Sexual dimorphism of estrogen-sensitized synoviocytes contributes to gender difference in temporomandibular joint osteoarthritis.

OBJECTIVES: Temporomandibular joint osteoarthritis (TMJOA) is approximately twice as prevalent in women than in men. Synoviocytes are believed to play a critical role in joint inflammation. However, it is unknown whether synoviocytes from different genders possess sexual dimorphisms that contribute to female-predominant TMJOA. MATERIALS AND METHODS: Freund's complete adjuvant combined with monosodium iodoacetate was used to induce TMJOA in female and male rats. Histologic and radiographic features were used to evaluate TMJOA. The expression of CD68, MCP-1, iNOS, and IL-1ß was detected by immunohistochemistry and real-time PCR. Primary fibroblast-like synoviocytes (FLSs) isolated from the synovial membrane of female and male rats were used for in vitro experiments. RESULTS: Female rats showed aggravated TMJOA features as compared to male rats. Increased expression of iNOS and IL-1ß was detected in synovial membrane from female TMJOA rats as compared to male rats. Furthermore, greater amounts of CD68-positive macrophage infiltration and increased MCP-1 expression around the synovial membrane were detected in female TMJOA rats compared to males. Primary cultured FLSs from female rats showed higher sensitivity to TNF-α treatment and recruited increased macrophage migration than male FLSs. More important, ovariectomy (OVX) by ablation in female rats repressed the sensitivity of female FLSs to TNF-α treatment due to the loss of estrogen production. Blockage of the estrogen receptor repressed estrogen-potentiated TNF-α-induced pro-inflammatory cytokine expression in OVX-FLSs. Moreover, the injection of estrogen receptor antagonists relieved the cartilage destruction and bone deterioration of TMJOA in female rats. CONCLUSION: Estrogen-sensitized synoviocytes in female rats may contribute to gender differences in the incidence and progression of TMJOA.

Progesterone attenuates temporomandibular joint inflammation through inhibition of NF-κB pathway in ovariectomized rats.

Sex hormones may contribute to the symptomatology of female-predominant temporomandibular disorders (TMDs) inflammatory pain. Pregnant women show less symptoms of TMDs than that of non-pregnant women. Whether progesterone (P4), one of the dominant sex hormones that regulates multiple biological functions, is involved in symptoms of TMDs remains to be explored. Freund's complete adjuvant were used to induce joint inflammation. We evaluated the behavior-related and histologic effects of P4 and the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 in the synovial membrane. Primary TMJ synoviocytes were treated with TNF-α or IL-1ß with the combination of P4. Progesterone receptor antagonist RU-486 were further applied. We found that P4 replacement attenuated TMJ inflammation and the nociceptive responses in a dose-dependent manner in the ovariectomized rats. Correspondingly, P4 diminished the DNA-binding activity of NF-κB and the transcription of its target genes in a dose-dependent manner in the synovial membrane of TMJ. Furthermore, P4 treatment showed decreased mRNA expression of proinflammatory cytokines, and partially reversed TNF-α and IL-1ß induced transcription of proinflammatory cytokines in the primary synoviocytes. Moreover, progesterone receptor antagonist RU-486 partially reversed the effects of P4 on NF-κB pathway. In conclusion, progesterone ameliorated TMJ inflammation through inhibition of NF-κB pathway.

Mineralized Collagen Regulates Macrophage Polarization During Bone Regeneration.

The host immune response to bone biomaterials is vital in determining the fate of scaffolds and also the outcomes of bone regeneration. Mineralized collagen is an ideal tissue-engineering scaffold for bone repair; however, little is known about its immunomodulatory properties after implantation. In this study, extrafibrillarly-mineralized collagen (EMC) and intrafibrillarly-mineralized collagen (IMC) scaffolds with different nanostructures were fabricated and their immunomodulatory properties via macrophage polarization during bone regeneration were investigated. Micro-CT findings showed that the IMC scaffold yielded more new bone formation than the EMC scaffold. In the defect area, more CD68 + CD163 + M2-like macrophages were observed in the IMC group, while M1-like macrophages positive for CD68 and inducible nitric oxide synthase (iNOS) increased dramatically in the EMC group. We further demonstrated, from the protein and RNA levels, that M2-associated anti-inflammatory cytokines interleukin (IL)-10 and arginase-1 were highly expressed in the macrophages seeded on the IMC scaffold, while those seeded on the EMC scaffold expressed more M1-related genes iNOS and IL-6. Moreover, the macrophage polarization in response to the nanostructure of mineralized collagen scaffolds influenced the osteogenesis of human bone marrow stromal cells. These findings suggest that the nanostructure of mineralized collagen scaffolds affects macrophage functional polarization during bone regeneration. The immunomodulatory properties of biomaterial scaffolds can be a dictator of bone regeneration outcomes.

Synovial TRPV1 is upregulated by 17-ß-estradiol and involved in allodynia of inflamed temporomandibular joints in female rats.

