The study is about the regulatory effects of estrogen and parathyroid hormone (PTH) on sclerostin, a protein that inhibits the Wnt/β-catenin pathway. The results indicate that estrogen may down-regulate sclerostin expression and that estrogen displays synergistic action with PTH. These results provide a new perspective on the relationship between estrogen and bone.

OBJECTIVE

To investigate whether estrogen can down-regulate SOST and MEF2 (myocyte enhancer factor 2) expression and whether co-treatment with estrogen and PTH has a stronger effect on suppressing SOST than PTH applied alone in ovariectomized rats.

METHODS

Forty-three-month-old virgin female Sprague-Dawley (SD) rats were ovariectomized and divided into four groups (n = 10). Another ten age-matched rats received sham operations as controls. After allowing 8 weeks for the development of vertebral osteopenia, the rats were administered the drug intervention. For this intervention, the estrogen group was subcutaneously injected with 17β-estradiol at 25 μg/kg body weight, the PTH group was injected with 80 μg/kg synthetic human PTH (1-34), and the co-treatment group was concurrently treated with PTH and estrogen at the above dosage. The OVX group and sham group were treated with vehicle. The drug treatment was conducted for 12 weeks. After the lumbar spine bone mineral density (BMD) was measured, the rats were sacrificed, and the lumbar spine and blood were collected for qPCR, Western blot, immunohistochemistry and other tests.

RESULTS

Estrogen can down-regulate MEF2 and sclerostin expression, and co-treatment with estrogen and PTH has a stronger effect on suppressing MEF2 and SOST mRNA than PTH alone. The co-treatment group displayed slightly higher bone mass and biomechanical properties than the PTH group, but the differences were not significant.

CONCLUSIONS

Estrogen appears to be a regulator of sclerostin, and the effect may involve suppressing MEF2s. Combined treatment with PTH and estrogen is not more beneficial for vertebral bone mass and strength than treatment with PTH alone in ovariectomized rats.

Non-syndromic craniosynostosis (NSC) is a frequent congenital malformation in which one or more cranial sutures fuse prematurely. Mutations causing rare syndromic craniosynostoses in humans and engineered mouse models commonly increase signaling of the <em>Wnt</em>, bone morphogenetic protein (BMP), or Ras/ERK pathways, converging on shared nuclear targets that promote bone formation. In contrast, the genetics of NSC is largely unexplored. More than 95% of NSC is sporadic, suggesting a role for de novo mutations. Exome sequencing of 291 parent-offspring trios with midline NSC revealed 15 probands with heterozygous damaging de novo mutations in <em>12</em> negative regulators of <em>Wnt</em>, BMP, and Ras/ERK signaling (10.9-fold enrichment, P = 2.4 × 10-11). SMAD6 had 4 de novo and 14 transmitted mutations; no other gene had more than 1. Four familial NSC kindreds had mutations in genes previously implicated in syndromic disease. Collectively, these mutations contribute to 10% of probands. Mutations are predominantly loss-of-function, implicating haploinsufficiency as a frequent mechanism. A common risk variant near BMP2 increased the penetrance of SMAD6 mutations and was overtransmitted to patients with de novo mutations in other genes in these pathways, supporting a frequent two-locus pathogenesis. These findings implicate new genes in NSC and demonstrate related pathophysiology of common non-syndromic and rare syndromic craniosynostoses. These findings have implications for diagnosis, risk of recurrence, and risk of adverse neurodevelopmental outcomes. Finally, the use of pathways identified in rare syndromic disease to find genes accounting for non-syndromic cases may prove broadly relevant to understanding other congenital disorders featuring high locus heterogeneity.

