Hypoxia exacerbates cardiomyocyte injury via upregulation of Wnt3a and inhibition of Sirt3.

Ischemic injury is a major cause of several cardiovascular diseases, such as myocardial infarction, cardiac hypertrophy, and ventricular remodeling. Using an in vitro hypoxia model to mimic ischemia, we found that hypoxia stimulated Wnt3a expression. A mechanistic study showed that hypoxia-inducible factor 1α (HIF-1α) was directly recruited to the Wnt3a promoter. Wnt3a overexpression significantly decreased cell viability, promoted the generation of apoptotic cells, and enhanced hypoxia-induced injury in neonatal rat cardiomyocytes. This was partially through the upregulation of Caspase-3 mRNA levels and cleaved PARP-1 protein levels. In addition, we observed that Wnt3a exacerbated hypoxia-induced mitochondrial dysfunction and cytosolic release of cytochrome C. Furthermore, we found that Sirt3, a mitochondrial NAD+-dependent deacetylase that modulates mitochondrial metabolism and homeostasis, was negatively regulated by Wnt3a. Conversely, Sirt3 overexpression repressed Wnt3a expression and ameliorated the hypoxia-induced mitochondrial dysfunction. Overall, our findings suggest that the hypoxia-Wnt3a-Sirt3 regulatory axis might be a potential target for cell protection in cardiac ischemia and hypoxia.

Dihydrotestosterone inhibits arylsulfatase B and Dickkopf Wnt signaling pathway inhibitor (DKK)-3 leading to enhanced Wnt signaling in prostate epithelium in response to stromal Wnt3A.

BACKGROUND: In tissue microarrays, immunostaining of the enzyme arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) was less in recurrent prostate cancers and in cancers with higher Gleason scores. In cultured prostate stem cells, decline in ARSB increased Wnt signaling through effects on Dickkopf Wnt Signaling Pathway Inhibitor (DKK)3. The effects of androgen exposure on ARSB and the impact of decline in ARSB on Wnt signaling in prostate tissue were unknown. METHODS: Epithelial and stromal tissues from malignant and normal human prostate were obtained by laser capture microdissection. mRNA expression of ARSB, galactose-6-sulfate-sulfatase (GALNS) and Wnt-signaling targets was determined by QPCR. Non-malignant human epithelial and stromal prostate cells were grown in tissue culture, including two-cell layer cultures. ARSB was silenced by specific siRNA, and epithelial cells were treated with stromal spent media following treatment with IWP-2, an inhibitor of Wnt secretion, and by exogenous recombinant human Wnt3A. Promoter methylation was detected using specific DKK3 and ARSB promoter primers. The effects of DHT and of ARSB overexpression on DKK expression were determined. Cell proliferation was assessed by BrdU incorporation. RESULTS: Normal stroma showed higher expression of vimentin, ARSB, and Wnt3A than epithelium. Normal epithelium had higher expression of E-cadherin, galactose 6-sulfate-sulfatase (GALNS), and DKK3 than stroma. In malignant epithelium, expression of ARSB and DKK3 declined, and expression of GALNS and Wnt signaling targets increased. In cultured prostate epithelial cells, Wnt-mediated signaling was greatest when ARSB was silenced and cells were exposed to exogenous Wnt3A. Exposure to 5α-dihydrotestosterone (DHT) increased ARSB and DKK3 promoter rmethylation, and effects of DHT on DKK3 expression were reversed when ARSB was overexpressed. CONCLUSIONS: Androgen-induced declines in ARSB and DKK3 may contribute to prostate carcinogenesis by sustained activation of Wnt signaling in prostate epithelium in response to stromal Wnt3A.

Involvement of miR-140-3p in Wnt3a and TGFß3 signaling pathways during osteoblast differentiation in MC3T3-E1 cells.

