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. Author manuscript; available in PMC: 2023 Sep 1.
Published in final edited form as: J Bone Miner Res. 2022 Jun 16:10.1002/jbmr.4637. doi: 10.1002/jbmr.4637

Inhibition of HMGB1/RAGE signaling reduces the incidence of Medication-Related Osteonecrosis of the Jaw (MRONJ) in mice

Ioannis Gkouveris 1,#,@, Danny Hadaya 1,#, Naseim Elzakra 1, Akrivoula Soundia 1, Olga Bezouglaia 1, Sarah M Dry 2, Flavia Pirih 3, Tara Aghaloo 1,*, Sotirios Tetradis 1,*
PMCID: PMC9474692  NIHMSID: NIHMS1817012  PMID: 35711109

Abstract

Medication-related Osteonecrosis of the jaws (MRONJ) is a severe complication of antiresorptive or antiangiogenic medications, used in the treatment of bone malignancy or osteoporosis. Bone necrosis, mainly represented by osteocytic death is always present in MRONJ sites, however the role of osteocyte death in MRONJ pathogenesis is unknown. High Mobility Group Box 1 (HMGB1) is a non-histone nucleoprotein that in its acetylated form accumulates in the cytoplasm, while non-acetylated HMGB1 localizes in the nucleus. SIRT1 deacetylase regulates cellular localization of HMGB1. Interestingly, HMGB1 is released during cell necrosis, and promotes inflammation through signaling cascades including activation of the RAGE receptor. Here, we utilized a well-established mouse MRONJ model that utilizes ligature-induced experimental periodontitis (EP) and treatment with either vehicle or Zolendronic Acid (ZA). Initially, we evaluated HMGB1-SIRT1 expression in osteocytes at 1, 2 and 4 weeks of treatment. Significantly increased cytoplasmic and perilacunar HMGB1 expression was observed at EP-sites of ZA vs. Veh animals at all timepoints. SIRT1 co-localized with cytoplasmic HMGB1 and presented a statistically significant increased expression at the EP sites of ZA animals for all timepoints. RAGE expression was significantly higher in the submucosal tissues EP sites of ZA animals compared to those in vehicle group. To explore the significance of increased cytoplasmic and extracellular HMGB1 and increased RAGE expression in MRONJ pathogenesis, we used pharmacologic inhibitors of these molecules. Combined HMGB1/RAGE inhibition resulted in lower MRONJ incidence with statistically significant decrease in osteonecrotic areas and bone exposure vs non-inhibitor treated ZA-animals. Together, our data point to the role of HMGB1 as a central alarmin, overexpressed at early phase of MRONJ pathogenesis during osteocytic death. Moreover, HMGB1-RAGE pathway may represent a new promising therapeutic target in patients at high risk of MRONJ.

INTRODUCTION

Medication-related Osteonecrosis of the jaws (MRONJ) is a severe complication of antiresorptive or antiangiogenic medications prescribed mainly to patients with bone malignancies or osteoporosis [1, 2]. MRONJ is characterized by exposed bone or bone that can be probed through an intraoral or extraoral fistula in the maxillofacial region that lasts for more than 8 weeks [1, 2]. Development of MRONJ may be accompanied by pain, eating discomfort, irritability, and decreased life satisfaction that overall have a negative impact on patients’ everyday life [3].

Although MRONJ was described in the literature nearly two decades ago, the precise mechanisms of MRONJ pathophysiology are still obscure. Apart from impaired jaw remodeling due to the antiresorptive effects, several hypotheses implicate local trauma, local or systemic infection/inflammation, blood supply attenuation, and/or soft-tissue toxicity. Nevertheless, none of these hypotheses is adequate to explain the full extent of the disease spectrum [4, 5].

An important dilemma in MRONJ pathogenesis is whether the primary event is a breakdown of the oral soft tissues followed by bone exposure and osteonecrosis or bone death is the initiating event, followed by mucosal deterioration. The existence of subclinical Stage 0 MRONJ, without bone exposure, in patients on antiresorptives [6–8], as well as translational studies in MRONJ animal models demonstrating a higher frequency of histologic bone necrosis versus bone exposure [9–11], strongly support bone necrosis as an early event and point to a key role of osteocyte death in MRONJ pathogenesis. However, research of osteocyte death during MRONJ is limited.

