Eosinophils infiltration and releasing TGF-<i>β</i>1 in the airways has been implicated in the pathogenesis of asthma, especially during acute episodes provoked by an allergen. TGF-<i>β</i>1 is a major mediator involved in pro-inflammatory responses and fibrotic tissue remodeling in asthma. We aimed to evaluate the effect of in vivo allergen-activated eosinophils on the expression of <i>COL1A1</i> and <i>FN</i> in ASM cells in asthma. A total of <em>12</em> allergic asthma patients and 11 healthy subjects were examined. All study subjects underwent bronchial challenge with <i>D. pteronyssinus</i> allergen. Eosinophils from peripheral blood were isolated before and 24 h after the bronchial allergen challenge using high-density centrifugation and magnetic separation. Individual co-cultures of blood eosinophils and immortalized human ASM cells were prepared. The TGF-<i>β</i>1 concentration in culture supernatants was analyzed using ELISA. Gene expression was analyzed using qRT-PCR. Eosinophils integrins were suppressed with linear RGDS peptide before co-culture with ASM cells. Results: The expression of <i>TGF-β1</i> in asthmatic eosinophils significantly increased over non-activated asthmatic eosinophils after allergen challenge, <i>p</i> < 0.001. The TGF-<i>β</i>1 concentration in culture supernatants was significantly higher in samples with allergen-activated asthmatic eosinophils compared to baseline, <i>p</i> < 0.05. The effect of allergen-activated asthmatic eosinophils on the expression of <i>TGF-β1</i>, <i>COL1A1</i>, and <i>FN</i> in ASM cells was more significant compared to non-activated eosinophils, <i>p</i> < 0.05, however, no difference was found on <i><em>WNT</em>-5A</i> expression. The incubation of allergen-activated asthmatic eosinophils with RGDS peptide was more effective compared to non-activated eosinophils as the gene expression in ASM cells was downregulated equally to the same level as healthy eosinophils.

Dynamic suspension culture of human pluripotent stem cells (hPSCs) in stirred bioreactors provides a valuable scalable culture platform for integrated differentiation toward different lineages for potential research and therapeutic applications. However, current protocols for scalable and integrated differentiation of hPSCs limited due to high cost of growth factors and technical challenges. Here, hPSCs aggregates primed with 6 and <em>12</em> μM of CHIR99021 (CHIR), a <em>Wnt</em> agonist, in combination with different concentrations of high cost Activin A (10, 25, 50, 100 ng/mL). We sought to determine the appropriate treatment duration for efficient and cost-effective differentiation protocol for foregut definitive endoderm production in a dynamic suspension culture. Afterward, we evaluated the impact of the initial hPSC aggregate sizes (small: 86 ± 18 μm; medium: 142 ± 32 μm; large: 214 ± 34 μm) as critical bioprocess parameter on differentiation efficacy at the beginning of induction. The results indicated that 1-day priming of hPSCs as 3D aggregates (hPSpheres) with 6 μM CHIR followed by treatment with a low concentration of Activin (10 ng/mL) for 2 days resulted in efficient differentiation to definitive endoderm. This finding confirmed by the presence of ≥70% SOX17/FOXA2-double positive cells that highly expressed the anterior endodermal marker HEX. These endodermal cells differentiated efficiently into mature functional hepatocytes [60% albumin (ALB)-positive cells]. The results showed that the initial size of hPSC aggregates significantly impacted on the efficacy of differentiation. The medium sized-hPSpheres resulted in higher productivity and differentiation efficiency for scalable hepatocytes production, whereas small aggregates resulted in significant cell-loss after CHIR treatment and large aggregates had less efficacious endodermal differentiation. Differentiated cells exhibited multiple characteristics of primary hepatocytes as evidenced by expressions of liver-specific markers, indocyanine green and low-density lipoprotein uptake, and glycogen storage. Thus, this platform could be employed for scalable production of hPSC-derived hepatocytes for clinical and drug discovery applications.

The androgen receptor is one of the key targets for prostate cancer treatment. Despite its less satisfactory effects, chemotherapy is the most common treatment option for metastatic and/or castration-resistant patients. There are constant needs for novel anti-prostate cancer therapeutic/prevention agents. Curcumin, a known chemo-preventive agent, was shown to inhibit prostate cancer cell growth. This study aimed to unravel the inhibitory effect of curcumin in prostate cancer through analyzing the alterations of expressions of curcumin targeting genes clusters in androgen-dependent LNCaP cells and androgen-independent metastatic C4-2B cells. Hierarchical clustering showed the highest number of differentially expressed genes at <em>12</em> h post treatment in both cells, suggesting that the androgen-dependent/independent manner of curcumin impacts on prostate cancer cells. Evaluation of significantly regulated top canonical pathways highlighted that Transforming growth factor beta (TGF-β), Wingless-related integration site (<em>Wnt</em>), Phosphoinositide 3-kinase/Protein Kinase B/ mammalian target of rapamycin (PIK3/AKT(PKB)/mTOR), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) signaling were primarily inhibited, and Phosphatase and tensin homolog (PTEN) dependent cell cycle arrest and apoptosis pathways were elevated with curcumin treatment. The short term (3-24 h) and long term (48 h) effect of curcumin treatment revealed 31 and four genes modulated in both cell lines. TGF-β signaling, including the androgen/TGF-β inhibitor Prostate transmembrane protein androgen-induced 1 (<i>PMEPA1</i>), was the only pathway impacted by curcumin treatment after 48 h. Our findings also established that <i>MYC</i> Proto-Oncogene, basic helix-loop-helix (bHLH) Transcription Factor (MYC) signaling was down-regulated in curcumin-treated cell lines. This study established, for the first time, novel gene-networks and signaling pathways confirming the chemo-preventive and cancer-growth inhibitory nature of curcumin as a natural anti-prostate cancer compound.

