The β-catenin signaling pathway is deregulated in nearly all colon cancers. Nonhypercalcemic vitamin D3 (1α,25-dehydroxyvitamin D3) analogues are candidate drugs to treat this neoplasia. We show that these compounds promote the differentiation of human colon carcinoma SW480 cells expressing vitamin D receptors (VDRs) (SW480-ADH) but not that of a malignant subline (SW480-R) or metastasic derivative (SW620) cells lacking VDR. 1α,25(OH)2D3 induced the expression of E-cadherin and other adhesion proteins (occludin, Zonula occludens [ZO]-1, ZO-2, vinculin) and promoted the translocation of β-catenin, plakoglobin, and ZO-1 from the nucleus to the plasma membrane. Ligand-activated VDR competed with T cell transcription factor (TCF)-4 for β-catenin binding. Accordingly, 1α,25(OH)2D3 repressed β-catenin–TCF-4 transcriptional activity. Moreover, VDR activity was enhanced by ectopic β-catenin and reduced by TCF-4. Also, 1α,25(OH)2D3 inhibited expression of β-catenin–TCF-4-responsive genes, c-myc, peroxisome proliferator-activated receptor δ, Tcf-1, and CD44, whereas it induced expression of ZO-1. Our results show that 1α,25(OH)2D3 induces E-cadherin and modulates β-catenin–TCF-4 target genes in a manner opposite to that of β-catenin, promoting the differentiation of colon carcinoma cells.

Cajal bodies (CBs) are nuclear suborganelles involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). In addition to snRNPs, they are highly enriched in basal transcription and cell cycle factors, the nucleolar proteins fibrillarin (Fb) and Nopp140 (Nopp), the survival motor neuron (SMN) protein complex, and the CB marker protein, p80 coilin. We report the generation of knockout mice lacking the COOH-terminal 487 amino acids of coilin. Northern and Western blot analyses demonstrate that we have successfully removed the full-length coilin protein from the knockout animals. Some homozygous mutant animals are viable, but their numbers are reduced significantly when crossed to inbred backgrounds. Analysis of tissues and cell lines from mutant animals reveals the presence of extranucleolar foci that contain Fb and Nopp but not other typical nucleolar markers. These so-called “residual” CBs neither condense Sm proteins nor recruit members of the SMN protein complex. Transient expression of wild-type mouse coilin in knockout cells results in formation of CBs and restores these missing epitopes. Our data demonstrate that full-length coilin is essential for proper formation and/or maintenance of CBs and that recruitment of snRNP and SMN complex proteins to these nuclear subdomains requires sequences within the coilin COOH terminus.

ADAR1 and ADAR2 are editing enzymes that deaminate adenosine to inosine in long double stranded RNA duplexes and specific pre-mRNA transcripts. Here, we show that full-length and N-terminally truncated forms of ADAR1 are simultaneously expressed in HeLa and COS7 cells owing to the usage of alternative starting methionines. Because the N-terminus of ADAR1 contains a nuclear export signal, the full-length protein localizes predominantly in the cytoplasm, whereas the N-terminally truncated forms are exclusively nuclear and accumulate in the nucleolus. ADAR2, which lacks a region homologous to the N-terminal domain of ADAR1, localizes exclusively to the nucleus and similarly accumulates in the nucleolus.Within the nucleolus, ADAR1 and ADAR2 co-localize in a novel compartment. Photobleaching experiments demonstrate that, in live cells, ADAR1 and ADAR2 are in constant flux in and out of the nucleolus. When cells express the editing-competent glutamate receptor GluR-B RNA, endogenous ADAR1 and ADAR2 de-localize from the nucleolus and accumulate at sites where the substrate transcripts accumulate. This suggests that ADAR1 and ADAR2 are constantly moving through the nucleolus and might be recruited onto specific editing substrates present elsewhere in the cell.

