P2Y2R­mediated transactivation of VEGFR2 through Src phosphorylation is associated with ESM­1 overexpression in radiotherapy­resistant­triple negative breast cancer cells.

We previously reported that radiotherapy­resistant (RT­R) triple negative breast cancer (TNBC) cells upregulate the expression of endothelial­specific molecule­1 (ESM­1) compared with TNBC cells. In addition, ESM­1 is involved in an increased proliferation and invasion of RT­R­TNBC cells compared with TNBC cells. It was further identified that, in RT­R­TNBC cells, P2Y2 purinergic receptor (P2Y2R)­mediated activation of p21­activated kinase 1 (PAK1), protein kinase C (PKC), c­Jun N­terminal kinase (JNK) and p38 MAPKs is related to ESM­1 expression via forkhead box O1 (FoxO1) regulation. Notably, it has been reported that P2Y2R mediates the transactivation of vascular epithelial growth factor receptor 2 (VEGFR2), and VEGFR2 is known to be involved in ESM­1 expression. Therefore, in the present study, the involvement of VEGFR2 in the P2Y2R­mediated ESM­1 upregulation in RT­R­TNBC cells and the relationship between P2Y2R and VEGFR2 activation was further examined. Western blotting and reverse transcription­PCR were used to monitor the expression of ESM­1, and the results demonstrated that extracellular ATP treatment regulated the expression of ESM­1 in a P2Y2R­dependent manner in RT­R­MDA­MB­231 cells. In addition, extracellular ATP activated Src and VEGFR2 after 5 min of incubation, which was abolished by knockdown of P2Y2R expression. VEGFR2 activation in response to ATP was also decreased by inhibiting Src activity, suggesting that ATP­activated P2Y2R regulates VEGFR2 phosphorylation via Src activation. Furthermore, ATP­induced ESM­1 expression was decreased by transfection with VEGFR2 small interfering RNA (siRNA). ESM­1­related signaling molecules, PAK1, PKC, JNK and p38 MAPKs, and the transcriptional regulator, FoxO1, which were activated by ATP, were also decreased following transfection with VEGFR2 siRNA. These results suggest that P2Y2R­mediated transactivation of VEGFR2 through Src phosphorylation is associated with ESM­1 overexpression in RT­R­TNBC cells.

High concentrations of soluble endoglin can inhibit BMP9 signaling in non-endothelial cells.

Endoglin (ENG) is a single-pass transmembrane protein highly expressed on vascular endothelial cells, although low expression levels can be detected in many other cell types. Its extracellular domain can be found in circulation known as soluble endoglin (sENG). Levels of sENG are elevated in many pathological conditions, in particular preeclampsia. We have shown that while loss of cell surface ENG decreases BMP9 signaling in endothelial cells, knocking down ENG in blood cancer cells enhances BMP9 signaling. Despite sENG binding to BMP9 with high affinity and blocking the type II receptor binding site on BMP9, sENG did not inhibit BMP9 signaling in vascular endothelial cells, but the dimeric form of sENG inhibited BMP9 signaling in blood cancer cells. Here we report that in non-endothelial cells such as human multiple myeloma cell lines and the mouse myoblast cell line C2C12, both monomeric and dimeric forms of sENG inhibit BMP9 signaling when present at high concentrations. Such inhibition can be alleviated by the overexpression of ENG and ACVRL1 (encoding ALK1) in the non-endothelial cells. Our findings suggest that the effects of sENG on BMP9 signaling is cell-type specific. This is an important consideration when developing therapies targeting the ENG and ALK1 pathway.

Sushi domain-containing protein 4 binds to epithelial growth factor receptor and initiates autophagy in an EGFR phosphorylation independent manner.

