Downregulation of histone deacetylase 8 (HDAC8) alleviated the progression of oral submucous fibrosis

Abstract

Background/purpose

Oral submucous fibrosis (OSF) is a premalignant disorder that is associated with betel nut chewing. The purpose of the study was to establish the role of histone deacetylase (HDAC) 8, one of histone deacetylases, in the regulation of fibrotic conditions to provide a therapeutic potential for OSF.

Materials and methods

First, we examined the expression of HDAC8 in fibrotic buccal mucosal fibroblasts (fBMFs) and OSF tissues. Markers of myofibroblasts and TGF-β signaling were conducted in fBMFs with HDAC8 knockdown were examined. Furthermore, epithelial–mesenchymal transition (EMT) markers, collagen gel contraction and migration ability were also examined in fBMFs transfected with sh-HDAC8. HDAC8 inhibitor was used to analyze the collagen gel contraction and wound healing ability in fBMFs.

Results

We observed the mRNA expression of HDAC8 was significantly increased in fBMFs. Compared to normal tissues, the protein level of HDAC8 was upregulated in OSF. Next, mRNA and protein expression of HDAC8 was significantly decreased, accompanying downregulation of α-SMA and COL1A1 in fBMFs infected with sh-HDAC8. To determine the critical role of HDAC8 in OSF fibrogenesis, results revealed that TGF-β secretion and the expression of EMT transcription factor SNAIL and p-Smad were significantly decreased in HDAC8-knockdown fBMFs. We further demonstrated that collagen gel contraction and migration ability were significantly decreased in fBMFs transfected with sh-HDAC8. Last, results revealed that significantly reduced collagen gel contraction and wound healing ability in fBMFs with HDAC8 inhibitor treatment.

Conclusion

We concluded that downregulation of HDAC8 alleviated the activities of myofibroblasts and TGF-β/Smad signaling pathway in OSF.

Introduction

Fibrosis is a repairing process when tissues or organs were damaged, it becomes an illness when collagen accumulated excessively. Oral submucous fibrosis (OSF) is a progressive and inflammatory fibrotic disorder, which is prevalent in Southeast Asia and its histologic feature is the excessive accumulation of fibrous connective tissues. The habit of betel nut chewing is closely associated with the occurrence of OSF.¹ The clinical symptoms of OSF are burning sensation, mouth opening limitation, and the possibility of malignant changes in oral cavity.² The association between OSF and betel nut chewing has been validated by patients with OSF in Taiwan.3, 4, 5 The correlation between betel nut extract, arecoline, and fibrosis had also been established according to former research.⁶ Mounting evidence suggests that arecoline-induced hyperactivity of myofibroblasts can lead to overexpression of α-smooth muscle actin (α-SMA), collagen1A1 (COL1A1) and result in abundant extracellular matrix (ECM) accumulation.⁷ Among numerous fibrotic pathways had been elucidated, arecoline-stimulated TGF-β is the major contributor to the development of OSF. Although many in vitro studies have shown promising results, no definitive drug for treating OSF is available so far.

Histone deacetylases (HDACs) modulate various cellular signaling which is associated with different fibrotic diseases.8, 9, 10 HDACs are enzymes that remove acetyl group from N-acetyl-l-lysine amino acid of histone which modulates epigenetic modifications. Currently, The HDACs superfamily were identified and divided into Zn⁺-dependent HDACs (classes I, IIa, IIb, and IV) and NAD⁺-dependent class III HDACs. HDAC8 belongs to class I HDACs was reported to promote tumorigenesis of oral squamous cell carcinoma (OSCC). Inhibition of class I HDAC suppress the initiation and recurrence of OSF.^11,12 Since OSF has been considered a premalignant state, class I HDAC inhibition might have the potential to attenuate OSF. Emerging data have established the antifibrotic potential of HDAC inhibitiors.^8,9,13 It is still uncertain whether HDACs have an influence on OSF. Moreover, the cell microenvironment may diversely influence the potency and efficacy of HDAC.

Therefore, the objective of this study is to investigate whether targeting HDAC8 can inhibit the development of OSF. The experiment was conducted to measure the expression of HDAC8 in fBMFs and OSF specimens. Myofibroblast associated markers, TGF-β/Smad signaling, collagen gel contraction were measured in fBMFs transfected with HDAC8 shRNA. fBMFs were also treated with HDAC8 inhibitor to examine the ability of collagen gel contraction and wound healing assay.

