Elevating CLIC4 in Multiple Cell Types Reveals a TGF- Dependent Induction of a Dominant Negative Smad7 Splice Variant.

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-β) elevates CLIC4, which enhances TGF-β signaling through CLIC4 mediated stabilization of phospho-Smad2/3. CLIC4 is essential for TGF-β induced conversion of fibroblasts to myofibroblasts and expression of matrix proteins, signaling via the p38MAPK pathway. Therefore, regulation of TGF-β 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-β pathway. Overexpression of CLIC4 in keratinocytes, mouse embryonic fibroblasts and other mouse and human cell types increases the expression of Smad7Δ, a novel truncated form of Smad7. The alternatively spliced Smad7Δ 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-β signaling inhibitory MH2 domain of Smad7. Treatment with exogenous TGF-β1 also enhances expression of Smad7Δ that is amplified in the presence of CLIC4. While Smad7 expression inhibits TGF-β signaling, exogenously expressed Smad7Δ does not inhibit TGF-β signaling as determined by TGF-β dependent proliferation, reporter assays and phosphorylation of Smad proteins. Instead, exogenous Smad7Δ acts as a dominant negative inhibitor of Smad7, thus increasing TGF-β signaling. This discovery adds another dimension to the myriad ways by which CLIC4 modifies TGF-β signaling

cDNA and predicted protein sequence of Smad7Δ.

<p>A. cDNA sequence of part of exon 4 of Smad7 showing the region missing in Smad7Δ in red. B. Protein sequence of Smad7 predicted upon <i>in silico</i> translation of Smad7 cDNA sequence. In red is the predicted C terminus amino acid sequence of Smad7Δ. C. Primary keratinocytes were transduced with adenoviruses expressing Smad7 or Smad7Δ and immunoblotted using an N-terminal Smad7 antibody. Bands appear at expected sizes of 45 and 31kD for Smad7 and Smad7Δ respectively.Smad7Δ transduced cells also show Smad7 protein at higher exposure, not shown here.</p

Smad7Δ is an alternately spliced isoform of Smad7, occurs constitutively, is highly inducible and associated with pathology.

<p>A. Shown is the genomic view of the Smad7 locus using RNAseq data in MEF and HOS cells. The spliced part of exon 4 of the Smad7 gene was enhanced to illustrate Smad7Δ and Smad7. Blue thick bar shows un-spliced Smad7 while red thick bar the Smad7Δ variant. Blue thin bars indicate un-split tags for the normal variant while red thin bars the split tags for Smad7Δ. The ratio of split tags and un-split tags is 2% in MEFs and 1.2% in HOS cells. Raw data are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161410#pone.0161410.s001" target="_blank">S1B Fig</a>. Primary MEFs were transduced with Vector (Control) or CLIC4 expressing adenoviruses with or without 30 min pretreatment with the 5μM ALK5 blocker SB431542. Smad7Δ expression was analyzed via real time PCR and is presented as fold change over untreated Vector only (Control) transduced MEFs. C. Primary MEFs were treated with 0.05 and 1ng TGF-β1 for 2h in serum free media and Smad7Δ and Smad7 levels analyzed by real time PCR. B,C, Statistical comparison was performed as indicated. *p<0.05, **p<0.005. D. Real time PCR analyses for Smad7Δ,Smad7 and CLIC4 were performed on RNA isolated from normal skin, squamous papillomas and squamous cell carcinomas arising from orthografts of oncogenic ras transduced primary mouse keratinocytes. Each bar represents tumor or skin from a separate mouse normalized to first papilloma for Smad7Δ and first normal skin sample for Smad7 and CLIC4.</p

TGF-β induces expression of Smad7Δ.

<p>A. Real time PCR for Smad7Δ was carried out on primary keratinocytes transduced with Vector (Control) or CLIC4 expressing adenoviruses +/- 30 min pretreatment with 5μM ALK5 blocker SB431542. B. Primary keratinocytes treated with indicated doses of TGF-β for 2h in serum free media were analyzed for Smad7Δ by real time PCR. C. Indicated cell types were transduced with empty vector (Control) or CLIC4 expressing adenovirus and subsequently treated or not with 1ng/ml TGF-β1 for 2h. Total RNA was reverse transcribed and analyzed for Smad7Δ expression by q-pcr. D. Primary keratinocytes from WT or CLIC4 KO mice treated with indicated doses of TGF- β1 for 2h in serum free media were analyzed for Smad7Δ by real time PCR. Statistical analysis compared each treated sample with its respective untreated control. A,B,C,D. Smad7Δ levels were normalized to GAPDH content and are presented as relative to control untreated sample. A,B,C,D. Statistical comparisons were carried out as indicated. *p<0.05, **p<0.005, ***p<0.0005.</p

Smad7Δ functions as a dominant inhibitor of Smad7.

<p>A. Primary keratinocytes were transfected with p3TP luciferase and pRLTK plasmids for 24h, then transduced with Smad7 and Smad7Δ adenoviruses as indicated and treated with TGF-β for 16h. Luciferase assays were performed and represented here as fold change relative to that of Vector transduced control sample. B. Primary keratinocytes were transduced with Vector (Control), Smad7, Smad7Δ or a combination of both for 16h, subsequently treated with TGF-β1 for 1h and analyzed on a TGF-β signaling PCR array by real time PCR. Data are presented as fold change over control sample transduced with empty vector. C. Primary keratinocytes were transduced as in B, treated with TGF-β1 for 1h and immunoblotted for p-Smad2. β-actin was used as loading control. D. Primary keratinocytes were transduced as in B, treated with TGF-β for 16h and subsequently exposed to ³H-thymidine for 3h. ³H-Thymidine incorporation was determined by scintillation counting of lysates and displayed as raw counts normalized to cell number. A, D Statistical comparison was performed as indicated. *p<0.05, **p<0.005, ***p<0.0005.</p

Comparison of Smad7 and Smad7Δ domains.

<p>A. Exon arrangement of Smad7 and location of MH1 and MH2 domains. B. Exon arrangement of Smad7Δ compared to that of Smad7 and the resulting MH1 and absence of MH2 domain predicted based on sequence analysis.</p

CLIC4 overexpression induces expression of Smad7Δ.

<p>A. Primary keratinocytes transduced with Vector (Control) or CLIC4 expressing adenoviruses for 16h were subjected to RT-PCR using Smad7 primers. B. Primary keratinocytes transduced with Vector (Control) or CLIC4 expressing adenoviruses for indicated periods of time were analyzed for Smad7 and CLIC4 by RT-PCR. GAPDH was used as control for A and B. C. Genomic organization of Smad7 gene showing the region deleted in SmadΔ in blue, based on sequencing analysis. D. To validate Smad7Δ specific primers, real time PCR was carried out on reverse transcribed RNA from control adenovirus, Smad7 or Smad7Δ adenovirus transduced keratinocytes or Smad7 plasmid DNA. Results are expressed relative to control sample which is assigned an arbitrary value of 1.0. E. Real time PCR for Smad7Δ was carried out on samples from Vector (Control), CLIC4 or GFP transduced keratinocytes and presented here as relative to control sample. Statistical comparisons were performed as indicated. **p<0.005.</p