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Macrophages are important regulators of bone remodeling, and M1 polarization is observed in the setting of medication-related osteonecrosis of the jaws (MRONJ). Here, we characterize the phenotype of macrophages during early stages of MRONJ development in zoledronate (ZA)–treated mice with periodontal disease and explore the role of rosiglitazone, a drug that has been reported to lower the M1/M2 macrophage ratio, in MRONJ burden. Mice received ZA, and experimental periodontal disease (EPD) was induced around their second left maxillary molar. The mice were euthanized 1, 2, or 4 wk later. Micro–computed tomography and histologic and immunohistochemical analyses were carried out. In a separate experiment, mice were treated with ZA in the absence or presence of rosiglitazone, EPD was induced for 5 wk, and the MRONJ burden was assessed. An M1 predilection was noted in ZA versus vehicle (Veh) mice at 1, 2, or 4 wk after ligature placement. M1 cells were found to be positive for MMP-13, and their presence coincided with disruption of the surrounding collagen network in ZA mice. Rosiglitazone caused a reversal in the M1/M2 polarization in Veh and ZA mice. Rosiglitazone did not cause significant radiographic changes 5 wk after EPD in Veh or ZA animals. Importantly, percentage osteonecrosis and bone exposure were decreased in the rosiglitazone-treated versus nontreated ZA sites 5 wk after EPD. Our data point to an important role of M1 macrophage polarization with an overexpression of MMP-13 in the early phases of MRONJ development and provide insight into the use of interventional approaches promoting an M2 phenotype as a preventative means to alleviate MRONJ burden. [ABSTRACT FROM AUTHOR]

Osteonecrosis of the jaws (ONJ) is a rare but severe complication of antiresorptive medications, such as bisphosphonates, used in the treatment of bone malignancy or osteoporosis. Tooth extraction and dental disease have been strongly associated with ONJ development. Here, we investigated molecular and cellular markers of socket healing after extraction of healthy or teeth with experimental periodontitis (EP) in Wistar-Han rats treated with zoledronic acid (ZA). We included 4 experimental groups: vehicle-treated animals with extraction of healthy teeth or teeth with ligature-induced EP and ZA-treated animals with extraction of healthy teeth or teeth with EP. Animals were pretreated with vehicle or ZA for a week, and EP was induced. Four weeks later, the second maxillary molars were extracted; sockets were allowed to heal for 4 wk; animals were euthanized; and maxillae were isolated. Radiographically, extraction sockets in groups 1, 2, and 3 demonstrated normal healing. Contrary incomplete socket healing was noted after extraction of teeth with EP in ZA-treated rats of group 4. Histologically, persistent inflammation and extensive osteonecrosis were seen in group 4. Disorganization of the collagen network, collagen type III predominance, and lack of collagen fiber insertion in the necrotic bone were associated with impaired socket healing. Cells positive for MMP-9, MMP-13, and α-SMA expression were present at the areas of epithelial invagination and adjacent to osteonecrotic bone. Importantly, human biopsies from patients with ONJ showed similar findings. Our data emphasize the importance of dental disease and tooth extraction in ONJ pathogenesis and help delineate an altered profile in wound-healing markers during ONJ development. [ABSTRACT FROM AUTHOR]

Langerhans cell histiocytosis ( LCH) is a group of rare and enigmatic disorders of the reticuloendothelial system characterised by abnormal, possibly neoplastic, proliferation of Langerhans cells. Oral lesions, in particular osteolytic lesions of the jaws, may be the primary or the sole manifestation of the disease. A 44 year old woman with a history of eosinophilic granuloma on the lower left canine region excised 25 years ago presented with an asymptomatic, ulcerated gingival lesion, extending lingual and distal to the right mandibular second premolar tooth, which was noticed approximately 6 months ago. The underlying bone was not involved. Histopathologic and immunohistochemical examination showed an LCH, consistent with eosinophilic granuloma. Radiotherapy was given, and 10 months later she developed three small painful ulcers located on the palatal gingivae around the right canine, between the second premolar and first molar, and around the right third molar, also diagnosed as eosinophilic granuloma. Eosinophilic granuloma may present in the gingival mucosa, in the absence of bone involvement, and may run a protracted course with remissions, reactivation and progression from single system to multi-system involvement. Therefore, long-term periodical follow-up and restaging is mandatory. The presence of a high proportion of plasma cells expressing lambda-light chains may cause a diagnostic dilemma with plasma cell malignancy would be interesting to be evaluated in additional cases. [ABSTRACT FROM AUTHOR]

