Cullin3 Aggravates The In ammatory Response of PDLSCs Via Regulation of Shh Signaling and Nrf2


 Objective: Sonic Hedgehog (Shh) was found to be correlated with inflammation degree of patients with periodontitis. Cullin3 is an important ubiquitin ligase for controlling Shh signaling. In this study, we exerted ourselves to clarify the roles of Shh and Cullin3 in P. gingivalis-LPS (Pg-LPS)-treated periodontal ligament stem cells (PDLSCs). Methods: Cell viability was detected using cell counting kit-8 (CCK-8). The inflammatory cytokines of PDLSCs were estimated by enzyme-linked immunosorbent assay (ELISA). The protein levels of Shh, Gli1 and NF-E2-related factor2 (Nrf2) were determined via western blots. Alkaline phosphatase staining and Alizarin red staining were performed to evaluate the differentiation and mineralization capabilities of PDLSCs. The apoptotic cells were screened by TUNEL staining. Results: Pg-LPS inhibited cell viability and triggered inflammation of PDLSCs. Overexpression of Cullin3 impeded the differentiation and mineralization capabilities of PDLSCs. Moreover, Cullin3 overexpression aggravated inflammation and cell apoptosis induced by Pg-LPS. Of note, while the protein levels of Shh, Gli1 and Nrf2 were elevated in PDLSCs treated with Pg-LPS, overexpression of Cullin3 decreased the expressions of them. Conclusion: Shh/Gli1 and Nrf2 were involved in the inflammation and cell apoptosis of PDLSCs, which was dominated by Cullin3.


Introduction
Periodontitis is a prevalent oral disease worldwide. Epidemiological survey shows that over 50 % adults suffer from periodontal disease (1,2). It is generally accepted that plaque bacteria, excessively accumulating on the tooth surface, invades periodontal tissue and triggers the in ammatory response, further resulting in the destruction of the periodontal tissue connection and the resorption of alveolar bone (3). Ultimately, the supporting structure of the tooth is lost (4). However, periodontal ligament has a good regeneration ability in physiological conditions in that alveolar bone and periodontal ligament are always in dynamic reconstruction, maintaining the integrity of periodontal support structure (5).
At present, there are rarely any effective therapies for periodontitis (6). The biological role of periodontal ligament stem cells (PDLSCs) is a hotspot in the treatment of periodontal disease. PDLSCs are crucial in maintaining the dynamic balance of periodontal tissues and are also the basis of periodontal tissue regeneration due to their multiple differentiation capabilities (7). Nevertheless, Osteogenic differentiation of PDLSCs was inhibited during the progression of periodontitis (8).
Sonic Hedgehog (Shh) is a member of Hedgehog (Hh) family and is widely distributed in multiple organs (9). Hh signal is usually silent in adults, even though it is involved in tissue maintenance and regeneration by modulating the regeneration and differentiation of stem cells (10). High expression of Shh was observed in gingival crevicular uid of patients with periodontitis (11), indicating that Shh may participate in the in ammatory response of periodontitis.
Cullin3 is a kind of E3 ubiquitin ligase with RING domain (12), which is different from other Cullins in E3 ligase family because it has no bridging protein. And a unique 3-box domain is needed to stabilize the binding sites. Therefore, there are not many active Cullin3 ubiquitin ligase complexes (13,14). Cullin3 represented a common signaling node for controlling Shh signaling pathways (15). As a key transcription factor responsible for Shh signal, Gli1 could be degraded by Cullin3 (16,17). In addition, Keleh-like ECHassociated protein 1 (Keap1) could interact with Cullin3, mediating the ubiquitination and degradation of NF-E2-related factor2 (Nrf2) (18,19). Keap1/Nrf2 is a crucial signaling pathway that orchestrates in ammation and oxidative stress (20,21). In this study, we aimed to illustrate the roles of Shh signaling pathways in LPS-induced PDLSCs and evaluate the effect of Cullin3 in the in ammatory process.

CCK-8 assay
PDLSCs were seeded into a 96-well plate at a density of 1 × 10 3 per well. Cell viability was estimated at 0 h, 24 h and 48 h at rst, after which 10 µl CCK-8 solution (Dojindo, Kumamoto, Japan) was added into each well, followed by incubation for 1 h. The absorbance value was at last recorded at 450 nm.

