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P73 tumor suppressor and its targets, p21 and PUMA, are required for madin-darby canine kidney cell morphogenesis by maintaining an appropriate level of epithelial to mesenchymal transition.

Zhang Y, Young A, Zhang J, Chen X - Oncotarget (2015)

Bottom Line: Due to the usage of two promoters, p73 is expressed as two isoforms, TAp73 and ∆Np73, with opposing functions.Finally, we showed knockdown of TAp73, p21 or PUMA induces epithelial to mesenchymal transition (EMT) with a decrease in E-cadherin and an increase in EMT transcription factors.Together, our data suggest that TAp73, p21 and PUMA play a critical role in modulating MDCK cell morphogenesis by maintaining an appropriate level of the EMT.

View Article: PubMed Central - PubMed

Affiliation: Comparative Oncology Laboratory, Schools of Medicine and Veterinary Medicine, University of Californian at Davis, Davis, CA, USA.

ABSTRACT
P73, a member of p53 tumor suppressor family, plays a crucial role in tumor suppression and neural development. Due to the usage of two promoters, p73 is expressed as two isoforms, TAp73 and ∆Np73, with opposing functions. Here, we investigated the potential role of p73 in epithelial polarity and morphogenesis by using Madin-Darby canine kidney (MDCK) cells as a model system. We found that knockdown of TAp73 enhances, whereas knockdown of ∆Np73 inhibits, MDCK cell proliferation and migration in two-dimensional (2-D) culture. We also found that knockdown of TAp73, but not ∆Np73, disrupts cyst formation of MDCK cells in three-dimensional (3-D) culture. Interestingly, we found that p21 and PUMA, both of which are induced by TAp73 but repressed by ∆Np73, are required for suppressing cell proliferation and migration in 2-D culture and for MDCK ce ll morphogenesis in 3-D culture. Finally, we showed knockdown of TAp73, p21 or PUMA induces epithelial to mesenchymal transition (EMT) with a decrease in E-cadherin and an increase in EMT transcription factors. Together, our data suggest that TAp73, p21 and PUMA play a critical role in modulating MDCK cell morphogenesis by maintaining an appropriate level of the EMT.

No MeSH data available.


Related in: MedlinePlus

Knockdown of p21 alters MDCK cell morphogenesis in 2-D and 3-D culturesA. Knockdown of TAp73 decreases, whereas knockdown of ΔNp73 increases, the expression of p21 and PUMA. The levels of p21, PUMA, and actin were measured in parental MDCK, MDCK-TAp73-KD, and MDCK-ΔNp73-KD cells by Western blotting. B. Generation of MDCK cell lines in which p21 was stably knocked down. Parental MDCK and MDCK-p21-KD cells were mock-treated or treated with camptothecin for 18h and the level of p21 and actin protein was measured by Western blotting. C. Representative microscopic images of parental MDCK and MDCK-p21-KD cells in 2-D cultures. D. Top panel: colony formation assay was performed with parental MDCK or MDCK-p21-KD cells. Bottom panel: the number of colonies was counted and presented as Mean ± S.D. from three separate experiments. E. Wound healing assay was performed with parental MDCK and MDCK-p21-KD cells. Top panel: cell migration was determined by visual assessment of cells migrating into the wound for 24 h using a phase-contrast microscopy. The dashed lines indicate the wound edge. Bottom panel: the time required for would closure was measured and presented as Mean ± s.d. from three separate experiments. F. Representative images of parental MDCK and MDCK-p21-KD cells in 3-D culture for 6 d or 12 d. Scale bar: 50 μM.
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Figure 3: Knockdown of p21 alters MDCK cell morphogenesis in 2-D and 3-D culturesA. Knockdown of TAp73 decreases, whereas knockdown of ΔNp73 increases, the expression of p21 and PUMA. The levels of p21, PUMA, and actin were measured in parental MDCK, MDCK-TAp73-KD, and MDCK-ΔNp73-KD cells by Western blotting. B. Generation of MDCK cell lines in which p21 was stably knocked down. Parental MDCK and MDCK-p21-KD cells were mock-treated or treated with camptothecin for 18h and the level of p21 and actin protein was measured by Western blotting. C. Representative microscopic images of parental MDCK and MDCK-p21-KD cells in 2-D cultures. D. Top panel: colony formation assay was performed with parental MDCK or MDCK-p21-KD cells. Bottom panel: the number of colonies was counted and presented as Mean ± S.D. from three separate experiments. E. Wound healing assay was performed with parental MDCK and MDCK-p21-KD cells. Top panel: cell migration was determined by visual assessment of cells migrating into the wound for 24 h using a phase-contrast microscopy. The dashed lines indicate the wound edge. Bottom panel: the time required for would closure was measured and presented as Mean ± s.d. from three separate experiments. F. Representative images of parental MDCK and MDCK-p21-KD cells in 3-D culture for 6 d or 12 d. Scale bar: 50 μM.

