Correction: Elevating CLIC4 in Multiple Cell Types Reveals a TGF-? Dependent Induction of a Dominant Negative Smad7 Splice Variant.
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ABSTRACT:
SUBMITTER: PLOS ONE Staff
PROVIDER: S-EPMC5153298 | biostudies-literature | 2016
REPOSITORIES: biostudies-literature
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Correction: Elevating CLIC4 in Multiple Cell Types Reveals a TGF-β Dependent Induction of a Dominant Negative Smad7 Splice Variant.
PloS one 20161212 12
[This corrects the article DOI: 10.1371/journal.pone.0161410.]. ...[more]
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Project description:CLIC4 (Chloride intracellular channel 4) belongs to a family of putative intracellular chloride channel proteins expressed ubiquitously in multiple tissues. CLIC4 is predominantly soluble and traffics between the cytoplasm and nucleus and participates in cell cycle control and differentiation. Transforming growth factor beta (TGF-?) elevates CLIC4, which enhances TGF-? signaling through CLIC4 mediated stabilization of phospho-Smad2/3. CLIC4 is essential for TGF-? induced conversion of fibroblasts to myofibroblasts and expression of matrix proteins, signaling via the p38MAPK pathway. Therefore, regulation of TGF-? signaling is a major mechanism by which CLIC4 modifies normal growth and differentiation. We now report that elevated CLIC4 alters Smad7 function, a feedback inhibitor of the TGF-? pathway. Overexpression of CLIC4 in keratinocytes, mouse embryonic fibroblasts and other mouse and human cell types increases the expression of Smad7?, a novel truncated form of Smad7. The alternatively spliced Smad7? variant is missing 94bp in exon 4 of Smad 7 and is conserved between mouse and human cells. The deletion is predicted to lack the TGF-? signaling inhibitory MH2 domain of Smad7. Treatment with exogenous TGF-?1 also enhances expression of Smad7? that is amplified in the presence of CLIC4. While Smad7 expression inhibits TGF-? signaling, exogenously expressed Smad7? does not inhibit TGF-? signaling as determined by TGF-? dependent proliferation, reporter assays and phosphorylation of Smad proteins. Instead, exogenous Smad7? acts as a dominant negative inhibitor of Smad7, thus increasing TGF-? signaling. This discovery adds another dimension to the myriad ways by which CLIC4 modifies TGF-? signaling.
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Project description:The telomerase catalytic subunit (hTERT) is an essential component of the holoenzyme complex that adds telomeric repeats to the ends of human chromosomes. Maintenance of telomeres by telomerase or another mechanism is required for cell immortalization, and loss of telomeric DNA has been proposed as a trigger for cellular senescence. Available evidence suggests that regulation of telomerase activity primarily depends on transcriptional control of hTERT. However, several human tissues as well as some normal cell strains have been shown to express low levels of hTERT mRNA even though they lack telomerase activity. We have previously identified six splice variants of hTERT, including a "deletion" variant (hTERTalpha) that is missing conserved residues from the catalytic core of the protein. Several of the deletion variants have been detected in normal and developing human tissues. We now show that hTERTalpha inhibits endogenous telomerase activity, which results in telomere shortening and chromosome end-to-end fusions. Telomerase inhibition induced a senescence-like state in HT1080 cells and apoptosis in a jejunal fibroblast cell line. These results suggest a possible role for hTERT splice variants in the regulation of telomerase activity.
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Project description:ObjectiveTo identify causative mutations in a patient affected by ataxia and spastic paraplegia.MethodsWhole-exome sequencing (WES) and whole-genome sequencing (WGS) were performed using patient's DNA sample. RT-PCR and cDNA Sanger sequencing were performed on RNA extracted from patient's fibroblasts, as well as western blot.ResultsA novel missense variant in SPG7 (c.2195T> C; p.Leu732Pro) was first found by whole-exome sequencing (WES), while the second, also unreported, deep intronic variant (c.286 + 853A>G) was identified by whole-genome sequencing (WGS). RT-PCR confirmed the in silico predictions showing that this variant activated a cryptic splice site, inducing the inclusion of a pseudoexon into the mRNA sequence, which encoded a premature stop codon. Western blot showed decreased SPG7 levels in patient's fibroblasts.InterpretationIdentification of a deep intronic variant in SPG7, which could only have been detected by performing WGS, led to a diagnosis in this HSP patient. This case challenges the notion of an autosomal dominant inheritance for SPG7, and illustrates the importance of performing WGS subsequently or alternatively to WES to find additional mutations, especially in patients carrying one variant in a gene causing a predominantly autosomal recessive disease.
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Project description:Two-pore domain potassium (K(2P)) channels modulate neuronal excitability throughout the entire CNS. The stretch-activated channel TREK1 (K(2P)2.1) is expressed widely in brain and has been linked to depression, neuroprotection, pain perception, and epilepsy. Little, however, is known about the regulation of TREK1 expression on the transcriptional and translational level or about its trafficking to the plasma membrane. Here we have used PCR techniques to identify a splice variant of TREK1 expressed in the brain, which encodes a heavily truncated TREK1 protein retaining a single transmembrane domain. Functional expression of this splice variant TREK1?Ex4 in tsA201 cells in the presence or absence of wild type TREK1 revealed that TREK1?Ex4 has no channel activity itself but reduced TREK1 whole cell current amplitude. Confocal analysis of the expression of fluorescently tagged TREK1 variants revealed that TREK1?Ex4 is translated, but it is retained in the intracellular compartment. Additionally, TREK1?Ex4 reduced the level of TREK1 expression in the plasma membrane. Long and short forms of TREK1 derived from alternative translation initiation are differentially affected by TREK1?Ex4, with the short form (lacking the first 41 amino acids at its N terminus) unaffected. This differential regulatory role of TREK1?Ex4 will alter the functional profile of TREK1 current in neurons where they are expressed. These results indicate that the N-terminal domain and first transmembrane domain of TREK1 are likely to be important for channel dimerization and trafficking to the plasma membrane.
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Project description:Cancer stroma has a profound influence on tumor development and progression. The conversion of fibroblasts to activated myofibroblasts is a hallmark of reactive tumor stroma. Among a number of factors involved in this conversion, transforming growth factor (TGF)-? has emerged as a major regulator. CLIC4, an integral protein in TGF-? signaling, is highly upregulated in stroma of multiple human cancers, and overexpression of CLIC4 in stromal cells enhances the growth of cancer xenografts. In this study, we show that conditioned media from tumor cell lines induces expression of both CLIC4 and the myofibroblast marker alpha smooth muscle actin (?-SMA) in stromal fibroblasts via TGF-? signaling. Genetic ablation of CLIC4 in primary fibroblasts prevents or reduces constitutive or TGF-?-induced expression of ?-SMA and extracellular matrix components that are markers of myofibroblasts. CLIC4 is required for the activation of p38 map kinase by TGF-?, a pathway that signals myofibroblast conversion in stromal cells. This requirement involves the interaction of CLIC4 with PPM1a, the selective phosphatase of activated p38. Conditioned media from fibroblasts overexpressing CLIC4 increases tumor cell migration and invasion in a TGF-?-dependent manner and promotes epithelial to mesenchymal transition indicating that high stromal CLIC4 serves to enhance tumor invasiveness and progression. Thus, CLIC4 is significantly involved in the development of a nurturing tumor microenvironment by enhancing TGF-? signaling in a positive feedback loop. Targeting CLIC4 in tumor stroma should be considered as a strategy to mitigate some of the tumor enhancing effects of the cancer stroma.
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