Expression of Heat Shock Proteins in Periapical Granulomas

doi:10.1016/j.joen.2013.10.021

Journal of Endodontics

Volume 40, Issue 6, June 2014, Pages 830-836

https://doi.org/10.1016/j.joen.2013.10.021 Get rights and content

Abstract

Introduction

Cells from virtually all organisms respond to a variety of stresses by the rapid synthesis of a highly conserved set of polypeptides termed heat shock proteins (HSPs). HSPs protect cells under adverse conditions such as infection, inflammation, and disease. We hypothesize that endodontic infection might result in an imbalance in the expression of heat shock genes, accounting for different clinical outcomes in periapical lesions.

Methods

We analyzed the expression of 44 HSPs genes using a pathway-specific real-time polymerase chain reaction array in 93 human periapical granulomas and 24 healthy periodontal ligament tissues collected postoperatively. Observed variations in the expression of HSP genes were also analyzed based on the classification of periapical granulomas as active or inactive. In addition, U937 cells were differentiated into macrophages, infected with different concentrations of purified Escherichia coli lipopolysaccharide (LPS), and used as templates for the HSP gene array. Protein expression was assessed by immunohistochemistry.

Results

The expression of HSP genes was significantly increased in granulomas compared with healthy periodontal ligament (P < .00001). Among the 44 HSP genes, DNAJC3, HSPA4, HSPA6, and HSPB1 showed the highest expression levels in both granulomas and LPS-treated macrophages. DNAJC3, HSPA6, and HSPB1 were highly expressed in active lesions, whereas HSPA4 expression was higher in inactive lesions (P < .005). Higher concentrations of LPS led to increased HSP expression in macrophages (P < .0001). Immunocytochemistry confirmed the expression and colocalization of HSPB1 and HSPA6 proteins in the cytoplasm of LPS-infected macrophages.

Conclusions

The observed differential expression patterns of HSPs in periapical granulomas and LPS-infected macrophages suggest that HSP genes and proteins are involved in periapical lesion development and may account for different clinical outcomes. Understanding the role of the heat shock response might provide additional insights into the process of periapical lesion development.

Section snippets

Subjects and Samples

This study was approved by the Committee for Protection of Human Subjects at the University of Texas Health Science Center at Houston, Houston, TX. Subjects were patients (age, 15–57 years; average = 38.2 years) presenting with periapical lesions characterized radiographically as rarefaction lesions with the disappearance of the periodontal ligament space and discontinuity of the lamina dura; patients were referred for endodontic surgery when teeth failed to heal after conventional root canal

Gene Expression Analysis

The expression of all 44 HSP genes was significantly increased in the pool of periapical granulomas compared with the pool of healthy periodontal ligaments (P < .00001). DNAJC3, HSPA4, HSPA6, and HSPB1 showed the highest expression levels in the periapical granulomas (Fig. 2A). Similarly, the expression of HSP genes was significantly higher in LPS-treated macrophages in comparison with macrophages without LPS treatment (P < .0001), with DNAJC3, HSPA4, HSPA6, and HSPB1 being the most highly

Discussion

In this study, we hypothesized that HSPs could play a role in the development process of apical periodontitis and that macrophages could be a source of HSPs in this process. HSPs have numerous functions, including the facilitation of protein folding and antigen-presenting properties to the immune system 13, 14. Furthermore, extracellular HSPs can stimulate the release of TNF-α; IL-1β, -6 and -12; nitric oxide; and chemokines by monocytes/macrophages (15), which can contribute to the development

Acknowledgments

Supported in part by UTSD startup funds to R.M.S., AAE Foundation to S.C.G., Fundaçāo de Amparo a Pesquisa do Estado de Sāo Paulo (FAPESP) to G.P.G, and Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES), BEX 1099/12-4, Brazil to L.C.S.

The authors thank the individuals that agreed to participate in this study and Pasha Goodman for invaluable help in the critical review of the manuscript.

The authors deny any conflicts of interest related to this study.

