Mediator Of Wnt Signaling Research Articles

The effect of histone deacetylase inhibitor trichostatin A on porcine mesenchymal stem cell transcriptome

The use of histone deacetylase inhibitors such as trichostatin A (TSA) for epigenetic transformation of mesenchymal stem cells (MSCs), whose nuclei will be transferred into enucleated oocytes, is a novel approach in research involving somatic cell cloning of pigs and other mammalian species. Although the effectiveness of TSA in cloning applications was confirmed, processes and mechanisms underlying achieved effects are not yet fully understood, especially for pig MSCs. To contribute to this knowledge, in this study we performed a comprehensive transcriptome analysis using high-throughput sequencing of pig bone-marrow derived MSCs, both treated and untreated with TSA, and evaluated the effect of TSA administration on their transcription profile after 24 h of in vitro culture. The expression of selected positive and negative mesenchymal surface antigens was also evaluated in these cells by flow cytometry. Subsequently, the stability of induced expression changes was evaluated after another 55–72 h of culture without TSA. The results of this study showed that TSA does not affect the expression of the selected surface antigens related to MSC mesenchymal stemness origin, namely: CD90 (positive marker), CD31 and CD34 (negative markers) and has a wide stimulating effect on MSCs transcription, affecting genes across the whole genome with some minor signs of site-specific acting in regions on SSC2 and SSC6. TSA turned out to have a higher impact on already expressed genes with only minor abilities to induce expression of silenced genes. Genes with expression affected by TSA were related to a wide range of biological processes, however, we found some evidence for specific stimulation of genes associated with development, differentiation, neurogenesis or myogenesis. TSA also seemed to interfere with Wnt signaling pathways by upregulation of several engaged genes. The analysis of cell transcriptome after prolonged culture following the TSA removal, showed that the expression level of majority of genes affected by TSA is restored to the initial level. Nonetheless, the set of about six hundred genes responsible for e.g. adhesion, signal transduction and cell communication was altered even after 55–72 h of culture without TSA. TSA also enhanced expression of some of pluripotency marker genes (FGF2, LIF, TERT) but their expression was stabilized during further culture without TSA. The detailed analysis of factors connected with neuron-like differentiation allowed us to assume that TSA mostly stimulates neurogenic differentiation pathway in the pig MSCs possibly through interaction with Wnt-mediated signaling and thus triggers mechanisms conducive to epigenetic reprograming.

Effect of sodium salt of Butrin, a novel compound isolated from Butea monosperma flowers on suppressing the expression of SIRT1 and Aurora B kinase-mediated apoptosis in colorectal cancer cells

The infrequent manifestation of SIRT1 and Aurora B kinase has shown to play a pivotal role in colorectal cancer (CRC) progression by regulating Wnt signaling pathway. The present study investigates the signaling events that regulate the modulation of SIRT1 and Aurora B kinase expression and it’s mediated cell proliferation in SW480 human primary adenocarcinoma CRC cells using Butea monosperma floral compounds (BMFC). In this, cell viability, mitochondrial mediated apoptosis, cell cycle arrest and inhibition of Wnt pathway were examined. Interestingly, the active novel compound, sodium salt of butrin, from BMFC significantly enhances the apoptosis activity, where SIRT1 and Aurora B kinase were ectopically overexpressed in CRC cells. Moreover, mRNA and protein expressions analysis indicates that the expression of GSK-3β, β-catenin, cyclin D1, pAKT, TGF-3β, SIRT1 and Aurora B kinase were down regulated in BMFC treated cells. These findings provide valuable information that the active BMFC having great impact on SIRT1 and Aurora B kinase mediated Wnt signaling down regulation in SW480 CRC cells.

Dishevelled stability is positively regulated by PKCζ-mediated phosphorylation induced by Wnt agonists

