During bone remodeling, degradation of skeletal connective tissue is regulated, at least in part, by the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinase (TIMPs), their natural inhibitors. Recently, the <em>Wnt</em> signaling pathway has been demonstrated to play a crucial role in the regulation of bone formation. Here, we investigated a potential role for <em>Wnt</em> signaling and functional cross-talk with bone morphogenetic protein (BMP)-2 in mRNA expression of MMPs, TIMPs and bone matrix proteins in pluripotent C2C<em>12</em> cells. To assess the functional contribution of <em>Wnt</em> signaling, we have generated C2C<em>12</em> cell lines stably over-expressing <em>Wnt</em>3a or <em>Wnt</em>5a, and then treated these cells with BMP-2 for 24 h. In these cultures, MMP-13 mRNA expression was induced by BMP-2 in <em>Wnt</em>3a over-expressing C2C<em>12</em> (<em>Wnt</em>3a-C2C<em>12</em>) cells but not in either <em>Wnt</em>5a over-expressing C2C<em>12</em> (<em>Wnt</em>5a-C2C<em>12</em>) cells or vehicle-transfected C2C<em>12</em> cells. MMP-13 mRNA was induced in these cells by addition of BMP-2 for <em>12</em> h and the enhancement lasted up to 48 h. These effects were observed in a dose-dependent manner. Enzymatic activity of MMP-13 also induced in <em>Wnt</em>3a-C2C<em>12</em> cells by addition of BMP-2. However, membrane type-1 matrix metalloproteinase (MT1-MMP) and MMP-2 mRNA expression was not affected by either <em>Wnt</em>3a or BMP-2. In contrast, TIMP-1 mRNA expression was suppressed by BMP-2 in <em>Wnt</em>3a-C2C<em>12</em> cells but not in <em>Wnt</em>5a-C2C<em>12</em> cells. Our results show that expression of MMP-13 and TIMP-1 is regulated by <em>Wnt</em> signaling combined with BMP-2 in osteoblastic differentiation, and this signaling may in part mediate MMP-13 and TIMP-1 production during bone formation and/or remodeling.

BACKGROUND

One of the key questions in developmental biology is how, from a relatively small number of conserved signaling pathways, is it possible to generate organs displaying a wide range of shapes, tissue organization, and function. The dentition and its distinct specific tooth types represent a valuable system to address the issues of differential molecular signatures. To identify such signatures, we performed a comparative transcriptomic analysis of developing murine lower incisors, mandibular molars and maxillary molars at the developmental cap stage (E14.5).

RESULTS

231 genes were identified as being differentially expressed between mandibular incisors and molars, with a fold change higher than 2 and a false discovery rate lower than 0.1, whereas only 96 genes were discovered as being differentially expressed between mandibular and maxillary molars. Numerous genes belonging to specific signaling pathways (the Hedgehog, Notch, <em>Wnt</em>, FGF, TGFβ/BMP, and retinoic acid pathways), and/or to the homeobox gene superfamily, were also uncovered when a less stringent fold change threshold was used. Differential expressions for 10 out of <em>12</em> (mandibular incisors versus molars) and 9 out of 10 selected genes were confirmed by quantitative reverse transcription-PCR (qRT-PCR). A bioinformatics tool (Ingenuity Pathway Analysis) used to analyze biological functions and pathways on the group of incisor versus molar differentially expressed genes revealed that 143 genes belonged to 9 networks with intermolecular connections. Networks with the highest significance scores were centered on the TNF/NFκB complex and the ERK1/2 kinases. Two networks ERK1/2 kinases and tretinoin were involved in differential molar morphogenesis.

CONCLUSIONS

These data allowed us to build several regulatory networks that may distinguish incisor versus molar identity, and may be useful for further investigations of these tooth-specific ontogenetic programs. These programs may be dysregulated in transgenic animal models and related human diseases leading to dental anomalies.