ELISA assay
The supernatants of PDLSCs were collected and centrifuged at 4°C (1000 g) for 10 min. The concentration of TNF-α, IL-6, IL-1β and IL-10 was determined by ELISA kits (Beyotime, Jiangsu, China) according to the manufacturer's instruction. The absorbance value was recorded at 450 nm.

Western blots
Total protein from cells were extracted using RIPA lysis buffer (Solarbio, Beijing, China). And the protein concentration was determined by a BCA assay kit (Beyotime, Jiangsu, China). Afterwards, SDS-PAGE was prepared to separate proteins, which were then transferred onto polyvinylidene di uoride (PVDF) membranes (EMD Millipore, MA, USA). PVDF membranes were incubated with primary antibodies, anti-Shh, anti-Gli1, anti-Cullin3 and anti-Nrf2 (Abcam, Cambridge, UK), which were removed the second day.
And secondary antibodies (Sigma-Aldrich) were prepared for incubation with PVDF membranes. The protein bands were visualized and analyzed using a chemiluminescence system (Bio-Rad, CA, USA).

ALP staining
PDLSCs were seeded into a 24-well plate at a density of 2 × 10 4 per well. Osteogenic-inducing medium was prepared to culture PDLSCs for 7 d. The medium was refreshed every 3 days, which was removed later on the 7th day. PDLSCS were subsequently cultured with added x solution (MKBio, Shanghai, China) for 4 min at room temperature. Then with 0.6 ml dye (MKBio) added into each well, the cells were cultured at room temperature for 10 min in the dark. Cell observation was done under a light microscope (Carl Zeiss, Jena, Germany) at last.

Alizarin red staining
The mineralization of PDLSCs was evaluated by Alizarin red S. PDLSCs were brie y seeded into a 24-well plate at a density of 2 × 10 4 per well. Osteogenic-inducing medium was prepared to culture PDLSCs for TUNEL assay Apoptotic cells were detected by a TUNEL staining kit (KeyGEN, Jiangsu, China). 4% paraformaldehyde was used to x PDLSCs for 30 min at room temperature, after which Proteinase K was added for incubation for another 30 min at 37°C. Subsequently, treatment of 1% Triton X-100 was added for incubation for 5 min. The cells were then incubated likewise for 30 min at 37°C in the dark after Streptavidin-HRP was added to the plate. Color reaction was produced by adding DAB solution. And then the cells were re-dyed with hematoxylin. Finally, the cells were observed under a light microscope (Carl Zeiss, Jena, Germany).

Statistical analysis
Data was presented as mean ± SD and was analyzed by GraphPad Prism 6.0. Student's t-test and oneway analysis of variance tests followed by Tukey's post hoc test were used to compare differences between groups. P < 0.05 was considered as statistical signi cance.

Pg-LPS impairs cell viability and triggers in ammtion of PDLSCs
PDLSCs were challenged to different concentrations of Pg-LPS, the viability of which was examined after 24 and 72 h. It was observed that cell viability slightly decreased when cells were exposed to 0.1 µg/ml Pg-LPS. Of note, cell viability was signi cantly reduced when the concentration of Pg-LPS reached 1 µg/ml (Fig. 1A). And Pg-LPS at 10 µg/ml represented more severe damage on cell viability. In addition, the productions of pro-in ammatory cytokines including TNF-α, IL-6 and IL-1β were elevated when PDLSCs were exposed to 1 µg/ml, whereas the level of anti-in ammatory cytokines IL-10 also increased following exposure to 0.1 µg/ml or 1 µg/ml Pg-LPS (Fig. 1B). However, it was noticed that IL-10 was markedly reduced when the concentration of Pg-LPS reached 10 µg/ml, indicating that high concentration of Pg-LPS triggered predominantly the secretions of pro-in ammatory cytokines.
Shh signaling and Nrf2 are activated in Pg-LPS-treated PDLSCs Furthermore, it was found that the protein levels of Shh and Gli1 were elevated when cells were exposed to Pg-LPS, accompanied by the increase of Nrf2 ( Fig. 2A and 2B), suggesting a stressful increase of Shh, Gli1 and Nrf2 in response to Pg-LPS challenge. Given that Cullin3 plays a vital role in degrading Gli1 and Nrf2, overexpression plasmids pcDNA 3.1-Cullin3 was transfected into PDLSCs. Results showed that pcDNA 3.1-Cullin3 effectively upregulated the expression of Cullin3 (Fig. 3A). Overexpression of Cullin3 further impaired cell viability in the presence of 1 µg/ml Pg-LPS (Fig. 3B).

