Toll-like receptor and C-type lectin receptor agonists attenuate osteogenic differentiation in human dental pulp stem cells.

BACKGROUND: This study aimed to investigate the effects of various toll-like receptor (TLR) and C-type lectin receptor (CLR) ligands on osteogenic differentiation in human dental pulp stem cells (hDPSCs). METHODS: hDPSCs were cultured and treated with various concentrations (0.01, 0.1, 1.0, and 10 µg/mL) of TLR or CLR agonists (PG-LPS, E.coli LPS, poly(I:C), Pam3CSK4, Furfurman, and Zymosan). Cell viability was determined by MTT assay. The effects of TLR and CLR agonists on osteogenic differentiation of hDPSCs were measured by alkaline phosphatase (ALP) activity, Alizarin Red S staining, and Von Kossa staining. In addition, the mRNA expression of osteogenesis-related genes (ALP, COL1A1, RUNX2, OSX, OCN and DMP1) was examined by RT-qPCR. A non-parametric analysis was employed for the statistical analyses. The statistically significant difference was considered when p < 0.05. RESULTS: Treatment with TLR and CLR agonists was associated with an increase in hDPSCs' colony-forming unit ability. Compared with the control group, TLR and CLR agonists significantly inhibited the osteogenic differentiation of hDPSCs by decreasing the ALP activity, mineralised nodule formation, and mRNA expression levels of osteogenesis-related genes (ALP, COL1A1, RUNX2, OSX, OCN and DMP1). The inhibition of TRIF but not Akt signalling rescued the effects of TLR and CLR agonist attenuating hDPSCs' mineralisation. CONCLUSIONS: The activation of TLRs or CLRs exhibited an inhibitory effect on osteogenic differentiation of hDPSCs via the TRIF-dependent signalling pathway.

Hypoxia Attenuates Colonic Innate Immune Response and Inhibits TLR4/NF-κB Signaling Pathway in Lipopolysaccharide-Induced Colonic Epithelial Injury Mice.

High altitude hypoxia can lead to a spectrum of gastrointestinal problems. As the first line of host immune defense, innate immune response in the intestinal mucosa plays a pivotal role in maintaining intestinal homeostasis and protecting against intestinal injury at high altitude. This study aimed to investigate the effect of hypoxia on the colonic mucosal barrier and toll-like receptor 4 (TLR4)-mediated innate immune responses in the colon. The mice were exposed to a hypobaric chamber to simulate a 5,000 m plateau environment for 7 days, and the colonic mucosa changes were recorded. At the same time, the inflammation model was established by lipopolysaccharide (LPS) to explore the effects of hypoxia on the TLR4/nuclear factor kappa B (NF-κB) signaling pathway and its downstream inflammatory factors [tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, and interferon (IFN)-γ] in the colon. We found that hypoxic exposure caused weight loss and structural disturbance of the colonic mucosa in mice. Compared with the control group, the protein levels of TLR4 [fold change (FC) = 0.75 versus FC = 0.23], MyD88 (FC = 0.80 versus FC = 0.30), TIR-domain-containing adaptor protein inducing interferon-ß (TRIF: FC = 0.89 versus FC = 0.38), and NF-κB p65 (FC = 0.75 versus FC = 0.24) in the colon of mice in the hypobaric hypoxia group were significantly decreased. LPS-induced upregulation of the TLR4/NF-κB signaling and its downstream inflammatory factors was inhibited by hypoxia. Specifically, compared with the LPS group, the protein levels of TLR4 (FC = 1.18, FC = 0.86), MyD88 (FC = 1.20, FC = 0.80), TRIF (FC = 1.20, FC = 0.86), and NF-κB p65 (FC = 1.29, FC = 0.62) and the mRNA levels of IL-1ß (FC = 7.38, FC = 5.06), IL-6 (FC = 16.06, FC = 9.22), and IFN-γ (FC = 2.01, FC = 1.16) were reduced in the hypobaric hypoxia plus LPS group. Our findings imply that hypoxia could lead to marked damage of the colonic mucosa and a reduction of TLR4-mediated colonic innate immune responses, potentially reducing host defense responses to colonic pathogens.

Effects of acupuncture on microglial polarization and the TLR4/TRIF/MyD88 pathway in a rat model of traumatic brain injury.

