A highly conserved Wnt-dependent TCF4 binding site within the proximal enhancer of the anti-myogenic Msx1 gene supports expression within Pax3-expressing limb bud muscle precursor cells

The product of the Msx1 gene is a potent inhibitor of muscle differentiation. Msx1 is expressed in muscle precursor cells of the limb bud that also express Pax3. It is thought that Msx1 may facilitate distal migration by delaying myogenesis in these cells. Despite the role played by Msx1 in inhibiting muscle differentiation, nothing is known of the mechanisms that support the expression of the Msx1 gene within limb bud muscle precursor cells. In the present study we have used a combination of comparative genomics, mouse transgenic analysis, in situ hybridisation and immunohistochemistry to identify a highly conserved and tissue-specific regulatory sub-domain within the previously characterised Msx1 gene proximal enhancer element that supports the expression of the Msx1 gene in Pax3-expressing mouse limb pre-muscle masses. Furthermore, using a combination of in situ hybridisation, in vivo ChIP assay and transgenic explant culture analysis we provide evidence that Msx1 expression in limb bud muscle precursor cells is dependent on the canonical Wnt/TCF signalling pathway that is important in muscle shape formation. The results of these studies provide evidence of a mechanistic link between the Wnt/TCF and the Msx1/Pax3/MyoD pathways within limb bud muscle precursor cells.     

Efficient inducible Cre-mediated recombination in Tcf21 cell lineages in the heart and kidney

Tcf21 is a Class II bHLH family member with essential roles in the formation of the lungs, kidneys, gonads, spleen, and heart. Here, we report the utility of a mouse line with targeted insertion of a tamoxifen-inducible Cre recombinase, MerCreMer at the Tcf21 locus. This mouse line will permit the inducible expression of Cre recombinase in Tcf21-expressing cells. Using ROSA26 reporter mice, we show that Cre recombinase is specifically and robustly activated in multiple Tcf21-expressing tissues during embryonic and postnatal development. The expression profile in the kidney is particularly dynamic with the ability to cause recombination in mesangial cells at one time of induction and podocytes at another time. These features make the Tcf21-driven inducible Cre line (Tcf21(iCre) ) a valuable genetic tool for spatiotemporal gene function analysis and lineage tracing of cells in the heart, kidney, cranial muscle, and gonads.     

Negative regulation of beta-catenin/Tcf signaling by naringenin in AGS gastric cancer cell

Functional activation of beta-catenin/Tcf signaling plays an important role in early events in carcinogenesis. We examined the effect of naringenin against beta-catenin/Tcf signaling in gastric cancer cells. Reporter gene assay showed that naringenin inhibited beta-catenin/Tcf signaling efficiently. In addition, the inhibition of beta-catenin/Tcf signaling by naringenin in HEK293 cells transiently transfected with constitutively mutant beta-catenin gene, whose product is not phosphorylated by GSK3beta, indicates that its inhibitory mechanism was related to beta-catenin itself or downstream components. To investigate the precise inhibitory mechanism, we performed immunofluorescence, Western blot, and EMSA. As a result, our data revealed that the beta-catenin distribution and the levels of nuclear beta-catenin and Tcf-4 proteins were unchanged after naringenin treatment. Moreover, the binding activities of Tcf complexes to consensus DNA were not affected by naringenin. Taken together, these data suggest that naringenin inhibits beta-catenin/Tcf signaling in gastric cancer with unknown mechanisms.     

Inhibition of protein-protein interactions: the discovery of druglike beta-catenin inhibitors by combining virtual and biophysical screening

The interaction between beta-catenin and Tcf family members is crucial for the Wnt signal transduction pathway, which is commonly mutated in cancer. This interaction extends over a very large surface area (4800 A(2)), and inhibiting such interactions using low molecular weight inhibitors is a challenge. However, protein surfaces frequently contain "hot spots," small patches that are the main mediators of binding affinity. By making tight interactions with a hot spot, a small molecule can compete with a protein. The Tcf3/Tcf4-binding surface on beta-catenin contains a well-defined hot spot around residues K435 and R469. A 17,700 compounds subset of the Pharmacia corporate collection was docked to this hot spot with the QXP program; 22 of the best scoring compounds were put into a biophysical (NMR and ITC) screening funnel, where specific binding to beta-catenin, competition with Tcf4 and finally binding constants were determined. This process led to the discovery of three druglike, low molecular weight Tcf4-competitive compounds with the tightest binder having a K(D) of 450 nM. Our approach can be used in several situations (e.g., when selecting compounds from external collections, when no biochemical functional assay is available, or when no HTS is envisioned), and it may be generally applicable to the identification of inhibitors of protein-protein interactions.     

A contemporary atlas of the mouse diaphragm: myogenicity, vascularity, and the pax3 connection

The thoracic diaphragm is a unique skeletal muscle composed of costal, crural, and central tendon domains. Although commonly described in medical textbooks, newer insights into the diaphragm cell composition are scarce. Here, using reporter mice, combined with gene expression analyses of whole tissues and primary cultures, we compared the diaphragm domains and their myogenic progenitors (i.e., Pax3/7 satellite cells). The outcomes of these analyses underscore the similarities between the myogenic aspects of the costal and crural domains. Expression levels of all myogenic genes examined (except Pax3) were strongly affected in mdx (dystrophin-null) mice and accompanied by an increase in fibrosis- and adiposity-related gene expression. Cell culture studies further indicated the presence of a non-myogenic Pax3-expressing population, potentially related to vascular mural cells. We additionally investigated the diaphragm vasculature. XLacZ4 and Sca1-GFP transgenes allowed a fine definition of the arterial and microvasculature network based on reporter expression in mural cells and capillary endothelium, respectively. We also provide insights into the organization of the diaphragm venous system, especially apparent in the central tendon and exhibiting arcades lined with fat-containing cells. The novel information in this "contemporary atlas" can be further explored in the context of diaphragm pathology and genetic disorders.     

Differential effect of NF-kappaB activity on beta-catenin/Tcf pathway in various cancer cells

beta-Catenin/Tcf and NF-kappaB pathways play an important role in biological functions. We determined the underlying mechanisms of differential interaction between two pathways in various human cancer cell lines. NF-kappaB positively regulated beta-catenin/Tcf pathways in human glioblastoma, whereas it has an opposite effect on beta-catenin/Tcf pathways in colon, liver, and breast cancer cells. Expression of lucine zipper tumor suppressor 2 (lzts2) was positively regulated by NF-kappaB activity in colon, liver, and breast cancer cells, whereas negatively regulated in glioma cells. Downregulation of lzts2 increased the beta-catenin/Tcf promoter activity and inhibited NF-kappaB-induced modulation of the nuclear translocation of beta-catenin. These data indicate that the differential crosstalk between beta-catenin/Tcf and NF-kappaB pathway in various cancer cells is resulted from the differences in the regulation of NF-kappaB-induced lzts2 expression.