A conserved ZFX/WNT3 axis modulates the growth and imatinib response of chronic myeloid leukemia stem/progenitor cells.

BACKGROUND: Zinc finger protein X-linked (ZFX) has been shown to promote the growth of tumor cells, including leukemic cells. However, the role of ZFX in the growth and drug response of chronic myeloid leukemia (CML) stem/progenitor cells remains unclear. METHODS: Real-time quantitative PCR (RT-qPCR) and immunofluorescence were used to analyze the expression of ZFX and WNT3 in CML CD34+ cells compared with normal control cells. Short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats/dead CRISPR-associated protein 9 (CRISPR/dCas9) technologies were used to study the role of ZFX in growth and drug response of CML cells. Microarray data were generated to compare ZFX-silenced CML CD34+ cells with their controls. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were performed to study the molecular mechanisms of ZFX to regulate WNT3 expression. RT-qPCR and western blotting were used to study the effect of ZFX on ß-catenin signaling. RESULTS: We showed that ZFX expression was significantly higher in CML CD34+ cells than in control cells. Overexpression and gene silencing experiments indicated that ZFX promoted the in vitro growth of CML cells, conferred imatinib mesylate (IM) resistance to these cells, and enhanced BCR/ABL-induced malignant transformation. Microarray data and subsequent validation revealed that WNT3 transcription was conservatively regulated by ZFX. WNT3 was highly expressed in CML CD34+ cells, and WNT3 regulated the growth and IM response of these cells similarly to ZFX. Moreover, WNT3 overexpression partially rescued ZFX silencing-induced growth inhibition and IM hypersensitivity. ZFX silencing decreased WNT3/ß-catenin signaling, including c-MYC and CCND1 expression. CONCLUSION: The present study identified a novel ZFX/WNT3 axis that modulates the growth and IM response of CML stem/progenitor cells.

Impact of genetic variations in the WNT family members and RUNX2 on dental and skeletal maturation: a cross-sectional study.

BACKGROUND: This study evaluated if genetic variations in the WNT family members and RUNX2 are associated with craniofacial maturation, investigating dental and skeletal maturity in children and teenagers. METHODS: Radiographs from pre-orthodontic treatment of Brazilian patients (7 to 17 years-old) were used to assess dental (panoramic radiographs) and skeletal maturity (cephalometric radiographs). The chronological age (CA) was calculated based on the date of birth and the time the radiographs were performed. For the dental maturity analysis, the Demirjian (1973) method was used and a delta [dental age - chronological age (DA-CA)] was calculated. For the skeletal maturity analysis, the Baccetti et al. (2005) method was used and the patients were classified as "delayed skeletal maturation", "advanced skeletal maturation" or "normal skeletal maturation". DNA isolated from buccal cells was used for genotyping of two genetic variations in WNT family genes: rs708111 (G > A) in WNT3A and rs1533767 (G > A) in WNT11; and two genetic variations in RUNX2: rs1200425 (G > A) and rs59983488 (G > T). A statistical analysis was performed and values of p < 0.05 indicated a significant difference. RESULTS: There were no associations between dental maturity and genotypes (p > 0.05). In the skeletal maturity analysis, the allele A in the rs708111 (WNT3A) was statistically more frequent in patients with delayed skeletal maturation (Prevalence Ratio = 1.6; 95% Confidence Interval = 1.00 to 2.54; p-value = 0.042). CONCLUSIONS: The rs708111 in the WNT3A gene impacts on skeletal maturation.

(Pro)renin receptor promotes colorectal cancer progression through inhibiting the NEDD4L-mediated Wnt3 ubiquitination and modulating gut microbiota.