BACKGROUND: Sushi domain-containing protein 4 (SUSD4) is a recently discovered protein with unknown cellular functions. We previously revealed that SUSD4 can act as complement inhibitor and as a potential tumor suppressor. METHODS: In a syngeneic mouse model of breast cancer, tumors expressing SUSD4 had a smaller volume compared with the corresponding mock control tumors. Additionally, data from three different expression databases and online analysis tools confirm that for breast cancer patients, high mRNA expression of SUSD4 in the tumor tissue correlates with a better prognosis. In vitro experiments utilized triple-negative breast cancer cell lines (BT-20 and MDA-MB-468) stably expressing SUSD4. Moreover, we established a cell line based on BT-20 in which the gene for EGFR was knocked out with the CRISPR-Cas9 method. RESULTS: We discovered that the Epithelial Growth Factor Receptor (EGFR) interacts with SUSD4. Furthermore, triple-negative breast cancer cell lines stably expressing SUSD4 had higher autophagic flux. The initiation of autophagy required the expression of EGFR but not phosphorylation of the receptor. Expression of SUSD4 in the breast cancer cells led to activation of the tumor suppressor LKB1 and consequently to the activation of AMPKα1. Finally, autophagy was initiated after stimulation of the ULK1, Atg14 and Beclin-1 axis in SUSD4 expressing cells. CONCLUSIONS: In this study we provide novel insight into the molecular mechanism of action whereby SUSD4 acts as an EGFR inhibitor without affecting the phosphorylation of the receptor and may potentially influence the recycling of EGFR to the plasma membrane.

Brain-Derived Neurotrophic Factor Regulates Differentiation of Stem Cells From Human Exfoliated Deciduous Teeth Through Growth Factor Receptor-Bound Protein 2-Extracellular Signal-Regulated Kinase Signaling Pathway.

OBJECTIVES: Stem cells from human exfoliated deciduous teeth are a population of multipotent mesenchymal stem cells that can self-renew and actively secrete a broad spectrum of trophic and immunomodulatory factors.Brain-derivedneurotrophic factor is able to induce stem cells to neural differentiation to repair the nervous system. However,the mechanism ofbrain-derivedneurotrophic factor-induced neural differentiation in stem cells from human exfoliated deciduous teeth remains unclear. MATERIALS AND METHODS: In this study, we focused on brain-derived neurotrophic factor and investigated its effects on neural differentiation in stem cells from human exfoliated deciduous teeth. We cultured stem cells from human exfoliated deciduous teeth under various different brain-derived neurotrophic factor concentrations. We then analyzed the effects of brain-derived neurotrophic factor to the neural differen-tiation and associated signaling pathways in stem cells from human exfoliated deciduous teeth. RESULTS: We demonstrated that a high concentration of brain-derived neurotrophic factor could induce neural differentiation in stem cells from human exfoliated deciduous teeth, and brain-derived neurotrophic factor also increased the expression levels of neural differentiation-related proteins in stem cells from human exfoliated deciduous teeth, which was associated with the suppression of growth factor receptor-bound protein 2/extracellular signal-regulated kinase 1 and 2 signaling pathways. CONCLUSIONS: Knockdown of growth factor receptor- bound protein 2 inhibited the neural differentiation of stem cells from human exfoliated deciduous teeth via changes in growth factor receptor-bound protein 2/extracellular signal-regulated kinase signaling pathways, but this phenotype could be rescued by overexpression of extracellular signal-regulated kinase. Our findings suggested that brain-derived neurotrophic factor can regulate the differentiation of stem cells from human exfoliated deciduous teeth through the growth factor receptor-bound protein 2/extracellular signal-regulated kinase signaling pathway.

Evaluation of the relationship between miR-1271 and GRB2 gene in endometriosis.

BACKGROUND: Endometriosis is a common gynecological condition with a substantial economic burden on society. It is known that both genetic and environmental factors are contributing to the phenotypic development of the disease. MicroRNAs have a vital role in the pathogenesis of endometriosis. miR-1271 and its direct target gene, GRB2 (growth factor receptor-bound protein 2), expression have been studied in gynecologic cancers, while their role in endometriosis has not been studied. OBJECTIVE: We measured miR-1271 and GRB2 gene expression in the eutopic and ectopic tissues of patients (endometrial tissues) in contrast to the control samples from healthy women. MATERIALS AND METHODS: In this study, a total of 45 samples (15 control samples, 15 eutopic samples and 15 ectopic samples) were collected. We used qRT-PCR (quantitative polymerase chain reaction) to evaluate the expression levels of the miR-1271 and GRB2 gene. RESULTS: We observed inverse expression of miR-1271 and GRB2 gene. MiR-1271 expression was significantly reduced in patients with endometriosis compared with healthy women. While there was a noticeable increase in the expression level of its target gene, GRB2, in tissues of endometriosis patients compared with normal control samples. CONCLUSION: We have shown an inverse relationship between the reduction of miR-1271 expression level and increase in the expression level of GRB2, therefore, increased GRB2 expression in endometriosis tissues can be due to decreased expression of this microRNA. Our findings suggested that miR-1271 maybe play a role as a biomarker in the diagnosis of patients with endometriosis.