OBJECTIVE: Neuroinflammation caused by traumatic brain injury (TBI) can lead to neurological deficits. Acupuncture can inhibit neuroinflammation and promote nerve repair; however, the specific mechanism is still unclear. The purpose of this study was to explore whether acupuncture could modulate the M1 and M2 phenotypic polarization of microglia in a rat model of TBI via the toll-like receptor 4 (TLR4)/intracellular toll-interleukin-1 receptor (TIR) domain-containing adaptor inducing interferon-ß (TRIF)/myeloid differentiation factor 88 (MyD88) pathway. METHODS: A total of 90 adult male Sprague-Dawley (SD) rats, SPF grade, were randomly divided into a normal group, model group and acupuncture group. Each group was further divided into three subgroups (first, third, and fifth day groups) according to the treatment time (n = 10 rats/subgroup). We used the modified neurological severity score (mNSS) method to quantify neurological deficits before and after modeling. We used Nissl staining to observe the pathological changes in brain tissue, flow cytometry to detect the proportion of M1 and M2 polarized microglia in the injured area on the first, third and fifth day, and co-immunoprecipitation (Co-IP) to examine TLR4/TRIF/MyD88 expression in microglia on the first, third and fifth day, as well as expression of the amount of binding of TLR4 with TRIF and MyD88. RESULTS: Compared to the model group, mNSS in the acupuncture group gradually decreased and pathological morphology improved. The proportion of CD11b/CD86 positive cells was decreased, while that of CD11b/CD206 was increased in the acupuncture group. Expression of IP TLR4, IP TRIF and IP MyD88 also decreased in the acupuncture group. CONCLUSION: The results of this study demonstrate that one of the mechanisms through which acupuncture mitigates neuroinflammation and promotes nerve repair in TBI rats may be inhibition of M1 phenotypic polarization and promotion of M2 phenotypic polarization through inhibition of the TLR4/TRIF/MyD88 signaling pathway.

Triptolide Alleviates Neuropathic Pain by Inhibiting the Activation of Microglial Toll-Like Receptor 3.

BACKGROUND: Current data indicates the incidence of neuropathic pain after surgical nerve injury is as high as 50%, thus representing a major problem for patients and for the medical system. Triptolide, a traditional Chinese herb, has anti-inflammatory effects on various neurodegenerative and neuroinflammatory diseases. This agent also reduces peripheral nerve injury-induced neuropathic pain, although the mechanism underlying this effect is still unknown. MATERIALS AND METHODS: The effects of triptolide on spinal nerve ligation (SNL) injury-induced neuropathic pain was studied in an animal model using behavioral, morphological and molecular biological methods. RESULTS: Repeated administration of intrathecal triptolide was found to alleviate SNL- or Poly(I:C) (toll-like receptor 3 agonist) injection-induced mechanical allodynia without any motor impairment. The mechanism by which triptolide reduces SNL- and Poly(I:C) injection-induced microglial activation appears to be via the inhibition of OX42 expression, which is a microglial-specific marker. Intrathecal triptolide also suppressed SNL- and Poly(I:C) injection-induced expression of spinal TRIF. TRIF transmits signals from activated TLR3 and is the downstream adaptor of TLR3 in microglia. In addition, intrathecal triptolide inhibited the expression of spinal pro-inflammatory IL-1 ß following SNL or Poly(I:C) injection. CONCLUSIONS: Intrathecal triptolide can suppress the TLR3/TRIF/IL-1 ß pathway in spinal microglia following SNL. This could be the underlying mechanism by which triptolide alleviate neuropathic pain induced by peripheral nerve injury.

Preventive Mechanism of Lycopene on Intestinal Toxicity Caused by Cyclophosphamide Chemotherapy in Mice by Regulating TLR4-MyD88/TRIF-TRAF6 Signaling Pathway and Gut-Liver Axis.