Dishevelled (Dvl) proteins are central mediators of both canonical and non-canonical Wnt signaling. It is well known that, upon Wnt stimulation, Dvl becomes phosphorylated. However, how Wnt-induced phosphorylation of Dvl is regulated and its consequences are poorly understood. Here we found that Dvl proteins are overexpressed in colon cancer cells. In addition, we found that Wnt3a treatment rapidly induces hyperphosphorylation and stabilization of Dvl2 and Dvl3. The latter can be blocked by inhibition of Protein Kinase C (PKC)α, PKCδ, and PKCζ isoforms. We also found that Wnt3a-induced phosphorylation of Dvl3 by PKCζ is required to avoid Dvl3 degradation via proteasome. This demonstrated, to our knowledge for the first time, that hyperphosphorylation of Dvl by PKCζ results in Dvl stabilization. This is clear contrast with the consequences reported to date of CK1δ/ε-mediated Dvl phosphorylation upon Wnt treatment. Mapping the interaction domain between PKCζ and Dvl3 indicated that, although the Dvl-DIX domain is required to stabilize PKCζ-phosphorylated Dvl, it is not the region phosphorylated by this kinase. Our data show that the Dvl-DEP domain, required for specific interaction with PKCζ, is the site phosphorylated by this kinase, and also probably the Dvl-C terminus. Our findings suggest a model of positive regulation of PKCζ-mediated Dvl signaling activity, to produce a strong and sustained response to Wnt3a treatment by stabilizing Dvl protein levels.

Microtubule actin cross-linking factor 1, a novel target in glioblastoma.

Genetic heterogeneity is recognized as a major contributing factor of glioblastoma resistance to clinical treatment modalities and consequently low overall survival rates. This genetic diversity results in variations in protein expression, both intratumorally and between individual glioblastoma patients. In this regard, the spectraplakin protein, microtubule actin cross-linking factor 1 (MACF1), was examined in glioblastoma. An expression analysis of MACF1 in various types of brain tumor tissue revealed that MACF1 was predominately present in gradeIII-IV astroctyomas and gradeIV glioblastoma, but not in normal brain tissue, normal human astrocytes and lower grade brain tumors. Subsequent genetic inhibition experiments showed that suppression of MACF1 selectively inhibited glioblastoma cell proliferation and migration in cell lines established from patient derived xenograft mouse models and immortalized glioblastoma cell lines that were associated with downregulation of the Wnt-signaling mediators, Axin1 and β-catenin. Additionally, concomitant MACF1 silencing with the chemotherapeutic agent temozolomide (TMZ) used for the clinical treatment of glioblastomas cooperatively reduced the proliferative capacity of glioblastoma cells. In conclusion, the present study represents the first investigation on the functional role of MACF1 in tumor cell biology, as well as demonstrates its potential as a unique biomarker that can be targeted synergistically with TMZ as part of a combinatorial therapeutic approach for the treatment of genetically multifarious glioblastomas.

CK1δ: a pharmacologically tractable Achilles' heel of Wnt-driven cancers?

Aberrant Wnt signaling has been widely accepted to be a key driver of a subset of human cancers and a heavily scrutinized molecular pathway for the development of personalized medicine. In a recently published issue of Science Translational Medicine, Rosenberg and coworkers reported that the delta isoform of the CK1 family of serine/threonine kinases (CK1δ), an important mediator of intracellular Wnt signaling, is amplified and overexpressed in human breast tumors. They further demonstrated that pharmacological inhibition of CK1δ is efficacious for these cancers and implicate β-catenin signaling as a key target of CK1δ. In this perspective, we will discuss the salient features of this novel anti-cancer therapeutic approach and the challenges that lie ahead to translate it into a viable treatment option for cancer patients.

BET bromodomain is a novel regulator of TAZ and its activity

Transcriptional coactivator with PDZ-binding motif (TAZ) is a key transcriptional mediator of Hippo signaling that has been recently reported to mediate Wnt-activated transcription and serve as a component to suppress canonical Wnt/β-catenin activity. The Bromodomain and Extra-terminal domain (BET) family of proteins can recognize the acetylated lysine chain on histones and plays a critical role in transcriptional regulation. However, the mechanisms underlying transcriptional repression by the BET bromodomain are poorly understood. Here, we found that BET bromodomain inhibition upregulated TAZ protein and its transcriptional output, independent of its well-established role as a mediator of Hippo and Wnt signaling. Additionally, JQ1, a synthetic BET inhibitor, suppressed Wnt/β-catenin activity by upregulating TAZ. Although JQ1 upregulated TAZ, which is known to promote cell proliferation, it drastically suppressed the growth of colon cancer cells by inducing cell cycle arrest. Collectively, our study identified an unexpected transcriptional repression function of the BET bromodomain and a novel mechanism for TAZ upregulation.

PTHrP-Derived Peptides Restore Bone Mass and Strength in Diabetic Mice: Additive Effect of Mechanical Loading.