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Cited by (20)

Heat shock proteins in infection
2019, Clinica Chimica Acta
Citation Excerpt :
Interestingly, members of the HSP70 family were highly expressed in both periapical granulomas and LPS-treated macrophages. A positive correlation between HspA6/HspA7 and DNAJC3 with IL-6 mRNA levels, DNAJC3, HspA6/HspA7 and HspB1 with IL-1b mRNA levels, and DNAJC3 and HspB1 with RANKL and TNF-α mRNA levels was observed for major role of HSPs as immune response activators in inducing the host immune defense against pathogens [15]. As known, innate immunity is the first line of defense against pathogens and is necessary for survival of the infected host.
Heat shock proteins (HSPs) are constitutively expressed under physiological conditions in most organisms but their expression can significantly enhance in response to four types of stimuli including physical (e.g., radiation or heat shock), chemical and microbial (e.g., pathogenic bacteria, viruses, parasites and fungi) stimuli, and also dietary. These proteins were identified for their role in protecting cells from high temperature and other forms of stress. HSPs control physiological activities or virulence through interaction with various regulators of cellular signaling pathways. Several roles were determined for HSPs in the immune system including intracellular roles (e.g., antigen presentation and expression of innate receptors) as well as extracellular roles (e.g., tumor immunosurveillance and autoimmunity). It was observed that exogenously administered HSPs induced various immune responses in immunotherapy of cancer, infectious diseases, and autoimmunity. Moreover, virus interaction with HSPs as molecular chaperones showed important roles in regulating viral infections including cell entry and nuclear import, viral replication and gene expression, folding/assembly of viral protein, apoptosis regulation, and host immunity. Viruses could regulate host HSPs at different levels such as transcription, translation, post-translational modification and cellular localization. In this review, we attempt to overview the roles of HSPs in a variety of infectious diseases.
Role of HSP70 protein in human periodontal ligament cell function and physiology
2019, Annals of Anatomy
Citation Excerpt :
Yoshimatsu et al. reported on an enhanced expression of HSP47, which is actively involved in the regulation of collagen I synthesis, in the tension zone during orthodontic tooth movement (Yoshimatsu et al., 2008). Finally, a differential expression pattern of HSPs has been described in different endodontic periapical lesions (Goodman et al., 2014). However, information on the function of HSP in hPDL cell physiology, also in conditions requiring its inhibition for medical reasons, is still missing.
Heat pre-treatment of mechanically loaded human periodontal ligament cells (hPDL) dampens the inflammatory cellular response, as evidenced by a reduced expression of pro-inflammatory cytokines, inhibition of monocyte adhesion and osteoclastic differentiation. These findings imply heat shock proteins (HSP) as cell protective molecules acting in the PDL that are up-regulated upon ischemia caused by mechanical loading. HSP70 and its inhibition by VER155008 as the active agent in several pharmaceuticals are established targets and strategies, respectively, in the treatment of neoproliferative diseases. However, the effect of both players on periodontal remodeling in unknown. Therefore, we analyzed the role of HSP70 and its frequently used inhibitor VER155008 in the regulation of physiological hPDL cell functions and immune cell interaction.
Fifth passage hPDL cells were cultured in the presence of 25 μm HSP70 inactivating agent VER155008. At harvest, HSP70 expression, cell proliferation, and parameters of cell interaction, colony formation and wound healing were analyzed by means of real-time PCR, immunohistochemistry, Western blot, biochemical MTS assay, microscopy, and functional assays for monocyte adhesion and differentiation.
Basal HSP70 expression and hPDL cell morphology were not affected by HSP70 inhibitor VER155008. In contrast, cell proliferation, tissue defect healing, and colony formation were reduced significantly following HSP70 inhibition, whereas apoptosis and necrosis, monocyte adhesion and osteoclastic differentiation were markedly increased.
The present data indicate a regulatory role for HSP70 protein in hPDL cell biology.
These findings identify HSP70 as a promising target in the attempt to modify periodontal remodeling and point to potential periodontal side effects of HSP70 pharmaceutical usage.
Proteomic Profiling and Differential Messenger RNA Expression Correlate HSP27 and Serpin Family B Member 1 to Apical Periodontitis Outcomes
2017, Journal of Endodontics
Citation Excerpt :
Furthermore, the observed overexpression of the HSP27 protein in periapical granulomas is in line with our previously published findings in which HSP27 mRNA was significantly up-regulated in periapical granulomas and in lipopolysaccharide-stimulated macrophages (15). HSP27 expression was significantly higher in inactive lesions, implicating a potential role for this molecule in regulating lesion progression (15). Nevertheless, additional mechanistic studies are needed to determine a functional role for HSP27 in the pathogenesis of apical periodontitis.
