Aberrant activation of Wnt/β-catenin signaling plays an important role in the pathogenesis of breast cancer. DACT1 (Dapper/Frodo) has been identified as involved in antagonizing Wnt/β-catenin signaling through interacting with Dishevelled (Dvl), a central mediator of Wnt signaling, whereas its role in breast tumorigenesis remains unclear.
We examined DACT1 expression in breast cancer cell lines and primary tumors with semiquantitative or quantitative RT-PCR and immunochemistry, and further evaluated the promoter methylation of DACT1 with methylation-specific PCR (MSP). We also explored the tumor-suppressive functions of DACT1 in vivo and in vitro, and its related mechanism in breast cancer.
We identified DACT1 as a methylated target in our breast cancer epigenome study. Here, we further investigated DACT1 expression in multiple breast cell lines and primary tumors, and further studied its function and molecular mechanisms. We found that DACT1 expression was silenced in eight (88.9%) of nine breast cancer cell lines, and its protein levels were obviously reduced in breast tumors compared with paired surgical-margin tissues. Promoter CpG methylation of DACT1 was detected in five (55.6%) of nine breast cancer cell lines and 40 (29.9%) of 134 primary tumors, but not in surgical-margin tissues and normal breast tissues. Demethylation treatment of breast cancer cell lines restored DACT1 expression along with promoter demethylation, suggesting that an epigenetic mechanism mediates DACT1 silencing in breast cancer. Functional assays showed that ectopic expression of DACT1 could inhibit breast tumor cell proliferation in vivo and in vitro through inducing apoptosis, and further suppress tumor cell migration through antagonizing the Wnt/β-catenin signaling pathway.
Our study demonstrates that DACT1 could function as a tumor suppressor but was frequently downregulated in breast cancer.
The Wnt/β-catenin signaling pathway plays important roles in the progression of colon cancer. DACT1 has been identified as a modulator of Wnt signaling through its interaction with Dishevelled (Dvl), a central mediator of both the canonical and noncanonical Wnt pathways. However, the functions of DACT1 in the WNT/β-catenin signaling pathway remain unclear. Here, we present evidence that DACT1 is an important positive regulator in colon cancer through regulating the stability and sublocation of β-catenin. We have shown that DACT1 promotes cancer cell proliferation in vitro and tumor growth in vivo and enhances the migratory and invasive potential of colon cancer cells. Furthermore, the higher expression of DACT1 not only increases the nuclear and cytoplasmic fractions of β-catenin, but also increases its membrane-associated fraction. The overexpression of DACT1 leads to the increased accumulation of nonphosphorylated β-catenin in the cytoplasm and particularly in the nuclei. We have demonstrated that DACT1 interacts with GSK-3β and β-catenin. DACT1 stabilizes β-catenin via DACT1-induced effects on GSK-3β and directly interacts with β-catenin proteins. The level of phosphorylated GSK-3β at Ser9 is significantly increased following the elevated expression of DACT1. DACT1 mediates the subcellular localization of β-catenin via increasing the level of phosphorylated GSK-3β at Ser9 to inhibit the activity of GSK-3β. Taken together, our study identifies DACT1 as an important positive regulator in colon cancer and suggests a potential strategy for the therapeutic control of the β-catenin-dependent pathway.
Dact proteins belong to the Dapper/Frodo protein family and function as cytoplasmic attenuators in Wnt and TGFβ signaling. Previous studies show that Dact1 is a potent Wnt signaling inhibitor by promoting degradation of β-catenin. We report a new mechanism for Dact2 function as an inhibitor of the canonical Wnt signaling pathway by interacting with PITX2. PITX2 is a downstream transcription factor in Wnt/β-catenin signaling, and PITX2 synergizes with Lef-1 to activate downstream genes. Immunohistochemistry verified the expression of Dact2 in the tooth epithelium, which correlated with Pitx2 epithelial expression. Dact2 loss of function and PITX2 gain of function studies reveal a feedback mechanism for controlling Dact2 expression. Pitx2 endogenously activates Dact2 expression and Dact2 feeds back to repress Pitx2 transcriptional activity. A Topflash reporter system was employed showing PITX2 activation of Wnt signaling, which is attenuated by Dact2. Transient transfections demonstrate the inhibitory effect of Dact2 on critical dental epithelial differentiation factors during tooth development. Dact2 significantly inhibits PITX2 activation of the Dlx2 and amelogenin promoters. Multiple lines of evidence conclude the inhibition is achieved by the physical interaction between Dact2 and Pitx2 proteins. The loss of function of Dact2 also reveals increased cell proliferation due to up-regulated Wnt downstream genes, cyclinD1 and cyclinD2. In summary, we have identified a novel role for Dact2 as an inhibitor of the canonical Wnt pathway in embryonic tooth development through its regulation of cell proliferation and differentiation.