There is an unmet need to understand the mechanisms underlying skeletal deterioration in diabetes mellitus (DM) and to develop therapeutic approaches to treat bone fragility in diabetic patients. We demonstrate herein that mice with type 1 DM induced by streptozotocin exhibited low bone mass, inferior mechanical and material properties, increased bone resorption, decreased bone formation, increased apoptosis of osteocytes, and increased expression of the osteocyte-derived bone formation inhibitor Sost/sclerostin. Further, short treatment of diabetic mice with parathyroid hormone related protein (PTHrP)-derived peptides corrected these changes to levels undistinguishable from non-diabetic mice. In addition, diabetic mice exhibited reduced bone formation in response to mechanical stimulation, which was corrected by treatment with the PTHrP peptides, and higher prevalence of apoptotic osteocytes, which was reduced by loading or by the PTHrP peptides alone and reversed by a combination of loading and PTHrP peptide treatment. In vitro experiments demonstrated that the PTHrP peptides or mechanical stimulation by fluid flow activated the survival kinases ERKs and induced nuclear translocation of the canonical Wnt signaling mediator β-catenin, and prevented the increase in osteocytic cell apoptosis induced by high glucose. Thus, PTHrP-derived peptides cross-talk with mechanical signaling pathways to reverse skeletal deterioration induced by DM in mice. These findings suggest a crucial role of osteocytes in the harmful effects of diabetes on bone and raise the possibility of targeting these cells as a novel approach to treat skeletal deterioration in diabetes. Moreover, our study suggests the potential therapeutic efficacy of combined pharmacological and mechanical stimuli to promote bone accrual and maintenance in diabetic subjects. © 2016 American Society for Bone and Mineral Research.

Distinct hydrophobic “patches” in the N- and C-tails of beta-catenin contribute to nuclear transport

β-catenin is a key mediator of Wnt signaling and its deregulated nuclear accumulation can drive cancer progression. While the central armadillo (Arm) repeats of β-catenin stimulate nuclear entry, the N- and C-terminal “tail” sequences are thought to regulate turnover and transactivation. We show here that the N- and C-tails are also potent transport sequences. The unstructured tails of β-catenin, when individually fused to a GFP-reporter, could enter and exit the nucleus rapidly in live cells. Proximity ligation assays and pull-down assays identified a weak interaction between the tail sequences and the FG-repeats of nucleoporins, consistent with a possible direct translocation of β-catenin through the nuclear pore complex. Extensive alanine mutagenesis of the tail sequences revealed that nuclear translocation of β-catenin was dependent on specific uniformly distributed patches of hydrophobic residues, whereas the mutagenesis of acidic amino acids had no effect. Moreover, the mutation of hydrophobic patches within the N-tail and C-tail of full length β-catenin reduced nuclear transport rate and diminished its ability to activate transcription. We propose that the tail sequences can contribute to β-catenin transport and suggest a possible similar role for hydrophobic unstructured regions in other proteins.

Effect of concentrated growth factors on function and Wnt3a expression of human periodontal ligament cells in vitro

Concentrated growth factors (CGFs), the new generation of platelet concentrate products, appear to exhibit superior potential for tissue regeneration. However, there are only a few studies supporting this. This study was designed to investigate the effect of CGFs on proliferation and alkaline phosphatase (ALP) activity of human periodontal ligament cells (hPDLCs) in vitro. Furthermore, as bone homeostasis is fundamentally controlled by Wnt-mediated signals, we also investigated Wnt3a expression of hPDLCs after treatment of CGFs. hPDLCs and CGFs were obtained from the same volunteer. CGFs or combination of recombined human TGF-β1 (rhTGF-β1) and PDGF-AB (rhPDGF-AB) were added to hPDLCs in different concentrations. The rate of proliferation was analyzed by an MTT assay. ALP activity was assessed using p-NPP assay. Quantitative RT-PCR was used to evaluate the gene expression of Wnt3a. In a range of concentrations, CGFs significantly promoted the proliferation of hPDLCs in a dose-dependent manner. ALP activity was also enhanced by CGFs in a dose-dependent and time-dependent manner. The stimulatory effect of CGFs was much greater than rhTGF-β1 and rhPDGF-AB combination. Quantitative RT-PCR results showed that Wnt3a mRNA expression was increased at 24 h in hPDLCs treated by CGFs. CGFs can enhance hPDLCs proliferation and ALP activity and may have great potential in clinical and biotechnological applications.

Divergent effects of Porcupine and Wntless on WNT1 trafficking, secretion, and signaling

Loss-of-function studies have identified Porcupine (PORCN) and Wntless (WLS) as essential mediators of Wnt secretion and signaling. Whereas PORCN is thought to palmitoylate Wnt proteins, WLS is believed to transport palmitoylated Wnt proteins to the cell surface. However, little is known about how these two proteins cooperate to regulate Wnt palmitoylation, trafficking, secretion, and signaling. We first investigated possible interactions between PORCN, WLS, and WNT1, by carrying out co-immunoprecipitation studies. These studies demonstrate the existence of a complex containing PORCN and WLS. They further show that PORCN and WLS compete for binding to WNT1. Then, we used gain-of-function studies to investigate the cooperation between PORCN and WLS as well as possible biochemical interactions between PORCN, WLS, and WNT1. Consistent with the proposed roles for PORCN and WLS, we show that overexpression of PORCN promotes palmitoylation of WNT1 while overexpression of WLS does not. Overexpression of PORCN enhances the ability of WLS to promote WNT1 trafficking to the cell surface as well as secretion, but decreases the ability of WLS to activate WNT1 signaling in target cell. These observations suggest that the levels of WNT1 on the cell surface and in the media are not the sole determinants of the activation of Wnt signaling in target cells.

WISP1, a downstream mediator of canonical WNT signaling, induces pathology in experimental osteoarthritis and predicts disease progression in early osteoarthritis patients.

WISP1, a downstream mediator of canonical WNT signaling, induces pathology in experimental osteoarthritis and predicts disease progression in early osteoarthritis patients.

Protective LRRK2 R1398H Variant Enhances GTPase and Wnt Signaling Activity.

Mutations in LRRK2 are a common cause of familial and idiopathic Parkinson’s disease (PD). Recently, the LRRK2 GTPase domain R1398H variant was suggested in genetic studies to confer protection against PD but mechanistic data supporting this is lacking. Here, we present evidence that R1398H affects GTPase function, axon outgrowth, and Wnt signaling in a manner opposite to pathogenic LRRK2 mutations. LRRK2 R1398H GTPase domain dimerization and GTP hydrolysis were increased whereas GTP binding was reduced, leading to a decrease in active GTP-bound LRRK2. This protective variant also increased axon length of primary cortical neurones in comparison to wild-type LRRK2, whereas the R1441G LRRK2 pathogenic mutant decreased axon outgrowth. Importantly, R1398H enhanced the stimulatory effect of LRRK2 on canonical Wnt signaling whereas the G2385R risk variant, in accordance with all previously tested pathogenic LRRK2 mutants, had the opposite effect. Molecular modeling placed R1398H in close proximity to PD-causing mutations suggesting that this protective LRRK2 variant, like familial mutations, affects intramolecular RocCOR domain interactions. Thus, our data suggest that R1398H LRRK2 is a bona fide protective variant. The opposite effects of protective versus PD associated LRRK2 variants on GTPase function and canonical Wnt signaling activity also suggests that regulation of these two basic signaling mechanisms is important for neuronal function. We conclude that LRRK2 mediated Wnt signaling and GTPase function are fundamental in conferring disease susceptibility and have clear implications for therapeutic target identification.

Wnt activation protects against neomycin-induced hair cell damage in the mouse cochlea.

Recent studies have reported the role of Wnt/β-catenin signaling in hair cell (HC) development, regeneration, and differentiation in the mouse cochlea; however, the role of Wnt/β-catenin signaling in HC protection remains unknown. In this study, we took advantage of transgenic mice to specifically knockout or overactivate the canonical Wnt signaling mediator β-catenin in HCs, which allowed us to investigate the role of Wnt/β-catenin signaling in protecting HCs against neomycin-induced damage. We first showed that loss of β-catenin in HCs made them more vulnerable to neomycin-induced injury, while constitutive activation of β-catenin in HCs reduced HC loss both in vivo and in vitro. We then showed that loss of β-catenin in HCs increased caspase-mediated apoptosis induced by neomycin injury, while β-catenin overexpression inhibited caspase-mediated apoptosis. Finally, we demonstrated that loss of β-catenin in HCs led to increased expression of forkhead box O3 transcription factor (Foxo3) and Bim along with decreased expression of antioxidant enzymes; thus, there were increased levels of reactive oxygen species (ROS) after neomycin treatment that might be responsible for the increased aminoglycoside sensitivity of HCs. In contrast, β-catenin overexpression reduced Foxo3 and Bim expression and ROS levels, suggesting that β-catenin is protective against neomycin-induced HC loss. Our findings demonstrate that Wnt/β-catenin signaling has an important role in protecting HCs against neomycin-induced HC loss and thus might be a new therapeutic target for the prevention of HC death.

