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. 2016 Jan 25:6:19821.
doi: 10.1038/srep19821.

Blocking protein phosphatase 2A signaling prevents endothelial-to-mesenchymal transition and renal fibrosis: a peptide-based drug therapy

Yuanjun Deng  1 , Yanyan Guo  1 , Ping Liu  1 , Rui Zeng  1 , Yong Ning  1 , Guangchang Pei  1 , Yueqiang Li  1 , Meixue Chen  1 , Shuiming Guo  1 , Xiaoqing Li  1 , Min Han  1 , Gang Xu  1
Affiliations

Blocking protein phosphatase 2A signaling prevents endothelial-to-mesenchymal transition and renal fibrosis: a peptide-based drug therapy

Yuanjun Deng et al. Sci Rep. .

Abstract

Endothelial-to-mesenchymal transition (EndMT) contributes to the emergence of fibroblasts and plays a significant role in renal interstitial fibrosis. Protein phosphatase 2A (PP2A) is a major serine/threonine protein phosphatase in eukaryotic cells and regulates many signaling pathways. However, the significance of PP2A in EndMT is poorly understood. In present study, the role of PP2A in EndMT was evaluated. We demonstrated that PP2A activated in endothelial cells (EC) during their EndMT phenotype acquisition and in the mouse model of obstructive nephropathy (i.e., UUO). Inhibition of PP2A activity by its specific inhibitor prevented EC undergoing EndMT. Importantly, PP2A activation was dependent on tyrosine nitration at 127 in the catalytic subunit of PP2A (PP2Ac). Our renal-protective strategy was to block tyrosine127 nitration to inhibit PP2A activation by using a mimic peptide derived from PP2Ac conjugating a cell penetrating peptide (CPP: TAT), termed TAT-Y127WT. Pretreatment with TAT-Y127WT was able to prevent TGF-β1-induced EndMT. Administration of the peptide to UUO mice significantly ameliorated renal EndMT level, with preserved density of peritubular capillaries and reduction in extracellular matrix deposition. Taken together, these results suggest that inhibiting PP2Ac nitration using a mimic peptide is a potential preventive strategy for EndMT in renal fibrosis.

