Publications by authors named "Keith L Kirkwood"

59 Publications

Subgingival microbiome is associated with alveolar bone loss measured 5-years later in postmenopausal women.

J Periodontol 2020 Nov 3. Epub 2020 Nov 3.

Department of Epidemiology and Environmental Health, School of Public Health and Health Professions, University at Buffalo - SUNY, Buffalo, New York, USA.

Background: The aim of this study was to quantify the association between subgingival microbiota and periodontal disease progression in older women, for which limited published data exist.

Methods: A total of 1016 postmenopausal women, aged 53 to 81 years, completed baseline (1997 to 2001) and 5-year (2002 to 2006) dental exams that included probing depth, clinical attachment level, gingival bleeding, and radiographic alveolar crestal height (ACH). Baseline microbiota were measured in subgingival plaque using 16S rRNA sequencing. Associations between 52 microbiota we previously found statistically significantly associated with clinical periodontal disease at baseline, were examined with disease progression. The traditional Socransky microbiota complexes also were evaluated. Side-by-side radiograph comparisons were used to define progression as ≥2 teeth with ≥1 mm ACH loss or ≥1 new tooth loss to periodontitis. The association between baseline centered log(2) ratio transformed microbial relative abundances and 5-year periodontal disease progression was measured with generalized linear models.

Results: Of 36 microbiota we previously showed were elevated in moderate/severe disease at baseline, 24 had statistically significantly higher baseline mean relative abundance in progressing compared with non-progressing women (P < .05, all); which included all Socransky red bacteria (P. gingivalis, T. forsythia, T. denticola). Of 16 microbiota elevated in none/mild disease at baseline, five had statistically significantly lower baseline abundance in non-progressing compared with progressing women (P < 0.05, all), including one Socransky yellow bacteria (S. oralis). When adjusted for baseline age, socioeconomic status, and self-rated general health status, odds ratios for 5-year progression ranged from 1.18 to 1.51 (per 1-standard deviation increment in relative abundance) for microbiota statistically significantly (P < 0.05) positively associated with progression, and from 0.77 to 0.82 for those statistically significantly (P < 0.05) inversely associated with progression. These associations were similar when stratified on baseline levels of pocket depth, gingival bleeding, ACH, and smoking status.

Conclusions: These prospective results affirm clearly that subgingival microbiota are measurably elevated several years prior to progression of alveolar bone loss, and include antecedent elevations in previously undocumented taxa additional to known Socransky pathogenic complexes.
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http://dx.doi.org/10.1002/JPER.20-0445DOI Listing
November 2020

The p38/MKP-1 signaling axis in oral cancer: Impact of tumor-associated macrophages.

Oral Oncol 2020 04 10;103:104591. Epub 2020 Feb 10.

Department of Oral Biology, School of Dental Medicine, University at Buffalo, Buffalo, NY, USA; Department of Head and Neck/Plastic and Reconstructive Surgery, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA. Electronic address:

Oral squamous cell carcinomas (OSCC) constitute over 95% of all head and neck malignancies. As a key component of the tumor microenvironment (TME), chronic inflammation contributes towards the development, progression, and regional metastasis of OSCC. Tumor associated macrophages (TAMs) associated with OSSC promote tumorigenesis through the production of cytokines and pro-inflammatory factors that are critical role in the various steps of malignant transformation, including tumor growth, survival, invasion, angiogenesis, and metastasis. The mitogen-activated protein kinases (MAPKs) can regulate inflammation along with a wide range of cellular processes including cell metabolism, proliferation, motility, apoptosis, survival, differentiation and play a crucial role in cell growth and survival in physiological and pathological processes including innate and adaptive immune responses. Dual specificity MAPK phosphatases (MKPs) deactivates MAPKs. MKPs are considered as an important feedback control mechanism that limits MAPK signaling and subsequent target gene expression. This review outlines the role of MKP-1, the founding member of the MKP family, in OSCC and the TME. Herein, we summarize recent progress in understanding the regulation of p38 MAPK/MKP-1 signaling pathways via TAM-related immune responses in OSCC development, progression and treatment outcomes.
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http://dx.doi.org/10.1016/j.oraloncology.2020.104591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7136140PMC
April 2020

Acid sphingomyelinase deficiency exacerbates LPS-induced experimental periodontitis.

Oral Dis 2020 Apr 30;26(3):637-646. Epub 2020 Jan 30.

Division of Endocrinology, Diabetes and Medical Genetics, Department of Medicine, College of Medicine, Medical University of South Carolina, Charleston, South Carolina.

Background: Mutation of the gene for acid sphingomyelinase (ASMase) causes Niemann-Pick disease. However, the effect of ASMase deficiency on periodontal health is unknown. Periodontal disease is a disease resulting from infection and inflammation of periodontal tissue and alveolar bone that support the teeth. The goal of this study was to determine the role of ASMase deficiency in periodontal inflammation and alveolar bone loss.

Methods: We induced periodontitis in wild-type and ASMase-deficient (ASMase ) mice with periodontal lipopolysaccharide (LPS) injection and compared the alveolar bone loss and periodontal inflammation between these mice.

Results: Results showed that ASMase deficiency did not significantly change metabolic parameters, but exacerbated LPS-induced alveolar bone loss, osteoclastogenesis, and periodontal tissue inflammation. To understand the mechanisms by which ASMase deficiency aggravates LPS-induced periodontitis, we analyzed sphingolipids in periodontal tissues. Results showed that ASMase deficiency led to increases in not only sphingomyelin, but also ceramide (CER), a bioactive sphingolipid known to promote inflammation. Results further showed that ASMase deficiency increased CER de novo synthesis.

Conclusion: ASMase deficiency exacerbated LPS-induced alveolar bone loss and periodontal inflammation. ASMase deficiency leads to an unexpected CER increase by stimulating de novo synthesis CER, which is likely to be involved in the ASMase deficiency-exacerbated periodontitis.
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http://dx.doi.org/10.1111/odi.13268DOI Listing
April 2020

Myeloid-Derived Suppressor Cells at the Intersection of Inflammaging and Bone Fragility.

Immunol Invest 2018 Nov;47(8):844-854

c Division of Geriatrics and Palliative Medicine , University at Buffalo, Research Service, Western New York Veterans Affairs Healthcare Service , Buffalo , New York , USA.

Age-related alteration of the immune system with aging, or immunosenescence, plays a major role in several age-associated conditions, including loss of bone integrity. Studies over the past several years have clearly established the immune system is chronically activated with advanced aging, termed inflammaging, and is characterized by elevated levels of proinflammatory cytokines in response to physiological or environmental cues that essentially result in an arrested immune system that maintains a low-level state of activation. This age-associated inflammation impacts several biological systems including the innate immune system, where aging results in a skewing of the hematopoiesis toward the myeloid lineage, including the expansion of myeloid-derived suppressor cells (MDSCs). This heterogeneous population of myeloid cells classically displays immunosuppressive capacity but they also have the ability to directly differentiate into osteoclasts. This review explores the possibility of inflammaging to be involved in reduction of bone microarchitecture and loss of bone mass/strength through the expansion of MDSCs and the osteoclastogenic capacity and activity.
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http://dx.doi.org/10.1080/08820139.2018.1552360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039312PMC
November 2018

Inflammaging.

Authors:
Keith L Kirkwood

Immunol Invest 2018 11;47(8):770-773

a Department of Oral Biology, School of Dental Medicine , University at Buffalo, The State University of New York , Buffalo , New York , USA.