Prickle1 mutation causes planar cell polarity and directional cell migration defects associated with cardiac outflow tract anomalies and other structural birth defects.

ABSTRACTPlanar cell polarity (PCP) is controlled by a conserved pathway that regulates directional cell behavior. Here, we show that mutant mice harboring a newly described mutation termed Beetlejuice (Bj) in Prickle1 (Pk1), a PCP component, exhibit developmental phenotypes involving cell polarity defects, including skeletal, cochlear and congenital cardiac anomalies. Bj mutants die neonatally with cardiac outflow tract (OFT) malalignment. This is associated with OFT shortening due to loss of polarized cell orientation and failure of second heart field cell intercalation mediating OFT lengthening. OFT myocardialization was disrupted with cardiomyocytes failing to align with the direction of cell invasion into the outflow cushions. The expression of genes mediating Wnt signaling was altered. Also noted were shortened but widened bile ducts and disruption in canonical Wnt signaling. Using an in vitro wound closure assay, we showed Bj mutant fibroblasts cannot establish polarized cell morphology or engage in directional cell migration, and their actin cytoskeleton failed to align with the direction of wound closure. Unexpectedly, Pk1 mutants exhibited primary and motile cilia defects. Given Bj mutant phenotypes are reminiscent of ciliopathies, these findings suggest Pk1 may also regulate ciliogenesis. Together these findings show Pk1 plays an essential role in regulating cell polarity and directional cell migration during development.

Natural Functions of PLIN2 Mediating Wnt/LiCl Signaling and Glycogen Synthase Kinase 3 (GSK3)/GSK3 Substrate-Related Effects Are Modulated by Lipid.

Belonging to the PLIN family, PLIN2 associates with lipid storage droplets (LSDs), but other functions of PLIN2 remain unclear. Here, we suggest that PLIN2 mediates Wnt signaling because PLIN2 small interfering RNA (siRNA) suppresses activation of Wnt/coreceptor pathways. The mediation in the Wnt/Frizzled pathway seems to occur from Dishevelleds to axin/glycogen synthase kinase 3(GSK3)/β-catenin complexes (AGβC) as Wnt decreases Dishevelled/PLIN2 but increases AGβC/PLIN2 associations. Augmenting cellular LSDs that affect PLIN2 associations with these proteins, oleic acid (OA) treatment inhibits Wnt-increased AGβC/PLIN2 associations and β-catenin T-cell factor signaling (β-CTS). Revealing that PLIN2 is a GSK3-associated protein, the study explored PLIN2-mediated effects on GSK3/GSK3 substrates. PLIN2 siRNA reduces inhibitory GSK3 levels and lithium chloride (LiCl)-upregulated β-catenin or CCAAT/enhancer binding protein α (c/EBPα) expression. OA treatment decreases LiCl-increased c/EBPα via PLIN2-c/EBPα dissociation. In addition to PLIN2 overexpression increasing β-CTS, PLIN2 depletion or overexpression drops or adds expression of GSK3 substrates, such as β-catenin, c/EBPα,c-Myc, cyclin D1, and insulin receptor substrate 1, and cell growth/survival. PLIN2 N or C terminus overexpression that is associated with higher levels of the substrates suggests that those substrates bind to specific regions of PLIN2. Mimicking the possible high lipid concentrations in cells in the human body under conditions of hyperlipidemia/obesity, OA-treated cells gain or reduce GSK3 substrate expression in parallel with a decrease (a Wnt-like effect) or increase in GSK3 activity, likely regulated by GSK3/PLIN2/GSK3 substrate associations.

The 1918 Influenza Virus PB2 Protein Enhances Virulence through the Disruption of Inflammatory and Wnt-Mediated Signaling in Mice.