Cyclophosphamide (CYC) is the first-line chemotherapy drug for cancer in clinical practice, and its intestinal toxicity seriously affects the treatment effect and prognosis of patients. Lycopene (LP) is the main pigment of ripe tomatoes and has strong antioxidant activity. However, the mechanism by which LP prevents CYC-induced intestinal injury remains unclear. The aim of this study was to investigate the mechanism of LP in preventing intestinal toxicity caused by CYC chemotherapy in mice. The results showed that LP significantly prevented spleen and thymus atrophy induced by CYC. In terms of intestinal injury, LP significantly increased the levels of superoxide dismutase (SOD), secretory immunoglobulin A (sIgA), interleukin (IL)-4, IL-12, and interferon (IFN)-γ, decreased the content of lipid oxidation (MDA), upregulated the protein expressions of toll-like receptors 4 (TLR4), myeloid differentiation factor 88 (MyD88), tumor necrosis factor receptor-associated factor 6 (TRAF6), toll/IL-1receptor domain containing adaptor protein inducing IFN-ß (TRIF), p-P38 MAPK (P38), and p-nuclear factor kappa-B (NF-κB) p65, and improved the small intestine tissue injury induced by CYC. In terms of liver injury, LP significantly increased the content of glutathione (GSH), decreased the contents of MDA, nitric oxide (NO), IL-1ß, IL-6, and tumor necrosis factor (TNF)-α, and repaired the liver tissue injury induced by CYC. Importantly, 10 mg/kg LP significantly prevented intestinal microbiota dysregulation in CYC mice. These results suggested that LP significantly prevented intestinal injury induced by CYC in mice by regulating the TLR4-MyD88/TRIF-TRAF6 signaling pathway and gut-liver axis.

Developmental exposure to chlorpyrifos causes neuroinflammation via necroptosis in mouse hippocampus and human microglial cell line.

Neurodevelopmental exposure to chlorpyrifos (CPF) could increase risks for neurological disorders, such as autism spectrum disorder, cognitive impairment, or attention deficit hyperactivity disorder. The potential involvement of microglia reactive to inflammatory stimuli in these neurological disorders has been generally reported. However, the concrete effects and potential mechanisms of microglia dysfunction triggered by developmental CPF exposure remain unclear. Therefore, we established mouse and human embryonic microglial cells (HMC3 cell) models of developmental CPF exposure to evaluate the effects of developmental CPF exposure on neuroinflammation and underlying mechanisms. The results showed that developmental exposure to CPF enhanced the expression of Iba1 in hippocampus. CPF treatment increased inflammatory cytokines levels and TSPO expression in hippocampus and HMC3 cells. The levels of necroptosis and necroptosis-related signaling RIPK/MLKL were increased in hippocampus and HMC3 cells following CPF exposure. Furthermore, the expression of TLR4/TRIF signaling was increased in hippocampus and HMC3 cells subjected to CPF exposure. Notably, the increased levels of TLR4/TRIF signaling, RIPK/MLKL signaling, necroptosis and pro-inflammatory cytokines induced by CPF treatment were remarkably inhibited by TAK-242 (a specific TLR4 inhibitor). Additionally, the necroptosis and pro-inflammatory cytokines production induced by CPF treatment were significantly relieved by Nec-1 (a specific RIPK1 inhibitor). In general, the above results suggested that activated microglia in hippocampus subjected to developmental CPF exposure underwent RIPK1/MLKL-mediated necroptosis regulated by TLR4/TRIF signaling.

Therapeutic effect of Qinghuayin against chronic atrophic gastritis through the inhibition of toll or interleukin-1 receptor domain-containing adaptor inducing interferon-ß signaling pathway.

OBJECTIVE: To examine the efficacy of Qinghuayin (, QHY) in rat chronic atrophic gastritis (CAG) models and explored the molecular mechanism of QHY in treating CAG. METHODS: In total, 65 Wistar rats were randomly divided into the control (= 10) and CAG groups ( = 55). CAG model rats were further divided into five groups: model ( = 10), vitacoenzyme ( = 10), low-dose QHY ( = 10), medium-dose QHY ( = 10), and high-dose QHY groups ( = 10). We analyzed histopathological changes using hematoxylin and eosin staining and measured interleukin (IL)-6 and IL-8 levels in serum using enzyme-linked immunosorbent assay (ELISA) (Boster Bio, Pleasanton, USA). In addition, gastrin (GAS), pepsinogen I (PGI), and PGII expressions were evaluated using ELISA. The protein and mRNA expression of toll-like receptor 4 (TLR4) and toll or interleukin-1 receptor domain-containing adaptor inducing interferon-ß (TRIF) was detected by Western blotting and quantitative reverse transcription-polymerase chain reaction, respectively. RESULTS: Our results revealed that histopathological changes in CAG model rates could be restored by low-, medium-, and high-dose QHY. The changes in GAS and PGI/II expression demonstrated that QHY improved CAG. Serum IL-6 and IL-levels were decreased by QHY administration. TLR4 and TRIF were upregulated at the mRNA and protein levels in the model group but downregulated by QHY administration. CONCLUSION: We concluded that QHY could effectively improve the histopathological changes of the gastric mucosa induced by CAG in rats. The therapeutic mechanism of QHY may be related to inhibition of the inflammatory factors IL-6 and IL-8 and suppression of TLR4/TRIF mRNA and protein expression.

