Balanced collagen deposition, reinstated tissue integrity and productive tissue remodeling are prerequisites for a dynamic wound healing [1]. Wound healing is composed of three classic processes: inflammatory responses, the formation of granulation tissue and re-epithelialization [2]. Neutrophils, macrophages, and lymphocytes take part in inflammatory reactions in wounds. Keratinocytes and stem cells play crucial roles in re-epithelialization to repair wounds and to promote proliferation and differentiation [3]. Fibroblasts and myofibroblasts, the key functional cells, have been shown to be involved in the formation of granulation tissue and wound contraction [4]. The modulation of inflammatory responses, cellular growth and adhesion, extracellular matrix (ECM) deposition and tissue remodeling at wound sites portrays the numerous biological processes regulated by transforming growth factor-beta (TGF-β) [5]. ECM reconstruction of scar tissue with collagen and fibronectin and the reduction of cell components are the main features of the remodeling phase [6]. ECM debris is opsonized by fibronectin in the inflammatory phase. Additionally, fibronectin triggers macrophages to phagocytize the ECM debris [6]. The intermediate filament protein vimentin contributes to various cellular functions [7,8]. Vimentin is involved in fibroblast proliferation and keratinocyte differentiation in wound healing via TGF-β signaling [9]. A previous study demonstrated the impaired migration and impeded contraction of fibroblasts during wound closure in vimentin knockout mice [10]. The cell-cell adhesion molecule E-cadherin modulates epithelial cells, and the migration of epithelial cells relies on the redistribution of E-cadherin [11].

Smad3 appears to have a primary role in wound healing [12]. The absence of Smad3 in in vivo and in vitro has revealed the specific targets of this transcription factor [13]. Smad3 deletion in mice causes rapid wound healing and attenuated inflammatory reactions [14]. The inhibition of Smad3 can promote irregular TGF-β reactions in fibrosis [6].

The transcription factor sex-determining region Y-box 2 (Sox2) is a key player that regulates embryonic development and stem cell maintenance [15]. Sox2 is involved with regulating the differentiation of tongue taste buds [16]. The repair of injured bronchiolar epithelium is associated with the induction of Sox2 [17]. Even though these findings suggest the involvement of Sox2 in regulating cell differentiation in numerous organs, the prerequisite for Sox2 in tongue wound healing is obscure.

Our previous study demonstrated that Smad3-deficient mice have accelerated re-epithelialization and tissue remodeling during tongue wound repair, and this was accompanied by a decreased expression of the transcription factor differentiated embryonic chondrocyte gene 1 (Dec1) compared with Smad3^+/+ mice [18]. Here we further dissect the requirements of Smad3 signaling in mouse tongue injuries and explore the links between extrinsic Smad3 signaling and the intrinsic transcriptional regulators of tongue wound healing.

Since Smad3 is involved in modulating ECM homeostasis, its regulation in wound healing appears to modulate the expression of ECM-related genes. We thus hypothesized that Smad3 acts as a major transcription factor regulating the wound healing process via the modulation of ECM homeostasis. Here we developed a mouse tongue injury model using Smad3^–/– mice and report that a Smad3 deficiency promotes wound healing and regulates the expression of ECM-related genes. Based on these results, we propose that Smad3 might be a favorable remedial agent for tissue injury. A profound comprehension of the cellular functions of Smad3 may consequently disclose its complete therapeutic potential.

Animals

All animal experiments were carried out in line with the protocols provided by the Animal Care and Use Committee of the Wakayama Medical University (Approval number: 660) as described previously [18]. Six to eight-week-old Smad3^+/+ and Smad3^–/– mice were sacrificed at zeitgeber time 6 (ZT6; six hr into the light phase), and their tongues were excised and examined three days after wounding as previously described [18]. Tongue tissues from the mice were fixed in 4% paraformaldehyde in PBS (Wako, Osaka, Japan) and subjected to immunohistochemistry and hematoxylin-eosin (H&E) staining.