Dapper homolog 1 (DACT1) is a disheveled partner in the planar cell polarity pathway. By using genome-wide promoter methylation screening, dapper homolog 1 (DACT1) was found to be frequently methylated in gastric cancer. We aim to clarify its epigenetic inactivation, biological function and clinical implication in gastric cancer. We demonstrated that DACT1 was silenced in 7 of 10 gastric cancer cell lines and in primary gastric cancers. Transcriptional gene silence of DACT1 was mainly regulated by promoter hypermethylation. Ectopic expression of DACT1 in silenced gastric cancer cell lines (AGS, BGC823 and MGC803) by stable transfection suppressed colony formation (P < 0.001), induced cell apoptosis (P < 0.01) and retarded tumorigenesis in nude mice (P < 0.001). The tumor suppressive effect of DACT1 was further confirmed by loss of DACT1 function experiment. The proapoptotic and antiproliferative effect by DACT1 was associated with inhibition of nuclear factor (NF)-κB activation and its downstream factors, including B-cell CLL/lymphoma-2, Bcl-X, interleukin-8 and tumor necrosis factor-α. Moreover, promoter methylation of DACT1 was detected in 29.3% (60/205) of primary gastric tumors. DACT1 methylation was significantly associated with tumor metastasis (P < 0.05), invasion (P < 0.05) and advanced tumor stage (P < 0.0005). These findings provided insight into the role of DACT1 as a novel functional tumor suppressor in gastric cancer through inhibiting NF-κB signaling pathway. Promoter methylation of DACT1 is associated with tumor aggressiveness.
Dapper, Dishevelled-associated antagonist of β-catenin (DACT), is a key regulator of Wnt signaling pathway. The purpose of this study is to explore the epigenetic changes and the function ofDACT2 in human gastric cancer (GC). Eight human gastric cancer cell lines, 167 cases of primary gastric cancer and 8 cases of normal gastric mucosa were involved in this study. In addition, methylation Specific PCR (MSP), semi-quantitative RT-PCR, colony formation assay, flow cytometry assay, siRNA, immunofluorescence techniques and xenograft mice models were employed. The results indicate that DACT2 is frequently methylated in human primary gastric cancer (55.7%), and that methylation of DACT2 is associated with lost or reduction in its expression (X2 test, P<0.01). We found that DACT2 expression was regulated by promoter region hypermethylation. Methylation of DACT2 is associated with tumor differentiation, invasion and intravascular cancerous emboli (X2 test, P<0.05, P<0.05 and P<0.05). In gastric cancer patients treated with 5-FU and cisplatin, the five-year survival rates are higher in DACT2 methylated cases. DACT2 inhibits cell proliferation, migration and invasion in gastric cancer cells and suppresses gastric cancer xenografts in mice. Restoration of DACT2 expression inhibits both canonical and noncanonical WNT signaling in SGC7901 cells. Restoration of DACT2 expression sensitized gastric cancer cells to paclitaxel and 5-FU. In conclusion, DACT2 is frequently methylated in human gastric cancer and DACT2 expression is silenced by promoter region hypermethylation. DACT2 suppressed gastric cancer proliferation, invasion and metastasis by inhibiting Wnt signaling both in vitro and in vivo.
Gastric cancer; DACT2; DNA methylation; Wnt signaling pathway; chemo-sensitivity
The Dact family of scaffold proteins was discovered by virtue of binding to Dvl proteins central to Wnt and Planar Cell Polarity (PCP) signaling. Subsequently Dact proteins have been linked to a growing list of potential partners implicated in β-catenin-dependent and β-catenin-independent forms of Wnt and other signaling. To clarify conserved and non-conserved roles for this protein family, we systematically compared molecular interactions of all three murine Dact paralogs by co-immunoprecipitation of proteins recombinantly expressed in cultured human embryonic kidney cells.
Every Dact paralog readily formed complexes with the Vangl, Dvl, and CK1δ/ε proteins of species ranging from fruit flies to humans, as well as with PKA and PKC. Dact proteins also formed complexes with themselves and with each other; their conserved N-terminal leucine-zipper domains, which have no known binding partners, were necessary and sufficient for this interaction, suggesting that it reflects leucine-zipper-mediated homo- and hetero-dimerization. We also found weaker, though conserved, interactions of all three Dact paralogs with the catenin superfamily member p120ctn. Complex formation with other previously proposed partners including most other catenins, GSK3, LEF/TCF, HDAC1, and TGFβ receptors was paralog-specific, comparatively weak, and/or more sensitive to empirical conditions.
Combined with published functional evidence from targeted knock-out mice, these data support a conserved role for Dact proteins in kinase-regulated biochemistry involving Vangl and Dvl. This strongly suggests that a principal role for all Dact family members is in the PCP pathway or a molecularly related signaling cascade in vertebrates.