Frizzled 2 acts as a 7-transmembrane receptor in the <em>Wnt</em>-Dishevelled signal transduction cascade. Among others, this cascade has been associated with neural crest cell proliferation and early migration during development in mammals. The genes for some components of this cascade are located in chromosomal regions that are deleted in human syndromes associated with neural crest cell defects, like DiGeorge and Velo-Cardio-Facial Syndrome. These syndromes are often accompanied by abnormalities in cardiac morphology. Furthermore, we have reported in previous studies the upregulation of the tissue polarity gene frizzled 2 in myofibroblasts during their migration into the necrotic area after myocardial infarction in the adult heart. It is known that genes that are upregulated during cardiac remodeling due to pathology often play a role during development. To investigate whether frizzled 2 can be associated with the process of cardiac morphogenesis we studied its expression in the thoracic arterial system and heart of mouse embryo's of 10, <em>12</em>, 14, 16 and 18 days after conception by means of in situ hybridization. At day 10 after conception signal could be found in the pharyngeal arches and arch arteries. The outflow tract, the ascending aorta and the pulmonary trunk were positive for frizzled 2 from day <em>12</em> on. This expression decreased with time and at day 18 only some signal could be detected in the aorta and pulmonary trunk. In contrast, in coronary and pulmonary arteries no expression was observed at any time point. Minor myocardial expression was observed in the ventricular septum at days <em>12</em> and 14. Atrial expression, although considerably lower than ventricular expression, could be detected somewhat later at days 14 and 16. Our results indicate that there is transient expression of frizzled 2 in areas that are invested by neural crest cells. This expression is downregulated upon neural crest cell differentiation. The frizzled 2 expression supports a role for the <em>Wnt</em>-frizzled pathway in neural crest-related disorders.

Notch encodes a transmembrane protein that functions in intercellular signaling. Although there is one Notch gene in Drosophila, vertebrates have three or more with overlapping patterns of embryonic expression. We cloned the entire 7575-bp coding region of an amphioxus Notch gene (AmphiNotch), encoding 2524 amino acids, and obtained the exon/intron organization from a genomic cosmid clone. Southern blot and PCR data indicate that AmphiNotch is the only Notch gene in amphioxus. AmphiNotch, like Drosophila Notch and vertebrate Notch1 and Notch2, has 36 EGF repeats, 3 Notch/lin-<em>12</em> repeats, a transmembrane region, and 6 ankyrin repeats. Phylogenetic analysis places it at the base of all the vertebrate genes, suggesting it is similar to the ancestral gene from which the vertebrate Notch family genes evolved. AmphiNotch is expressed in all three embryonic germ layers in spatiotemporal patterns strikingly similar to those of all the vertebrate homologs combined. In the developing nerve cord, AmphiNotch is first expressed in the posteriormost part of the neural plate, then it becomes more broadly expressed and later is localized dorsally in the anteriormost part of the nerve cord corresponding to the diencephalon. In late embryos and larvae, AmphiNotch is also expressed in parts of the pharyngeal endoderm, in the anterior gut diverticulum, and, like AmphiPax2/5/8, in the rudiment of Hatschek's kidney. A comparison with Notch1 and Pax5 and Pax8 expression in the embryonic mouse kidney helps support homology of the amphioxus and vertebrate kidneys. AmphiNotch is also an early marker for presumptive mesoderm, transcripts first being detectable at the gastrula stage in a ring of mesendoderm just inside the blastopore and subsequently in the posterior mesoderm, notochord, and somites. As in sea urchins and vertebrates, these domains of AmphiNotch expression overlap with those of several <em>Wnt</em> genes and brachyury. These relationships suggest that amphioxus shares with other deuterostomes a common mechanism for patterning along the anterior/posterior axis involving a posterior signaling center in which the Notch and <em>Wnt</em> pathways and brachyury interact.

The <em>Wnt</em> molecules are a family of secretory glycoproteins implicated in proliferation and differentiation of both normal and malignant cells. Despite extensive investigation of the <em>WNT</em> genes expression profile in various tumors, little is known about their expression in chronic lymphocytic leukemia (CLL). In this study, the expression profile of 14 <em>WNT</em> genes was investigated in a large number of Iranian patients with CLL. Semi-quantitative RT-PCR was performed on peripheral blood leukemic cells obtained from 62 patients with CLL. Peripheral blood mononuclear cells isolated from 11 age matched normal subjects served as control to determine baseline expression level of these genes. Our results have demonstrated significant up-regulation of <em>WNT</em>-3, <em>WNT</em>-4, <em>WNT</em>-5B, <em>WNT</em>-7B, <em>WNT</em>-9A, <em>WNT</em>-10A, and <em>WNT</em>-16B in patients with CLL compared to normal subjects (p < 0.05 to p < 0.0001). <em>WNT</em> gene expression analysis in different CLL subtypes showed a similar pattern of expression in progressive and indolent clinical subtypes. Over-expression of <em>WNT</em>-5A and <em>WNT</em>-9A genes was observed in patients with no mutation in their immunoglobulin (Ig) variable region heavy chain (Ig VH) genes compared to those with mutated Ig VH genes. Comparison between patients expressing VH1 (n = 9), VH3 (n = 40) and VH4 (n = <em>12</em>) gene families, revealed down-regulation of <em>WNT</em>-3 and <em>WNT</em>-9A in VH3 positive patients. Our results indicate up-regulation of many members of the <em>WNT</em> gene family in CLL suggesting involvement of the <em>Wnt</em> canonical and/or noncanonical signaling pathways in CLL tumorigenesis.