[Effect of activation of Toll-like receptor signaling pathway of peripheral blood mononuclear cell in recombinant hepatitis B surface antigen immune response].

Objective: To explore the effect and mechanism of activation of peripheral blood mononuclear cell (PBMC) Toll-like receptor (TLR3) signaling pathway in recombinant HBsAg (rHBsAg) immune response. Methods: White blood cells were collected from peripheral blood of 13 healthy donors in the preparation of blood products. PBMC was isolated and treated with Poly I:C (Poly I:C group) and PBS (control group) respectively. 48 h later, some cells were collected and the expressions of TLR3 signaling pathway proteins were detected by flow cytometry. After activating (Poly I:C group)/inactivating (control group) TLR3 signaling pathway, rHBsAg was given to both groups for 72 h, and the proportions of DC, T, B cells and their subsets in PBMC were detected by flow cytometry. Paired t-test, paired samples wilcoxon signed-rank test and canonical correlation analyses were used for statistical analysis. Results: The percentage of TLR3 protein-positive cells (19.21%) and protein expression (8 983.95), NF-κB protein expression (26 193.13), the percentage of pNF-κB protein-positive cells (13.73%) and its proportion in NF-κB (16.03%), and the percentage of pIRF3 protein-positive cells (12.64%) and its proportion in IRF3 (21.80%) in Poly I:C group were higher than those in control group (11.54%, 8 086.00, 22 340.66, 8.72%, 9.71%, 9.57%, 19.12%) (P<0.05), and the percentage of TRIF protein-positive cells (89.75%) and protein expression (304 219.54) were higher in Poly I:C group than in the control group (89.64%, 288 149.72) (P>0.05). After PBMC stimulation by rHBsAg, the proportions of mDC (2.90%), pDC (1.80%), B cell (5.31%) and plasma cell (67.71%) in Poly I:C group were significantly higher than those in the control group (1.83%, 0.81%, 4.23%, 58.82%) (P<0.05). Results of canonical correlation analysis showed that the expression of TLR3 protein was positively correlated with the proportions of plasma cells, the expression of pIRF3 protein was positively correlated with the proportions of plasma cells and mDC, and the percentage of pNF-κB protein-positive cells and the percentage of pIRF3 protein-positive cells were positively correlated with the proportion of CD4+T cells. Conclusions: Poly I:C can activate TLR3/TRIF/NF-κB and TLR3/TRIF/IRF3 signaling pathway, promote the function of downstream signaling molecules, and then promote the maturation of DC, induce the immune responses of CD4+T cell, and promote the maturation and activation of B cells and the immune response of rHBsAg.

Variations within Toll-like receptor (TLR) and TLR signaling pathway-related genes and their synergistic effects on the risk of Guillain-Barré syndrome.