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http://dx.doi.org/10.1080/08820139.2018.1552392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7250180PMC
November 2018

Functionalized nanoparticles containing MKP-1 agonists reduce periodontal bone loss.

J Periodontol 2019 08 13;90(8):894-902. Epub 2019 Mar 13.

Department of Oral Biology, University at Buffalo, Buffalo, NY, USA.

Background: Progress over of the past several years has elucidated a role for mitogen-activated protein kinase phosphatase to regulate periodontal inflammation yielding new possibilities for treatment of periodontal diseases. These studies aimed to determine if nanoparticles (NPs) loaded with a pharmacological agent that induces mitogen-activated protein kinase phosphatase have potential clinical utility for management of periodontal inflammation and alveolar bone.

Methods: Polyethylene glycol (PEG)-polylactide (PLA) (PEG-PLA) NPs were loaded with auranofin (ARN), an antirheumatic drug, to induce mitogen-activated protein kinase phosphatase (MKP)-1 expression in vitro and in vivo. Release kinetics of ARN from NPs was performed by high performance liquid chromatography (HPLC). Fluorescent-labeled NPs were used to show uptake into macrophages by flow cytometry. Real-time quantitative polymerase chain reaction (qPCR) was used to determine dual specificity protein phosphatase (Dusp)-1 mRNA induction by Auranofin-loaded nanoparticles (ARN-NPs) and viability of ARN-NPs was determined by colorimetric in vitro assays. Functional in vitro assays were used to measure functional MKP-1 induction and preclinical models using Aggregatibacter actinomycetemcomitans lipopolysaccharide-induced alveolar bone loss and microcomputed tomography was used to determine in vivo efficacy of functionalized ARN-NPs.

Results: Data indicated that ARN-NPs had reduced cytotoxicity compared with free ARN and Dusp1 mRNA and MKP-1 activity was significantly increased by ARN-NPs in vitro. Flow cytometry indicated rapid uptake into macrophages. Finally, significant bone loss reduction was observed with ARN-NPs compared with control NPs in vivo using an lipopolysaccharide-induced rat model of periodontitis.

Conclusion: Results from these studies suggest that developing NPs functionalized with ARN have anti-inflammatory activities and may be a novel adjuvant therapeutic strategy to significantly improve periodontitis therapy and outcomes.
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http://dx.doi.org/10.1002/JPER.18-0572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7112177PMC
August 2019

Activation of vitamin D in the gingival epithelium and its role in gingival inflammation and alveolar bone loss.

J Periodontal Res 2019 Aug 25;54(4):444-452. Epub 2019 Feb 25.

Department of Oral Biology, University of Florida, Gainesville, Florida.

Background And Objective: Both chronic and aggressive periodontal disease are associated with vitamin D deficiency. The active form of vitamin D, 1,25(OH) D , induces the expression of the antimicrobial peptide LL-37 and innate immune mediators in cultured human gingival epithelial cells (GECs). The aim of this study was to further delineate the mechanism by which vitamin D enhances the innate defense against the development of periodontal disease (PD).

Materials And Methods: Wild-type C57Bl/6 mice were made deficient in vitamin D by dietary restriction. Cultured primary and immortalized GEC were stimulated with 1,25(OH) D , followed by infection with Porphyromonas gingivalis, and viable intracellular bacteria were quantified. Conversion of vitamin D to 25(OH)D and 1,25(OH) D was quantified by ELISA. Effect of vitamin D on basal IL-1α expression in mice was determined by topical administration to the gingiva of wild-type mice, followed by qRT-PCR.

Results: Dietary restriction of vitamin D led to alveolar bone loss and increased inflammation in the gingiva in the mouse model. In primary human GEC and established human cell lines, treatment of GEC with 1,25(OH) D inhibited the intracellular growth of P. gingivalis. Cultured GEC expressed two 25-hydroxylases (CYP27A1 and CYP2R1), as well as 1-α hydroxylase, enabling conversion of vitamin D to both 25(OH)D and 1,25(OH) D . Topical application of both vitamin D and 1,25(OH) D to the gingiva of mice led to rapid inhibition of IL-1α expression, a prominent pro-inflammatory cytokine associated with inflammation, which also exhibited more than a 2-fold decrease from basal levels in OKF6/TERT1 cells upon 1,25(OH) D treatment, as determined by RNA-seq.

Conclusion: Vitamin D deficiency in mice contributes to PD, recapitulating the association seen in humans, and provides a unique model to study the development of PD. Vitamin D increases the activity of GEC against the invasion of periodontal pathogens and inhibits the inflammatory response, both in vitro and in vivo. GEC can convert inactive vitamin D to the active form in situ, supporting the hypothesis that vitamin D can be applied directly to the gingiva to prevent or treat periodontal disease.
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http://dx.doi.org/10.1111/jre.12646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6626553PMC
August 2019

Inhibition of the histone demethylase KDM4B leads to activation of KDM1A, attenuates bacterial-induced pro-inflammatory cytokine release, and reduces osteoclastogenesis.

Epigenetics 2018 7;13(5):557-572. Epub 2018 Aug 7.

a Department of Drug Discovery and Biomedical Sciences , Medical University of South Carolina , Charleston , South Carolina, USA.

Periodontal disease (PD) afflicts 46% of Americans with no effective adjunctive therapies available. While most pharmacotherapy for PD targets bacteria, the host immune response is responsible for driving tissue damage and bone loss in severe disease. Herein, we establish that the histone demethylase KDM4B is a potential drug target for the treatment of PD. Immunohistochemical staining of diseased periodontal epithelium revealed an increased abundance of KDM4B that correlates with inflammation. In murine calvarial sections exposed to Aggregatibacter actinomycetemcomitans lipopolysaccharide (Aa-LPS), immunohistochemical staining revealed a significant increase in KDM4B protein expression. The 8-hydroxyquinoline ML324 is known to inhibit the related demethylase KDM4E in vitro, but has not been evaluated against any other targets. Our studies indicate that ML324 also inhibits KDM4B (IC50: 4.9 μM), and decreases the pro-inflammatory cytokine response to an Aa-LPS challenge in vitro. Our results suggest that KDM4B inhibition-induced immunosuppression works indirectly, requiring new protein synthesis. In addition, fluorescence-stained macrophages exhibited a significant decrease in global monomethyl histone 3 lysine 4 (H3K4me) levels following an Aa-LPS challenge that was prevented by KDM4B inhibition, suggesting this effect is produced through KDM1A-mediated demethylation of H3K4. Finally, ML324 inhibition of KDM4B in osteoclast progenitors produced a significant reduction in Aa-LPS-induced osteoclastogenesis. These data link histone methylation with host immune response to bacterial pathogens in PD, and suggest a previously unreported, alternative mechanism for epigenetic control of the host inflammatory environment. As such, KDM4B represents a new therapeutic target for treating hyper-inflammatory diseases that result in bone destruction.
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http://dx.doi.org/10.1080/15592294.2018.1481703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6260135PMC
February 2019

Mean annual attachment, bone level, and tooth loss: A systematic review.

J Periodontol 2018 06;89 Suppl 1:S120-S139

Biostatistics Unit, University College London Eastman Dental Institute, London, UK.

Background: Rate of progression of periodontitis has been used to inform the design of classifications of periodontal diseases. However, the evidence underpinning this topic is unclear and no systematic review has yet been conducted.

Objectives: The focused question for this systematic review was: in adults, what is the progression of periodontitis in terms of clinical attachment loss, radiographic bone loss, and tooth loss?

Data Sources: Highly sensitive electronic search was conducted for published data in MEDLINE, EMBASE, LILACS, and unpublished grey literature in OpenGrey up to February 2016. Reference lists of retrieved studies for full-text screening and reviews were hand-searched for potentially eligible studies.

Study Eligibility Criteria And Participants: Prospective, longitudinal observational studies with follow-up of at least 12 months and presenting data on the primary outcome, change in clinical attachment level, in adults (age ≥18 years). Secondary outcomes, tooth loss and bone level change, were only assessed in studies reporting the primary outcome. Studies investigating specific disease populations or only on treated periodontitis patients were excluded.

Study Appraisal And Synthesis Methods: Risk of bias and methodology were assessed using the Newcastle-Ottawa Scale with two additional questions on security of outcome assessment. Studies were pooled by abstracting or estimating mean annual attachment or bone level change and annual tooth loss. Random effects meta-analysis was conducted with investigation of effect of potential modifiers where possible.

Results: A total 11,482 records were screened for eligibility; 33 publications of 16 original studies reporting on more than 8,600 participants were finally included as eligible for the review. The studies represented populations from both developing and developed economies. Mean annual attachment loss was 0.1 mm per year (95% CI 0.068, 0.132; I = 99%) and mean annual tooth loss was 0.2 teeth per year (95% CI 0.10, 0.33; I = 94%). Observational analysis of highest and lowest mean attachment change quintiles suggested substantial differences between groups with minimal annual change in the lowest quintile and an average deterioration of 0.45 mm mean attachment loss per year in the highest group. This value increased to 0.6 mm per year with periodontitis alone. There was surprisingly little effect of age or gender on attachment level change. Geographic location, however, was associated with more than three times higher mean annual attachment loss in Sri Lanka and China (0.20 mm, 95% CI 0.15, 0.27; I  = 83%) vs North America and Europe (0.056 mm, 95% CI 0.025, 0.087; I  = 99%) P < 0.001.

Limitations: There were a limited number of studies (N = 16), high variability of design in key study components (sampling frames, included ages, data analyses), and high statistical heterogeneity that could not be explained.

Conclusions: Within the limitations of the research, the data show that mean annual attachment level change varies considerably both within and between populations. Overall, the evidence does not support or refute the differentiation between forms of periodontal diseases based upon progression of attachment level change.
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http://dx.doi.org/10.1002/JPER.17-0062DOI Listing
June 2018

Periodontitis: Consensus report of workgroup 2 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions.

J Periodontol 2018 06;89 Suppl 1:S173-S182

University of Hong Kong, Hong Kong, SAR China.

A new periodontitis classification scheme has been adopted, in which forms of the disease previously recognized as "chronic" or "aggressive" are now grouped under a single category ("periodontitis") and are further characterized based on a multi-dimensional staging and grading system. Staging is largely dependent upon the severity of disease at presentation as well as on the complexity of disease management, while grading provides supplemental information about biological features of the disease including a history-based analysis of the rate of periodontitis progression; assessment of the risk for further progression; analysis of possible poor outcomes of treatment; and assessment of the risk that the disease or its treatment may negatively affect the general health of the patient. Necrotizing periodontal diseases, whose characteristic clinical phenotype includes typical features (papilla necrosis, bleeding, and pain) and are associated with host immune response impairments, remain a distinct periodontitis category. Endodontic-periodontal lesions, defined by a pathological communication between the pulpal and periodontal tissues at a given tooth, occur in either an acute or a chronic form, and are classified according to signs and symptoms that have direct impact on their prognosis and treatment. Periodontal abscesses are defined as acute lesions characterized by localized accumulation of pus within the gingival wall of the periodontal pocket/sulcus, rapid tissue destruction and are associated with risk for systemic dissemination.
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http://dx.doi.org/10.1002/JPER.17-0721DOI Listing
June 2018

Periodontitis: Consensus report of workgroup 2 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions.

J Clin Periodontol 2018 06;45 Suppl 20:S162-S170

University of Hong Kong, Hong Kong, SAR China.

A new periodontitis classification scheme has been adopted, in which forms of the disease previously recognized as "chronic" or "aggressive" are now grouped under a single category ("periodontitis") and are further characterized based on a multi-dimensional staging and grading system. Staging is largely dependent upon the severity of disease at presentation as well as on the complexity of disease management, while grading provides supplemental information about biological features of the disease including a history-based analysis of the rate of periodontitis progression; assessment of the risk for further progression; analysis of possible poor outcomes of treatment; and assessment of the risk that the disease or its treatment may negatively affect the general health of the patient. Necrotizing periodontal diseases, whose characteristic clinical phenotype includes typical features (papilla necrosis, bleeding, and pain) and are associated with host immune response impairments, remain a distinct periodontitis category. Endodontic-periodontal lesions, defined by a pathological communication between the pulpal and periodontal tissues at a given tooth, occur in either an acute or a chronic form, and are classified according to signs and symptoms that have direct impact on their prognosis and treatment. Periodontal abscesses are defined as acute lesions characterized by localized accumulation of pus within the gingival wall of the periodontal pocket/sulcus, rapid tissue destruction and are associated with risk for systemic dissemination.
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http://dx.doi.org/10.1111/jcpe.12946DOI Listing
June 2018

Mean annual attachment, bone level, and tooth loss: A systematic review.

J Clin Periodontol 2018 06;45 Suppl 20:S112-S129

Biostatistics Unit, University College London Eastman Dental Institute, London, UK.

Background: Rate of progression of periodontitis has been used to inform the design of classifications of periodontal diseases. However, the evidence underpinning this topic is unclear and no systematic review has yet been conducted.

Objectives: The focused question for this systematic review was: in adults, what is the progression of periodontitis in terms of clinical attachment loss, radiographic bone loss, and tooth loss?

Data Sources: Highly sensitive electronic search was conducted for published data in MEDLINE, EMBASE, LILACS, and unpublished grey literature in OpenGrey up to February 2016. Reference lists of retrieved studies for full-text screening and reviews were hand-searched for potentially eligible studies.

Study Eligibility Criteria And Participants: Prospective, longitudinal observational studies with follow-up of at least 12 months and presenting data on the primary outcome, change in clinical attachment level, in adults (age ≥18 years). Secondary outcomes, tooth loss and bone level change, were only assessed in studies reporting the primary outcome. Studies investigating specific disease populations or only on treated periodontitis patients were excluded.

Study Appraisal And Synthesis Methods: Risk of bias and methodology were assessed using the Newcastle-Ottawa Scale with two additional questions on security of outcome assessment. Studies were pooled by abstracting or estimating mean annual attachment or bone level change and annual tooth loss. Random effects meta-analysis was conducted with investigation of effect of potential modifiers where possible.

Results: A total 11,482 records were screened for eligibility; 33 publications of 16 original studies reporting on more than 8,600 participants were finally included as eligible for the review. The studies represented populations from both developing and developed economies. Mean annual attachment loss was 0.1 mm per year (95% CI 0.068, 0.132; I = 99%) and mean annual tooth loss was 0.2 teeth per year (95% CI 0.10, 0.33; I = 94%). Observational analysis of highest and lowest mean attachment change quintiles suggested substantial differences between groups with minimal annual change in the lowest quintile and an average deterioration of 0.45 mm mean attachment loss per year in the highest group. This value increased to 0.6 mm per year with periodontitis alone. There was surprisingly little effect of age or gender on attachment level change. Geographic location, however, was associated with more than three times higher mean annual attachment loss in Sri Lanka and China (0.20 mm, 95% CI 0.15, 0.27; I  = 83%) vs North America and Europe (0.056 mm, 95% CI 0.025, 0.087; I  = 99%) P < 0.001.

Limitations: There were a limited number of studies (N = 16), high variability of design in key study components (sampling frames, included ages, data analyses), and high statistical heterogeneity that could not be explained.

Conclusions: Within the limitations of the research, the data show that mean annual attachment level change varies considerably both within and between populations. Overall, the evidence does not support or refute the differentiation between forms of periodontal diseases based upon progression of attachment level change.
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http://dx.doi.org/10.1111/jcpe.12943DOI Listing
June 2018

Hematopoietic Stem Cells as a Novel Source of Dental Tissue Cells.

Sci Rep 2018 05 23;8(1):8026. Epub 2018 May 23.

Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC, 29425, USA.

While earlier studies have suggested that cells positive for hematopoietic markers can be found in dental tissues, it has yet to be confirmed. To conclusively demonstrate this, we utilized a unique transgenic model in which all hematopoietic cells are green fluorescent protein (GFP). Pulp, periodontal ligament (PDL) and alveolar bone (AvB) cell culture analysis demonstrated numerous GFP cells, which were also CD45 (indicating hematopoietic origin) and co-expressed markers of cellular populations in pulp (dentin matrix protein-1, dentin sialophosphoprotein, alpha smooth muscle actin [ASMA], osteocalcin), in PDL (periostin, ASMA, vimentin, osteocalcin) and in AvB (Runx-2, bone sialoprotein, alkaline phosphatase, osteocalcin). Transplantation of clonal population derived from a single GFP hematopoietic stem cell (HSC), into lethally irradiated recipient mice, demonstrated numerous GFP cells within dental tissues of recipient mice, which also stained for markers of cell populations in pulp, PDL and AvB (used above), indicating that transplanted HSCs can differentiate into cells in dental tissues. These hematopoietic-derived cells deposited collagen and can differentiate in osteogenic media, indicating that they are functional. Thus, our studies demonstrate, for the first time, that cells in pulp, PDL and AvB can have a hematopoietic origin, thereby opening new avenues of therapy for dental diseases and injuries.
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http://dx.doi.org/10.1038/s41598-018-26258-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5966408PMC
May 2018

Should Dental Schools Invest in Training Predoctoral Students for Academic Careers? Two Viewpoints: Viewpoint 1: Dental Schools Should Add Academic Careers Training to Their Predoctoral Curricula to Enhance Faculty Recruitment and Viewpoint 2: Addition of Academic Careers Training for All Predoctoral Students Would Be Inefficient and Ineffective.

J Dent Educ 2018 Apr;82(4):379-387

Dr. Fung is Associate Professor and Assistant Dean for Curriculum Integration, Western University of Health Sciences College of Dental Medicine; Dr. Fatahzadeh is Professor of Oral Medicine, Rutgers School of Dental Medicine; Dr. Kirkwood is Professor, Department of Oral Biology, State University of New York at Buffalo; Dr. Hicks is Professor, University of Texas Health Science Center at San Antonio School of Dentistry; and Dr. Timmons is Associate Professor and Program Director, Department of Oral Pathology, Radiology, and Medicine, University of Iowa College of Dentistry & Dental Clinics.

This Point/Counterpoint considers whether providing dental students with academic career training and teaching experiences during their predoctoral education would be valuable to recruit dental academicians. While training the next generation of dentists continues to be the primary focus for dental schools, the cultivation and recruitment of dental faculty members from the pool of dental students remain challenges. Viewpoint 1 supports the position that providing dental students with exposure to academic career opportunities has positive value in recruiting new dental faculty. The advantages of academic careers training as a required educational experience in dental schools and as a potential means to recruit dental students into the ranks of faculty are described in this viewpoint. In contrast, Viewpoint 2 contends that such career exposure has limited value and argues that, across the board, allocation of resources to support preparation for academic careers would have a poor cost-benefit return on investment. Adding a requirement for educational experiences for all students would overburden institutions, students, and faculty according to this viewpoint. The authors agree that research is needed to determine how and where to make predoctoral curricular changes that will have maximum impact on academic recruitment.
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http://dx.doi.org/10.21815/JDE.018.035DOI Listing
April 2018

Commensal Gut Microbiota Immunomodulatory Actions in Bone Marrow and Liver have Catabolic Effects on Skeletal Homeostasis in Health.

Sci Rep 2017 07 18;7(1):5747. Epub 2017 Jul 18.

Department of Oral Health Sciences and Center for Oral Health Research, College of Dental Medicine, Medical University of South Carolina, Charleston, South Carolina, 29425, USA.

Despite knowledge the gut microbiota regulates bone mass, mechanisms governing the normal gut microbiota's osteoimmunomodulatory effects on skeletal remodeling and homeostasis are unclear in the healthy adult skeleton. Young adult specific-pathogen-free and germ-free mice were used to delineate the commensal microbiota's immunoregulatory effects on osteoblastogenesis, osteoclastogenesis, marrow T-cell hematopoiesis, and extra-skeletal endocrine organ function. We report the commensal microbiota has anti-anabolic effects suppressing osteoblastogenesis and pro-catabolic effects enhancing osteoclastogenesis, which drive bone loss in health. Suppression of Sp7(Osterix) and Igf1 in bone, and serum IGF1, in specific-pathogen-free mice suggest the commensal microbiota's anti-osteoblastic actions are mediated via local disruption of IGF1-signaling. Differences in the RANKL/OPG Axis in vivo, and RANKL-induced maturation of osteoclast-precursors in vitro, indicate the commensal microbiota induces sustained changes in RANKL-mediated osteoclastogenesis. Candidate mechanisms mediating commensal microbiota's pro-osteoclastic actions include altered marrow effector CD4T-cells and a novel Gut-Liver-Bone Axis. The previously unidentified Gut-Liver-Bone Axis intriguingly implies the normal gut microbiota's osteoimmunomodulatory actions are partly mediated via immunostimulatory effects in the liver. The molecular underpinnings defining commensal gut microbiota immunomodulatory actions on physiologic bone remodeling are highly relevant in advancing the understanding of normal osteoimmunological processes, having implications for the prevention of skeletal deterioration in health and disease.
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http://dx.doi.org/10.1038/s41598-017-06126-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5515851PMC
July 2017

Mitogen-Activated Protein Kinase 2 Signaling Shapes Macrophage Plasticity in Aggregatibacter actinomycetemcomitans-Induced Bone Loss.

Infect Immun 2017 Jan 29;85(1). Epub 2016 Dec 29.

Department of Oral Health Sciences and the Center for Oral Health Research, Medical University of South Carolina, Charleston, South Carolina, USA

Aggregatibacter actinomycetemcomitans is associated with aggressive periodontal disease, which is characterized by inflammation-driven alveolar bone loss. A. actinomycetemcomitans activates the p38 mitogen-activated protein kinase (MAPK) and MAPK-activated protein kinase 2 (MK2) stress pathways in macrophages that are involved in host responses. During the inflammatory process in periodontal disease, chemokines are upregulated to promote recruitment of inflammatory cells. The objective of this study was to determine the role of MK2 signaling in chemokine regulation during A. actinomycetemcomitans pathogenesis. Utilizing a murine calvarial model, Mk2 and Mk2 mice were treated with live A. actinomycetemcomitans bacteria at the midsagittal suture. MK2 positively regulated the following macrophage RNA: Emr1 (F4/80), Itgam (CD11b), Csf1r (M-CSF Receptor), Itgal (CD11a), Tnf, and Nos2 Additionally, RNA analysis revealed that MK2 signaling regulated chemokines CCL3 and CCL4 in murine calvarial tissue. Utilizing the chimeric murine air pouch model, MK2 signaling differentially regulated CCL3 and CCL4 in the hematopoietic and nonhematopoietic compartments. Bone resorption pits in calvaria, observed by micro-computed tomography, and osteoclast formation were decreased in Mk2 mice compared to Mk2 mice after A. actinomycetemcomitans treatment. In conclusion, these data suggest that MK2 in macrophages contributes to regulation of chemokine signaling during A. actinomycetemcomitans-induced inflammation and bone loss.
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http://dx.doi.org/10.1128/IAI.00552-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5203644PMC
January 2017

Sexual Dimorphism in MAPK-Activated Protein Kinase-2 (MK2) Regulation of RANKL-Induced Osteoclastogenesis in Osteoclast Progenitor Subpopulations.

PLoS One 2015 6;10(5):e0125387. Epub 2015 May 6.

Department of Oral Health Sciences and the Center for Oral Health Research, Medical University of South Carolina, Charleston, South Carolina, United States of America.

Osteoclasts (OCs) are bone-resorptive cells critical for maintaining skeletal integrity through coupled bone turnover. OC differentiation and activation requires receptor activator of NF-kB ligand (RANKL) signaling through the p38 MAPK pathway. However the role of the p38 MAPK substrate, MAPK-activated protein kinase 2 (MK2), is not clearly delineated. Within the bone marrow exists a specific subpopulation of defined osteoclast progenitor cells (dOCPs) with surface expression of B220(-)Gr1(-)CD11b(lo/-)CD115(+) (dOCP(lo/-)). In this study, we isolated dOCPs from male and female mice to determine sex-specific effects of MK2 signaling in osteoclastogenesis (OCgen). Male Mk2(-/-) mice display an increase in the dOCP(lo) cell population when compared to Mk2(+/+) mice, while female Mk2(-/-) and Mk2(+/+ )mice exhibit no difference. Defined OCPs from male and female Mk2(+/+) and Mk2(-/-) bone marrow were treated with macrophage colony stimulation factor (M-CSF) and RANKL cytokines to promote OCgen. RANKL treatment of dOCP(lo) cells stimulated p38 and MK2 phosphorylation. Tartrate-resistant acid phosphatase (TRAP) assays were used to quantify OC number, size, and TRAP enzyme activity post-RANKL stimulation. MK2 signaling was critical for male dOCP(lo) OCgen, yet MK2 signaling regulated OCgen from female dOCP- and CD11b(hi) subpopulations as well. The functional gene, Ctsk, was attenuated in both male and female Mk2(-/-) dOCP(lo)-derived OCs. Conversely, MK2 signaling was only critical for gene expression of pre-OC fusion genes, Oc-stamp andTm7sf4, in male OCgen. Therefore, these data suggest there is a sexual dimorphism in MK2 signaling of OCP subpopulations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0125387PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4422514PMC
April 2016

DUSP1 phosphatase regulates the proinflammatory milieu in head and neck squamous cell carcinoma.

Cancer Res 2014 Dec 13;74(24):7191-7. Epub 2014 Oct 13.

Department of Oral Health Sciences and Center for Oral Health Research, Medical University of South Carolina (MUSC), Charleston, South Carolina.

DUSP1 is a dual-specificity phosphatase that regulates mitogen-activated protein (MAP) kinase activity. Studies have associated loss of DUSP1 expression with certain cancers, but there has been no report of a mechanism by which this supports tumor progression. In this study, we found DUSP1 mRNA and protein decreased in human head and neck squamous cell carcinoma tissues compared with adjacent nontumor controls. To evaluate the impact of this difference, we compared the susceptibility of Dusp1-deficient mice with oral squamous carcinogenesis induced by 4-nitroquinoline 1-oxide. Dusp1-deficient mice displayed enhanced disease progression, characterized by advanced onset, histologic stage, and tumor burden. In a syngeneic model of tumor progression, subcutaneous injection of EO771 cells formed faster-growing tumors in Dusp1-deficient mice, an effect abrogated by inhibition of p38 MAP kinase with SB203580. Histologic and quantitative assessments demonstrated increased inflammation and deregulated chemokine and cytokine expression in Dusp1-deficient tumor tissues. Specifically, proinflammatory cytokine IL1β was elevated. IL1β production was recapitulated ex vivo in primary bone marrow-derived macrophages from Dusp1-deficient mice. Together, our results clearly establish the role of Dusp1 as a tumor suppressor gene that regulates cancer-associated inflammation.
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http://dx.doi.org/10.1158/0008-5472.CAN-14-1379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4268021PMC
December 2014

Critical role of MKP-1 in lipopolysaccharide-induced osteoclast formation through CXCL1 and CXCL2.

Cytokine 2015 Jan 27;71(1):71-80. Epub 2014 Sep 27.

Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC, USA. Electronic address:

Unlabelled: Osteoclast (OC) progenitors (OCP) have been defined in the bone marrow (BM) as CD3(-)CD45R(B220)(-)GR1(-)CD11b(lo/)(-)CD115(+) (dOCP) and more recently in the peripheral blood (PB) as Lym(-)Ly6G(-)CD11b(+)Ly6C(+). These progenitors respond to stimuli, including LPS from periopathogenic Aggregatibacter actinomycetemcomitans, activating MAPK signaling, resulting in cytokine/chemokine-mediated osteoclastogenesis. Intracellular negative signaling pathways, including MAPK phosphatase-1 (MKP-1, gene Dusp1) deactivate MAPK pathways (p-p38 and p-JNK) and reduce inflammatory cytokines/chemokines.

Objective: To delineate the role of MKP-1 in chemokine-mediated OC formation using defined OC progenitor populations. Given its role in innate immune inflammatory signaling, we hypothesize that MKP-1 regulates LPS-induced OC formation from BM OCP through deregulated chemokines.

Methods: BM and PB from WT and Dusp1(-/-) female mice (8-12weeks) was obtained and sorted into defined progenitor populations. BM sorted dOCP were primed with MCSF and RANKL (48h), blocked with vehicle or chemokine blocking antibodies and stimulated with LPS (48-96h). TRAP assay and OC activity were measured for OC formation and activity following treatments. NanoString Array and qPCR were utilized for gene expression analysis.

Results: Dusp1(-/-) dOCPs formed more and larger osteoclasts from CD11b(hi) and dOCP compared to matched WT (P<0.05 each). PB-derived dOCP produced larger and more functional osteoclasts from Dusp1(-/-) mice compared to WT controls. NanoString array data revealed significant deregulation in chemokine expression from Dusp1(-/-) versus WT cells. qPCR validation of target genes revealed that Dusp1 deficient CD11b(+) populations display 1.5-3.5-fold greater expression of CXCL1 and 2-3-fold greater expression of CXCL2 compared to WT in CD11b(hi) and dOCP (P<0.05 each). Antibody blocking studies using anti-CXCL1 and CXCL2 antibodies blunted osteoclastogenesis in Dusp1(-/-) cells.

Conclusion: MKP-1 negatively regulates chemokine-driven OC formation and subsequent bone resorption in response to LPS stimulation. Collectively, these data provide useful insight into mechanisms potentially leading to the development of therapeutic treatment of periodontal disease.
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http://dx.doi.org/10.1016/j.cyto.2014.08.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4254057PMC
January 2015

The G protein-coupled receptor GALR2 promotes angiogenesis in head and neck cancer.

Mol Cancer Ther 2014 May 25;13(5):1323-33. Epub 2014 Feb 25.

Authors' Affiliations: Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry; Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan; Department of Craniofacial Biology, The Medical University of South Carolina, Columbia, South Carolina.

Squamous cell carcinoma of the head and neck (SCCHN) is an aggressive disease with poor patient survival. Galanin receptor 2 (GALR2) is a G protein-coupled receptor that induces aggressive tumor growth in SCCHN. The objective of this study was to investigate the mechanism by which GALR2 promotes angiogenesis, a critical oncogenic phenotype required for tumor growth. The impact of GALR2 expression on secretion of proangiogenic cytokines in multiple SCCHN cell lines was investigated by ELISA and in vitro angiogenesis assays. Chemical inhibitor and genetic knockdown strategies were used to understand the key regulators. The in vivo impact of GALR2 on angiogenesis was investigated in mouse xenograft, chick chorioallantoic membrane, and the clinically relevant mouse orthotopic floor-of-mouth models. GALR2 induced angiogenesis via p38-MAPK-mediated secretion of proangiogenic cytokines, VEGF, and interleukin-6 (IL-6). Moreover, GALR2 activated small-GTP-protein, RAP1B, thereby inducing p38-mediated inactivation of tristetraprolin (TTP), which functions to destabilize cytokine transcripts. This resulted in enhanced secretion of proangiogenic cytokines and angiogenesis in vitro and in vivo. In SCCHN cells overexpressing GALR2, inactivation of TTP increased secretion of IL-6 and VEGF, whereas inhibition of p38 activated TTP and decreased cytokine secretion. Here, we report that GALR2 stimulates tumor angiogenesis in SCCHN via p38-mediated inhibition of TTP with resultant enhanced cytokine secretion. Given that p38 inhibitors are in clinical use for inflammatory disorders, GALR2/p38-mediated cytokine secretion may be an excellent target for new adjuvant therapy in SCCHN.
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http://dx.doi.org/10.1158/1535-7163.MCT-13-0904DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4023835PMC
May 2014

MKP-1 signaling events are required for early osteoclastogenesis in lineage defined progenitor populations by disrupting RANKL-induced NFATc1 nuclear translocation.

Bone 2014 Mar 20;60:16-25. Epub 2013 Nov 20.

Department of Craniofacial Biology, Center for Oral Health Research, Medical University of South Carolina, Charleston, SC 29425, USA; Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC 29425, USA. Electronic address:

Cytokine-directed osteoclastogenesis is initiated in response to macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) to drive formation of osteoclasts (OC), large bone resorptive cells of hematopoietic origin. RANKL-induced signaling activates the MAPK pathways, which initiates nuclear translocation of the master regulator of osteoclast formation, transcription factor NFATc1. Proper control over these signaling events is essential to normal OC formation response to stimuli. MAPK phosphatase 1 (MKP-1), a serine and tyrosine phosphatase encoded by the gene Dusp1, functions to dephosphorylate and subsequently inactivate MAPK (p38 and JNK) signaling essential in osteoclastogenesis. Here, we explored the role of MKP-1 during RANKL-driven osteoclastogenesis from defined (B220/CD45(-)GR1(-)CD11b(lo/-)CD115(+)) OC progenitor (dOCP) populations using WT and Dusp1(-/-) global knockout mice. Sorted cells were driven to OC by M-CSF pre-treatment followed by RANKL stimulation for 3days. OC formation and qPCR products were analyzed for maturation. Results indicate that Dusp1(-/-) dOCP form less numerous, significantly smaller and less functional OC compared to WT controls. These data were corroborated by mRNA expression of the key OC genes, Nfatc1 and Tm7sf4 (DC-STAMP), which were significantly reduced in early osteoclastogenesis in OC progenitor from Dusp1(-/-) mice. Intriguingly, our data reveals that MKP-1 may positively control OC formation in response to RANKL by regulating NFATc1 nuclear translocation. Collectively, this report supports the idea that MKP-1 signaling is essential in early osteoclastogenesis in response to RANKL-induced signaling.
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http://dx.doi.org/10.1016/j.bone.2013.11.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3945035PMC
March 2014

Differential expression of mitogen activating protein kinases in periodontitis.

J Clin Periodontol 2013 Aug 7;40(8):757-64. Epub 2013 Jun 7.

Department of Periodontics and Oral Medicine, School of Dentistry, University of Michigan, Ann Arbor, MI, USA.

Aim: Following toll-like receptor (TLR) engagement, lipopolysaccharide (LPS) can stimulate the expression of pro-inflammatory cytokines thus activating the innate immune response. The production of inflammatory cytokines results, in part, from the activation of kinase-induced signalling cascades and transcriptional factors. Of the four distinct classes of mitogen-activated protein kinases (MAPK) described in mammals, p38, c-Jun N-terminal activated kinases (JNK1-3) and extracellular activated kinases (ERK1,2) are the best studied. Previous data have established that p38 MAPK signalling is required for inflammation and bone loss in periodontal disease pre-clinical animal models.

Materials & Methods: In this study, we obtained healthy and diseased periodontal tissues along with clinical parameters and microbiological parameters. Excised fixed tissues were immunostained with total and phospho-specific antibodies against p38, JNK and ERK kinases.

Results: Intensity scoring from immunostained tissues was correlated with clinical periodontal parameters. Rank correlations with clinical indices were statistically significantly positive (p-value < 0.05) for total p38 (correlations ranging 0.49-0.68), phospho-p38 (range 0.44-0.56), and total ERK (range 0.52-0.59) levels, and correlations with JNK levels also supported association (range 0.42-0.59). Phospho-JNK and phospho-ERK showed no significant positive correlation with clinical parameters of disease.

Conclusion: These data strongly implicate p38 MAPK as a major MAPK involved in human periodontal inflammation and severity.
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http://dx.doi.org/10.1111/jcpe.12123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4091899PMC
August 2013

Inactivation or loss of TTP promotes invasion in head and neck cancer via transcript stabilization and secretion of MMP9, MMP2, and IL-6.

Clin Cancer Res 2013 Mar 24;19(5):1169-79. Epub 2013 Jan 24.

Periodontics and Oral Medicine, Oral Maxillofacial Surgery, University of Michigan School of Dentistry, Ann Arbor, Michigan 48109, USA.

Purpose: Invasion is the critical step in progression of a precancerous lesion to squamous cell carcinoma of the head and neck (HNSCC). Invasion is regulated by multiple proinflammatory mediators. Tristetraprolin (TTP) is an mRNA-degrading protein that regulates multiple proinflammatory mediators. TTP may serve as an excellent treatment target. Rap1 is a ras-like oncoprotein that induces critical signaling pathways. In this study, the role of rap1 in TTP-mediated invasion was investigated.

Experimental Design: Using complementary approaches, we modulated TTP and altered expression of interleukin (IL)-6 and matrix metalloproteinase (MMP) 2/9, which were quantified by ELISA and zymogram. Invasion was evaluated in vitro using the oral-cancer-equivalent (OCE) three-dimensional model and in vivo in the chick chorioallantoic membrane (CAM). The role of rap1 and p38 were established using knockdown strategies.

Results: Downregulation of TTP significantly increased invasion via secretion of MMP9/2 and IL-6. In the novel OCE and CAM invasion models of HNSCC, cells with downregulated TTP destroyed the basement membrane to invade the underlying connective tissue. Rap1 induces p38 mitogen-activated protein kinase (p38)-mediated inactivation of TTP. Inactive TTP enhances transcript stability via binding to the 3'-untranslated region (UTR). High IL-6 and MMP9 are prognostic for poor clinical outcomes in patients with HNSCC.

Conclusions: Targeting the rap1-p38-TTP cascade is an attractive novel treatment strategy in HNSCC to concurrently suppress multiple mediators of invasion.
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http://dx.doi.org/10.1158/1078-0432.CCR-12-2927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3594656PMC
March 2013

Loss of expression and function of SOCS3 is an early event in HNSCC: altered subcellular localization as a possible mechanism involved in proliferation, migration and invasion.

PLoS One 2012 18;7(9):e45197. Epub 2012 Sep 18.

Department of Diagnosis and Surgery, School of Dentistry at Araraquara, Universidade Estadual Paulista (UNESP), Araraquara, São Paulo, Brazil.

Background: Suppressor of cytokine signaling 3 (SOCS3) is an inducible endogenous negative regulator of signal transduction and activator of transcription 3 (STAT3). Epigenetic silencing of SOCS3 has been shown in head and neck squamous cell carcinoma (HNSCC), which is associated with increased activation of STAT3. There is scarce information on the functional role of the reduction of SOCS3 expression and no information on altered subcellular localization of SOCS3 in HNSCC.

Methodology/principal Findings: We assessed endogenous SOCS3 expression in different HNSCC cell lines by RT-qPCR and western blot. Immunofluorescence and western blot were used to study the subcellular localization of endogenous SOCS3 induced by IL-6. Overexpression of SOCS3 by CMV-driven plasmids and siRNA-mediated inhibition of endogenous SOCS3 were used to verify the role of SOCS3 on tumor cell proliferation, viability, invasion and migration in vitro. In vivo relevance of SOCS3 expression in HNSCC was studied by quantitative immunohistochemistry of commercially-available tissue microarrays. Endogenous expression of SOCS3 was heterogeneous in four HNSCC cell lines and surprisingly preserved in most of these cell lines. Subcellular localization of endogenous SOCS3 in the HNSCC cell lines was predominantly nuclear as opposed to cytoplasmic in non-neoplasic epithelial cells. Overexpression of SOCS3 produced a relative increase of the protein in the cytoplasmic compartment and significantly inhibited proliferation, migration and invasion, whereas inhibition of endogenous nuclear SOCS3 did not affect these events. Analysis of tissue microarrays indicated that loss of SOCS3 is an early event in HNSCC and was correlated with tumor size and histological grade of dysplasia, but a considerable proportion of cases presented detectable expression of SOCS3.

Conclusion: Our data support a role for SOCS3 as a tumor suppressor gene in HNSCC with relevance on proliferation and invasion processes and suggests that abnormal subcellular localization impairs SOCS3 function in HNSCC cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0045197PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3445460PMC
February 2013

Oral squamous carcinoma cells secrete RANKL directly supporting osteolytic bone loss.

Oral Oncol 2013 Feb 16;49(2):119-28. Epub 2012 Sep 16.

Department of Craniofacial Biology, Center for Oral Health Research Medical University of South Carolina, Charleston, SC, USA.

Objective: Local invasion of bone is a frequent complication of oral squamous cell carcinoma (OSCC). Development of these osteolytic lesions is mediated by osteoclasts. Receptor activation of NF-κB ligand (RANKL) signaling, counteracted by osteoprotegerin (OPG), regulates osteoclastogenesis. Previous studies in rodent models have demonstrated that inhibition of RANKL decreases tumor growth and lesions within bone. However, the contributory role of OSCC cells to this disease process has yet to be defined.

Methods: RANKL expression was assessed in a panel of OSCC cell lines by qPCR, flow cytometry, and ELISA. Induction of osteoclastogenesis was assessed by co-culture with macrophages or with OSCC-derived conditioned medium. In an animal model of bone invasion, nude mice were injected intratibially with UMSCC-11B cells expressing a RANKL luciferase promoter to detect tumor-derived RANKL activity. Osteolytic lesions were analyzed by X-ray, micro-CT, and histological methods. RANKL expression was assessed in human OSCC tissues by immunohistochemistry.

Results: We demonstrated that OSCCs express varied levels of all RANKL isoforms, both membrane-bound and soluble RANKL. Both co-culture and treatment with OSCC-conditioned media induced osteoclastogenesis. In mice, we demonstrated human RANKL promoter activity during bone invasion. Over the course of the experiment, animals suffered osteolytic lesions as RANKL-driven luciferase expression increased with time. After 8weeks, human-derived RANKL was detected in areas of bone resorption by immunohistochemistry. Similar epithelial RANKL expression was detected in human OSCC tissues.

Conclusion: These data demonstrate the ability of OSCCs to produce RANKL, directly altering the tumor microenvironment to increase osteoclastogenesis and mediate local bone invasion.
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http://dx.doi.org/10.1016/j.oraloncology.2012.08.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3527639PMC
February 2013

MKP-1 is essential for canonical vitamin D-induced signaling through nuclear import and regulates RANKL expression and function.

Mol Endocrinol 2012 Oct 16;26(10):1682-93. Epub 2012 Aug 16.

Department of Craniofacial Biology, Medical University of South Carolina, Charleston, 173 Ashley Avenue, BSB 449, Charleston, South Carolina 29425, USA.

Vitamin D(3,) and its most active form, 1,25(OH)(2)D(3), are well known to stimulate osteoclastogenesis through stromal cell induction of the receptor activator of nuclear factor-κB ligand (RANKL). MAPK phosphatase-1 (MKP-1) is a phosphatase classically known to negatively regulate the innate immune response through dephosphorylation of p38, ERK, and c-Jun N-terminal kinase activity. This paper describes a new function of MKP-1 in permitting genomic 1,25(OH)(2)D(3) signaling and downstream osteoclastogenesis through RANKL. Initially, quantitative RT-PCR (qRT-PCR) and immunoblot analysis comparing bone marrow stromal cells (BMSC) revealed that 1,25(OH)(2)D(3)-induced vitamin D receptor (VDR), cytochrome P 45024a1, and RANKL mRNA expression and protein were significantly attenuated or absent in MKP-1(-/-) BMSC. Immunoblot analysis from cellular fractions of wild type and MKP-1(-/-) BMSC stimulated with 10(-7) m 1,25(OH)(2)D(3) revealed retinoid X receptor (RXR)α nuclear import was impaired in MKP-1(-/-) BMSC, whereas VDR import was not. Proximity ligation assays revealed that baseline VDR-RXRα heterodimer translocation was unchanged, yet 1,25(OH)(2)D(3)-induced nuclear translocation of VDR-RXRα heterodimers was reduced in MKP-1(-/-) BMSC. A functional consequence was observed as BMSC from MKP-1(-/-) mice treated with 1,25(OH)(2)D(3) and cocultured with RAW 264.7 cells had a 91% decrease in osteoclastogenesis and a 94.5% decrease in mineralized matrix resorption compared with wild-type cocultures (P < 0.01). These results reveal an unexpected, permissive role for MKP-1 in canonical 1,25(OH)(2)D(3) signaling via VDR-RXRα heterodimer nuclear import and downstream osteoclastogenesis through stromal cell RANKL expression.
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http://dx.doi.org/10.1210/me.2012-1033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3458222PMC
October 2012

Sexual dimorphism in periapical inflammation and bone loss from mitogen-activated protein kinase phosphatase-1 deficient mice.

J Endod 2012 Aug 8;38(8):1097-100. Epub 2012 Jun 8.

Department of Oral Rehabilitation, Division of Endodontics, Medical University of South Carolina, 173 Ashley Ave., Charleston, SC 29425, USA.

Introduction: Mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) has been shown to be a key negative regulator of the MAPK pathways of the innate immune system. The impact of MKP-1 in an endodontic model has yet to be studied. Thus, the purpose of this study was to determine the role of MKP-1 in a bacterial-driven model of pathologic endodontic bone loss.

Methods: Pulps were exposed in both lower first molars of 10-week-old mkp-1(+/+) and mkp-1(-/-) mice and left open to the oral environment for either 3 or 8 weeks. At death, mandibles were harvested and scanned by micro-computed tomography (μCT) to determine periapical bone loss. Histopathologic scoring was then performed on the samples to determine the amount of inflammatory infiltrate within the periapical microenvironment.

Results: Significant bone loss and inflammatory infiltrate were found in all experimental groups when compared with control. No statistical difference was found between mkp-1(+/+) and mkp-1(-/-) at either time point with respect to bone loss or inflammatory infiltrate. At 8 weeks, male mkp-1(-/-) mice were found to have significantly more bone loss and inflammatory infiltrate when compared with female mkp-1(-/-) mice. There was also a significant correlation between an increase in bone loss and increase in inflammatory infiltrate.

Conclusions: A sexual dimorphism exists in the periapical inflammatory process, where male mkp-1(-/-) mice have more inflammation than female mkp-1(-/-) mice. The increase in inflammatory infiltrate correlates to more bone loss in the male mice.
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http://dx.doi.org/10.1016/j.joen.2012.04.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3399764PMC
August 2012

MAPK usage in periodontal disease progression.

J Signal Transduct 2012 23;2012:308943. Epub 2012 Jan 23.

Department of Endodontics, Periodontics and Oral Medicine, The First People's Hospital of Yunnan Province, Kunming, Yunnan 650032, China.

In periodontal disease, host recognition of bacterial constituents, including lipopolysaccharide (LPS), induces p38 MAPK activation and subsequent inflammatory cytokine expression, favoring osteoclastogenesis and increased net bone resorption in the local periodontal environment. In this paper, we discuss evidence that the p38/MAPK-activated protein kinase-2 (MK2) signaling axis is needed for periodontal disease progression: an orally administered p38α inhibitor reduced the progression of experimental periodontal bone loss by reducing inflammation and cytokine expression. Subsequently, the significance of p38 signaling was confirmed with RNA interference to attenuate MK2-reduced cytokine expression and LPS-induced alveolar bone loss. MAPK phosphatase-1 (MKP-1), a negative regulator of MAPK activation, was also critical for periodontal disease progression. In MPK-1-deficient mice, p38-sustained activation increased osteoclast formation and bone loss, whereas MKP-1 overexpression dampened p38 signaling and subsequent cytokine expression. Finally, overexpression of the p38/MK2 target RNA-binding tristetraprolin (TTP) decreased mRNA stability of key inflammatory cytokines at the posttranscriptional level, thereby protecting against periodontal inflammation. Collectively, these studies highlight the importance of p38 MAPK signaling in immune cytokine production and periodontal disease progression.
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http://dx.doi.org/10.1155/2012/308943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3270463PMC
August 2012

Kaposi sarcoma-associated herpesvirus (KSHV) induces a functional tumor-associated phenotype for oral fibroblasts.

Cancer Lett 2012 May 17;318(2):214-20. Epub 2011 Dec 17.

Department of Medicine, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425, United States.

The Kaposi sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi sarcoma (KS), the most common HIV/AIDS-associated tumor worldwide. Involvement of the oral cavity portends a poor prognosis for patients with KS, but mechanisms for KSHV regulation of the oral tumor microenvironment are largely unknown. Infiltrating fibroblasts are found with KS lesions, and KSHV establishes latent infection within human primary fibroblasts in vitro, but contributions for KSHV-infected fibroblasts to the KS microenvironment have not been previously characterized. Secretion of pro-migratory factors and intratumoral invasion are characteristics of tumor-associated fibroblasts (TAF) found in the microenvironment of non-viral malignancies. In the present study, we show that latent KSHV infection of primary human fibroblasts isolated from the oral cavity enhances their secretion of KS-promoting cytokines and intrinsic invasiveness through VEGF-dependent mechanisms. Moreover, we find that KSHV induces these effects through Sp1- and Egr2-dependent transcriptional activation of the Extracellular Matrix MetalloPRoteinase INducer (emmprin). These data implicate KSHV activation of emmprin in the induction of a "TAF-like" phenotype for oral fibroblasts in the KS microenvironment and support the potential utility of targeting TAFs and/or emmprin in the treatment of oral KS.
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http://dx.doi.org/10.1016/j.canlet.2011.12.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3303930PMC
May 2012

Is monocyte chemotactic protein 1 elevated in aseptic loosening of TKA? A pilot study.

Clin Orthop Relat Res 2012 Jul;470(7):1879-84

Department of Orthopaedics, Louisiana State University Health Sciences Center, 1542 Tulane Avenue, New Orleans, LA 70115, USA.

Background: Failure of TKA from aseptic loosening is a growing concern, as TKA is performed with increasing frequency. Loosening is multifactorial and may be associated with elevated inflammatory cytokines in addition to biomechanical failure.

Questions/purposes: We asked whether proinflammatory cytokines and chemokines are elevated in synovial fluid from patients undergoing revision surgery as compared to those with osteoarthritis (OA) or rheumatoid arthritis (RA).

Methods: We obtained synovial fluid samples from 20 patients: six with aseptic loosening of TKA (all with bone loss), 10 with primary OA, and four with RA. A panel of cytokines/chemokines was screened using a SearchLight(®) Array (Pierce Biotechnology, Rockford, IL, USA) in one revision sample. Using these data, we assayed the synovial fluids for monocyte chemotactic protein 1 (MCP-1) by ELISA.

Results: We observed an increase in synovial MCP-1 levels in samples from patients planned for TKA revision compared to those with OA or RA. In patients undergoing revision arthroplasty, the mean (± SD) MCP-1 concentration was 21,233 ± 18,966 pg/mL (range, 1550-50,657 pg/mL; n = 6). In patients with OA, the mean MCP-1 level was 3012 ± 3321 pg/mL. In patients with RA, the mean MCP-1 concentration was 690 ± 561 pg/mL.

Conclusions: All patients undergoing revision TKA showed elevated concentrations of MCP-1 compared to patients with OA and RA, suggesting MCP-1 may serve as a potential marker or predictor of bone loss in patients undergoing revision surgery.

Clinical Relevance: MCP-1 may be a novel biomarker in patients showing early symptoms of aseptic loosening of TKA.
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http://dx.doi.org/10.1007/s11999-011-2191-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3369079PMC
July 2012