The Wnt/ß-catenin signaling and TGFß signaling pathways play a key role in osteoblast differentiation. The miRNAs play important roles in regulating gene expression at the post-transcriptional level through fine-tuning of protein-encoding gene expression. However, involvement of miRNAs is not established for Wnt3a and TGFß signaling pathways in osteoblast differentiation. Here, we examined the role of miRNAs expressed differentially after Wnt3a expression during osteoblast differentiation. Over-expression of the Wnt3a gene increased ALP transcription, but decreased Col1, Runx2, and OCN transcription in osteoblastic MC3T3-E1 cells. Expression profiling and quantitative PCR for miRNAs showed that miR-140-3p decreased in Wnt3a-over-expressing osteoblastic cells. Wnt3a over-expression increased TGFß3 expression, whereas transfection of the miR-140-3p mimic into MC3T3-E1 cells significantly inhibited TGFß3 expression. Luciferase assay for the TGFß3 transcript showed that TGFß3 was a direct target of miR-140-3p. miR-140-3p mimic transfection resulted in significantly increased OCN transcription, but did not affect ALP, Col1, and Runx2 transcription in MC3T3-E1 cells. rTGFß3 treatment decreased OCN transcription in MC3T3-E1 cells. These results suggest that the miR-140-3p is involved in osteoblast differentiation as a critical regulatory factor between Wnt3a and TGFß3 signaling pathways.

Wnt signaling induces neurite outgrowth in mouse retinal ganglion cells.

Wingless-type (Wnt) signaling pathways mediate axonal growth and remodeling in the embryonic optic nerve, brain and spinal cord. Recent studies demonstrated that the canonical Wnt/ß-catenin signaling pathway also induces axonal regeneration after injury in the optic nerve of adult animals. However, the molecular mechanisms of Wnt-mediated axonal growth are not well understood. Additionally, because Wnt signaling is stimulated in neurons as well as neighboring non-neuronal cells, the cell type(s) responsible for Wnt-induced axonal regeneration are not known. The objectives of this study were to investigate potential mechanisms and target cells of Wnt3a stimulated neurite growth using primary retinal ganglion cell (RGC) cultures. We demonstrated that Wnt3a ligand induced dose-dependent increases in average neurite length and number of neurites in RGCs. QPCR analysis of candidate mediators showed that Wnt3a-dependent neurite growth was associated with lower expression of Ripk1 and Ripk3 genes. Additionally, inhibiting Ripk1 signaling with Necrostatin-1s led to increased neurite number per cell but not increased neurite length. Therefore, Ripk signaling may be involved in mediating the effects of Wnt3a on neurite number but Ripk activity does not seem to be required for Wnt3a-dependent regulation of neurite length. This study shows that RGCs are direct cellular targets of Wnt3a-induced axonal growth, and we identified a novel association between Wnt signaling and Rip kinases in neurite formation.

Beta-elemene increases chemosensitivity to 5-fluorouracil through down-regulating microRNA-191 expression in colorectal carcinoma cells.

Colorectal carcinoma is a common malignant tumor occurring in the alimentary system. Despite developments of modern medicine, developed resistance to 5-fluorouracil (5-FU) may lead to poor prognosis. Herein, we aimed to explore the effects of beta-elemene on colorectal carcinoma cells (HCT116 and HT29) as well as the underlying mechanisms. Beta-elemene reduced cell viability and induced apoptosis in HCT116 and HT29 cells. Increased apoptosis following beta-elemene exposure was due to enhanced sensitivity to 5-FU through down-regulating miR-191. Expression of key kinases, including Wnt3a, and ß-catenin, were down-regulated by beta-elemene through a miR-191 mechanism. Moreover, beta-elemene might improve resistance of colorectal carcinoma cells to 5-FU by down-regulating miR-191, thereby inhibiting the Wnt/ß-catenin pathway.

Glycogen synthase kinase-3ß inhibition enhances myelination in preterm newborns with intraventricular hemorrhage, but not recombinant Wnt3A.

