A Tight Control of Non-Canonical TGF-β Pathways and MicroRNAs Downregulates Nephronectin in Podocytes

Nephronectin (NPNT) is an extracellular matrix protein in the glomerular basement membrane that is produced by podocytes and is important for the integrity of the glomerular filtration barrier. Upregulated transforming growth factor beta (TGF-beta) and altered NPNT are seen in different glomerular diseases. TGF-beta downregulates NPNT and upregulates NPNT-targeting microRNAs (miRs). However, the pathways involved were previously unknown. By using selective inhibitors of the canonical, SMAD-dependent, and non-canonical TGF-beta pathways, we investigated NPNT transcription, translation, secretion, and regulation through miRs in podocytes. TGF-beta decreased NPNT mRNA and protein in cultured human podocytes. TGF-beta-dependent regulation of NPNT was meditated through intracellular signaling pathways. Under baseline conditions, non-canonical pathways predominantly regulated NPNT post-transcriptionally. Podocyte NPNT secretion, however, was not dependent on canonical or non-canonical TGF-beta pathways. The canonical TGF-beta pathway was also dispensable for NPNT regulation after TGF-beta stimulation, as TGF-beta was still able to downregulate NPNT in the presence of SMAD inhibitors. In contrast, in the presence of different non-canonical pathway inhibitors, TGF-beta stimulation did not further decrease NPNT expression. Moreover, distinct non-canonical TGF-beta pathways mediated TGF-beta-induced upregulation of NPNT-targeting miR-378a-3p. Thus, we conclude that post-transcriptional fine-tuning of NPNT expression in podocytes is mediated predominantly through non-canonical TGF-beta pathways.

Automated summary

The study reveals the significance of transforming growth factor beta and Nephronectin in various glomerular diseases. It demonstrates that transforming growth factor beta regulates Nephronectin expression through non-canonical signaling pathways and mediates the upregulation of microRNA-378a-3p targeting Nephronectin.