Materials and methods

Cells extraction, culture condition, and clinical tissues

Extraction of fBMFs and BMFs were from the normal buccal mucosa in OSF patients recruited in Chung Shan Medical University Hospital. All operations were under the adherence to the precepts of the Helsinki Declaration and evaluation by Chung Shan Medical University's Institutional Review Committee. fBMFs and BMFs were cultured as previously reported, having cell cultures used between the third and eighth passages.¹⁴ Histological fibrotic mucosa or normal tissues were obtained from OSF patients or normal subjects recruited at the Department of Dentistry, Chung Shan Medical University Hospital. Normal oral mucosa (N) samples were obtained from healthy individuals without areca-chewing habits. The final diagnoses by two experienced pathologists were recruited for this study.¹⁴

Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from cells using Trizol reagent as directed by the manufacturer (Invitrogen Life Technologies, Carlsbad, CA, USA). The Superscript III first-strand synthesis system (Invitrogen Life Technologies) was used to reverse-transcribe qRT–PCRs of mRNAs. ABI StepOne™ Real-Time PCR Systems (Applied Biosystems, Waltham, MA, USA) will be used to run qRT-PCR experiments on the resultant cDNAs.¹⁵

Lentiviral-mediated knockdown HDAC8

The pLV-RNAi vector was purchased from Biosettia Inc. (Biosettia, San Diego, CA, USA). The method of cloning the double-stranded shRNA sequence was described in the manufacturer's protocol. Oligonucleotide sequence of lentiviral vectors expressing shRNA that targets HDAC8 was synthesized and cloned into pLVRNAi to generate a lentiviral expression vector.¹⁶

Western blot

Western blot analysis was performed as previously described. The primary antibody against HDAC8, α-SMA, and COL1A1 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). GAPDH was used as an internal control.¹⁶

TGF-Β ELISA ELISA analysis

ELISA kit was used to detect the concentrations of TGF-Β secretion (R&D Systems, Minneapolis, MN, USA). On a microplate reader, absorbance was measured via a 450 nm filter (MRX; Dynatech Laboratories, Chantilly, VA, USA). Each sample was tested three times.¹⁷

Collagen gel contraction analysis

2 × 10⁵ cells of fBMFs with Sh-Luc. or Sh-HDAC8 were mixed with 2 times concentrated DMEM and 2 mg/mL of collagen solution (Sigma–Aldrich, St. Louis, MO, USA). The mixture of cells and collagen was added into a 24-transwell-plate (Corning, somerville, MA, USA) followed by incubation at 37 °C for 2 h. The polymerized collagen gels were detached from the well using a sterile scraper and then added 0.5 mL serum free DMEM medium. After 48 h incubation, the contraction of the gels was photographed and the contraction index was calculated using ImageJ software (National Institutes of Health, Bethesda MD, USA).¹⁸

Migration assays

The Transwell system with a polycarbonate filter membrane of 8-μm pore size (Corning Costar, Acton, MA, USA) was utilized to assess the migration capacities. Serum-containing media (10% FBS) was used as the chemoattractant in the lower chamber. Cells on the other side of the membrane were stained with 0.1% crystal violet (Sigma–Aldrich) after fixation. These cells were counted from five different visual areas of 100-fold magnification under a microscope.¹⁹

Statistical analysis

Data were presented as mean ± SD. Statistical differences were evaluated by one-way ANOVA and P < 0.05 was considered significant.

Results

Results demonstrated that mRNA level of HDAC8 was significantly increased in fBMFs (n = 5) relative to BMFs (n = 5) (Fig. 1A). The protein level of HDAC8 were upregulated in OSF (n = 2) as compare to normal tissues (n = 2) (Fig. 1B). To ascertain the critical role of HDAC8 in OSF, we utilized lentiviral vectors expressing shRNA to knockdown the expression of HDAC8 in fBMFs. Results revealed the mRNA (Fig. 2A) and protein expressions (Fig. 2B) of HDAC8 were significantly decreased and markers of myofibroblast markers including α-SMA and COL1A1 were also inhibited in HDAC8-kncockdown fBMFs (Fig. 2B). Since EMT is essential in the development of fibrosis, EMT-associated markers were examined. Fig. 3A revealed that the EMT transcription factor SNAIL and phospho-Smad (pSmad) is notably diminished in fBMFs with HDAC8 knockdown. Fig. 3B showed that the secretion of TGF-β significantly decreased as well. Results demonstrated that TGF-β/SNAIL/Smad signaling is significantly inhibited in fBMFs with sh-HDAC8 knockdown.

Figure 1.

The relative expression of HDAC8 is upregulated in fBMFs and OSF.

The mRNA level of HDAC8 in fBMFs (A) and protein level of HDAC8 in OSF tissues (B) were analyzed.

Figure 2.

HDAC8-shRNA mediated knockdown inhibits the expression of myofibroblast markers.

The relative mRNA level of HDAC8 (A) and the protein level of myofibroblast-associated marker (B) were analyzed in non-transfected (sh-Luc.) and HDAC8 shRNA transfected (sh-HDAC8) fBMFs. Mean ± SD of triplicate samples from one representative experiment. ∗P < 0.05 compared with the Sh-Luc. group.

Figure 3.

HDAC8 silencing inhibits TGF-β1/Smad signaling pathway sh-HDAC8 fBMFs.