Understanding endogenous mechanisms of neuroprotection may have important clinical applications. It is well established that brain tissue becomes more resistant to ischemic injury following a sublethal ischemic insult. This process, called ischemic preconditioning (IPC), can be induced in adult rat hippocampal slice cultures by a brief oxygen-glucose deprivation (OGD) [Hassen, G.W., Tian, D., Ding, D., Bergold, P.J., 2004. A new model of ischemic preconditioning using young adult hippocampal slice cultures. Brain Res. Brain Res. Protoc. 13, 135-143]. We have analyzed the changes in gene expression brought about by IPC in this model in order to understand the mechanisms involved. Total RNA was isolated at different time points following a brief OGD (3, 6 and <em>12</em> h) and used to probe genome-wide expression microarrays. Genes were identified that were significantly up- or down-regulated relative to controls. We placed genes that were differentially expressed into statistically significant groups based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and gene ontology (GO) terms. Genes involved in signal transduction, transcription, and oxidative phosphorylation are differentially expressed at each time point. The analysis demonstrates that alterations in signaling pathways (TGF-beta, <em>Wnt</em>, MAPK, ErbB, Toll-like receptor, JAK-STAT, VEGF) consistently accompany IPC. RT-PCR was used to confirm that members of these signaling pathways are regulated as predicted by the microarray analysis. We verified that protein translation following OGD is necessary for IPC. We also found that blocking the NMDA receptor during OGD does not significantly inhibit IPC in this model or produce large changes in gene expression. Our data thus suggests that changes in signaling pathways and their down-stream targets play an important role in triggering endogenous neuroprotection.

OBJECTIVE

While their negative impact on bone health is well established, the effects of aromatase inhibition (AI) on Wnt inhibitors and osteoprotegerin (OPG) are unknown. The aim of the study was to investigate the effects of AI on serum levels of sclerostin, DKK-1 and OPG, as well as their associations with PINP and CTX as markers of bone turnover and bone mineral density (BMD) assessed by DXA.

METHODS

We conducted a prospective longitudinal analysis of 70 postmenopausal women with hormone receptor-positive early breast cancer (BC) treated with anastrozole. All measurements were performed at baseline, 12 and 24 months of treatment. We measured the association of the investigated variables with circulating bone turnover markers, as well as with the BMD.

RESULTS

After 24 months of AI therapy, sclerostin and OPG concentrations increased from 29.5 pmol/l (SD = 15.1) and 6.8 pmol/l (SD = 2.2) at baseline to 43.2 pmol/l (SD = 20.6) (p < 0.001) and 7.4 pmol/l (SD = 2.2) (p = 0.028), respectively. DKK-1 levels decreased from 34.3 pmol/l (SD = 13.5) at baseline to 29.7 pmol/l (SD = 12.3) at the 24-month visit (p = 0.005). Sclerostin levels significantly correlated with PTH, OPG and BMD of the lumbar spine, while DKK-1 correlated with the BMD of the femoral neck and of the total hip.

CONCLUSIONS

The observed increase in sclerostin levels indicates a central role of osteocytes in bone turnover in women with BC.

The aim of our study was to investigate the sex- and age-related changes of serum Dickkopf-1 (Dkk-1), a soluble inhibitor of the <em>Wnt</em> signaling pathway, in healthy individuals and in patients with breast cancer (BC) and bone metastases (BM) using a new ELISA. Association of serum Dkk-1 with markers of bone turnover was also investigated. Serum Dkk-1 measurements were performed using a commercial sandwich ELISA in 150 healthy men, 175 healthy pre- and postmenopausal women (20-65 years), 22 women with BC and BM (mean age 63 years), and 16 women with BC and metastases at sites other than bone (mean age 53 years). Intra- and interassay coefficients of variation were below 7% and <em>12</em>%, respectively. The detection limit was determined to be 0.018 microg/L. In healthy women and men, Dkk-1 did not change with age. Serum Dkk-1 modestly correlated with serum bone alkaline phosphatase (r = 0.19, P = 0.013) and serum C-terminal cross-linking telopeptide of type I collagen (r = 0.19, P = 0.014) in women but not in men. Dkk-1 levels were higher in women with BC and BM (5.57 +/- 5.50 microg/L) than in healthy age-matched controls (3.47 +/- 1.47 microg/L, P < 0.0001) and women with metastases at sites other than bone (3.57 +/- 1.66 microg/L, P = 0.0003). In conclusion, serum Dkk-1 is stable with age in healthy women and men and increases in patients with BC and BM. Measurements of circulating Dkk-1 with this new ELISA may be useful for the clinical investigation of patients with malignant bone diseases.

