to investigate the intervention effect of tetramethylpyrazine(TMP) combined with transplantation of neural stem cells(NSCs) on middle cerebral artery occlusion(MCAO) rat model and to explore the mechanism of TMP combined with NSCs transplantation on ischemic stroke based on the regulation of stem cell biological behavior. MCAO rats were randomly divided into a model group, a TMP group, an NSCs transplantation group, and a TMP combined with NSCs transplantation group according to neurological function scores. A sham group was set up at the same time. The neurological function score was used to evaluate the improvement of neurological function in MCAO rats after TMP combined with NSCs transplantation. The proliferation, migration, and differentiation of NSCs were evaluated by BrdU, BrdU/DCX, BrdU/NeuN, and BrdU/GFAP immunofluorescence labeling. The protein expression of stromal cell-derived factor 1(SDF-1), C-X-C motif chemokine receptor 4(CXCR4), as well as oxidative stress pathway proteins nuclear factor erythroid 2-related factor 2(Nrf2), Kelch-like ECH-associated protein 1(KEAP1), heme oxygenase 1(HO-1), NAD(P)H quinone oxidoreductase 1(NQO1) was detected by Western blot to study the migration mechanism of TMP combined with NSCs. The results showed that TMP combined with NSCs transplantation significantly improved the neurological function score in MCAO rats. Immunofluorescence staining showed a significant increase in the number of BrdU~+, BrdU~+/DCX~+, BrdU~+/NeuN~+, and BrdU~+/GFAP~+ cells in the TMP, NSCs transplantation, and combined treatment groups, with the combined treatment group showing the most significant increase. Further Western blot analysis revealed significantly elevated expression of CXCR4 protein in the TMP, NSCs transplantation, and combined treatment groups, along with up-regulated protein expression of Nrf2, HO-1, and NQO1, and decreased KEAP1 protein expression. This study showed that both TMP and NSCs transplantation can promote the recovery of neurological function by promoting the proliferation, migration, and differentiation of NSCs, and the effect of TMP combined with NSCs transplantation is superior. The mechanism of action may be related to the activation of the Nrf2/HO-1/CXCR4 pathway.

[Mechanism of tetramethylpyrazine intervention with ischemia-reperfusion rats based on Nrf2/HO-1/CXCR4 pathway through regulating neural stem cell migration].

This study aims to decipher the mechanism of tetramethylpyrazine(TMP) in regulating the migration of neural stem cells(NSCs) in the rat model of middle cerebral artery occlusion(MCAO) via the nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase 1(HO-1)/C-X-C motif chemokine receptor 4(CXCR4) pathway. SD rats were randomized into sham, MCAO(model), and tetramethylpyrazine(TMP, 20 mg·kg~(-1) and 40 mg·kg~(-1)) groups. The neurological impairment was assessed by the modified neurological severity score(mNSS). The immunofluorescence assay was employed to detect the cells stained with both 5-bromodeoxyuridine(BrdU) and doublecortin(DCX) in the brain tissue. The effect of TMP on the migration of C17.2 cells was observed. Western blot was employed to determine the protein levels of Nrf2, HO-1, p62, NAD(P)H quinone oxidoreductase 1(NQO1), stromal cell-derived factor 1(SDF-1), and CXCR4 in the brain tissue and C17.2 cells. The results showed that after 7 days and 21 days of mode-ling, the mNSS and BrdU~+/DCX~+ cells were increased, and the expression of Nrf2 and CXCR4 in the brain tissue was up-regulated. Compared with the model group, TMP(40 mg·kg~(-1)) reduced the mNSS, increased the number of BrdU~+/DCX~+ cells, and up-regulated the expression of Nrf2, CXCR4, and SDF-1. In addition, TMP promoted the migration of C17.2 cells and up-regulated the expression of p62, Nrf2, HO-1, and NQO1 in a time-and dose-dependent manner. The expression was the highest at the time point of 12 h in the TMP(50 µg·mL~(-1)) group(P<0.01). In conclusion, TMP activates the Nrf2/HO-1/CXCR4 pathway to promote the migration of NSCs to the ischemic area, thus exerting the therapeutic effect on the ischemia-reperfusion injury. This study provides experimental support for the application of TMP in ischemic stroke.