Hematoxylin-eosin staining

The tongue tissues were fixed with 4% paraformaldehyde for 24 hr, then embedded in paraffin and 4 μm sections were stained with H&E.

Immunohistochemical staining

Antigen retrieval (10× Citrate Buffer pH 6.0; Abcam plc, Cambridge, UK) and peroxidase blocking (DAKO, Santa Clara, CA, USA) were performed in accordance with the manufacturers’ protocols. Antibodies to Sox2 (ab97959, 1:100, Abcam), Phospho-E-cadherin (ab76319, 1:50, Abcam), PMN (CLAD31140, 1:1,000; CEDARLANE, Burlington, NC, USA), Fibronectin (ab45688, 1:200, Abcam) and Vimentin (ab92547, 1:100, Abcam) were used as primary antibodies. The specimens were treated using the same 3,3′-diaminobenzidine (DAB) reaction conditions. Images were captured using a microscope (OLYMPUS, Tokyo, Japan).

Cell culture and siRNA transfection

HGFs (ScienCell, Carlsbad, CA, USA) were seeded in 6-well plates for treatment with a scrambled siRNA (negative control) or a Smad3 siRNA (Qiagen, Hilden, Germany) using RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA). The scratch wound healing was performed with a 1 mL tip after transfection for 24 hr. The sense and antisense siRNA sequences used were as follows: Smad3 siRNA, 5′-r (GAGAUUCGAAUGACGGUAATT)-3′ and 5′-r (UUACCGUCAUUCGAAUCUCTT)-3′. Cell lysates were collected 48 hr after transfection.

Wound healing assay

HGFs were harvested after transfection with a scrambled siRNA or a Smad3 siRNA from monolayer cultures at 80% confluence by brief trypsinization and were seeded in Ibidi wound healing dishes (Ibidi, Bavaria, Germany) where they were grown to confluence in serum-free medium for 24 hr. The insert walls of the Ibidi wound healing dishes were removed after 24 hr, and the migration of HGFs into scraped areas was observed and photographed at 24 hr and 48 hr. An inverted light microscope with a grid reticle in the microscope ocular was used to measure the closure of the gap, which was measured at six marked sites. The percentage of the starting distance at the wound edges was set to determine the unhealed areas. All experiments were performed three times on separate days. Data from these independent experiments are reported as mean±standard deviation.

Quantitative real-time PCR

Total RNAs were extracted by QIAZOL according to the manufacturer’s protocol (Qiagen, Hilden, Germany). A TURBO DNA-free Kit (Applied Biosystems, Foster City, CA, USA) was used to remove contaminating DNA from the RNA preparations. First-strand cDNAs were synthesized from 1 μg total RNA using High Capacity RNA-to -cDNA Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) and were reverse-transcribed using High Capacity RNA-to-cDNA Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). TaqMan probes of target genes, including Smad3 (Assay ID: Hs00969210_m1), Sox2 (Assay ID: Hs04234836_s1), E-cadherin (Assay ID: Hs01023895_m1), Fibronectin (Assay ID Hs01549976_m1) and Vimentin (Assay ID Hs00958111_m1) were used for RT-PCR analysis. These experiments were performed in triplicate.

Statistical analysis

Independent two-tailed Student’s t-test or analysis of variance (ANOVA) was performed using SPSS 16.0 (SPSS Inc., Chicago, IL, USA) for statistical analysis. p-values<0.05 are considered statistically significant.

Tongue wound healing is promoted by the up-regulation of polymorphonuclear neutrophils, Sox2, E-cadherin, vimentin, and fibronectin in Smad3^-/- mice

We examined how the number and distribution of polymorphonuclear neutrophils (PMNs) and the expression of Sox2, E-cadherin, vimentin and fibronectin were affected by wound-healing in Smad3^+/+ and in Smad3^–/– mice. On day 3 after wounding, positive staining for Sox2 and phospho-E-cadherin was observed in the epithelium of Smad3^–/– mice compared to Smad3^+/+ mice (Fig. 1A). Positive staining of PMNs, vimentin and fibronectin was observed in stromal cells of Smad3^–/– mice (Fig. 1B).

