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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

Comparison of E-cadherin expression in HPT and HK-2 cell cultures.A) Expression of E-cadherin mRNA in three independent HPT isolates (human proximal tubule cells) compared to that in HK-2 cells growth in three different media formulation. HPT cells were grown in the 20/12EGF formulation. B) Levels of E-cadherin protein measured quantitatively using an ELISA. C) Western analysis of E-cadherin in the identical cultures as in A and B. D) Graphical representation of the integrated optical densities of the western shown in C. Significant differences between HK-2 and each HPT isolate are designated ***p < 0.0001, of ** p< 0.001 as determined by one-way ANOVA with Tukey’s post-hoc test
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pone.0120132.g005: Comparison of E-cadherin expression in HPT and HK-2 cell cultures.A) Expression of E-cadherin mRNA in three independent HPT isolates (human proximal tubule cells) compared to that in HK-2 cells growth in three different media formulation. HPT cells were grown in the 20/12EGF formulation. B) Levels of E-cadherin protein measured quantitatively using an ELISA. C) Western analysis of E-cadherin in the identical cultures as in A and B. D) Graphical representation of the integrated optical densities of the western shown in C. Significant differences between HK-2 and each HPT isolate are designated ***p < 0.0001, of ** p< 0.001 as determined by one-way ANOVA with Tukey’s post-hoc test

Mentions: The expression of E- and N-cadherin was compared between the HK-2 and HPT cells at the level of mRNA and protein expression. Since the results of western analysis can be influenced by the development time used to produce the blot, an ELISA-based method was employed to quantify the amount of E- and N-cadherin that was present in both the HK-2 and HPT cells. Three independent isolates of HPT cells were used in the experiments and these were grown on 20/12EGF. The HK-2 cells were grown on KSFM, 20/12FCS, and 20/12EGF. The results of this analysis confirmed that the HPT cells produced E-cadherin mRNA in substantially higher amounts than the HK-2 cells regardless of growth media composition (Fig. 5A). The levels of E-cadherin mRNA in HPT cells was over 50 fold higher than levels in HK-2 cells. This difference in mRNA expression did translate to the amount of E-cadherin protein, with ELISA analysis showing a similar large (>50 fold) increase in E-cadherin protein in the HPT cells compared to the HK-2 cell line (Fig. 5B). This difference in protein level was also exemplified on a standard western as shown in Fig. 5C & D. An identical analysis of N-cadherin expression confirmed that N-cadherin mRNA expression was higher in the HK-2 cell line compared to the HPT cells (Fig. 6A). An analysis of N-cadherin protein expression also demonstrated that this difference did translate to the differences in the N-cadherin protein (Fig. 6B, C and D). However, the magnitude of the difference between N-cadherin protein expression between the HPT and HK-2 cell lines was much less (4 to 10 fold) than that found for the E-cadherin protein (Fig. 5B versus 6B), albeit, the smaller difference did not manifest on the western in Fig. 6C and D most probably due to the higher sensitivity of the ELISA in detecting low levels of N-cadherin in the HPT samples. Overall, the results demonstrate that the level of E-cadherin mRNA and protein is significantly higher in HPT cells compared to HK-2 cells and that the expression pattern is reversed for the expression of N-cadherin between the cell lines.


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)

Comparison of E-cadherin expression in HPT and HK-2 cell cultures.A) Expression of E-cadherin mRNA in three independent HPT isolates (human proximal tubule cells) compared to that in HK-2 cells growth in three different media formulation. HPT cells were grown in the 20/12EGF formulation. B) Levels of E-cadherin protein measured quantitatively using an ELISA. C) Western analysis of E-cadherin in the identical cultures as in A and B. D) Graphical representation of the integrated optical densities of the western shown in C. Significant differences between HK-2 and each HPT isolate are designated ***p < 0.0001, of ** p< 0.001 as determined by one-way ANOVA with Tukey’s post-hoc test
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120132.g005: Comparison of E-cadherin expression in HPT and HK-2 cell cultures.A) Expression of E-cadherin mRNA in three independent HPT isolates (human proximal tubule cells) compared to that in HK-2 cells growth in three different media formulation. HPT cells were grown in the 20/12EGF formulation. B) Levels of E-cadherin protein measured quantitatively using an ELISA. C) Western analysis of E-cadherin in the identical cultures as in A and B. D) Graphical representation of the integrated optical densities of the western shown in C. Significant differences between HK-2 and each HPT isolate are designated ***p < 0.0001, of ** p< 0.001 as determined by one-way ANOVA with Tukey’s post-hoc test
Mentions: The expression of E- and N-cadherin was compared between the HK-2 and HPT cells at the level of mRNA and protein expression. Since the results of western analysis can be influenced by the development time used to produce the blot, an ELISA-based method was employed to quantify the amount of E- and N-cadherin that was present in both the HK-2 and HPT cells. Three independent isolates of HPT cells were used in the experiments and these were grown on 20/12EGF. The HK-2 cells were grown on KSFM, 20/12FCS, and 20/12EGF. The results of this analysis confirmed that the HPT cells produced E-cadherin mRNA in substantially higher amounts than the HK-2 cells regardless of growth media composition (Fig. 5A). The levels of E-cadherin mRNA in HPT cells was over 50 fold higher than levels in HK-2 cells. This difference in mRNA expression did translate to the amount of E-cadherin protein, with ELISA analysis showing a similar large (>50 fold) increase in E-cadherin protein in the HPT cells compared to the HK-2 cell line (Fig. 5B). This difference in protein level was also exemplified on a standard western as shown in Fig. 5C & D. An identical analysis of N-cadherin expression confirmed that N-cadherin mRNA expression was higher in the HK-2 cell line compared to the HPT cells (Fig. 6A). An analysis of N-cadherin protein expression also demonstrated that this difference did translate to the differences in the N-cadherin protein (Fig. 6B, C and D). However, the magnitude of the difference between N-cadherin protein expression between the HPT and HK-2 cell lines was much less (4 to 10 fold) than that found for the E-cadherin protein (Fig. 5B versus 6B), albeit, the smaller difference did not manifest on the western in Fig. 6C and D most probably due to the higher sensitivity of the ELISA in detecting low levels of N-cadherin in the HPT samples. Overall, the results demonstrate that the level of E-cadherin mRNA and protein is significantly higher in HPT cells compared to HK-2 cells and that the expression pattern is reversed for the expression of N-cadherin between the cell lines.

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