Inflammation, Vol. 42, No. 4, August 2019 (# 2019) DOI: 10.1007/s10753-019-00977-4 ORIGINAL ARTICLE Glycoprotein Nonmetastatic Melanoma Protein B (GPNMB) Ameliorates the Inflammatory Response in Periodontal Disease Rong Song1,2 and Lexun Lin 2,3 Abstract— Glycoprotein nonmetastatic melanoma protein B (GPNMB) is a type I trans- membrane protein that can modulate osteoblasts and bone mineralization. Periodontal disease (PD) is characterized by gum inflammation, alveolar bone resorption, and tooth loss. In this study, we found that GPNMB is highly expressed in inflamed periodontal tissue through microarray and immunohistochemistry (IHC) assays. The role of GPNMB in the pathogen- esis of PD was evaluated with primary human periodontal ligament cells (hPDLCs) treated with lipopolysaccharide (LPS) and a GPNMB-expressing lentivirus (lenti-GP). In the hPDLCs treated with LPS and lenti-GP, the expression of tumor necrosis factor (TNF)-α and interleukin (IL)-6 was suppressed and that of IL-10 was upregulated. GPNMB signifi- cantly decreased apoptosis in the hPDLCs treated with LPS. GPNMB could upregulate the expression of Jumonji domain-containing protein 3 (Jmjd3), a histone 3 lysine 27 (H3K27) demethylase that is linked to the modulation of the inflammatory response and apoptosis. Taken together, our data find that GPNMB is highly expressed in gum tissue with PD and may be an anti-inflammatory player in the pathogenesis of PD. KEY WORDS: periodontal disease; GPNMB; inflammatory cytokine; Jmjd3. INTRODUCTION [3]. In this study, our initial intention was to probe the molecular basis for bone destruction during PD. A micro- Periodontal disease (PD) is a chronic oral disease that array screening bone metabolism factors was used to assess is highly prevalent in adults. PD is mainly characterized by gum tissue samples collected from patients who suffered inflammation in the gums, periodontal pocket formation, from PD. In the microarray, glycoprotein nonmetastatic alveolar bone resorption, and tooth loosening and displace- melanoma protein B (GPNMB), monocyte chemotactic ment [1, 2]. There are two hallmark events in PD: inflam- protein 1 (MCP-1), macrophage inflammatory protein-1α mation and alveolar bone resorption/tooth loss [2, 3]. The (MIP-1α), and macrophage colony-stimulating factor inflammation event includes gingivitis and periodontitis (MCSF) showed varied expression patterns between the healthy and inflamed periodontal tissues. MCP-1, MIP-1α, 1 Department of Prosthodontics, First Affiliated Hospital of Harbin Med- and MCSF are well established as inflammatory cytokines ical University, Harbin, 150001, China [4–7]. To date, there are no data available on the connec- 2 Department of Microbiology, Harbin Medical University, Har- tion between GPNMB and the pathogenesis of PD. bin, 150081, China GPNMB, a type I transmembrane protein with 560– 3 To whom correspondence should be addressed at Department of Micro- 572 amino acids, is also known as osteoactivin (OA) in rats biology, Harbin Medical University, Harbin, 150081, China. E-mail: [email protected] and dendritic cell-heparin integrin ligand (DC-HIL) in mice [8–10]. The GPNMB gene is located on chromosome 1170 0360-3997/19/0400-1170/0 # 2019 Springer Science+Business Media, LLC, part of Springer Nature GPNMB Ameliorates Inflammatory Response in Periodontal Disease 1171 7p15.1 and contains 11 exons [10]. GPNMB is expressed The tissue samples were assessed with a human bone in the bone, liver, and kidneys as well as numerous cell metabolism-related protein array (QAH-BMA-1000, types, including osteoclasts, macrophages, dendritic cells, RayBiotech Guangzhou, Guangzhou, China). Briefly, all and tumor cells [9–11]. GPNMB acts as a downstream tissue samples were lysed with cell lysis buffer to extract mediator of bone morphogenetic protein 2 (BMP-2) and total protein. The extracts were loaded onto the glass array affects the differentiation, maturation, adhesion, and mi- and detected with primary antibodies and a Cy3- gration of osteoblasts and thus affects bone mineralization streptavidin-labeled secondary antibody with standard [12]. An accumulating number of studies have suggested washing procedures. The fluorescence of the array was that GPNMB is also a negative regulator of inflammation detected with a laser scanner (Innoscan 300 Microarray [11]. GPNMB is expressed at high levels in liver inflam- Scanner, Innopsys, Carbonne, France). matory cells, suggesting a critical role in acute liver inflam- mation [13, 14]. OA in rats shows early-phase upregulation Immunohistochemistry in the kidney tubular epithelium when renal injury occurs Sixty-nine periodontal tissue samples were collected and may trigger renal interstitial fibrosis [15]. GPNMB is from untreated patients (mean age, 46.4 ± 7.63; range, 30– highly expressed in aggressive breast cancers and facili- 59 years) with moderate to severe PD. The patients were tates the metastasis of breast cancer to bone [16]. selected based on the following diagnostic criteria: pocket Jumonji domain-containing protein 3 (JMJD3), which is depth > 4 mm, clinical attachment loss ≥ 3 mm, and