TGF-β1 is involved in senescence-related pathways in glomerular endothelial cells via p16 translocation and p21 induction - PubMed (original) (raw)

Sayo Ueda et al. Sci Rep. 2021.

Abstract

p16 inhibits cyclin-dependent kinases and regulates senescence-mediated arrest as well as p21. Nuclear p16 promotes G1 cell cycle arrest and cellular senescence. In various glomerular diseases, nuclear p16 expression is associated with disease progression. Therefore, the location of p16 is important. However, the mechanism of p16 trafficking between the nucleus and cytoplasm is yet to be fully investigated. TGF-β1, a major cytokine involved in the development of kidney diseases, can upregulate p21 expression. However, the relationship between TGF-β1 and p16 is poorly understood. Here, we report the role of podocyte TGF-β1 in regulating the p16 behavior in glomerular endothelial cells. We analyzed podocyte-specific TGF-β1 overexpression mice. Although p16 was found in the nuclei of glomerular endothelial cells and led to endothelial cellular senescence, the expression of p16 did not increase in glomeruli. In cultured endothelial cells, TGF-β1 induced nuclear translocation of p16 without increasing its expression. Among human glomerular diseases, p16 was detected in the nuclei of glomerular endothelial cells. In summary, we demonstrated the novel role of podocyte TGF-β1 in managing p16 behavior and cellular senescence in glomeruli, which has clinical relevance for the progression of human glomerular diseases.

© 2021. The Author(s).

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Figure 1

Characterization and pathological changes in podocyte-specific TGF-β1 overexpression mice. (a) TGF-β1 was expressed in podocytes. HA-tag was conjugated with bioactive porcine TGF-β1 in PodCre(+) TGF mice. HA-tag merged with nephrin (podocyte marker). (b) Diffuse expression of HA-tag conjugated TGF-β1 was observed in the kidney of PodCre(+) TGF mice. (c) Representative pictures of western blot analysis of glomeruli protein. The bands immunoblotted with Smad3 increased in size when phosphorylated (arrow). Smad3 was phosphorylated in glomeruli in PodCre(+) TGF mice. (d) Urine albumin excretion was significantly increased in PodCre(+) TGF mice. (N = 6 in control mice, N = 8 in PodCre(+) TGF mice). *P < 0.01 (Mann–Whitney’s U test). (e) Representative pictures of periodic acid-Schiff (PAS) stain and collagen IV immunohistochemistry in PodCre(+) TGF mice. PodCre(+) TGF mice showed a significant increase in collagen IV immunostained area (N = 6 in control mice, N = 8 in PodCre(+) TGF mice). *P < 0.01 (t-test). (f) Representative pictures of electron microscopy. PodCre(+) TGF mice showed mesangial expansion (white arrow) and diffuse foot process effacement (black arrow). Scale bar: 10 µm. (g) Representative pictures of glomerular basement membrane by using electron microscopy and quantitative evaluation of glomerular basement membrane width. PodCre(+) TGF mice showed a significant thickening of glomerular basement membrane (N = 3 in control mice, N = 4 in PodCre(+) TGF mice). Scale bar: 2 µm. **P < 0.05 (t-test). (−): control mice. (+): Podocyte-specific TGF-β1 overexpression mice (PodCre(+) TGF mice). Po.: positive control. pSmad3: phosphorylated Smad3. n.s.: not significant.

Figure 2

Figure 2

Detection of the markers for cellular senescence in podocyte-specific TGF-β1 overexpression mice. (a) Representative pictures of senescence-associated β-galactosidase staining. The nuclei were counterstained with propidium iodide. Senescence-associated β-galactosidase activity was significantly increased in PodCre(+) TGF mice. (N = 3 in control mice, N = 4 in PodCre(+) TGF mice). **P < 0.05 (t-test). (b) Representative pictures of western blot analysis of Rb2 and p27 expression in glomeruli. PodCre(+) TGF mice had significant expression levels of Rb2 and p27. (N = 4 in control mice, N = 6 in PodCre(+) TGF mice). **P < 0.05 (t-test). (c) Representative pictures of p16 immunohistochemistry. PodCre(+) TGF mice had p16 expression mainly in endothelial cells. PodCre(+) TGF mice showed a significant increase in p16 immunostained nuclei in endothelial and mesangial cells. (N = 6 in control mice, N = 8 in PodCre(+) TGF mice). **P < 0.05 (t-test). (d) Representative pictures of p21 immunohistochemistry. PodCre(+) TGF mice had p21 expression mainly in endothelial cells. PodCre(+) TGF mice showed a significant increase in p21 immunostained nuclei in endothelial cells. (N = 6 in control mice, N = 8 in PodCre(+) TGF mice). **P < 0.05 (t-test). (e) Representative pictures of western blot analysis of p16 and p21 expression in glomeruli. PodCre(+) TGF mice had a significant expression of p21, but not that of p16. (N = 4 in control mice, N = 6 in PodCre(+) TGF mice). **P < 0.05 (t-test). (−): control mice. (+): Podocyte-specific TGF-β1 overexpression mice (PodCre(+) TGF mice). PI: Propidium iodide. SA-β-gal: Senescence-associated β-galactosidase. Col IV: Collagen IV. Endo.: Endothelial cell. Mes.: Mesangial cell. n.s.: not significant.