N-glycosylation of GDF15 abolishes its inhibitory effect on EGFR in AR inhibitor-resistant prostate cancer cells.

Castration-resistance of prostate cancer is one of the most challenging clinical problems. In the present study, we have performed proteomics and glycomics using LNCaP model. Growth differentiation factor-15 (GDF15) level is increased in androgen receptor (AR) inhibitor-resistant cells and the inhibitory effect of GDF15 on epithelial growth factor receptor (EGFR) pathway is relieved by GDF15 N70 glycosylation. Interference of GDF15 (siRNA or N70Q dominant negative) or EGFR pathway (inhibitor or siRNA for EGFR, SRC or ERK) decreases the resistant-cell survival in culture and tumor growth in mice. Our study reveals a novel regulatory mechanism of prostate cancer AR inhibitor resistance, raises the possibility of AR/SRC dual-targeting of castration-resistance of prostate cancer, and lays foundation for the future development of selective inhibitors of GDF15 glycosylation.

Integrated DNA Copy Number and Expression Profiling Identifies IGF1R as a Prognostic Biomarker in Pediatric Osteosarcoma.

Osteosarcoma is a primary malignant bone tumor arising from bone-forming mesenchymal cells in children and adolescents. Despite efforts to understand the biology of the disease and identify novel therapeutics, the survival of osteosarcoma patients remains dismal. We have concurrently profiled the copy number and gene expression of 226 osteosarcoma samples as part of the Strategic Partnering to Evaluate Cancer Signatures (SPECS) initiative. Our results demonstrate the heterogeneous landscape of osteosarcoma in younger populations by showing the presence of genome-wide copy number abnormalities occurring both recurrently among samples and in a high frequency. Insulin growth factor receptor 1 (IGF1R) is a receptor tyrosine kinase which binds IGF1 and IGF2 to activate downstream pathways involved in cell apoptosis and proliferation. We identify prevalent amplification of IGF1R corresponding with increased gene expression in patients with poor survival outcomes. Our results substantiate previously tenuously associated copy number abnormalities identified in smaller datasets (13q34+, 20p13+, 4q35-, 20q13.33-), and indicate the significance of high fibroblast growth factor receptor 2 (FGFR2) expression in distinguishing patients with poor prognosis. FGFR2 is involved in cellular proliferation processes such as division, growth and angiogenesis. In summary, our findings demonstrate the prognostic significance of several genes associated with osteosarcoma pathogenesis.

Tribbles homolog 3 contributes to high glucose-induced injury in retinal pigment epithelial cells via binding to growth factor receptor-bound 2.

Diabetic retinopathy (DR) is the most typical complication of diabetes, which severely threatens sight. Tribbles homolog 3 (TRB3), a kind of pseudokinase, is discovered to be highly expressed in diabetes and retinas after retinal detachment. TRB3 expression in human retinal pigment epithelial (hRPE) cells exposed to different concentrations of glucose was tested by RT-qPCR and western blot. Then, cells were induced with 30 mM high glucose (HG) to establish a DR cell model. Following TRB3 knockdown, cell viability estimation employed CCK-8 assay. The mRNA levels of inflammatory factors were detected by RT-qPCR. Reactive oxygen species (ROS) level was measured by DCFH-DA assay, and levels of oxidative stress markers were evaluated applying corresponding kits. Cell apoptosis was assayed by TUNEL assay and western blot. Following, the growth factor receptor-bound 2 (GRB2) expression was also examined by RT-qPCR and western blot. The interaction between TRB3 and GRB2 was verified by Co-IP assay. After GRB2 was overexpressed in HG-induced hRPE cells transfected with shRNA-TRB3, functional experiments were conducted again. The results manifested that TRB3 expression was elevated under HG conditions. Deficiency of TRB3 enhanced the viability while alleviated inflammation, oxidative stress, and apoptosis in HG-induced hRPE cells. GRB2 was also increased in HG-exposed hRPE cells. Moreover, GRB2 had a strong affinity with TRB3 and positively regulated by TRB3. After GRB2 overexpression, the effects of TRB3 knockdown on HG-stimulated hRPE cells were all reversed. Briefly, this study confirmed the promoting role of TRB3/GRB2 axis in the progression of DR.