bleed- also known as lysine-specific demethylase 6B (KDM6B), is a ing upon probing. During their treatment for PD, the pa- histone 3 lysine 27 (H3K27) demethylase [17]. JMJD3 is tients had their granulation tissue on the inner wall of the essential for the polarization of M2 macrophages [18]andis deep-seated periodontitis site scraped under local anesthe- also a negative regulator during inflammation [19]. A recent sia. Healthy periodontal tissue was collected from 13 peo- study showed that JMJD3 can upregulate the expression of ple (mean age, 13.5 ± 1.33; range, 12–15 years) who had insulin-like growth factor binding protein 5 (IGFBP5), which their premolars removed for orthodontic reduction (study promotes periodontal tissue repair [20]. inclusion required complete roots, no cavities, and no In this study, we found that GPNMB is highly expressed obvious periodontal tissue inflammation) and served as in inflamed gum tissue, significantly upregulates the expres- negative control samples. The teeth were wiped with sion of Jmjd3 and may be a suppressor of periodontal inflam- 75% ethanol and washed twice with phosphate-buffered mation and a protective mediator during the progression of PD. saline (PBS), and the surrounding periodontal tissue was gently peeled off for use as part of the negative control group. The tissue samples were paraffinized, sectioned, MATERIALS AND METHODS and stained following a standard immunohistochemistry (IHC) protocol. An anti-GPNMB rabbit antibody (20338- Tissue Samples and a Microarray 1-AP) was purchased from Wuhan Sanying Biotechnology (Wuhan, China). A goat anti-rabbit antibody was pur- Inflamed gingival specimens were collected from four chased from ZhongshanJinqiao Biotechnology (Beijing, subjects (mean age, 42 ± 2.16; range, 40–45 years) with un- China). Stained areas of interest (AOIs) were selected from treated moderate PD. The PD patients were selected based on the IHC image, and the integral optical density (IOD) of the diagnostic criteria adapted from the criteria for the classification selected AOIs was determined. All subjects were free of of periodontal diseases and conditions as follows: pocket depth systemic diseases. between > 4 mm and ≤ 6 mm; 3–4 mm of clinical attachment loss; and bleeding upon probing. During treatment, four pieces Cell Culture of granulation tissue from the inner wall of the deep periodon- tal pocket were collected from each patient under local anes- Premolars were collected from 12- to 15-year-old thesia. Four healthy tissue samples from the other side of the healthy donors undergoing orthodontic treatment with the periodontal pocket were also collected from each patient to informed consent of the donor and their parents. The teeth serve as negative controls. All patients were free of systemic were washed twice with PBS, and the attached periodontal diseases other than PD. Informed consent was obtained from ligament tissue was peeled off with a scalpel. The tissue each participant in the study. The procedure for tissue sample samples were cut into pieces (1–2mm3) and digested with collection was approved by the Ethics Committee of Harbin type I collagenase for 40 min at 37 °C. The separated Medical University. primary human periodontal ligament cells (hPDLCs) were 1172 Song, and Lin harvested with centrifugation at 1000 r/min for 5 min. The subjected to reverse transcription. The cDNA template cells were cultured with α-MEM complete medium was used to amplify target mRNAs with specific primers (Gibco, Carlsbad, CA) supplemented with 10% fetal bo- by using a LightCycler 2.0 (Roche, Basel, Switzerland). β- vine serum (FBS), streptomycin (50 μg/ml), and penicillin Actin was used as the reference to normalize the mRNA (100 U/ml). The cells were passaged at least three times expression. The 2-ΔΔCt method was used to calculate the before being used in subsequent experiments. relative mRNA abundance. GPNMB-Expressing Lentivirus Flow Cytometry The coding region (1619 bp) of the GPNMB gene Treated hPDLCs were collected using 0.25% trypsin, (NM_001005340) was amplified and ligated into a centrifuged, and washed with Dulbecco’smodifiedEagle’s lentiviral vector. A total of 293 T cells were cotransfected medium (DMEM). The cells were incubated with annexin with the GPNMB-expressing plasmid (lenti-GP) and two V-FITC and presidiums iodide (PI) at room temperature in packaging plasmids. The supernatant containing lenti-GP the dark for 15 min, and approximately 20,000 cells were was collected after 48 h of culture. analyzed by flow cytometry (BD Bioscience, San Jose, CA). AnnexinV-FITC/PI detection kit (WLA001a) was Enzyme-Linked ImmunoSorbent Assay obtained from Wanleibio. The levels of interleukin (IL)-6, IL-10, and tumor Statistical Analysis necrosis factor (TNF)-α in the supernatants of hPDLCs given various treatments were measured with a human IL-6 Cell experiments were repeated at least three times. enzyme-linked immunosorbent assay (ELISA) detection Student’s t test was used for data analysis. The data are kit (WLE04, Wanleibio, China), a human IL-10 ELISA expressed as the mean ± standard deviation (SD).