According to Nomenclature Committee on Cell Death (NCCD), cell death can be divided, based on functional attributes, into Accidental (ACD) and Regulated (RCD) cell death, [12]. ACD involves an unexpected attack or injury that overcomes control processes. On the other hand, RCD includes specific control signaling cascades, and based on its molecular aspects, it can be further classified into multiple subtypes. Apoptosis is the main RCD and remains immunologically silent, while necrotic death represents a lytic, inflammatory RCD process [12, 13].

Our research has focused on early events during MRONJ development [4]. We observed increased caspase 3 expression in the submucosa around developing MRONJ lesions, as early as one week after disease initiation [4]. However, no significant increase in caspase 3 expression was seen in osteocytes in areas of osteonecrosis (not shown), suggesting that apoptosis might not be a leading pathway in osteocyte death during MRONJ onset.

A central molecule in cell necrosis is HMGB1 [14–16]. HMGB1, a non-histone chromatin-associated protein, also known as amphoterin or HMG1 [17], is the most abundant non-histone nucleoprotein of the HMGB gene family, but is also found in the cytoplasm, shuttling back and forth from the cytoplasm to the nucleus [18]. At the acetylated status, HMGB1 accumulates in the cytoplasm, while non-acetylated HMGB1 localizes in the nucleus[17]. HMGB1 is released in cell necrosis, and promotes inflammation in multiple settings [19–24]. Inflammatory stimulation results in HMGB1 release through necrotic death [21, 22], while in vivo and in vitro models of necrosis demonstrate increased HMGB1 release [14, 15, 23]. Sirtuin1 or SIRT1, a NAD+-dependent class III protein deacetylase, deacetylates and regulates cellular localization and release of HMGB1 [25–28]. Importantly, exome-wide association studies identified the single nucleotide polymorphism (SNP) rs932658 on chromosome 10 of the SIRT1 gene significantly associated with lower risk of MRONJ [29, 30].

Given the genetic linkage of SIRT1 with MRONJ incidence and the regulation of HMGB1 localization by SIRT1, in the current manuscript we explored the presence of SIRT1-HMGB1 in osteocytic death and their role as key regulators of MRONJ development.

MATERIALS AND METHODS

Animal care

This study was approved by the UCLA Chancellor’s Animal Research Committee (ARC). Four-week old male C57BL/6 mice (Charles River Laboratories, Raleigh, NC) were utilized for these experiments, following ARRIVE guidelines. A standard diet (NIH-31 Modified Open Formula, ENVIGO, Madison, WI, USA) and water were provided ad libitum. Animals received intraperitoneal (IP) injections of Veh (endotoxin-free saline), or 200 μg/kg Zoledronic Acid (LKT Laboratories, St Paul, MN) two times weekly through the duration of the experiment.

MRONJ mouse model

Animals were pre-treated with Veh or ZA for 1 week. Then, 6–0 silk sutures were ligated around the right maxillary second molar (M2) to induce experimental periodontitis (EP). We have previously used this approach to initiate ONJ in mice [4]. Animals continued to receive Veh or ZA through the duration of the experiment. Twelve mice were used per group, per time-point. Animals were evaluated at one-week, two-weeks, or four-weeks following initiation of experimental periodontal disease. Upon termination of the experiment, mice were euthanized, maxillae were harvested and imaged at 40x magnification (Keyonce VHX-100, Osaka, Japan). A total of 72 mice were used.

Pharmacologic Inhibition of HMGB1 and RAGE signaling

Animals were pre-treated for 1 week prior to ligature placement with Veh or ZA, and 2 days prior to ligature placement in the absence or presence of 10 mg/kg Tanshinone IIA (Tan, Sigma), and 3 mg/kg receptor for advanced glycation end products (RAGE) Antagonist Peptide (RAP, Calbiochem). Tan and RAP were administered by i.p. injection. Tan is a cytokine-releasing inhibitory drug that prevents HMGB1 release from the nucleus [31]. RAP is a competitive antagonist for the RAGE that disrupts the association of RAGE with its ligands [32–34]. Then, 6–0 silk sutures were ligated around the right maxillary second molar to induce experimental periodontitis (EP), and animals continued to receive Veh, ZA or inhibitors for 6 weeks, after which, they were euthanized and evaluated for clinical, radiographic, and histologic signs of MRONJ (Fig. 6 A). Twelve mice were used per group.