Glucocorticoids paradoxically promote steroid resistance in B cell acute lymphoblastic leukemia through CXCR4/PLC signaling.

Glucocorticoid (GC) resistance in childhood relapsed B-cell acute lymphoblastic leukemia (B-ALL) represents an important challenge. Despite decades of clinical use, the mechanisms underlying resistance remain poorly understood. Here, we report that in B-ALL, GC paradoxically induce their own resistance by activating a phospholipase C (PLC)-mediated cell survival pathway through the chemokine receptor, CXCR4. We identify PLC as aberrantly activated in GC-resistant B-ALL and its inhibition is able to induce cell death by compromising several transcriptional programs. Mechanistically, dexamethasone (Dex) provokes CXCR4 signaling, resulting in the activation of PLC-dependent Ca2+ and protein kinase C signaling pathways, which curtail anticancer activity. Treatment with a CXCR4 antagonist or a PLC inhibitor improves survival of Dex-treated NSG mice in vivo. CXCR4/PLC axis inhibition significantly reverses Dex resistance in B-ALL cell lines (in vitro and in vivo) and cells from Dex resistant ALL patients. Our study identifies how activation of the PLC signalosome in B-ALL by Dex limits the upfront efficacy of this chemotherapeutic agent.

Cross-species single-cell analysis uncovers the immunopathological mechanisms associated with IgA nephropathy progression.

IgA nephropathy (IgAN) represents the main cause of renal failure, while the precise pathogenetic mechanisms have not been fully determined. Herein, we conducted a cross-species single-cell survey on human IgAN and mouse and rat IgAN models to explore the pathogenic programs. Cross-species single-cell RNA sequencing (scRNA-Seq) revealed that the IgAN mesangial cells (MCs) expressed high levels of inflammatory signatures CXCL12, CCL2, CSF1, and IL-34 and specifically interacted with IgAN macrophages via the CXCL12/CXCR4, CSF1/IL-34/CSF1 receptor, and integrin subunit alpha X/integrin subunit alpha M/complement C3 (C3) axes. IgAN macrophages expressed high levels of CXCR4, PDGFB, triggering receptor expressed on myeloid cells 2, TNF, and C3, and the trajectory analysis suggested that these cells derived from the differentiation of infiltrating blood monocytes. Additionally, protein profiling of 21 progression and 28 nonprogression IgAN samples revealed that proteins CXCL12, C3, mannose receptor C-type 1, and CD163 were negatively correlated with estimated glomerular filtration rate (eGFR) value and poor prognosis (30% eGFR as composite end point). Last, a functional experiment revealed that specific blockade of the Cxcl12/Cxcr4 pathway substantially attenuated the glomerulus and tubule inflammatory injury, fibrosis, and renal function decline in the mouse IgAN model. This study provides insights into IgAN progression and may aid in the refinement of IgAN diagnosis and the optimization of treatment strategies.

Comprehensive Analysis of CXCR4, JUNB, and PD-L1 Expression in Circulating Tumor Cells (CTCs) from Prostate Cancer Patients.

CXCR4, JUNB and PD-L1 are implicated in cancer progression and metastasis. The current study investigated these biomarkers in CTCs isolated from metastatic prostate cancer (mPCa) patients at the RNA and protein levels. CTCs were isolated from 48 mPCa patients using the Ficoll density gradient and ISET system (17 out of 48). The (CK/PD-L1/CD45) and (CK/CXCR4/JUNB) phenotypes were identified using two triple immunofluorescence stainings followed by VyCAP platform analysis. Molecular analysis was conducted with an EpCAM-dependent method for 25/48 patients. CK-8, CK-18, CK-19, JUNB, CXCR4, PD-L1, and B2M (reference gene) were analyzed with RT-qPCR. The (CK+/PD-L1+/CD45-) and the (CK+/CXCR4+/JUNB+) were the most frequent phenotypes (61.1% and 62.5%, respectively). Furthermore, the (CK+/CXCR4+/JUNB-) phenotype was correlated with poorer progression-free survival [(PFS), HR: 2.5, p = 0.049], while the (CK+/PD-L1+/CD45-) phenotype was linked to decreased overall survival [(OS), HR: 262.7, p = 0.007]. Molecular analysis revealed that 76.0% of the samples were positive for CK-8,18, and 19, while 28.0% were positive for JUNB, 44.0% for CXCR4, and 48.0% for PD-L1. Conclusively, CXCR4, JUNB, and PD-L1 were highly expressed in CTCs from mPCa patients. The CXCR4 protein expression was associated with poorer PFS, while PD-L1 was correlated with decreased OS, providing new biomarkers with potential clinical relevance.