<AbstractText>Osteoporosis and cardiovascular (CV) diseases are closely correlated. RANKL/RANK/OPG pathway and <em>Wnt</em> signalling pathway both implicated in the pathogenesis of osteoporosis and cardiovascular diseases. We aimed to investigate the effect of denosumab or romosozumab therapy on cardiovascular outcomes in patients with primary osteoporosis.</AbstractText><AbstractText>PubMed, Cochrane library, and EMBASE databases were systematically searched from the inception dates to June 4, 2019. Randomized clinical trials evaluating the effect of denosumab or romosozumab versus active comparators or placebo for at least 6 months in patients with primary osteoporosis or osteopenia were included. Two investigators independently extracted data for study characteristics, outcomes of interest, and risk of bias in accordance with PRISMA guidelines.</AbstractText><AbstractText>17 relevant studies (denosumab: n = 11, 13615 participants; romosozumab: n = 6, <em>12</em>219 participants) were included. No associations between denosumab therapy and risk of a composite cardiovascular outcome (1.06 [95 % CI, 0.88-1.28], p = 0.54), three-point major adverse cardiovascular event (3P MACE, 1.01 [95 % CI, 0.83-1.23], p = 0.93), and four-point major adverse cardiovascular event (4P MACE, 0.99 [95 % CI, 0.83-1.18], p = 0.89) were identified. Romosozumab therapy did not increase the risk of composite cardiovascular outcome (1.26 [95 % CI, 0.95-1.68], p = 0.11), and 3P MACE (1.41 [95 % CI, 0.99-2.02], p = 0.06), while increased the risk of 4P MACE (1.39 [95 % CI, 1.01-1.90], p = 0.04) among elderly men and postmenopausal woman with osteoporosis over a period of <em>12</em>-36 months. Denosumab or romosozumab did not increase or reduce specific cardiovascular outcomes, including CV death or death, myocardial infarction, stroke, atrial fibrillation, heart failure, aortic and intracranial aneurysm, aortic dissection, aortic valve disease and hypertension (all p＞0.05). Sensitivity analysis conducted by random effects model altered the result of 4 P MACE in romosozumab (1.36 [0.99-1.87], p = 0.06). No other significant difference was detected in the sensitivity analyses and subgroup analyses.</AbstractText><AbstractText>Denosumab therapy was not associated with any risk of composite and specific cardiovascular outcomes among patients with primary osteoporosis than active comparators or placebo, while romosozumab therapy might increase the risk of 4 P MACE.</AbstractText>

BACKGROUND

Medulloblastomas in children can be categorized into 4 molecular subgroups with differing clinical characteristics, such that subgroup determination aids in prognostication and risk-adaptive treatment strategies. Magnetic resonance spectroscopy (MRS) is a widely available, noninvasive tool that is used to determine the metabolic characteristics of tumors and provide diagnostic information without the need for tumor tissue. In this study, we investigated the hypothesis that metabolite concentrations measured by MRS would differ between molecular subgroups of medulloblastoma and allow accurate subgroup determination.

METHODS

MRS was used to measure metabolites in medulloblastomas across molecular subgroups (SHH = <em>12</em>, Groups 3/4 = 17, <em>WNT</em> = 1). Levels of 14 metabolites were analyzed to determine those that were the most discriminant for medulloblastoma subgroups in order to construct a multivariable classifier for distinguishing between combined Group 3/4 and SHH tumors.

RESULTS

Medulloblastomas across molecular subgroups revealed distinct spectral features. Group 3 and Group 4 tumors demonstrated metabolic profiles with readily detectable taurine, lower levels of lipids, and high levels of creatine. SHH tumors showed prominent choline and lipid with low levels of creatine and little or no evidence of taurine. A 5-metabolite subgroup classifier inclusive of creatine, myo-inositol, taurine, aspartate, and lipid 13a was developed that could discriminate between Group 3/4 and SHH medulloblastomas with excellent accuracy (cross-validated area under the curve [AUC] = 0.88).

CONCLUSIONS

The data show that medulloblastomas of Group 3/4 differ metabolically as measured using MRS when compared with SHH molecular subgroups. MRS is a useful and accurate tool to determine medulloblastoma molecular subgroups.

While the surgical procedure of distraction osteogenesis (DO) is very successful in the treatment of orthopedic conditions, its major limitation of slow bone formation in the distracted gap has prompted numerous attempts to understand and accelerate this slow bone formation. Interestingly, <em>WNT</em>/FZD signaling has been identified as a critical pathway in mediating bone formation and regeneration but has not yet been studied in the context of DO. The objective of this study was to determine the spatial and temporal localization of endogenous <em>WNT</em> signaling proteins at various times of bone formation in a wild-type mouse model of DO. In this study, the DO protocol performed on mice consisted of three phases: latency (5 days), distraction (<em>12</em> days), and consolidation (34 days). Our immunohistochemical findings of distracted bone specimens show an increased expression of <em>WNT</em> ligands (<em>WNT</em>4 and <em>WNT</em>10A), receptors (FZD1 and 2, LRP5 and 6), β-catenin, and pathway antagonizers (DKK1; CTBP1 and 2; sFRP1, 2, and 4) during the distraction phase, which were then down-regulated during consolidation. This is the first published report to show an activation of the <em>WNT</em> pathway in DO and could help identify <em>WNT</em> as a potential therapeutic target in accelerating bone regeneration during DO.