Curcuminoids Inhibit Angiogenic Behaviors of Human Umbilical Vein Endothelial Cells via Endoglin/Smad1 Signaling.

BACKGROUND: Angiogenesis is primarily attributed to the excessive proliferation and migration of endothelial cells. Targeting the vascular endothelial growth factor (VEGF) is therefore significant in anti-angiogenic therapy. Although these treatments have not reached clinical expectations, the upregulation of alternative angiogenic pathways (endoglin/Smad1) may play a critical role in drug (VEGF-neutralizing agents) resistance. Enhanced endoglin expression following a VEGF-neutralizing therapy (semaxanib®) was noted in patients. Treatment with an endoglin-targeting antibody augmented VEGF expression in human umbilical vein endothelial cells (HUVECs). Therefore, approaches that inhibit both the androgen and VEGF pathways enhance the HUVECs cytotoxicity and reverse semaxanib resistance. The purpose of this study was to find natural-occurring compounds that inhibited the endoglin-targeting pathway. METHODS: Curcuminoids targeting endoglin were recognized from two thousand compounds in the Traditional Chinese Medicine Database@Taiwan (TCM Database@Taiwan) using Discovery Studio 4.5. RESULTS: Our results, obtained using cytotoxicity, migration/invasion, and flow cytometry assays, showed that curcumin (Cur) and demethoxycurcumin (DMC) reduced angiogenesis. In addition, Cur and DMC downregulated endoglin/pSmad1 phosphorylation. CONCLUSIONS: The study first showed that Cur and DMC demonstrated antiangiogenic activity via the inhibition of endoglin/Smad1 signaling. Synergistic effects of curcuminoids (i.e., curcumin and DMC) and semaxanib on HUVECs were found. This might be attributed to endoglin/pSmad1 downregulation in HUVECs. Combination treatment with curcuminoids and a semaxanib is therefore expected to reverse semaxanib resistance.

EWI2 promotes endolysosome-mediated turnover of growth factor receptors and integrins to suppress lung cancer.

As a partner of tetraspanins, EWI2 suppresses glioblastoma, melanoma, and prostate cancer; but its role in lung cancer has not been investigated. Bioinformatics analysis reveals that EWI2 gene expression is up regulated in lung adenocarcinoma and higher expression of EWI2 mRNA may predict poorer overall survival. However, experimental analysis shows that EWI2 protein is actually downregulated constantly in the tissues of lung adenocarcinoma and lung squamous cell carcinoma. Forced expression of EWI2 in human lung adenocarcinoma cells reduces total cellular and cell surface levels of various integrins and growth factor receptors, which initiates the outside-in motogenic and mitogenic signaling. These reductions result in the decreases in 1) cell-matrix adhesion, cell movement, and cell transformation in vitro and 2) tumor growth, burden, and metastasis in vivo, and result from the increases in lysosomal trafficking and proteolytic degradation of theses membrane receptors. EWI2 elevates lysosome formation by promoting nuclear retention of TFEB, the master transcription factor driving lysosomogenesis. In conclusion, EWI2 as a lung cancer suppressor attenuates lung cancer cells in a comprehensive fashion by inhibiting both tumor growth and tumor metastasis; EWI2 as an endolysosome regulator promotes lysosome activity to enhance lysosomal degradation of growth factor receptors and integrins and then reduce their levels and functions; and EWI2 can become a promising therapeutic candidate given its accessibility at the cell surface, dual inhibition on growth factor receptors and integrins, and broad-spectrum anti-cancer activity. More importantly, our observations also provide a novel therapeutic strategy to bypass the resistance to EGFR inhibitors.

FOXD1 Regulates the Sensitivity of Cetuximab by Regulating the Expression of EGFR in Head and Neck Squamous Cell Cancer.