The 1918-1919 influenza pandemic remains the single greatest infectious disease outbreak in the past century. Mouse and nonhuman primate infection models have shown that the 1918 virus induces overly aggressive innate and proinflammatory responses. To understand the response to viral infection and the role of individual 1918 genes on the host response to the 1918 virus, we examined reassortant avian viruses nearly identical to the pandemic 1918 virus (1918-like avian virus) carrying either the 1918 hemagglutinin (HA) or PB2 gene. In mice, both genes enhanced 1918-like avian virus replication, but only the mammalian host adaptation of the 1918-like avian virus through reassortment of the 1918 PB2 led to increased lethality. Through the combination of viral genetics and host transcriptional profiling, we provide a multidimensional view of the molecular mechanisms by which the 1918 PB2 gene drives viral pathogenicity. We demonstrate that 1918 PB2 enhances immune and inflammatory responses concomitant with increased cellular infiltration in the lung. We also show for the first time, that 1918 PB2 expression results in the repression of both canonical and noncanonical Wnt signaling pathways, which are crucial for inflammation-mediated lung regeneration and repair. Finally, we utilize regulatory enrichment and network analysis to define the molecular regulators of inflammation, epithelial regeneration, and lung immunopathology that are dysregulated during influenza virus infection. Taken together, our data suggest that while both HA and PB2 are important for viral replication, only 1918 PB2 exacerbates lung damage in mice infected with a reassortant 1918-like avian virus. As viral pathogenesis is determined in part by the host response, understanding the key host molecular driver(s) of virus-mediated disease, in relation to individual viral genes, is a promising approach to host-oriented drug efforts in preventing disease. Previous studies have demonstrated the importance of host adaptive genes, HA and PB2, in mediating disease although the mechanisms by which they do so are still poorly understood. Here, we combine viral genetics and host transcriptional profiling to show that although both 1918 HA and 1918 PB2 are important mediators of efficient viral replication, only 1918 PB2 impacts the pathogenicity of an avian influenza virus sharing high homology to the 1918 pandemic influenza virus. We demonstrate that 1918 PB2 enhances deleterious inflammatory responses and the inhibition of regeneration and repair functions coordinated by Wnt signaling in the lungs of infected mice, thereby promoting virus-associated disease.

Abstract 18494: WNT Mediated Regulation Signals IN Chamber-Specific Neonatal Heart Maturation and Disease

Background: Chamber-specific and temporally orchestrated perinatal cardiac growth and maturation is critical to functional adaptation of heart to the dramatic changes in hemodynamic load and nutrient environment, and perturbation of this process may have major implications in congenital heart defects (CHDs). However, the responsible molecular regulatory mechanisms are still poorly understood. Objective: To determine the factors that control chamber specificity of neonatal heart growth during perinatal maturation Methods: Total transcriptome was analyzed from deep RNA-seq in male newborn mouse (C57BL) left ventricle (LV) and right ventricle (RV) at postnatal day 0, 3 and 7. Functional validation was performed in Neonatal Rat Ventricular Myocytes (NRVMs) and intact neonatal mice. Results: Chamber specific- regulation of cell cycle genes was correlated with chamber specific- expression of the non-canonical Wnt11. Exogenous expression of Wnt11 induced maturation markers and suppressed cell cycle genes in myocytes. In contrast, targeted Wnt11 inhibition induced myocyte proliferation associated with upregulated cell cycle genes both in cultured NRVMs and in the intact heart. Notably, Wnt inhibition also significantly enhanced β-catenin phosphorylation without impacting JNK, PKCα, or p38- activities. Remarkably, both Wnt mediated signals and cell cycle markers were altered in heart samples from infants cyanotic CHDs. Furthermore, hypoxia exposure suppressed Wnt11 and induced cell cycle genes in cultured cardiomyocytes. Conclusions: Differential expression of Wnt11 and downstream cell cycle genes are novel molecular basis for chamber specific maturation. Wnt11 mediated-signaling has potential impact on cyanotic CHDs. Differential role of Wnt signaling in chamber specific post-natal heart maturation can lead to potential chamber-specific diagnoses and therapies for congenital heart diseases.