The <em>Wnt</em> pathway is a major signaling pathway that regulates corneal epithelial stem cells. However, little is known about how the ultrastructure of the limbal epithelial basement membrane (EBM) affects <em>Wnt</em> activity. Due to its enhanced matrix stability, the cross-linked amniotic membrane (AM) has gained increasing interest in the field of regenerative medicine. For the first time, we used EDC/NHS cross-linked denuded AM (CLDAM) as a simulated EBM substrate to investigate this mechanism. Human limbal epithelial (HLE) cells were cultured on dishes (HLE/dish), denuded AM (HLE/DAM) or CLDAM (HLE/CLDAM). Compared with HLE/dish or HLE/DAM cultures, HLE/CLDAM cultures showed greater BrdU retention and colony formation efficiency and expressed higher levels of p63, ABCG2, integrin β1, and integrin-linked kinase (ILK). Nuclear β-catenin and TCF-4 levels were higher in HLE/CLDAM cultures compared with HLE cells cultured on collagen IV, laminin, Matrigel, or DAM. Silencing of ILK in HLE/CLDAM cultures resulted in decreased levels of nuclear β-catenin, TCF-4 and deltaNp63α, whereas cytokeratin <em>12</em> expression increased. Over-expression of ILK in HLE/dish cultures had the opposite effects. Accordingly, we proposed that the CLDAM matrix, with its higher rigidity and rougher ultrastructure, better preserved HLE progenitor cells in vitro, possibly by activating integrin β1/ILK, which indirectly activated <em>Wnt</em>/β-catenin and subsequently deltaNp63α. Crosstalk between the integrin β1/ILK and <em>Wnt</em>/β-catenin pathways appears to play a crucial role in limbal progenitor cell survival on EBM.

UNASSIGNED

We demonstrated the superior capability of carbodiimide cross-linked denuded amniotic membrane (CLDAM) than natural DAM to preserve limbo-corneal epithelial progenitor cells in vitro, then we used CLDAM as a simulated epithelial basement membrane (EBM) to study how EBM maintains limbal epithelial stem cells (LESCs). We found that integrin-linked kinase (ILK) is an important mediator that transfers survival signals detected by integrin β1 to the Wnt/β-catenin pathway, which in turn up-regulates deltaNp63α, a master gene that regulates LESC function. The rougher surface of the limbal EBM suggests that the surface complexity of the LESC niche may be important in regulating LESC function, which is triggered by the recognition of topographic cues by integrin β1, followed by activation of the ILK/Wnt/β-catenin/p63 cascade.

Oral Squamous Cell Carcinoma (OSCC) is the sixth most common cancer worldwide. OSCC invasion into the lymph nodes and mandible correlates with increased rates of recurrence and lower overall survival. Tumors that infiltrate mandibular bone proliferate rapidly and induce bone destruction. While survival rates have increased <em>12</em>% over the last 20 years, this improvement is attributed to general advances in prevention, earlier detection, and updated treatments. Additionally, despite decades of research, the molecular mechanisms of OSCC invasion into the mandible are not well understood. Parathyroid Hormone-related Protein (PTHrP), has been shown to be essential for mandibular invasion in OSCC animal models, and our previous studies demonstrate that the transcription factor Gli2 increases PTHrP expression in tumor metastasis to bone. In OSCC, we investigated regulators of Gli2, including Hedgehog, TGFβ, and <em>Wnt</em> signaling to elucidate how PTHrP expression is controlled. Here we show that canonical Hedgehog and TGFβ signaling cooperate to increase PTHrP expression and mandibular invasion in a Gli2-dependent manner. Additionally, in an orthotopic model of mandibular invasion, inhibition of Gli2 using shRNA resulted in a significant decrease of both PTHrP expression and bony invasion. Collectively, our findings demonstrate that multiple signaling pathways converge on Gli2 to mediate PTHrP expression and bony invasion, highlighting Gli2 as a therapeutic target to prevent bony invasion in OSCC.

Valproic acid (VPA), a widely used anticonvulsant, inhibits glycogen synthase kinase 3β and activates the <em>Wnt</em>/β-catenin pathway, which is associated with hair growth cycle and anagen induction. To assess the efficacy of topical VPA for treating androgenetic alopecia (AGA), we performed a randomized, double-blind, placebo-controlled clinical trial. Male patients with moderate AGA underwent treatment with either VPA (sodium valproate, 8.3%) or placebo spray for 24 weeks. The primary end-point for efficacy was the change in hair count during treatment, which was assessed by phototrichogram analysis. Of the 40 patients enrolled in the study, 27 (n = 15, VPA group; n = <em>12</em>, placebo group) completed the entire protocol with good compliance. No statistical differences in age, hair loss duration and total hair count at baseline were found between the groups. The mean change in total hair count was significantly higher in the VPA group than in the placebo group (P = 0.047). Both groups experienced mostly mild and self-limited adverse events, but their differences in prevalence rates were similar between the two groups (P = 0.72). A subject treated with topical VPA developed ventricular tachycardia, but it did not seem to be related to the VPA spray. Topical VPA increased the total hair counts of our patients; therefore, it is a potential treatment option for AGA.

