Analysis of an alternative human CD133 promoter reveals the implication of Ras/ERK pathway in tumor stem-like hallmarks

<p>Abstract</p> <p>Background</p> <p>An increasing number of studies support the presence of stem-like cells in human malignancies. These cells are primarily responsible for tumor initiation and thus considered as a potential target to eradicate tumors. CD133 has been identified as an important cell surface marker to enrich the stem-like population in various human tumors. To reveal the molecular machinery underlying the stem-like features in tumor cells, we analyzed a promoter of <it>CD133 </it>gene using human colon carcinoma Caco-2 and synovial sarcoma Fuji cells, which endogenously express <it>CD133 </it>gene.</p> <p>Results</p> <p>A reporter analysis revealed that P5 promoter, located far upstream in a human <it>CD133 </it>gene locus, exhibits the highest activity among the five putative promoters (P1 to P5). Deletion and mutation analysis identified two ETS binding sites in the P5 region as being essential for its promoter activity. Electrophoretic mobility shift assays demonstrated the specific binding between nuclear factors and the ETS binding sequence. Overexpression of dominant-negative forms of Ets2 and Elk1 resulted in the significant decrease of P5 activity. Furthermore, treatment of Fuji cells with a specific MEK/ERK inhibitor, U0126, also markedly decreased CD133 expression, but there was no significant effect in Caco-2 cells, suggesting cell type-specific regulation of CD133 expression. Instead, the side population, another hallmark of TSLCs, was dramatically diminished in Caco-2 cells by U0126. Finally, Ras-mediated oncogenic transformation in normal human astrocytes conferred the stem-like capability to form neurosphere-like colonies with the increase of <it>CD133 </it>mRNA expression.</p> <p>Conclusions</p> <p>In conclusion, the Ras/ERK pathway at least in part contributes to the maintenance and the acquisition of stem-like hallmarks, although the extent of its contribution is varied in a cell type-specific manner. These findings could help our comprehensive understanding of tumor stemness, and also improve the development of eradicative therapies against human malignancies.</p

Diverse dystonin gene mutations cause distinct patterns of

Loss-of-function mutations in dystonin (DST) can cause hereditary sensory and autonomic neuropathy type 6 (HSAN-VI) or epidermolysis bullosa simplex (EBS). Recently, DST-related diseases were recognized to be more complex than previously thought because a patient exhibited both neurological and skin manifestations, whereas others display only one or the other. A single DST locus produces at least three major DST isoforms: DST-a (neuronal isoform), DST-b (muscular isoform) and DST-e (epithelial isoform). Dystonia musculorum (dt) mice, which have mutations in Dst, were originally identified as spontaneous mutants displaying neurological phenotypes. To reveal the mechanisms underlying the phenotypic heterogeneity of DST-related diseases, we investigated two mutant strains with different mutations: a spontaneous Dst mutant (Dstdt-23Rbrcmice) and a gene-trap mutant (DstGt mice). The Dstdt-23Rbrc allele possesses a nonsense mutation in an exon shared by all Dst isoforms. The DstGt allele is predicted to inactivate Dst-a and Dst-bisoforms but not Dst-e There was a decrease in the levels of Dst-a mRNA in the neural tissue of both Dstdt-23Rbrc and DstGt homozygotes. Loss of sensory and autonomic nerve ends in the skin was observed in both Dstdt-23Rbrc and DstGt mice at postnatal stages. In contrast, Dst-e mRNA expression was reduced in the skin of Dstdt-23Rbrc mice but not in DstGt mice. Expression levels of Dst proteins in neural and cutaneous tissues correlated with Dst mRNAs. Because Dst-e encodes a structural protein in hemidesmosomes (HDs), we performed transmission electron microscopy. Lack of inner plaques and loss of keratin filament invasions underneath the HDs were observed in the basal keratinocytes of Dstdt-23Rbrc mice but not in those of DstGt mice; thus, the distinct phenotype of the skin of Dstdt-23Rbrc mice could be because of failure of Dst-e expression. These results indicate that distinct mutations within the Dst locus can cause different loss-of-function patterns among Dst isoforms, which accounts for the heterogeneous neural and skin phenotypes in dt mice and DST-related diseases

Biallelic Variants in UBA5 Link Dysfunctional UFM1 Ubiquitin-like Modifier Pathway to Severe Infantile-Onset Encephalopathy