Spinal cord injury (SCI) has been associated with a marked increase in bone loss and bone remodeling, especially short-term after injury. The absence of mechanical load, mediated by osteocyte mechanosensory function, seems to be a causative factor related to bone loss in this condition. However, the pathogenesis and clinical management of this process remain unclear. Therefore, the aim of the study was to analyze the effect of recent SCI on the <em>Wnt</em> pathway antagonists, sclerostin and Dickkopf (Dkk-1), and their relationship with bone turnover and bone mineral density (BMD) evolution. Forty-two patients (aged 35 ± 14yrs) with a recent (<6months) complete SCI were prospectively included. Sclerostin and Dkk-1, bone turnover markers (bone formation: PINP, bone ALP; resorption: sCTx) and BMD (lumbar spine, proximal femur, total body and lower extremities [DXA]) were assessed at baseline and at 6 and <em>12</em> months. The results were compared with a healthy control group. 22/42 patients completed the <em>12</em>-month follow-up. At baseline, SCI patients showed a marked increase in bone markers (PINP and sCTx), remaining significantly increased at up to 6 months of follow-up. Additionally, they presented significantly increased Dkk-1 values throughout the study, whereas sclerostin values did not significantly change. BMD markedly decreased at the proximal femur (-20.2 ± 5.4%, p < 0.01), total body (-5.7 ± 2.2%, p = 0.02) and lower extremities (-13.1 ± 4.5%, p = 0.01) at <em>12</em> months. Consequently, 59% of patients developed densitometric osteoporosis at <em>12</em> months. Patients with higher Dkk-1 values (>58 pmol/L) at baseline showed higher sublesional BMD loss. In conclusion, this study shows that short-term after SCI there is a marked increase in bone turnover and bone loss, the latter associated with an increase in Dkk-1 serum levels. The persistence of increased levels of this <em>Wnt</em> antagonist throughout the study and their relationship with the magnitude of bone loss suggests a contributory role of this mediator in this process.

OBJECTIVE

Orthotopic liver transplantation (OLT) can be an effective treatment option for certain patients with early stage hepatocellular carcinoma (HCC) meeting Milan, UCSF, or Hangzhou criteria. However, HCC recurrence rates post-OLT range from 20 to 40 %, with limited follow-up options. Elucidating genetic drivers common to primary and post-OLT recurrent tumors may further our understanding and help identify predictive biomarkers of recurrence-both to ultimately help manage clinical decisions for patients undergoing OLT.

METHODS

Whole exome and RNA sequencing in matched primary and recurrent tumors, normal adjacent tissues, and blood from four Chinese HCC patients was conducted. SiRNA knockdown and both qRT-PCR and Western assays were performed on PLCPRF5, SNU449 and HEPG2 cell lines; immunohistochemistry and RNA Sequencing were conducted on the primary tumors of Chinese HCC patients who experienced tumor recurrence post-OLT (n = 9) or did not experience tumor recurrence (n = <em>12</em>).

RESULTS

In three independent HCC studies of patients undergoing transplantation (n = 21) or surgical resection (n = 242, n = 44) of primary tumors (total n = 307), HERC5 mRNA under-expression correlated with shorter: time to tumor recurrence (p = 0.007 and 0.02) and overall survival (p = 0.0063 and 0.023), even after adjustment for relevant clinical variables. HERC5 loss drives CCL20 mRNA and protein over-expression and associates with regulatory T cell infiltration as measured by FOXP3 expression. Further, matched primary and recurrent tumors from the 4 HCC patients indicated clonal selection advantage of Wnt signaling activation and CDKN2A inactivation.

CONCLUSIONS

HERC5 plays a crucial role in HCC immune evasion and has clinical relevance as a reproducible prognostic marker for risk of tumor recurrence and survival in patients.

The genetic architecture of adolescent idiopathic scoliosis (AIS) remains poorly understood. Here we present the result of a 4-stage genome-wide association study composed of 5,953 AIS patients and 8,137 controls. Overall, we identified three novel susceptible loci including rs7593846 at 2p14 near MEIS1 (Pcombined = 1.19 × 10-13, OR = 1.21, 95% CI = 1.10-1.32), rs7633294 at 3p14.1 near MAGI1 (Pcombined = 1.85 × 10-<em>12</em>, OR = 1.20, 95% CI = 1.09-1.32), and rs9810566 at 3q26.2 near TNIK (Pcombined = 1.14 × 10-11, OR = 1.19, 95% CI = 1.08-1.32). We also confirmed a recently reported region associated with AIS at 20p11.22 (Pcombined = 1.61 × 10-15, OR = 1.22, 95% CI = 1.<em>12</em>-1.34). Furthermore, we observed significantly asymmetric expression of <em>Wnt</em>/beta-catenin pathway in the bilateral paraspinal muscle of AIS patients, including beta-catenin, TNIK, and LBX1. This is the first study that unveils the potential role of <em>Wnt</em>/beta-catenin pathway in the development of AIS, and our findings may shed new light on the etiopathogenesis of AIS.