Women with reproductive capability are more likely to suffer from temporomandibular disorders (TMD), with orofacial pain as the most common complaint. In the past, we focused on the role of estradiol in TMD pain through the nervous system. In this study, we explored estradiol's influence on synoviocyte gene expressions involved in the allodynia of the inflamed TMJ. The influence of 17-ß-estradiol on NGF and TRPV1 expression in TMJ synovium was determined in vivo and in vitro and analyzed by Western blot and real-time PCR. Complete Freund's adjuvant (CFA) injection into the TMJ was used to induce TMJ arthritis. Capsazepine served as a TRPV1 antagonist. Head withdrawal threshold was examined using a von Frey Anesthesiometer. We observed that estradiol upregulated the expressions of TRPV1 and NGF in a dose-dependent manner. In the primary cultured synoviocytes, TRPV1 was upregulated by lipopolysaccharide (LPS), estradiol, and NGF, while NGF antibodies fully blocked LPS and estradiol-induced upregulation of TRPV1. Activation of TRPV1 in the primary synoviocytes with capsaicin, a TRPV1 agonist, dose-dependently enhanced COX-2 transcription. Moreover, intra-TMJ injection of TRPV1 antagonist, capsazepine, significantly attenuated allodynia of the inflamed TMJ induced by intra-TMJ injection of CFA in female rats. This article presents a possible local mechanism for estradiol that may be involved in TMJ inflammation or pain in the synovial membrane through the pain-related gene TRPV1. This finding could potentially help clinicians understand the sexual dimorphism of TMD pain.

Estradiol promotes M1-like macrophage activation through cadherin-11 to aggravate temporomandibular joint inflammation in rats.

Macrophages play a major role in joint inflammation. Estrogen is involved in rheumatoid arthritis and temporomandibular disorders. However, the underlying mechanism is still unclear. This study was done to verify and test how estrogen affects M1/M2-like macrophage polarization and then contributes to joint inflammation. Female rats were ovariectomized and treated with increasing doses of 17ß-estradiol for 10 d and then intra-articularly injected with CFA to induce temporomandibular joint (TMJ) inflammation. The polarization of macrophages and expression of cadherin-11 was evaluated at 24 h after the induction of TMJ inflammation and after blocking cadherin-11 or estrogen receptors. NR8383 macrophages were treated with estradiol and TNF-α, with or without blocking cadherin-11 or estrogen receptors, to evaluate the expression of the M1/M2-like macrophage-associated genes. We found that estradiol increased the infiltration of macrophages with a proinflammatory M1-like predominant profile in the synovium of inflamed TMJ. In addition, estradiol dose-dependently upregulated the expressions of the M1-associated proinflammatory factor inducible NO synthase (iNOS) but repressed the expressions of the M2-associated genes IL-10 and arginase in NR8383 macrophages. Furthermore, estradiol mainly promoted cadherin-11 expression in M1-like macrophages of inflamed TMJ. By contrast, blockage of cadherin-11 concurrently reversed estradiol-potentiated M1-like macrophage activation and TMJ inflammation, as well as reversed TNF-α-induced induction of inducible NO synthase and NO in NR8383 macrophages. The blocking of estrogen receptors reversed estradiol-potentiated M1-like macrophage activation and cadherin-11 expression. These results suggested that estradiol could promote M1-like macrophage activation through cadherin-11 to aggravate the acute inflammation of TMJs.

Estradiol-potentiated cadherin-11 in synovial membrane involves in temporomandibular joint inflammation in rats.

Estrogen is involved in inflammation/pain of temporomandibular joint (TMJ), but the underlying mechanisms are largely unknown. Cadherin-11 plays an essential role in synovial inflammation. This study examined whether estrogen could potentiate cadherin-11 in synoviocytes and contribute to TMJ inflammatory pain. Female rats were ovariectomized, treated with increasing doses of 17ß-estradiol for 10 days, and injected intra-articularly with complete Freund's adjuvant to induce TMJ inflammation. The expression of cadherin-11 in synovial membrane was evaluated. TMJ pain was blocked with intra-articular injection of anti-cadherin-11 antibody and evaluated by head withdrawal threshold. Primary TMJ synoviocytes were treated with estradiol and tumor necrosis factor (TNF)-α or blocked with anti-cadherin-11 antibody to assess the expression of cadherin-11, interleukin (IL)-6, cyclooxygenase 2 (COX-2), and inducible nitric oxide synthase (iNOS). We observed that estradiol potentiated the inflammation-induced expression of cadherin-11 in the synoviocytes of synovial membrane from inflamed TMJ. Estradiol induced cadherin-11 expression in a dose- and time-dependent manner in primary synoviocytes and further potentiated the induction of cadherin-11 by TNF-α in synoviocytes. Furthermore, an estrogen receptor antagonist or a NF-κB inhibitor partially blocked the effects of estradiol on cadherin-11 induction in the synovial membrane. Blocking cadherin-11 partially reversed the TMJ inflammatory pain and estradiol-potentiated proliferation of synovial lining cells accompanied with iNOS expression. In addition, blocking cadherin-11 reversed TNF-α-induced and estradiol-potentiated transcription of IL-6, COX-2, and iNOS in primary synoviocytes. These results suggest that estrogen aggravated TMJ inflammatory pain partially through cadherin-11-mediated release of proinflammatory cytokines and enzymes in the synoviocytes.