Synaptogenesis has been extensively studied along with dendritic spine development in glutamatergic pyramidal neurons, however synapse development in cortical interneurons, which are largely aspiny, is comparatively less well understood. Dact1, one of 3 paralogous Dact (Dapper/Frodo) family members in mammals, is a scaffold protein implicated in both the Wnt/β-catenin and the Wnt/Planar Cell Polarity pathways. We show here that Dact1 is expressed in immature cortical interneurons. Although Dact1 is first expressed in interneuron precursors during proliferative and migratory stages, constitutive Dact1 mutant mice have no major defects in numbers or migration of these neurons. However, cultured cortical interneurons derived from these mice have reduced numbers of excitatory synapses on their dendrites. We selectively eliminated Dact1 from mouse cortical interneurons using a conditional knock-out strategy with a Dlx-I12b enhancer-Cre allele, and thereby demonstrate a cell-autonomous role for Dact1 during postsynaptic development. Confirming this cell-autonomous role, we show that synapse numbers in Dact1 deficient cortical interneurons are rescued by virally-mediated re-expression of Dact1 specifically targeted to these cells. Synapse numbers in these neurons are also rescued by similarly targeted expression of the Dact1 binding partner Dishevelled-1, and partially rescued by expression of Disrupted in Schizophrenia-1, a synaptic protein genetically implicated in susceptibility to several major mental illnesses. In sum, our results support a novel cell-autonomous postsynaptic role for Dact1, in cooperation with Dishevelled-1 and possibly Disrupted in Schizophrenia-1, in the formation of synapses on cortical interneuron dendrites.
Thyroid cancer is the most common endocrine malignant disease and the incidence is increasing. DACT2 was found frequently methylated in human lung cancer and hepatocellular carcinoma. To explore the epigenetic change and the role of DACT2 in thyroid cancer, 7 thyroid cancer cell lines, 10 cases of non-cancerous thyroid tissue samples and 99 cases of primary thyroid cancer samples were involved in this study. DACT2 was expressed and unmethylated in K1, SW579, FTC-133, TT, W3 and 8505C cell lines. Loss of expression and complete methylation was found in TPC-1 cells. Restoration of DACT2 expression was induced by 5-aza-2′deoxycytidine treatment. It demonstrates that the expression of DACT2 was regulated by promoter region methylation. In human primary papillary thyroid cancer, 64.6% (64/99) was methylated and methylation of DACT2 was related to lymph node metastasis (p<0.01). Re-expression of DACT2 suppresses cell proliferation, invasion and migration in TPC-1 cells. The activity of TCF/LEF was inhibited by DACT2 in wild-type or mutant β-catenin cells. The activity of TCF/LEF was increased by co-transfecting DACT2 and Dvl2 in wild-type or mutant β-catenin cells. Overexpression of wild-type β-catenin promotes cell migration and invasion in DACT2 stably expressed cells. The expression of β-catenin, c-myc, cyclinD1 and MMP-9 were decreased and the level of phosphorylated β-catenin (p-β-catenin) was increased after restoration of DACT2 expression in TPC-1 cells. The expression of β-catenin, c-myc, cyclinD1 and MMP-9 were increased and the level of p-β-catenin was reduced after knockdown of DACT2 in W3 and SW579 cells. These results suggest that DACT2 suppresses human papillary thyroid cancer growth and metastasis by inhibiting Wnt signaling. In conclusion, DACT2 is frequently methylated in papillary thyroid cancer. DACT2 expression was regulated by promoter region methylation. DACT2 suppresses papillary thyroid cancer proliferation and metastasis by inhibiting Wnt signaling.
In mice, genetically engineered knockout of the Dapper Antagonist of Catenin-1 (Dact1) locus, which encodes a scaffold protein involved in Wnt signaling, leads to decreased excitatory input formation on dendrites of developing forebrain neurons. We have previously demonstrated this in both (excitatory, glutamatergic) pyramidal neurons of the hippocampus and in (inhibitory GABAergic) interneurons of the cortex. We have also demonstrated that knockout of the Dact1 locus leads to decreased dendrite complexity in cultured hippocampal pyramidal neurons, and to decreased spine formation on dendrites of forebrain pyramidal neurons in vitro and in vivo. Synapse phenotypes resulting from Dact1 loss in cultured cortical interneurons can be rescued by recombinant overexpression of the Dact1 binding partner, Dishevelled-1 (Dvl1), but not by recombinant expression of a constitutively active form of the small GTPase Rac1. This contrasts with dendrite spine phenotypes resulting from Dact1 loss in cultured hippocampal pyramidal neurons, which can be fully rescued by recombinant expression of activated Rac1. Taken together, these data suggest that in maturing forebrain neurons there are molecularly separate requirements for Dact1 in dendrite arborization/spine formation vs. synaptogenesis. Here, we show that the developmental requirement for Dact1 during dendrite arborization, which we previously demonstrated only in hippocampal pyramidal neurons, is also present in cortical interneurons, and we discuss mechanistic implications of this finding.