Intraventricular hemorrhage (IVH) in preterm infants results in reduced proliferation and maturation of oligodendrocyte progenitor cells (OPCs), and survivors exhibit reduced myelination and neurological deficits. Wnt signaling regulates OPC maturation and myelination in a context dependent manner. Herein, we hypothesized that the occurrence of IVH would downregulate Wnt signaling, and that activating Wnt signaling by GSK-3ß inhibition or Wnt3A recombinant human protein (rh-Wnt3A) treatment might promote maturation of OPCs, myelination of the white matter, and neurological recovery in premature rabbits with IVH. These hypotheses were tested in autopsy samples from preterm infants and in a rabbit model of IVH. Induction of IVH reduced expressions of activated ß-catenin, TCF-4, and Axin2 transcription factors in preterm newborns. Both AR-A014418 (ARA) and Wnt-3A treatment activated Wnt signaling. GSK-3ß inhibition by intramuscular ARA treatment accelerated maturation of OPCs, myelination, and neurological recovery in preterm rabbits with IVH compared to vehicle controls. In contrast, intracerebroventricular rh-Wnt3A treatment failed to enhance myelination and neurological function in rabbits with IVH. ARA treatment reduced microglia infiltration and IL1ß expression in rabbits with IVH relative to controls, whereas Wnt3A treatment elevated TNFα, IL1ß, and IL6 expression without affecting microglia density. GSK-3ß inhibition downregulated, while rh-Wnt3A treatment upregulated Notch signaling; and none of the two treatments affected the Sonic-Hedgehog pathway. The administration of ARA or rh-Wnt3A did not affect gliosis. The data suggest that GSK-3ß inhibition promoted myelination by suppressing inflammation and Notch signaling; and Wnt3A treatment failed to enhance myelination because of its pro-inflammatory activity and synergy with Notch signaling. GSK-3ß inhibitors might improve the neurological outcome of preterm infants with IVH.

Wnt3α and transforming growth factor-ß induce myofibroblast differentiation from periodontal ligament cells via different pathways.

Myofibroblasts are specialized cells that play a key role in connective tissue remodeling and reconstruction. Alpha-smooth muscle actin (α-SMA), vimentin and tenascin-C are myofibroblast phenotype, while α-SMA is the phenotypic marker. The observation that human periodontal ligament cells (hPDLCs) differentiate into myofibroblasts under orthodontic force has provided a new perspective for understanding of the biological and biomechanical mechanisms involved in orthodontic tooth movement. However, the cell-specific molecular mechanisms leading to myofibroblast differentiation in the periodontal ligament (PDL) remain unclear. In this study, we found that expression of Wnt3α, transforming growth factor-ß1 (TGF-ß1), α-SMA and tenascin-C increased in both tension and compression regions of the PDL under orthodontic load compared with unloaded control, suggesting that upregulated Wnt3α and TGF-ß1 signaling might have roles in myofibroblast differentiation in response to orthodontic force. We reveal in vitro that both Wnt3α and TGF-ß1 promote myofibroblast differentiation from hPDLCs. Dickkopf-1 (DKK1) impairs Wnt3α-induced myofibroblast differentiation in a ß-catenin-dependent manner. TGF-ß1 stimulates myofibroblast differentiation via a JNK-dependent mechanism. DKK1 has no significant effect on TGF-ß1-induced myofibroblastic phenotype.

Wnt5a Suppresses ß-catenin Signaling during Hair Follicle Regeneration.

Hair follicles display periodic growth. Wnt signaling is a critical regulator for hair follicle regeneration. Previously, we reported that Wnt5a inhibits the telogen-to-anagen transition of hair follicles, but the mechanism by which this process occurs has not yet been reported. Here, we determined the expression patterns of Wnt signaling pathway molecules by quantitative reverse transcription polymerase chain reaction, western blot, and immunohistochemistry and found that ß-catenin signaling was suppressed by Wnt5a. We then compared the phenotypes and expression patterns following ß-catenin knockdown and Wnt5a overexpression during hair follicle regeneration induced by hair depilation and observed similar patterns. In addition, we performed a rescue experiment in the JB6 cell line and found that the inhibitory effect of Wnt5a on cell proliferation could be rescued by the addition of Wnt3a. Our data reveal that Wnt5a suppresses the activation of ß-catenin signaling during hair follicle regeneration.