Multiplex Detection of Fluorescent Chemokine Binding to CXC Chemokine Receptors by NanoBRET.

NanoLuc-mediated bioluminescence resonance energy transfer (NanoBRET) has gained popularity for its ability to homogenously measure ligand binding to G protein-coupled receptors (GPCRs), including the subfamily of chemokine receptors. These receptors, such as ACKR3, CXCR4, CXCR3, play a crucial role in the regulation of the immune system, are associated with inflammatory diseases and cancer, and are seen as promising drug targets. The aim of this study was to optimize NanoBRET-based ligand binding to NLuc-ACKR3 and NLuc-CXCR4 using different fluorescently labeled chemokine CXCL12 analogs and their use in a multiplex NanoBRET binding assay of two chemokine receptors at the same time. The four fluorescent CXCL12 analogs (CXCL12-AZD488, -AZD546, -AZD594, -AZD647) showed high-affinity saturable binding to both NLuc-ACKR3 and NLuc-CXCR4, with relatively low levels of non-specific binding. Additionally, the binding of all AZDye-labeled CXCL12s to Nluc receptors was inhibited by pharmacologically relevant unlabeled chemokines and small molecules. The NanoBRET binding assay for CXCL10-AZD488 binding to Nluc-CXCR3 was also successfully established and successfully employed for the simultaneous measurement of the binding of unlabeled small molecules to NLuc-CXCR3 and NLuc-CXCR4. In conclusion, multiplexing the NanoBRET-based competition binding assay is a promising tool for testing unlabeled (small) molecules against multiple GPCRs simultaneously.

SDF-1 promotes metastasis of NSCLC by enhancing chemoattraction of megakaryocytes through the PI3K/Akt signaling pathway.

Lung cancer (LC) is the leading cause of cancer-associated deaths worldwide, among which non-small-cell lung cancer (NSCLC) accounts for 80%. Stromal cell-derived factor-1 (SDF-1) inhibition results in a significant depletion of NSCLC metastasis. Additionally, SDF-1 is the only natural chemokine known to bind and activate the receptor CXCR4. Thus, we attempted to clarify the molecular mechanism of SDF-1 underlying NSCLC progression. Transwell migration, adhesion, and G-LISA assays were used to assess megakaryocytic chemotaxis in vitro and in vivo in terms of megakaryocytic migration, adherence, and RhoA activation, respectively. Western blotting was used to assess PI3K/Akt-associated protein abundances in MEG-01 cells and primary megakaryocytes under the indicated treatment. A hematology analyzer and flow cytometry were used to assess platelet counts in peripheral blood and newly formed platelet counts in Lewis LC mice under different treatments. Immunochemistry and flow cytometry were used to measure CD41+ megakaryocyte numbers in Lewis LC mouse tissue under different treatments. ELISA was used to measure serum TPO levels, and H&E staining was used to detect NSCLC metastasis.SDF-1 receptor knockdown suppressed megakaryocytic chemotaxis in Lewis LC mice. SDF-1 receptor inhibition suppressed megakaryocytic chemotaxis via the PI3K/Akt pathway. SDF-1 receptor knockdown suppressed CD41+ megakaryocyte numbers in vivo through PI3K/Akt signaling. SDF-1 receptor inhibition suppressed CD41+ megakaryocytes to hinder NSCLC metastasis. SDF-1 facilitates NSCLC metastasis by enhancing the chemoattraction of megakaryocytes via the PI3K/Akt signaling pathway, which may provide a potential new direction for seeking therapeutic plans for NSCLC.

Effects of CD44 siRNA on inhibition, survival, and apoptosis of breast cancer cell lines (MDA-MB-231 and 4T1).

BACKGROUND: Breast cancer (BC) is one of the most common cancers in the world. Despite the many advances that have been made in treating patients, many patients are still resistant to treatment. CD44 is one of the surface glycoproteins of BC cells that plays an important role in the proliferation of these cells and inhibition of their apoptosis. Therefore, targeting it can be a treatment way for BC patients. METHODS: In this study, the effect of anti-CD44 siRNA on the proliferation, apoptosis, and migration rate of MDA-MB-231 and 4T1 cells was investigated. The techniques used in this study were MTT assay, RT-PCR, and flow cytometry. RESULTS: The apoptosis and proliferation rates in CD44 siRNA-treated cells were higher and lower, respectively, compared to untreated cells. Also, cell migration was less in treated cells compared to untreated cells. CD44 siRNA also decreased the expression of CXCR4, c-myc, Vimentin, ROCK, and MMP-9. CONCLUSION: Finally, CD44 targeting can be a good treatment option to make BC cells more sensitive to apoptosis.

Danshen injection mitigated the cerebral ischemia/reperfusion injury by suppressing neuroinflammation via the HIF-1α/CXCR4/NF-κB signaling pathway.

Danshen injection (DI) is effective in treating cardiovascular and cerebrovascular diseases, including ischemic stroke (IS), including IS, but its mechanism is unclear. A middle cerebral artery occlusion model was used to simulate ischemia/reperfusion (I/R) injury in SD rats. Overexpression of hypoxia-inducible factor 1α (HIF-1α) was achieved by AAV-HIF-1α. Rats were treated with DI or saline. Neurological scores and infarction rates were assessed. I/R damage was examined by HE, 2,3,5-triphenyltetrazolium and Nissl stainings. Expression levels of relative proteins [TNF-α, IL-6, IL-1ß, SOD, MDA, ROS, HIF-1α, CXC chemokine receptor 4 (CXCR4) and NF-κB] were measured. DI treatment improved neurological scores and reduced infarction rates, suggesting that it inhibits inflammation and oxidative stress. The expression levels of HIF-1α, CXCR4 and NF-κB were decreased. However, the effectiveness of DI on inflammation inhibition was lost after HIF-1α overexpression. DI may directly target HIF-1α to suppress neuroinflammation and reduce I/R injury by suppressing the HIF-1α/CXCR4/NF-κB signaling pathway.

Circadian-clock-controlled endocrine and cytokine signals regulate multipotential innate lymphoid cell progenitors in the bone marrow.

Innate lymphoid cells (ILCs), strategically positioned throughout the body, undergo population declines over time. A solution to counteract this problem is timely mobilization of multipotential progenitors from the bone marrow. It remains unknown what triggers the mobilization of bone marrow ILC progenitors (ILCPs). We report that ILCPs are regulated by the circadian clock to emigrate and generate mature ILCs in the periphery. We found that circadian-clock-defective ILCPs fail to normally emigrate and generate ILCs. We identified circadian-clock-controlled endocrine and cytokine cues that, respectively, regulate the retention and emigration of ILCPs at distinct times of each day. Activation of the stress-hormone-sensing glucocorticoid receptor upregulates CXCR4 on ILCPs for their retention in the bone marrow, while the interleukin-18 (IL-18) and RORα signals upregulate S1PR1 on ILCPs for their mobilization to the periphery. Our findings establish important roles of circadian signals for the homeostatic efflux of bone marrow ILCPs.

Clostridioides difficile toxin B subverts germinal center and antibody recall responses by stimulating a drug-treatable CXCR4-dependent mechanism.

Recurrent Clostridioides difficile infection (CDI) results in significant morbidity and mortality. We previously established that CDI in mice does not protect against reinfection and is associated with poor pathogen-specific B cell memory (Bmem), recapitulating our observations with human Bmem. Here, we demonstrate that the secreted toxin TcdB2 is responsible for subversion of Bmem responses. TcdB2 from an endemic C. difficile strain delayed immunoglobulin G (IgG) class switch following vaccination, attenuated IgG recall to a vaccine booster, and prevented germinal center formation. The mechanism of TcdB2 action included increased B cell CXCR4 expression and responsiveness to its ligand CXCL12, accounting for altered cell migration and a failure of germinal center-dependent Bmem. These results were reproduced in a C. difficile infection model, and a US Food and Drug Administration (FDA)-approved CXCR4-blocking drug rescued germinal center formation. We therefore provide mechanistic insights into C. difficile-associated pathogenesis and illuminate a target for clinical intervention to limit recurrent disease.