OBJECTIVES: Previous experiments have shown that growth factor receptors play important role in tumor proliferation, metastasis, and therapeutic effect of chemotherapeutic drugs. At the same time, forkhead box D1 (FOXD1) plays an important role in a variety of signal transmission, but its expression profile was known little about head and neck cancer. The purpose of this experiment was to explore the regulation of FOXD1 on the tumor progression of head and neck cancer and to explore the correlation of FOXD1 on the expression of growth factor receptors (EGFR). METHODS: The bioinformatics online database analyzed the expression of FOXD1 and EGFR in tumor tissues and nontumor tissues. Real-time quantitative PCR was used to detect the FOXD1 and EGFR expression in 45 tumor tissues and 15 nontumor tissues. The plasmid was used to construct FOXD1 overexpressing head and neck squamous cell cancer lines and observe the clonal formation and invasion of tumor cells under the intervention of EGFR-specific antibody-cetuximab. RESULTS: The expression of FOXD1 and EGFR in tumor tissues was higher than that in nontumor tissues. The higher expression of FOXD1 and EGFR was not conducive to the prognosis of patients. The expression of FOXD1 and EGFR was positively correlated, and immunohistochemical analysis showed the high expression of FOXD1 and EGFR has close relation to the advanced stage of the tumor. In vitro cell experiments proved that overexpression of FOXD1 can partially offset the cloning ability of cetuximab on head and neck tumor cells. CONCLUSION: FOXD1 has an important regulatory role in the progression of head and neck cancer, and its abnormally high expression was not conducive to the prognosis of cancer patients. FOXD1 can regulate the expression of growth factor receptors in head and neck cancer, which provides a new idea for the better use of tumor growth factor receptor-specific antibodies for collaborative therapy.

Growth factor receptor and ß1 integrin signaling differentially regulate basal clonogenicity and radiation survival of fibroblasts via a modulation of cell cycling.

Cell adhesion to extracellular matrix proteins mediates resistance to radio- and chemotherapy by activating integrin signaling. In addition, mutual and cooperative interactions between integrin and growth factor receptor signaling contribute to the cellular radiation response. Here, we investigate to which extend the crosstalk between ß1 integrins and growth factor receptor signaling determines the cellular radiation response of fibroblasts by assessing clonogenic survival and cell cycling. By utilizing growth factor signaling competent and either ß1 integrin wildtype GD25ß1A fibroblasts or ß1 integrin mutant, signaling incompetent GD25ß1B fibroblasts, we show basal clonogenic survival to depend on growth factor receptor but not integrin signaling. Our data further suggest the cooperation between ß1 integrins and growth factor receptors to be critical for enhancing the radiation-induced G2/M cell cycle block leading to improved clonogenic radiation survival. By pharmacological inhibition of EGFR and PI3K, we additionally show that the essential contribution of EGFR signaling to radiogenic G2/M cell cycle arrest depends on the co-activation of the ß1 integrin signaling axis, but occurs independent of PI3K. Taken together, elucidation of the signaling circuitry underlying the EGFR/ß1 integrin crosstalk may support the development of advanced molecular targeted therapies for radiation oncology.

Kinase inhibitor-induced cardiotoxicity assessed in vitro with human pluripotent stem cell derived cardiomyocytes.

Many small molecule kinase inhibitors (SMKIs), used predominantly in cancer therapy, have been implicated in serious clinical cardiac adverse events, which means that traditional preclinical drug development assays were not sufficient for identifying these cardiac liabilities. To improve clinical cardiac safety predictions, the effects of SMKIs targeting many different signaling pathways were studied using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) in combined assays designed for the detection of both electrophysiological (proarrhythmic) and non-electrophysiological (non-proarrhythmic) drug-induced cardiotoxicity. Several microplate-based assays were used to quantitate cell death, apoptosis, mitochondrial damage, energy depletion, and oxidative stress as mechanism-based non-electrophysiological cardiomyocyte toxicities. Microelectrode arrays (MEA) were used to quantitate in vitro arrhythmic events (iAEs), field potential duration (FPD) prolongation, and spike amplitude suppression (SAS) as electrophysiological effects. To enhance the clinical relevance, SMKI-induced cardiotoxicities were compared by converting drug concentrations into multiples of reported clinical maximum therapeutic plasma concentration, "FoldCmax", for each assay. The results support the conclusion that the combination of the hPSC-CM based electrophysiological and non-electrophysiological assays have significantly more predictive value than either assay alone and significantly more than the current FDA-recommended hERG assay. In addition, the combination of these assays provided mechanistic information relevant to cardiomyocyte toxicities, thus providing valuable information on potential drug-induced cardiotoxicities early in drug development prior to animal and clinical testing. We believe that this early information will be helpful to guide the development of safer and more cost-effective drugs.