Guillain-Barré syndrome (GBS) is the commonest post-infectious polyradiculopathy. Although genetic background of the host seems to play an important role in the susceptibility to GBS, genes conferring major risk are not yet known. Dysregulation of Toll-like receptor (TLR) molecules exacerbates immune-inflammatory responses and the genetic variations within TLR pathway-related genes contribute to differential risk to infection. The aim of this study was to delineate the impact of genetic variations within TLR2, TLR3, and TLR4 genes as well as TLR signaling pathway-related genes such as MyD88, TRIF, TRAF3, TRAF6, IRF3, NFκß1, and IκBα on risk of developing GBS. Fourteen polymorphisms located within TLR2 (rs3804099, rs111200466), TLR3 (rs3775290, rs3775291), TLR4 (rs1927911, rs11536891), MyD88 (rs7744, rs4988453), TRIF (rs8120), TRAF3 (rs12147254), TRAF6 (rs4755453), IRF3 (rs2304204), NFκß1 (rs28362491), and IκBα (rs696) genes were genotyped in 150 GBS patients and 150 healthy subjects either by PCR-RFLP or TaqMan Allelic Discrimination Assay. Genotypes of two polymorphic variants, Del/Del of rs111200466 insertion and deletion (INDEL) polymorphism of TLR2 gene and TT of rs3775290 single nucleotide polymorphism (SNP) of TLR3 gene had significantly higher frequencies among GBS patients, while the frequencies of TT genotype of rs3804099 SNP of TLR2 gene and TT genotype of rs11536891 SNP of TLR4 gene were significantly higher in controls. Gene-gene interaction study by Multifactor Dimensionality Reduction analysis also suggested a significant combined effect of TLR2, and NFκß1 genes on the risk of GBS. The SNPs in the IκBα and IRF3 genes correlated with severity of GBS. The genes encoding TLRs and TLR signaling pathway-related molecules could serve as crucial genetic markers of susceptibility and severity of GBS.

Sevoflurane attenuates cognitive dysfunction and NLRP3-dependent caspase-1/11-GSDMD pathway-mediated pyroptosis in the hippocampus via upregulation of SIRT1 in a sepsis model.

Septic encephalopathy (SE) is a devastating consequence of sepsis, a hyper-triggered host response against infectious challenge, which ultimately leads to brain damage. The present study examined whether sevoflurane (SVF), a volatile anaesthetic, can counteract the perturbation of homeostasis in a caecal ligation and puncture (CLP)-induced mouse model of SE. SVF enhances neurocognition in terms of spatial memory improvement via counter-regulation of activated oxidative-inflammatory stress and pyroptotic processes in SE. Further, the beneficial effects of SVF against SE are mediated by activation of silent information regulator 1 (SIRT1)-mediated reduction of reactive oxygen species (ROS) level, regulation of thioredoxin (TXN) and thioredoxin interacting protein (TIP) levels, reduction of inflammatory-pyroptotic signalling (NLRP3, caspase 1/11, GSDMD, TLR4 and TRIF) proteins, as well as a reduction of inflammatory cytokine (IL-1ß and IL-18) levels. These findings suggest that SVF may have therapeutic potential for the treatment of SE and associated cognitive malfunction.

Isobavachalcone suppresses the TRIF-dependent signaling pathway of Toll-like receptors.

Toll-like receptors (TLRs) are integral membrane-bound receptors that are central to innate and adaptive immune responses. They are known to activate a cascade of downstream signals to induce the secretion of inflammatory cytokines, chemokines, and type I interferons. Dysregulated activation of TLR signaling pathways can induce the activation of various transcription factors, such as nuclear factor kappa B (NF-κB) and interferon regulatory factor 3 (IRF3). TLRs act via MyD88- and TRIF-mediated pathways to induce inflammatory responses. To evaluate the therapeutic potential of isobavachalcone (IBC), a natural chalcone component of Angelica keiskei, we examined its effects on signal transduction via TLR signaling pathways. IBC inhibited the activation of NF-κB and IRF3 induced by TLR agonists and their target genes. IBC also inhibited the activation of NF-κB and IRF3 induced by overexpression of downstream signaling components of TLR signaling pathways. These results suggest that IBC can regulate both MyD88- and TRIF-dependent signaling pathways of TLRs, resulting in a dramatic increase of new therapeutic options for various inflammatory diseases involving TLRs.

Sortilin regulates keratinocyte proliferation and apoptosis through the PI3K-AKT signaling pathway.

Sortilin is found to regulate proliferation and death of different cells, while its role in regulating keratinocyte proliferation and apoptosis is still unknown. In this study, we found that sortilin levels significantly increased in psoriasis patients, and sortilin suppression eliminated the proliferation of HaCaT cells induced by M5 cocktail solution and enhanced the levels of cleaved caspase 3 protein and the Bax/Bcl-2 ratio; however, levels of p-PI3K and p-AKT were decreased. In addition, sortilin silencing remitted the characteristic changes associated with psoriasis-like skin lesions. In summary, suppressed sortilin expression helped inhibit keratinocyte proliferation in HaCaT cells by inactivating PI3K/AKT signaling, which provides a new target for the therapy of psoriasis.