Understanding protein expression profiles of apical periodontitis may contribute to the discovery of novel diagnostic or therapeutic molecular targets.
Periapical tissue samples (n = 5) of patients with lesions characterized as nonhealing were submitted for proteomic analysis. Two differentially expressed proteins (heat shock protein 27 [HSP27] and serpin family B member 1 [SERPINB1]) were selected for characterization, localization by immunofluorescence, and association with known biomarkers of acute inflammatory response in human apical periodontitis (n = 110) and healthy periodontal ligaments (n = 26). Apical periodontitis samples were categorized as stable/inactive (n = 70) or progressive/active (n = 40) based on the ratio of expression of receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG). Next, the expression of HSP27, SERPINB1, C-X-C motif Chemokine Receptor 1 (CXCR1), matrix metalloproteinase 8 (MMP8), myeloperoxidase (MPO), and cathepsin G (CTSG) messenger RNA was evaluated using real-time polymerase chain reaction. Data analysis was performed using the Shapiro-Wilk test, analysis of variance, and the Pearson test. P values <.05 were considered statistically significant.
Proteomic analysis revealed 48 proteins as differentially expressed in apical periodontitis compared with a healthy periodontium, with 30 of these proteins found to be expressed in all 4 lesions. The expression of HSP27 and SERPINB1 was ∼2-fold higher in apical periodontitis. Next, an increased expression of HSP27 was detected in epithelial cells, whereas SERPINB1 expression was noted in neutrophils and epithelial cells. HSP27 and SERPINB1 transcripts were highly expressed in stable/inactive lesions (P < .05). Significant negative correlations were found between the expression of HSP27 and SERPINB1 with biomarkers of acute inflammation including CXCR1, MPO, and CTSG.
Our data suggest HSP27 and SERPINB1 as potential regulators of the inflammatory response in apical periodontitis. Additional functional studies should be performed to further characterize the role of these molecules during the development/progression of apical periodontitis.
Periapical Lesions Increase Macrophage Infiltration and Inflammatory Signaling in Muscle Tissue of Rats
2017, Journal of Endodontics
Our previous studies have shown that periapical lesions (PLs) in rats cause systemic disorders such as increased tumor necrosis factor-α plasma levels, insulin resistance, and impairment in insulin signal transduction in muscle tissue. However, the mechanisms involved in these alterations are not fully understood. Under chronic inflammatory conditions such as obesity, it has been shown that the skeletal muscle is affected by inflammation, and the number of resident macrophages that are associated with impairments of insulin action and sensitivity is increased. This study aimed to investigate the presence of macrophages, activation of inflammatory pathways in muscle tissue, glycemia, and insulinemia of rats with PLs.
Sixty Wistar rats were distributed into a control group; a group with 1 PL (1PL), which was induced in the right maxillary first molar; and a group with 4 PLs (4PL), which were induced in the right upper and lower first and second molars. We quantified macrophage content by immunohistochemistry for the F4/80 protein. We evaluated Jun N-terminal kinase and IKKα/β phosphorylation status in the muscle tissue by Western blotting. Serum levels of lipopolysaccharide (LPS) and HSP70 and plasma levels of glucose and insulin were assessed by using commercial kits.
The 1PL and 4PL groups showed increase in macrophage content, IKKα/β, and Jun N-terminal kinase phosphorylation status, serum LPS and HSP70 levels, and insulin resistance and no changes in glycemia and insulinemia compared with the control group. There was no difference in these parameters between the 1PL and 4PL groups.
PLs promoted an increase in macrophage infiltration, activation of inflammatory pathways in muscle tissue, and serum concentrations of HSP70 and LPS in rats. The present study improves the knowledge on the impact of oral inflammations on the development of systemic alteration, which can induce insulin resistance.
Heat Shock 70 Protein Genes and Genetic Susceptibility to Apical Periodontitis
2016, Journal of Endodontics
Citation Excerpt :
Other studies have suggested that HSPA6 is a secondary regulator of stress at low expression levels, possibly depending on the cell type and growth conditions under non-stressed conditions (36). Members of the HSP70 gene family play a crucial role in the maintenance of infection and subsequent tissue destruction in autoimmune diseases and cancer (37), as well as in apical periodontitis (21). Furthermore, HSP70 proteins have known roles in the induction of proinflammatory cytokines and thereby may contribute to the pathogenesis of autoimmune disease and chronic inflammation (38).