Recently, we identified a patient with an infantile sacrococcygeal teratoma and a constitutional t(<em>12</em>;15)(q13;q25). Here, we show that, as a result of this chromosomal translocation, the SUMO/Sentrin-specific protease 1 gene (SENP1) on chromosome <em>12</em> and the embryonic polarity-related mesoderm development gene (MESDC2) on chromosome 15 are disrupted and fused. Both reciprocal SENP1-MESDC2 (SEME) and MESDC2-SENP1 (MESE) fusion genes are transcribed in tumor-derived cells and their open reading frames encode aberrant proteins. As a consequence of this, and in contrast to wild-type (WT) MESDC2, the translocation-associated SEME protein is no longer targeted to the endoplasmatic reticulum, leading to a presumed loss-of-function as a chaperone for the <em>WNT</em> co-receptors LRP5 and/or LRP6. Ultimately, this might lead to abnormal development and/or routing of germ cell tumor precursor cells. SUMO, a post-translational modifier, plays an important role in several cellular key processes and is cleaved from its substrates by WT SENP1. Using a PML desumoylation assay, we found that translocation-associated MESE proteins exhibit desumoylation capacities similar to those observed for WT SENP1. We speculate that spatio-temporal disturbances in desumoylating activities during critical stages of embryonic development might have predisposed the patient. Together, the constitutional t(<em>12</em>;15)(q13;q25) translocation revealed two novel candidate genes for neonatal/infantile GCT development: MESDC2 and SENP1.

Colorectal traditional serrated adenomas (TSAs) are often associated with precursor polyps, including hyperplastic polyps and sessile serrated adenoma/polyps. To elucidate the molecular mechanisms involved in the progression from precursor polyps to TSAs, the present study analyzed 15 precursor polyp-associated TSAs harboring <em>WNT</em> pathway gene mutations. Laser microdissection-based sequencing analysis showed that BRAF or KRAS mutations were shared between TSA and precursor polyps in all lesions. In contrast, the statuses of <em>WNT</em> pathway gene mutations were different between the 2 components. In 8 lesions, RNF43, APC, or CTNNB1 mutations, were exclusively present in TSA. RNF43 mutations were shared between the TSA and precursor components in 3 lesions; however, they were heterozygous in the precursor polyps whereas homozygous in the TSA. In 4 lesions with PTPRK-RSPO3 fusions, RNA in situ hybridization demonstrated that overexpression of RSPO3, reflecting PTPRK-RSPO3 fusion transcripts, was restricted to TSA components. Consistent with the results of the genetic and in situ hybridization analyses, nuclear β-catenin accumulation and MYC overexpression were restricted to the TSA component in 13 and <em>12</em> lesions, respectively. These findings indicate that the <em>WNT</em> pathway gene alterations are acquired during the progression from the precursor polyps to TSAs and that the activation of the <em>WNT</em> pathway plays a critical role in the development of TSA rather than their progression to high-grade lesions.

CWP232291 (CWP291) is a small-molecule inhibitor of <em>Wnt</em> signaling that causes degradation of β-catenin via apoptosis induction through endoplasmic reticulum stress activation. This first-in-human, open-label, dose-escalation study to evaluate the safety, maximum tolerated dose (MTD), and preliminary efficacy of CWP291 enrolled 69 patients with hematologic malignancies (acute myeloid leukemia [AML], n = 64; myelodysplastic syndrome, n = 5) in 15 dose-escalation cohorts of 4 to 334 mg/m2 using a modified 3+3 design and 1 dose-expansion cohort. CWP291 was administered IV daily for 7 days every 21 days. The most common treatment-emergent adverse events (TEAEs) were nausea (n = 44, 64%), vomiting (n = 32, 46%), diarrhea (n = 25, 36%), and infusion-related reactions (n = 20, 29%). Grade ≥3 TEAEs in >3 patients (5%) were pneumonia (n = 8, <em>12</em>%); hypophosphatemia (n = 6, 8%); leukocytosis, nausea, cellulitis, sepsis, and hypokalemia (n = 5 each, 7% each); and hypertension (n = 4, 6%). Dose-limiting toxicities included nausea (n = 3) and abdominal pain, anaphylactic reaction, myalgia, and rash (n = 1, each); the MTD was defined at 257 mg/m2. CWP232204, the active metabolite of CWP291, showed pharmacokinetic linearity on both days 1 and 7, and a terminal half-life of ∼<em>12</em> hours. Among 54 response-evaluable AML patients, there was one complete response at a dose of 153 mg/m2 and one partial response at 198 mg/m2; bone marrow blast percentage reduced from a median of 58.3% to 3.5% and 15.0% to 4.2%, respectively. Future studies will explore CWP291, with a mechanism of action aimed at eradication of earlier progenitors via <em>Wnt</em> pathway blockade, as combination therapy. This trial was registered at www.clinicaltrials.gov as #NCT01398462.

Genomic alterations of small bowel cancers remain poorly understood due to the rarity of these diseases. In the current study, the authors report the identification of somatic mutations from patients with duodenal adenocarcinoma by whole-exome sequencing.

Whole-exome sequencing and follow-up analysis were conducted in <em>12</em> matched tumor-normal tissue duodenal adenocarcinoma tissue pairs to examine the genetic characteristics of this disease. Somatic mutations (single-nucleotide variants and short insertion/deletions) were obtained and filtered and then searched for recurrently mutated genes and pathways.

An excess of C-to-T transitions at the CpG dinucleotide was observed in the substitution of bases. The authors identified recurrent mutations in tumor protein p53 (TP53), KRAS, catenin (cadherin-associated protein) β-1 (CTNNB1), AT-rich interactive domain 2 (ARID2), adenomatous polyposis coli (APC), erb-b2 receptor tyrosine kinase 2 (ERBB2), ARID1A, cadherin-related family member 1 (CDHR1), NRAS, Bcl-2-related ovarian killer (BOK), radial spoke head 14 homolog (chlamydomonas) (RTDR1), cell division cycle 27 (CDC27), catalytic subunit of phosphoinositide-3-kinase (PIK3CA), and SMAD family member 4 (SMAD4). Pathway scan indicated that the Wnt signaling pathway, regulation of the actin cytoskeleton pathway, ErbB signaling pathway, and the pathway of focal adhesion were the most extensively affected pathways.

This genomic characterization of duodenal adenocarcinoma provides researchers with insight into its somatic landscape and highlights the vital role of the Wnt/β-catenin signaling pathway. The study data also indicate that duodenal adenocarcinomas have a genetic resemblance to gastric and colorectal cancers. These discoveries may benefit the future development of molecular diagnosis and personalized therapies. Cancer 2016;<em>12</em>2:1689-96. © 2016 American Cancer Society.

The majority of colon tumors are driven by aberrant <em>Wnt</em> signaling in intestinal stem cells, which mediates an efficient route toward initiating intestinal cancer. Natural lipophilic polyphenols and long-chain polyunsaturated fatty acids (PUFAs) generally suppress <em>Wnt</em>- and NF-κB- (nuclear factor-κ light-chain enhancer of activated B-cell) related pathways. However, the effects of these extrinsic agents on colonic leucine-rich repeat-containing G-protein-coupled receptor 5-positive (Lgr5+) stem cells, the cells of origin of colon cancer, have not been documented to date. Therefore, we examined the effect of n-3 PUFA and polyphenol (curcumin) combination on Lgr5+ stem cells during tumor initiation and progression in the colon compared with an n-6 PUFA-enriched control diet. Lgr5-EGFP-IRES-creERT2 knock-in mice were fed diets containing n-6 PUFA (control), n-3 PUFA, n-6 PUFA+curcumin or n-3 PUFA+curcumin for 3 weeks, followed by 6 azoxymethane (AOM) injections, and terminated 17 weeks after the last injection. To further elucidate the effects of the dietary bioactives at the tumor initiation stage, Lgr5+ stem cells were also assessed at <em>12</em> and 24 h post AOM injection. Only n-3 PUFA+curcumin feeding reduced nuclear β-catenin in aberrant crypt foci (by threefold) compared with control at the progression time point. n-3 PUFA+curcumin synergistically increased targeted apoptosis in DNA-damaged Lgr5+ stem cells by 4.5-fold compared with control at <em>12</em> h and maximally reduced damaged Lgr5+ stem cells at 24 h, down to the level observed in saline-treated mice. Finally, RNAseq analysis indicated that p53 signaling in Lgr5+ stem cells from mice exposed to AOM was uniquely upregulated only following n-3 PUFA+curcumin cotreatment. These novel findings demonstrate that Lgr5+ stem cells are uniquely responsive to external dietary cues following the induction of DNA damage, providing a therapeutic strategy for eliminating damaged Lgr5+ stem cells to reduce colon cancer initiation.

Electrical excitability by membrane depolarization is crucial for survival and maturation of newborn cells in the dentate gyrus of the hippocampus. However, traditional technology for membrane depolarization lacks temporal and spatial precision. Optogenetics can be used to activate channelrhodopsin-2 (ChR2), allowing cationic current to depolarize genetically targeted cells. In this study, we used ChR2-EGFP driven by doublecortin (DCX) to promote survival and maturation of newborn cells in the dentate gyrus after traumatic brain injury (TBI). C57BL/6 mice underwent lateral fluid percussion TBI. TBI mice were transfected with a lentivirus carrying the DCX-ChR2-EGFP gene. We observed that not only immature neurons but also type-2b intermediate progenitor (IPs) and neuroblasts expressed DCX-EGFP, indicating that DCX-expressing newborn cells could provide a long time window for electrical activity regulation. Quantitative results showed that the number of EGFP-expressing cells began to rise at 3 days after TBI and peaked at 9 days after TBI. By optical depolarization of DCX-EGFP-expressing cells between 3 and <em>12</em> days, we observed significantly improved cognitive deficits after TBI with enhanced survival and maturation of newborn cells in the dentate gyrus. We also investigated the role of optical depolarization in neural stem cells transfected with a lentivirus carrying the ChR2-DCX-EGFP gene in vitro. By administrating verapamil to block L-type calcium channels, we verified that the up-regulation of MAP2, NeuN, Neurog2, NeuroD1 and GluR2 in newborn cells was mediated by ChR2-elicted depolarization. By using β-catenin inhibitor Dkk1, we demonstrated that optical depolarization of DCX-EGFP-expressing cells facilitated survival and maturation probably through the <em>Wnt</em>/β-catenin signaling cascade.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system characterized by myelin loss and neuronal dysfunction. Although the majority of patients do not present familial aggregation, Mendelian forms have been described. We performed whole-exome sequencing analysis in 132 patients from 34 multi-incident families, which nominated likely pathogenic variants for MS in <em>12</em> genes of the innate immune system that regulate the transcription and activation of inflammatory mediators. Rare missense or nonsense variants were identified in genes of the fibrinolysis and complement pathways (PLAU, MASP1, C2), inflammasome assembly (NLRP<em>12</em>), <em>Wnt</em> signaling (UBR2, CTNNA3, NFATC2, RNF213), nuclear receptor complexes (NCOA3), and cation channels and exchangers (KCNG4, SLC24A6, SLC8B1). These genes suggest a disruption of interconnected immunological and pro-inflammatory pathways as the initial event in the pathophysiology of familial MS, and provide the molecular and biological rationale for the chronic inflammation, demyelination and neurodegeneration observed in MS patients.

Estrogen deficiency, which mainly occurs in postmenopausal women, is a primary reason for osteoporosis in clinical diagnosis. However, the molecular regulation of osteoporosis in menopausal females is still not adequately explained in the literature, with the diagnosis and treatment for osteoporosis being limited. Herein, exosomal microRNAs (miRNAs) were used to evaluate their diagnosis and prediction effects in menopausal females with osteoporosis. In this study, 6 menopausal females without osteoporosis and <em>12</em> menopausal females with osteoporosis were enrolled. The serum exosomes were isolated, and the miRNA expression was detected by miRNA high-throughput sequencing. Exosomal miRNA effects were analyzed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. The miRNA-targeted genes were evaluated by Targetscan 7.2 and the protein-protein interactions (PPI) by STRING. Hub genes were analyzed by the CytoHubba app of Cytoscape. The results showed that 191 aberrant miRNAs were found in the group of menopausal females with osteoporosis, including 72 upregulated miRNAs and <em>12</em>1 downregulated miRNAs. Aberrant miRNAs were involved in many signaling pathways, such as the <em>Wnt</em>, MAPK, and Hippo pathways. Based on PPI network analysis, FBXL3, FBXL13, COPS2, UBE2D3, DCUN1D1, DCUN1D4, CUL3, FBXO22, ASB6, and COMMD2 were the 10 most notable genes in the PPI network. In conclusion, aberrant serum exosomal miRNAs were associated with an altered risk of osteoporosis in menopausal females and may act as potential biomarkers for the prediction of risk of osteoporosis in menopausal females.

Keywords: biomarker; diagnosis; exosome; osteoporosis.

To investigate the mitigation effect and mechanism of oestrogen and PTH on disc degeneration in rats after ovariectomy, as well as on <em>Wnt</em>/β-catenin pathway activity, thirty 3-month-old rats were ovariectomized and divided into three groups. Ten additional rats were used as controls. Eight weeks later, the rats were administered oestrogen or PTH for <em>12</em> weeks, and then discs were collected for tests. Results showed that nucleus pulposus cells in the Sham group were mostly notochord cells, while in the OVX group, cells gradually developed into chondrocyte-like cells. Oestrogen or PTH could partly recover the notochord cell number. After ovariectomy, the endplate roughened and endplate porosity decreased. After oestrogen or PTH treatment, the smoothness and porosity of endplate recovered. Compared with the Sham group, Aggrecan, Col2a and <em>Wnt</em>/β-catenin pathway expression in OVX group decreased, and either oestrogen or PTH treatment improved their expression. The biomechanical properties of intervertebral disc significantly changed after ovariectomy, and oestrogen or PTH treatment partly recovered them. Disc degeneration occurred with low oestrogen, and the underlying mechanisms involve nutrition supply disorders, cell type changes and decreased <em>Wnt</em>/β-catenin pathway activity. Oestrogen and PTH can retard disc degeneration in OVX rats and enhance <em>Wnt</em>/β-catenin pathway activity in nucleus pulposus.