Figure 6:

Figure 6:

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Histologic assessment of Veh and ZA treated animals in the absence or presence of HMGB1 and RAGE inhibitors. A) Experimental timeline. B) Representative coronal H&E sections of Veh and ZA treated animals with or without inhibitors 6 weeks after EP. Aqua double arrows indicate the epithelial to alveolar crest distance, red arrows the ligature, fuchsia arrows the bone exposure, yellow dotted line the area of osteonecrosis in ZA treated animals in absence of inhibitors, and aqua dotted line the area of osteonecrosis in ZA treated animals in the presence of inhibitors. C) magnified area of alveolar bone, indicated by black squares in (B), in Veh and ZA treated animals in the absence or presence of HMGB1 and RAGE inhibitors. Quantification of D) the epithelium to crest distance (μm), E) percent empty osteocytic lacunae, F) percent of osteonecrosis, G) percent of MRONJ prevalence (% of animals) at 6-week time point, and H) the number of TRAP+ cells. Data in D), E), F), and H) represent mean ± SEM. **** p < 0.0001, ** p < 0.01.

μCT-Imaging

Maxillae were fixed and imaged by ex-vivo micro-computed tomography (μCT) at 10μm resolution, as previously described [35, 36]. For linear measurements, axial slices were converted to DICOM format and imported in the Dolphin Imaging software (Chatsworth, CA, USA). Periodontal bone loss and and cemento-enamel junction (CEJ) to alveolar bone crest (ABC) distance were measured as described [35, 36].

Histologic Analysis

Maxillae were decalcified, sectioned, and H&E stained [37]. The epithelium to alveolar crest distance, total number of osteocytic lacunae, number of empty lacunae, and osteonecrotic area, and number of osteoclasts were quantified, as previously described [37].

Immunofluorescence staining analysis

Immunofluorescence staining of tissue sections were probed with rabbit polyclonal antibody against HMGB1 (Abcam, ab 18256, 1:200), mouse monoclonal antibody against RAGE (Santa Cruz, sc 365154, 1:200) and mouse monoclonal antibody against SIRT1 (Abcam, Ab 157401 1:200). DAPI staining was performed to detect nuclei (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-24941). For negative control, the staining procedure was carried out without the use of primary antibody. Photomicrographs were obtained using a fluorescence optical microscopy (Mantra, PerkinElmer, USA).

Immunofluorescence quantification of HMGB1 and SIRT1 positive osteocytes as well as identification of cellular location were performed manually using Image J (NIH) software. For immunofluorescence evaluation initial 40X followed by 100X magnification for further confirmation were applied. The ROI was defined as the alveolar bone from the alveolar crest to the root apex. For RAGE expression, immunofluorescence quantification was performed using Image J (NIH). The ROI was defined as the submucosal area from the the basal membrane of keratinized epithelium to the alveolar crest under 40X magnification. A minimal and maximal threshold were set and total expression was calculated. The adjusted threshold for positive expression was kept the same for all samples in all groups.

Statistics

Raw data were analyzed using GraphPad Prism (GraphPad Software, Inc. La Jolla, CA). Descriptive statistics were used to calculate the mean and the standard error of the mean (SEM). Data were analyzed by two-way ANOVA and post-hoc Tukey’s test for multiple comparisons, with statistical significance of 0.05. Socket healing was analyzed using the Fischer’s exact test.

RESULTS

Clinical and radiographic assessment

The average initial body weight of all mice was 19.4 g ± 2.1 (mean ± SEM). The average body weight for all mice necropsied at the 1 w, 2 w, and 4 w time points were 20.4 g ± 1.8, 22.4 g ± 2.3, and 25.2 g ± 2.6, respectively. A statistically significant increase was noted between the initial body weight and the body weight at the 4w time point. However, no differences in weight were observed among Veh vs. ZA groups at any time point.