BACKGROUND: We previously found that (pro)renin receptor ((P)RR) augments Wnt3 protein without affecting Wnt3 gene transcription in colorectal cancer (CRC) cells, thus contributes to CRC initiation. The present study aims to investigate whether (P)RR further promotes CRC progression following oncogenesis and the related mechanisms. Notably, we deeply elaborate how (P)RR affects Wnt3 protein level and the key enzyme that mediates this process. METHODS: Immunohistochemistry, western blotting and immunofluorescence were performed to detect protein expression status. A kind of gastrointestinal epithelium-specific ATP6AP2 ((P)RR encoding gene) knock-in mice were generated using Crispr/Cas9 system. RESULTS: We found that increased (P)RR expression in primary CRC lesions is positively associated with higher Wnt3 protein level and disease progression. Progressive CRC presents less colocalization of Wnt3 and an E3 ubiquitin ligase NEDD4L in primary lesions than non-progressive CRC. In colon cancer cells, (P)RR dramatically inhibits the NEDD4L-mediated Wnt3 protein ubiquitination. ATP6AP2 knock-in mice show more diminished Wnt3-NEDD4L colocalization in their gut epithelium in comparison to wildtype mice. They also have abnormal gut bacterial flora distribution. Especially, Lachnospiraceae_NK4A136 and Bacteroides genus, which are generally protective against CRC, are suppressed in guts of ATP6AP2 knock-in mice. CONCLUSIONS: Collectively, (P)RR promotes CRC progression through inhibiting the NEDD4L-mediated Wnt3 ubiquitination and modulating gut microbiota. Video Abstract.

Achyranthoside D attenuates chondrocyte loss and inflammation in osteoarthritis via targeted regulation of Wnt3a.

BACKGROUND: Achyranthes bidentata Blume (A. bidentata) is a common Chinese herb used to treat osteoarthritis (OA). Achyranthoside D (Ach-D) is a glucuronide saponin isolated from A. bidentata. PURPOSE: To assess the mechanisms of action of Ach-D and its effects on OA. METHODS: The effects of Ach-D were evaluated in rats underwent anterior cruciate ligament transection (ACLT) with medial meniscectomy (MMx) and in interleukin (IL)-1ß-induced chondrocytes. Histological changes in rat cartilage tissues were detected using Safranin O-Fast green and haematoxylin-eosin staining. Immunohistochemical staining, qRT-PCR, ELISA, immunoblotting, and immunofluorescence were conducted to examine cartilage degeneration-related and inflammation-related factor expression. CCK-8, LDH assay, and EdU staining were performed to detect chondrocyte death. RESULTS: Ach-D dose-dependently reduced the Osteoarthritis Research Society International (OARSI) scores, alleviated cartilage injury, and decreased the serum concentrations of CTX-II and COMP in ACLT-MMx models. Ach-D increased the expression levels of collagen II and aggrecan and decreased the levels of cartilage degeneration-related proteins, ADAMTS-5, MMP13, and MMP3, in rat cartilage tissues. Additionally, nod-like receptor protein 3 (NLRP3)-related inflammation was reduced by Ach-D, as shown by the significantly inhibited expression levels of NLRP3, ASC, GSDMD, IL-6, TNF-α, IL-1ß, and IL-18 in rat cartilage tissues. In primary rat chondrocytes, Ach-D protected against IL-1ß-induced viability loss and LDH release. Wnt3a is the target protein of Ach-D. Mechanistically, Ach-D alleviated OA by inhibiting Wnt signalling. CONCLUSION: ACH-D may reduce inflammation and cartilage degeneration by inhibiting the Wnt signalling pathway, thereby reducing OA.

LINC01023 Promotes the Hepatoblastoma Tumorigenesis via miR-378a-5p/WNT3 Axis.