DAAM1 and DAAM2 are co-required for myocardial maturation and sarcomere assembly

Wnt ligands regulate heart morphogenesis but the underlying mechanisms remain unclear. Two Formin-related proteins, DAAM1 and 2, were previously found to bind the Wnt effector Disheveled. Here, since DAAM1 and 2 nucleate actin and mediate Wnt-induced cytoskeletal changes, a floxed-allele of Daam1 was used to disrupt its function specifically in the myocardium and investigate Wnt-associated pathways. Homozygous Daam1 conditional knockout (CKO) mice were viable but had misshapen hearts and poor cardiac function. The defects in Daam1 CKO mice were observed by mid-gestation and were associated with a loss of protrusions from cardiomyocytes invading the outflow tract. Further, these mice exhibited noncompaction cardiomyopathy (NCM) and deranged cardiomyocyte polarity. Interestingly, Daam1 CKO mice that were also homozygous for an insertion disrupting Daam2 (DKO) had stronger NCM, severely reduced cardiac function, disrupted sarcomere structure, and increased myocardial proliferation, suggesting that DAAM1 and DAAM2 have redundant functions. While RhoA was unaffected in the hearts of Daam1/2 DKO mice, AKT activity was lower than in controls, raising the issue of whether DAAM1/2 are only mediating Wnt signaling. Daam1-floxed mice were thus bred to Wnt5a null mice to identify genetic interactions. The hearts of Daam1 CKO mice that were also heterozygous for the null allele of Wnt5a had stronger NCM and more severe loss of cardiac function than Daam1 CKO mice, consistent with DAAM1 and Wnt5a acting in a common pathway. However, deleting Daam1 further disrupted Wnt5a homozygous-null hearts, suggesting that DAAM1 also has Wnt5a-independent roles in cardiac development.

Nicotine reduces the levels of surfactant proteins A and D via Wnt/β-catenin and PKC signaling in human airway epithelial cells

A deficiency of surfactant proteins A and D has been proposed as a mechanism in airway remodeling, which is one characteristic of chronic obstructive pulmonary disease (COPD). We recently showed that in vitro nicotine exposure induces Wnt3a/β-catenin activation, which is a pathway that has also been implicated in altering levels of SP-A and SP-D. Nicotine induced activation of protein kinase C(PKC), and the involvement of PKC in mediating Wnt signaling has been demonstrated previously. The main aim of this study was to investigate whether human bronchial epithelial cells reduce levels of SP-A and SP-D in vitro following nicotine stimulation via the Wnt3a/β-catenin and PKC signaling pathway. We showed that nicotine activated the Wnt3a/β-catenin and PKC signaling pathway, and this activation was accompanied by a decrease in SP-A and SP-D expression. Knockdown of Wnt3a with small interfering RNA (siRNA) prevented translocation of β-catenin into the nucleus and reduction levels of SP-A and SP-D. Furthermore, a PKC inhibitor partially prevented these effects,which suggests in HBECs, Wnt3a/β-catenin and PKC pathways interact during nicotine-reduced levels of SP-A and SP-D. These results suggest that HBECs reduce the levels of surfactant proteins A and D in vitro via the Wnt3a/β-catenin and PKC signaling pathway upon nicotine stimulation.

Lgr6 marks nail stem cells and is required for digit tip regeneration

The tips of the digits of some mammals, including human infants and mice, are capable of complete regeneration after injury. This process is reliant on the presence of the overlaying nail organ and is mediated by a proliferative blastema. Epithelial Wnt/β-catenin signaling has been shown to be necessary for mouse digit tip regeneration. Here, we report on Lgr5 and Lgr6 (leucine-rich repeat-containing G protein-coupled receptor 5 and 6), two important agonists of the Wnt pathway that are known to be markers of several epithelial stem cell populations. We find that Lgr5 is expressed in a dermal population of cells adjacent to the specialized epithelia surrounding the keratinized nail plate. Moreover, Lgr5-expressing cells contribute to this dermis, but not the blastema, during digit tip regeneration. In contrast, we find that Lgr6 is expressed within cells of the nail matrix portion of the nail epithelium, as well as in a subset of cells in the bone and eccrine sweat glands. Genetic lineage analysis reveals that Lgr6-expressing cells give rise to the nail during homeostatic growth, demonstrating that Lgr6 is a marker of nail stem cells. Moreover, Lgr6-expressing cells contribute to the blastema, suggesting a potential direct role for Lgr6-expressing cells during digit tip regeneration. This role is confirmed by analysis of Lgr6-deficient mice, which have both a nail and bone regeneration defect.