Clinical examination and photographs confirmed the presence of the ligature around the crown of the maxillary right second molar (M2) in all groups. μCT examination was conducted at the one, two, and four-week necropsy time points to investigate the effects of EP in Veh and ZA treated animals. Periodontal bone loss was assessed by measuring the distance between the cemento-enamel junctions (CEJ) and the alveolar bone crest (ABC). No difference was found in the CEJ-ABC distance at the healthy site of Veh vs. ZA animals at any time points (not shown). Ligature placement increased the CEJ-ABC distance in Veh animals at all time points. In contrast, the CEJ-ABC distance in the ligature site of ZA animals did not increase during the experiment, and was significantly lower than the CEJ-ABC distance of the ligature site in Veh animals (Fig. 1).

Figure 1:

Figure 1:

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Radiographic assessment of Veh and ZA treated animals. Sagittal and coronal sections of Veh or ZA animals at A) 1-week, B) 2-week and C) 4-week timepoints after EP. D) Quantification of EP-induced alveolar bone loss indicated by the CEJ-alveolar bone crest (ABC) distance, at the 1-week, 2-week, and 4-week time points. Data represent mean ± SEM. **** p < 0.0001, ** p < 0.01.

Histologic assessment

H&E staining of histologic sections of the healthy sites of Veh and ZA treated animals demonstrated normal epithelium, submucosal tissues, alveolar bone and periodontal ligament (PDL, Fig. 2A). In contrast, in EP sites abundant inflammatory infiltrate and bone resorption in the ligated areas of Veh treated animals was observed. In the ZA treated groups, inflammatory infiltrate was present, however the alveolar crest retained its height (Fig. 2A, yellow and aqua arrows). The epithelium-alveolar crest distance (Fig. 2, orange line) decreased significantly, in ZA vs Veh animals (Fig. 2A and 2B). Importantly, a statistically significant increase at EP sites in the percent of empty osteocytic lacunae (Fig. 2A, green arrows) between the ZA vs. Veh animals was observed (Fig. 2C). Histologic examination revealed no empty lacunae and normal epithelial, submucosal and osseous architecture in non-EP sites of both veh and ZA treated animals (not shown). A progressive increase in MRONJ prevalence, defined as histologic bone exposure (Fig. 2A, fuchsia arrows), was observed in ZA vs. Veh treated animals at EP sites that was maximal by 4 weeks of treatment (Fig. 2D).

Figure 2:

Figure 2:

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Histologic assessment of Veh and ZA treated animals. A) Representative coronal H&E images of healthy and EP sites at 1-week, 2-week and 4-week timepoints of Veh and ZA treated animals. Yellow arrows point to inflammatory infiltrate, aqua arrows to bone levels, green arrows to necrotic bone, fuchsia arrows to bone exposure, red arrows to ligature and orange line to epithelium to alveolar crest distance. B) Quantification of epithelium to alveolar crest distance (μm), C) percent osteonecrosis and D) MRONJ prevalence (% of animals) at 1-week, 2-week and 4-week timepoints. Data in B) and C) represent mean ± SEM. *** p < 0.001, ** p < 0.01, * p < 0.05.

Immunofluorescence analysis

To test HMGB1expression in osteocytes, EP was induced for 1, 2, and 4 w in Veh or ZA animals and immunofluorescence (IF) was performed. In healthy sites of Veh and ZA animals low expression levels of HMGB1 were noted (Fig. 3A). In EP sites of Veh mice, osteocytes demonstrated low, mostly nuclear, HMGB1 expression up to 4 w (Fig. 3 B, C, D yellow arrows). In contrast, in ZA mice HMGB1 was mostly localized in the cytoplasm of osteocytes. Several empty osteocytic lacunae exhibited perilacunar HMGB1, suggesting its release during osteocyte death (Fig. 3, B, C, D white arrows). Quantification of osteocytes with cytoplasmic HMGB1 expression showed that EP sites had higher expression from healthy sites in both Veh and ZA mice. In EP sites of Veh animals, the percent of osteocytes with HMGB1 cytoplasmic expression was low at the 1w time point and expression declined thereafter. In contrast, in EP sites of ZA animals HMGB1 cytoplasmic expression in osteocytes continued to increase throughout the experiment and was maximal at the 4w timepoint. Importantly, HMGB1 expression was statistically significantly higher in ZA vs. Veh animals in all timepoints (Fig. 3, E).

