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Cadherin expression, vectorial active transport, and metallothionein isoform 3 mediated EMT/MET responses in cultured primary and immortalized human proximal tubule cells.

Slusser A, Bathula CS, Sens DA, Somji S, Sens MA, Zhou XD, Garrett SH - PLoS ONE (2015)

Bottom Line: It was shown, based on the pattern of cadherin expression, connexin expression, vectorial active transport, and transepithelial resistance, that the HK-2 cell line has already undergone many of the early features associated with EMT.The HK-2 cell line, transfected with MT-3, may be an effective model to study the process of MET.The study implicates the unique C-terminal sequence of MT-3 in the conversion of HK-2 cells to display an enhanced epithelial phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND, United States of America.

ABSTRACT

Background: Cultures of human proximal tubule cells have been widely utilized to study the role of EMT in renal disease. The goal of this study was to define the role of growth media composition on classic EMT responses, define the expression of E- and N-cadherin, and define the functional epitope of MT-3 that mediates MET in HK-2 cells.

Methods: Immunohistochemistry, microdissection, real-time PCR, western blotting, and ELISA were used to define the expression of E- and N-cadherin mRNA and protein in HK-2 and HPT cell cultures. Site-directed mutagenesis, stable transfection, measurement of transepithelial resistance and dome formation were used to define the unique amino acid sequence of MT-3 associated with MET in HK-2 cells.

Results: It was shown that both E- and N-cadherin mRNA and protein are expressed in the human renal proximal tubule. It was shown, based on the pattern of cadherin expression, connexin expression, vectorial active transport, and transepithelial resistance, that the HK-2 cell line has already undergone many of the early features associated with EMT. It was shown that the unique, six amino acid, C-terminal sequence of MT-3 is required for MT-3 to induce MET in HK-2 cells.

Conclusions: The results show that the HK-2 cell line can be an effective model to study later stages in the conversion of the renal epithelial cell to a mesenchymal cell. The HK-2 cell line, transfected with MT-3, may be an effective model to study the process of MET. The study implicates the unique C-terminal sequence of MT-3 in the conversion of HK-2 cells to display an enhanced epithelial phenotype.

No MeSH data available.


Related in: MedlinePlus

Effect of altered domains of metallothionein-3 on the expression of connexin 32 in HK-2 cells.A) Messenger RNA of connexin 32 assessed with real-time PCR and expressed as fold change in expression versus HK-2 cells transfected with the blank vector. The change in connexin 32 expression was normalized to the change in β-actin expression. Significant differences from HK-2 (BV) are designated as *** p < 0.0001. B) Western analysis of connexin 32 was conducted in identical cultures as in (A). β-actin was used as a loading control and for densitometric normalization. Densitometry is shown beside the corresponding blot.
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pone.0120132.g011: Effect of altered domains of metallothionein-3 on the expression of connexin 32 in HK-2 cells.A) Messenger RNA of connexin 32 assessed with real-time PCR and expressed as fold change in expression versus HK-2 cells transfected with the blank vector. The change in connexin 32 expression was normalized to the change in β-actin expression. Significant differences from HK-2 (BV) are designated as *** p < 0.0001. B) Western analysis of connexin 32 was conducted in identical cultures as in (A). β-actin was used as a loading control and for densitometric normalization. Densitometry is shown beside the corresponding blot.

Mentions: The results of this determination demonstrated that the C-terminal sequence of MT-3 is required for the establishment of vectorial active transport when MT-3 is transfected and expressed in the HK-2 cell line. First, the mutation of the N-terminal sequence of MT-3 had no effect on the ability of HK-2 cells to form domes or generate a transepithelial resistance (Fig. 9 A,B; Table 1). In contrast, mutation of the C-terminal sequence of MT-3 abolished both dome formation and the transepithelial resistance of the monolayer (Fig. 9A,C; Table 1). The MT-1E isoform of MT does not contain either of the unique C-terminal or N-terminal sequences of MT-3 and transfection of HK-2 cells with MT-1E do not form domes in culture or develop a transepithelial resistance (Fig. 9D). The insertion of the N-terminal sequence of MT-3 into the MT-1E gene and subsequent transfection into the HK-2 cells does not result in dome formation or the generation of a transepithelial resistance (Fig. 9E, Table 1). In contrast, the insertion of the C-terminal sequence of MT-3 into the MT-1E gene and subsequent transfection into the HK-2 cells results in both dome formation and the generation of a transepithelial resistance (Fig. 9F; Table 1). The expression of E- and N-cadherin in HK-2 cells transfected with each MT-3 mutant construct was assessed (Fig. 10). While the expression the constructs that produced domes in culture expressed high levels of E-cadherin, constructs containing only the N-terminal domain of MT-3 were also able to highly express this cadherin, despite the lack of the ability to form domes. The repression of N-cadherin, however, required the presence of the C-terminal domain (Fig. 10). The expression of connexin 32 required the presence of both domains with each domain being able to support intermediate levels of connexin 32 (Fig. 11).