The protein level of Snail, phosphorylation of Smad, total Smad (A) as well as TGF-β secretion (B) were analyzed in non-transfected (sh-Luc.) and HDAC8 shRNA transfected (sh-HDAC8) fBMFs. Mean ± SD of triplicate samples from one representative experiment. ∗P < 0.05 compared with the Sh-Luc. group.

Collagen gel contraction and Transwell migration assays were performed to investigate the role of HDAC8 in the regulation of fibrotic behaviors of fBMFs. Fig. 4A showed the collagen gel contractility is significantly reduced in fBMFs transfected sh-HDAC8 compared to sh-Luc. These implicated that the abilities of ECM secretion and fibrotic change in fBMFs may due to HDAC8 inhibition. Migration abilities of fBMFs transfected with HDAC8 were also considerably reduced (Fig. 4B). We concluded that the fibrotic properties of fBMFs were downregulated in HDAC8-silencing fBMFs. To confirmed the results above, we utilized potential therapeutic agent, PCI34051 as a HDAC8 inhibitor. PCI34051 diminished collagen gel contraction ability and wound healing ability in fBMFs (Fig. 5). Briefly, our results suggested that upregulation of HDAC8 in fBMFs increased the activities of myofibroblasts which may contribute to fibrogenesis and malignant progression of the oral mucosa.

Figure 4.

Downregulation of HDAC8 attenuates collagen gel contraction and migration ability in sh-HDAC8 fBMFs.

Relative gel area (A) and migration ability (B) was analyzed in non-transfected (sh-Luc.) and HDAC8 shRNA transfected (sh-HDAC8) fBMFs. Mean ± SD of triplicate samples from one representative experiment. ∗P < 0.05 compared with the Sh-Luc. group.

Figure 5.

PCI34051 inhibits collagen gel contraction and wound healing ability in fBMFs.

Relative gel area (A) and wound healing ability (B) was utilized to examine fBMFs treated with PCI34051. Mean ± SD of triplicate samples from one representative experiment. ∗P < 0.05 compared with the control group.

Discussion

In this study, the expression of HDAC8 was increased in both fBMFs and OSF tissues, while downregulation of HDAC8 may reverse the transition of fibroblast to myofibroblast to combat OSF. We are the first to elucidate that HDAC8 has anti-fibrotic effects in OSF. The mechanisms of fibrosis were explored in various aspects over the past decades. Multiple signaling pathways are involved in regulating fibroblast and myofibroblast transition, particularly TGF-β which mediated major pro-fibrotic signals and was regarded as the master regulator of fibrosis.²⁰ The significance of TGF-β in modulating fibrosis has already been shown in numerous fibrotic diseases such as lung, ischemic heart disease and renal fibrosis.^21,22 EMT pathways play pivotal roles in regulating the progression of fibrosis. Modulating of OSF via EMT is also investigated in multiple studies.^23,24 Besides, transcription factors like Slug/Snail, ZEB and Twist were also involved in mediating EMT.

As HDACs have shown remarkable therapeutic capacities and the role of HDACs has delineated in detail. Recently, it was reported that the aberrant expression of HDACs in fibrotic diseases. Hence, numerous studies indicated that HDACI has anti-fibrotic properties in cardiac and pulmonary diseases. TGF-β-induced myofibroblast differentiation was attenuated while inhibiting the phosphorylation of HDAC2 in vitro.^9,25 Likewise, the former study demonstrated that inhibition of HDAC8 which then downregulated the expression of TGF-β and myofibroblast-associated markers improved pulmonary fibrosis.²⁶ Sander et al. revealed that HDACI inhibited pulmonary fibroblast.²⁷ Aside from the previous study, HDACI induced cell cycle and apoptosis of hepatic stellate cells and improved liver fibrosis in bile duct ligation mice model.²⁸

In our study, we found that relative HDAC8 expressions were significantly higher in OSF specimens compared to normal mucosa in both mRNA and protein level. The suppressions of HDAC8 by the transfection of sh-HDAC8s to these OSF specimens also gave rise to the attenuated expressions of HDAC8, the protein level of α-SMA and COL1A1 as well as TGF-β/pSmad/Snail pathway. In sh-HDAC8-transfected fBMFs, the collagen gel contraction and migration abilities were significantly reduced. These indicated the crucial role of HDAC8 in regulating the expressions of fibrotic features of OSF specimens. Moreover, the application of potential therapeutic agent targeting HDAC8, PCI34051 as a HDAC8 inhibitor, also provided an encouraging preliminary results on downregulation of fibrotic characters of OSF.

Overall, these results concluded that suppression of HDAC8 in OSF tissues reduced the expression of several fibrosis-related molecules as well as the features of fibrotic fibroblasts. We provided new insights into the significant role of HDAC8 in the progression of fibrosis in the oral mucosa.

Declaration of competing interest

The authors have no conflicts of interest to this article.