Hepatoblastoma is the most common type of hepatic tumors occurring in children between 0 and 5 years. And the exact pathophysiology of the disease is still mysterious. Accumulating studies on LncRNA have shown its pivotal role in the development and progression of distinct human cancers. However, the role of LINC01023 in hepatoblastoma is unknown. The relative expression of LINC01023, miR-378a-5p, and Wnt3 on hepatoblastoma tissue and cell lines was determined by quantitative polymerase chain reaction (qRT-PCR). The effect of LINC01023 downregulation and upregulation on cell proliferation, colony formation and apoptosis activities in HUH6 and HepG2 Cells was assessed by CKK8, clonogenic and flow cytometry analysis, respectively. Dual luciferase, RNA immunoprecipitation (RIP), and RNA pull-down were performed to confirm the interaction between LINC01023 and miR-378a-5p. Similarly, Dual luciferase assay was performed to confirmed the interaction between Wnt3 and miR-378a-5p. The xenograft tumorgenicity test was performed to elucidate the tumorgenicity potential of LINC01023. LINC01023 was significantly upregulated in hepatoblastoma tissue and cell lines rather than in adjacent normal hepatic tissue and QSG7701 cell lines. LINC01023 silencing attenuated cell proliferation, colony formation and increased cell apoptosis. Conversely, LINC01023 upregulation results in significant increase in cell proliferation, and colony formation activities however, a significant reduction in apoptosis activity was reported. Interaction between the LINC01023 and WNT3 was confirmed by dual luciferase assay. Xenograft animal tumorgenicity test confirmed the in-vivo tumorigenesis potential of LINC01203. To the best of our knowledge, this study is the first study demonstrating the role of LINC01023 in hepatoblastoma tumorigenesis through the LINC01023/miR-378a-5p/Wnt3 axis. It could be a potential therapeutic target and a prognostic biomarker in hepatoblastoma.

Chronic GPER activation prompted the proliferation of ileal stem cell in ovariectomized mice depending on Paneth cell-derived Wnt3.

Menopausal women often face long-term estrogen treatment. G protein-coupled estrogen receptor (GPER) expressed in intestinal crypt was activated by estrogen therapy, but it was unclear whether chronic GPER activation during menopause had an effect on intestinal stem cells (ISCs). We tested the effect of chronic GPER activation on ISCs of ovariectomized (OVX) mice by injection of the selective GPER agonist G-1 for 28 days, or G-1 stimulation of organoids derived from crypts of OVX mice. G-1 up-regulated crypt depth, the number of Ki67+, bromodeoxyuridine+ cells and Olfm4+ ISCs, and the expression of ISCs marker genes (Lgr5, Olfm4 and Axin2). G-1 administration promoted organoid growth, increased the number of EdU+ cells per organoid and protein expression of Cyclin D1 and cyclin B1 in organoids. After G-1 treatment in vivo or in vitro, Paneth cell-derived Wnt3, Wnt3 effector ß-catenin and Wnt target genes c-Myc and Cyclin D1 increased in ileum or organoids. Once blocking the secretion of Wnt3 from Paneth cells, the effects of G-1 on organoids growth, ISCs marker genes and Wnt/ß-catenin signaling were abolished. G-1 did not affect the number of Paneth cells in ex vivo organoids, while activated Mmp7/cryptdin program in Paneth cells, promoted their maturation, and increased the expression of lysozyme protein. G-1 pretreatment in OVX mice inhibited radiation-induced ISCs proliferation injury and enhanced the resistance of mice to intestinal injury. In conclusion, chronic GPER activation prompted the Wnt3 synthesis in Paneth cells, thus increased the proliferation of ISCs via activation of Wnt3/ß-catenin signaling in OVX mice.

The aOECs Facilitate the Neuronal Differentiation of Neural Stem Cells in the Inflammatory Microenvironment Through Up-Regulation of Bioactive Factors and Activation of Wnt3/ß-Catenin Pathway.

The therapeutic application of neural stem cells (NSCs) in the central nerve system (CNS) injury is a promising strategy for combating irreversible neuronal loss. However, a variety of obvious inflammatory responses following nerve injury rapidly create an unfavorable microenvironment for survival and neuronal differentiation of NSCs in lesion area, limiting the efficacy of NSC-based therapy for CNS injury. It remained unknown how to effectively increase the neuronal differentiation efficiency of NSCs through transplantation. Here, we demonstrated that curcumin (CCM)-activated olfactory ensheathing cells (aOECs) effectively promoted neuronal differentiation of NSCs in the activated microglial inflammatory condition, and co-transplantation of aOECs and NSCs improved neurological recovery of rats after spinal cord injury (SCI), as evidenced by higher expression levels of neuronal markers and lower expression levels of glial markers in the differentiated cells, greater number of Tuj-1-positive cells as well as higher Basso, Beattie, and Bresnahan (BBB) locomotor scale, compared to the corresponding controls. Pathologically, hematoxylin and eosin (HE) staining and immunostaining also showed that aOECs remarkably enhanced the in vivo neuronal differentiation of NSCs and migration, and nerve repair. Further analysis revealed that the underlying mechanisms of aOECs potentiating the neuronal conversion of NSCs under inflammatory environment were tightly associated with up-regulation of anti-inflammatory cytokines and neurotrophic factors in OECs, and importantly, the activation of Wnt3/ß-catenin pathway was likely involved in the mechanisms underlying the observed cellular events. Therefore, this study provides a promising strategy for SCI repair by co-transplantation of aOECs and NSCs.