Dact1; Rho GTPase; Sholl analysis; Wnt; dendrite; interneuron; planar cell polarity
DACT2 (Dapper, Dishevelled-associated antagonist of β-catenin homolog 2) is a member of the DACT family involved in the regulation of embryonic development. Human DACT2 is localized on 6q27, a region of frequent loss of heterozygosity in human cancers. However, the regulation of DACT2 expression and function in hepatocellular carcinoma (HCC) remains unclear. In this study, genetic and epigenetic changes of DACT2 were analyzed in HCC cell lines and primary cancer. We found no single-nucleotide polymorphism (SNP) associated with HCC. Promoter region methylation was correlated with loss or reduction of DACT2 expression, and restoration of DACT2 expression was induced by 5-aza-2’-deoxycytidine (5-AZA) in HCC cell lines. Promoter region methylation was found in 54.84% of primary HCC. Reduction of DACT2 expression was associated with promoter hypermethylation, and expression of DACT2 was inversely related to β-catenin expression in primary HCC. DACT2 suppressed cell proliferation, induced G2-M arrest in cell lines and inhibited tumor growth in xenograft nude mice. The transcriptional activity of TCF-4 and the expression of Wnt signaling downstream genes were suppressed by DACT2 re-expression and reactivated by depletion of DACT2. In conclusion, DACT2 is frequently methylated in HCC and its expression is regulated by promoter hypermethylation. DACT2 suppresses HCC by inhibiting Wnt signaling in human HCC.
DACT2; hepatocellular carcinoma; epigenetics; DNA methylation; Wnt signaling
Dacts are multi-domain adaptor proteins. They have been implicated in Wnt and Tgfβ signaling and serve as a nodal point in regulating many cellular activities. Dact genes have so far only been identified in bony vertebrates. Also, the number of Dact genes in a given species, the number and roles of protein motifs and functional domains, and the overlap of gene expression domains are all not clear. To address these problems, we have taken an evolutionary approach, screening for Dact genes in the animal kingdom and establishing their phylogeny and the synteny of Dact loci. Furthermore, we performed a deep analysis of the various Dact protein motifs and compared the expression patterns of different Dacts.
Our study identified previously not recognized dact genes and showed that they evolved late in the deuterostome lineage. In gnathostomes, four Dact genes were generated by the two rounds of whole genome duplication in the vertebrate ancestor, with Dact1/3 and Dact2/4, respectively, arising from the two genes generated during the first genome duplication. In actinopterygians, a further dact4r gene arose from retrotranscription. The third genome duplication in the teleost ancestor, and subsequent gene loss in most gnathostome lineages left extant species with a subset of Dact genes. The distribution of functional domains suggests that the ancestral Dact function lied with Wnt signaling, and a role in Tgfβ signaling may have emerged with the Dact2/4 ancestor. Motif reduction, in particular in Dact4, suggests that this protein may counteract the function of the other Dacts. Dact genes were expressed in both distinct and overlapping domains, suggesting possible combinatorial function.
The gnathostome Dact gene family comprises four members, derived from a chordate-specific ancestor. The ability to control Wnt signaling seems to be part of the ancestral repertoire of Dact functions, while the ability to inhibit Tgfβ signaling and to carry out specialized, ortholog-specific roles may have evolved later. The complement of Dact genes coexpressed in a tissue provides a complex way to fine-tune Wnt and Tgfβ signaling. Our work provides the basis for future structural and functional studies aimed at unraveling intracellular regulatory networks.
Dact; Dapper; Frodo; Wnt signaling; Tgfβ signaling; Evolution; Protein motifs; Expression; Chordates; Vertebrates
Dapper, Dishevelled-associated antagonist of β-catenin (DACT), is involved in Xenopus embryonic development. Human DACT2 is localized on chromosome 6q27, a region of frequent loss of heterozygosity (LOH) in human cancers. However, the function and regulation of DACT2 in human lung cancer remain unclear. DNA sequencing, methylation-specific PCR (MSP), semi-quantitative RT-PCR, western blotting, and xenograft models were employed in this study. Eight lung cancer cell lines, 106 cases of primary lung cancer, four specimens of normal lung from patients without cancer, and 99 blood samples from healthy individuals were examined. We found that while there was no SNP related to lung cancer, the DACT2 promoter region is frequently methylated in human lung cancer. DACT2 is silenced by promoter region hypermethylation and re-expressed by 5-aza-2′-deoxyazacytidine treatment of lung cancer cell lines. Methylation of DACT2 was associated with poor differentiation of lung cancer. Loss of DACT2 expression was associated with promoter region hypermethylation in primary lung cancer, and was associated with increased β-catenin expression. Restoration of DACT2 expression suppressed tumour proliferation both in vitro and in vivo. DACT2 expression was down-regulated by siRNA knockdown in H727 cells. DACT2 inhibited T-cell factor/lymphoid enhancer factor (TCF/LEF) and its downstream genes. In conclusion, DACT2 methylation is a potential lung cancer detection marker. DACT2 is regulated by promoter region hypermethylation. DACT2 inhibits lung cancer proliferation by suppressing the Wnt signalling pathway in lung cancer.
DACT2; chromosome 6q27; SNP; DNA methylation; epigenetics; lung cancer
OBJECTIVE—Wnt signaling inhibits adipogenesis, but its regulation, physiological relevance, and molecular effectors are poorly understood. Here, we identify the Wnt modulator Dapper1/Frodo1 (Dact1) as a new preadipocyte gene involved in the regulation of murine and human adipogenesis.