Mentions: Cell cycle arrest and apoptosis are known to be required for cyst formation in 3-D culture of MDCK cells [17]. Notably, we found that knockdown of TAp73 enhances whereas knockdown of ΔNp73 inhibits, MDCK cell proliferation (Figures 1D and 2C). These results prompted us to speculate that the enhanced cell proliferation leads to irregular cyst formation for MDCK-TAp73-KD cells in 3-D culture potentially via p21 and PUMA. Both p21 and PUMA are p53 family target genes, and are key regulators of growth inhibition. p21 mediates cell cycle arrest whereas PUMA mediates apoptosis [18, 19]. Indeed, we found that the level of p21 and PUMA was decreased in MDCK-TAp73-KD cells, but increased in MDCK-ΔNp73-KD cells (Figure 3A). To determine the role of p21 in cell morphogenesis, we generated stable MDCK cell lines in which p21 was knocked down. Two representative clones (clones #5 and #10) were shown in Figure 3B. As expected, the level of p21 was significantly reduced in MDCK-p21-KD cells as compared to that in parental MDCK cells regardless of camptothecin treatment. Interestingly, in 2-D culture, MDCK-p21-KD cells exhibited an elongated morphology (Figure 3C). These cells also proliferated and migrated faster than parental MDCK cells (Figure 3D-3E). Furthermore, MDCK-p21-KD cells were unable to form regular cyst structure in 3-D culture (Figure 3F).


P73 tumor suppressor and its targets, p21 and PUMA, are required for madin-darby canine kidney cell morphogenesis by maintaining an appropriate level of epithelial to mesenchymal transition.

Zhang Y, Young A, Zhang J, Chen X - Oncotarget (2015)