Neuroblastoma (NB) is a childhood solid malignant tumor originating from precursor cells of the peripheral nervous system. We have previously established a risk classification system based on DNA copy number profiles. To further explore the pathogenesis of NBs in distinct risk groups, we performed whole-exome sequencing analysis of 57 primary and 7 recurrent/metastatic tumors with unique chromosomal aberration profiles as categorized by our genomic sub-grouping system. Overall, a low frequency of somatic mutations was found. Besides ALK (4/64, 6.3%), SEMA6C, SLIT1 and NRAS, genes involved in the axon guidance pathway, were identified as recurrently mutated in 6 of 64 tumors (9.4%). Pathway enrichment analysis revealed enrichment of 25 mutated genes in the mitogen-activated protein kinase (MAPK) pathway, 13 genes in the <em>Wnt</em> pathway, and <em>12</em> genes in the axon guidance pathway. Genomic analyses demonstrated that primary and matched recurrent or metastatic tumors obtained from sporadic and monozygotic twin NBs were clonally related with variable extents of genetic heterogeneity. Monozygotic twin NBs displayed different evolutionary trajectories. These results indicate the involvement of the axon guidance, MAPK and <em>Wnt</em> pathways in NB and demonstrate genomic diversity with NB progression.

Gamma-tocotrienol (GT3), a naturally occurring vitamin E isomer, a promising radioprotector, has been shown to protect mice against radiation-induced hematopoietic and gastrointestinal injuries. We analyzed changes in protein expression profiles of spleen tissue after GT3 treatment in mice exposed to gamma radiation to gain insights into the molecular mechanism of radioprotective efficacy. Male CD2F1 mice, <em>12</em>-to-14 weeks old, were treated with either vehicle or GT3 at 24 h prior to 7 Gy total-body irradiation. Nonirradiated vehicle, nonirradiated GT3 and age-matched naïve animals were used as controls. Blood and tissues were harvested on days 0, 1, 2, 4, 7, 10 and 14 postirradiation. High-resolution mass-spectrometry-based radioproteomics was used to identify differentially expressed proteins in spleen tissue with or without drug treatment. Subsequent bioinformatic analyses helped delineate molecular markers of biological pathways and networks regulating the cellular radiation responses in spleen. Our results show a robust alteration in spleen proteomic profiles including upregulation of the <em>Wnt</em> signaling pathway and actin-cytoskeleton linked proteins in mediating the radiation injury response in spleen. Furthermore, we show that 24 h pretreatment with GT3 attenuates radiation-induced hematopoietic injury in the spleen by modulating various cell signaling proteins. Taken together, our results show that the radioprotective effects of GT3 are mediated, via alleviation of radiation-induced alterations in biochemical pathways, with wide implications on overall hematopoietic injury.

<AbstractText>The positive effects of denosumab (DMAb) on bone mineral density (BMD) are quickly reversible after its discontinuation. We investigated whether this rebound was associated with dysregulation of the <em>Wnt</em> canonical pathway and/or by the increase in the receptor-activator of nuclear factor-kappa B ligand (RANKL) serum levels.</AbstractText><AbstractText>The study included patients (n = 15) with postmenopausal osteoporosis to whom DMAb was administered for 78 months and then discontinued. We collected BMD data at baseline/month 0 (M0), M60, M84 (6 months after last DMAb administration, coinciding when the next DMAb dose would typically be due), and after 3 and <em>12</em> months of follow-up (FU-M3 and FU-M<em>12</em>, respectively). Serum C-terminal telopeptide of type 1 collagen (CTX-I), Dickkopf-1 (Dkk-1), and sclerostin were measured at M0, M60, M84, FU-M3, and FU-M<em>12</em>. Serum N-terminal propeptide of type 1 procollagen (PINP) and RANKL were dosed at M60, M84, FU-M3, and FU-<em>12</em>.</AbstractText><AbstractText>We found a significant decrease in the T-score at all sites at FU-M<em>12</em>, when compared to M84 (-0.51 ± 0.91 at the lumbar spine; -0.72 ± 0.33 at the total hip; and -0.42 ± 0.27 at the femoral neck, p < 0.05). After DMAb discontinuation (M84 vs FU M<em>12</em>) CTX-I, PINP increased already at FU-M3 (+0.921 ± 0.482 ng/mL, +<em>12</em>6.60 ± 30.36 ng/mL, respectively, p < 0.01), RANKL increased at FU-M<em>12</em> (+0.041 ± 0.062 ng/mL, p < 0.05), while Dkk-1 and sclerostin decreased at FU-M<em>12</em> (-10.90 ± 11.80 and - 13.00 ± 10.52 pmol/L, respectively, p < 0.01). No changes in BMD or any of the markers were found between M60 and M84.</AbstractText><AbstractText>RANKL serum levels progressively increased after discontinuation of long-term DMAb while Dkk-1 and sclerostin serum levels decreased. The increase in RANKL serum levels supports the hypothesis of a sudden loss of inhibition of the resting osteoclast line after DMAb clearance, with a hyperactivation of these cells. Our results suggest that the changes in serum <em>Wnt</em> inhibitors after DMAb suspension might represent a mere feedback response to the increased bone turnover.</AbstractText>

This study evaluated the effects of deficient IGF-I expression in osteocytes on fracture healing. Transgenic mice with conditional knockout (cKO) of Igf1 in osteocytes were generated by crossing Dmp1-Cre mice with Igf1 flox mice. Fractures were created on the mid-shaft of tibia of <em>12</em>-week-old male cKO mice and wild-type (WT) littermates by three-point bending. At 21 and 28days post-fracture healing, the increases in cortical bone mineral density, mineral content, bone area, and thickness, as well as sub-cortical bone mineral content at the fracture site were each greater in cKO calluses than in WT calluses. There were 85% decrease in the cartilage area and >2-fold increase in the number of osteoclasts in cKO calluses at 14days post-fracture, suggesting a more rapid remodeling of endochondral bone. The upregulation of mRNA levels of osteoblast marker genes (cbfa1, alp, Opn, and Ocn) was greater in cKO calluses than in WT calluses. μ-CT analysis suggested an accelerated bony union of the fracture gap in cKO mice. The Sost mRNA level was reduced by 50% and the Bmp2 mRNA level was increased 3-fold in cKO fractures at 14days post-fracture, but the levels of these two mRNAs in WT fractures were unchanged, suggesting that the accelerated fracture repair may in part act through the <em>Wnt</em> and/or BMP signaling. In conclusion, conditional deletion of Igf1 in osteocytes not only did not impair, but unexpectedly enhanced, bony union of the fracture gap. The accelerated bony union was due in part to upregulation of the <em>Wnt</em> and BMP2 signaling in response to deficient osteocyte-derived IGF-I expression, which in turn favors intramembranous over endochondral bone repair.

Photobiomodulation therapy (PBMT) has been demonstrated as regulating osteoblast proliferation. MicroRNAs (miRNAs) are involved in various pathophysiologic processes in osteoblast, but the role of miRNAs in the PBMT-based promotion of osteoblast proliferation remains unclear. This study aimed to investigate the effects of PBMT treatment (3.75 J/cm2) on mouse pre-osteoblast cell line MC3T3-E1 proliferation and apoptosis via the miR-503/<em>Wnt</em>3a pathway; meanwhile, detect the expressions of miR-503 and <em>Wnt</em>3a after PBMT treatment and the role of miR-503 in regulating <em>Wnt</em> signaling molecules <em>Wnt</em>3a, β-catenin, Runx2, apoptotic proteins caspase-3, and Bcl-2 in vitro. The PBMT parameters were as follows: 808 nm continuous wavelength, 0.401 W output power, 0.042 W/cm2 power density, 9.6 cm2 spot size, 36 J energy, 3.75 J/cm2 energy density, 90 s irradiation for three times per <em>12</em> h, 14.5 cm distance of the laser source and the angle of divergence of the laser beam was 7°. In our present study, the target relationship was predicted and verified by bioinformatics analysis and luciferase reporter assays. Gene mRNA and protein expressions were examined by qPCR and western blot analysis. The MTT method was used to evaluate the effect of miR-503 on MC3T3-E1 cells proliferation. And cell apoptosis was examined by flow cytometry. The results showed that PBMT treatment reduced the expression of miR-503 and increased the level of <em>Wnt</em>3a (p < 0.01). Bioinformatics analysis and luciferase reporter assays revealed that <em>Wnt</em>3a was a target of miR-503, and <em>Wnt</em>3a was regulated by miR-503. Furthermore, miR-503 was found to functionally inhibit proliferation and promote apoptosis (p < 0.01). And during this process, <em>Wnt</em>3a, β-catenin, Runx2, and Bcl-2 expressions were significantly inhibited (p < 0.01); however, caspase-3 level was upregulated (p < 0.01). These results suggest that miR-503 plays a role in osteoblast proliferation and apoptosis in response to PBMT, which is potentially amenable to therapeutic manipulation for clinical application.

Patients with anterior cruciate ligament (ACL) rupture are two times as likely to develop posttraumatic osteoarthritis (PTOA). Annually, there are ∼900,000 knee injuries in the United States, which account for ∼<em>12</em>% of all osteoarthritis (OA) cases. PTOA leads to reduced physical activity, deconditioning of the musculoskeletal system, and in severe cases requires joint replacement to restore function. Therefore, treatments that would prevent cartilage degradation post-injury would provide attractive alternatives to surgery. Sclerostin (Sost), a <em>Wnt</em> antagonist and a potent negative regulator of bone formation, has recently been implicated in regulating chondrocyte function in OA. To determine whether elevated levels of Sost play a protective role in PTOA, we examined the progression of OA using a noninvasive tibial compression overload model in SOST transgenic (SOSTTG ) and knockout (Sost-/- ) mice. Here we report that SOSTTG mice develop moderate OA and display significantly less advanced PTOA phenotype at 16 weeks post-injury compared with wild-type (WT) controls and Sost-/- . In addition, SOSTTG built ∼50% and ∼65% less osteophyte volume than WT and Sost-/- , respectively. Quantification of metalloproteinase (MMP) activity showed that SOSTTG had ∼2-fold less MMP activation than WT or Sost-/- , and this was supported by a significant reduction in MMP2/3 protein levels, suggesting that elevated levels of SOST inhibit the activity of proteolytic enzymes known to degrade articular cartilage matrix. Furthermore, intra-articular administration of recombinant Sost protein, immediately post-injury, also significantly decreased MMP activity levels relative to PBS-treated controls, and Sost activation in response to injury was TNFα and NF-κB dependent. These results provide in vivo evidence that sclerostin functions as a protective molecule immediately after joint injury to prevent cartilage degradation. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