RESEARCH DESIGN AND METHODS—Changes in Dact1 expression were investigated in three in vitro models of adipogenesis. In vitro gain- and loss-of-function studies were used to investigate the mechanism of Dact1 action during adipogenesis. The in vivo regulation of Dact1 and Wnt/β-catenin signaling were investigated in murine models of altered nutritional status, of pharmacological stimulation of in vivo adipogenesis, and during the development of dietary and genetic obesity.
RESULTS—Dact1 is a preadipocyte gene that decreases during adipogenesis. However, Dact1 knockdown impairs adipogenesis through activation of the Wnt/β-catenin signaling pathway, and this is reversed by treatment with the secreted Wnt antagonist, secreted Frizzled-related protein 1 (Sfrp1). In contrast, constitutive Dact1 overexpression promotes adipogenesis and confers resistance to Wnt ligand-induced antiadipogenesis through increased expression of endogenous Sfrps and reduced expression of Wnts. In vivo, in white adipose tissue, Dact1 and Wnt/β-catenin signaling also exhibit coordinated expression profiles in response to altered nutritional status, in response to pharmacological stimulation of in vivo adipogenesis, and during the development of dietary and genetic obesity.
CONCLUSIONS—Dact1 regulates adipogenesis through coordinated effects on gene expression that selectively alter intracellular and paracrine/autocrine components of the Wnt/β-catenin signaling pathway. These novel insights into the molecular mechanisms controlling adipose tissue plasticity provide a functional network with therapeutic potential against diseases, such as obesity and associated metabolic disorders.
The purpose of this study was to determine the relationship between hypermethylation of DACT1 gene promoter and lower mRNA expression in bladder urothelial carcinoma tissue. The methylation status of 29 urothelial carcinoma samples and 29 normal tissue samples were examined by methylation-specific polymerase chain reaction (MSP). The DACT1 mRNA transcript levels and DACT1 protein levels in all samples were then evaluated to define the relationship between the methylation status of the DACT1 promoter and its expression at the transcriptional and translational levels. Decreased expression of DACT1 was detected in 89.66% of urothelial carcinomas (26/29; P < 0.005). Promoter hypermethylation was found in 58.62% (17/29) urothelial carcinomas and 25% (7/29) normal tissues, respectively (P < 0.05). DACT1 expression was lower in tissues where the DACT1 gene promoter was hypermethylated than in unmethylated tissues (0.25±0.17 vs 0.69±0.30, P < 0.05). DACT1 gene hypermethylation was closely related to tumor size, grade and stage (P < 0.05). Our results indicate that silencing and downregulation of DACT1 mRNA may be implicated in carcinogenesis and the progression of bladder urothelial carcinoma, and may be a potential prognostic factor.
DNA methylation; DACT1; hypermethylation; bladder urothelial carcinoma
Genetic and epigenetic defects in Wnt/β-catenin signaling play important roles in colorectal cancer progression. Here we identify DACT3, a member of the DACT (Dpr/Frodo) gene family, as a negative regulator of Wnt/β-catenin signaling that is transcriptionally repressed in colorectal cancer. Unlike other Wnt signaling inhibitors that are silenced by DNA methylation, DACT3 repression is associated with bivalent histone modifications. Remarkably, DACT3 expression can be robustly de-repressed by a pharmacological combination that simultaneously targets both histone methylation and deacetylation, leading to strong inhibition of Dishevelled (Dvl)-mediated Wnt/β-catenin signaling and massive apoptosis of colorectal cancer cells. Our study identifies DACT3 as an important regulator of Wnt/β-catenin signaling in colorectal cancer and suggests a potential strategy for therapeutic control of Wnt/β-catenin signaling in colorectal cancer.
We previously reported that Sestd1 KO phenocopies Dact1 KO in mice, consistent with a model in which Sestd1 and Dact1 act together to form a crucial functional complex that regulates Vangl2 in the Wnt/Planar Cell Polarity (PCP) pathway. Here, we show that Dvl2, a binding partner of Dact1, also forms complexes with Sestd1, and does so independently of both Dact1 and Vangl2. In cell-based assays, whereas Sestd1 does not alter Dvl2 activation of the Wnt/β-catenin signaling pathway, Dvl2 enhances activation of Rho family GTPases by Dact1 and Sestd1, consistent with a role in the PCP pathway. In mice, although Dvl2 KO is recessive in an otherwise wild type background, it leads to dominant embryonic lethality in either the Sestd1 or Dact1 KO background. This genetic synergy stands in contrast to the epistasis we have previously reported between Sestd1 and Dact1 KO, and suggests independent or semi-independent functions for Dvl2 vs. Sestd1/Dact1 in the regulation of the PCP pathway during development. In conclusion, biochemical and genetic interactions between Dvl2, Sestd1, and Dact1, in addition to prior reported interactions between these same molecules and Vangl2, suggest that all these gene products can form complexes together and regulate the PCP pathway during mammalian development. However, Sestd1 and Dact1 have a closely allied function in the post-translational regulation of Vangl2 that is at least partially distinct from the functions of Dvl2 in this pathway.
