WNT16B is a new marker of cellular senescence that regulates p53 activity and the phosphoinositide 3-kinase/AKT pathway

Abstract

Senescence is a tumor suppression mechanism that is induced by several stimuli, including oncogenic signaling and telomere shortening, and controlled by the p53/p21(WAF1) signaling pathway. Recently, a critical role for secreted factors has emerged, suggesting that extracellular signals are necessary for the onset and maintenance of senescence. Conversely, factors secreted by senescent cells may promote tumor growth. By using expression profiling techniques, we searched for secreted factors that were overexpressed in fibroblasts undergoing replicative senescence. We identified WNT16B, a member of the WNT family of secreted proteins. We found that WNT16B is overexpressed in cells undergoing stress-induced premature senescence and oncogene-induced senescence in both MRC5 cell line and the in vivo murine model of K-Ras(V12)-induced senescence. By small interfering RNA experiments, we observed that both p53 and WNT16B are necessary for the onset of replicative senescence. WNT16B expression is required for the full transcriptional activation of p21(WAF1). Moreover, WNT16B regulates activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway. Overall, we identified WNT16B as a new marker of senescence that regulates p53 activity and the PI3K/AKT pathway and is necessary for the onset of replicative senescence.

Figure 1.

Expression of WNT16 in young (PDL26) and replicative senescent (PDL64) MRC5 human fibroblasts. A, MRC5 fibroblasts PDL26 and PDL64 were stained for SA-β-Gal expression and quantified. Columns, mean; bars, SD. B, left, WNT16A, WNT16B, and p21 mRNA expression was analyzed by RT-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as control. Right, WNT16B and p21 mRNA expression by qRT-PCR. mRNA expression in MRC5 PDL26 fibroblasts was normalized to 1. Columns, mean; bars, SD. C, Western blot analysis of WNT16, p53, and p21 protein expression. β-Actin expression was used as a loading control. D, TCA precipitation of proteins from the culture medium of MRC5 fibroblasts and Western blot analysis of WNT16 protein secretion.

Figure 2.

Expression of WNT16B in SIPS and OIS. A, MRC5 fibroblasts were stained for SA-β-Gal expression and quantified. Columns, mean; bars, SD. B, WNT16B mRNA expression was analyzed by qRT-PCR in MRC5 PDL26 fibroblasts exposed to H₂O₂ or X-rays (left) or infected with a retrovirus coding for H-Ras^V12 (right). mRNA expression in MRC5 PDL26 fibroblasts was normalized to 1. Columns, mean; bars, SD. C, left and middle, Western blot analysis of WNT16B, p53, and p21 protein expression in MRC5 PDL26 fibroblasts exposed to H₂O₂ or X-rays; right, WNT16, Rb, and phosphorylated ERK protein expression was analyzed in MRC5 PDL26 fibroblasts infected with a retrovirus coding for H-Ras^V12. β-Actin was used as a loading control. D, expression of WNT16B during OIS in vivo. Immunohistochemical analysis of WNT16B, p19^ARF, p16^INK4A, and Ki-67 in serial sections of K-Ras^V12–induced lung adenomas. Note strong positive staining of WNT16B in the cytoplasm of most tumor cells [adenomas (Ad)] but weak or negative staining in the pocket of nontumoral cells located in the bottom left corner [normal lung (NL)]. Most tumor cells showed nuclear staining of p19^ARF often with intense signal in the nucleoli. A fraction of tumor cells gave positive nuclear staining for p16^INK4A, whereas the cytoplasmic staining could be nonspecific. Scale bar, 50 μm.

Figure 3.

Inhibition of WNT16B or p53 expression in presenescent fibroblasts. A, control senescent cells (PDL64), WNT16B^kd cells, and p53^kd MRC5 fibroblasts were stained for SA-β-Gal activity and quantified. Columns, mean; bars, SD. B, left, 5,000 control senescent cells (PDL64), WNT16B^kd cells, and p53^kd cells were seeded into 10-cm² plates for 3 wk and stained with methylene blue; right, proliferation curves of control senescent cells (PDL64), WNT16B^kd cells, and p53^kd cells. C, left, WNT16B, p53, p21, WIG1, PERP, and PAI-1 mRNA expression was analyzed by qRT-PCR in WNT16B^kd MRC5 fibroblasts. mRNA expression in MRC5 PDL26 fibroblasts was normalized to 1. Columns, mean; bars, SD. Right, WNT16, p53, and p21 protein expression was analyzed using α-tubulin as a loading control. D, left, WNT16B, p53, and p21 mRNA expression in young (PDL26), senescent (PDL64), or p53^kd MRC5 fibroblasts was analyzed by qRT-PCR. mRNA expression in MRC5 PDL26 fibroblasts was normalized to 1. Columns, mean; bars, SD. Right, WNT16, p53, and p21 protein expression was analyzed using α-tubulin as a loading control.

Figure 4.

Regulation of p21 transcription by WNT16B. A, qRT-PCR analysis of WNT16B and p53 expression in MRC5-hTERT cells transfected with a control siRNA (siCT) and a siRNA targeting WNT16B (siWNT16B) or p53 (siP53). B, MRC5-hTERT fibroblasts expressing siCT or siWNT16B and treated with H2O2 were stained for SA-β-Gal expression and quantified. Columns, mean; bars, SD. C, qRT-PCR analysis of p21 expression after H2O2 treatment (left) or X radiations (right). MRC5-hTERT cells were transfected with siCT, siWNT16B, or siP53. D, Western blot analysis of WNT16, p53, and p21 expression and p53 and AKT activation after X radiations. α-Tubulin was used as a loading control. The film was overexposed to detect WNT16 basal expression and evaluate the siWNT16B efficiency. Note the presence of a cross-reacting band seen only in hTERT-immortalized fibroblasts.

Figure 5.

Regulation of AKT by WNT16B. A, Western blot analysis of AKT activation in dividing (PDL28), senescent (PDL64), WNT16B^kd, and p53^kd MRC5 fibroblasts. α-Tubulin was used as a loading control. B, Western blot analysis of AKT activation and WNT16, p53, and p21 protein expression in pBabe and pBabe-WNT16B MRC5 fibroblasts. α-Tubulin was used as a loading control. C, analysis of AKT activation in pBabe-WNT16B MRC5 fibroblasts by Western blot after treatment with LY294002.