Overexpression of Cullin3 exacerbates in ammation and apoptosis
The in ammatory response of PDLSCs following transfection of pcDNA 3.1-Cullin3 was evaluated using ELISA kits. It was noticed that 1 µg/ml Pg-LPS triggered high levels of TNF-α, IL-6 and IL-1β, and Cullin3 exacerbated the in ammatory response. By contrast, the level of IL-10 signi cantly decreased following transfection of pcDNA 3.1-Cullin3 (Fig. 5A). Moreover, TUNEL staining demonstrated that LPS induced cell apoptosis was further promoted by Cullin3 overexpression (Fig. 5B and 5C). Additionally, it was found that Shh, Gli1 and Nrf2 were upregulated in response to 1 µg/ml Pg-LPS, while Cullin3 overexpression ameliorated these effects (Fig. 6A and 6B), indicating that Cullin3 promoted in ammatory response and cell apoptosis likely through downregulating the expression of Shh, Gli1 and Nrf2.

Discussion
PDLSCs have the ability of self-renewal and differentiation, which play a major role in repairing damaged dental tissues and are considered to be most useful for periodontal reconstruction and regeneration (24).
In the current study, we found that cell viability of PDLSCs was signi cantly reduced when exposed to 1 µg/ml Pg-LPS. Furthermore, numerous pro-in ammatory cytokines including TNF-α, IL-6 and IL-1β were produced following stimulation of Pg-LPS. Shh is reported to be potentially a new therapeutic target for periodontitis and periodontal regeneration (25), the expression of which along with that of Gli1 in PDLSCs was examined in this study and was found to have an obvious increase after Pg-LPS stimulation.
In basal condition, Keap1 binds Nrf2 to its BTB domain, and connects Cullin3 to its Kelch domain. Nrf2 is maintained at a low level through ubiquitination degradation mediated by Cullin3 (18). NRF escapes from degradation in response to stimulus and is thus released, regulating the expression of downstream target genes (26). Our results showed that the expression of Nrf2 increased in response to Pg-LPS stimulation. It was also noticed that overexpression of Cullin3 in PDLSCs impaired cell viability and inhibited the differentiation and mineralization of PDLSCs. Moreover, in ammatory response was exacerbated and apoptotic cells increased following Cullin3 overexpression in the presence of Pg-LPS. Of note, the levels of Shh, Gli1 and Nrf2 in PDLSCs decreased when transfected with pcDNA3.1-Cullin3, suggesting that Shh/Gli1 and Nrf2 mediated by Cullin3 might participate in the in ammatory response, and thus affect the differentiation and mineralization of PDLSCs.
In summary, Cullin3 was found to be involved in the in ammatory response of PDLSCs via orchestrating the expression of Shh signaling and Nrf2. However, the present study is limited by inadequate exploration of the nuclear regulation of Nrf2. And it is of necessity to further investigate the speci c regulatory mechanism of Gli1 in future study.

Declarations
Ethics approval and consent to participate Not Applicable.

Consent for publication
All authors agreed to publish.

Availability of data and materials
The data that support the ndings of this study are available from the corresponding author upon reasonable request.

Competing interests
The authors declare that they have no competing interests. Funding This work was supported by grants from the Liaoning Natural Science Foundation (No.20180550563 to Correspondence). Authors' contributions Chun Shi contributed to conception and design, analysis and interpretation of the data, critically revised the article for important intellectual content. Wanhong Chen contributed to design and analysis of the data, drafted and revised the manuscript. Jiangling Su and Shixiong Cai substantially contributed to conception and design, acquisition, analysis, and interpretation of data; drafted and critically revised the article for important intellectual content. All authors approved the nal version of the article and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.