Figure 3:

Figure 3:

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HMGB1 localization in osteocytes of Veh and ZA treated animals. Representative HMGB1 immunofluorescence and DAPI sections at 40X and 100X magnification of healthy (A) and EP sites at 1-week (B), 2-week (C) and 4-week (D) timepoints of Veh and ZA treated animals. Yellow arrows point to nuclear HMGB1 localization and white arrows to cytoplasmic and perilacunar HMGB1 localization. D) Quantification of cells with cytoplasmic HMGB1 expression. Data represent mean ± SEM. # p < 0.001 compared to Veh healthy site, & p < 0.001 compared to ZA healthy site, **** p < 0.001, ** p < 0.01.

SIRT1 deacetylase interacts with HMGB1 and restrains its pro-inflammatory efficacy through nuclear retention. Having observed the increased cytoplasmic and perilacunar HMGB1 localization in osteocytes at EP sites of animals treated with ZA, and given the close functional relationship between SIRT1 and HMGB1, we examined osteocyte expression of both SIRT1 and HMGB1 (Fig 4). In healthy sites of Veh and ZA mice SIRT1 levels were low (Fig. 4A). EP sites of Veh mice showed a mild increase of SIRT1 levels (Fig. 4 B, C, D). In contrast, in ZA mice, SIRT1 was high for all timepoints (Fig. 4, B, C, D yellow arrows), colocalizing with cytoplasmic HMGB1 (Fig. 4, white arrows). Quantification of osteocytes with both HMGB1 and SIRT1 expression revealed a statistically significant increase of all EP sites compared to healthy sites for both Veh and ZA animals. Importantly, quantification of osteocytes with both HMGB1 and SIRT1 expression was significantly higher at the EP sites of ZA vs. Veh animals for all timepoints (Fig. 4E).

Figure 4:

Figure 4:

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SIRT1 and HMGB1 co-localization in osteocytes of Veh and ZA treated animals. Representative SIRT1, HMGB1 immunofluorescence and DAPI sections at 100X magnification of healthy (A) and EP sites at 1-week (B), 2-week (C) and 4-week (D) timepoints of Veh and ZA treated animals. D) Quantification of cells with both HMGB1 and SIRT1 expression at 1-week, 2-week and 4-week timepoints. Yellow arrows point to SIRT1 and white arrows to SIRT1-HMGB1 colocalization. Data represent mean ± SEM. # p < 0.01 compared to Veh healthy site, & p < 0.001 compared to ZA healthy site,* p < 0.05.

To explore signaling downstream of released HMGB-1, we assessed RAGE expression levels. RAGE interacts with AGE and Damage-Associated Molecular Pattern (DAMP) molecules, and is one of the main receptors activated by extracellularly released HMGB1 [16]. RAGE expression levels in the periodontal tissues of healthy sites of Veh and ZA animals were low. EP induced RAGE expression in the periodontal tissues of Veh animals at 1 w, and in the periodontal tissues of ZA animals as 1, 2 and 4 w. Importantly, RAGE expression was statistically significantly higher at the EP sites of ZA vs. Veh animals for all timepoints (Fig. 5 A, B).

Figure 5:

Figure 5:

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RAGE expression in submucosal tissues of Veh and ZA treated animals. A) Representative RAGE immunofluorescence sections of healthy and EP sites at 1-week, 2-week and 4-week timepoints of Veh and ZA treated animals. The aqua dotted line shows the position of the ligature, while the white dotted line shows the position of the alveolar bone. B) Quantification of RAGE expression at 1-week, 2-week and 4-week time points. Data represents mean ± SEM. # p < 0.05 compared to Veh healthy site, & p < 0.001 compared to ZA healthy site * p < 0.05.