Different Impact Of Antiretroviral Drugs On Bone Differentiation In An In Vitro Model.

Recently increasing emphasis is placed on preventive health and management of chronic comorbidities avoiding long-term toxicities of antiretroviral therapy (ART). Drawing from this background we decided to use the Saos-2, osteosarcoma cell line, as a cellular model, to evaluate the effects of some antiretroviral drugs such as abacavir (ABC), tenofovir (TDF), efavirenz (EFV), etravirine (ETR), and darunavir (DRV), on bone differentiation related pathways. According to our observation, treatment with TDF and ABC affects the ability of the cells to produce calcium deposits with a reduced expression of type I collagen gene and p21 mRNA, also increasing the activity of Wnt3a related pathway. On the other hand treatment with EFV and DRV was not related to any significant reduction of calcium deposits but displayed a decrease in the expression of Wnt3a at day 14 and Type I Collagen at day 7 compared with untreated cells, even if this last down regulation was not confirmed at day 14. Instead ETR administration to Saos-2 cells increases the calcium deposits collagen type I production, as a result of Wnt3a mRNA overexpression, and of an upregulation of collagen type I expression, being also the only drug able to increase the expression of p21 cdk inhibitor as further marker of terminal differentiation. In summary these data suggest the potential negative interference of TDF and ABC on bone differentiation. DRV and EFV partially affect collagen type I production, instead ETR facilitates a positive bone balance as a result of an increased osteoblasts terminal differentiation.

Cortical gyrification induced by fibroblast growth factor 2 in the mouse brain.

Gyrification allows an expanded cortex with greater functionality to fit into a smaller cranium. However, the mechanisms of gyrus formation have been elusive. We show that ventricular injection of FGF2 protein at embryonic day 11.5-before neurogenesis and before the formation of intrahemispheric axonal connections-altered the overall size and shape of the cortex and induced the formation of prominent, bilateral gyri and sulci in the rostrolateral neocortex. We show increased tangential growth of the rostral ventricular zone (VZ) but decreased Wnt3a and Lef1 expression in the cortical hem and adjacent hippocampal promordium and consequent impaired growth of the caudal cortical primordium, including the hippocampus. At the same time, we observed ectopic Er81 expression, increased proliferation of Tbr2-expressing (Tbr2(+)) intermediate neuronal progenitors (INPs), and elevated Tbr1(+) neurogenesis in the regions that undergo gyrification, indicating region-specific actions of FGF2 on the VZ and subventricular zone (SVZ). However, the relative number of basal radial glia-recently proposed to be important in gyrification-appeared to be unchanged. These findings are consistent with the hypothesis that increased radial unit production together with rapid SVZ growth and heightened localized neurogenesis can cause cortical gyrification in lissencephalic species. These data also suggest that the position of cortical gyri can be molecularly specified in mice. In contrast, a different ligand, FGF8b, elicited surface area expansion throughout the cortical primordium but no gyrification. Our findings demonstrate that individual members of the diverse Fgf gene family differentially regulate global as well as regional cortical growth rates while maintaining cortical layer structure.

Overexpression of Wnt3a facilitates the proliferation and neural differentiation of neural stem cells in vitro and after transplantation into an injured rat retina.