Dapagliflozin diminishes memory and cognition impairment in Streptozotocin induced diabetes through its effect on Wnt/ß-Catenin and CREB pathway.

Diabetic neuropathy is a chronic condition that affects a significant number of individuals with diabetes. Streptozotocin injection intraperitoneally to rodents produces pancreatic islet ß-cell destruction causing hyperglycemia, which affect the brain leading to memory and cognition impairment. Dapagliflozin may be able to reverse beta-cell injury and alleviate this impairment. This effect may be via neuroprotective effect or possible involvement of the antioxidant, and anti-apoptotic properties. Forty rats were divided into four groups as follows: The normal control group, STZ-induced diabetes group, STZ-induced diabetic rats followed by treatment with oral dapagliflozin group and normal rats treated with oral dapagliflozin. Behavioral tests (Object location memory task and Morris water maze) were performed. Serum biomarkers (blood glucose and insulin) were measured and then the homeostatic model assessment for insulin resistance (HOMA-IR) was calculated. In the hippocampus the followings were determined; calmodulin, ca-calmodulin kinase Ⅳ (CaMKIV), protein kinase A (PKA) and cAMP-responsive element-binding protein to determine the transcription factor CREB and its signaling pathway also Wnt signaling pathway and related parameters (WnT, B-catenin, lymphoid enhancer binding factor LEF, glycogen synthase kinase 3ß). Moreover, nuclear receptor-related protein-1, acetylcholine and its hydrolyzing enzyme acetylcholine esterase, oxidative stress parameter malondialdehyde (MDA) and apoptotic parameter caspase-3 were determined. STZ was able to cause destruction to pancreatic ß-cells which was reflected on glucose levels causing diabetes. Diabetic neuropathy was clear in the rats performing the behavioral tests. Memory and cognition parameters in the hippocampus were negatively affected. Oxidative stress and apoptotic parameter were elevated while the electrical activity was declined. Dapagliflozin was able to reverse the previously mentioned parameters and behavior. Thus, to say dapagliflozin significantly showed neuroprotective action along with antioxidant, and anti-apoptotic properties.

Transport and Secretion of the Wnt3 Ligand by Motor Neuron-like Cells and Developing Motor Neurons.

The vertebrate neuromuscular junction (NMJ) is formed by a presynaptic motor nerve terminal and a postsynaptic muscle specialization. Cumulative evidence reveals that Wnt ligands secreted by the nerve terminal control crucial steps of NMJ synaptogenesis. For instance, the Wnt3 ligand is expressed by motor neurons at the time of NMJ formation and induces postsynaptic differentiation in recently formed muscle fibers. However, the behavior of presynaptic-derived Wnt ligands at the vertebrate NMJ has not been deeply analyzed. Here, we conducted overexpression experiments to study the expression, distribution, secretion, and function of Wnt3 by transfection of the motor neuron-like NSC-34 cell line and by in ovo electroporation of chick motor neurons. Our findings reveal that Wnt3 is transported along motor axons in vivo following a vesicular-like pattern and reaches the NMJ area. In vitro, we found that endogenous Wnt3 expression increases as the differentiation of NSC-34 cells proceeds. Although NSC-34 cells overexpressing Wnt3 do not modify their morphological differentiation towards a neuronal phenotype, they effectively induce acetylcholine receptor clustering on co-cultured myotubes. These findings support the notion that presynaptic Wnt3 is transported and secreted by motor neurons to induce postsynaptic differentiation in nascent NMJs.