Figure 3

Figure 3

Expression of p16 and p21 induced by the activation of the TGF-β1-Smad3 pathway in endothelial cells. (a) Representative pictures of western blot analysis of p16 and p21 expression in endothelial cells induced by the stimulation of the TGF-β1-Smad3 pathway. (b, c) Activation of the TGF-β1-Smad3 pathway can increase the expression of p21 in 24 h (late phase), but not that of p16 in endothelial cells (N = 3). C: control. T: TGF-β1. S: SB431542. CA: constitutive active Smad3. pSmad3: phosphorylated Smad3. n.s.: not significant. **P < 0.05 (t-test).

Figure 4

Figure 4

Nuclear translocation of p16 induced by the activation of the TGF-β1-Smad3 pathway in endothelial cells. (a) Representative pictures of western blot analysis of p16 and p21 expression in the nucleus and cytoplasm of endothelial cells induced by the stimulation of the TGF-β1-Smad3 pathway. (b, c) Activation of the TGF-β1-Smad3 pathway can translocate p16 to the nuclei in 30 min (early phase), while it can increase the expression of p21 in endothelial cells in 24 h (late phase) (N = 4). C: control. T: TGF-β1. S: SB431542. CA: constitutive active Smad3. nuc.: nucleus. cyto.: cytoplasm. n.s.: not significant. *P < 0.01. **P < 0.05 (t-test).

Figure 5

Figure 5

Expression of β-galactosidase and p16 in the glomeruli of patients with kidney diseases. (a) β-galactosidase was detected in glomeruli of patients with kidney disease. (b) p16 is expressed in endothelial, mesangial cells, and extracapillary cells/podocytes in patients with various human kidney diseases. Representative pictures of each pattern are shown. β-gal: β-galactosidase. Col IV: Collagen IV. Cont.: Control (N = 3). MCD: Minimal change disease (N = 3). Lupus III, IV: Systemic lupus nephritis class III or IV (N = 6). Lupus V: Systemic lupus nephritis class V (N = 3). IgA: IgA nephropathy (N = 6). Purp.: Purpura nephritis (N = 4). MPGN: Membranoproliferative glomerulonephritis (N = 3). MN: Membranous nephropathy (N = 3). ANCA: ANCA glomerulonephritis (N = 6). DN: Diabetic nephropathy (N = 6). White column: Endothelial cells. Grey column: Mesangial cells. Black column: Extracapillary cells and podocytes.

Similar articles

Cited by

References

    1. Serrano M, Hannon GJ, Beach D. A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature. 1993;366:704–707. doi: 10.1038/366704a0. - DOI - PubMed
    1. Serrano M. The tumor suppressor protein p16INK4a. Exp. Cell Res. 1997;237:7–13. doi: 10.1006/excr.1997.3824. - DOI - PubMed
    1. McHugh D, Gil J. Senescence and aging: Causes, consequences, and therapeutic avenues. J. Cell Biol. 2018;217:65–77. doi: 10.1083/jcb.201708092. - DOI - PMC - PubMed
    1. Kuilman T, Michaloglou C, Mooi WJ, Peeper DS. The essence of senescence. Genes Dev. 2010;24:2463–2479. doi: 10.1101/gad.1971610. - DOI - PMC - PubMed
    1. Salama R, Sadaie M, Hoare M, Narita M. Cellular senescence and its effector programs. Genes Dev. 2014;28:99–114. doi: 10.1101/gad.235184.113. - DOI - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources