Cadherins are a superfamily of calcium-binding membrane glycoproteins that mediate homotypic cell-cell interactions and are involved in morphogenetic processes such as cell compaction, epithelial differentiation and cell migration. Cadherin-16 (CDH-16) was identified as a tissue-specific cadherin found exclusively in the kidney (therefore originally named Ksp-cadherin) and co-expressed with E-cadherin in the epithelial cells of the distal tubules and of the collecting ducts. Recently, it has been clearly demonstrated that CDH-16 is also expressed in the thyroid, both during embryonic development and in the adult, and its expression is modulated in thyroid cancer (L. Nitsch, unpublished observations). The restricted expression pattern of CDH-16 prompted us to investigate a possible role for the transcription factor Pax8 in its transcriptional regulation. Pax8 is a member of the Pax family of paired domain-containing genes, and is expressed in the developing and adult kidney and thyroid, placenta, urogenital tract and developing neural tube. Moreover, Pax8 has been shown to be a master gene for thryoid development and differention. In this study, we analyzed the role of the transcription factor Pax8 in the regulation of CDH-16 expression by different means. In particular, by reporter gene assays we demonstrated that the region of DNA identified as the minimal promoter required for the expression of CDH-16 gene is transcriptionally active in thyroid cells as well as in kidney cells, while it is not active in other cell types. At the same time, we observed that in transactivation experiments conducted in HeLa cells, Pax8 is able to activate transcription from a reporter construct carrying the CDH-16 minimal promoter. These results were further strenghtend by Chromatin-immunoprecipitation assays performed with a specific anti-PAX8 antibody on genomic DNA prepared from rat thyroid cells showing that indeed Pax8 is able to bind in vivo the promoter region of the CDH-16 gene. We then used the MatInspector analysis software to identify the Pax8 binding site(s) within the minimal promoter region and the analysis revealed the presence of one predicted Pax8 binding site within this region. The ability of Pax8 to bind to this binding site was demonstrated by EMSA assays. In addition, the mutagenesis of this binding site in the context of the CDH-16 minimal promoter abrogates the ability of Pax8 to activate transcription from it. Finally, we show that Pax8 silencing in FRTL-5 thyroid cells causes a significant down-regulation of the expression of CDH-16. Therefore, we conclude that CDH-16 is a novel downstream target of the transcription factor Pax8, likely since the early steps of thyroid development.
CDH-16 is a novel target of the transcription factor Pax8
Tiziana de Cristofaro;Tina Di Palma;Mariastella Zannini
2010
Abstract
Cadherins are a superfamily of calcium-binding membrane glycoproteins that mediate homotypic cell-cell interactions and are involved in morphogenetic processes such as cell compaction, epithelial differentiation and cell migration. Cadherin-16 (CDH-16) was identified as a tissue-specific cadherin found exclusively in the kidney (therefore originally named Ksp-cadherin) and co-expressed with E-cadherin in the epithelial cells of the distal tubules and of the collecting ducts. Recently, it has been clearly demonstrated that CDH-16 is also expressed in the thyroid, both during embryonic development and in the adult, and its expression is modulated in thyroid cancer (L. Nitsch, unpublished observations). The restricted expression pattern of CDH-16 prompted us to investigate a possible role for the transcription factor Pax8 in its transcriptional regulation. Pax8 is a member of the Pax family of paired domain-containing genes, and is expressed in the developing and adult kidney and thyroid, placenta, urogenital tract and developing neural tube. Moreover, Pax8 has been shown to be a master gene for thryoid development and differention. In this study, we analyzed the role of the transcription factor Pax8 in the regulation of CDH-16 expression by different means. In particular, by reporter gene assays we demonstrated that the region of DNA identified as the minimal promoter required for the expression of CDH-16 gene is transcriptionally active in thyroid cells as well as in kidney cells, while it is not active in other cell types. At the same time, we observed that in transactivation experiments conducted in HeLa cells, Pax8 is able to activate transcription from a reporter construct carrying the CDH-16 minimal promoter. These results were further strenghtend by Chromatin-immunoprecipitation assays performed with a specific anti-PAX8 antibody on genomic DNA prepared from rat thyroid cells showing that indeed Pax8 is able to bind in vivo the promoter region of the CDH-16 gene. We then used the MatInspector analysis software to identify the Pax8 binding site(s) within the minimal promoter region and the analysis revealed the presence of one predicted Pax8 binding site within this region. The ability of Pax8 to bind to this binding site was demonstrated by EMSA assays. In addition, the mutagenesis of this binding site in the context of the CDH-16 minimal promoter abrogates the ability of Pax8 to activate transcription from it. Finally, we show that Pax8 silencing in FRTL-5 thyroid cells causes a significant down-regulation of the expression of CDH-16. Therefore, we conclude that CDH-16 is a novel downstream target of the transcription factor Pax8, likely since the early steps of thyroid development.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