Various populations of cancer stem cells (CSCs) have been identified in hepatocellular carcinoma (HCC). <em>Wnt</em> signaling is variably activated in HCC and regulates CSCs and tumorigenesis. We explored cell-to-cell <em>Wnt</em> and stemness heterogeneity in HCC by labeling freshly isolated cancer cells with a <em>Wnt</em>-specific reporter, thereby identifying a small subset (0.4%-8.9%) of <em>Wnt</em>-activity^high cells. Further cellular subset analysis identified a refined subset of <em>Wnt</em>-activity^highALDH1⁺EpCAM⁺ triple-positive (TP) cells as the most stem-like, phenotypically plastic, and tumorigenic among all putative CSC populations. These TP "superpotent CSCs" (spCSCs) specifically upregulate the expression of dishevelled 1 (DVL1) through the antagonism between abnormal spindle-like microcephaly-associated (ASPM) and the ubiquitin ligase complex Cullin-3/KLHL-<em>12</em>. Subsequent functional and molecular studies revealed the role of DVL1 in controlling spCSCs and their tumorigenic potential. These findings provide the mechanistic basis of the <em>Wnt</em> and stemness heterogeneity in HCC and highlight the important role of DVL1^high spCSCs in tumor progression.

Polycystic ovarian syndrome (PCOS) is a common endocrinologic disorder in women of reproductive age characterized by polycystic ovaries, oligo/anovulation, and hyperandrogenism. Not only anovulation but also endometrial dysfunction can reduce fertility in PCOS patients. <em>Wnt</em> pathway is responsible for endometrial proliferation which be strongly regulated by estradiol. To determine the effects of clomiphene citrate (CC) and letrozole, we measured the expression of some main ligands of <em>Wnt</em>/β-catenin signaling including <em>Wnt</em>7a, <em>Wnt</em>3, and <em>Wnt</em>8b in the endometrial samples taken from PCOS women on day <em>12</em> of the menses who received 100 mg CC or 5 mg letrozole as well as from women without treatment. Significantly, the mean estrogen and progesterone concentration were lower and higher, respectively, in letrozole than CC. The mean endometrial thickness (ET) was significantly greater in letrozole compared to CC. Assessment of the mRNA and protein expression of <em>Wnt</em>7a, <em>Wnt</em>3, and <em>Wnt</em>8b showed significantly lower expression in CC than the letrozole and control groups. Collectively, letrozole provided a better molecular response in the endometrium of PCOS patients during the proliferative phase, similar to natural cycles, compared to CC. CC decreased the ligands expression of <em>Wnt</em>3, <em>Wnt</em>7a, and <em>Wnt</em>8b, resulting in endometrial dysfunction.

The cancer stem cell marker, EpCAM, is an important indicator of <em>Wnt</em>/β-catenin signaling activation and a functional component of hepatocellular tumor-initiating cells. A high-throughput screening assay was developed to identify inhibitors of EpCAM-dependent growth of hepatocellular carcinoma (HCC) cells. EpCAM(+) and EpCAM(-) HCC cell lines were assessed for differential sensitivity to a <em>Wnt</em>/β-catenin pathway inhibitor. Libraries comprising 22 668 pure compounds and 107 741 crude or partially purified natural product extracts were tested, and <em>12</em> pure compounds and 67 natural product extracts were identified for further study. Three active compounds and the positive control were further characterized in terms of effects on EpCAM expression. Treatment of EpCAM(+) Hep3B cells resulted in loss of EpCAM expression as assessed by flow cytometry. This reduction was incomplete (most cells continued to express EpCAM), but resulted in generation of cell populations expressing lower levels of EpCAM. Sublethal concentrations (~IC50 ) reduced median EpCAM expression to 28% of control after 1 day and 19% of control after 2 days. Reduction in EpCAM expression preceded growth inhibition suggesting that a threshold of EpCAM expression may be required for growth of EpCAM-dependent cells. The identification of compounds with a variety of possible molecular targets suggests a likelihood of multiple mechanisms for modulation of EpCAM-dependent cell growth.