OBJECTIVE

The aim of this study is to identify gene expression signatures that accompany dedifferentiation at the cancer invasion front in colorectal cancer.

METHODS

Two types of colorectal cancer were selected. Both types were well-differentiated adenocarcinomas at the superficial lesion. One type showed a dedifferentiated phenotype at the invasion front (type A, 13 samples); the other showed almost no dedifferentiated cancer cells at the invasion front (type B, <em>12</em> samples). Laser microdissection was combined with a cDNA microarray analysis to investigate the superficial lesions and the invasion front in colorectal cancers.

RESULTS

Eighty-three genes were differentially expressed between types A and B in the superficial lesions, and the samples of superficial lesions were divided correctly into two clusters by these genes. Interestingly, the samples of the invasion front were also divided into the two same clusters by these genes. The text mining method selected 10 genes involved in potential mechanisms causing dedifferentiation of cancer cells at the invasion front. The potential mechanisms include the networks of transforming growth factor-beta, Wnt, and Hedgehog signals. The expression levels of 10 genes were calculated by quantitative reverse transcription-PCR and 8 genes were confirmed to be significantly differentially expressed between two types (P < 0.05). The gene expression profiles of 8 genes divided <em>12</em> test cases into two clusters with one misclassification.

CONCLUSIONS

The molecular mechanisms constructed with 8 genes from three networks of transforming growth factor-beta, Wnt, and Hedgehog signals were found to correlate with dedifferentiation at the invasion front of colorectal cancer.

Previous genome-wide association studies have identified common variants in genes associated with bone mineral density (BMD) and risk of fracture. Recently, we identified single nucleotide polymorphisms (SNPs) in Wingless-type mouse mammary tumor virus integration site (<em>WNT</em>)16 that were associated with peak BMD in premenopausal women. To further identify the role of Wnt16 in bone mass regulation, we created transgenic (TG) mice overexpressing human <em>WNT</em>16 in osteoblasts. We compared bone phenotypes, serum biochemistry, gene expression, and dynamic bone histomorphometry between TG and wild-type (WT) mice. Compared with WT mice, <em>WNT</em>16-TG mice exhibited significantly higher whole-body areal BMD and bone mineral content (BMC) at 6 and <em>12</em> weeks of age in both male and female. Microcomputer tomography analysis of trabecular bone at distal femur revealed 3-fold (male) and 14-fold (female) higher bone volume/tissue volume (BV/TV), and significantly higher trabecular number and trabecular thickness but lower trabecular separation in TG mice compared with WT littermates in both sexes. The cortical bone at femur midshaft also displayed significantly greater bone area/total area and cortical thickness in the TG mice in both sexes. Serum biochemistry analysis showed that male TG mice had higher serum alkaline phosphatase, osteocalcin, osteoprotegerin (OPG), OPG to receptor activator of NF-kB ligand (tumor necrosis family ligand superfamily, number 11; RANKL) ratio as compared with WT mice. Also, lower carboxy-terminal collagen cross-link (CTX) to tartrate-resistant acid phosphatase 5, isoform b (TRAPc5b) ratio was observed in TG mice compared with WT littermates in both male and female. Histomorphometry data demonstrated that both male and female TG mice had significantly higher cortical and trabecular mineralizing surface/bone surface and bone formation rate compared with sex-matched WT mice. Gene expression analysis demonstrated higher expression of Alp, OC, Opg, and Opg to Rankl ratio in bone tissue in the TG mice compared with WT littermates. Our data indicate that <em>WNT</em>16 is critical for positive regulation of both cortical and trabecular bone mass and structure and that this molecule might be targeted for therapeutic interventions to treat osteoporosis.

During gastrulation in Xenopus convergence and extension movements, mediated by mediolateral intercalations, are the driving force for early neural plate morphogenesis. Here we show that the winged helix transcriptional regulator, Xfd-<em>12</em>' is dynamically expressed in medial neural plate precursors that undergo convergence and extension movements. These medial neuraxial progenitors are specified in and beyond the Spemann organizer prior to specification of the basal anlage of the neural plate. The initiation of Xfd-<em>12</em>' expression coincides with the induction of mesendoderm by Nodal-related growth factors at the late blastula stage. Comparative expression analysis suggests that cellular rearrangements at the pre-gastrulation stage account for regionalization of the Spemann organizer into head and trunk organizer compartments, the latter in which medial neural plate progenitors reside. While the maintenance of Xfd-<em>12</em>' expression in the dorsal non-involuting marginal zone requires FGF signalling, its subsequent positioning along the medial aspect of the neuraxis depends on signalling by <em>Wnt</em> and Nodal-related family members. Based on these findings we propose that XFD-<em>12</em>' is a trunk organizer component that might control convergence and extension movements of medial neural plate precursors during gastrulation.