Dact1; Dvl2; Rho GTPase; Sestd1; Vangl2; planar cell polarity
Dact1 (Dapper/Frodo), an intracellular phosphoprotein that binds Dishevelled, catenins, and other signaling proteins, is expressed in the developing and mature mammalian central nervous system, but its function there is unknown. Dact1 colocalized with synaptic markers and partitioned to postsynaptic fractions from cultured mouse forebrain neurons. Hippocampal neurons from Dact1 knockout mice had simpler dendritic arbors and fewer spines than hippocampal neurons from wild type littermates. This correlated with reductions in excitatory synapses and miniature excitatory postsynaptic currents, whereas inhibitory synapses were not affected. Loss of Dact1 resulted in a decrease in activated Rac, and recombinant expression of either Dact1 or constitutively active Rac, but not Rho or Cdc42, rescued dendrite and spine phenotypes in Dact1 mutant neurons. Our findings suggest that during neuronal differentiation Dact1 plays a critical role in a molecular pathway promoting Rac activity underlying the elaboration of dendrites and the establishment of spines and excitatory synapses.
Dendritic spine; Synapse; Rac; Dendrite; Forebrain; Hippocampus
Limited information is available regarding mechanisms by which miRNAs contribute to pulmonary carcinogenesis. The present study was undertaken to examine expression and function of miRNAs induced by cigarette smoke condensate (CSC) in normal human respiratory epithelia and lung cancer cells.
Micro-array and quantitative RT-PCR (qRT-PCR) techniques were used to assess miRNA and host gene expression in cultured cells, and surgical specimens. Software-guided analysis, RNA cross-link immunoprecipitation (CLIP), 3′ UTR luciferase reporter assays, qRT-PCR, focused super-arrays and western blot techniques were used to identify and confirm targets of miR-31. Chromatin immunoprecipitation (ChIP) techniques were used to evaluate histone marks and transcription factors within the LOC554202 promoter. Cell count and xenograft experiments were used to assess effects of miR-31 on proliferation and tumorigenicity of lung cancer cells.
CSC significantly increased miR-31 expression and activated LOC554202 in normal respiratory epithelia and lung cancer cells; miR-31 and LOC554202 expression persisted following discontinuation of CSC exposure. miR-31 and LOC554202 expression levels were significantly elevated in lung cancer specimens relative to adjacent normal lung tissues. CLIP and reporter assays demonstrated direct interaction of miR-31 with Dickkopf-1 (Dkk-1) and DACT-3. Over-expression of miR-31 markedly diminished Dkk-1 and DACT3 expression levels in normal respiratory epithelia and lung cancer cells. Knock-down of miR-31 increased Dkk-1 and DACT3 levels, and abrogated CSC-mediated decreases in Dkk-1 and DACT-3 expression. Furthermore, over-expression of miR-31 diminished SFRP1, SFRP4, and WIF-1, and increased Wnt-5a expression. CSC increased H3K4Me3, H3K9/14Ac and C/EBP-β levels within the LOC554202 promoter. Knock-down of C/EBP-β abrogated CSC-mediated activation of LOC554202. Over-expression of miR-31 significantly enhanced proliferation and tumorigenicity of lung cancer cells; knock-down of miR-31 inhibited growth of these cells.
Cigarette smoke induces expression of miR-31 targeting several antagonists of cancer stem cell signaling in normal respiratory epithelia and lung cancer cells. miR-31 functions as an oncomir during human pulmonary carcinogenesis.
Wnt/β-catenin signaling plays key roles in tooth development, but how this pathway intersects with the complex interplay of signaling factors regulating dental morphogenesis has been unclear. We demonstrate that Wnt/β-catenin signaling is active at multiple stages of tooth development. Mutation of β-catenin to a constitutively active form in oral epithelium causes formation of large, misshapen tooth buds and ectopic teeth, and expanded expression of signaling molecules important for tooth development. Conversely, expression of key morphogenetic regulators including Bmp4, Msx1 and Msx2 is down-regulated in embryos expressing the secreted Wnt inhibitor Dkk1 which blocks signaling in epithelial and underlying mesenchymal cells. Similar phenotypes are observed in embryos lacking epithelial β-catenin, demonstrating a requirement for Wnt signaling within the epithelium. Inducible Dkk1 expression after the bud stage causes formation of blunted molar cusps, down-regulation of the enamel knot marker p21, and loss of restricted ectodin expression, revealing requirements for Wnt activity in maintaining secondary enamel knots. These data place Wnt/β-catenin signaling upstream of key morphogenetic signaling pathways at multiple stages of tooth development and indicate that tight regulation of this pathway is essential both for patterning tooth development in the dental lamina, and for controlling the shape of individual teeth.