Tumor Necrosis Factor (TNF)-α and Interleukin (IL)-6 play a fundamental role in bone loss in rheumatoid arthritis (RA), partly due to the inhibition of the <em>Wnt</em> canonical pathway. The aim of our study was to investigate the short-term effects of three different treatments on <em>Wnt</em> inhibitors (Dkk-1 and sclerostin) and on bone turnover markers (BTMs): N-propeptide of type I collagen (PINP) and C-terminal telopeptide of type I collagen (β-CTX-I). We performed a retrospective analysis of prospectively collected data. We enrolled women affected by early RA (< <em>12</em> months) with active disease (DAS28 ≥ 2.6) despite a 6-month treatment with methotrexate (10-15 mg/week), who then started certolizumab pegol, tocilizumab, or methyl-prednisolone (8 mg/daily). Patients were divided into three groups according to the treatment. Blood samples were collected at baseline, week 1, and week 4. We selected 14 patients treated with certolizumab pegol, 14 patients with tocilizumab, and 20 patients with methyl-prednisolone. No difference between any of the tested parameters was found at baseline. β-CTX-I, Dkk-1, and sclerostin decreased after 1 week of treatment with certolizumab pegol (- 27% ± 21.5, - 50% ± 13.2, and - 30% ± 30.4, respectively, p < 0.05). Methyl-prednisolone induced similar changes, albeit less marked, on β-CTX-I and <em>Wnt</em> inhibitors, with a decrease in PINP (- 16.1% ± 16.5, p < 0.05). Tocilizumab did not significantly affect BTMs or <em>Wnt</em> inhibitors. No significant changes were found for PTH and 25OHD. In the first four weeks of treatment, TNFα inhibition showed strong effects on BTMs and <em>Wnt</em> inhibitors, differently from IL-6 blockade. Glucocorticoids induced similar changes; nonetheless, they showed undesired effects on bone formation.

Colorectal cancer is a common cancer with high mortality rate. Despite being the standard anti-colorectal cancer drug, 5-fluorouracil (5-FU) exhibits only limited therapeutic benefits. Herein, we investigated whether paricalcitol, a synthetic vitamin D analogue with potential antitumor properties, would enhance the chemopreventive efficacy of 5-FU on an intermediate-term (15 weeks) model of colorectal tumors induced by azoxymethane (AOM) in rats. After AOM injection, 5-FU was administered during the 9th and 10th weeks (<em>12</em> mg/kg/day for 4 days, then 6 mg/kg every other day for another 4 doses), whereas paricalcitol (2.5 μg/kg/day; 3 days/week) was given from the 7th to the 15th week. At week 15, the animals were euthanized and their resected colons were examined macroscopically and microscopically. Quantitative RT-PCR was used to measure the transcription activities of <em>Wnt</em>, β-catenin, DKK-1, CDNK-1A, NF-κB, and COX-2 genes, and ELISA was used to quantify the protein levels of β-catenin, COX-2, HSP90, and VEGF. IHC was additionally used to measure β-catenin, HSP90, and inducible nitric oxide synthase (iNOS). Compared with their individual therapy, combination of 5-FU and paricalcitol showed more significant reducing effect on numbers of grown tumors and large aberrant crypts foci. Mechanistically, paricalcitol and 5-FU had cooperated together to repress the expression of procancerous <em>Wnt</em>, β-catenin, NF-κB, COX-2, iNOS, VEGF, and HSP-90 more, and to upregulate the expression of antitumorigenesis DKK-1 and CDNK-1A, compared with their monotherapies. Our findings suggest that combined use of paricalcitol with 5-FU exhibits an augmenting chemopreventive effect against colorectal tumors, and might potentially be useful for chemoprevention in colorectal cancer patients. Cancer Prev Res; 9(6); 491-501. ©2016 AACR.

Epidermal homeostasis relies on a well-defined transcriptional control of keratinocyte proliferation and differentiation, which is critical to prevent skin diseases such as atopic dermatitis, psoriasis or cancer. We have recently shown that the homeobox transcription factor DLX3 and the tumor suppressor p53 co-regulate cell cycle-related signaling and that this mechanism is functionally involved in cutaneous squamous cell carcinoma development. Here we show that DLX3 expression and its downstream signaling depend on protein kinase C α (PKCα) activity in skin. We found that following <em>12</em>-O-tetradecanoyl-phorbol-13-acetate (TPA) topical treatment, DLX3 expression is significantly upregulated in the epidermis and keratinocytes from mice overexpressing PKCα by transgenic targeting (K5-PKCα), resulting in cell cycle block and terminal differentiation. Epidermis lacking DLX3 (DLX3cKO), which is linked to the development of a DLX3-dependent epidermal hyperplasia with hyperkeratosis and dermal leukocyte recruitment, displays enhanced PKCα activation, suggesting a feedback regulation of DLX3 and PKCα. Of particular significance, transcriptional activation of epidermal barrier, antimicrobial peptide and cytokine genes is significantly increased in DLX3cKO skin and further increased by TPA-dependent PKC activation. Furthermore, when inhibiting PKC activity, we show that epidermal thickness, keratinocyte proliferation and inflammatory cell infiltration are reduced and the PKC-DLX3-dependent gene expression signature is normalized. Independently of PKC, DLX3 expression specifically modulates regulatory networks such as <em>Wnt</em> signaling, phosphatase activity and cell adhesion. Chromatin immunoprecipitation sequencing analysis of primary suprabasal keratinocytes showed binding of DLX3 to the proximal promoter regions of genes associated with cell cycle regulation, and of structural proteins and transcription factors involved in epidermal differentiation. These results indicate that Dlx3 potentially regulates a set of crucial genes necessary during the epidermal differentiation process. Altogether, we demonstrate the existence of a robust DLX3-PKCα signaling pathway in keratinocytes that is crucial to epidermal differentiation control and cutaneous homeostasis.