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THOC5, a member of the mRNA export complex, contributes to processing of a subset of wingless/integrated (Wnt) target mRNAs and integrity of the gut epithelial barrier.

Saran S, Tran DD, Klebba-Färber S, Moran-Losada P, Wiehlmann L, Koch A, Chopra H, Pabst O, Hoffmann A, Klopfleisch R, Tamura T - BMC Cell Biol. (2013)

Bottom Line: Depletion of the THOC5 gene did not cause pathological alterations in liver or kidney.Furthermore, THOC5 is dispensable for general mRNA export in terminally differentiated organs, indicating that multiple mRNA export pathways exist.These data imply that THOC5 may be a useful tool for studying intestinal stem cells, for modifying the differentiation processes and for cancer therapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut fuer Biochemie, Medizinische Hochschule Hannover, OE4310 Carl-Neuberg-Str, 1, Hannover D-30623, Germany. tamura.teruko@MH-Hannover.de.

ABSTRACT

Background: THO (Suppressors of the transcriptional defects of hpr1 delta by overexpression) complex 5 (THOC5), an mRNA export protein, is involved in the expression of only 1% of all genes. Using an interferon inducible knockout mouse system, we have previously shown that THOC5 is an essential element in the maintenance of hematopoietic stem cells and cytokine-mediated hematopoiesis in adult mice. Here we interrogate THOC5 function in cell differentiation beyond the hematopoietic system and study pathological changes caused by THOC5 deficiency.

Results: To examine whether THOC5 plays a role in general differentiation processes, we generated tamoxifen inducible THOC5 knockout mice. We show here that the depletion of THOC5 impaired not only hematopoietic differentiation, but also differentiation and self renewal of the gut epithelium. Depletion of the THOC5 gene did not cause pathological alterations in liver or kidney. We further show that THOC5 is indispensable for processing of mRNAs induced by Wnt (wingless/integrated) signaling which play key roles in epithelial cell differentiation/proliferation. A subset of Wnt target mRNAs, SRY-box containing gene 9 (Sox9), and achaete-scute complex homolog 2 (Ascl2), but not Fibronectin 1 (Fn1), were down-regulated in THOC5 knockout intestinal cells. The down-regulated Wnt target mRNAs were able to bind to THOC5. Furthermore, pathological alterations in the gastrointestinal tract induced translocation of intestinal bacteria and caused sepsis in mice. The bacteria translocation may cause Toll-like receptor activation. We identified one of the Toll-like receptor inducible genes, prostaglandin-endoperoxidase synthase 2 (Ptgs2 or COX2) transcript as THOC5 target mRNA.

Conclusion: THOC5 is indispensable for processing of only a subset of mRNAs, but plays a key role in processing of mRNAs inducible by Wnt signals. Furthermore, THOC5 is dispensable for general mRNA export in terminally differentiated organs, indicating that multiple mRNA export pathways exist. These data imply that THOC5 may be a useful tool for studying intestinal stem cells, for modifying the differentiation processes and for cancer therapy.

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Depletion of the THOC5 gene causes severe pathological alteration in the gastrointestinal tract. Rosa26-CreERT2 and Rosa26-CreERT2:THOC5 (flox/flox) mice were treated with (+) or without (−) tamoxifen and jejunum (A-E), caecum (E), and colon (E) were fixed in formalin. Paraffin sections were stained by hematoxylin and eosin (HE) (A) or were supplied for immunohistochemical staining using THOC5 (B, E), Ki67 (C), and cleaved caspase 3 (Cl. Caspase 3) (D) specific antibodies as indicated. Bars represent 400 μm (HE) or 20 μm (immunohistochemical staining).
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Figure 5: Depletion of the THOC5 gene causes severe pathological alteration in the gastrointestinal tract. Rosa26-CreERT2 and Rosa26-CreERT2:THOC5 (flox/flox) mice were treated with (+) or without (−) tamoxifen and jejunum (A-E), caecum (E), and colon (E) were fixed in formalin. Paraffin sections were stained by hematoxylin and eosin (HE) (A) or were supplied for immunohistochemical staining using THOC5 (B, E), Ki67 (C), and cleaved caspase 3 (Cl. Caspase 3) (D) specific antibodies as indicated. Bars represent 400 μm (HE) or 20 μm (immunohistochemical staining).