Uterine fibroids, or leiomyoma, are the most common benign tumors in women of reproductive age. In this work, the effect of silencing the mediator complex subunit <em>12</em> (Med<em>12</em>) gene in human uterine fibroid cells was evaluated. The role of Med<em>12</em> in the modulation of <em>Wnt</em>/β-catenin and cell proliferation-associated signaling was evaluated in human uterine fibroid cells. Med<em>12</em> was silenced in the immortalized human uterine fibroid cell line (HuLM) using a lentivirus-based Med<em>12</em> gene-specific RNA interference strategy. HuLM cells were infected with lentiviruses carrying Med<em>12</em>-specific short hairpin RNA (shRNA) sequences or a nonfunctional shRNA scrambled control with green fluorescence protein. Stable cells that expressed low levels of Med<em>12</em> protein were characterized. <em>Wnt</em>/β-catenin signaling, sex steroid receptor signaling, cell cycle-associated, and fibrosis-associated proteins were measured. Med<em>12</em> knockdown cells showed significantly (P < 0.05) reduced levels of <em>Wnt</em>4 and β-catenin proteins as well as cell proliferation, as compared with scrambled control cells. Med<em>12</em> knockdown cells also showed reduced levels of cell cycle-associated cyclin D1, Cdk1, and Cdk2 proteins as well as reduced activation of p-extracellular signal-regulated kinase, p-protein kinase B, and transforming growth factor (TGF)-β signaling pathways as compared with scrambled control cells. Moreover, TGF-β-regulated fibrosis-related proteins such as fibronectin, collagen type 1, and plasminogen activator inhibitor-1 were significantly (P < 0.05) reduced in Med<em>12</em> knockdown cells as compared with scrambled control cells. Together, these results suggest that Med<em>12</em> plays a key role in the regulation of HuLM cell proliferation through the modulation of <em>Wnt</em>/β-catenin, cell cycle-associated, and fibrosis-associated protein expression.

TC1 (C8orf4) is associated with aggressive behavior and poor survival in cancer. We have recently reported that it is a target gene of NF-kappaB and regulates the <em>Wnt</em>/beta-catenin pathway. Here, we show that TC1 is upregulated by various cellular stresses and mediates heat shock response. Heat shock and other cellular stresses including H2O2, <em>12</em>-O-tetradecanoylphorbol 13-acetate (TPA), lipopolysaccharide (LPS), and UV enhance TC1 transcription in HeLa, KATO-III, HEK293T, and HK cells. TC1 protein then moves into the nuclei independently of NF-kappaB activation. TC1 upregulates heat shock proteins, and TC1-knockdown inhibits stress-induced downstream regulation significantly. Heat shock factor 1(HSF1) and TC1 upregulate each other, suggesting a potential positive feedback in the heat shock response regulation. Our data suggest that TC1 is a novel heat shock response regulator.

Morphogenesis of the mammalian secondary palate requires coordination of cell migration, proliferation, differentiation, apoptosis and synthesis of extracellular matrix molecules by numerous signal transduction pathways. Recent evidence suggests a role for members of the <em>Wnt</em> family of secreted cytokines in orofacial development. However, no study has systematically or comprehensively examined the expression of <em>Wnts</em> in embryonic orofacial tissue. We thus conducted a survey of the expression of all known <em>Wnt</em> genes in the developing murine secondary palate. Using an RT-PCR strategy to assay gene expression, <em>12</em> of the 19 known members of the <em>Wnt</em> family were found to be expressed in embryonic palatal tissue during key phases of its development. The expression of 5 <em>Wnt</em> family members was found to be temporally regulated. Moreover, these <em>Wnts</em> had unique spatio-temporal patterns of expression which suggested possible roles in palatal ontogeny.