Endoglin Is an Important Mediator in the Final Common Pathway of Chronic Kidney Disease to End-Stage Renal Disease.

Chronic kidney disease (CKD) is a slow-developing, progressive deterioration of renal function. The final common pathway in the pathophysiology of CKD involves glomerular sclerosis, tubular atrophy and interstitial fibrosis. Transforming growth factor-beta (TGF-ß) stimulates the differentiation of fibroblasts towards myofibroblasts and the production of extracellular matrix (ECM) molecules, and thereby interstitial fibrosis. It has been shown that endoglin (ENG, CD105), primarily expressed in endothelial cells and fibroblasts, can function as a co-receptor of TGF signaling. In several human organs, endoglin tends to be upregulated when chronic damage and fibrosis is present. We hypothesize that endoglin is upregulated in renal interstitial fibrosis and plays a role in the progression of CKD. We first measured renal endoglin expression in biopsy samples obtained from patients with different types of CKD, i.e., IgA nephropathy, focal segmental glomerulosclerosis (FSGS), diabetic nephropathy (DN) and patients with chronic allograft dysfunction (CAD). We showed that endoglin is upregulated in CAD patients (p < 0.001) and patients with DN (p < 0.05), compared to control kidneys. Furthermore, the amount of interstitial endoglin expression correlated with eGFR (p < 0.001) and the amount of interstitial fibrosis (p < 0.001), independent of the diagnosis of the biopsies. Finally, we investigated in vitro the effect of endoglin overexpression in TGF-ß stimulated human kidney fibroblasts. Overexpression of endoglin resulted in an enhanced ACTA2, CCN2 and SERPINE1 mRNA response (p < 0.05). It also increased the mRNA and protein upregulation of the ECM components collagen type I (COL1A1) and fibronectin (FN1) (p < 0.05). Our results suggest that endoglin is an important mediator in the final common pathway of CKD and could be used as a possible new therapeutic target to counteract the progression towards end-stage renal disease (ESRD).

P75NTR Exacerbates SCI-induced Mitochondrial Damage and Neuronal Apoptosis Depending on NTRK3.

OBJECTIVE: The aim of the study was to investigate the mechanism by which p75 neurotrophin receptor (p75NTR) affects mitochondrial damage and neuronal apoptosis in spinal cord injury (SCI). METHODS: After the establishment of SCI rat models, short hairpin (sh) RNA of p75NTR and control sh-RNA were injected into SCI rats, respectively. On days 1, 7 and 21 after SCI, the severity of SCI and cell apoptosis in SCI rats were determined as well as the recovery of hind limb performance and p75NTR expression. After spinal cord neurons were transfected with p75NTR overexpression plasmid or empty plasmid vector or cotransfected with overexpression plasmids of p75NTR and neurotrophic tyrosine receptor kinase3 (NTRK3), the expression levels of p75NTR and NTRK3 were quantified. Moreover, we detected the apoptosis and proliferation rates of the neurons in addition to the levels of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) in the neurons. The binding between p75NTR and NTRK3 was confirmed via Co-immunoprecipitation (Co-IP). RESULTS: The rat spinal cords in the Model group were notably damaged after SCI accompanied by increased apoptosis and decreased locomotor function. The expression of p75NTR was significantly upregulated after SCI. The aforementioned injuries were remarkably ameliorated in response to injection of sh-p75NTR. p75NTR overexpression induced mitochondrial damage and neuronal apoptosis in spinal cord neurons, while the promotive effects were perturbed by NTRK3 overexpression. Furthermore, p75NTR directly bound to and downregulated NTRK3. CONCLUSION: Both in vivo and in vitro experiments showed that p75NTR aggravates mitochondrial damage and neuronal apoptosis in SCI through downregulating NTRK3.

