Are mTOR and Endoplasmic Reticulum Stress Pathway Genes Associated with Oral and Bone Diseases?

The purpose of this cohort study was to identify associations between combined oral and bone disease phenotypes and genes present in cell regulatory pathways. The studied pathways play important roles in cellular growth, proliferation, differentiation, and homeostasis. DNA samples extracted from whole saliva of 3,912 individuals were genotyped and these data analyzed according to dental caries experience, periapical lesions, periodontitis, osteoporosis, or temporomandibular joint discomfort. Samples were obtained from the Dental Registry and DNA Repository project at the University of Pittsburgh. Twenty-seven polymorphisms in eight genes related to mTOR or endoplasmic reticulum stress pathways were selected for genotyping. Allele frequencies and Hardy-Weinberg equilibrium were calculated. Analyses were performed comparing genotypes between affected and unaffected individuals for each phenotype, as well as for the associated phenotypes combined. For all analyses, we used the software PLINK with an alpha of 0.002. Borderline associations with multiple variants of several genes were found, suggesting that both pathways may be involved in the susceptibility to multiple conditions affecting the oral cavity and bones. When combining patients that had concomitant dental caries, periodontitis, and periapical pathology, several markers in RHEB showed statistically significant association. Multiple conditions affecting bone and teeth (i.e., dental caries, periodontitis, periapical lesion formation, and osteoporosis) appear to share similar underlying genetic etiological factors, which allow us to hypothesize that instead of individually, they should be studied in conjunction in human populations.

Role of estrogen related receptor beta (ESRRB) in DFN35B hearing impairment and dental decay.

BACKGROUND: Congenital forms of hearing impairment can be caused by mutations in the estrogen related receptor beta (ESRRB) gene. Our initial linkage studies suggested the ESRRB locus is linked to high caries experience in humans. METHODS: We tested for association between the ESRRB locus and dental caries in 1,731 subjects, if ESRRB was expressed in whole saliva, if ESRRB was associated with the microhardness of the dental enamel, and if ESRRB was expressed during enamel development of mice. RESULTS: Two families with recessive ESRRB mutations and DFNB35 hearing impairment showed more extensive dental destruction by caries. Expression levels of ESRRB in whole saliva samples showed differences depending on sex and dental caries experience. CONCLUSIONS: The common etiology of dental caries and hearing impairment provides a venue to assist in the identification of individuals at risk to either condition and provides options for the development of new caries prevention strategies, if the associated ESRRB genetic variants are correlated with efficacy.

DNA damage drives accelerated bone aging via an NF-κB-dependent mechanism.

Advanced age is one of the most important risk factors for osteoporosis. Accumulation of oxidative DNA damage has been proposed to contribute to age-related deregulation of osteoblastic and osteoclastic cells. Excision repair cross complementary group 1-xeroderma pigmentosum group F (ERCC1-XPF) is an evolutionarily conserved structure-specific endonuclease that is required for multiple DNA repair pathways. Inherited mutations affecting expression of ERCC1-XPF cause a severe progeroid syndrome in humans, including early onset of osteopenia and osteoporosis, or anomalies in skeletal development. Herein, we used progeroid ERCC1-XPF-deficient mice, including Ercc1-null (Ercc1(-/-)) and hypomorphic (Ercc1(-/Δ)) mice, to investigate the mechanism by which DNA damage leads to accelerated bone aging. Compared to their wild-type littermates, both Ercc1(-/-) and Ercc1(-/Δ) mice display severe, progressive osteoporosis caused by reduced bone formation and enhanced osteoclastogenesis. ERCC1 deficiency leads to atrophy of osteoblastic progenitors in the bone marrow stromal cell (BMSC) population. There is increased cellular senescence of BMSCs and osteoblastic cells, as characterized by reduced proliferation, accumulation of DNA damage, and a senescence-associated secretory phenotype (SASP). This leads to enhanced secretion of inflammatory cytokines known to drive osteoclastogenesis, such as interleukin-6 (IL-6), tumor necrosis factor α (TNFα), and receptor activator of NF-κB ligand (RANKL), and thereby induces an inflammatory bone microenvironment favoring osteoclastogenesis. Furthermore, we found that the transcription factor NF-κB is activated in osteoblastic and osteoclastic cells of the Ercc1 mutant mice. Importantly, we demonstrated that haploinsufficiency of the p65 NF-κB subunit partially rescued the osteoporosis phenotype of Ercc1(-/Δ) mice. Finally, pharmacological inhibition of the NF-κB signaling via an I-κB kinase (IKK) inhibitor reversed cellular senescence and SASP in Ercc1(-/Δ) BMSCs. These results demonstrate that DNA damage drives osteoporosis through an NF-κB-dependent mechanism. Therefore, the NF-κB pathway represents a novel therapeutic target to treat aging-related bone disease.

