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Possible involvement of NEDD4 in keloid formation; its critical role in fibroblast proliferation and collagen production.

Chung S, Nakashima M, Zembutsu H, Nakamura Y - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2011)

Bottom Line: Activation of NEDD4 affected subcellular localization and protein stability of p27 which was implied its critical role in contact inhibition.It also induced accumulation of β-catenin in the cytoplasm and activated the TCF/β-catenin transcriptional activity.Furthermore, NEDD4 upregulated expressions of fibronectin and type 1 collagen and contributed to the excessive accumulation of extracellular matrix.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Japan.

ABSTRACT
Keloid represents overgrowth of granulation tissue, which is characterized by collection of atypical fibroblasts with excessive deposition of extracellular matrix components, after skin injury, but its etiology is still largely unknown. We recently performed genome-wide association study (GWAS) of keloid and identified NEDD4 to be one of candidate molecules associated with keloid susceptibility. Here we demonstrate a possible mechanism of NEDD4 involvement in keloid formation through enhancement of the proliferation and invasiveness of fibroblasts as well as upregulation of type 1 collagen expression. Activation of NEDD4 affected subcellular localization and protein stability of p27 which was implied its critical role in contact inhibition. It also induced accumulation of β-catenin in the cytoplasm and activated the TCF/β-catenin transcriptional activity. Furthermore, NEDD4 upregulated expressions of fibronectin and type 1 collagen and contributed to the excessive accumulation of extracellular matrix. Our findings provide new insights into mechanism developing keloid and can be applied for development of a novel treatment for keloid.

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NEDD4 activated Akt signaling pathway through diminished PTEN protein level in fibroblasts. (A) In vitro ubiquitination assay of PTEN by NEDD4. NEDD4 ubiquitinated PTEN directly. (B) NEDD4 ubiquitinated PTEN directly and promoted the protein degradation. Immunoblot analysis for endogenous PTEN in NEDD4 over-expressing NIH3T3 cells. Control or NEDD4 expression vector were transfected and incubated for 48 h. β-actin (ACTB) was blotted as the loading control. (C) Over-expression of NEDD4 in NIH3T3 cells enhanced the phosphorylation level of Akt. Control or NEDD4 expression vector were transfected and incubated for 48 h. Phosphor-Akt and total Akt were detected by immunoblotting. β-actin (ACTB) was blotted as the loading control.
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fig04: NEDD4 activated Akt signaling pathway through diminished PTEN protein level in fibroblasts. (A) In vitro ubiquitination assay of PTEN by NEDD4. NEDD4 ubiquitinated PTEN directly. (B) NEDD4 ubiquitinated PTEN directly and promoted the protein degradation. Immunoblot analysis for endogenous PTEN in NEDD4 over-expressing NIH3T3 cells. Control or NEDD4 expression vector were transfected and incubated for 48 h. β-actin (ACTB) was blotted as the loading control. (C) Over-expression of NEDD4 in NIH3T3 cells enhanced the phosphorylation level of Akt. Control or NEDD4 expression vector were transfected and incubated for 48 h. Phosphor-Akt and total Akt were detected by immunoblotting. β-actin (ACTB) was blotted as the loading control.

Mentions: NEDD4 is characterized as an E3 ubiquitin ligase with a HECT domain, involved in ubiquitin-mediated protein degradation.18) Recently, NEDD4 was demonstrated to directly ubiquitinate PTEN (phosphatase and tensin homolog deleted on chromosome 10) and regulate its stability.33,34) p27 and β-catenin are known to be targets of the Akt protein which is regulated by PTEN. Interestingly, germline PTEN mutation associated with multiple hamartoma syndrome, also known as PTEN hamartoma tumor syndrome (PHTS), and the patient of PHTS tends to form keloids.35,36) Hence, we performed in vitro ubiquitination assay using human NEDD4 and PTEN recombinant proteins. We incubated them with E1 and E2 enzymes, and examined the ubiquitination of PTEN by anti-Ub antibody. The ubiquitination of PTEN was detected in the presence of the NEDD4 recombinant protein, while it was not without NEDD4 (Fig. 4A). Subsequently, we also found that the drastic reduction of the PTEN protein level in NEDD4 over-expressing cells, compared with the parental cells, by western blot analysis (Fig. 4B).


Possible involvement of NEDD4 in keloid formation; its critical role in fibroblast proliferation and collagen production.

Chung S, Nakashima M, Zembutsu H, Nakamura Y - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2011)

NEDD4 activated Akt signaling pathway through diminished PTEN protein level in fibroblasts. (A) In vitro ubiquitination assay of PTEN by NEDD4. NEDD4 ubiquitinated PTEN directly. (B) NEDD4 ubiquitinated PTEN directly and promoted the protein degradation. Immunoblot analysis for endogenous PTEN in NEDD4 over-expressing NIH3T3 cells. Control or NEDD4 expression vector were transfected and incubated for 48 h. β-actin (ACTB) was blotted as the loading control. (C) Over-expression of NEDD4 in NIH3T3 cells enhanced the phosphorylation level of Akt. Control or NEDD4 expression vector were transfected and incubated for 48 h. Phosphor-Akt and total Akt were detected by immunoblotting. β-actin (ACTB) was blotted as the loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3313695&req=5

fig04: NEDD4 activated Akt signaling pathway through diminished PTEN protein level in fibroblasts. (A) In vitro ubiquitination assay of PTEN by NEDD4. NEDD4 ubiquitinated PTEN directly. (B) NEDD4 ubiquitinated PTEN directly and promoted the protein degradation. Immunoblot analysis for endogenous PTEN in NEDD4 over-expressing NIH3T3 cells. Control or NEDD4 expression vector were transfected and incubated for 48 h. β-actin (ACTB) was blotted as the loading control. (C) Over-expression of NEDD4 in NIH3T3 cells enhanced the phosphorylation level of Akt. Control or NEDD4 expression vector were transfected and incubated for 48 h. Phosphor-Akt and total Akt were detected by immunoblotting. β-actin (ACTB) was blotted as the loading control.
Mentions: NEDD4 is characterized as an E3 ubiquitin ligase with a HECT domain, involved in ubiquitin-mediated protein degradation.18) Recently, NEDD4 was demonstrated to directly ubiquitinate PTEN (phosphatase and tensin homolog deleted on chromosome 10) and regulate its stability.33,34) p27 and β-catenin are known to be targets of the Akt protein which is regulated by PTEN. Interestingly, germline PTEN mutation associated with multiple hamartoma syndrome, also known as PTEN hamartoma tumor syndrome (PHTS), and the patient of PHTS tends to form keloids.35,36) Hence, we performed in vitro ubiquitination assay using human NEDD4 and PTEN recombinant proteins. We incubated them with E1 and E2 enzymes, and examined the ubiquitination of PTEN by anti-Ub antibody. The ubiquitination of PTEN was detected in the presence of the NEDD4 recombinant protein, while it was not without NEDD4 (Fig. 4A). Subsequently, we also found that the drastic reduction of the PTEN protein level in NEDD4 over-expressing cells, compared with the parental cells, by western blot analysis (Fig. 4B).

Bottom Line: Activation of NEDD4 affected subcellular localization and protein stability of p27 which was implied its critical role in contact inhibition.It also induced accumulation of β-catenin in the cytoplasm and activated the TCF/β-catenin transcriptional activity.Furthermore, NEDD4 upregulated expressions of fibronectin and type 1 collagen and contributed to the excessive accumulation of extracellular matrix.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Japan.

ABSTRACT
Keloid represents overgrowth of granulation tissue, which is characterized by collection of atypical fibroblasts with excessive deposition of extracellular matrix components, after skin injury, but its etiology is still largely unknown. We recently performed genome-wide association study (GWAS) of keloid and identified NEDD4 to be one of candidate molecules associated with keloid susceptibility. Here we demonstrate a possible mechanism of NEDD4 involvement in keloid formation through enhancement of the proliferation and invasiveness of fibroblasts as well as upregulation of type 1 collagen expression. Activation of NEDD4 affected subcellular localization and protein stability of p27 which was implied its critical role in contact inhibition. It also induced accumulation of β-catenin in the cytoplasm and activated the TCF/β-catenin transcriptional activity. Furthermore, NEDD4 upregulated expressions of fibronectin and type 1 collagen and contributed to the excessive accumulation of extracellular matrix. Our findings provide new insights into mechanism developing keloid and can be applied for development of a novel treatment for keloid.

Show MeSH
Related in: MedlinePlus