VHL ameliorates arecoline-induced oral submucosal fibrosis by promoting HDAC6 ubiquitination and blocking NF-κB pathway

Abstract

The chronic illness known as oral submucous fibrosis (OSF) results in tissue fibrosis, precancerous lesions, and scarring. It usually manifests itself in the buccal mucosa. It frequently occurs in the buccal mucosa. Von Hippel-Lindau (VHL) is an essential component of E3 ubiquitin ligase complex. The loss of VHL led to reduced fibrotic responses, accompanied by ameliorated fiber deposition. However, the precise impact of VHL on OSF is yet unclear. OSF tissues and normal mucosal tissues were applied to analyze the distinct expression of VHL and histone deacetylase 6 (HDAC6). Oral fibroblasts were treated to arecoline to simulate OSF in vitro, and molecular biological experiments were conducted to identify the role of VHL in buccal mucosa fibroblasts (BMFs). VHL was downregulated and HDAC6 was upregulated in OSF tissues and BMFs. Overexpression of VHL inhibited fibrosis in arecoline-treated BMFs. VHL inhibits the level of HDAC6 by inducing the ubiquitination of HDAC6. Knockdown of HDAC6 reduces the fibrogenic ability of BMFs. Furthermore, overexpression of HDAC6 contributes to the activation of NF-κB signaling in BMFs. HDAC6 selective inhibitor ACY-1215 inhibited the NF-κB signaling pathway. VHL attenuated arecoline-induced OSF by inhibiting the ubiquitination of HDAC6 and blocking NF-κB pathway. As a result, our study offers new perspectives into the discovery of novel tactics that can be employed against OSF.

Keywords: HDAC6; NF-κB; Oral submucosal fibrosis; Ubiquitination; Von Hippel-Lindau.

Fig. 1

VHL and HDAC6 expression in normal mucosal tissues and OSF tissues. (A) The immunohistochemical staining image displayed the VHL and HDAC6 protein levels in both OSF and normal tissues. Scale bars, 50 μm. (B) RT-qPCR results showed VHL and HDAC6 mRNA levels in OSF and normal tissues. (C) The viability of BMFs using an MTT assay. (D) The immunofluorescence staining for α-SMA (green) and DAPI (blue) was captured. Scale bars, 20 μm. (E) WB of VHL and HDAC6 expression in BMFs and fBMFs. *P<0.5, **P<0.01, ***P<0.001.

Fig. 2

Upregulation of VHL inhibits arecoline induced fibrosis. (A, B) The VHL plasmid was transfected into BMFs. The level of VHL was analyzed by RT-qPCR and WB. (C) The contraction activity of BMFs was determined by collagen gel contraction assay. (D) Effects of VHL overexpression on wound closure in arecoline -treated BMFs. Light microscopic images of BMFs were taken at 0 h and 24 h after the infliction of a scratch. Magnification: × 5. Scale bars: 100 μm. (E) Changes of fibrosis protein markers expression in arecoline-treated BMFs. *P<0.5, **P<0.01, ***P<0.001.

Fig. 3

VHL enhances HDAC6 ubiquitination and inhibits arecoline -induced BMFs activation. (A) BMFs harboring overexpression of VHL were treated with 100 µg/ml CHX for 2, 4, 6 h. The treated cells were lysed and subjected to WB analysis with the antibodies indicated. The relative ratios of HDAC6/β-actin normalized to one at t = 0 time point were quantified. (B) Overexpressing VHL cells were treated with ALLN for 24 h before being collected for WB. (C) BMFs were transfected with HA-Ubiquitin, Flag-HDAC6, or overexpressing VHL, then immunoprecipitated with anti-Flag beads, followed by immunoblotting. (D) The mRNA level of HDAC6 was analyzed by qPCR. (E) sh-NC and sh-HDAC6 BMFs were treated with arecoline and the gel contraction capacity of sh-NC and sh-HDAC6 cells was measure by collagen contraction assay. (F) The migration capacity was examined by wound healing assay. (G) The protein levels of α-SMA, COL1A1 and FN1 were determined by WB. *P<0.5, **P<0.01, ***P<0.001.

Fig. 4

HDAC6 activates of NF-κB pathway. (A) The effects of overexpression of HDAC6 on the expression of HDAC6, p-IκBα, IκBα, p-p65, p65 were examined by WB analysis. (B) The effects of HDAC6 inhibitor (ACY1215) on the expression of HDAC6, p-IκBα, IκBα, p-p65, p65. *P<0.5, **P<0.01, ***P<0.001.

Fig. 5

VHL regulated oral submucosal fibrosis by modulating HDAC6. (A) The mRNA expression of VHL and HDAC6 was measured by qPCR. (B) The protein expression of VHL, HDAC6, p-IκBα, IκBα, p-p65, p65 was detected by WB. (C) The levels of IL-6 and TNF-α were measured by ELISA. (D) Effects of VHL and HDAC6 on arecoline -induced collagen gel contraction in BMFs expressing NC, oe-VHL, oe-VHL + HDAC6 and oe-VHL + BAY 11-7082. (E) Representative images of the wound area in BMFs expressing NC, oe-VHL, oe-VHL + HDAC6 and oe-VHL + BAY 11-7082. (F) The expression levels of fibrotic-associated proteins (α-SMA, COL1A1 and FN1) were analyzed by WB. *P<0.5, **P<0.01, ***P<0.001.