Sortilin-derived peptides promote pancreatic beta-cell survival through CREB signaling pathway.

Deterioration of insulin secretion and pancreatic beta-cell mass by inflammatory attacks is one of the main pathophysiological features of type 2 diabetes (T2D). Therefore, preserving beta-cell mass and stimulating insulin secretion only in response to glucose for avoiding the hypoglycemia risks, are the most state-of-the-art option for the treatment of T2D. In this study we tested two correlated hypothesis that 1/ the endogenous peptide released from sortilin, known as PE, that stimulates insulin secretion only in response to glucose, protects beta-cells against death induced by cytokines, and 2/ Spadin and Mini-Spadin, two synthetic peptides derived from PE, that mimic the effects of PE in insulin secretion, also provide beneficial effect on beta-cells survival. We show that PE and its derivatives by inducing a rise of intracellular calcium concentration by depolarizing the membrane protect beta-cells against death induced by Interleukin-1ß. Using biochemical, confocal imaging and cell biology techniques, we reveal that the protective effects of PE and its derivatives rely on the activation of the CaM-Kinase pathway, and on the phosphorylation and activation of the transcription factor CREB. In addition, Mini-Spadin promotes beta-cell proliferation, suggesting its possible regenerative effect. This study highlights new possible roles of PE in pancreatic beta-cell survival and its derivatives as pharmacological tools against diabetes.

LncRNA STXBP5-AS1 suppresses stem cell-like properties of pancreatic cancer by epigenetically inhibiting neighboring androglobin gene expression.

Previous studies suggest the tumor suppressor role of long non-coding RNA (lncRNA) STXBP5-AS1 in cervical and gastric cancer, but its expression pattern and functional mechanism are still elusive in pancreatic cancer (PC). Relative expression of STXBP5-AS1 in PC both in vivo and in vitro was analyzed by real-time PCR. IC50 of Gemcitabine was determined by the MTT assay. Cell proliferation in response to drug treatment was investigated by colony formation assay. Cell apoptosis was measured by both caspase-3 activity and Annexin V/PI staining. Cell invasion capacity was scored by the transwell assay in vitro, and lung metastasis was examined with the tail vein injection assay. Cell stemness was determined in vitro by sphere formation and marker profiling, respectively, and in vivo by limited dilution of xenograft tumor incidence. Subcellular localization of STXBP5-AS1 was analyzed with fractionation PCR. Association between STXBP5-AS1 and EZH2 was investigated by RNA-immunoprecipitation. The binding of EZH2 on ADGB promoter was analyzed by chromatin immunoprecipitation. The methylation was quantified by bisulfite sequencing. We showed downregulation of STXBP5-AS1 in PC associated with poor prognosis. Ectopic STXBP5-AS1 inhibited chemoresistance and metastasis of PC cells. In addition, STXBP5-AS1 compromised stemness of PC cells. Mechanistically, STXBP5-AS1 potently recruited EZH2 and epigenetically regulated neighboring ADGB transcription, which predominantly mediated the inhibitory effects of STXBP5-AS1 on stem cell-like properties of PC cells. Our study highlights the importance of the STXBP5-EZH2-ADGB axis in chemoresistance and stem cell-like properties of PC.

Regulation of progranulin expression and location by sortilin in oxygen-glucose deprivation/reoxygenation injury.