Expression of XBP1s in bone marrow stromal cells is critical for myeloma cell growth and osteoclast formation.

BM stromal cells (BMSCs) are key players in the microenvironmental support of multiple myeloma (MM) cell growth and bone destruction. A spliced form of the X-box-binding protein-1 (XBP1s), a major proximal effector of unfolded protein response signaling, is highly expressed in MM cells and plays an indispensable role in MM pathogenesis. In the present study, we found that XBP1s is induced in the BMSCs of the MM microenvironment. XBP1s overexpression in healthy human BMSCs enhanced gene and/or protein expression of VCAM-1, IL-6, and receptor activator of NF-κB ligand (RANKL), enhancing BMSC support of MM cell growth and osteoclast formation in vitro and in vivo. Conversely, deficiency of XBP1 in healthy donor BMSCs displayed a range of effects on BMSCs that were opposite to those cells with overexpression of XBP1s. Knock-down of XBP1 in MM patient BMSCs greatly compromised their increased VCAM-1 protein expression and IL-6 and RANKL secretion in response to TNFα and reversed their enhanced support of MM-cell growth and osteoclast formation. Our results demonstrate that XBP1s is a pathogenic factor underlying BMSC support of MM cell growth and osteoclast formation and therefore represents a therapeutic target for MM bone disease.

Resveratrol triggers the pro-apoptotic endoplasmic reticulum stress response and represses pro-survival XBP1 signaling in human multiple myeloma cells.

OBJECTIVE: Resveratrol, trans-3, 4', 5,-trihydroxystilbene, suppresses multiple myeloma (MM). The endoplasmic reticulum (ER) stress response component inositol-requiring enzyme 1α (IRE1α)/X-box binding protein 1 (XBP1) axis is essential for MM pathogenesis. We investigated the molecular action of resveratrol on IRE1α/XBP1 axis in human MM cells. MATERIALS AND METHODS: Human MM cell lines ANBL-6, OPM2, and MM.1S were utilized to determine the molecular signaling events following treatment with resveratrol. The stimulation of IRE1α/XBP1 axis was analyzed by Western blot and reverse transcription polymerase chain reaction. The effect of resveratrol on the transcriptional activity of spliced XBP1 was assessed by luciferase assays. Chromatin immunoprecipitation was performed to determine the effects of resveratrol on the DNA binding activity of XBP1 in MM cells. RESULTS: Resveratrol activated IRE1α as evidenced by XBP1 messenger RNA splicing and phosphorylation of both IRE1α and its downstream kinase c-Jun N-terminal kinase in MM cells. These responses were associated with resveratrol-induced cytotoxicity of MM cells. Resveratrol selectively suppressed the transcriptional activity of XBP1s while it stimulated gene expression of the molecules that are regulated by the non-IRE1/XBP1 axis of the ER stress response. Luciferase assays indicated that resveratrol suppressed the transcriptional activity of XBP1s through sirtuin 1, a downstream molecular target of resveratrol. Chromatin immunoprecipitation studies revealed that resveratrol decreased the DNA binding capacity of XBP1 and increased the enrichment of sirtuin 1 at the XBP1 binding region in the XBP1 promoter. CONCLUSIONS: Resveratrol exerts its chemotherapeutic effect on human MM cells through mechanisms involving the impairment of the pro-survival XBP1 signaling and the activation of pro-apoptotic ER stress response.

IMiD immunomodulatory compounds block C/EBP{beta} translation through eIF4E down-regulation resulting in inhibition of MM.