HMGB1/RAGE inhibition

To explore the significance of HMGB1/RAGE signaling in MRONJ pathogenesis, we treated animals with a combined pharmacologic inhibition of HMGB1 by 10 mg/kg Tanshinone IIA (Tan) to prevent HMGB1 release from the nucleus [38] and 3 mg/kg RAGE antagonist peptide (RAP). Specifically, animals were initially treated with Veh or ZA for 3 days followed by introduction of HMGB1 and RAGE inhibitors. After 4 days, EP was induced and pharmacologic treatments continued for 6 more weeks (Fig. 6A). Histology demonstrated an increased submucosal width in Veh animals (Fig. 6 B, aqua double arrow), that was significantly decreased in ZA animals (Fig. 6B) with bone penetrating through the epithelium and exposed to the oral cavity (Fig. 6B, white arrows). Inhibitors did not affect the submucosa dimensions in Veh, but significantly increased the submucosal thickness in ZA animals (Fig. 6B). No osteonecrotic areas were present in Veh animals without or with inhibitor treatment. As expected, empty osteocytic lacunae (Fig. 6C, yellow arrows) and areas of osteonecrosis (Fig. 6B), yellow outlined area were present at the alveolar crest of EP sites in ZA animals. Importantly, inhibitor treatment decreased the area of osteonecrosis in ZA animals (Fig. 6B, aqua outlined area and 6C). Quantification of the histologic findings confirmed the above observations and demonstrated a statistically significant increase in the epithelial to alveolar bone crest distance (Fig. 6D), a statistically significant decrease in osteonecrotic area (Fig. 6E), and a statistically significant decrease in bone exposure (Fig. 6 A, fuchsia arrows) to the oral cavity (Fig. 6F) in inhibitor treated vs non-treated ZA animals. Quantification of TRAP positive cells demonstrated a decrease in the osteoclast number in the Veh animals treated with inhibitors vs. the Veh control animals that did not reach statistical significance (P=0.06).

DISCUSSION

MRONJ is a rare but significant side effect of antiresorptive medications prescribed for the management of bone malignancy or osteoporosis[39]. MRONJ most commonly occurs after extraction of clinically non-restorable teeth or teeth with periapical or periodontal disease or around such teeth prior to extraction [1, 40–42]. Therefore, infection/inflammation associated with pre-existing dental disease, is present in most MRONJ cases.

Delineating early events during MRONJ initiation is crucial to uncover points of early diagnosis and intervention and to understand mechanisms of disease onset and development. For these studies, we utilized a model of MRONJ that employs administration of high-dose ZA and EP, but not tooth extraction [4]. We have previously demonstrated that experimental periapical or periodontal disease and high dose antiresorptives, (ZA and RANKL inhibitors) are sufficient to induce MRONJ-like changes in mice and rats [4, 39]. This model avoids the severe traumatic impact of tooth extraction that compounds healing of soft and osseous structures and complicates distinction of normal vs. pathologic tissue responses.

We were able to follow changes in the mucosal and osseous alveolar structures as early as one week and observed that periodontal bone loss advanced during the experimental timepoints in veh treated animals. In contrast, ZA administration inhibited periodontal bone loss, and progressively increased osteonecrosis, decreased epithelial to alveolar crest distance and induced bone exposure. We have previously shown that these early changes are paralleled by disruption of the mucosal vasculature and altered responses to hypoxia, cell adhesion, oxidative stress and cell apoptosis [4].

Interestingly, in MRONJ animal models utilizing both dental disease or tooth extraction in the presence of antiresorptives, the incidence of histologic osteonecrosis is higher than the presence of bone exposure to oral cavity [9–11]. Furthermore, Stage 0 MRONJ is a well-described phase of the disease in patients with non-specific symptoms in the absence of bone exposure [2]. More than 50% of these patients develop clinically exposed bone within 4–5 months after diagnosis [7]. These translational and clinical observations support the notion that osteocytic death is an early event in MRONJ pathogenesis that precedes soft tissue breakdown.