Progranulin is a secreted glycoprotein expressed in neurons and microglial cells that is involved in maintaining physiological functions. Many studies have found that progranulin may play a protective role against ischemic brain injury, but little is known about how the expression level and cellular localization status of progranulin is regulated after hypoxia-ischemia. Research has confirmed that sortilin, encoded by SORT1, can bind with progranulin and deliver a mature secretory isoform of progranulin to lysosomes, and progranulin is then cleaved. In the present study, we aimed to figure out whether sortilin could affect the expression and cellular localization of progranulin and regulate cell apoptosis during hypoxia-ischemia. In this study, oxygen-glucose deprivation/reoxygenation (OGD/R) in primary cortical neurons was used to mimic hypoxic-ischemic episodes. After OGD/R, the neuroprotective effects of progranulin against hypoxia-ischemia were examined, and primary cortical neurons were transduced with a SORT1 knockdown lentivirus to inhibit the expression of sortilin. The results showed that sortilin inhibition increased PGRN expression and alleviated cell injury induced by hypoxia-ischemia. Additionally, sortilin inhibition was associated with less PGRN localization in lysosomes. All of these findings suggest that sortilin can regulate the expression of PGRN, most likely by transporting it to lysosomes and affecting the cell injury in hypoxia-ischemia.

Sortilin promotes glioblastoma invasion and mesenchymal transition through GSK-3ß/ß-catenin/twist pathway.

High aggressiveness is a hallmark of glioblastoma and predicts poor prognosis of patients with glioblastoma. The expression level of sortilin has been preliminarily reported to be elevated in high-grade glioma; however, the potential significance of sortilin in glioblastoma progression has not been elucidated. In this study, we investigated the oncogenic effect of sortilin in glioblastoma. Increased levels of sortilin were noted in the mesenchymal subtype of glioblastoma and highly aggressive subtypes of glioblastoma tissues and cell lines. In addition, high levels of sortilin predicted poor prognoses in patients with glioblastoma. Sortilin knockdown or inhibition with AF38469 (an orally bioavailable inhibitor of sortilin) significantly suppressed migration and invasion by inhibiting EMT-like mesenchymal transition in glioblastoma cells. Furthermore, we proved that sortilin promoted cell invasion mainly via Glycogen synthase kinase 3 beta (GSK-3ß)/ß-catenin/Twist-induced EMT-like mesenchymal transition in glioblastoma. Taken together, our results demonstrate a critical role of sortilin in glioblastoma invasion and EMT-like mesenchymal transition, indicating that sortilin contributes to glioblastoma progression. These data also highlight the dramatic antitumor effects of AF38469 in glioblastoma, suggesting that AF38469 is a potentially powerful antitumor agent for sortilin-overexpressing human glioblastoma.

Understanding the mechanism of bias signaling of the insulin-like growth factor 1 receptor: Effects of LL37 and HASF.

The development of biased agonist drugs is widely recognized to be important for the treatment of many diseases, including cardiovascular disease. While GPCR biased agonism has been heavily characterized there is a distinct lack of information with respect to RTK biased agonism both in the identification of biased agonists as well as their attendant mechanisms. One such RTK, the Insulin-like Growth Factor 1 Receptor (IGF1R) plays an important role in a range of biological and disease processes. The micropeptide LL37 has been described as a biased agonist of the IGF1R. We were interested to further understand the mechanism by which LL37 promotes biased signaling through the IGF1R. We found that LL37 biased agonism is dependent on ß-arrestin 2. Moreover, BRET assays indicated that LL37 biased agonism is explained by the inability of LL37 to promote the recruitment of IRS1 to the IGF1R compared to IGF1. LL37 promotes an altered association of IGF1R with GRK6, which could also serve as an explanation for bias. We also demonstrated a functional consequence of this bias by showing that while LL37 can promote cell proliferation, it does not induce protein synthesis, unlike IGF1, which does both. We have recently identified HASF, a natural protein released by mesenchymal stem cells, as a novel ligand of the IGF1R. HASF is a paracrine factor with potent cardioprotective and cardio-regenerative properties which also acts via IGF1R biased signaling, preferentially activated ERK over Akt.

ProBDNF inhibits proliferation, migration and differentiation of mouse neural stem cells.