Knockdown of p21 alters MDCK cell morphogenesis in 2-D and 3-D culturesA. Knockdown of TAp73 decreases, whereas knockdown of ΔNp73 increases, the expression of p21 and PUMA. The levels of p21, PUMA, and actin were measured in parental MDCK, MDCK-TAp73-KD, and MDCK-ΔNp73-KD cells by Western blotting. B. Generation of MDCK cell lines in which p21 was stably knocked down. Parental MDCK and MDCK-p21-KD cells were mock-treated or treated with camptothecin for 18h and the level of p21 and actin protein was measured by Western blotting. C. Representative microscopic images of parental MDCK and MDCK-p21-KD cells in 2-D cultures. D. Top panel: colony formation assay was performed with parental MDCK or MDCK-p21-KD cells. Bottom panel: the number of colonies was counted and presented as Mean ± S.D. from three separate experiments. E. Wound healing assay was performed with parental MDCK and MDCK-p21-KD cells. Top panel: cell migration was determined by visual assessment of cells migrating into the wound for 24 h using a phase-contrast microscopy. The dashed lines indicate the wound edge. Bottom panel: the time required for would closure was measured and presented as Mean ± s.d. from three separate experiments. F. Representative images of parental MDCK and MDCK-p21-KD cells in 3-D culture for 6 d or 12 d. Scale bar: 50 μM.
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Figure 3: Knockdown of p21 alters MDCK cell morphogenesis in 2-D and 3-D culturesA. Knockdown of TAp73 decreases, whereas knockdown of ΔNp73 increases, the expression of p21 and PUMA. The levels of p21, PUMA, and actin were measured in parental MDCK, MDCK-TAp73-KD, and MDCK-ΔNp73-KD cells by Western blotting. B. Generation of MDCK cell lines in which p21 was stably knocked down. Parental MDCK and MDCK-p21-KD cells were mock-treated or treated with camptothecin for 18h and the level of p21 and actin protein was measured by Western blotting. C. Representative microscopic images of parental MDCK and MDCK-p21-KD cells in 2-D cultures. D. Top panel: colony formation assay was performed with parental MDCK or MDCK-p21-KD cells. Bottom panel: the number of colonies was counted and presented as Mean ± S.D. from three separate experiments. E. Wound healing assay was performed with parental MDCK and MDCK-p21-KD cells. Top panel: cell migration was determined by visual assessment of cells migrating into the wound for 24 h using a phase-contrast microscopy. The dashed lines indicate the wound edge. Bottom panel: the time required for would closure was measured and presented as Mean ± s.d. from three separate experiments. F. Representative images of parental MDCK and MDCK-p21-KD cells in 3-D culture for 6 d or 12 d. Scale bar: 50 μM.
Mentions: Cell cycle arrest and apoptosis are known to be required for cyst formation in 3-D culture of MDCK cells [17]. Notably, we found that knockdown of TAp73 enhances whereas knockdown of ΔNp73 inhibits, MDCK cell proliferation (Figures 1D and 2C). These results prompted us to speculate that the enhanced cell proliferation leads to irregular cyst formation for MDCK-TAp73-KD cells in 3-D culture potentially via p21 and PUMA. Both p21 and PUMA are p53 family target genes, and are key regulators of growth inhibition. p21 mediates cell cycle arrest whereas PUMA mediates apoptosis [18, 19]. Indeed, we found that the level of p21 and PUMA was decreased in MDCK-TAp73-KD cells, but increased in MDCK-ΔNp73-KD cells (Figure 3A). To determine the role of p21 in cell morphogenesis, we generated stable MDCK cell lines in which p21 was knocked down. Two representative clones (clones #5 and #10) were shown in Figure 3B. As expected, the level of p21 was significantly reduced in MDCK-p21-KD cells as compared to that in parental MDCK cells regardless of camptothecin treatment. Interestingly, in 2-D culture, MDCK-p21-KD cells exhibited an elongated morphology (Figure 3C). These cells also proliferated and migrated faster than parental MDCK cells (Figure 3D-3E). Furthermore, MDCK-p21-KD cells were unable to form regular cyst structure in 3-D culture (Figure 3F).

Bottom Line: Due to the usage of two promoters, p73 is expressed as two isoforms, TAp73 and ∆Np73, with opposing functions.Finally, we showed knockdown of TAp73, p21 or PUMA induces epithelial to mesenchymal transition (EMT) with a decrease in E-cadherin and an increase in EMT transcription factors.Together, our data suggest that TAp73, p21 and PUMA play a critical role in modulating MDCK cell morphogenesis by maintaining an appropriate level of the EMT.

View Article: PubMed Central - PubMed

Affiliation: Comparative Oncology Laboratory, Schools of Medicine and Veterinary Medicine, University of Californian at Davis, Davis, CA, USA.

ABSTRACT
P73, a member of p53 tumor suppressor family, plays a crucial role in tumor suppression and neural development. Due to the usage of two promoters, p73 is expressed as two isoforms, TAp73 and ∆Np73, with opposing functions. Here, we investigated the potential role of p73 in epithelial polarity and morphogenesis by using Madin-Darby canine kidney (MDCK) cells as a model system. We found that knockdown of TAp73 enhances, whereas knockdown of ∆Np73 inhibits, MDCK cell proliferation and migration in two-dimensional (2-D) culture. We also found that knockdown of TAp73, but not ∆Np73, disrupts cyst formation of MDCK cells in three-dimensional (3-D) culture. Interestingly, we found that p21 and PUMA, both of which are induced by TAp73 but repressed by ∆Np73, are required for suppressing cell proliferation and migration in 2-D culture and for MDCK ce ll morphogenesis in 3-D culture. Finally, we showed knockdown of TAp73, p21 or PUMA induces epithelial to mesenchymal transition (EMT) with a decrease in E-cadherin and an increase in EMT transcription factors. Together, our data suggest that TAp73, p21 and PUMA play a critical role in modulating MDCK cell morphogenesis by maintaining an appropriate level of the EMT.

No MeSH data available.


Related in: MedlinePlus