Mentions: We next examined the gastrointestinal tract. Gastrointestinal tissues were isolated from 6- to 9-week old Rosa26-CreERT2 and Rosa26-CreERT2:THOC5 (flox/flox) mice on day 0 (no tamoxifen) to day 6 after tamoxifen treatment. Notably, 6 days after tamoxifen treatment the length of small intestine from Rosa26-CreERT2:THOC5 (flox/flox) mice was reduced to half (from 32–33 cm to 17–18 cm), and the length of colon was slightly reduced (by approximately 20%). Depletion of THOC5 caused severe degenerative lesions in the small intestine (Figure 5A). The epithelial villi (Figure 5A, arrows) were disordered and shortened, and the crypts (area of the intestinal stem cells) in THOC5 depleted mice show severe dilatation and necrosis (Figure 5A, asterisk), and sometimes sloughing of cells (crypt abscesses). It should be noted that the changes are minimally associated with influx of inflammatory cells. We then examined THOC5 expression in intestinal epithelial cells. THOC5 is expressed in the nucleus of most intestinal epithelial cells, including the most prevalent enterocytes, in control mice and in non-treated Rosa26-CreERT2:THOC5 (flox/flox) mice, however 3 days after tamoxifen treatment, THOC5 was drastically reduced in the intestinal epithelial cells from Rosa26-CreERT2:THOC5 (flox/flox) mice (Figure 5B). We next stained serial sections with Ki67 and cleaved caspase 3 specific antibodies. Upon depletion of THOC5, Ki67 positive cells were drastically reduced (Figure 5C), while cleaved caspase 3 positive cells were detected in the crypt lesion 3 days after tamoxifen treatment, and 5 days after tamoxifen treatment cleaved caspase 3 positive cells were distributed throughout whole villi (Figure 5D), suggesting that the structural breakdown of stem cell niche, and/or bacterial infection causes apoptosis of more differentiated epithelial cells.


THOC5, a member of the mRNA export complex, contributes to processing of a subset of wingless/integrated (Wnt) target mRNAs and integrity of the gut epithelial barrier.

Saran S, Tran DD, Klebba-Färber S, Moran-Losada P, Wiehlmann L, Koch A, Chopra H, Pabst O, Hoffmann A, Klopfleisch R, Tamura T - BMC Cell Biol. (2013)

Depletion of the THOC5 gene causes severe pathological alteration in the gastrointestinal tract. Rosa26-CreERT2 and Rosa26-CreERT2:THOC5 (flox/flox) mice were treated with (+) or without (−) tamoxifen and jejunum (A-E), caecum (E), and colon (E) were fixed in formalin. Paraffin sections were stained by hematoxylin and eosin (HE) (A) or were supplied for immunohistochemical staining using THOC5 (B, E), Ki67 (C), and cleaved caspase 3 (Cl. Caspase 3) (D) specific antibodies as indicated. Bars represent 400 μm (HE) or 20 μm (immunohistochemical staining).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4222586&req=5

