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miR-744 enhances type I interferon signaling pathway by targeting PTP1B in primary human renal mesangial cells - PubMed

  • ️Thu Jan 01 2015

miR-744 enhances type I interferon signaling pathway by targeting PTP1B in primary human renal mesangial cells

Xiaoyan Zhang et al. Sci Rep. 2015.

Abstract

Renal mesangial cells (RMCs) constitute a population of cells in glomerular mesangium. Inflammatory cytokines produced by RMCs play a vital role in renal inflammation. miRNAs are key regulators of inflammatory cytokine expression. The abnormal expression of renal miRNAs and the consequent changes in inflammatory signal transduction are closely associated with renal inflammation. However, our knowledge of the functions of renal miRNAs is still limited. In this study, we investigated the role of miR-744 in type I interferon (IFN) signaling pathway in primary human RMCs. We show that overexpression of miR-744 enhances IFN-induced CCL2, CCL5, CXCL10, and IL6 expression specifically in RMCs. We found that the activation of TYK2, STAT1 and STAT3 was significantly enhanced by miR-744. miR-744 also enhanced the activation of non-classical signal components, such as ERK and p38. We then identified PTP1B, a ubiquitously expressed phosphatase, as the target of miR-744 that is responsible for enhancing type I IFN response. Finally, miR-744 expression was induced by type I IFN in RMCs. Collectively, our data indicate that by targeting PTP1B, miR-744 plays a feed-forward role in regulating type I IFN signaling pathway. These findings give us new insights into the functions of renal miRNAs in regulating important signaling pathways.

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Figures

Figure 1
Figure 1. miR-744 enhances the expression of type-I-IFN-inducible genes in primary human RMCs.

RMCs were transfected with an miR-744 mimic (200 nM) or negative control (NC) mimic (200 nM) 24 h before type I IFN was added. The cells were harvested after 6 h. The relative expression of type-I-IFN-inducible genes was detected with real-time PCR: CCL2, CCL5, CXCL10, and IL6 are shown in (A); MX1 and IFIT3 are shown in (B). Supernatants harvested at 6 h were used to detect protein levels of CCL2, CXCL10, and IL6 (C); Supernatants harvested at 24 h were used to detect protein levels of CCL5 with an ELISA (D). RMCs were treated with an miR-744 inhibitor (400 nM) or control inhibitor (400 nM) for 48 h, then the cells were incubated for 6 hours with type I IFN. The relative expression of type-I-IFN-inducible genes was measured with real-time PCR: CCL2, CCL5, CXCL10, and IL6 are shown in (E); MX1 and IFIT3 are shown in (F). Supernatants harvested at 6 h were used to measure protein levels of CCL2, CXCL10, and IL6 (G); Supernatants harvested at 24 h were used to measure protein levels of CCL5 with an ELISA (H).*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; NS, not significant.

Figure 2
Figure 2. miR-744 enhances the classical JAK–STAT pathway and non-classical MAPK and NF-κB pathways downstream of type I IFN.

Cells were transfected with miR-744 mimic or NC mimic (200 nM) for 24 h, then stimulated with type I IFN for 0 min or 15 min. Proteins were harvested and analyzed with western blotting to detect the phosphorylation of TYK2, STAT1, JAK1, and STAT2 (A) in the JAK–STAT pathway and p38, ERK, and STAT3 in the non-classical pathway (B) (The full-length blots/gels of TYK2, STAT1, JAK1, STAT2, p38, ERK and STAT3 are presented in Supplementary Fig. S4, Fig. S5, Fig. S6, Fig. S7, Fig. S8, Fig. S9, Fig. S10, respectively). RMCs were pretreated with SB203580 (p38 inhibitor), PD98059 (ERK inhibitor), or PDTC (NF-κB inhibitor) for 0.5 h before stimulation with type I IFN for 6 h. RNA was then harvested and the expression of IFN-inducible inflammatory genes was detected (C,D). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; NS, not significant.

Figure 3
Figure 3. miR-744 targets PTP1B, which is responsible for its regulation of type I IFN signaling pathway.