Medication-related osteonecrosis of the jaw (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, whereas 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 versus vehicle (Veh) animals at all time points. SIRT1 colocalized with cytoplasmic HMGB1 and presented a statistically significant increased expression at the EP sites of ZA animals for all time points. RAGE expression was significantly higher in the submucosal tissues EP sites of ZA animals compared with 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 versus 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. © 2022 American Society for Bone and Mineral Research (ASBMR)., (© 2022 American Society for Bone and Mineral Research (ASBMR).)

Antiresorptive agents such as bisphosphonates (BP) and denosumab are commonly prescribed for the management of primary bone malignancy, bone metastasis, osteoporosis, Paget disease, or other bone disorders. Medication-related osteonecrosis of the Jaws (MRONJ) is a rare but significant complication of antiresorptive medications. Duration, dose, and antiresorptive potency as well as concomitant diseases, additional medications, and local factors affect MRONJ incidence and severity. MRONJ pathophysiology is still poorly understood. Nevertheless, decreased bone resorption due to osteoclastic inhibition along with trauma, infection/inflammation, or blood supply inhibition are considered synergistic factors for disease development. In addition, previous data research examined the effects of antiresorptive medication on immune system components and introduced potential alterations on immune response as novel elements in MRONJ pathogenesis. Considering that macrophages are the first cells in the nonspecific immune response, it is not surprising that these multifaceted players attracted increased attention in MRONJ research recently. This current review attempted to elucidate the effects of antiresorptive medications on several aspects of macrophage activity in relation to the complex inflammatory microenvironment of MRONJ. Collectively, unravelling the mode of action and extent of macrophages' potential contribution in MRONJ occurrence will provide novel insight in disease pathogenesis and potentially identify intrinsic therapeutic targets.

Medication related osteonecrosis of the Jaws (MRONJ) is a severe complication of antiresorptive and anti-angiogenic medications. Osteoclast inhibition is central in MRONJ pathogenesis. Here, we investigated if local application of RANKL (a key molecule in osteoclast activation) could enhance osteoclast generation and improve extraction socket healing in the presence of bisphosphonates. Thirty Wistar-Han rats received one saline or 66 μg/kg zoledronate (ZA) i.p. dose before surgery. A week later, mandibular molars were extracted bilaterally. Collagen tapes infused with water or RANKL were placed in the extraction sockets of 60 hemimandibles of veh (veh/RANKL-, veh/RANKL+) or ZA treated rats (ZA/RANKL-, ZA/RANKL+). Rats were euthanized 3 or 12 days after surgery. Animals euthanized at 12 days received two additional veh or ZA injections. Clinical, radiographic and histologic assessments were performed. Visually, at the 3-day timepoint, no sockets demonstrated complete healing. At the 12-day timepoint, sockets of veh/RANKL- and veh/RANKL+ rats showed intact mucosa, while mucosal defects were noted in ZA/RANKL- rats. Importantly, ZA/RANKL+ sockets showed absence of bone exposure. RANKL delivery increased bone healing in the ZA/RANKL+ sites 12 days after extraction compared to the ZA/RANKL- sites. Histologically, at the 3-day timepoint, ZA/RANKL- sockets demonstrated extensive bone exposure and osteonecrosis. In contrast, ZA/RANKL+ rats showed granulation tissue coverage and significantly reduced osteonecrosis, similar to the veh groups. Importantly, in the ZA/RANKL+ group, osteoclasts attached to the bone surface and osteoclast numbers were higher compared to ZA/RANKL- sites. At the 12-day timepoint, persistent osteonecrosis and bone exposure were detected in the sockets of ZA/RANKL- animals. Contrary, ZA/RANKL+ rats demonstrated socket epithelialization and reduced osteonecrosis. Significantly more total and bony attached osteoclasts persisted in the ZA/RANKL+ vs the ZA/RANKL- group. We present a novel approach towards improving socket healing, in the presence of ZA, by enhancing osteoclastic numbers and attachment through local RANKL application. Our approach is clinically applicable and could improve treatment outcomes of patients on high-dose ZA therapy., (Copyright © 2021. Published by Elsevier Inc.)