Figure 5: Depletion of the THOC5 gene causes severe pathological alteration in the gastrointestinal tract. Rosa26-CreERT2 and Rosa26-CreERT2:THOC5 (flox/flox) mice were treated with (+) or without (−) tamoxifen and jejunum (A-E), caecum (E), and colon (E) were fixed in formalin. Paraffin sections were stained by hematoxylin and eosin (HE) (A) or were supplied for immunohistochemical staining using THOC5 (B, E), Ki67 (C), and cleaved caspase 3 (Cl. Caspase 3) (D) specific antibodies as indicated. Bars represent 400 μm (HE) or 20 μm (immunohistochemical staining).
Mentions: We next examined the gastrointestinal tract. Gastrointestinal tissues were isolated from 6- to 9-week old Rosa26-CreERT2 and Rosa26-CreERT2:THOC5 (flox/flox) mice on day 0 (no tamoxifen) to day 6 after tamoxifen treatment. Notably, 6 days after tamoxifen treatment the length of small intestine from Rosa26-CreERT2:THOC5 (flox/flox) mice was reduced to half (from 32–33 cm to 17–18 cm), and the length of colon was slightly reduced (by approximately 20%). Depletion of THOC5 caused severe degenerative lesions in the small intestine (Figure 5A). The epithelial villi (Figure 5A, arrows) were disordered and shortened, and the crypts (area of the intestinal stem cells) in THOC5 depleted mice show severe dilatation and necrosis (Figure 5A, asterisk), and sometimes sloughing of cells (crypt abscesses). It should be noted that the changes are minimally associated with influx of inflammatory cells. We then examined THOC5 expression in intestinal epithelial cells. THOC5 is expressed in the nucleus of most intestinal epithelial cells, including the most prevalent enterocytes, in control mice and in non-treated Rosa26-CreERT2:THOC5 (flox/flox) mice, however 3 days after tamoxifen treatment, THOC5 was drastically reduced in the intestinal epithelial cells from Rosa26-CreERT2:THOC5 (flox/flox) mice (Figure 5B). We next stained serial sections with Ki67 and cleaved caspase 3 specific antibodies. Upon depletion of THOC5, Ki67 positive cells were drastically reduced (Figure 5C), while cleaved caspase 3 positive cells were detected in the crypt lesion 3 days after tamoxifen treatment, and 5 days after tamoxifen treatment cleaved caspase 3 positive cells were distributed throughout whole villi (Figure 5D), suggesting that the structural breakdown of stem cell niche, and/or bacterial infection causes apoptosis of more differentiated epithelial cells.

Bottom Line: Depletion of the THOC5 gene did not cause pathological alterations in liver or kidney.Furthermore, THOC5 is dispensable for general mRNA export in terminally differentiated organs, indicating that multiple mRNA export pathways exist.These data imply that THOC5 may be a useful tool for studying intestinal stem cells, for modifying the differentiation processes and for cancer therapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut fuer Biochemie, Medizinische Hochschule Hannover, OE4310 Carl-Neuberg-Str, 1, Hannover D-30623, Germany. tamura.teruko@MH-Hannover.de.

ABSTRACT

Background: THO (Suppressors of the transcriptional defects of hpr1 delta by overexpression) complex 5 (THOC5), an mRNA export protein, is involved in the expression of only 1% of all genes. Using an interferon inducible knockout mouse system, we have previously shown that THOC5 is an essential element in the maintenance of hematopoietic stem cells and cytokine-mediated hematopoiesis in adult mice. Here we interrogate THOC5 function in cell differentiation beyond the hematopoietic system and study pathological changes caused by THOC5 deficiency.

Results: To examine whether THOC5 plays a role in general differentiation processes, we generated tamoxifen inducible THOC5 knockout mice. We show here that the depletion of THOC5 impaired not only hematopoietic differentiation, but also differentiation and self renewal of the gut epithelium. Depletion of the THOC5 gene did not cause pathological alterations in liver or kidney. We further show that THOC5 is indispensable for processing of mRNAs induced by Wnt (wingless/integrated) signaling which play key roles in epithelial cell differentiation/proliferation. A subset of Wnt target mRNAs, SRY-box containing gene 9 (Sox9), and achaete-scute complex homolog 2 (Ascl2), but not Fibronectin 1 (Fn1), were down-regulated in THOC5 knockout intestinal cells. The down-regulated Wnt target mRNAs were able to bind to THOC5. Furthermore, pathological alterations in the gastrointestinal tract induced translocation of intestinal bacteria and caused sepsis in mice. The bacteria translocation may cause Toll-like receptor activation. We identified one of the Toll-like receptor inducible genes, prostaglandin-endoperoxidase synthase 2 (Ptgs2 or COX2) transcript as THOC5 target mRNA.

Conclusion: THOC5 is indispensable for processing of only a subset of mRNAs, but plays a key role in processing of mRNAs inducible by Wnt signals. Furthermore, THOC5 is dispensable for general mRNA export in terminally differentiated organs, indicating that multiple mRNA export pathways exist. These data imply that THOC5 may be a useful tool for studying intestinal stem cells, for modifying the differentiation processes and for cancer therapy.

Show MeSH
Related in: MedlinePlus