Fig. 1. (A) Representative images of hematoxylin-eosin (H&E) staining and immunohistochemistry for sex-determining region Y-box 2 (Sox2) and phospho-E-cadherin in the tongues of Smad3^+/+ and of Smad3^–/– mice three days after wounding. Positive staining for Sox2 and phospho-E-cadherin in the squamous epithelium was observed in Smad3^–/– mice. 40×, scale bars=50 µm; 60×, scale bars=20 µm. (B) Positive staining for polymorphonuclear neutrophils (PMNs), vimentin and fibronectin was observed in stromal cells of Smad3^–/– mice. 40×, scale bars=50 µm; 60×, scale bars=20 µm.

The wound healing process is accelerated by a Smad3 deficiency

To further verify the effects of Smad3 on the wound healing process, cell motility of HGFs was evaluated in wound healing assays after transfection of HGFs with Smad3 siRNA. As shown in Fig. 2A, B, compared with the control group, the cell migration ability was significantly increased in HGFs at 24 hr after Smad3 knockdown in the experimental group. There was no significant difference between the control group and the experimental group at 48 hr.

Fig. 2. (A) Smad3 knockdown promotes the migration of HGFs. The cell migration ability of HGFs is induced by Smad3 knockdown at 24 hr, compared with the control group. Scale bars=20 μm. (B) The unhealed area of HGFs is reduced after Smad3 knockdown at 24 hr. Data shown represent mean±standard deviation; *p<0.05. Results are representative of at least three independent experiments. HGFs, human gingival fibroblasts; siRNA, small interfering RNA; ns, not significant.

Smad3 knockdown promotes wound healing via the upregulation of Sox2, E-cadherin, vimentin, and fibronectin

The mRNA expression levels of Sox2, E-cadherin, fibronectin and vimentin were significantly increased after transfection of HGFs with Smad3 siRNA compared with controls (Fig. 3). Silencing of Smad3 suppressed the Smad3 mRNA expression (Fig. 3).

Fig. 3. Smad3 knockdown promotes wound healing via the upregulation of Sox2, E-cadherin, vimentin, and fibronectin. The expression of Sox2, E-cadherin, vimentin and fibronectin were significantly upregulated after Smad3 knockdown in HGFs compared to controls. Silencing of Smad3 suppressed the Smad3 mRNA expression. Data shown represent mean±standard deviation; *p<0.05. All results are representative of at least three independent experiments. SoX2, sex-determining region Y-box 2; HGFs, human gingival fibroblasts; siRNA, small interfering RNA.

This study used Smad3^–/– mice to demonstrate that a Smad3 deficiency stimulates tongue wound healing and mitigates scar formation. Such implications were achieved by a complex series of actions that triggered the accumulation of PMNs, augmented Sox2 and E-cadherin expression levels and increased vimentin and fibronectin expression in the growth of tongue fibroblasts. Consequently, these findings suggest that Smad3 signaling has consequences for very diverse features of reactions to tongue injuries and eventually to traumatic injuries. Our study gives insights into the effects of a Smad3 deficiency via biological intermediates, Sox2 and E-cadherin, that can change the course of the recovery from epithelial injury. In accordance with the rapid repair and provoked expression of ECM proteins in Smad3-deficient mice, implementation of a targeted siRNA can be indicative of a useful therapeutic tool to enhance wound healing in a wide spectrum of clinical applications. A Smad3 deficiency will aid in the assessment of treatments and biological imaging to enhance understanding and ameliorate tongue remodeling after injury.