tooth; mouse; embryo; Wnt; molar; incisor; β-catenin; dental development
The primary metabolite of the herbicide atrazine (ATRA), diaminochlorotriazine (DACT), has been suggested to cause disruption in the hypothalamic-pituitary-gonadal axis leading to inhibition of luteinizing hormone (LH) release. DACT is a reactive electrophile known to form covalent protein adducts both in vitro and in vivo following ATRA exposure and maybe targeting proteins involved in GnRH-induced calcium signaling and subsequent LH release. To test this hypothesis, LβT2 pituitary cells were exposed to 300 μM DACT for 24 hrs and examined by fluorescence microscopy for GnRH-induced changes in intracellular calcium and LH release. LβT2 cells exposed to DACT had markedly diminished GnRH-induced intracellular calcium transients and a significant decreased LH release in response to GnRH. DACT appeared to cause a selective decrease in caffeine-sensitive ryanodine receptor-operated calcium stores in LβT2 cells, rather than in thapsigargin-sensitive ER calcium stores. This sensitivity correlated with the formation of covalent protein adducts by DACT, as determined by mass spectrometry. ERp57 was identified by mass spectrometry as a target of DACT adduction in the ER that could potentially mediate the effects of DACT on inhibition of GnRH-induced calcium signaling and inhibition of LH release. Intracellular calcium responses to GnRH and release of LH were restored in DACT-treated cells with the addition of a calcium ionophore (A23187). These data suggest that DACT forms adducts on proteins involved in calcium handling within the ER and that dysfunction in this critical signaling system is associated with loss of normal sensitivity to GnRH and subsequent decreased release of LH.
atrazine; chlorotriazines; DACT; protein adducts; brain
Classical tissue recombination studies demonstrated that initiation of tooth development depends on activation of odontogenic potential in the mesenchyme by signals from the presumptive dental epithelium. Although several members of the Wnt family of signaling molecules are expressed in the presumptive dental epithelium at the beginning of tooth initiation, whether Wnt signaling is directly involved in the activation of the odontogenic mesenchyme has not been characterized. In this report, we show that tissue-specific inactivation of β-catenin, a central component of the canonical Wnt signaling pathway, in the developing tooth mesenchyme caused tooth developmental arrest at the bud stage in mice. We show that mesenchymal β-catenin function is required for expression of Lef1 and Fgf3 in the developing tooth mesenchyme and for induction of primary enamel knot in the developing tooth epithelium. Expression of Msx1 and Pax9, two essential tooth mesenchyme transcription factors downstream of Bmp and Fgf signaling, respectively, were not altered in the absence of β-catenin in the tooth mesenchyme. Moreover, we found that constitutive stabilization of β-catenin in the developing palatal mesenchyme induced aberrant palatal epithelial invaginations that resembled early tooth buds both morphologically and in epithelial molecular marker expression, but without activating expression of Msx1 and Pax9 in the mesenchyme. Together, these results indicate that activation of the mesenchymal odontogenic program during early tooth development requires concerted actions of Bmp, Fgf and Wnt signaling from the presumptive dental epithelium to the mesenchyme.
β-catenin; Cre/lox; epithelial-mesenchymal interaction; induction; odontogenic potential; signaling; tooth development; Wnt
NUMB is a multifunctional protein implicated to function in self-renewal and differentiation of progenitors in several tissues. To characterize the transcripts and to analyze the expression pattern of NUMB in odontogenesis, we isolated 2 full-length clones for NUMB from mouse dental pulp mRNA. One novel sequence contained 200 bp insertion in the phosphotyrosine binding domain (PTB). Confocal microscopy analysis showed strong NUMB expression in human dental pulp stem cells (hDPSC) and preameloblasts. Western blot analysis indicated that NUMB isoforms were differentially expressed in various dental tissues. Immunohistochemical analysis showed that in postnatal mouse tooth germs, NUMB was differentially expressed in the preameloblasts, odontoblasts, cervical loop region, and in the dental pulp stem cells during development. Interestingly, overexpression of NUMB in HAT-7, a preameloblast cell line, had dramatic antagonizing effects on the protein expression level of activated Notch 1. Further analysis of the Notch signaling pathway showed that NUMB significantly downregulates sonic hedgehog (Shh) expression in preameloblasts. Therefore, we propose that NUMB maintains ameloblast progenitor phenotype at the cervical loop by downregulating the activated Notch1 protein and thereby inhibiting the mRNA expression of Shh.