Since cell death can be both a consequence and a cause of inflammation that propagates tissue damage [43], we focused our studies on osteocytic death at early stages of MRONJ occurrence. Apoptosis is a type of regulated cell death (RCD) that remains immunologically silent, whereas non-apoptotic RCDs including necrosis may activate variable inflammatory response [13]. Impaired balance of a single or mixed types of cell death could drive to autoimmune or infectious diseases as well neurodegeneration disorders and cancer [44, 45]. Since our preliminary data (not shown) did not reveal a significant difference in osteocytic apoptosis in ZA vs. control animals with EP, we examined the potential that other RCD processes, and particularly necrosis, might affect osteocytes during MRONJ pathogenesis.

HMGB1 is a nuclear, non-histone, chromatin-associated protein. During cellular stress HMGB1 is overexpressed and accumulates in the cytosol. Excessive cytosolic accumulation may result to either active cellular secretion [46] or direct release following membrane lysis of cells dying via necrosis[17, 47]. Indeed, HMGB1 release through necrotic cell death has been reported in numerous chronic inflammatory and autoimmune diseases, including sepsis, rheumatoid arthritis, atherosclerosis, chronic kidney disease and systemic lupus erythematosus (SLE) [48–52]. In addition, HMGB1 is released from cancer cells undergoing chemotherapy or radiotherapy [53], as a result of necrotic cell death [54]. In our studies, ZA treated animals with EP showed increased HMGB1 expression that was preferentially non-nuclear in osteocytes at areas of developing osteonecrosis, at one week that was further enhanced by four weeks. In empty osteocytic lacunae, HMGB1 demonstrated a perilacunar localization, suggesting its release and persistence after osteocytic death. This perilacunar and extracellular presence of osteocytic HMGB1 as early as one week points to its potential role in the early phases of MRONJ setting and subsequent propagation.

This HMGB1 non-nuclear increase was accompanied by high cytoplasmic levels of the SIRT1 deacetylase during the experimental timepoints. SIRT1 deacetylates HMGB1 at multiple N-terminal domains and promotes HMGB1 nuclear localization [25]. This cytoplasmic SIRT1 expression parallels the high levels of cytoplasmic HMGB1 likely in an effort of the cell to deacetylate HMGB1. Increased expression of both HMGB1 and SIRT1 has been reported in settings of hypoxia and hypoxia-induced chemotherapy resistance in non-small cell lung cancer patients [55] [56] [57]. Similar to our findings, Sirt1 increased expression coincides with increased intracellular Sirt1-HMGB1 binding and HMGB1 extracellular secretion in a hypoxia-reperfusion injury model [58]. It appears that in early timepoints osteocytes attempt to negotiate cellular stress by expressing both SIRT1 and HMGB1 likely in an effort to deacetylate HMGB1. However, the perilacunar localization of HMGB1 in empty osteocytic lacunae suggests the failure of SIRT1 to redirect HMGB1 to the nucleus. Importantly, two exome-wide association studies pointed that the single nucleotide polymorphism (SNP) rs932658 on the SIRT1/HERC4 chromosome 10 of the SIRT1 gene is accompanied by lower MRONJ risk, ostensibly by favoring enhanced SIRT1 expression [29, 30]. The genomic patient-based studies and our translational findings point to a central role of the SIRT1-HMGB1 necrotic pathway in MRONJ pathogenesis and suggest potential treatment intervention for MRONJ burden that targets this pathway.

Extracellular HMGB1 is a common Damage-Associated Molecular Patterns (DAMP) molecule, that acts as a prototypical alarmin to activate innate immunity and inflammatory responses [16, 18]. Several of the HMGB1 effects are mediated by activation of RAGE and subsequent NF-kB activation followed by increased cytokine formation and release [16], including Kawasaki disease with coronary artery injury, inflammatory heart disease and Alzheimer’s Disease [59–61].

RAGE, a multi-ligand receptor belonging to the immunoglobulin superfamily, is expressed on multiple cell types. RAGE is highly expressed during embryogenesis, but is found at low levels in adults. RAGE overexpression is associated with various pathological conditions, such as inflammation, diabetes, cardiovascular diseases, neurodegenerative disorders, and cancer [62]. Importantly, RAGE is significantly overexpressed in gingival tissues of patients with severe periodontitis, with or without diabetes [63]. Indeed, EP induced RAGE expression in the gingival tissues of control mice, however, RAGE expression was significantly higher in animals treated with ZA at all timepoints, suggesting participation of RAGE signaling at the MRONJ setting.