The ubiquitin fold modifier 1 (UFM1) cascade is a recently identified evolutionarily conserved ubiquitin-like modification system whose function and link to human disease have remained largely uncharacterized. By using exome sequencing in Finnish individuals with severe epileptic syndromes, we identified pathogenic compound heterozygous variants in UBAS, encoding an activating enzyme for UFM1, in two unrelated families. Two additional individuals with biallelic UBAS variants were identified from the UK-based Deciphering Developmental Disorders study and one from the Northern Finland Intellectual Disability cohort. The affected individuals (n = 9) presented in early infancy with severe irritability, followed by dystonia and stagnation of development. Furthermore, the majority of individuals display postnatal microcephaly and epilepsy and develop spasticity. The affected individuals were compound heterozygous for a missense substitution, c.1111G>A (p.A1a371Thr; allele frequency of 0.28% in Europeans), and a nonsense variant or c.164G>A that encodes an amino acid substitution p.Arg5SHis, but also affects splicing by facilitating exon 2 skipping, thus also being in effect a loss-of-function allele. Using an in vitro thioester formation assay and cellular analyses, we show that the p.A1a371Thr variant is hypomorphic with attenuated ability to transfer the activated UFM1 to UFC1. Finally, we show that the CNS-specific knockout of Ufml in mice causes neonatal death accompanied by microcephaly and apoptosis in specific neurons, further suggesting that the UFM1 system is essential for CNS development and function. Taken together, our data imply that the combination of a hypomorphic p.A1a371Thr variant in trans with a loss-of-function allele in UBAS underlies a severe infantile-onset encephalopathy.Peer reviewe

Endoplasmic Reticulum-Localized Transmembrane Protein Dpy19L1 Is Required for Neurite Outgrowth

<div><p>The endoplasmic reticulum (ER), including the nuclear envelope, is a continuous and intricate membrane-bound organelle responsible for various cellular functions. In neurons, the ER network is found in cell bodies, axons, and dendrites. Recent studies indicate the involvement of the ER network in neuronal development, such as neuronal migration and axonal outgrowth. However, the regulation of neural development by ER-localized proteins is not fully understood. We previously reported that the multi-transmembrane protein Dpy19L1 is required for neuronal migration in the developing mouse cerebral cortex. A Dpy19L family member, Dpy19L2, which is a causative gene for human Globozoospermia, is suggested to act as an anchor of the acrosome to the nuclear envelope. In this study, we found that the patterns of exogenous Dpy19L1 were partially coincident with the ER, including the nuclear envelope in COS-7 cells at the level of the light microscope. The reticular distribution of Dpy19L1 was disrupted by microtubule depolymerization that induces retraction of the ER. Furthermore, Dpy19L1 showed a similar distribution pattern with a ER marker protein in embryonic mouse cortical neurons. Finally, we showed that Dpy19L1 knockdown mediated by siRNA resulted in decreased neurite outgrowth in cultured neurons. These results indicate that transmembrane protein Dpy19L1 is localized to the ER membrane and regulates neurite extension during development.</p></div

Endoplasmic reticulum (ER) localization of Dpy19L1 in COS-7 cells.

<p>COS-7 cells were transfected with a Dpy19L1-GFP plasmid. After 24 h, the subcellular localization of Dpy19L1 was observed by immunofluorescence for GFP and Calreticulin, a marker for the ER. (A) Confocal images of Dpy19L1-GFP (green) and Calreticulin (magenta). Right panel is the merged image. Dpy19L1-GFP shows a similar pattern with Calreticulin. Arrows indicate localization of Dpy19L1 around the nucleus. (B) Double staining of Dpy19L1 and Calreticulin. Lower image indicates intensity profile analysis along a white line in the upper images. (C) Colocalization analysis. An example of scatter plot of red and green pixel intensities of the Dpy19L1-GFP/Calreticulin double-labeled cell shown in Fig 1A. Intensities were measured from fifteen COS-7 cells. (D) Dynamics of Dpy19L1 and the ER were observed by time-lapse imaging. The movies are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167985#pone.0167985.s006" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167985#pone.0167985.s007" target="_blank">S2</a> Movies. COS-7 cells were transfected with a Dpy19L1-GFP plasmid together with pER-DsRed2. Upper and lower images show perinuclear (N) and peripheral (P) regions, respectively. Time-lapse images were recorded from twenty-five COS-7 cells. Results shown are representative of at least three independent culture experiments. Scale bars: 20 μm in A and C.</p

Dpy19L1 distribution in embryonic cortical neurons.