Ras GTPases-H-Ras, N-Ras, and K-Ras 4B/4A-operate as key molecular switches that convey extracellular signals from surface receptors to the interior of the cell, thereby regulating essential processes including proliferation, differentiation, and survival (15,34). It is well known that Ras must be attached to the inner leaflet of the plasma membrane (PM) to be functional (50). This is accomplished by lipidic additions to the protein C terminus (33), which contains the essential signal for localizing Ras to membranes: the CAAX box (where C is cysteine, A is alyphatic amino acid, and X is serine/methionine). This motif undergoes posttranslational modifications that make it more hydrophobic. The cysteine is farnesylated, the AAX sequence is proteolyzed, and the newly C-terminal cysteine is carboxymethylated (50). However, a second signal is necessary for efficiently positioning Ras in the membrane. This is accomplished by palmitoylation of cysteine 181 in N-Ras, and cysteines 181 and 184 in H-Ras. In the case of K-Ras 4B the second signal is attained by a polybasic motif of six lysines (175 to 180) that interacts electrostatically with the negatively charged membrane (24-26).Recently, a new twist has been provided by findings indicating that Ras isoforms are distinctively segregated in different PM microdomains with unique biochemical and physicochemical characteristics, H-Ras can be found in bulk membrane and in lipid rafts, both caveolar and noncaveolar. K-Ras is exclusively located in bulk membrane, whereas N-Ras can only be detected in noncaveolar lipid rafts (35,(38)(39)(40). Furthermore, recent reports indicate that Ras is also present in endomembranes such as endosomes, endoplasmic reticulum (ER), and the Golgi complex (10,37,45). The significance of this distribution seems to go beyond that of a transient event associated to transport and/or recycling. Instead, a pool of Ras appears to reside in these organelles, and therein Ras can productively engage downstream effectors (10,11,37,45). Moreover, at these endomembranes Ras regulation is undertaken by proteins that operate in a location-specific fashion. As such, the guanine nucleotide exchange factor RasGRP specifically regulates Ras activation at the Golgi complex (7, 9), whereas SOS and RasGRF undertake Ras regulation at the ER. Likewise, stimuli such as lysophosphatidic acid preferentially activate the Ras pool at the ER, whereas calcium ionophores are more effective in activating PM Ras (4).The fact that exogenous stimuli activate Ras distinctively depending on its localization and that Ras regulation at different sites requires the participation of specific intermediaries hints at the necessity for a location-specific control. This, in term, may imply that Ras functions at its different sites may not be totally redundant. Thus, a selective activation of Ras at each of its locations could be intended to generate variability in its biochemical and biological outputs. It is known that Ras regulates numerous cellular functions through the activation of an...

Trigeminal ganglion neurons comprise three main cell body-size types. This cell size heterogeneity provides an excellent neuronal model to study the cell size-dependent organization and dynamics of the nucleoli, Cajal (coiled) bodies (CBs), and nuclear speckles of pre-mRNA splicing factors, nuclear structures that play a key role in the normal neuronal physiology. We have analyzed the number of nucleoli and CBs and the structural and molecular organization of CBs and nuclear speckles in the three neuronal types by using immunofluorescence with antibodies that recognize nucleoli (fibrillarin), CBs (coilin), and nuclear speckles (snRNPs), confocal microscopy, and electron microscopy. Whereas the mean number of nucleoli per neuron decreases as a function of cell size, the number of CBs per cell significantly increases in large neurons in comparison with the small ones. In addition, large neurons have a higher proportion of CBs associated with the nucleolus. In all neuronal types, CBs concentrate coilin, fibrillarin, snRNPs, and the survival motor neuron protein (SMN). Immunostaining for snRNPs shows small speckle domains and extensive areas of diffuse nucleoplasmic signal in large neurons, in contrast with the large nuclear speckles found in small neurons. Furthermore, flow cytometric analysis shows that all neurons are in the range of diploid cells. These findings indicate that the fusion behavior of nucleoli, the formation of CBs and their relationships with the nucleolus, as well as the compartmentalization of the pre-mRNA splicing machinery, is related to cell body size in the trigeminal ganglion neurons. Because transcriptional activity is a basic determinant mechanism of cell size in diploid cells, we suggest that our findings reflect a distinct transcription-dependent organization of the nucleolus and splicing machinery in the three cell types of trigeminal ganglion neurons.