Abstract: FR-PO711

MicroRNA-181a (miR-181a) Drives Deptor Downregulation by TGFbeta (TGFb) to Induce Mesangial Cell (MC) Hypertrophy and Fibronectin Expression

Session Information

Category: Glomerular

  • 1002 Glomerular: Basic/Experimental Pathology

Authors

  • Maity, Soumya, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States
  • Das, Falguni, UTHSCSA, SAN ANTONIO, Texas, United States
  • Ghosh-choudhury, Nandini, UTHSCSA, SAN ANTONIO, Texas, United States
  • Kasinath, Balakuntalam S., University of Texas Health Science Center, San Antonio, Texas, United States
  • Ghosh-Choudhury, Goutam, University of Texas Health Science Center, San Antonio, Texas, United States
Background

TGFb-stimulated noncanonical mTOR signaling contributes to MC hypertrophy and matrix protein expansion. The molecular mechanism of mTOR activation is not known. Deptor is a common inhibitory subunit for both mTORC1 and mTORC2.

Methods

MCs, qRT-PCR, reporter transfection, immunoblotting, anti-miR-181a and miR-181 mimic transfection, protein synthesis and hypertrophy assays were used.

Results

In MCs, TGFb reduced the expression of deptor in a time-dependent and prolonged manner. This deptor downregulation was associated with increased activation of both mTORC1 and mTORC2 as determined by the phosphorylation of S6 kinase and Akt (Ser-473), respectively. To study the mechanism of repression of deptor, we considered microRNA-mediated post-transcriptional regulation. Bioinformatic analysis revealed the presence of miR-181a recognition element in the 3’UTR (untranslated region) of deptor mRNA. In MCs, TGFb significantly increased the expression of miR-181a in time-dependent manner. Overexpression of miR-181a mimic inhibited the expression of deptor. To determine the responsiveness of the miR-181a recognition element, we used a reporter construct in which deptor 3’ UTR was fused to the luciferase gene (3’UTR-Luc). Co-transfection of miR-181a mimic with this reporter plasmid showed significant reduction in luciferase activity, indicating the responsiveness of the recognition element. Incubation of 3’UTR-Luc-transfected MCs with TGFb significantly decreased the luciferase activity. Importantly, transfection of anti-miR-181a inhibited TGFb-induced phosphorylation of S6 kinase and Akt (Ser-473), two substrates of mTORC1 and mTORC2, respectively. In contrast transfection of miR-181a mimic increased the activity of mTORC1 and mTORC2. Furthermore, anti-miR-181a significantly inhibited TGFb-induced protein synthesis and hypertrophy of mesangial cells. Similarly, anti-miR-181a attenuated TGFb-stimulated fibronectin expression.

Conclusion

Our results provide the first evidence for the mechanism of deptor downregulation by TGFb, involving miR-181a. Furthermore, we demonstrate for the first time that miR-181a contributes to TGFb-induced mesangial cell hypertrophy and matrix protein expression. Use of anti-miR-181a may be beneficial in TGFb-mediated fibrotic kidney disease.

Funding

  • NIDDK Support