Cellular migration is a fundamental process required for myofibroblasts to repair the injured tissue in wound healing. Smad3 is a critical regulator of TGF-β signaling and immediately stimulates transcriptional regulation and signal transduction of target genes [19]. Smad3 is also engaged in regulating cellular proliferation [20] and the migration of transformed keratinocytes [20,21]. Silencing the Smad3 pathway can impede the phenomenon of EMT [22]. A Smad3 deficiency facilitated the production of proinflammatory cytokines in macrophages in response to LPS treatment [23]. Smad3^–/– fibroblasts had a partial resistance to TGF-β mediated growth arrest [24] and decreased cellular migration in myocardial infarction [24,25]. Compared to wild-type mice, cutaneous wound-healing was considerably increased in Smad3-deficient mice [26], thus pointing out the involvement of Smad3 in the regulation of cell migration.

Cellular alterations in the wound environment have an important role with reference to injury. Our current understanding of squamous epithelium wound repair and tissue homeostasis is attributed to mouse models. Our results showed wound closure at a greater rate and increased ECM deposition that occurred in Smad3-deficient mice, thereby signifying an important role of Smad3-dependent pathways in the physiological response to trauma. The functional absence of Smad3 signaling could explicitly or implicitly affect immune cell migration, thus generating many PMN-positive cells in localized wounds and enabling an inhibitory effect of Smad3 signaling on immune cell growth after injury. Smad3-deficient mice had diminished scarring, quick healing, and the expression of many diverse ECM proteins. Previous results on cutaneous incisions [14] were like our results, demonstrating that a Smad3 deficiency could stimulate the recovery from traumatic injury.

The dysregulation of cytokines in wounds affects actin and fibronectin during the transition of fibroblasts to myofibroblasts [26]. Hence, induced fibronectin expression in Smad3-deficient mice after wounding showed a direct implication of Smad3 signaling pivotal to the initiation of fibronectin, reminiscent of a distinct function of Smad3 to induce fibronectin in fibroblasts. In embryonic development, various studies have highlighted the function of the transcription factor Sox2 in Smad-mediated TGF-β signal transduction [27]. Consequently, it is conceivable that impaired Smad3 signaling can affect Sox2 function linked to cell renewal. The major finding in this study is the direct function of Smad3 on the expression of Sox2 in tongue wounds. We discovered that Smad3 is a hub gene linked to the functions of Sox2, E-cadherin and ECM proteins in tongue injuries. Notably, the Smad3 siRNA regulated Sox2 levels (Fig. 3), signifying a key role for Smad3 in Sox2 regulation. Overall, these results indicate that both the Smad3 function and intrinsic cellular properties significantly affect Sox2, offering details on altering the balance of Smad3 in a tongue injury environment that can communicate the therapeutic potential of those factors in traumatic injury.

Additional characterization identified a high incidence of re-epithelialization, a substantial contribution to neutrophil infiltration and reduced granulation tissue formation at the wound. We previously used Smad3-deficient mice to elucidate Dec1/Smad3 signaling functions in tongue wound healing, which suggested that the suppression of cell migration and proliferation by Smad3 may occur via Dec1 [18]. Following the observation that Smad3-deficient mice have an increased neutrophil infiltration into wound sites and considering that Sox2 is expressed in epithelial cells, we surmised that high levels of Sox2 expression in Smad3- deficient mice can facilitate the wound healing process.

There is a known limitation of our study. The wound healing process lasts for several days. Since this study was restricted to a 3-day period, we did not entirely replicate the process of tongue scarring. Supplemental extended studies are needed to confirm our findings. In accordance with the rapid repair and increased Sox2 and E-cadherin expression in Smad3-deficient mice, in vitro studies of the molecular mechanism involved are required to verify and expand our findings favoring the importance of the Smad3-dependent pathway in the repair of tongue injuries.

In summary, our findings reveal for the first time the expression of the transcription factor Smad3 and its important role in modulating different processes involved in tongue wound healing, such as Sox2, E-cadherin and ECM protein expression, and the migration of fibroblasts.

We thank Mr. Toshiki Uematsu for technical assistance and the staff of the animal facility for care of the mice.

This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by Nihon University Multidisciplinary Research Grant for 2018.

The authors declare that they have no competing interests.