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
Publications
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:BackgroundTranscription factors YAP and TAZ function as the primary mediators of the Hippo pathway. Yet, crosstalk of YAP and TAZ with other signaling pathways remains relatively unexplored. We have explored the impact of YAP and TAZ levels on the TGF-β/Smad signaling pathway in human skin dermal fibroblasts.MethodsYAP and TAZ levels in dermal fibroblasts were reduced in dermal fibroblasts by siRNA-mediated knockdown. The effects of YAP and TAZ reduction on TGF-β/Smad signaling were examined by quantitative real-time PCR, Western analysis, and immunostaining. Luciferase reporter assays and electrophoretic mobility shift assays were conducted to investigate the transcription factor DNA-binding and transcriptional activities.ResultsKnockdown of both YAP and TAZ (YAP/TAZ), but not either separately, impaired TGF-β1-induced Smad3 phosphorylation and Smad3 transcriptional activity, thereby inhibiting the expression of TGF-β target genes. This reduction by reduced levels of YAP/TAZ results from induction of inhibitory Smad7, which inhibits Smad3 phosphorylation and activity by TGF-β1. Conversely, prevention of Smad7 induction restores Smad3 phosphorylation and Smad3 transcriptional activity in fibroblasts that have reduced YAP/TAZ. In agreement with these findings, inhibition of YAP/TAZ transcriptional activity, similar to the reduction of YAP/TAZ levels, also significantly induced Smad7 and impaired TGF-β/Smad signaling. Further investigations revealed that reduced levels of YAP/TAZ led to induction of activator protein-1 (AP-1) activity, Activated AP-1 bound to DNA sequences in the Smad7 gene promoter, and deletion of these AP-1 binding sequences substantially reduced Smad7 promoter reporter activity.ConclusionYAP/TAZ functions in concert with transcription factor AP-1 and Smad7 to regulate TGF-β signaling, in human dermal fibroblasts. Reduction of YAP/TAZ levels leads to activation of AP-1 activity, which induces Smad7. Smad7 suppresses the TGF-β pathway.
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