Neural stem cell-based therapy is a promising option for repair after injury. However, poor stem cell proliferation and insufficient differentiation of the stem cells into neurons are still difficult problems. The present study investigated whether transplantation of neural stem cells (NSCs) genetically modified to express Wnt3a is a promising approach to overcome these difficulties. We explored the possibility that Wnt3a might contribute to the therapeutic effect of NSC transplantation in retinal repair. The relative promotion of proliferation and neural differentiation by modified NSCs was investigated in a rat model of optic nerve crush. A recombinant lentivirus (Lenti-Wnt3a) was engineered to express Wnt3a. NSCs infected with control lentivirus (Lenti-GFP) or Lenti-Wnt3a were transplanted into the subretinal space immediately after the optic nerve crush. The proliferation and neural differentiation activity of the NSCs were assessed in vitro and in vivo. Overexpression of Wnt3a in NSCs induced activation of Wnt signaling, promoted proliferation, and directed the differentiation of the NSCs into neurons both in vitro and in vivo. Our study suggests that Wnt3a can potentiate the therapeutic benefits of NSC-based therapy in the injured retina.

Wnt3a expression is associated with MMP-9 expression in primary tumor and metastatic site in recurrent or stage IV colorectal cancer.

BACKGROUND: The wnt/ß-catenin signaling pathway is known to affect in cancer oncogenesis and progression by interacting with the tumor microenvironment. However, the roles of wnt3a and wnt5a in colorectal cancer (CRC) have not been thoroughly studied. In the present study, we investigated the expression of wnt protein and the concordance rate in primary tumor and metastatic sites in CRC. To determine the relationship of wnt proteins with invasion related protein, we also analyzed the association between wnt protein expression and the expression of matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor receptor-2 (VEGFR-2). METHODS: Tumor tissue was obtained from eighty-three paraffin- embedded blocks which were using resected tissue from both the primary tumor and metastatic sites for each patient. We performed immunohistochemical staining for wnt3a, wnt5a, ß-catenin, MMP-9 and VEGFR-2. RESULTS: Wnt3a, wnt5a, ß-catenin, and MMP-9 expression was high; the proteins were found in over 50% of the primary tumors, but the prevalence was lower in tissue from metastatic sites. The concordance rates between the primary tumor and metastatic site were 76.2% for wnt5a and 79.4% for wnt3a and ß-catenin, but VEGFR-2 was expressed in 67.4% of the metastatic sites even when not found in the primary tumor. Wnt3a expression in primary tumors was significantly associated with lymph node involvement (p = 0.038) and MMP-9 expression in the primary tumor (p = 0.0387), mesenchyme adjacent to tumor (p = 0.022) and metastatic site (p = 0.004). There was no other relationship in the expression of these proteins. Vascular invasion in primary tumor tissue may be a potential prognostic marker for liver metastasis, but no significant association was observed among the wnt protein, MMP-9, and VEGFR-2 for peritoneal seeding. In survival analysis, ß-catenin expression was significantly correlated with overall survival (p = 0.05). CONCLUSIONS: Wnt3a and wnt5a expression had a concordance rate higher than 60% with a high concordance rate between the primary tumor and metastatic site. Wnt3a expression is associated with the expression of MMP-9 in primary tumor tissue adjacent mesenchymal tissue, and at the metastatic site. As a prognostic marker, only ß-catenin expression showed significant relation with survival outcome.

Effect of the Jianpi Bushen Prescription on the expression of SHP-1, Wnt3a, and AP-1 proteins in chemically damaged mice.