Regulation of the p75 neurotrophin receptor attenuates neuroinflammation and stimulates hippocampal neurogenesis in experimental Streptococcus pneumoniae meningitis.

BACKGROUND: Streptococcus pneumoniae meningitis is a destructive central nervous system (CNS) infection with acute and long-term neurological disorders. Previous studies suggest that p75NTR signaling influences cell survival, apoptosis, and proliferation in brain-injured conditions. However, the role of p75NTR signaling in regulating pneumococcal meningitis (PM)-induced neuroinflammation and altered neurogenesis remains largely to be elucidated. METHODS: p75NTR signaling activation in the pathological process of PM was assessed. During acute PM, a small-molecule p75NTR modulator LM11A-31 or vehicle was intranasally administered for 3 days prior to S. pneumoniae exposure. At 24 h post-infection, clinical severity, histopathology, astrocytes/microglia activation, neuronal apoptosis and necrosis, inflammation-related transcription factors and proinflammatory cytokines/mediators were evaluated. Additionally, p75NTR was knocked down by the adenovirus-mediated short-hairpin RNA (shRNA) to ascertain the role of p75NTR in PM. During long-term PM, the intranasal administration of LM11A-31 or vehicle was continued for 7 days after successfully establishing the PM model. Dynamic changes in inflammation and hippocampal neurogenesis were assessed. RESULTS: Our results revealed that both 24 h (acute) and 7, 14, 28 day (long-term) groups of infected rats showed increased p75NTR expression in the brain. During acute PM, modulation of p75NTR through pretreatment of PM model with LM11A-31 significantly alleviated S. pneumoniae-induced clinical severity, histopathological injury and the activation of astrocytes and microglia. LM11A-31 pretreatment also significantly ameliorated neuronal apoptosis and necrosis. Moreover, we found that blocking p75NTR with LM11A-31 decreased the expression of inflammation-related transcription factors (NF-κBp65, C/EBPß) and proinflammatory cytokines/mediators (IL-1ß, TNF-α, IL-6 and iNOS). Furthermore, p75NTR knockdown induced significant changes in histopathology and inflammation-related transcription factors expression. Importantly, long-term LM11A-31 treatment accelerated the resolution of PM-induced inflammation and significantly improved hippocampal neurogenesis. CONCLUSION: Our findings suggest that the p75NTR signaling plays an essential role in the pathogenesis of PM. Targeting p75NTR has beneficial effects on PM rats by alleviating neuroinflammation and promoting hippocampal neurogenesis. Thus, the p75NTR signaling may be a potential therapeutic target to improve the outcome of PM.

Therapeutic Neuroprotection by an Engineered Neurotrophin that Selectively Activates Tropomyosin Receptor Kinase (Trk) Family Neurotrophin Receptors but Not the p75 Neurotrophin Receptor.

The neurotrophin growth factors bind and activate two types of cell surface receptors: the tropomyosin receptor kinase (Trk) family and p75. TrkA, TrkB, and TrkC are bound preferentially by nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3 (NT3), respectively, to activate neuroprotective signals. The p75 receptors are activated by all neurotrophins, and paradoxically in neurodegenerative disease p75 is upregulated and mediates neurotoxic signals. To test neuroprotection strategies, we engineered NT3 to broadly activate Trk receptors (mutant D) or to reduce p75 binding (mutant RK). We also combined these features in a molecule that activates TrkA, TrkB, and TrkC but has reduced p75 binding (mutant DRK). In neurodegenerative disease mouse models in vivo, the DRK protein is a superior therapeutic agent compared with mutant D, mutant RK, and wild-type neurotrophins and protects a broader range of stressed neurons. This work rationalizes a therapeutic strategy based on the biology of each type of receptor, avoiding activation of p75 toxicity while broadly activating neuroprotection in stressed neuronal populations expressing different Trk receptors. SIGNIFICANCE STATEMENT: The neurotrophins nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3 each can activate a tropomyosin receptor kinase (Trk) A, TrkB, or TrkC receptor, respectively, and all can activate a p75 receptor. Trks and p75 mediate opposite signals. We report the engineering of a protein that activates all Trks, combined with low p75 binding, as an effective therapeutic agent in vivo.

Characterization of human FDCs reveals regulation of T cells and antigen presentation to B cells.

Stromal-derived follicular dendritic cells (FDCs) are essential for germinal centers (GCs), the site where B cells maturate their antibodies. FDCs present native antigen to B cells and maintain a CXCL13 gradient to form the B cell follicle. Yet despite their essential role, the transcriptome of human FDCs remains undefined. Using single-cell RNA sequencing and microarray, we provided the transcriptome of these enigmatic cells as a comprehensive resource. Key genes were validated by flow cytometry and microscopy. Surprisingly, marginal reticular cells (MRCs) rather than FDCs expressed B cell activating factor (BAFF). Furthermore, we found that human FDCs expressed TLR4 and can alter antigen availability in response to pathogen-associated molecular patterns (PAMPs). High expression of PD-L1 and PD-L2 on FDCs activated PD1 on T cells. In addition, we found expression of genes related to T cell regulation, such as HLA-DRA, CD40, and others. These data suggest intimate contact between human FDCs and T cells.

Histone deacetylase inhibitor panobinostat induces antitumor activity in epithelioid sarcoma and rhabdoid tumor by growth factor receptor modulation.

BACKGROUND: Epithelioid sarcomas and rhabdoid tumors are rare, aggressive malignancies with poor prognosis. Both are characterized by INI1 alterations and deregulation of growth factor receptors albeit their interaction has not been elucidated. METHODS: In this study, we investigated the activity of a panel of epigenetic modulators and receptor tyrosine kinase inhibitors in vitro on respective cell lines as well as on primary patient-derived epithelioid sarcoma cells, and in vivo on xenografted mice. Focusing on histone deacetylase (HDAC) inhibitors, we studied the mechanism of action of this class of agents, its effect on growth factor receptor regulation, and changes in epithelial-to-mesenchymal transition by using cell- and RT-qPCR-based assays. RESULTS: Pan-HDAC inhibitor panobinostat exhibited potent anti-proliferative activity at low nanomolar concentrations in A204 rhabdoid tumor, and VAESBJ/GRU1 epithelioid sarcoma cell lines, strongly induced apoptosis, and resulted in significant tumor growth inhibition in VAESBJ xenografts. It differentially regulated EGFR, FGFR1 and FGFR2, leading to downregulation of EGFR in epithelioid sarcoma and to mesenchymal-to-epithelial transition whereas in rhabdoid tumor cells, EGFR was strongly upregulated and reinforced the mesenchymal phenotype. All three cell lines were rendered more susceptible towards combination with EGFF inhibitor erlotinib, further enhancing apoptosis. CONCLUSIONS: HDAC inhibitors exhibit significant anticancer activity due to their multifaceted actions on cytotoxicity, differentiation and drug sensitization. Our data suggest that the tailored, tissue-specific combination of HDAC inhibitors with therapeutics which target cellular salvage mechanisms might increase their therapeutic relevance.

2,5-Hexanedione induced apoptosis in rat spinal cord neurons and VSC4.1 cells via the proNGF/p75NTR and JNK pathways.

Increasing evidence suggests that n-hexane induces nerve injury via neuronal apoptosis induced by its active metabolite 2,5-hexanedione (HD). However, the underlying mechanism remains unknown. Studies have confirmed that pro-nerve growth factor (proNGF), a precursor of mature nerve growth factor (mNGF), might activate apoptotic signaling by binding to p75 neurotrophin receptor (p75NTR) in neurons. Therefore, we studied the mechanism of the proNGF/p75NTR pathway in HD-induced neuronal apoptosis. Sprague-Dawley (SD) rats were injected with 400 mg/kg HD once a day for 5 weeks, and VSC4.1 cells were treated with 10, 20, and 40 mM HD in vitro. Results showed that HD effectively induced neuronal apoptosis. Moreover, it up-regulated proNGF and p75NTR levels, activated c-Jun N-terminal kinase (JNK) and c-Jun, and disrupted the balance between B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax). Our findings revealed that the proNGF/p75NTR signaling pathway was involved in HD-induced neuronal apoptosis; it can serve as a theoretical basis for further exploration of the neurotoxic mechanisms of HD.