<em>Wnt</em>5a is a secreted pleiotropic glycoprotein produced in an inflammatory state by a wide spectrum of ubiquitous cell populations. Recently, we demonstrated that <em>Wnt</em>5a skews the differentiation of human monocyte derived dendritic cells (moDCs) to a tolerogenic functional state. In this study we focus our interest on the role of this <em>Wnt</em> ligand after DC differentiation, during their maturation and function. We show that the expression of <em>Wnt</em> receptors is tightly regulated during the life cycle of DCs suggesting a differential responsiveness to <em>Wnt</em> signaling conditioned by their differentiation stage and the maturational stimuli. Furthermore, we confirm that <em>Wnt</em>5a is the main non-canonical <em>Wnt</em> protein expressed by DCs and its production increases upon specific stimuli. Exogenous <em>Wnt</em>5a improved the endocytic capacity of immature DCs but it is not a stimulatory signal on its own, slightly affecting the maturation and function of DCs. However, knocking down <em>Wnt</em>5a gene expression in maturing DCs demonstrates that DC-derived <em>Wnt</em>5a is necessary for normal IL-<em>12</em> secretion and plays a positive role during the development of Th1 responses. <em>Wnt</em>5a acts both in autocrine and paracrine ways. Thus, human naive CD4(+) T cells express <em>Wnt</em> receptors and, the addition of <em>Wnt</em>5a during CD3/CD28 stimulation enhances IL-2 and IFN-γ production. Taken together these results suggest a time-dependent role for <em>Wnt</em>5a during inflammatory responses conditioned by the differentiation stage of cellular targets.

During pregnancy, the uterus shows marked morphological and physiological changes under the regulation of ovarian steroid. To elucidate the molecular cues of these changes, we tried to identify the transcripts differentially expressed in the pregnant rat uterus by using the suppression subtractive hybridization method. Seven independent clones were isolated and one of the up-regulated genes was secreted frizzled-related protein 4 (sFRP4). sFRP4 contains a <em>Wnt</em>-binding domain and belongs to the secreted frizzled protein family whose members are assumed to function as modulators of the <em>Wnt</em> signal. The expression level of sFRP4 mRNA reached a peak in the pregnant uterus on day <em>12</em>, when uterine decidualization was almost complete in the rat. In situ hybridization histochemistry revealed that sFRP4 transcripts were observed in the decidual cells. In addition, proliferating cell nuclear antigen (PCNA)-positive cells were shown to be overlapped in decidua, suggesting that sFRP4 mRNA expression was accompanied by the late phase of decidual cell proliferation. Moreover, sFRP4 and estrogen receptor-alpha transcripts were co-localized. Furthermore, we analyzed the regulation of sFRP4 by estrogen using 17 beta-estradiol-treated ovariectomized rats. sFRP4 mRNA was detected in the uterus at 48 h after estrogen treatment, especially in endometrial stroma where PCNA-positive cells were also observed. The results in this study led us to the notion that sFRP4 mRNA may be up-regulated after estrogen treatment in the late phase of uterine cell proliferation.

OBJECTIVE

The current study aimed to explore the effect ofcurcumin on androgen receptor (AR) expression in endometrial carcinoma cells, as well as the underlying mechanisms.

METHODS

Endometrial carcinoma cells were treated with curcumin (10, 50, and 100 micromol/l) for <em>12</em>, 24, and 48 hours. Their growth curves were drawn using MTT assays and their apoptotic rates were determined using flow cytometry. The mRNA and protein expression of AR was detected using PCR and that of the <em>Wnt</em> signal related nucleopro- tein beta-cantenin was observed using western blot analysis. The influence of beta-cantenin on the action of curcumin was observed.

RESULTS

Curcumin downregulated the proliferation and apoptosis of human endometrial carcinoma cells in concentration and time-dependent manners. It downregulated the expression of AR and beta-cantenin in the cells. r<em>Wnt</em>3a partially cancelled the effects of curcumin on the proliferation and apoptosis of human endometrial carcinoma cells as well as the AR expression-downregulating effect of curcumin.

CONCLUSIONS

Curcumin inhibits the proliferation and apoptosis of human endometrial carcinoma cells by downregulating their AR expression through the Wnt signal pathway.

Cancer develops through a multistep process in which normal cells progress to malignant tumors via the evolution of their genomes as a result of the acquisition of mutations in cancer driver genes. The number, identity and mode of action of cancer driver genes, and how they contribute to tumor evolution is largely unknown. This study deployed the Mouse Mammary Tumor Virus (MMTV) as an insertional mutagen to find both the driver genes and the networks in which they function. Using deep insertion site sequencing we identified around 31000 retroviral integration sites in 604 MMTV-induced mammary tumors from mice with mammary gland-specific deletion of Trp53, Pten heterozygous knockout mice, or wildtype strains. We identified 18 known common integration sites (CISs) and <em>12</em> previously unknown CISs marking new candidate cancer genes. Members of the <em>Wnt</em>, Fgf, Fgfr, Rspo and Pdgfr gene families were commonly mutated in a mutually exclusive fashion. The sequence data we generated yielded also information on the clonality of insertions in individual tumors, allowing us to develop a data-driven model of MMTV-induced tumor development. Insertional mutations near <em>Wnt</em> and Fgf genes mark the earliest "initiating" events in MMTV induced tumorigenesis, whereas Fgfr genes are targeted later during tumor progression. Our data shows that insertional mutagenesis can be used to discover the mutational networks, the timing of mutations, and the genes that initiate and drive tumor evolution.

