Abstract
ADAMTS16 (a disintegrin and metalloproteinase with thrombospondin motifs) is a secreted mammalian metalloproteinase with unknown function. We report here that murine Adamts16 is co-expressed with the Wilms tumor protein, Wt1, in the developing glomeruli of embryonic kidneys. Adamts16 mRNA levels were significantly reduced upon transfection of embryonic murine kidney explants with Wt1 antisense vivo-morpholinos. Antisense knockdown of Adamts16 inhibited branching morphogenesis in kidney organ cultures. Adamts16 was detected by in situ mRNA hybridization and/or immunohistochemistry also in embryonic gonads and in spermatids and granulosa cells of adult testes and ovaries, respectively. Silencing of Wt1 by transfection with antisense vivo-morpholinos significantly increased Adamts16 mRNA in cultured embryonic XY gonads (11.5 and 12.5 days postconception), and reduced Adamts16 transcripts in XX gonads (12.5 and 13.5 days postconception). Three predicted Wt1 consensus motifs could be identified in the promoter and the 5'-untranslated region of the murine Adamts16 gene. Binding of Wt1 protein to these elements was verified by EMSA and ChIP. A firefly luciferase reporter gene under control of the Adamts16 promoter was activated ∼8-fold by transient co-transfection of human granulosa cells with a Wt1 expression construct. Gradual shortening of the 5'-flanking sequence successively reduced and eventually abrogated Adamts16 promoter activation by Wt1. These findings demonstrate that Wt1 differentially regulates the Adamts16 gene in XX and XY embryonic gonads. It is suggested that Adamts16 acts immediately downstream of Wt1 during murine urogenital development. We propose that Adamts16 is involved in branching morphogenesis of the kidneys in mice.
Highlights
ADAMTS16 is a mammalian metalloproteinase with unknown function
Adamts16 and Wt1 Proteins Are Expressed in Gonads and Kidneys— Adamts16 mRNA has been detected by Reverse Transcription (RT)-PCR in several organs, including kidneys and gonads [1], very little is known about the protein localization in these tissues
Our initial microarray gene expression analysis showed that Adamts16 transcripts were strongly reduced upon RNAi silencing of the Wilms tumor protein Wt1 in mesonephros-derived M15 cells
Summary
Results: Transcription of the Adamts gene is regulated by Wilms tumor protein Wt1, and knockdown of Adamts reduces branching morphogenesis in cultured embryonic kidneys. It is not known whether Egr and Sp1 are physiological regulators of ADAMTS16 in vivo; nor have other trans-acting factors been ascertained far In view of this background, our study was aimed at providing novel insights into the gene regulatory mechanisms underlying the characteristic expression pattern of ADAMTS16. Knockdown of Adamts by transfection with antisense vivo-morpholinos impaired branching morphogenesis in murine embryonic kidney cultures. These findings suggest a role for Adamts in the development of the genitourinary system in mice
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