Androgenetic alopecia (AGA) is a highly heritable condition, and the most common form of hair loss in men. The phenotype is characterized by an androgen-dependent, progressive loss of hair from the scalp, which may commence during puberty. Up to 80% of European men experience some degree of androgen-dependent hair loss during their lifetime. Current treatment options for AGA have limited efficacy, and improved understanding of the underlying biological causes is required to facilitate novel therapeutic approaches. To date, molecular genetic studies have implicated <em>12</em> genomic regions in AGA and identified a number of candidate genes. The latter include those encoding the androgen receptor (AR), the histone deacetylases (HDAC) 4 and 9, and the <em>WNT</em> molecule <em>WNT</em>10A. However, the majority of contributing genetic risk factors still await identification. This review describes the current status of AGA genetic research. We discuss the strength of the genetic approach and anticipated developments in the field, and how these will facilitate the systematic unravelling of AGA pathobiology, a process which may lead to the identification of new therapeutic targets.

Glioblastoma (GBM) is one of the most aggressive and lethal types of brain tumor. Despite the advancements in conventional or targeted therapies, median survival of GBM patients is less than <em>12</em> months. Amongst various signaling pathways aberrantly activated in glioma, active <em>Wnt</em>/β-catenin signaling pathway is one of the crucial oncogenic players. β-catenin, an important mediator of <em>Wnt</em> signaling pathway, gets phosphorylated by GSK3β complex. Phosphorylated β-catenin is specifically recognized by β-Trcp1, a F-box/WD40-repeat protein and with the help of Skp1 it plays a central role in recruiting phosphorylated β-catenin for degradation. In GBM, expression of β-TrCP1 and its affinity for β catenin is reported to be very low. Hence, we investigated whether any other members of the E3 ubiquitin ligase family could be involved in degradation of nuclear β-catenin. We here report that FBXO16, a component of SCF E3 ubiquitin ligase complex, is an interacting protein partner for β-catenin and mediates its degradation. Next, we show that FBXO16 functions as a tumor suppressor in GBM. Under normal growth conditions, FBXO16 proteasomally degrades β-catenin in a GSK-3β independent manner. Specifically, the C-terminal region of FBXO16 targets the nuclear β-catenin for degradation and inhibits TCF4/LEF1 dependent <em>Wnt</em> signaling pathway. The nuclear fraction of β-catenin undergoes K-48 linked poly-ubiquitination in presence of FBXO16. In summary, we show that due to low expression of FBXO16, the β-catenin is not targeted in glioma cells leading to its nuclear accumulation resulting in active <em>Wnt</em> signaling. Activated <em>Wnt</em> signaling potentiates the glioma cells toward a highly proliferative and malignant state.

Few studies have utilized microarray analysis to understand the genome wide changes involved in the development of the hypothalamus despite its overall importance to basic physiology. Gene expression profiling of immortalized, clonal hypothalamic neurons, embryonic-derived mHypoE-46 and adult-derived mHypoA-2/<em>12</em>, reveals that the expression of <em>12</em>25 probes was significantly changed between the two neuronal models. Further comparison of the gene expression profiles identified two categories of genes that were confirmed with qRT-PCR: (i) genes implicated in the <em>Wnt</em> signaling pathway; and (ii) transcription factors previously implicated in the development of the central nervous system. Yet, functional analysis of the two cell lines, including hormonal responses and secretion, indicate that they are comparable despite their developmental origin. This study provides a comprehensive analysis of embryonic- and adult-derived hypothalamic neuronal cell models that both express neuropeptide Y, and identifies novel genes as candidates for mediating the development of specific hypothalamic neurons.

The aims of this study were to evaluate the osteoprotective effect of aqueous extract from Rhizoma Dioscoreae (RDE) on rats with ovariectomy- (OVX-) induced osteopenia. Our results show that RDE could inhibit bone loss of OVX rats after a <em>12</em>-week treatment. The microarray analysis showed that 68 genes were upregulated and that 100 genes were downregulated in femurs of the RDE group rats compared to those in the OVX group. The Ingenuity Pathway Analysis (IPA) showed that several downregulated genes had the potential to code for proteins that were involved in the <em>Wnt</em>/ β -catenin signaling pathway (Sost, Lrp6, Tcf7l2, and Alpl) and the RANKL/RANK signaling pathway (Map2k6 and Nfatc4). These results revealed that the mechanism for an antiosteopenic effect of RDE might lie in the synchronous inhibitory effects on both the bone formation and the bone resorption, which is associated with modulating the <em>Wnt</em>/ β -catenin signaling and the RANKL/RANK signaling.