This study investigated the effect of the Jianpi Bushen Prescription (JBP) on the expression of 3 major proteins in chemically damaged model mice. The 3 proteins were the Wnt3a, the SHP-1, and the transcription factors (NF-E2, c-jun, and c-fos) of the AP-1 protein family. Kunming mice were randomly divided into chemically damaged group (N=48), which received an abdominal injection of (100 mg/kg) cyclophosphamide once a day for 3 consecutive days, and control group (N=12), which received the same amount of saline. Then, the chemically damaged mice were randomly divided into chemically damaged model group (N=12), which received 0.2 mL/10 g of saline twice a day for 9 days, positive control group (N=12), which received 0.2 mL/10 g of the e-jiao slurry (EJS) compound twice a day for 9 days, low dose JBP group (N=12), which received 0.1 g/kg suspension JBP (100% concentration) twice a day for 9 days and high dose JBP group (N=12), which received 0.1 g/kg suspension JBP (200% concentration) twice a day for 9 days. The bilateral femur and tibia bone marrow were collected from the mice in all groups. The protein expression of the specified proteins and transcription factors in the bone marrow mononuclear cells were detected by Western blot analysis. The results showed that the protein expression of Wnt3a was significantly downregulated in the chemically damaged model group compared to the control group (P<0.05). The low dose JBP, high dose JBP, and e-jiao slurry treatments significantly upregulated the protein expression of Wnt3a compared to the chemically damaged model group (P<0.05), with the low dose JBP producing the best results. Compared to the control group, the protein expressions of SHP-1, c-fos, c-jun, and NF-E2 were significantly higher in the chemically damaged model group (all P<0.05). The protein expressions of SHP-1, c-fos, c-jun, and NF-E2 were significantly lower in the chemically damaged model+the low dose JBP, chemically damaged model+high dose JBP, or chemically damaged model+EJS group compared to chemically damaged model (all P<0.05), with the low dose JBP producing the best results. These results indicate that JBP regulates the expressions of SHP-1, Wnt3a, and AP-1 proteins in chemically damaged mice.

Activation of NMDA receptors upregulates a disintegrin and metalloproteinase 10 via a Wnt/MAPK signaling pathway.

A disintegrin and metalloproteinase 10 (ADAM10) is the constitutive α-secretase that governs the nonamyloidogenic pathway of ß-amyloid precursor protein processing and is an attractive drug target for treating Alzheimer's disease. To date, little is known about the mechanism by which ADAM10 is regulated in neurons. Using mouse primary cortical neurons, we show here that NMDA receptor (NMDAR) activation led to upregulation of the genes encoding ADAM10 and ß-catenin proteins. Interestingly, the ADAM10 upregulation was abolished by inhibitors of Wnt/ß-catenin signaling. Conversely, activation of the Wnt/ß-catenin signaling pathway by recombinant Wnt3a stimulated ADAM10 expression. We further showed that both the NMDAR- and Wnt3a-induced ADAM10 upregulation was blocked by ERK inhibitors. We suggest that the NMDARs control ADAM10 expression via a Wnt/MAPK signaling pathway.

Activation of the Wnt/ß-catenin signaling pathway is associated with glial proliferation in the adult spinal cord of ALS transgenic mice.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the progressive and fatal loss of motor neurons. In ALS, there is a significant cell proliferation in response to neurodegeneration; however, the exact molecular mechanisms of cell proliferation and differentiation are unclear. The Wnt signaling pathway has been shown to be involved in neurodegenerative processes. Wnt3a, ß-catenin, and Cyclin D1 are three key signaling molecules of the Wnt/ß-catenin signaling pathway. We determined the expression of Wnt3a, ß-catenin, and Cyclin D1 in the adult spinal cord of SOD1(G93A) ALS transgenic mice at different stages by RT-PCR, Western blot, and immunofluorescence labeling techniques. We found that the mRNA and protein of Wnt3a and Cyclin D1 in the spinal cord of the ALS mice were upregulated compared to those in wild-type mice. In addition, ß-catenin translocated from the cell membrane to the nucleus and subsequently activated transcription of the target gene, Cyclin D1. BrdU and Cyclin D1 double-positive cells were increased in the spinal cord of these mice. Moreover, Wnt3a, ß-catenin, and Cyclin D1 were also expressed in both neurons and astrocytes. The expression of Wnt3a, ß-catenin or Cyclin D1 in mature GFAP(+) astrocytes increased. Moreover, BrdU/Cyclin D1/GFAP triple-positive cells were detected in the ALS mice. Our findings suggest that neurodegeneration activates the Wnt/ß-catenin signaling pathway, which is associated with glial proliferation in the adult spinal cord of ALS transgenic mice. This mechanism may be significant in clinical gene therapy.

Liposomal honokiol promotes hair growth via activating Wnt3a/ß-catenin signaling pathway and down regulating TGF-ß1 in C57BL/6N mice.

Liposomal honokiol isolated from the genus Magnolia has been found to have antiangiogenic, anti-inflammatory and antitumor properties. However, there has no report on its role in hair growth. Hair follicles are life-long cycled organelles that go through from anagen, catagen and telogen stages and are regulated by diverse signaling pathways, including Wnt/ß-catenin, Notch, Epidermal growth factor (EGF) and Sonic hegehog (SHH). Wnt signals are essential for the initiation of hair follicle placode development and a new potential target of hair loss treatment. This study was designed to investigate the effect of liposomal honokiol (Lip-honokiol) on inducing hair anagen. We identified the hair grew out in advance in the shaving area of C57BL/6N mice after the treatment of liposomal honokiol (Lip-honokiol) by daily abdominal injection. We first demonstrated that Lip-Honokiol activated the Wnt3a/ß-catenin pathway and downregulated the transforming growth factor-ß1 (TGF-ß1) to promote hair growth in mice via immunohistochemistry and immunofluorescence staining. These findings suggest that Lip-honokiol activated the Wnt/ß-catenin pathway and accelerated the transfer from the telogen to anagen stage and finally promoted the hair growth.

A model of neocortical area patterning in the lissencephalic mouse may hold for larger gyrencephalic brains.

In the mouse, two telencephalic signaling centers orchestrate embryonic patterning of the cerebral cortex. From the rostral patterning center in the telencephalon, the Fibroblast Growth Factor, FGF8, disperses as a morphogen to establish the rostral to caudal axis of the neocortical area map. FGF8 coordinates with Wnt3a from the cortical hem to regulate graded expression of transcription factors that position neocortical areas, and control hippocampal development. Whether similar signaling centers pattern the much larger cortices of carnivore and primate species, however, is unclear. The limited dispersion range of FGF8 and Wnt3a is inconsistent with patterning larger cortical primordia. Yet the implication that different mechanisms organize cortex in different mammals flies in the face of the tenet that developmental patterning mechanisms are conserved across vertebrate species. In the present study, both signaling centers were identified in the ferret telencephalon, as were expression gradients of the patterning transcription factor genes regulated by FGF8 and Wnt3a. Notably, at the stage corresponding to the peak period of FGF8 signaling in the mouse neocortical primordium (NP), the NP was the same size in ferret and mouse, which would allow morphogen patterning of the ferret NP. Subsequently, the size of ferret neocortex shot past that of the mouse. Images from online databases further suggest that NP growth in humans, too, is slowed in early cortical development. We propose that if early growth in larger brains is held back, mechanisms that pattern the neocortical area map in the mouse could be conserved across mammalian species.

A simple and scalable hydrogel-based system for culturing protein-producing cells.

Recombinant protein therapeutics have become important components of the modern medicine. Majority of them are produced with mammalian cells that are cultured either through adherent culturing, in which cells are cultured on substrates, or suspension culturing, in which cells are suspended and cultured in agitated cell culture medium in a culture vessel. The adherent cell culturing method is limited by its low yield. In suspension culturing, cells need extensive genetic manipulation to grow as single cells at high density, which is time- and labor-consuming. Here, we report a new method, which utilizes a thermoreversible hydrogel as the scaffold for culturing protein-expressing cells. The hydrogel scaffolds not only provide 3D spaces for the cells, but also act as physical barriers to prevent excessive cellular agglomeration and protect cells from the hydrodynamic stresses. As a result, cells can grow at high viability, high growth rate, and extremely high yield even without genetic manipulations. The cell yield in the hydrogels is around 20 times of the suspension culturing. In addition, the protein productivity per cell per day in the hydrogel is higher than the adherent culturing method. This new method is simple, scalable and defined. It will be of great value for both the research laboratories and pharmaceutical industry for producing proteins.