OBJECTIVE

To study the expression of Wnt during corneal wound healing and to understand the signaling mechanism involved.

METHODS

Rat cornea was demarcated on the central area by 4-mm trepine, and the epithelium within this area was removed by scalpel. The epithelium was scraped and isolated to extract RNA. To determine the proliferation of corneal epithelial cells, corneoscleral rims from human donors were treated with dispase II for 15 minutes, and epithelial cells were isolated. Cells were plated on a 3T3 feeder cells layer. The proliferation of corneal epithelial cells was evaluated by colony-forming efficiency.

RESULTS

Wnt 5b and 7a were rapidly induced in wounded cornea, and Wnt 7a promoted proliferation of corneal epithelial cells. In addition, matrix metalloproteinase (MMP)-12 was expressed in wounded rat corneal epithelium. Transcription of MMP-12 was responsive to Wnt/beta-catenin signaling. Function blockade of MMP-12 delayed Wnt 7a-induced cell proliferation.

CONCLUSIONS

These results indicate that Wnt proteins and MMP-12 regulate the proliferation of corneal epithelial cells and that Wnt signaling contributes to the resurfacing of defective areas during corneal wound healing.

Comparative studies of vulva development between Caenorhabditis elegans and other nematode species have provided some insight into the evolution of patterning networks. However, molecular genetic details are available only in C. elegans and Pristionchus pacificus. To extend our knowledge on the evolution of patterning networks, we studied the C. elegans male hook competence group (HCG), an equivalence group that has similar developmental origins to the vulval precursor cells (VPCs), which generate the vulva in the hermaphrodite. Similar to VPC fate specification, each HCG cell adopts one of three fates (1 degree, 2 degrees, 3 degrees), and 2 degrees HCG fate specification is mediated by LIN-<em>12</em>/Notch. We show that 2 degrees HCG specification depends on the presence of a cell with the 1 degree fate. We also provide evidence that <em>Wnt</em> signaling via the Frizzled-like <em>Wnt</em> receptor LIN-17 acts to specify the 1 degree and 2 degrees HCG fate. A requirement for EGF signaling during 1 degree fate specification is seen only when LIN-17 activity is compromised. In addition, activation of the EGF pathway decreases dependence on LIN-17 and causes ectopic hook development. Our results suggest that <em>WNT</em> plays a more significant role than EGF signaling in specifying HCG fates, whereas in VPC specification EGF signaling is the major inductive signal. Nonetheless, the overall logic is similar in the VPCs and the HCG: EGF and/or <em>WNT</em> induce a 1 degree lineage, and LIN-<em>12</em>/NOTCH induces a 2 degrees lineage. <em>Wnt</em> signaling is also required for execution of the 1 degree and 2 degrees HCG lineages. lin-17 and bar-1/beta-catenin are preferentially expressed in the presumptive 1 degree cell P11.p. The dynamic subcellular localization of BAR-1-GFP in P11.p is concordant with the timing of HCG fate determination.

To characterize the effects of an estrogen receptor (ER) agonist on the gene expressions in the uterus, immature female rats were administered once orally with 17alpha-ethynyl estradiol (EE, 3 mug/kg), a potent ER agonist. We focused on four categories of sex steroid hormone receptor genes: well-known estrogen target genes, <em>Wnt</em> genes, and beta-catenin/T-cell factor (TCF) target genes. ERalpha, ERbeta, progesterone receptor, and androgen receptor mRNAs were all downregulated at 24 and/or 48 h after EE administration. Complement C3 and insulin-like growth factor 1 mRNAs were markedly induced after EE administration. Although the time courses of <em>Wnt</em>4, <em>Wnt</em>5a, and <em>Wnt</em>7a mRNA status varied until <em>12</em> h after EE administration, all of them were simultaneously downregulated at 24 and 48 h. The remarkable downregulation of <em>Wnt</em>7a mRNA in response to EE was considered to be important to understand the various uterine phenomena affected by ER agonists. In the beta-catenin/TCF target genes, the downregulation of anti-Mullerian hormone type 2 receptor and bone morphogenetic protein 4 mRNA after EE administration appeared to be closely related to the downregulation of <em>Wnt</em>7a. The upregulation of cyclin D1 and follistatin mRNA at the early phase after EE administration was considered to have been affected by the upregulation of <em>Wnt</em>4. These results indicate that an ER agonist influences not only the mRNA expression of sex steroid hormone receptor genes and well-known estrogen target genes but also <em>Wnt</em> genes (<em>Wnt</em>4, <em>Wnt</em>5a, <em>Wnt</em>7a) and beta-catenin/TCF target genes in the uterus of immature rats, indicating that their molecules are the potential players affected by estrogenic stimuli.