We performed middle cerebral artery occlusion (MCAO) in rats to investigate the effect and some of the underlying mechanisms of radial extracorporeal shock wave therapy (rESWT) in cerebral ischemia rats. We measured neurological function and cerebral blood flow (CBF) using a full-field laser perfusion imager and brain infarct volume on days 3, <em>12</em>, and 30. Immunofluorescence, western blot, and real-time polymerase chain reaction (PCR) techniques were used to detect the expression of vascular endothelial growth factor (VEGF), neuron-specific enolase (NSE), nestin, <em>Wnt</em>3a, and β-catenin in the ischemic hemisphere. The dose of rESWT used on the head revealed remarkable advantages over sham rESWT, as demonstrated by improved neurological function scores, increased CBF, and reduced brain infarct volume. Furthermore, applying rESWT to the head and limbs enhanced short-term neurological function. Our results confirmed that rESWT can induce VEGF expression over an extended period with a profound effect, which may be the primary reason for CBF recovery. High NSE and nestin expression levels suggest that rESWT enhanced the number of neurons and neural stem cells (NSCs). <em>Wnt</em>3a and β-catenin expression were up-regulated in the ischemic hemisphere, indicating that rESWT promoted NSC proliferation and differentiation via the <em>Wnt</em>/β-catenin pathway. Overall, our findings suggest that an appropriate rESWT dose delivered to the head of rats helps restore neurological function and CBF, and additional application of rESWT to the limbs is more effective than treating the head alone.

The <em>Wnt</em>/β-catenin or canonical <em>Wnt</em> signaling pathway plays fundamental roles in early development and in maintaining adult tissue homeostasis. R-spondin 3 (Rspo3) is a secreted protein that has been implicated in activating the <em>Wnt</em>/β-catenin signaling in amphibians and mammals. Here we report that zebrafish Rspo3 plays a negative role in regulating the zygotic <em>Wnt</em>/β-catenin signaling. Zebrafish Rspo3 has a unique domain structure. It contains a third furin-like (FU3) domain. This FU3 is present in other four ray-finned fish species studied but not in elephant shark. In zebrafish, rspo3 mRNA is maternally deposited and has a ubiquitous expression in early embryonic stages. After <em>12</em> hpf, its expression becomes tissue-specific. Forced expression of rspo3 promotes dorsoanterior patterning and increases the expression of dorsal and anterior marker genes. Knockdown of rspo3 increases ventral-posterior development and stimulates ventral and posterior marker genes expression. Forced expression of rspo3 abolishes exogenous <em>Wnt</em>3a action and reduces the endogenous <em>Wnt</em> signaling activity. Knockdown of rspo3 results in increased <em>Wnt</em>/β-catenin signaling activity. Further analyses indicate that Rspo3 does not promote maternal <em>Wnt</em> signaling. Human RSPO3 has similar action when tested in zebrafish embryos. These results suggest that Rspo3 regulates dorsoventral and anteroposterior patterning by negatively regulating the zygotic <em>Wnt</em>/β-catenin signaling in zebrafish embryos.

Pancreatic adenocarcinoma (PA) is among the most aggressive human tumors with an overall 5-year survival rate of <5% and available treatments are only minimal effective. <em>WNT</em>/β-catenin signaling has been identified as one of <em>12</em> core signaling pathways that are commonly mutated in PA. To obtain more insight into the role of <em>WNT</em>/β-catenin signaling in PA we established human PA cell lines that are deficient of the central canonical <em>WNT</em> signaling protein β-catenin by using zinc-finger nuclease (ZFN) mediated targeted genomic disruption in the β-catenin gene (CTNNB1). Five individual CTNNB1 gene disrupted clones (BxPC3ΔCTNNB1) were established from a BxPC-3 founder cell line. Despite the complete absence of β-catenin, all clones displayed normal cell cycle distribution profiles, overall normal morphology and no elevated levels of apoptosis although increased doubling times were observed in three of the five BxPC3ΔCTNNB1 clones. This confirms that <em>WNT</em>/β-catenin signaling is not mandatory for long term cell growth and survival in BxPC-3 cells. Despite a normal morphology of the β-catenin deficient cell lines, quantitative proteomic analysis combined with pathway analysis showed a significant down regulation of proteins implied in cell adhesion combined with an up-regulation of plakoglobin. Treatment of BxPC3ΔCTNNB1 cell lines with siRNA for plakoglobin induced morphological changes compatible with a deficiency in the formation of functional cell to cell contacts. In addition, a re-localization of E-cadherin from membranous in untreated to accumulation in cytoplasmatic puncta in plakoglobin siRNA treated BxPC3ΔCTNNB1 cells was observed. In conclusion we describe in β-catenin deficient BxPC-3 cells a rescue function for plakoglobin on cell to cell contacts and maintaining the localization of E-cadherin at the cellular surface, but not on canonical <em>WNT</em> signaling as measured by TFC/LEF mediated transcription.

