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Deletion of Porcn in mice leads to multiple developmental defects and models human focal dermal hypoplasia (Goltz syndrome).

Liu W, Shaver TM, Balasa A, Ljungberg MC, Wang X, Wen S, Nguyen H, Van den Veyver IB - PLoS ONE (2012)

Bottom Line: Conditional Porcn inactivation by EIIa-driven or Hprt-driven Cre recombinase results in increased early embryonic lethality.Mesenchyme-specific Prx-Cre-driven inactivation of Porcn produces FDH-like limb defects, while ectodermal Krt14-Cre-driven inactivation produces thin skin, alopecia, and abnormal dentition.Furthermore, cell-based assays confirm that human PORCN mutations reduce WNT3A secretion.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT

Background: Focal Dermal Hypoplasia (FDH) is a genetic disorder characterized by developmental defects in skin, skeleton and ectodermal appendages. FDH is caused by dominant loss-of-function mutations in X-linked PORCN. PORCN orthologues in Drosophila and mice encode endoplasmic reticulum proteins required for secretion and function of Wnt proteins. Wnt proteins play important roles in embryo development, tissue homeostasis and stem cell maintenance. Since features of FDH overlap with those seen in mouse Wnt pathway mutants, FDH likely results from defective Wnt signaling but molecular mechanisms by which inactivation of PORCN affects Wnt signaling and manifestations of FDH remain to be elucidated.

Results: We introduced intronic loxP sites and a neomycin gene in the mouse Porcn locus for conditional inactivation. Porcn-ex3-7flox mice have no apparent developmental defects, but chimeric mice retaining the neomycin gene (Porcn-ex3-7Neo-flox) have limb, skin, and urogenital abnormalities. Conditional Porcn inactivation by EIIa-driven or Hprt-driven Cre recombinase results in increased early embryonic lethality. Mesenchyme-specific Prx-Cre-driven inactivation of Porcn produces FDH-like limb defects, while ectodermal Krt14-Cre-driven inactivation produces thin skin, alopecia, and abnormal dentition. Furthermore, cell-based assays confirm that human PORCN mutations reduce WNT3A secretion.

Conclusions: These data indicate that Porcn inactivation in the mouse produces a model for human FDH and that phenotypic features result from defective WNT signaling in ectodermal- and mesenchymal-derived structures.

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Cell-based WNT3A secretion assay.(A) Western blot showing WNT3A levels in total cell lysates after transient transfection. (NTC, non-transfected control). (B) Quantification of WNT3A levels in cell lysates. Co-expression of WNT3A with wild type PORCN (WNT3A+PORCN) results in disappearance of WNT3A from the cell lysate compared to WNT3A alone or WNT3A+GFP. In contrast, co-expression of mutant PORCN forms p.M1I and p.R124X with WNT3A causes WNT3A retention in cells, but other mutations, p.S136F, p.G168R, and p.Y359X, do not affect WNT3A secretion. We compared WNT3A levels in cells co-transfected by WNT3A and either wild type or mutant PORCN forms to WNT3A levels in cells expressing WNT3A+GFP, because these contain similar amounts of transfected DNA. Fold changes with standard deviation are shown; all data were normalized to β-tubulin; (*) indicates significant difference at p<0.05; the experiment was repeated 4 times.
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pone-0032331-g007: Cell-based WNT3A secretion assay.(A) Western blot showing WNT3A levels in total cell lysates after transient transfection. (NTC, non-transfected control). (B) Quantification of WNT3A levels in cell lysates. Co-expression of WNT3A with wild type PORCN (WNT3A+PORCN) results in disappearance of WNT3A from the cell lysate compared to WNT3A alone or WNT3A+GFP. In contrast, co-expression of mutant PORCN forms p.M1I and p.R124X with WNT3A causes WNT3A retention in cells, but other mutations, p.S136F, p.G168R, and p.Y359X, do not affect WNT3A secretion. We compared WNT3A levels in cells co-transfected by WNT3A and either wild type or mutant PORCN forms to WNT3A levels in cells expressing WNT3A+GFP, because these contain similar amounts of transfected DNA. Fold changes with standard deviation are shown; all data were normalized to β-tubulin; (*) indicates significant difference at p<0.05; the experiment was repeated 4 times.