Objectives: The aim of this study was to investigate the relationship between M1 and M2 macrophage polarization and clinical stage in patients with medication-related osteonecrosis of the jaw (MRONJ) who underwent treatment with bisphosphonates or denosumab., Materials and Methods: M1 and M2 macrophage density and expression of interleukin (IL)-6 and IL-10 were assessed on biopsies of mucosal tissues surrounding necrotic bone in 30 MRONJ patients with stages 1-3 and controls. For identification of M1 and M2 macrophages, double CD68/iNOS and CD68/CD206 immunofluorescence staining was conducted, respectively. Computer-assisted immunofluorescence quantification of markers was performed., Results: Early stage 1 MRONJ patients showed a switch toward the M2 phenotype, as indicated by the higher density of M2 macrophages, the decreased M1/M2 ratio, and the upregulation of IL-10. MRONJ patients with advanced stages 2 and 3 showed a shift toward M1-polarized macrophages, as suggested by the higher density of M1 macrophages, the increased M1/M2 ratio, and the overexpression of IL-6. The macrophage density of both M1 and M2 subsets was significantly enhanced in patients receiving bisphosphonates compared with those receiving denosumab., Conclusions: The M1-M2 macrophage polarization status in mucosal tissues bordering necrotic bone correlates with clinical stage of MRONJ. Patients with early-stage MRONJ show a switch toward M2-polarized macrophages, while MRONJ patients with advanced stage demonstrate a shift toward the M1 phenotype., Clinical Relevance: Therapeutic molecules targeting the inflammatory microenvironment via the regulation of either M1 or M2 macrophage polarization may represent a novel strategy for treatment of MRONJ.

Osteonecrosis of the jaw (ONJ), a rare, but potentially severe side effect of anti-resorptive medications, presents as exposed bone in the maxillofacial region lasting for at least 8 weeks. While clinical experience and animal models concur in finding that systemic antiresorptive treatment in conjunction with local risk factors, such as tooth extraction or dental disease may lead to ONJ development, the subclinical molecular changes that precede bone exposure remain poorly understood. The identification of these changes is not only important in understanding disease pathophysiology, but could provide potential for treatment development. Here, we evaluated the early stages of ONJ utilizing a model of experimental periodontitis (EP) in mice treated with two different types of antiresorptives, targeting potential changes in vasculature, hypoxia, oxidative stress, and apoptosis. Antiresorptive treatment in animals with EP increased levels of empty osteocytic lacunae and increased ONJ prevalence compared to Veh animals. The arteriole and venule network seen around EP areas was diminished in animals treated with antiresorptives. Higher levels of vascular endothelial growth factor A (VEGF-A) and vascular cell adhesion protein-1 (VCAM-1) were observed 1-week following EP in treated animals. Finally, levels of hypoxia, oxidative stress, and apoptosis remained high in antiresorptive treated animals with EP through the duration of the experiment. Together, our data point to subclinical vasculature organizational disturbances that subsequently affect levels of hypoxia, oxidative stress, and apoptosis in the area of developing ONJ., (Copyright © 2019. Published by Elsevier Inc.)