Our understanding of the mechanism(s) of pathogenesis and persistence of vertebrate parvoviruses remains incomplete. With the recent availability of the complete genome sequences of human, rat and mouse, and the ability to search these sequences and to locate matches to exact genomic regions, further insight into the interaction of parvoviruses with their human and rodent hosts is possible. To determine the extent and nature of sequence identity between candidate parvoviruses and their respective hosts, blast searches of the genome sequences of adeno-associated virus, parvovirus B19, mouse parvovirus, the prototype strain and immunosuppressant variant of minute virus of mouse, Kilham rat virus and rat parvovirus were performed against the genome(s) of their respective hosts (human, rat and mouse) using the resources of the NCBI and the Celera Discovery System. Regions of identity and similarity were mapped to their precise location in their particular host genome. For each virus, between one and <em>12</em> identical regions were found. Each identical region was 17-26 nt and was generally found at multiple sites within the particular host genome. These identical regions were predominantly located in non-coding regions of particular host genes and in intergenic regions. The ontology of host genes in which identical regions were found for each of the nine virus-host interactions highlighted several pathways/processes, including the cytoskeleton, cell adhesion and <em>Wnt</em> signalling. Within each virus species, these homologous regions were highly conserved (100 % identity in 16 out of 23 alignments where more than one sequence was available). All of these aspects suggest a particular advantage to the viruses of the presence of these sequences.

Notch proteins belong to a family of single pass transmembrane receptors that are activated after interactions with the membrane-bound ligands Delta and Jagged/Serrate. We determined the pathways responsible for the inhibitory effects of Notch on osteoblastogenesis and the contributions of the RAM domain and ankyrin repeats to this process in cells of the osteoblastic lineage.

BACKGROUND

Notch receptors play a role in osteoblast differentiation. Activation of Notch results in its cleavage and the release of its intracellular domain (NICD), which interacts with the CBF1/RBP-Jkappa, Suppressor of Hairless, Lag-1 (CSL) family of transcription factors. The interaction is presumably mediated by the RBP-Jkappa-associated module (RAM) of NICD, although the role of the ankyrin repeats is uncertain.

METHODS

To determine the contributions of the RAM domain and ankyrin repeats to the inhibitory effects of Notch on osteoblastogenesis, ST-2 and MC3T3-E1 cells were transfected or transduced with vectors expressing NICD, RAM (NICD DeltaRAM), and ankyrin (NICD DeltaANK) deletion mutants.

RESULTS

Notch increased the transactivation of transiently transfected <em>12</em>xCSL-Luc constructs, containing <em>12</em> repeats of an RBP-Jkappa/CSL binding site, and of the hairy and E (spl) (HES)-1 promoter. Deletion of the ankyrin repeats resulted in the loss of <em>12</em>xCSL-Luc and HES-1 promoter transactivation, whereas deletion of the RAM domain caused a partial loss of <em>12</em>xCSL-Luc and sustained HES-1 promoter transactivation. NICD overexpression inhibited osteocalcin mRNA levels and alkaline phosphatase activity in ST-2 cells, and deletion of the ankyrin repeats, and to a lesser extent of the RAM domain, resulted in loss of the NICD inhibitory effect. NICD inhibited <em>Wnt</em> signaling and deletion of ankyrin repeats or the RAM domain restored <em>Wnt</em> signaling activity.

CONCLUSIONS

The RAM domain and ankyrin repeats are required for Notch signaling and activity, and the CSL pathway is central to the inhibitory effect of Notch on osteoblastogenesis.

OBJECTIVE

Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease, and recent studies suggested that oxidative stress (OS) contributes to the cascade that leads to dopamine cell degeneration in PD. In this study, we hypothesized that salidroside (SDS) offers protection against OS injury in 6-hydroxydopamine (6-OHDA) unilaterally lesioned rats as well as the underlying mechanism.

METHODS

SDS and LiCl (activators of the <em>Wnt</em>/β-catenin signaling pathway) administration alone and in combination with 6-OHDA injection in rats was performed 3 days before modeling for 17 consecutive days to verify the regulatory mechanism by which SDS affects the <em>Wnt</em>/β-catenin signaling pathway as well as to evaluate the protective effect of SDS on PD in relation to OS in vivo. In addition, pheochromocytoma <em>12</em> (PC<em>12</em>) cells were incubated with 10 µmol/L SDS or LiCl alone or with both in combination for 1 h followed by a 24-h incubation with 100 µmol/L 6-OHDA to obtain in vitro data.