Mentions: It has been shown that Drosophila Porcupine and, in cell culture assays, its murine ortholog lipid-modify Wnt proteins (in particular Wnt3a) to promote their secretion from the ER and consequently from Wnt-producing cells, allowing them to act as signaling molecules that activate receptors on Wnt-responding cells [18], [22], [23]. However, a direct effect of Porcupine on WNT-protein secretion and accordingly a link between FDH-causing mutations and WNT signaling had not yet been demonstrated in human cells when we initiated our studies. We therefore performed a cell-based assay to investigate how human wild type (wt) and mutant PORCN influence secretion of WNT proteins (Figure 7). Co-expression of wt PORCN and WNT3A in human embryonic kidney (HEK293T) cells showed that, as expected, transiently overexpressed wt PORCN reduced WNT3A in the cell to virtually undetectable levels, indicating increased secretion of WNT3A. In contrast, PORCN with mutations p.M1I (c.3G>A) and p.R124X (c.370C>T), known to cause human FDH, resulted in WNT3A retention in cells compared to wt PORCN (Figure 7A). However, other PORCN mutations found in FDH, p.S136F (c.407C>T), p.G168R (c.502G>A), and p.Y359X (c.1077C>A), did not or only mildly affected WNT3A retention in cells. It remains to be investigated how these mutations affect WNT protein signaling. Interestingly, other recent studies also indicate that Porcupine proteins without lipid adducts are still secreted but have defective Wnt signaling [25], [46]. When the same assay was performed with overexpressed WNT1, which is also known to be modified by Porcupine, we found that disease-causing mutations of PORCN did not reduce WNT1 secretion as much as was seen with WNT3A (Figure S3). These results are in agreement with other non-human data demonstrating that the functional consequences of lipid modification of Wnt proteins by Porcupine varies between different Wnt ligands and that implications for Wnt signaling are complex and not limited to their secretion from the ER [19].


Deletion of Porcn in mice leads to multiple developmental defects and models human focal dermal hypoplasia (Goltz syndrome).

Liu W, Shaver TM, Balasa A, Ljungberg MC, Wang X, Wen S, Nguyen H, Van den Veyver IB - PLoS ONE (2012)

Cell-based WNT3A secretion assay.(A) Western blot showing WNT3A levels in total cell lysates after transient transfection. (NTC, non-transfected control). (B) Quantification of WNT3A levels in cell lysates. Co-expression of WNT3A with wild type PORCN (WNT3A+PORCN) results in disappearance of WNT3A from the cell lysate compared to WNT3A alone or WNT3A+GFP. In contrast, co-expression of mutant PORCN forms p.M1I and p.R124X with WNT3A causes WNT3A retention in cells, but other mutations, p.S136F, p.G168R, and p.Y359X, do not affect WNT3A secretion. We compared WNT3A levels in cells co-transfected by WNT3A and either wild type or mutant PORCN forms to WNT3A levels in cells expressing WNT3A+GFP, because these contain similar amounts of transfected DNA. Fold changes with standard deviation are shown; all data were normalized to β-tubulin; (*) indicates significant difference at p<0.05; the experiment was repeated 4 times.
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Related In: Results  -  Collection