Cadherin expression, vectorial active transport, and metallothionein isoform 3 mediated EMT/MET responses in cultured primary and immortalized human proximal tubule cells.

Slusser A, Bathula CS, Sens DA, Somji S, Sens MA, Zhou XD, Garrett SH - PLoS ONE (2015)

Effect of altered domains of metallothionein-3 on the expression of connexin 32 in HK-2 cells.A) Messenger RNA of connexin 32 assessed with real-time PCR and expressed as fold change in expression versus HK-2 cells transfected with the blank vector. The change in connexin 32 expression was normalized to the change in β-actin expression. Significant differences from HK-2 (BV) are designated as *** p < 0.0001. B) Western analysis of connexin 32 was conducted in identical cultures as in (A). β-actin was used as a loading control and for densitometric normalization. Densitometry is shown beside the corresponding blot.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4372585&req=5

pone.0120132.g011: Effect of altered domains of metallothionein-3 on the expression of connexin 32 in HK-2 cells.A) Messenger RNA of connexin 32 assessed with real-time PCR and expressed as fold change in expression versus HK-2 cells transfected with the blank vector. The change in connexin 32 expression was normalized to the change in β-actin expression. Significant differences from HK-2 (BV) are designated as *** p < 0.0001. B) Western analysis of connexin 32 was conducted in identical cultures as in (A). β-actin was used as a loading control and for densitometric normalization. Densitometry is shown beside the corresponding blot.
Mentions: The results of this determination demonstrated that the C-terminal sequence of MT-3 is required for the establishment of vectorial active transport when MT-3 is transfected and expressed in the HK-2 cell line. First, the mutation of the N-terminal sequence of MT-3 had no effect on the ability of HK-2 cells to form domes or generate a transepithelial resistance (Fig. 9 A,B; Table 1). In contrast, mutation of the C-terminal sequence of MT-3 abolished both dome formation and the transepithelial resistance of the monolayer (Fig. 9A,C; Table 1). The MT-1E isoform of MT does not contain either of the unique C-terminal or N-terminal sequences of MT-3 and transfection of HK-2 cells with MT-1E do not form domes in culture or develop a transepithelial resistance (Fig. 9D). The insertion of the N-terminal sequence of MT-3 into the MT-1E gene and subsequent transfection into the HK-2 cells does not result in dome formation or the generation of a transepithelial resistance (Fig. 9E, Table 1). In contrast, the insertion of the C-terminal sequence of MT-3 into the MT-1E gene and subsequent transfection into the HK-2 cells results in both dome formation and the generation of a transepithelial resistance (Fig. 9F; Table 1). The expression of E- and N-cadherin in HK-2 cells transfected with each MT-3 mutant construct was assessed (Fig. 10). While the expression the constructs that produced domes in culture expressed high levels of E-cadherin, constructs containing only the N-terminal domain of MT-3 were also able to highly express this cadherin, despite the lack of the ability to form domes. The repression of N-cadherin, however, required the presence of the C-terminal domain (Fig. 10). The expression of connexin 32 required the presence of both domains with each domain being able to support intermediate levels of connexin 32 (Fig. 11).

Bottom Line: It was shown, based on the pattern of cadherin expression, connexin expression, vectorial active transport, and transepithelial resistance, that the HK-2 cell line has already undergone many of the early features associated with EMT.The HK-2 cell line, transfected with MT-3, may be an effective model to study the process of MET.The study implicates the unique C-terminal sequence of MT-3 in the conversion of HK-2 cells to display an enhanced epithelial phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND, United States of America.

ABSTRACT

Background: Cultures of human proximal tubule cells have been widely utilized to study the role of EMT in renal disease. The goal of this study was to define the role of growth media composition on classic EMT responses, define the expression of E- and N-cadherin, and define the functional epitope of MT-3 that mediates MET in HK-2 cells.

Methods: Immunohistochemistry, microdissection, real-time PCR, western blotting, and ELISA were used to define the expression of E- and N-cadherin mRNA and protein in HK-2 and HPT cell cultures. Site-directed mutagenesis, stable transfection, measurement of transepithelial resistance and dome formation were used to define the unique amino acid sequence of MT-3 associated with MET in HK-2 cells.

Results: It was shown that both E- and N-cadherin mRNA and protein are expressed in the human renal proximal tubule. It was shown, based on the pattern of cadherin expression, connexin expression, vectorial active transport, and transepithelial resistance, that the HK-2 cell line has already undergone many of the early features associated with EMT. It was shown that the unique, six amino acid, C-terminal sequence of MT-3 is required for MT-3 to induce MET in HK-2 cells.

Conclusions: The results show that the HK-2 cell line can be an effective model to study later stages in the conversion of the renal epithelial cell to a mesenchymal cell. The HK-2 cell line, transfected with MT-3, may be an effective model to study the process of MET. The study implicates the unique C-terminal sequence of MT-3 in the conversion of HK-2 cells to display an enhanced epithelial phenotype.

No MeSH data available.


Related in: MedlinePlus