Antiresorptive agents, such as bisphosphonates and denosumab, are frequently used for the management of osteoporosis. Indeed, both medications decrease the risk of osteoporotic fractures; however, these medications are associated with rare but potentially severe side effects, such as osteonecrosis of the jaw (ONJ). ONJ, defined as an area of exposed bone in the maxillofacial region that lasts for 8 weeks, often presents with significant pain and infection and can lead to serious complications. Interestingly, other treatments for osteoporosis have been developed, such as antibodies against the osteocyte-secreted protein, sclerostin. Sclerostin functions to inhibit the Wnt signaling cascade, leading to inhibition of bone formation. In clinical trials, a sclerostin antibody (romosozumab, Amgen Inc., UCB Brussels) increases bone formation and lowers the risk of osteoporotic fractures. However, in conjunction with increased osteoblastic activity, a reduction in bone resorption markers is observed. This antiresorptive effect raises the concern of possible ONJ development in patients treated with sclerostin antibodies. Here, utilizing ligature-induced experimental periodontitis (EP), we evaluated the effects of sclerostin inhibition on the development of ONJ-like lesions in ovariectomized rats. Beginning 8 weeks post-ovariectomy, rats were treated for 22 weeks with weekly injections of vehicle (Veh), 200 μg/kg zoledronic acid (ZA), a potent bisphosphonate at 100-fold the osteoporosis dose, or 5 mg/kg sclerostin antibody (Scl-Ab) at the osteoporotic dose. EP was initiated at week 12 and maintained for the remainder of the study. Scl-Ab treatment transiently increased serum P1NP, a bone formation marker, increased BV/TV, and decreased eroded surfaces in lumbar vertebrae. ZA-treated rats developed histologic features of ONJ, whereas Veh-treated controls did not. Scl-Ab animals lost less periodontal bone in sites with EP. However, these animals presented with no histologic signs of ONJ. In conclusion, sclerostin inhibition enhanced structural bone parameters, without inducing ONJ-like lesions, in ovariectomized rats with EP. © 2018 American Society for Bone and Mineral Research., (© 2018 American Society for Bone and Mineral Research.)

Purpose: Medication-related osteonecrosis of the jaw (MRONJ) is a rare but severe side effect of antiresorptive medications. Most animal models use tooth extraction as an instigating local factor to induce MRONJ, with varied results. However, these teeth are healthy and absent of dental disease, a rare finding that does not reflect clinical practices. The authors hypothesized that extraction of teeth with periapical inflammation would lead to MRONJ in rats treated with high-dose bisphosphonates., Materials and Methods: Rats were pretreated with zoledronic acid (ZA) for 1 week. Pulp exposure (PE) was established by exposing the pulpal chamber of the first and second molars. Experimental periapical disease (EPD) was induced by PE and bacterial inoculation into pulp chambers of the first and second mandibular molars. The mandibular molars were extracted 4 weeks after PE or EPD, and animals were euthanized 4 weeks after tooth extraction. Extraction sockets were assessed clinically, radiographically, and histologically., Results: Clinically, radiographically, and histologically, socket healing was observed in all vehicle-treated animals and in ZA-treated animals after extraction of healthy teeth or teeth with PE. In contrast, bone exposure, lack of socket healing, and osteonecrosis were present in most ZA-treated animals after extraction of teeth with EPD. Bacterial presence was noted in areas of osteonecrotic alveolar bone., Conclusion: These data support a synergistic contribution of severe dental disease and tooth extraction to MRONJ pathogenesis. Importantly, this model is amenable to manipulation of methodologic conditions for the dissection of parameters involved in MRONJ pathogenesis., (Copyright © 2018 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.)

The expression of proinflammatory cytokines in various malignant neoplasms is widely considered to represent the host immune response to tumor development. The role of interferon (IFN)γ in head and neck squamous cell carcinoma, and its association with endoplasmic reticulum (ER) stress pathways, remains a subject of ongoing investigation. Dentin sialophosphoprotein (DSPP), which is a member of the small integrin‑binding N‑linked glycoproteins family, has been implicated in malignant transformation and invasion of oral squamous cell carcinoma (OSCC). Recent studies have established matrix metalloproteinase (MMP)20 as the cognate MMP partner of DSPP. The present study examined the effects of IFNγ treatment on DSPP and MMP20 expression, ER stress, the unfolded protein response (UPR), and calcium (Ca) homeostasis regulatory mechanisms in OSCC cells. The OSC2 OSCC cell line was treated with IFNγ at specific time‑points. At each time‑point, the mRNA expression levels of DSPP and MMP20, and those of ER‑stress‑, UPR‑ and Ca homeostasis‑associated proteins [78‑kDa glucose‑regulated protein (GRP78), sarco/endoplasmic reticulum Ca2+‑ATPase (SERCA2b), inositol 1,4,5‑trisphosphate receptor (IP3r), protein kinase R‑like ER kinase (PERK) and inositol‑requiring enzyme 1 (IRE1)], were assessed by reverse transcription‑quantitative polymerase chain reaction. The protein expression levels of B‑cell lymphoma 2 (Bcl‑2), Bcl‑2‑associated X protein (Bax), proliferating cell nuclear antigen (PCNA) and cytochrome c were analyzed by western blotting. Cell viability, apoptosis and migration were evaluated by MTT, Annexin V‑fluorescein isothiocyanate flow cytometry and wound‑healing assays, respectively. IFNγ treatment significantly downregulated the mRNA expression levels of the major ER stress regulator GRP78 and, to a lesser extent, the UPR‑associated molecule IRE1; however, IFNγ had no significant effect on PERK. With regards to ER Ca homeostasis molecules, treatment with IFNγ downregulated the mRNA expression levels of SERCA2b and upregulated those of IP3r. Furthermore, DSPP and MMP20 mRNA expression levels were significantly reduced following IFNγ treatment. Notably, treatment with IFNγ hampered OSC2 migration, reduced cell viability and PCNA protein expression, enhanced apoptosis, downregulated Bcl‑2, and upregulated Bax and cytochrome c. Overall, IFNγ inhibited OSCC cell viability and migration, and increased apoptosis, possibly by regulating ER stress and UPR mechanisms. In addition, IFNγ‑induced DSPP and MMP20 downregulation may correspond with alteration in ER Ca homeostasis.

Dentin sialophosphoprotein (DSPP) is upregulated in various human cancers, including head and neck squamous cell carcinoma. Cancer cells are commonly found under constant endoplasmic reticulum (ER) stress and exhibit increased levels of misfolded proteins, due to gene mutations and a stressful microenvironment. The present study examined the effects of DSPP silencing on the regulation of ER stress and the unfolded protein response (UPR) in oral cancer cells. A recently established stable DSPP short hairpin (sh)RNA-silenced OSC2 oral cancer cell line was used. The mRNA expression levels of ER stress-associated proteins, including 78 kDa glucose-regulated protein (GRP78), sarcoplasmic/endoplasmic reticulum calcium ATPase 2b (SERCA2b), inositol 1,4,5-trisphosphate receptor (IP3r), protein kinase R-like endoplasmic reticulum kinase (PERK), serine/threonine-protein kinase/endoribonuclease IRE1 (IRE1), activating transcription factor 6 (ATF6) and matrix metalloproteinase 20 (MMP20), were assessed by reverse transcription-quantitative polymerase chain reaction. The expression levels of apoptosis-related [B‑cell lymphoma 2 (Bcl2), Bcl2-associated X protein (Bax) and cytochrome c] and cell proliferation-related [proliferating cell nuclear antigen (PCNA)] proteins were analyzed by western blotting. Cell viability, apoptosis and migration were monitored by MTT assay, Annexin V-fluorescein isothiocyanate flow cytometry and wound-healing assay, respectively. In transiently transfected puromycin‑free OSC2 cells, DSPP silencing markedly downregulated the mRNA expression levels of major ER stress regulators, including GRP78, SERCA2b, PERK, IRE1 and ATF6, as well as MMP20. DSPP silencing also resulted in decreased cell viability and migration, and enhanced apoptosis. Furthermore, PCNA and Bcl2 levels were decreased, whereas Bax and cytochrome c protein levels were increased in DSPP-silenced OSC2 cells. Sustained puromycin treatment partially counteracted the effects of DSPP silencing on the mRNA expression levels of ER stress-related proteins and MMP20, and on the migratory capacity of OSC2 cells. However, following puromycin treatment of DSPP-silenced cells, cell viability was further reduced and apoptosis was enhanced. In conclusion, these data provide evidence to suggest that DSPP may be involved in ER stress mechanisms in oral squamous cell carcinoma, since its downregulation in OSC2 cells led to significant alterations in the levels of major ER stress-associated proteins, and subsequent collapse of the UPR system.