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pone-0032331-g007: Cell-based WNT3A secretion assay.(A) Western blot showing WNT3A levels in total cell lysates after transient transfection. (NTC, non-transfected control). (B) Quantification of WNT3A levels in cell lysates. Co-expression of WNT3A with wild type PORCN (WNT3A+PORCN) results in disappearance of WNT3A from the cell lysate compared to WNT3A alone or WNT3A+GFP. In contrast, co-expression of mutant PORCN forms p.M1I and p.R124X with WNT3A causes WNT3A retention in cells, but other mutations, p.S136F, p.G168R, and p.Y359X, do not affect WNT3A secretion. We compared WNT3A levels in cells co-transfected by WNT3A and either wild type or mutant PORCN forms to WNT3A levels in cells expressing WNT3A+GFP, because these contain similar amounts of transfected DNA. Fold changes with standard deviation are shown; all data were normalized to β-tubulin; (*) indicates significant difference at p<0.05; the experiment was repeated 4 times.
Mentions: It has been shown that Drosophila Porcupine and, in cell culture assays, its murine ortholog lipid-modify Wnt proteins (in particular Wnt3a) to promote their secretion from the ER and consequently from Wnt-producing cells, allowing them to act as signaling molecules that activate receptors on Wnt-responding cells [18], [22], [23]. However, a direct effect of Porcupine on WNT-protein secretion and accordingly a link between FDH-causing mutations and WNT signaling had not yet been demonstrated in human cells when we initiated our studies. We therefore performed a cell-based assay to investigate how human wild type (wt) and mutant PORCN influence secretion of WNT proteins (Figure 7). Co-expression of wt PORCN and WNT3A in human embryonic kidney (HEK293T) cells showed that, as expected, transiently overexpressed wt PORCN reduced WNT3A in the cell to virtually undetectable levels, indicating increased secretion of WNT3A. In contrast, PORCN with mutations p.M1I (c.3G>A) and p.R124X (c.370C>T), known to cause human FDH, resulted in WNT3A retention in cells compared to wt PORCN (Figure 7A). However, other PORCN mutations found in FDH, p.S136F (c.407C>T), p.G168R (c.502G>A), and p.Y359X (c.1077C>A), did not or only mildly affected WNT3A retention in cells. It remains to be investigated how these mutations affect WNT protein signaling. Interestingly, other recent studies also indicate that Porcupine proteins without lipid adducts are still secreted but have defective Wnt signaling [25], [46]. When the same assay was performed with overexpressed WNT1, which is also known to be modified by Porcupine, we found that disease-causing mutations of PORCN did not reduce WNT1 secretion as much as was seen with WNT3A (Figure S3). These results are in agreement with other non-human data demonstrating that the functional consequences of lipid modification of Wnt proteins by Porcupine varies between different Wnt ligands and that implications for Wnt signaling are complex and not limited to their secretion from the ER [19].

Bottom Line: Conditional Porcn inactivation by EIIa-driven or Hprt-driven Cre recombinase results in increased early embryonic lethality.Mesenchyme-specific Prx-Cre-driven inactivation of Porcn produces FDH-like limb defects, while ectodermal Krt14-Cre-driven inactivation produces thin skin, alopecia, and abnormal dentition.Furthermore, cell-based assays confirm that human PORCN mutations reduce WNT3A secretion.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT

Background: Focal Dermal Hypoplasia (FDH) is a genetic disorder characterized by developmental defects in skin, skeleton and ectodermal appendages. FDH is caused by dominant loss-of-function mutations in X-linked PORCN. PORCN orthologues in Drosophila and mice encode endoplasmic reticulum proteins required for secretion and function of Wnt proteins. Wnt proteins play important roles in embryo development, tissue homeostasis and stem cell maintenance. Since features of FDH overlap with those seen in mouse Wnt pathway mutants, FDH likely results from defective Wnt signaling but molecular mechanisms by which inactivation of PORCN affects Wnt signaling and manifestations of FDH remain to be elucidated.

Results: We introduced intronic loxP sites and a neomycin gene in the mouse Porcn locus for conditional inactivation. Porcn-ex3-7flox mice have no apparent developmental defects, but chimeric mice retaining the neomycin gene (Porcn-ex3-7Neo-flox) have limb, skin, and urogenital abnormalities. Conditional Porcn inactivation by EIIa-driven or Hprt-driven Cre recombinase results in increased early embryonic lethality. Mesenchyme-specific Prx-Cre-driven inactivation of Porcn produces FDH-like limb defects, while ectodermal Krt14-Cre-driven inactivation produces thin skin, alopecia, and abnormal dentition. Furthermore, cell-based assays confirm that human PORCN mutations reduce WNT3A secretion.

Conclusions: These data indicate that Porcn inactivation in the mouse produces a model for human FDH and that phenotypic features result from defective WNT signaling in ectodermal- and mesenchymal-derived structures.

Show MeSH
Related in: MedlinePlus