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

Abstract

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 β (TGF-β) and altered NPNT are seen in different glomerular diseases. TGF-β 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-β pathways, we investigated NPNT transcription, translation, secretion, and regulation through miRs in podocytes. TGF-β decreased NPNT mRNA and protein in cultured human podocytes. TGF-β-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-β pathways. The canonical TGF-β pathway was also dispensable for NPNT regulation after TGF-β stimulation, as TGF-β was still able to downregulate NPNT in the presence of SMAD inhibitors. In contrast, in the presence of different non-canonical pathway inhibitors, TGF-β stimulation did not further decrease NPNT expression. Moreover, distinct non-canonical TGF-β pathways mediated TGF-β-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-β pathways.

Keywords: TGF-β; microRNAs; nephronectin; podocytes; podocytopathies; post-transcriptional regulation.

Figure 1

Illustration of canonical and non-canonical TGF-β pathways and the respective inhibitors used for the blockade of separate parts of the pathways. TGF-β signaling is intracellularly mediated via a canonical, SMAD2- and SMAD3-dependent pathway or different non-canonical pathways, which signal via different routes, namely the Rho-ROCK, NFkB, PI3K-AKT-mTOR, and MAP-kinase pathways via JNK or p53 and Ras-Raf-MEK-ERK. Each signaling arm facilitates different reactions in the cell, such as proliferation, cell survival, epithelial-to-mesenchymal transition (EMT), or protein synthesis (adapted from [18]).

Figure 2

NPNT expression and localization in human podocytes. (a,b): NPNT mRNA (a) and protein (b) expression in cultured human podocytes, untreated and after TGF-β treatment. NPNT expression was normalized to HPRT and GAPDH, respectively, and is given as change compared to untreated cells. A representative Western blot picture is shown. n = 3–17 independent experiments, * p < 0.05, *** p < 0.001. (c): Change in NPNT protein concentration measured in the cell culture supernatant of untreated cultured human podocytes and after stimulation with TGF-β. n = 5 independent experiments.

Figure 3

TGF-β-induced downregulation of NPNT is mediated by TGF-βR1. (a,b): qPCR (a) and Western blot (b) for NPNT mRNA and protein expression, respectively, in cultured human podocytes, untreated and after TGF-βR1 inhibition with SD208. NPNT expression was normalized to HPRT and GAPDH, respectively, and is given as the change compared to untreated cells. A representative Western blot picture is shown. n = 6–8 independent experiments, ** p < 0.01. (c): NPNT protein concentration in cell culture supernatant of human podocytes, untreated and after treatment with SD208 (TGF-βR1 inhibitor), given as the change compared to untreated cells. n = 4 independent experiments.

Figure 4

Non-canonical pathways predominantly mediate post-transcriptional regulation of NPNT under baseline conditions. (a,b) qPCR for NPNT mRNA in cultured human podocytes, untreated and after treatment with inhibitors of the non-canonical pathways (a), PD169316, SP600125, PD98059, AKT inhibitor, MG-132, and Y-27632, or with SM16 and/or SIS3 for canonical pathway inhibition (b). NPNT mRNA expression normalized to HPRT is given as the change compared to untreated cells. n = 6–7 independent experiments; ** p < 0.01. (c,d) NPNT protein expression in cultured human podocytes, untreated and after treatment with different inhibitors of the non-canonical (c) and the canonical pathways (d) as described in (a,b). NPNT protein expression, normalized to GAPDH, is given as the change compared to untreated cells. A representative Western blot result is shown for each treatment. n = 6–11 independent experiments; n.s. = not significant, * p < 0.05. (e,f) Human podocytes were treated as described in (a,b). NPNT protein expression was measured by ELISA in the cell culture supernatant and plotted as the change compared to untreated controls. n = 3 independent experiments.

Figure 5

TGF-β-mediated downregulation of NPNT is dependent on the non-canonical signaling pathways in podocytes. (a,b) Western blot to determine NPNT protein expression in cultured human podocytes in the presence of (a) canonical (SM16 and SIS3) or (b) non-canonical (PD169316, SP600125, PD98059, AKT inhibitor, MG-132, and Y-27632) pathway inhibitors after stimulation with TGF-β. NPNT protein expression, normalized to GAPDH, is given as the change compared to stimulation with inhibitor without TGF-β. Representative Western blots are shown for each treatment. n = 5–6 independent experiments; * p < 0.05.

Figure 6

Post-transcriptional regulation of NPNT expression might be mediated by miR-378a-3p. (a,b): TaqMan PCR for miR-378a-3p detection in cultured human podocytes after treatment with inhibitors of the (a): non-canonical pathways (PD169316, SP600125, PD98059, AKT inhibitor, MG-132, and Y-27632) and (b): canonical pathway inhibitors (SM16 and SIS3), plotted as the change as compared to untreated cells. n = 4–6 independent experiments. * p < 0.05, ** p < 0.01 *** p < 0.001.

Figure 7

Proposed mechanism of podocyte NPNT regulation by TGF-β. We propose that podocyte NPNT expression is regulated primarily via the non-canonical TGF-β pathways. This regulation is mediated directly via inhibition of NPNT transcription and indirectly via miR-378a-3p. TGF-βRI-mediated phosphorylation of eukaryotic elongation factor 1A1 (eEF1A1) might further be involved in the regulation of protein translation [33]. Intracellular NPNT is either secreted and deposited into the glomerular basement membrane (GBM) or degraded. GEC = glomerular endothelial cell.