Immunomodulatory derivatives of thalidomide (IMiD compounds), such as pomalidomide and lenalidomide, are highly active in multiple myeloma (MM) treatment. However, the precise mechanisms of action and resistance in MM are unresolved. Here we show that IMiD compounds down-regulate CCAAT/enhancer-binding protein-ß (C/EBPß) resulting in abrogation of cell proliferation. Overexpression of C/EBPß rescued MM cells from IMiD-induced inhibition of proliferation, indicating that C/EBPß is critical in mediating antiproliferative effects. IMiD-induced decrease of C/EBPß protein led to impaired transcription of interferon regulatory factor 4 (IRF4). Down-regulation of IRF4 by lenalidomide was confirmed by longitudinal studies of bone marrow samples from 23 patients obtained before and during lenalidomide treatment using CD138⁺/IRF4⁺ double labeling. In contrast to down-regulation of C/EBPß protein, IMiD compounds did not alter C/EBPß mRNA levels or protein stability, suggesting translational regulation of C/EBPß. We could demonstrate that C/EBPß protein expression is under eIF4E-translational control in MM. Furthermore, inhibition of the eIF4E-C/EBPß axis by IMiD compounds was not observed in IMiD-resistant MM cells. However, targeting translation at a different level by inhibiting eukaryotic translation initiation factor 4E-binding protein 1 phosphorylation overcame resistance, suggesting that this pathway is critical and might be a target to overcome drug resistance.

Regulation of unfolded protein response modulator XBP1s by acetylation and deacetylation.

XBP1 (X-box-binding protein 1) is a key modulator of the UPR (unfolded protein response), which is involved in a wide range of pathological and physiological processes. The mRNA encoding the active spliced form of XBP1 (XBP1s) is generated from the unspliced form by IRE1 (inositol-requiring enzyme 1) during the UPR. However, the post-translational modulation of XBP1s remains largely unknown. In the present study, we demonstrate that XBP1s is a target of acetylation and deacetylation mediated by p300 and SIRT1 (sirtuin 1) respectively. p300 increases the acetylation and protein stability of XBP1s, and enhances its transcriptional activity, whereas SIRT1 deacetylates XBP1s and inhibits its transcriptional activity. Deficiency of SIRT1 enhances XBP1s-mediated luciferase reporter activity in HEK (human embryonic kidney)-293 cells and the up-regulation of XBP1s target gene expression under ER (endoplasmic reticulum) stress in MEFs (mouse embryonic fibroblasts). Consistent with XBP1s favouring cell survival under ER stress, Sirt1-/- MEFs display a greater resistance to ER-stress-induced apoptotic cell death compared with Sirt1+/+ MEFs. Taken together, these results suggest that acetylation/deacetylation constitutes an important post-translational mechanism in controlling protein levels, as well as the transcriptional activity, of XBP1s. The present study provides a novel insight into the molecular mechanisms by which SIRT1 regulates UPR signalling.

General transcription factor IIA-gamma increases osteoblast-specific osteocalcin gene expression via activating transcription factor 4 and runt-related transcription factor 2.

ATF4 (activating transcription factor 4) is an osteoblast-enriched transcription factor that regulates terminal osteoblast differentiation and bone formation. ATF4 knock-out mice have reduced bone mass (severe osteoporosis) throughout life. Runx2 (runt-related transcription factor 2) is a runt domain-containing transcription factor that is essential for bone formation during embryogenesis and postnatal life. In this study, we identified general transcription factor IIA gamma (TFIIA gamma) as a Runx2-interacting factor in a yeast two-hybrid screen. Immunoprecipitation assays confirmed that TFIIA gamma interacts with Runx2 in osteoblasts and when coexpressed in COS-7 cells or using purified glutathione S-transferase fusion proteins. Chromatin immunoprecipitation assay of MC3T3-E1 (clone MC-4) preosteoblast cells showed that in intact cells TFIIA gamma is recruited to the region of the osteocalcin promoter previously shown to bind Runx2 and ATF4. A small region of Runx2 (amino acids 258-286) was found to be required for TFIIA gamma binding. Although TFIIA gamma interacts with Runx2, it does not activate Runx2. Instead, TFIIA gamma binds to and activates ATF4. Furthermore, TFIIA gamma together with ATF4 and Runx2 stimulates osteocalcin promoter activity and endogenous mRNA expression. Small interfering RNA silencing of TFIIA gamma markedly reduces levels of endogenous ATF4 protein and Ocn mRNA in osteoblastic cells. Overexpression of TFIIA gamma increases levels of ATF4 protein. Finally, TFIIA gamma significantly prevents ATF4 degradation. This study shows that a general transcription factor, TFIIA gamma, facilitates osteoblast-specific gene expression through interactions with two important bone transcription factors ATF4 and Runx2.

Wnt10b increases postnatal bone formation by enhancing osteoblast differentiation.