Background: Recent findings indicate that dentin sialophosphoprotein (DSPP) and matrix metalloproteinase (MMP) 20 interact in oral squamous cell carcinoma (OSCC). The objective of this study was to determine the effects of DSPP/MMP20 gene silencing on oral cancer stem cell (OCSC) markers., Methods: The expression of well-established OCSC markers: ABCG2; ALDH1; CD133; CD44; BMI1; LGR4, and Podoplanin in DSPP/MMP20-silenced OSCC cell line, OSC2, and controls were assayed by western blot (WB), and flow cytometry techniques. The sensitivity of OSC2 cells to cisplatin following DSPP/MMP20 silencing was also determined., Results: DSPP/MMP20 silencing resulted in downregulation of OCSC markers, more profoundly ABCG2 (84%) and CD44 (81%), following double silencing. Furthermore, while treatment of parent (pre-silenced) OSC2 cells with cisplatin resulted in upregulation of OCSC markers, DSPP/MMP20-silenced OSC2 cells similarly treated resulted in profound downregulation of OCSC markers (72 to 94% at 50 μM of cisplatin), and a marked reduction in the proportion of ABCG2 and ALDH1 positive cells (~ 1%)., Conclusions: We conclude that the downregulation of OCSC markers may signal a reduction in OCSC population following MMP20/DSPP silencing in OSCC cells, while also increasing their sensitivity to cisplatin. Thus, our findings suggest a potential role for DSPP and MMP20 in sustaining OCSC population in OSCCs, possibly, through mechanism(s) that alter OCSC sensitivity to treatment with chemotherapeutic agents such as cisplatin., Competing Interests: The study was approved by the University of Texas Health Science Center-Houston’s Institutional Review Board for all experimental procedures including human tissue samples and cell lines.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Objectives: The aim of this study was to evaluate the expression levels of DNA damage response (DDR) markers in potentially preneoplastic oral epithelial lesions (PPOELs)., Study Design: Immunohistochemical expression of DDR markers (γΗ2 ΑΧ, pChk2, 53 BP1, p53, and phosphorylated at Ser 15 p53) was assessed in 41 oral leukoplakias, ranging from hyperplasia (H) to dysplasia (D) and in comparison with oral squamous cell carcinoma (OSCC) and normal mucosa (NM). Statistical and receiver operating characteristic curve analysis were performed., Results: γH2 AX immunoexpression demonstrated a gradual increase and upper layer extension from NM to H to higher D degrees to OSCC. pChk2 expression was minimal in NM, relatively low in PPOELs, with an increasing tendency from H to D, and higher in OSCC. 53 BP1 demonstrated higher levels in OSCC than in NM, whereas its expression in PPOELs was heterogeneous, gradually increasing according to D. p53 demonstrated progressively higher levels and upper layer extension from H to D to OSCC. Phosphorylated p53 was absent in NM and relatively low in PPOELs and OSCC., Conclusions: DDR markers' expression is variable in PPOELs, showing a tendency to increase along with dysplasia. Activated DDR mechanisms may play an important protective role at early stages of oral carcinogenesis, but probably suffer progressive deregulation, eventually failing to suppress malignant transformation., (Copyright © 2018. Published by Elsevier Inc.)

Calcific aortic vasculopathy correlates with bone loss in osteoporosis in an age-independent manner. Prior work suggests that teriparatide, the bone anabolic treatment for postmenopausal osteoporosis, may inhibit the onset of aortic calcification. Whether teriparatide affects the progression of preexisting aortic calcification, widespread among this patient population, is unknown. Female apolipoprotein E-deficient mice were aged for over 1 yr to induce aortic calcification, treated for 4.5 wk with daily injections of control vehicle (PBS), 40 µg/kg teriparatide (PTH40), or 400 µg/kg teriparatide (PTH400), and assayed for aortic calcification by microcomputed tomography (microCT) before and after treatment. In a followup cohort, aged female apolipoprotein E-deficient mice were treated with PBS or PTH400 and assayed for aortic calcification by serial microCT and micropositron emission tomography. In both cohorts, aortic calcification detected by microCT progressed similarly in all groups. Mean aortic 18 F-NaF incorporation, detected by serial micropositron emission tomography, increased in the PBS-treated group (+14 ± 5%). In contrast, 18 F-NaF incorporation decreased in the PTH400-treated group (-33 ± 20%, P = 0.03). Quantitative histochemical analysis by Alizarin red staining revealed a lower mineral surface area index in the PTH400-treated group compared with the PBS-treated group ( P = 0.04). Furthermore, Masson trichrome staining showed a significant increase in collagen deposition in the left ventricular myocardium of mice that received PTH400 [2.1 ± 0.6% vs. control mice (0.5 ± 0.1%), P = 0.02]. In summary, although teriparatide may not affect the calcium mineral content of aortic calcification, it reduces 18 F-NaF uptake in calcified lesions, suggesting the possibility that it may reduce mineral surface area with potential impact on plaque stability. NEW & NOTEWORTHY Parathyroid hormone regulates bone mineralization and may also affect vascular calcification, which is an important issue, given that its active fragment, teriparatide, is widely used for the treatment of osteoporosis. To determine whether teriparatide alters vascular calcification, we imaged aortic calcification in mice treated with teriparatide and control mice. Although teriparatide did not affect the calcium content of cardiovascular deposits, it reduced their fluoride tracer uptake.

Objective: Primary oral malignant melanoma (POMM) is a rare type of malignancy with a very poor prognosis, the molecular pathogenesis of which remains elusive. The aim of this study was to assess the expression status of signal transducer and activator of transcription (STAT) proteins in POMM., Study Design: Six POMMs were included in the study. Total protein levels of STAT1, STAT3, and STAT5a, as well as the tyrosine phosphorylated (activated) form of STAT3 (pSTAT3), were assessed immunohistochemically., Results: Immunohistochemical evaluation of total STAT3 revealed diffuse and strong cytoplasmic and nuclear expression in the majority of tumor cells of all cases, whereas activated pSTAT3 had mostly mild nuclear expression in 5%-40% of malignant melanocytes in all cases. Evaluation of STAT1 and STAT5a identified mainly mild cytoplasmic expression in the absence of nuclear localization., Conclusion: The identification of aberrant STAT3 expression and activation in oral malignant melanocytes supports a possible role of this molecule in POMM. In contrast, STAT5a has only limited cytoplasmic expression, mitigating against its involvement in POMM. Also, STAT1's low levels may have implications for POMM sensitivity to interferon-based therapeutic strategies, considering the role of this molecule in cutaneous melanoma immunotherapy., (Copyright © 2018. Published by Elsevier Inc.)

Signal transducer and activator of transcription 3 (STAT3) and mitogen activated protein kinases (MAPKs), including ERK and JNK, have been implicated in oral squamous cell carcinoma (OSCC) development and progression. Our purpose was to evaluate the levels of activated STAT3, ERK1/2 and JNK by immunohistochemistry in OSCC and to investigate possible correlations of these molecules with each other as well as with the degree of tumor differentiation. Immunohistochemical assessment of the phosphorylated levels of STAT3(tyrosine/ serine), ERK1/2 and JNK was performed in 60 OSCC, including well, moderately and poorly differentiated tumors. Semiquantitative scoring system was used, by calculating intensity of immunostaining, percentage of positive cells and combined scores. Statistics included Fisher's test, Student's T-Test and Kruskal-Wallis analysis, Spearman's correlation coefficient and multivariate logistic regression analyses. Immunohistochemical levels of both pSTAT3(tyr) and pERK1/2 showed statistically significant differences between well and poorly differentiated tumors with the latter receiving higher mean percentage, intensity and total scores. On the other hand, pJNK showed statistically significantly higher intensity levels in moderately compared to poorly differentiated tumors. pSTAT3(ser) immunoexpression did not appear to correlate with tumor differentiation. Between different molecules, more pronounced, pERK1/2 levels exhibited statistically significant positive correlation with pSTAT3(ser), pSTAT3(tyr) and pJNK expression. ERK1/2 and STAT3 activation (as assessed by tyrosine but not serine phosphorylation) could contribute to a less differentiated phenotype in OSCC, while JNK activation may have an opposite, although possibly less pronounced, effect. Positive correlations between MAPK and STAT3 levels may indicate a direct crosstalk and/or regulation by common upstream pathways.