<p>(A) Expression of <i>Dpy19L1</i> mRNA visualized by ISH on sagittal section of E14.5 mouse embryo. ctx, cerebral cortex; d, diencephalon; p, pons; mo, medulla oblongata; H, heart; Lu, lung; Li, liver; Ki; kidney. (B) Primary neurons were prepared from the E14.5 mouse cerebral cortex and cultured for 5 days. The distribution of Dpy19L1 was observed by immunostaining with anti-Dpy19L1 antibody (C-ter). (C) Double immunostaining for Dpy19L1 (green) and Calreticulin (magenta). Nucleus was labeled by DRAQ5 (blue). Lower right panel shows the merged image. Dpy19L1 is partially colocalized with the ER marker Calreticulin. Arrowheads show Dpy19L1 distribution around the nucleus. C′ and C″ show magnified images of boxed areas. Results shown were obtained from three independent cultures. Scale bars: 200 μm in A, 30 μm in B, and 20 μm in C.</p

Requirement of Dpy19L1 in neurite extension of cortical neurons.

<p>(A) COS-7 cells were transfected with either a Dpy19L1 siRNA or control siRNA along with a CAG-Dpy19L1 expression plasmid, followed by western blot analysis at 48 h after transfection. Dpy19L1 expression was efficiently reduced by Dpy19L1 downregulation. α-Tubulin was used as a control. (B) Either control siRNA or one of two Dpy19L1 siRNAs was transfected in embryonic cortical neurons, and 48 h later, the expression level was checked by RT-PCR. Each Dpy19L1 siRNA decreased <i>Dpy19L1</i> expression. β-actin was used as a control. (A, B) The blots (A) and the gels (B) were cropped from the same gels, and the full-length blots and gels are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167985#pone.0167985.s005" target="_blank">S5B and S5A Fig</a>, respectively. (C−E) E14.5 cortical neurons were transfected with control or Dpy19L1 siRNA and allowed to differentiate for 72 h. The lengths of the longest neurite were compared between control and Dpy19L1-downregulated neurons. Dpy19L1-downregulated neurons show reduced neurite length compared with that of control neurons. Scale bar: 100 μm. (D) Histogram shows distribution of neurite lengths of control and Dpy19L1-downregulated neurons. (E) Average neurite length of control siRNA and Dpy19L1 siRNA transfected neurons. (F) Comparison of the number of cleaved Caspase3-labeled cells between control and Dpy19L1-downregulated neurons at 48 h after transfection. All data shown here are from at least three independent culture experiments. Values are mean ± SEM. *<i>P</i> < 0.05.</p

Distribution patterns of Dpy19L1 along microtubules in COS-7 cells.

<p>(A-C) Double staining of Dpy19L1-GFP (green) and endogenous α-Tubulin (magenta) in COS-7 cells transfected with Dpy19L1-GFP. Nucleus was labeled by DRAQ5 (blue). Lower right panel shows the merged image. Boxed areas are magnified in B and C. Dpy19L1 is highly localized in a perinuclear region (arrows). The meshwork-like pattern of Dpy19L1 along the microtubule network is observed (yellow arrowheads). (D,E) COS-7 cells transfected with a pDpy19L1-GFP plasmid were treated by nocodazole, an inhibitor of microtubule assembly, for 3 h before fixation. (D) Immunostaining of GFP and α-Tubulin. (E) Immunostaining of GFP and Calreticulin. The cytoplasmic reticular staining of Dpy19L1 is severely disrupted by application of nocodazole. Nucleus was labeled by Hoechst 33342 (blue). Results shown here were obtained from at least three independent culture experiments. Scale bars: 20 μm.</p

Distribution of Calreticulin in neurites of Dpy19L1-downregulated neurons.

<p>Either control siRNA (A and C) or Dpy19L1 siRNA1 (B and D) was transfected in embryonic cortical neurons. Cortical neurons were allowed to differentiate for 72 h, subjected to immunocytochemistry for GFP and Calreticulin (A and B), or for GFP and α-Tubulin (C and D). Boxed areas are magnified in A’-D”. Calreticulin, an ER marker, was observed in neurites of both control and Dpy19L1-downregulated neurons. Results shown are representative of at least three independent culture experiments. Scale bars: 50 μm in A-D, 20 μm in A’-D”.</p