UNLABELLED: Overexpression of Wnt10b from the osteocalcin promoter in transgenic mice increases postnatal bone mass. Increases in osteoblast perimeter, mineralizing surface, and bone formation rate without detectable changes in pre-osteoblast proliferation, osteoblast apoptosis, or osteoclast number and activity suggest that, in this animal model, Wnt10b primarily increases bone mass by stimulating osteoblastogenesis. INTRODUCTION: Wnt signaling regulates many aspects of development including postnatal accrual of bone. Potential mechanisms for how Wnt signaling increases bone mass include regulation of osteoblast and/or osteoclast number and activity. To help differentiate between these possibilities, we studied mice in which Wnt10b is expressed specifically in osteoblast lineage cells or in mice devoid of Wnt10b. MATERIALS AND METHODS: Transgenic mice, in which mouse Wnt10b is expressed from the human osteocalcin promoter (Oc-Wnt10b), were generated in C57BL/6 mice. Transgene expression was evaluated by RNase protection assay. Quantitative assessment of bone variables was done by radiography, muCT, and static and dynamic histomorphometry. Mechanisms of bone homeostasis were evaluated with assays for BrdU, TUNEL, and TRACP5b activity, as well as serum levels of C-terminal telopeptide of type I collagen (CTX). The endogenous role of Wnt10b in bone was assessed by dynamic histomorphometry in Wnt10b(-/-) mice. RESULTS: Oc-Wnt10b mice have increased mandibular bone and impaired eruption of incisors during postnatal development. Analyses of femoral distal metaphyses show significantly higher BMD, bone volume fraction, and trabecular number. Increased bone formation is caused by increases in number of osteoblasts per bone surface, rate of mineral apposition, and percent mineralizing surface. Although number of osteoclasts per bone surface is not altered, Oc-Wnt10b mice have increased total osteoclast activity because of higher bone mass. In Wnt10b(-/-) mice, changes in mineralizing variables and osteoblast perimeter in femoral distal metaphyses were not observed; however, bone formation rate is reduced because of decreased total bone volume and trabecular number. CONCLUSIONS: High bone mass in Oc-Wnt10b mice is primarily caused by increased osteoblastogenesis, with a minor contribution from elevated mineralizing activity of osteoblasts.

TSC2 integrates Wnt and energy signals via a coordinated phosphorylation by AMPK and GSK3 to regulate cell growth.

Mutation in the TSC2 tumor suppressor causes tuberous sclerosis complex, a disease characterized by hamartoma formation in multiple tissues. TSC2 inhibits cell growth by acting as a GTPase-activating protein toward Rheb, thereby inhibiting mTOR, a central controller of cell growth. Here, we show that Wnt activates mTOR via inhibiting GSK3 without involving beta-catenin-dependent transcription. GSK3 inhibits the mTOR pathway by phosphorylating TSC2 in a manner dependent on AMPK-priming phosphorylation. Inhibition of mTOR by rapamycin blocks Wnt-induced cell growth and tumor development, suggesting a potential therapeutic value of rapamycin for cancers with activated Wnt signaling. Our results show that, in addition to transcriptional activation, Wnt stimulates translation and cell growth by activating the TSC-mTOR pathway. Furthermore, the sequential phosphorylation of TSC2 by AMPK and GSK3 reveals a molecular mechanism of signal integration in cell growth regulation.

Signaling by target of rapamycin proteins in cell growth control.

Target of rapamycin (TOR) proteins are members of the phosphatidylinositol kinase-related kinase (PIKK) family and are highly conserved from yeast to mammals. TOR proteins integrate signals from growth factors, nutrients, stress, and cellular energy levels to control cell growth. The ribosomal S6 kinase 1 (S6K) and eukaryotic initiation factor 4E binding protein 1(4EBP1) are two cellular targets of TOR kinase activity and are known to mediate TOR function in translational control in mammalian cells. However, the precise molecular mechanism of TOR regulation is not completely understood. One of the recent breakthrough studies in TOR signaling resulted in the identification of the tuberous sclerosis complex gene products, TSC1 and TSC2, as negative regulators for TOR signaling. Furthermore, the discovery that the small GTPase Rheb is a direct downstream target of TSC1-TSC2 and a positive regulator of the TOR function has significantly advanced our understanding of the molecular mechanism of TOR activation. Here we review the current understanding of the regulation of TOR signaling and discuss its function as a signaling nexus to control cell growth during normal development and tumorigenesis.