Wnt3a Neutralization Enhances T-cell Responses through Indirect Mechanisms and Restrains Tumor Growth.

The Wnt/ß-catenin pathway regulates T-cell functions, including the repression of effector functions to the advantage of memory development via Tcf1. In a companion study, we demonstrate that, in human cancers, Wnt3a/ß-catenin signaling maintains tumor-infiltrating T cells in a partially exhausted status. Here, we have investigated the effects of Wnt3a neutralization in vivo in a mouse tumor model. Abundant Wnt3a was released, mostly by stromal cells, in the tumor microenvironment. We tested whether Wnt3a neutralization in vivo could rescue the effector capacity of tumor-infiltrating T cells, by administering an antibody to Wnt3a to tumor-bearing mice. This therapy restrained tumor growth and favored the expansion of tumor antigen-specific CD8+ effector memory T cells with increased expression of Tbet and IFNγ and reduced expression of Tcf1. However, the effect was not attributable to the interruption of T-cell-intrinsic ß-catenin signaling, because Wnt3a/ß-catenin activation correlated with enhanced, not reduced, T-cell effector functions both ex vivo and in vitro Adoptively transferred CD8+ T cells, not directly exposed to the anti-Wnt3a antibody but infiltrating previously Wnt3a-neutralized tumors, also showed improved functions. The rescue of T-cell response was thus secondary to T-cell-extrinsic changes that likely involved dendritic cells. Indeed, tumor-derived Wnt3a strongly suppressed dendritic cell maturation in vitro, and anti-Wnt3a treatment rescued dendritic cell activities in vivo Our results clarify the function of the Wnt3a/ß-catenin pathway in antitumor effector T cells and suggest that Wnt3a neutralization might be a promising immunotherapy for rescuing dendritic cell activities. Cancer Immunol Res; 6(8); 953-64. ©2018 AACR.

Effect of BMPs and Wnt3a co-expression on the osteogenetic capacity of osteoblasts.

In the present study, third­generation autologous­inactivated bone morphogenic protein 2 (BMP2), BMP4, BMP6, BMP7, BMP9 and Wnt3a lentiviral vectors were constructed and integrated into the genome of MC3T3­E1 murine mesenchymal stem cells (MMSCs) to produce osteoinductive factor gene­modified MMSCs. The transfection efficiency of each osteoinductive factor was then determined by detecting the expression levels of runt related transcription factor 2 (Runx2) mRNA. The cotransfection with combinations of two lentiviruses was performed, and the expression levels of bone γ­carboxyglutamate protein and alkaline phosphatase in the MC3T3­E1 cell culture supernatant were detected. The expression level of Runx2 mRNA was detected by reverse transcription­polymerase chain reaction, and western blotting was performed to detect the protein expression levels of BMP2, BMP4, BMP6, BMP7, BMP9 and Wnt3a. The results demonstrated that the recombinant lentiviruses were successfully transfected into MC3T3­E1 cells. The relative expression levels of Runx2 mRNA were greatest in the BMP2 group, sequentially followed by the BMP4, BMP9, BMP7, Wnt3a and BMP6 groups. The results of cotransfection of MC3T3­E1 cells (a total of 8 groups) demonstrated that BMP­2 and BMP­7 exhibited the highest cotransfection efficiency. Western blot analysis demonstrated that following BMP2 and BMP7 cotransfection of MC3T3­E1 cells, the protein expression levels of BMP2, BMP4, BMP6, BMP7, BMP9 and Wnt3a were increased compared with control cells. In conclusion, the third­generation lentiviral vectors effectively improved the osteogenic efficiencies of MC3T3­E1 cells, which provided an important theoretical basis and therapeutic strategy for bone reconstruction and tissue engineering.