WNT3 hypomethylation counteracts low activity of the Wnt signaling pathway in the placenta of preeclampsia.

Preeclampsia is a hypertensive disorder of pregnancy. Many studies have shown that epigenetic mechanisms may play a role in preeclampsia. Moreover, our previous study indicated that the differentially methylated genes in preeclampsia were enriched in the Wnt/ß-catenin signaling pathway. This study aimed to identify differentially methylated Wnt/ß-catenin signaling pathway genes in the preeclamptic placenta and to study the roles of these genes in trophoblast cells in vitro. Using an Illumina Infinium HumanMethylation 850 K BeadChip, we found that the Wnt signaling pathway was globally hypermethylated in the preeclamptic group compared with the term birth group, but hypomethylated in the preeclamptic group compared with the preterm birth group. Among all Wnt/ß-catenin signaling pathway factors, WNT3 was the most significantly differentially expressed gene and was hypomethylated in the preeclamptic group compared to the nonhypertensive groups, namely, the preterm birth group and term birth group. This result was confirmed by pyrosequencing. Through quantitative real-time PCR and western blot analysis, the WNT3 gene was found to be highly expressed in preeclamptic placental tissues, in contrast to other WNT factors, which were previously reported to be expressed at low levels in placental tissues. Additionally, in the HTR8/SVneo cell line, knockdown of WNT3 suppressed the Wnt/ß-catenin signaling pathway, consistent with the findings for other WNT factors. These results prompted us to speculate that the WNT3 gene counteracts the low activation state of the Wnt signaling pathway in the preeclamptic placenta through methylation modification.

Aralia armata (Wall.) Seem Improves Intimal Hyperplasia after Vascular Injury by Downregulating the Wnt3α/Dvl-1/ß-Catenin Pathway.

The aim of the study is to examine the mechanism of Aralia armata (Wall.) Seem (AAS) in improving intimal hyperplasia after vascular injury in rats. Rats with femoral artery injury were randomly divided into three groups: the model group, AAS low-dose group (40 mg/kg), and AAS high-dose group (80 mg/kg). The sham operation group was used as a control group. HE staining was used to observe the changes in femoral artery vessels. Immunohistochemistry was adopted to detect α-SMA, PCNA, GSK-3ß, and ß-catenin proteins in femoral artery tissue. The CCK-8 test and wound healing assay were employed to analyze the effect of AAS on proliferation and migration of vascular smooth muscle cells (VSMCs) cultured in vitro. Western blotting (WB) and polymerase chain reaction (PCR) assays were used to evaluate the molecular mechanism. AAS reduced the stenosis of blood vessels and the protein expressions of α-SMA, PCNA, GSK-3ß, and ß-catenin compared to the model group. In addition, AAS (0-15 µg/mL) effectively inhibited the proliferation and migration of VSMCs. Moreover, the results of WB and PCR showed that AAS could inhibit the activation of ß-catenin induced by 15% FBS and significantly decrease the expression levels of Wnt3α, Dvl-1, GSK-3ß, ß-catenin, and cyclin D1 in the upstream and downstream of the pathway. AAS could effectively inhibit the proliferation and migration of neointima after vascular injury in rats by regulating the Wnt/ß-catenin signaling pathway.

Apoptotic cells represent a dynamic stem cell niche governing proliferation and tissue regeneration.

Stem cells (SCs) play a key role in homeostasis and repair. While many studies have focused on SC self-renewal and differentiation, little is known regarding the molecular mechanism regulating SC elimination and compensation upon loss. Here, we report that Caspase-9 deletion in hair follicle SCs (HFSCs) attenuates the apoptotic cascade, resulting in significant temporal delays. Surprisingly, Casp9-deficient HFSCs accumulate high levels of cleaved caspase-3 and are improperly cleared due to an essential caspase-3/caspase-9 feedforward loop. These SCs are retained in an apoptotic-engaged state, serving as mitogenic signaling centers by continuously releasing Wnt3 and instructing proliferation. Investigating the underlying mechanism, we reveal a caspase-3/Dusp8/p38 module responsible for Wnt3 induction, which operates in both normal and Casp9-deleted HFSCs. Notably, Casp9-deleted mice display accelerated wound repair and de novo hair follicle regeneration. Taken together, we demonstrate that apoptotic cells represent a dynamic SC niche, from which emanating signals drive SC proliferation and tissue regeneration.