Heat shock proteins (HSPs) protect cells under adverse conditions such as infection, inflammation, and disease. The differential expression of HSPs in human periapical granulomas suggests a potential role for these proteins in periapical lesion development, which may contribute to different clinical outcomes. Therefore, we hypothesized that polymorphisms in HSP genes leading to perturbed gene expression and protein function may contribute to an individual's susceptibility to periapical lesion development.
Subjects with deep carious lesions with or without periapical lesions (≥3 mm) were recruited at the University of Texas School of Dentistry at Houston and at the University of Pittsburgh. Genomic DNA samples of 400 patients were sorted into 2 groups: 183 cases with deep carious lesions and periapical lesions (cases) and 217 cases with deep carious lesions but without periapical lesions (controls). Eight single nucleotide polymorphisms (SNPs) in HSPA4, HSPA6, HSPA1L, HSPA4L, and HSPA9 genes were selected for genotyping. Genotypes were generated by end point analysis by using Taqman chemistry in a real-time polymerase chain reaction assay. Allele and genotype frequencies were compared among cases and controls by using χ² and Fisher exact tests as implemented in PLINK v.1.07. In silico analysis of SNP function was performed by using Polymorphism Phenotyping V2 and MirSNP software.
Overall, SNPs in HSPA1L and HSPA6 showed significant allelic association with cases of deep caries and periapical lesions (P < .05). We also observed altered transmission of HSPA1L SNP haplotypes (P = .03). In silico analysis of HSPA1L rs2075800 function showed that this SNP results in a glutamine-to-lysine substitution at position 602 of the protein and might affect the stability and function of the final protein.
Variations in HSPA1L and HSPA6 may be associated with periapical lesion formation in individuals with untreated deep carious lesions. Future studies could help predict host susceptibility to developing apical periodontitis.
Assessment of Apical Expression of Alpha-2 Integrin, Heat Shock Protein, and Proinflammatory and Immunoregulatory Cytokines in Response to Endodontic Infection
2015, Journal of Endodontics
Citation Excerpt :
However, no data for ITGA2 were found in the literature. Hsp44 genes were significantly increased in a pool of periapical granulomas compared with a pool of healthy periodontal ligaments (19). These authors indicated that the HSP40 gene family plays a crucial role in the maintenance of infection and tissue destruction in apical periodontitis.
The purpose of this study was to examine alpha-2 integrin, molecular mediators, cytokines, and chemokines from cells in periapical interstitial fluid from root canal infections before and after the reduction of the bacterial load using a cleaning procedure.
Subjects included 20 patients referred to the School of Dentistry at the Universidade Federal de Minas Gerais (Belo Horizonte, Minas Gerais, Brazil). Clinical samples were taken from teeth with pulp necrosis, and no patients had acute periapical symptoms at the time of the appointments. After cleaning and drying, 3 paper points were introduced into the root canal, passing passively through the root apex (2 mm) into the periapical tissues for 1 minute. The samples were collected immediately after root canal cleaning and 7 days later (restrained root canal bacterial load) to characterize those gene expressions using real-time polymerase chain reaction.
Significantly lower levels of tumor necrosis factor alpha, chemokine ligand 5 (CCL5), chemokine ligand 2/monocyte chemotactic protein 1 (CCL2/MCP-1), and interleukin (IL)-8 in teeth with restrained bacterial loads (second collection) compared with the first collection were observed (P < .05). Similarly, the messenger RNA expression of the integrins secreted phosphoprotein 1 (SSP1)/ostepontin and focal adhesion kinase (FAK) decreased in samples from the second collection (P < .05). The messenger RNA for the regulatory cytokine IL-10 was significant higher in samples from the second collection (day 7) compared with the first collection (day 0) (P < .05). Messenger RNA expression of IL-1β, IL-17A, interferon gamma, alpha-2 integrin, and Hsp47/SERPINH1 were similar at both time points (P > .05).
These findings suggest that after reducing the root canal bacterial load a decrease in the inflammatory response took place in the periapical lesions.

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Basic ResearchExpression of Heat Shock Proteins in Periapical Granulomas

Abstract

Introduction

Methods

Results

Conclusions

Section snippets

Subjects and Samples

Gene Expression Analysis

Discussion

Acknowledgments

J Biol Chem

Int J Biochem Cell Biol

Arch Oral Biol

J Endod

Methods

Pharmacol Ther

Immunity

Cell

Cell Signal

J Endod

Heat shock proteins: facts, thoughts, and dreams

Shock

Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators

Genes Dev

Basic Research
Expression of Heat Shock Proteins in Periapical Granulomas