ProBDNF, a precursor of brain-derived neurotrophic factor (BDNF), is an important regulator of neurodegeneration, hippocampal long-term depression, and synaptic plasticity. ProBDNF and its receptors pan-neurotrophin receptor p75 (p75NTR), vps10p domain-containing receptor Sortilin and tropomyosin receptor kinase B (TrkB) are expressed in neuronal and glial cells. The role of proBDNF in regulation of neurogenesis is not fully defined. This study aims to uncover the function of proBDNF in regulating the differentiation, migration and proliferation of mouse neural stem cells (NSCs) in vitro. We have found that proBDNF and its receptors are constitutively expressed in NSCs when assessed by immunocytochemistry and western blotting. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay showed that exogenous proBDNF treatment reduced mouse NSCs viability by 38% at 10ng/mL. The migration of NSCs was also reduced by exogenous proBDNF treatment in a concentration-dependent manner (by 90% at 10ng/mL) but increased by anti-proBDNF antibody treatment (by 50%). BrdU (5-Bromo-2'-Deoxyuridine) incorporation was performed for detection of newborn cells. We have found that proBDNF significantly inhibited proliferation of NSCs and reduced the number of differentiated neurons, oligodendrocytes and astrocytes, while anti-proBDNF antibody treatment promoted proliferation and differentiation of NSCs. In conclusion, proBDNF may oppose the functions of mature BDNF by inhibiting the proliferation, differentiation and migration of NSCs during development. Conversely, anti-proBDNF antibody treatment promoted proliferation and differentiation of NSCs.

Motif mimetic of epsin perturbs tumor growth and metastasis.

Tumor angiogenesis is critical for cancer progression. In multiple murine models, endothelium-specific epsin deficiency abrogates tumor progression by shifting the balance of VEGFR2 signaling toward uncontrolled tumor angiogenesis, resulting in dysfunctional tumor vasculature. Here, we designed a tumor endothelium-targeting chimeric peptide (UPI) for the purpose of inhibiting endogenous tumor endothelial epsins by competitively binding activated VEGFR2. We determined that the UPI peptide specifically targets tumor endothelial VEGFR2 through an unconventional binding mechanism that is driven by unique residues present only in the epsin ubiquitin-interacting motif (UIM) and the VEGFR2 kinase domain. In murine models of neoangiogenesis, UPI peptide increased VEGF-driven angiogenesis and neovascularization but spared quiescent vascular beds. Further, in tumor-bearing mice, UPI peptide markedly impaired functional tumor angiogenesis, tumor growth, and metastasis, resulting in a notable increase in survival. Coadministration of UPI peptide with cytotoxic chemotherapeutics further sustained tumor inhibition. Equipped with localized tumor endothelium-specific targeting, our UPI peptide provides potential for an effective and alternative cancer therapy.

C3orf58, a novel paracrine protein, stimulates cardiomyocyte cell-cycle progression through the PI3K-AKT-CDK7 pathway.

RATIONALE: The regenerative capacity of the heart is markedly diminished shortly after birth, coinciding with overall withdrawal of cardiomyocytes from cell cycle. Consequently, the adult mammalian heart has limited capacity to regenerate after injury. The discovery of factors that can induce cardiomyocyte proliferation is, therefore, of high interest and has been the focus of extensive investigation throughout the past years. OBJECTIVE: We have recently identified C3orf58 as a novel hypoxia and Akt induced stem cell factor (HASF) secreted from mesenchymal stem cells, which can promote cardiac repair through cytoprotective mechanisms. Here, we tested the hypothesis that HASF can also contribute to cardiac regeneration by stimulating cardiomyocyte division and proliferation. METHODS AND RESULTS: Neonatal ventricular cardiomyocytes were stimulated in culture for 7 days with purified recombinant HASF protein. Compared with control untreated cells, HASF-treated neonatal cardiomyocytes exhibited 60% increase in DNA synthesis as measured by bromodeoxyuridine incorporation. These results were confirmed by immunofluorescence confocal microscopy showing a 50% to 100% increase in the number of cardiomyocytes in the mitotic and cytokinesis phases. Importantly, in vivo cardiac overexpression of HASF in a transgenic mouse model resulted in enhanced level of DNA synthesis and cytokinesis in neonatal and adult cardiomyocytes. These proliferative effects were modulated by a phosphoinositide 3-kinase-protein kinase B-cycle-dependent kinase 7 pathway as revealed by the use of phosphoinositide 3-kinase -pathway-specific inhibitors and silencing of the Cdk7 gene. CONCLUSIONS: Our studies support the hypothesis that HASF induces cardiomyocyte proliferation via a phosphoinositide 3-kinase-protein kinase B-cycle-dependent kinase 7 pathway. The implications of this finding may be significant for cardiac regeneration biology and therapeutics.