(A) Schematic diagram of potential miR-744 binding sites in the 3′-UTR of PTP1B, predicted by RNAhybrid, and two mutant binding sites. Mutant 1 abolished the binding to miR-744 without changing the nucleotide composition of the sequence, while mutant 2 affected both the nucleotide composition of the sequence and the binding of the 3′-UTR to miR-744. (B) RMCs were simultaneously transfected with NC or miR-744 (200 nM) and the PTP1B 3′-UTR-containing vector (50 ng per well) or pSicheck2 vector (50 ng per well). Luciferase activity was measured 24 h after transfection. (C) RMCs were simultaneously transfected with NC or miR-744 (200 nM) and the PTP1B 3′-UTR-containing vector (50 ng per well) or PTP1B 3′-UTR mutant vector (50 ng per well). Luciferase activity was measured 24 h after transfection, quantified, and expressed as relative luciferase activity. (D) RMCs were transfected with miR-744 mimic or inhibitor and the corresponding control mimic or inhibitor for either 24 h for the mimics (left) or for 48 h for the inhibitors (right). The levels of PTP1B mRNA were detected after stimulation with type I IFN for 6 h. (E) RMCs were transfected with miR-744 mimic or inhibitor and the corresponding control mimic or inhibitor for either 24 h for the mimics or for 48 h for the inhibitors. PTP1B was detected in the whole-cell lysates with western blotting. The ratios of PTP1B to β-tubulin in the control-transfected cells was arbitrarily set at 1(The full-length blots/gels of PTP1B are presented in Supplementary Fig. S11, Fig. S12, respectively). (F) Induction of CCL5 after PTP1B was silenced with siRNA (200 nM) in RMCs. (G) Efficiency of siRNA measured with real-time PCR. (H) Western blotting analysis of the phosphorylation of STAT1 and STAT3 in PTP1B-silenced RMCs. Cells were treated with type I IFN (1000 U/mL) for the indicated times (The full-length blots/gels of STAT1 and STAT3 are presented in Supplementary Fig. S13, Fig. S14, respectively). At least three independent experiments were performed. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. NS, not significant.

Figure 4
Figure 4. miR-744 is induced by type I IFN and may act as a feed-forward regulator of the type I IFN signaling pathway.

(A) RMCs were stimulated with type I IFN (1000 U/mL) and the RNA was collected at the indicated time points. The expression of miR-744 was detected with qRT–PCR. Each point shows the mean relative expression level of miR-744 for three independent experiments. We used RNU48 as the reference gene. (B) Schematic diagram of the mechanisms by which miR-744 feed-forward regulates the IFN-activated classical JAK–STAT and non-classical MAPK and NF-κB signaling pathways. Type I IFN triggers an unknown transcription factor to induce miR-744 expression. miR-744 subsequently represses PTP1B expression, leading to the enhanced activation of TYK2, STAT1, STAT3, ERK, p38, and NF-κB, thus enhancing the expression of IFN-induced inflammatory genes.

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References

    1. Hawkins N. J., Wakefield D. & Charlesworth J. A. The role of mesangial cells in glomerular pathology. Pathology 22, 24–32 (1990). - PubMed
    1. Radeke H. & Resch K. The inflammatory function of renal glomerular mesangial cells and their interaction with the cellular immune system. Clin Investig 70, 825–842 (1992). - PubMed
    1. Veis J., Yamashita W., Liu Y. & Ooi B. The biology of mesangial cells in glomerulonephritis. Proc Soc Exp Biol Med 195, 160–167 (1990). - PubMed
    1. Flür K. et al. Viral RNA induces type I interferon-dependent cytokine release and cell death in mesangial cells via melanoma-differentiation-associated gene-5: Implications for viral infection-associated glomerulonephritis. Am J Physiol 175, 2014–2022 (2009). - PMC - PubMed
    1. Chan T.-M., Leung J. K.-H., Ho S. K.-N. & Yung S. Mesangial cell-binding anti-DNA antibodies in patients with systemic lupus erythematosus. J Am Soc Nephrol 13, 1219–1229 (2002). - PubMed

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