RESULTS

In vivo the administration of LiCl was found to ameliorate behavioral deficits and dopaminergic neuron loss; increase superoxide dismutase (SOA) activity, glutathione peroxidase (GSH-Px) levels, and glycogen synthase kinase 3β phosphorylation (GSK-3β-Ser9); reduce malondialdehyde (MDA) accumulation in the striatum and the GSK-3β mRNA level; as well as elevate β-catenin and cyclinD1 mRNA and protein levels in 6-OHDA-injected rats. This SDS treatment regimen was found to strengthen the beneficial effect of LiCl on 6-OHDA-injected rats. In vitro LiCl treatment decreased the toxicity of 6-OHDA on PC<em>12</em> cells and prevented apoptosis. Additionally, LiCl treatment increased SOA activity, GSH-Px levels, and GSK-3β-Ser9 phosphorylation; decreased MDA accumulation in the striatum and GSK-3β mRNA levels; as well as increased β-catenin and cyclinD1 mRNA and protein levels in 6-OHDA-treated PC<em>12</em> cells. Additionally, SDS treatment increased the protective effect of LiCl on 6-OHDA-treated PC<em>12</em> cells.

CONCLUSIONS

Evidence from experimental models suggested that SDS may confer neuroprotection against the neurotoxicity of 6-OHDA in response to OS injury and showed that these beneficial effects may be related to regulation of the Wnt/β-catenin signaling pathway. Therefore, SDS might be a potential therapeutic agent for treating PD.

Focal dermal hypoplasia (FDH) is an X-linked dominant disorder caused by mutations in the gene PORCN, which encodes a protein required for the secretion and signaling of <em>Wnt</em> proteins. While deletions are responsible for a small percentage of FDH-causing mutations, the vast majority of mutations are single-nucleotide substitutions or small deletions or insertions that can be identified by sequence analysis. In 2007, we implemented a PORCN gene sequencing test for individuals with a clinical diagnosis of FDH. To date, we have detected <em>12</em> novel PORCN mutations and 6 previously reported mutations in 53 such unrelated patients. The pathogenic PORCN mutations included nine nonsense mutations, three missense mutations, one small deletion, two small duplications, and three splice-site mutations. Of these mutations, two were found in affected men and were mosaic; one of these was found in three other affected women. The remaining 16 mutations were found only in women. All the mutations detected in women were presumed heterozygous. In addition to the disease-causing mutations, eight nucleotide variants of unknown significance were identified. Further characterization of these variants suggests that four of them are pathogenic mutations. These findings add to the heterogeneity of mutations in the PORCN gene that cause FDH.

BACKGROUND

Diabetic macular oedema (DMO) is the commonest cause of vision loss in people with diabetes. Laser photocoagulation can be effective in the treatment of DMO; however, its mechanism of action is still poorly understood. A better understanding of these mechanisms may allow the development of therapeutic approaches that could avoid the deleterious adverse events associated with photocoagulation.

METHODS

We have used proteomic techniques to identify the protein changes induced by threshold intensity retinal laser treatment in a rodent model of diabetic retinopathy. Retinae were obtained from diabetic Dark Agouti rats 8 weeks following laser treatment. Extracted proteins from lasered and non-lasered diabetic retinae were separated and compared using two-dimensional gel electrophoresis.

RESULTS

Image analysis revealed 24 protein spots with decreased expression after laser treatment and 9 spots with increased expression. On lasered retinal gels, four spots were uniquely expressed, with eight unique spots on non-lasered gels. Twenty-two protein spots of interest were identified using matrix-assisted desorption ionization-mass spectrometry with database matching. Following laser, <em>Wnt</em>-5 beta, LEK-1, GADPH, claudin-<em>12</em> and calretinin were significantly down-regulated in expression.

CONCLUSIONS

The present study provides a proteomic insight into the underlying biological basis for the therapeutic effects of laser for DMO. We provide further evidence of the involvement of <em>Wnt</em> pathway signalling in the neural retina in DMO, and for up to 2 months following laser treatment. Changes in LEK-1 and claudin-<em>12</em> may have effector roles, and changes in glyceraldehyde-3-phosphate dehydrogenase and calretinin may reflect the altered retinal microenvironment resulting from laser treatment.

BACKGROUND

Environmental factors contribute to the etiology of cleft palate (CP). Identification of genes that are methylated during development of the secondary palate will contribute to a better understanding of the gene-environment link contributing to CP.

METHODS

Genomic DNA fragments from secondary palate tissue from gestational days (GDs) <em>12</em> to 14 were subjected to Selective Enrichment of Methylated DNA (SEMD) and used to probe NimbleGen 2.1M mouse promoter arrays. Input (control) and SEMD samples were labeled with Cy3 and Cy5, respectively, and used for array hybridization (three arrays per GD). Data were analyzed using the Bioconductor package Ringo. Gene methylation was verified by pyrosequencing analysis and expression by quantitative real-time PCR.

