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, 95 (30), e4297

An Investigation of Crosstalk Between Wnt/β-catenin and Transforming Growth Factor-β Signaling in Androgenetic Alopecia

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An Investigation of Crosstalk Between Wnt/β-catenin and Transforming Growth Factor-β Signaling in Androgenetic Alopecia

Gui-Qing Lu et al. Medicine (Baltimore).

Abstract

Background: Wnt and transforming growth factor-β (TGF-β) signaling pathways are known to be involved in the pathogenesis of androgenetic alopecia (AGA). However, the way that Wnt and TGF-β signaling is altered in patients with AGA and whether there exists a crosstalk between them in pathogenetic process of AGA remain unclear.

Objectives: To investigate the expression of Wnt and TGF-β signaling and the crosstalk between these 2 signaling pathways in AGA.

Methods: Fifteen male patients with AGA were recruited for our research. Fifteen scalp specimens of the balding were collected from frontal areas, and 9 nonbalding were collected from occipital areas. We analyzed the expression and activation of downstream Wnt and TGF-β signaling molecules in both balding and nonbalding hair follicles isolated from scalp specimens. Furthermore, we evaluated the activation of Wnt and TGF-β signaling after either of them was blocked with the inhibitor in balding and nonbalding dermal papilla (DP) cells.

Results: Compared with the nonbalding counterparts, the mRNA level of Wnt10a and LEF1 was decreased. But TβRI and TβRII, and the protein expression of TGF-β1 was elevated in balding hair follicles. To investigate the crosstalk between Wnt and TGF-β signaling, we used SB431542 to inhibit the TGF-β signaling in balding DP cells and found that SB431542 significantly attenuated the phosphorylation of Smad2 and Akt. However, the mRNA level of Wnt10a, LEF1, and the nuclear translocation of β-catenin was increased. On the other hand, we suppressed the Wnt signaling by XAV939 in nonbalding DP cells, which displayed that the level of β-catenin and LEF1 was significantly inhibited; however, the level of active TGF-β1 and the phosphorylation of Smad2 and Akt were up-regulated.

Conclusions: These data indicate that crosstalk between Wnt/β-catenin and TGF-β signaling pathways may exist as one of the important mechanisms contributing to AGA.

Conflict of interest statement

The authors have no conflicts of interest to disclose.

Figures

Figure 1
Figure 1
Activation of Wnt/β-catenin signaling in AGA pathology. Fifteen specimens of the balding hair follicles were collected from the frontal scalp (lanes 10–24), and 9 specimens of nonbalding hair follicles were collected from occipital scalp (lanes 1–9). The mRNA level of Wnt10a and LEF1 was evaluated by RT-qPCR. The data showed that the average value of mRNA level of Wnt10a (A) and LEF1 (B) in balding hair follicles was much lower than the nonbalding. DP cells were isolated and cultured from balding and nonbalding scalp, respectively, and the β-catenin expression in DP cells was assessed by immunofluorescence (C) and western blotting (D and E). The result turned out that the activity of β-catenin in balding DP cells was lower compared with the nonbalding, not only in the cytoplasm but also in the nuclear (C–E). The cellular localization of β-catenin (green) with immunofluorescence was shown by using cyto tracer to stain cytoplasm and DAPI (4’, 6-diamidino-2-phenylindole) to stain nuclei, scar bar = 200 μm (n = 3 per group, data are presented as mean ± standard error of the mean). AGA = androgenitic alopecia, DAPI = 4′,6-diamidino-2-phenylindole, DP = dermal papilla.
Figure 2
Figure 2
Activation of TGF-β signaling in AGA pathology. To confirm the alteration of TGF-β signaling in AGA, the mRNA level of TβRI and TβRII, protein concentration of total and active TGF-β1, and protein level of p-Smad2, Smad2, p-Akt, and Akt were measured by RT-qPCR, ELISA, and western blotting methods, respectively. Compared with the nonbalding, the average mRNA level of TβRI (A) and TβRII (B) and the protein concentration of total and active TGF-β1 (C) were significantly increased in balding hair follicles. The phosphorylation of Smad2 and Akt was significantly up-regulated in the balding DP cells than the nonbalding (D) (n = 3 per group, data are presented as mean ± standard error of the mean, ∗∗∗P < 0.001). AGA = androgenitic alopecia, DP = dermal papilla, TGF-β = transforming growth factor-β.
Figure 3
Figure 3
Activation of Wnt/β-catenin signaling in balding DP cells treated with SB431542. Balding DP cells were cocultured with SB431542 (at a concentration of 20 μM) for 4 d to inhibit the TGF-β signaling and the activation of Wnt signaling was subsequently evaluated by the methods of western blotting and RT-qPCR. The significant down-regulation of phosphorylation level of Smad2 and Akt confirmed that SB431542 was sufficient to inhibit the TGF-β signaling in balding DP cells (A). The RT-qPCR data showed that mRNA level of Wnt10a and LEF1 was significantly increased in balding DP cells after SB431542 treatment (B). The β-catenin expression was further detected by immunofluorescence (C). The cellular localization of β-catenin (green) was shown by using cyto tracer to stain cytoplasm and DAPI (4’, 6-diamidino-2-phenylindole) to stain nuclei, scar bar = 200 μm. Results revealed that the total β-catenin expression was not significantly altered but the β-catenin nuclear accumulation was remarkably increased (n = 3 per group, data are presented as mean ± standard error of the mean, P < 0.05, ∗∗P < 0.01 compared with the bald). DAPI = 4′,6-diamidino-2-phenylindole, DP = dermal papilla, TGF-β = transforming growth factor-β.
Figure 4
Figure 4
Activation of TGF-β signaling in nonbalding DP cells treated with XAV939. The nonbalding DP cells were treated with XAV939 (at a concentration of 20 μM) for 4 d to inhibit the Wnt/β-catenin signaling, and activation of TGF-β signaling was assessed afterward. The expression of β-catenin (A) and mRNA level of LEF1 (B) was significantly inhibited by XAV939, but the mRNA level of Wnt10a (B) was not altered. The protein concentration of total and active TGF-β1, the mRNA level of TβRI and TβRII, and the protein expression of p-Smad2, Smad2, p-Akt, and Akt were tested by ELISA, RT-qPCR, and western blotting, respectively. With the significant suppression of Wnt signal-mediated transcription in nonbalding DP cells, XAV939 significantly increased the concentration of active TGF-β1 (C) but without affecting the total TGF-β1 (C) and the expression of TβRI and TβRII (D). The phosphorylation level of Smad2 and Akt was up-regulated with the increased phosphorylation of Smad2 and Akt (E) (n = 3 per group, data are presented as mean ± standard error of the mean and ∗∗∗P < 0.001 compared with the nonbald). DP = dermal papilla, ns = nonsignificant difference, TGF-β = transforming growth factor-β.

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