Expression of Wnt3, ß-catenin and MMP-7 in gastric cancer and precancerous lesions and their correlations with Helicobacter pylori infection. / Wnt3、ß-catenin、MMP-7åœ¨èƒƒç™Œå’Œç™Œå‰ç— å˜ä¸­çš„è¡¨è¾¾åŠå ¶ä¸Žå¹½é—¨èžºæ†èŒæ„ŸæŸ“çš„ç›¸å ³æ€§.

OBJECTIVES: To compare the expression of the Wnt signaling-associated proteins (Wnt3, ß-catenin, MMP-7) in gastric cancer and precancerous lesions with positive and negative Helicobacter pylori (H.pylori, Hp) infection, and to further explore the mechanisms underlying the Wnt signaling pathway involving in the formation of gastric cancer and its relationship with Hp infection. METHODS: The complete paraffin samples with pathologically confirmed diagnosis, who came from the First Hospital of Changsha from January 2018 to April 2020, were collected. All samples were randomly divided into a gastric cancer group (n=57), a precancerous lesion group (n=84), and a chronic superficial gastritis group (n=25). Improved Giemsa staining was used to detect Hp infection, and according the results of Hp infection the above groups were divided into a Hp positive subgroup and a negative subgroup. The expressions of Wnt3, ß-catenin and MMP-7 were examined with immunohistochemistry. RESULTS: The Wnt3, ß-catenin, and MMP-7 were highly expressed in the gastric cancer group and the gastric precancerous lesion group. The Wnt3 and MMP-7 were highly expressed in cytoplasm, and ß-catenin showed a tendency of cell membrane transferring to cytoplasm and nucleus, which was characterized by "nuclear translocation". The positive rates of the Wnt3, ß-catenin, and MMP-7 expressions in the gastric cancer group were higher than those in the precancerous lesion group and the chronic superficial gastritis group (all P<0.05), which showed a gradually increasing trend with the deterioration of differentiation degree. In addition, the expressions of Wnt3, ß-catenin, and MMP-7 in the Hp positive subgroup in the gastric cancer group and the precancerous lesion group were higher than those in the Hp negative subgroup (all P<0.05). CONCLUSIONS: Aberrant activation of Wnt signaling pathway is involved in the occurrence and development of precancerous lesions and gastric cancer, and which is related with Hp infection. Meanwhile, the Wnt3, ß-catenin and MMP-7 may be used as molecular markers for early diagnosis of gastric cancer and indicators to judge the degree of differentiation and malignancy of gastric cancer.

Paeonolide as a Novel Regulator of Core-Binding Factor Subunit Alpha-1 in Bone-Forming Cells.

Paeonia suffruticosa has been extensively used as a traditional medicine with various beneficial effects; paeonolide (PALI) was isolated from its dried roots. This study aimed to investigate the novel effects and mechanisms of PALI in pre-osteoblasts. Here, cell viability was evaluated using an MTT assay. Early and late osteoblast differentiation was examined by analyzing the activity of alkaline phosphatase (ALP) and by staining it with Alizarin red S (ARS). Cell migration was assessed using wound healing and Boyden chamber assays. Western blot and immunofluorescence analyses were used to examine the intracellular signaling pathways and differentiation proteins. PALI (0.1, 1, 10, 30, and 100 µM) showed no cytotoxic or proliferative effects in pre-osteoblasts. In the absence of cytotoxicity, PALI (1, 10, and 30 µM) promoted wound healing and transmigration during osteoblast differentiation. ALP staining demonstrated that PALI (1, 10, and 30 µM) promoted early osteoblast differentiation in a dose-dependent manner, and ARS staining showed an enhanced mineralized nodule formation, a key indicator of late osteoblast differentiation. Additionally, low concentrations of PALI (1 and 10 µM) increased the bone morphogenetic protein (BMP)-Smad1/5/8 and Wnt-ß-catenin pathways in osteoblast differentiation. Particularly, PALI (1 and 10 µM) increased the phosphorylation of ERK1/2 compared with BMP2 treatment, an FDA-approved drug for bone diseases. Furthermore, PALI-mediated early and late osteoblast differentiation was abolished in the presence of the ERK1/2 inhibitor U0126. PALI-induced RUNX2 (Cbfa1) expression and nuclear localization were also attenuated by blocking the ERK1/2 pathway during osteoblast differentiation. We suggest that PALI has biologically novel activities, such as enhanced osteoblast differentiation and bone mineralization mainly through the intracellular ERK1/2-RUNX2 signaling pathway, suggesting that PALI might have therapeutic action and aid the treatment and prevention of bone diseases, such as osteoporosis and periodontitis.

Calcium calmodulin kinase II activity is required for cartilage homeostasis in osteoarthritis.

WNT ligands can activate several signalling cascades of pivotal importance during development and regenerative processes. Their de-regulation has been associated with the onset of different diseases. Here we investigated the role of the WNT/Calcium Calmodulin Kinase II (CaMKII) pathway in osteoarthritis. We identified Heme Oxygenase I (HMOX1) and Sox-9 as specific markers of the WNT/CaMKII signalling in articular chondrocytes through a microarray analysis. We showed that the expression of the activated form of CaMKII, phospho-CaMKII, was increased in human and murine osteoarthritis and the expression of HMOX1 was accordingly reduced, demonstrating the activation of the pathway during disease progression. To elucidate its function, we administered the CaMKII inhibitor KN93 to mice in which osteoarthritis was induced by resection of the anterior horn of the medial meniscus and of the medial collateral ligament in the knee joint. Pharmacological blockade of CaMKII exacerbated cartilage damage and bone remodelling. Finally, we showed that CaMKII inhibition in articular chondrocytes upregulated the expression of matrix remodelling enzymes alone and in combination with Interleukin 1. These results suggest an important homeostatic role of the WNT/CaMKII signalling in osteoarthritis which could be exploited in the future for therapeutic purposes.

TREM-2 defends the liver against hepatocellular carcinoma through multifactorial protective mechanisms.

OBJECTIVE: Hepatocellular carcinoma (HCC) is a prevalent and aggressive cancer usually arising on a background of chronic liver injury involving inflammatory and hepatic regenerative processes. The triggering receptor expressed on myeloid cells 2 (TREM-2) is predominantly expressed in hepatic non-parenchymal cells and inhibits Toll-like receptor signalling, protecting the liver from various hepatotoxic injuries, yet its role in liver cancer is poorly defined. Here, we investigated the impact of TREM-2 on liver regeneration and hepatocarcinogenesis. DESIGN: TREM-2 expression was analysed in liver tissues of two independent cohorts of patients with HCC and compared with control liver samples. Experimental HCC and liver regeneration models in wild type and Trem-2-/- mice, and in vitro studies with hepatic stellate cells (HSCs) and HCC spheroids were conducted. RESULTS: TREM-2 expression was upregulated in human HCC tissue, in mouse models of liver regeneration and HCC. Trem-2-/- mice developed more liver tumours irrespective of size after diethylnitrosamine (DEN) administration, displayed exacerbated liver damage, inflammation, oxidative stress and hepatocyte proliferation. Administering an antioxidant diet blocked DEN-induced hepatocarcinogenesis in both genotypes. Similarly, Trem-2-/- animals developed more and larger tumours in fibrosis-associated HCC models. Trem-2-/- livers showed increased hepatocyte proliferation and inflammation after partial hepatectomy. Conditioned media from human HSCs overexpressing TREM-2 inhibited human HCC spheroid growth in vitro through attenuated Wnt ligand secretion. CONCLUSION: TREM-2 plays a protective role in hepatocarcinogenesis via different pleiotropic effects, suggesting that TREM-2 agonism should be investigated as it might beneficially impact HCC pathogenesis in a multifactorial manner.

Right, left and cilia: How asymmetry is established.

The initial breaking of left-right (L-R) symmetry in the embryo is controlled by a motile-cilia-driven leftward fluid flow in the left-right organiser (LRO), resulting in L-R asymmetric gene expression flanking the LRO. Ultimately this results in left- but not right-sided activation of the Nodal-Pitx2 pathway in more lateral tissues. While aspects of the initial breaking event clearly vary between vertebrates, events in the Lateral Plate Mesoderm (LPM) are conserved through the vertebrate lineage. Evidence from model systems and humans highlights the role of cilia both in the initial symmetry breaking and in the ability of more lateral tissues to exhibit asymmetric gene expression. In this review we concentrate on the process of L-R determination in mouse and humans.

Paneth cell-derived growth factors support tumorigenesis in the small intestine.

Paneth cells (PCs) are small intestinal epithelial cells that secrete antimicrobial peptides and growth factors, such as Wnt ligands. Intriguingly, the context in which PC-derived Wnt secretion is relevant in vivo remains unknown as intestinal epithelial ablation of Wnt does not affect homeostatic proliferation or restitution after irradiation injury. Considering the importance of growth factors in tumor development, we explored here the role of PCs in intestinal carcinogenesis using a genetic model of PC depletion through conditional expression of diphtheria toxin-α subunit. PC depletion in Apc Min mice impaired adenoma development in the small intestine and led to decreased Wnt3 expression in small bowel adenomas. To determine if PC-derived Wnt3 was required for adenoma development, we examined tumor formation after PC-specific ablation of Wnt3 We found that this was sufficient to decrease small intestinal adenoma formation; moreover, organoids derived from these tumors displayed slower growth capacity. Overall, we report that PC-derived Wnt3 is required to sustain early tumorigenesis in the small bowel and identify a clear role for PC-derived Wnt production in intestinal pathology.

ARE CDX2, BETA-CATENIN AND WNT IMMUNOMARCHERS USEFUL FOR EVALUATING THE CHANCE OF DISEASE PROGRESSION OR EVOLUTION TO DEATH IN PATIENTS WITH COLORECTAL CANCER?

BACKGROUND: Colorectal cancer (CRC) is one of the most common types of cancer in the world. Over time, intestinal epithelial cells undergo mutations that may lead to proliferative advantage and the emergence of cancer. Mutations in the beta-catenin pathway are amongst those described in the development of CRC. AIM: To verify the existence of a relation between the presence of Wnt3, beta-catenin and CDX2 in colorectal cancer samples and clinical outcomes such as disease progression or death. METHOD: Wnt3a, beta-catenin and CDX2 immunohistochemistry was performed on CRC tissue microarray samples (n=122), and analysis regarding the relation between biomarker expression and disease progression or death was performed. RESULTS: No significant difference was found between the presence or absence of CDX2, beta-catenin or Wnt3a expression and clinical stage, tumor grade, disease progression or death. CONCLUSION: CDX2, beta-catenin and Wnt3a are not useful to predict prognosis in patients with CRC.

Wnt3 distribution in the zebrafish brain is determined by expression, diffusion and multiple molecular interactions.

Wnt3 proteins are lipidated and glycosylated signaling molecules that play an important role in zebrafish neural patterning and brain development. However, the transport mechanism of lipid-modified Wnts through the hydrophilic extracellular environment for long-range action remains unresolved. Here we determine how Wnt3 accomplishes long-range distribution in the zebrafish brain. First, we characterize the Wnt3-producing source and Wnt3-receiving target regions. Subsequently, we analyze Wnt3 mobility at different length scales by fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. We demonstrate that Wnt3 spreads extracellularly and interacts with heparan sulfate proteoglycans (HSPG). We then determine the binding affinity of Wnt3 to its receptor, Frizzled1 (Fzd1), using fluorescence cross-correlation spectroscopy and show that the co-receptor, low-density lipoprotein receptor-related protein 5 (Lrp5), is required for Wnt3-Fzd1 interaction. Our results are consistent with the extracellular distribution of Wnt3 by a diffusive mechanism that is modified by tissue morphology, interactions with HSPG, and Lrp5-mediated receptor binding, to regulate zebrafish brain development.