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Figures

Figure 1
Figure 1. EndMT occurs in patients with primary and secondary nephropathy.
(a) Co-localization of α-SMA (red) and CD31(green) on serial sections of kidney from patients with renal fibrosis. These were representative photomicrographs from patients with IgAN, DN and LN compared with controls at a magnification of ×800 (n = 8, 5 hpf per mouse, 40 hpf per group). (b) Representative 3D reconstructed images from patients with IgA illustrated microvascular endothelial cells underwent endothelial-to-mesenchymal transition. (c) The bar graphs summarized the average of CD31+ PTC numbers per visual field and the percentage of α-SMA+CD31+ peritubular capillary (PTC) numbers in total CD31+ PTC numbers in each group. *P < 0.05. Bars represent means ± sd.
Figure 2
Figure 2. EndMT in the mouse model of UUO and TGF-β1-induced EndMT in HUVECs.
(a) Serial kidney sections from UUO and sham-operated mice were double stained with antibodies to α-SMA (green) and CD31 (red) at a magnification of ×800 (n = 8, 5 hpf per mouse, 40 hpf per group). (b) 3D reconstructed images illustrated microvascular endothelial cells underwent endothelial-to-mesenchymal transition in UUO mice. (c) The bar graphs summarized the average of CD31+ PTC numbers per visual field and the percentage of α-SMA+CD31+ PTC numbers in total CD31+ PTC numbers in each group. *P < 0.05. Bars represent means ± sd. (d) Phase contrast microscopy showing morphological changes in cells treated with TGF-β1 (10 ng/ml) for 72 h (upper). Cells were double labeled with α-SMA (green) and VE-cadherin (red), and cell nuclei were enhanced by staining with DAPI (middle). (e) Lysates were analyzed by western blot for protein expressions of α-SMA and VE-cadherin with densitometry analysis (lower). n ≥ 3. *P < 0.05 versus the control group. Bars represent means ± sd. Scale bar: 20 μm.
Figure 3
Figure 3. PP2A activates in UUO mice and TGF-β1-treated HUVECs.
(a) PP2A activity was measured using the Serine/Threonine Phosphatase Assay System in UUO kidney tissues and TGF-β1-treated HUVECs (n = 8). (b) OA prevents TGF-β1-induced EndMT. Representative western blot of α-SMA and VE-cadherin in each group with densitometry analysis of protein expression (n ≥ 3). (c) OA prevents TGF-β1-induced dephosphorylation of occludin. Cell lysates were subjected to immunoprecipitation (IP) with an anti-occludin anti-body, followed by immunoblotting (IB) with anti-p-threonine and anti-p-serine antibodies. The bargraph shows the average volume density normalized to occludin (n ≥ 3). *P < 0.05 versus the basal condition. Bars represent means ± sd.
Figure 4
Figure 4. PP2A activation depends on PP2Ac tyrosine nitration during EndMT.
(a) Representative immunohistochemical detection of PP2Ac in the kidney tissue of sham-operated and UUO mice (left). Co-localization of PP2Ac (green) with CD31(red) was visualized as yellow in the merged images from sham-operated and UUO-14d mice (n = 8). Scale bar: 20 μm. (b,c) Western blot analysis of PP2Ac expression in sham-operated and UUO-14d mice (n = 8) and in TGF-β1-treated HUVECs for the indicated periods with densitometry analysis (n ≥ 3). (d) HUVECs were incubated with or without TGF-β1 for 72 h and were harvested for immunoprecipitation analysis. Cell lysates were subjected to immunoprecipitation (IP) with an anti-PP2Ac anti-body, followed by immunoblotting (IB) with anti-3-nitrotyrosine (3-NT), anti-acetylation (acetyl), anti-phosphotyrosine (p-Tyr), and anti-methylation (unmethylated) antibodies, respectively. Densitometry analysis of 3-NT, acetyl, p-Tyr and methyl of PP2Ac in d. (e) Analysis of PP2A activity in immunoprecipitation of 3-NT-IP, Acetyl-IP, p-Tyr-IP and Unmethylated-IP, expressed as percentage of the value in control group, respectively (n ≥ 3). *P < 0.05 versus the basal condition. NS: no significance. Bars represent means ± sd.
Figure 5
Figure 5. Tyr-127 is critical for PP2Ac catalytic function.
(a) LC-ESI tandem MS (MS/MS) analysis by Q Exactive detected six peptides sequenced in the spectra. Summary of the PP2Ac modification sites (left). The amino acid sequence is based on NCBI accession number (NP_002706.1) for the mature PP2Acα protein. The numbering of all amino acid residues cited in this report refers to this amino acid sequence of the mature protein (right). (b) The figure is based on the secondary structure, deposited as PDB file (PDB accession code: 2IE4C). The helixes are colored orange, and the β-strands and loop switches are colored blue and grey, respectively. (c–g) Ribbon diagram of PP2Ac, with residues identified for PP2Ac nitration shown in yellow. (h) HUVECs were treated with TGF-β1, TGF-β1 + TAT-Y127WT/Y130WT/Y265WT Y267WT/Y284WT and cultured as untreated controls for 72 h. Representative western blot of VE-cadherin and α-SMA in each group with densitometry analysis (n ≥ 3).
Figure 6
Figure 6. The effect of TAT-Y127WT on EndMT in vitro.
HUVECs were treated with TGF-β1, TGF-β1+TAT-Y127WT, or TGF-β1+TAT-Y127Scr or were cultured as untreated controls for 72 h. (a) Representative western blot of VE-cadherin and α-SMA in each group with densitometry analysis of protein expression (n ≥ 3). (b) P-serine and p-threonine levels were measured in occludin immunoprecipitates by western blot analysis. Bar graphs present the densitometric analysis of p-serine and p-threonine (n ≥ 3). *P < 0.05 versus the TGF-β1 group. Bars represent means ± sd.
Figure 7
Figure 7. The pharmacokinetics and distribution of TAT-Y127WT in vivo.
(a) Mice were intravenously received 5nmol/g FITC-conjugated TAT-Y127WT. Representative whole-body fluorescence imaging was performed at 0, 0.5, 4 and 24 h post-injection. (b) Blood samples were used to detect peptide fluorescence in different time points (n = 6). (c) Mice were anesthetized at different time points and the heart, liver, lung, spleen and kidneys were removed and examined for fluorescence with whole body imaging. *P < 0.05 versus the basal condition. Bars represent means ± sd. Scale bar: 50 μm.
Figure 8
Figure 8. TAT-Y127WT maintains peritubular capillary density by inhibiting EndMT and exerts an anti-fibrotic effect in the UUO-induced renal fibrosis model.
(a) Colocalization of CD31 (red) with α-SMA (green)is visualized as yellow in the merged images and is marked by white arrowheads (n = 8). (b) The bar graphs summarized the average of CD31+ PTC numbers per visual field and the percentage of α-SMA+CD31+ PTC numbers in total CD31+ PTC numbers in each group at a magnification of ×800 (n = 8, 40 hpf per group). *P < 0.05 versus UUO group. Bars represent means ± sd. (c) Immunohistochemical staining for α-SMA and vimentin in the obstructed kidneys. (d) Fold change of α-SMA+ and vimentin+ areas in each group (n = 8). *P < 0.05 versus the UUO group. Bars represent means ± sd. Scale bar: 50 μm.
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References

    1. Zeisberg M. & Neilson E. G. Mechanisms of tubulointerstitial fibrosis. J Am Soc Nephrol 21, 1819–1834 (2010). - PubMed
    1. Steegh F. M. E. G. et al. Early loss of peritubular capillaries after kidney transplantation. J Am Soc Nephrol 22, 1024–1029 (2011). - PMC - PubMed
    1. Ohashi R. Peritubular Capillary regression during the progression of experimental obstructive nephropathy. J Am Soc Nephrol 13, 1795–1805 (2002). - PubMed
    1. Daehn I. & Bottinger E. P. Microvascular endothelial cells poised to take center stage in experimental renal fibrosis. J Am Soc Nephrol 26, 767–769 (2015). - PMC - PubMed
    1. Zeisberg E. M., Potenta S., Xie L., Zeisberg M. & Kalluri R. Discovery of Endothelial to Mesenchymal Transition as a Source for Carcinoma-Associated Fibroblasts. Cancer Res 67, 10123–10128 (2007). - PubMed

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