JNKs (c-Jun N-terminal kinases) belong to mitogen-activated protein kinases' family and become activated by several growth factors, stress, radiation, and other extracellular signals. In turn, JNK activation results in phosphorylation of downstream molecules involved in many normal cellular processes. Nevertheless, recent data have linked JNK signaling with several pathological conditions, including neurodegenerative diseases, inflammation, and cancer. The role of JNK in cancer remains controversial. Initially, JNK was thought to play a rather oncosuppressive role by mediating apoptosis in response to stress stimuli, inflammatory, or oncogenic signals. However, a number of studies have implicated JNK in malignant transformation and tumor growth. The contradictory functions of JNK in cancer may be due to the diversity of JNK upstream and downstream signaling and are under intensive investigation. This review summarizes current literature focusing on the significance of JNK pathway in cancer development and progression, particularly addressing its role in oral cancer. Understanding the complexity of JNK signaling has the potential to elucidate important molecular aspects of oral cancer, possibly leading to development of novel and individualized therapeutic strategies.

Mitogen-activated protein kinases (MAPKs) are a family of protein kinases that link extracellular stimuli with intracellular responses and participate in numerous cellular processes such as growth, proliferation, differentiation, inflammation and apoptosis. Persistent activation of signal transducer and activator of transcription 3 (STAT3), which is accompanied by increases in STAT3 tyrosine phosphorylation, is associated with cell proliferation, differentiation and apoptosis in oral squamous cell carcinoma (OSCC). The role and significance of the activation of MAPKs, particularly of c-Jun N-terminal kinase (JNK), on STAT3 signaling in OSCC have not been thoroughly investigated. The present study examines the effects of JNK1/2 modulation on STAT3 signaling and cellular activities in OSCC cells. The expression levels of STAT3 [total, tyrosine phosphorylated (p-Tyr) and serine phosphorylated (p-Ser)], JNK, c-Jun and cyclin D1 were assessed in the OSCC cell lines SCC25 and SCC9. Inhibition of JNK1/2 was achieved by pharmacological agents (SP600125) and by small interfering RNA (siRNA) silencing, while JNK1/2 was induced by active MAPK kinase 7. Cell proliferation and viability rates were also evaluated. Inhibition of JNK1/2 with either SP600125 treatment or specific siRNA silencing resulted in decreased levels of p-Ser STAT3 and increased levels of p-Tyr STAT3 and cyclin D1 in both cell lines. Furthermore, JNK1/2 inhibition resulted in a dose-dependent increase in cell growth and viability in both cell lines. Opposite results were observed with JNK1/2 induction in both cell lines. The present results are supportive of a potential tumor suppressive role of JNK1/2 signaling in OSCC, which may be mediated through negative crosstalk with the oncogenic STAT3 signaling pathway. The possible therapeutic implications of JNK1/2 inhibition for patients with OSCC require to be investigated.

Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) signaling pathway possesses confirmed oncogenic potential in oral squamous cell carcinoma (OSCC). Crosstalk with other molecular pathways contributes to STAT3 regulation in cancer. The effects of mitogen-activated protein kinases (MAPKs) and particularly extracellular signal-regulated kinase 1/2 (Erk1/2) on STAT3 signaling in OSCC have not been thoroughly investigated. The present study examined the effects of Erk1/2 modulation on STAT3 signaling and cell growth in OSCC cells. Constitutive expression levels of phosphorylated (tyrosine and serine) and total STAT3, Erk1/2 and cyclin D1 were assessed in OSCC cell lines. Erk1/2 modulation was achieved by pharmacological agents; siRNA silencing against Erk1/2 was also performed. Cell proliferation and viability were assessed. Erk1/2 inhibition with either U0126 treatment or specific siRNA silencing resulted in decreases in p-ser STAT3 and cyclin D1 levels and increases in p-tyr STAT3 in OSCC cells. Moreover, Erk1/2 inhibition resulted in a dose-dependent reduction in OSCC cell growth and viability. Erk1/2 induction had the opposite effects. Taken together, these results are supportive of an active crosstalk between the oncogenic Erk1/2 and STAT3 pathways in OSCC, the significance of which requires further investigation.