Tooth enamel is formed by epithelially-derived cells called ameloblasts, while the pulp dentin complex is formed by the dental mesenchyme. These tissues differentiate with reciprocal signaling interactions to form a mature tooth. In this study we have characterized ameloblast differentiation in human developing incisors, and have further investigated the role of extracellular matrix proteins on ameloblast differentiation. Histological and immunohistochemical analyses showed that in the human tooth, the basement membrane separating the early developing dental epithelium and mesenchyme was lost shortly before dentin deposition was initiated, prior to enamel matrix secretion. Presecretary ameloblasts elongated as they came into contact with the dentin matrix, and then shortened to become secretory ameloblasts. In situ hybridization showed that at the presecretory stage of odontoblasts expressed type I collagen mRNA, and also briefly expressed amelogenin mRNA. This was followed by upregulation of amelogenin mRNA expression in secretory ameloblasts. In vitro, amelogenin expression was up-regulated in ameloblast lineage cells cultured in Matrigel, and was further up-regulated when these cells/Matrigel were co-cultured with dental pulp cells. Co-culture also up-regulated type I collagen expression by the dental pulp cells. Type I collagen coated culture dishes promoted a more elongated ameloblast lineage cell morphology and enhanced cell adhesion via integrin α2β1. Taken together, these results suggest that the basement membrane proteins and signals from underlying mesenchymal cells coordinate to initiate differentiation of preameloblasts and regulate type I collagen expression by odontoblasts. Type I collagen in the dentin matrix then anchors the presecretary ameloblasts as they further differentiate to secretory cells. These studies show the critical roles of the extracellular matrix proteins in ameloblast differentiation.
basement membrane proteins; type I collagen; integrin; dental epithelial cells; dental pulp cells; ameloblast lineage cells
Four conserved signaling pathways, including the bone morphogenetic proteins (Bmp), fibroblast growth factors (Fgf), Sonic hedgehog (Shh), and Wingless-related (Wnt) pathways, are each repeatedly used throughout tooth development. Inactivation of any of these resulted in early tooth developmental arrest in mice. The mutations identified thus far in human patients with tooth agenesis also affect these pathways. Recent studies show that these signaling pathways interact through positive and negative feedback loops to regulate not only morphogenesis of individual teeth but also tooth number, shape, and spatial pattern. Increased activity of each of the Fgf, Shh, and canonical Wnt signaling pathways revitalizes development of the physiologically arrested mouse diastemal tooth germs whereas constitutive activation of canonical Wnt signaling in the dental epithelium is able to induce supernumerary tooth formation even in the absence of Msx1 and Pax9, two transcription factors required for normal tooth development beyond the early bud stage. Bmp4 and Msx1 act in a positive feedback loop to drive sequential tooth formation whereas the Osr2 transcription factor restricts Msx1-mediated expansion of the mesenchymal odontogenic field along both the buccolingual and anteroposterior axes to pattern mouse molar teeth in a single row. Moreover, the ectodermal-specific ectodysplasin (EDA) signaling pathway controls tooth number and tooth shape through regulation of Fgf20 expression in the dental epithelium, whereas Shh suppresses Wnt signaling through a negative feedback loop to regulate spatial patterning of teeth. In this article, we attempt to integrate these exciting findings in the understanding of the molecular networks regulating tooth development and patterning.
tooth development; dentition; signaling network; revitalization; Msx1; Osr2
The Wnt/β-catenin signalling is aberrantly activated in primary B cell chronic lymphocytic leukaemia (CLL). Epigenetic silencing of pathway inhibitor genes may be a mechanism for its activation. In this study, we investigated systematically and quantitatively the methylation status of 12 Wnt/β-catenin pathway inhibitor genes – CDH1, DACT1, DKK1, DKK2, DKK3, DKK4, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5 and WIF1 – in the cell lines EHEB and MEC-1 as well as patient samples.
Quantification of DNA methylation was performed by means of bisulphite pyrosequencing and confirmed by bisulphite Sanger sequencing. Gene expression was analysed by qPCR using GAPDH as internal control. E-cadherin and β-catenin protein quantification was carried out by microsphere-based immunoassays. Methylation differences observed between the patient and control groups were tested using generalised least squares models.
For 10 genes, a higher methylation level was observed in tumour material. Only DKK4 exhibited similarly high methylation levels in both tumour and normal specimens, while DACT1 was always essentially unmethylated. However, also for these inhibitors, treatment of cells with the demethylating agent 5-aza-2´-deoxycytidine resulted in an induction of their expression, as shown by quantitative PCR, suggesting an indirect epigenetic control of activity. While the degree of demethylation and its transcriptional consequences differed between the genes, there was an overall high correlation of demethylation and increased activity. Protein expression studies revealed that no constitutive Wnt/β-catenin signalling occurred in the cell lines, which is in discrepancy with results from primary CLL. However, treatment with 5-aza-2´-deoxycytidine caused accumulation of β-catenin. Simultaneously, E-cadherin expression was strongly induced, leading to the formation of a complex with β-catenin and thus demonstrating its epigenetically regulated inhibition effect.
The results suggest an epigenetic silencing mechanism of the Wnt/β-catenin pathway inhibitor genes in CLL. Hypermethylation and silencing of functionally related genes may not be completely stochastic but result from the tumour epigenome reprogramming orchestrated by Polycomb-group repressive complexes. The data are of interest in the context of epigenetic-based therapy.
B cell chronic lymphocytic leukaemia; Wnt/β-catenin pathway; Inhibitor genes; DNA hypermethylation; Epigenetic silencing; β-catenin