RESULTS

A total of 5577 methylated genes were identified during palate development: (1) 74% of genes were methylated on all three GDs; (2) CpG islands accounted for only 30% of methylated regions of interest (MRIs); (3) location of MRIs was more often observed in gene bodies (73%) than in promoters; (4) evaluation of MRIs on GDs <em>12</em>-14 revealed no significant differentially methylated regions; (5) DAVID analysis of MRIs revealed that the cadherin and Wnt signaling pathways, as well as pathways involved in proteoglycan synthesis, were significantly enriched for methylated genes.

CONCLUSIONS

Our prior studies identified differentially expressed mRNAs and microRNAs in the developing palate. The current study complements these studies by identifying genes whose expression may be altered as a result of DNA methylation.

Research into the drug romosozumab began with the investigation of patients with excess bone formation. The understanding of the wingless-type mouse mammary tumor virus integration site (<em>Wnt</em>) signaling pathway in bone metabolism identified the negative regulator of bone mass sclerostin as a potential target for the treatment of osteoporosis. Preclinical studies confirmed this idea because they showed that sclerostin antibodies have the potential to increase bone formation. Biochemical analyses of clinical studies showed a significant increase in bone formation markers, which then slowly decreased within a year. This was accompanied by a particularly initially pronounced decrease in bone resorption. This dual mechanism of action led to an increase in bone mineral density and a significant reduction in fracture risk. Clinical vertebral fractures decreased by between 28 and 36%, nonvertebral fractures shown in a post hoc analysis by 42%. Romosozumab is administered once a month in the form of two injections. At the puncture site, reactions occur in about 5%. The most significant side effects are cardiovascular. In phase III studies, the number of serious cardiovascular complications was not significantly, albeit numerically, higher than in the control group. In Japan, South Korea, Canada, Australia, and the USA, osteoporosis patients at a high risk of fracture may already be treated with romosozumab (Evenity). Approval in the European Union was granted by 2019-<em>12</em>-<em>12</em>.

A connection between colorectal carcinogenesis and inflammation is well known, but the underlying molecular mechanisms have not been elucidated. Chemically induced colitis-associated cancer (CAC) is an outstanding mouse model for studying the link between inflammation and cancer. Additionally, the CAC model is used for examining novel diagnostic, prognostic, and predictive markers for use in clinical practice. Here, a CAC model was established in less than 100 days using azoxymethane (AOM) with dextran sulfate sodium salt (DSS) in BALB/c mice. We examined the mRNA expression profiles of three groups: control untreated mice (K), DSS-induced chronic colitis mice (D), and AOM/DSS-induced CAC (AD) mice. We identified 6301 differentially expressed genes (DEGs) among the three groups, including 93 persistently upregulated genes and 139 persistently downregulated genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that the most persistent DEGs were significantly enriched in metabolic or inflammatory components in the tumor microenvironment. Furthermore, several associated DEGs were identified as potential DEGs by protein-protein interaction (PPI) network analysis. We selected 14 key genes from the DEGs and potential DEGs for further quantitative real-time PCR (qPCR) verification. Six persistently upregulated, 3 persistently downregulated DEGs, and the other 3 genes showed results consistent with the microarray data. We demonstrated the regulation of <em>12</em> key genes specifically involved in <em>Wnt</em> signaling, cytokine and cytokine receptor interactions, homeostasis, and tumor-associated metabolism during colitis-associated CRC. Our results suggest that a close relationship between metabolic and inflammatory mediators of the tumor microenvironment is present in CAC.

Integrative genetic changes were examined in relation to tumor growth and progression of sporadic colorectal cancers. Ninety-two sporadic colorectal cancer patients and <em>12</em> human colorectal cancer cell lines were evaluated. Genetic changes in representative steps of colorectal tumorigenesis were determined. Biological characteristics, i.e., clinicopathologic parameters, expression of invasion-associated molecules, and in vitro invasion and migration, in association with these changes were further analyzed. Adenomatous polyposis coli (APC) and/or <em>Wnt</em>-activated alterations occurred in 66% patients, whereas mismatch repair (MMR) defects and/or RAF-mediated alterations were identified in 47% patients. The crossover rate between these two alterations was 26%. Differential mRNA expression of ARK5 was closely associated with that of MMP2, MMP9, and S100A4 (P< or =0.044-0.001). Additionally, enhanced ARK5 mRNA expression was more frequent in tumors displaying RAF-mediated alterations and crossover pathways (P=0.01 and 0.03, respectively). Upregulation of CEA mRNA was more common in the advanced stages (P=0.034), while VEGF expression was greater in poorly differentiated or mucinous tumors (P=0.042). The high expressions of MMP2 and MMP9 were closely associated with invasion and migration of colorectal tumors and cell lines. Our results conclusively show that specific pathways of colorectal tumorigenesis are closely associated with characteristic tumor growth and invasion.