HMGB1 and RAGE expression is upregulated during periodontal disease [64–66]. Indeed, healthy sites of Veh and ZA treated animals showed a similar, low expression of RAGE and osteocytic HMGB1, while their levels increased in EP sites of Veh treated animals. However, in ZA treated animals in the presence of EP, the RAGE and osteocytic HMGB1 expression was heightened in all time points, as discussed above. Our data suggest that the increased HMGB1 and RAGE expression in sites of developing osteonecrosis is not the direct effect of ZA treatment, because the expression of the same markers would have increased in the healthy sites of the animals. This increased expression appears to be the synergistic result of resorption inhibition by ZA and localized inflammation by EP.

To explore the potential involvement of the HMGB1-RAGE signaling pathway in MRONJ pathogenesis and severity, we employed two inhibitors. Tan, a natural steroidal pigment, acts as a selective and effective HMGB1 inhibitor in in vitro and in vivo studies [22, 49, 51, 67, 68] and lowers lethal endotoxemia and reduces incidence of lethal sepsis[49], reduces myocardial ischemia reperfusion injury [68], protects brains against focal ischemia [31], attenuates sepsis-induced immunosuppression [38], and improve symptoms of rheumatoid arthritis destruction [67]. RAP blocks S100 and HMGB1 mediated RAGE activation and reduces growth and metastasis of MPanc-96 pancreatic cancer cells and C6-glioma cells [32], restores alveolar fluid clearance and attenuates lung injury in a pig model of acute respiratory distress syndrome [33], blunts airway reactivity, airway inflammation and goblet cell metaplasia in a mouse model of asthma [34] In our studies, the combined Tan/RAP treatment of ZA animals decreased MRONJ indices, as shown by the decreased bone exposure, increased epithelial to alveolar crest distance and decreased area of osteonecrosis.

Some limitations in our study need to be mentioned. The intent of the inhibitor studies was to provide proof of principle evidence of the HMGB1/RAGE pathway involvement in MRONJ development. Therefore, both inhibitors were administered to mice prior to EP induction, aiming to maximize the inhibition of these molecules. In a clinical scenario, this could be akin a patient on antiresorptives scheduled for a planned oral procedure. For a patient with existing symptoms, inhibition of the HMGB1/RAGE pathway would be applied after the onset of MRONJ. Other DAMPS might be induced and participate in MRONJ development. In our study we focused on HMGB1, because we were intrigued by its increase in osteocytes and empty osteocytic lacunae at areas of developing osteonecrosis. Indeed, inhibition of HMGB1/RAGE did not eliminate completely the MRONJ burden. Finally, although the HMGB1/RAGE inhibitor studies provide mechanistic insights, osteocytic death contribution towards the pathogenesis of MRONJ is complex and we have undertaken experiments towards dissecting these mechanisms in detail.

Collectively, our data introduce HMGB1 as a central alarmin, overexpressed at early phase of MRONJ pathogenesis during osteocytic death. The osteocytic increase of SIRT1, the cytoplasmic localization of HMGB1 in osteocytes, the extracellular perilacunar release of HMGB1, and the increased expression of RAGE in the submucosal tissues during the early phases of MRONJ initiation and progression point to the potentially key role this pathway plays in MRONJ pathogenesis. Importantly, the combined inhibition of the HMGB1 and RAGE attenuated disease burden and severity suggest that the HMGB1-RAGE pathway may represent a new promising therapeutic target in patients at high risk of MRONJ occurrence.

ACKNOWLEDGMENTS

This work was supported by the R01 DE019465 grant from NIH/NIDCR (ST). We gratefully acknowledge the Translational Pathology Core Laboratory (TPCL) at the David Geffen School of Medicine at UCLA for all histology and digital imaging services.

Disclosures

ST and TA have been paid consultants for Amgen Inc. (Thousand Oaks, CA). ST has received grant support from Amgen Inc. The other authors declare no potential conflicts of interest. Data available on request from the authors.

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