Acetylated Sp1 inhibits PTEN expression through binding to PTEN core promoter and recruitment of HDAC1 and promotes cancer cell migration and invasion.

Specificity protein 1 (Sp1) is often overexpressed in cancer cells. Its binding sites are known to exist in the phosphatase and tension homolog deleted on chromosome 10 (PTEN) promoter. In this study, we hypothesized that Sp1 negatively regulates PTEN expression. We used several cell lines to determine the effects of Sp1. The results showed that Sp1 overexpression inhibited the expression and promoter activity of PTEN and correspondingly upregulated AKT phosphorylation, whereas Sp1 knockdown upregulated the expression and promoter ability of PTEN and downregulated AKT phosphorylation. Moreover, a series of deletion and site-directed mutations of the PTEN promoter indicated that Sp1 can inhibit PTEN promoter activity through a specific Sp1-binding site at the PTEN core promoter in vivo. Meanwhile, non-acetylated Sp1, with its loss of DNA binding activity, failed to inhibit the expression and promoter activity of PTEN. Histone deacetylase 1 was necessary for Sp1 to inhibit PTEN expression. The inverse expression of Sp1 and PTEN was found in tongue cancer cells and salivary adenoid cystic cancer (SACC)-LM cells (possessing higher potential for lung metastasis than SACC-83) as compared with that in adjacent normal tissue and SACC-83 cells, respectively. Sp1 knockdown decreased the migration and invasion of SACC-LM cells, whereas Sp1 overexpression increased the migration and invasion of SACC-83 cells. Overall, these results suggest that Sp1 is involved in the development and invasiveness of cancer through inhibition of PTEN.

Hippocampal nerve growth factor potentiated by 17ß-estradiol and involved in allodynia of inflamed TMJ in rat.

UNLABELLED: The hippocampus is believed to play an important role in sex-based differences of pain perception. Whether estrogen potentiates allodynia in the inflamed temporomandibular joint (TMJ) through affecting the expressions of pain-related genes in the hippocampus remains largely unknown. Because the nerve growth factor (NGF) is an important gene related to inflammatory pain, we tested whether hippocampal NGF may be involved in TMJ inflammatory pain. Here we showed that the rat hippocampal NGF was upregulated by TMJ inflammation induced by complete Freund adjuvant. NGF upregulation was further potentiated by estradiol in a dose-dependent manner. In contrast, NGF transcription in the amygdala, prefrontal cortex, and thalamus was not affected by TMJ inflammation and estradiol. An intrahippocampal injection of NGF antibody or NGF receptor inhibitor K252a (inhibitor for tropomyosin receptor kinase A, TrkA) reduced the allodynia of inflamed TMJ in proestrous rats. Our data suggest that the hippocampal NGF is involved in estradiol-sensitized allodynia of inflammatory TMJ pain. PERSPECTIVE: We report that complete Freund adjuvant-induced temporomandibular joint (TMJ) inflammation upregulated hippocampal nerve growth factor (NGF) expression, and estradiol replacement potentiated this upregulation. These results propose that estradiol could modulate TMJ pain through the NGF signaling pathway in the hippocampus to exacerbate TMJ pain and offer a possible mechanism of sexual dimorphism of temporomandibular disorder pain.

17ß-estradiol aggravates temporomandibular joint inflammation through the NF-κB pathway in ovariectomized rats.

OBJECTIVE: Women of childbearing age are more likely than men to experience temporomandibular disorders, with pain as the main symptom. Temporomandibular joint (TMJ) inflammation is believed to be a major reason for TMJ pain. Whether sex hormones are involved in the sexual dimorphism of TMJ inflammation and pain remains to be elucidated. The aim of this study was to examine whether estrogen affects TMJ inflammation and pain via the NF-κB pathway. METHODS: Female rats were divided into 6 groups: the control group, the sham-ovariectomized group, and 4 groups of ovariectomized rats treated with 17ß-estradiol at a dosage of 0 µg/day, 20 µg/day, 80 µg/day, and 200 µg/day, respectively, for 10 days and then injected intraarticularly with Freund's complete adjuvant to induce TMJ inflammation. The behavior-related and histologic effects of 17ß-estradiol were evaluated 24 hours after the induction of TMJ inflammation. NF-κB activity in the synovial membrane was examined by electrophoretic mobility shift assay. The expression of the NF-κB target genes tumor necrosis factor α, interleukin-1ß (IL-1ß), IL-6, cyclooxygenase 2, and inducible nitric oxide synthase in the synovial membrane was examined by real-time polymerase chain reaction. RESULTS: Treatment with estradiol potentiated TMJ inflammation in a dose-dependent manner and also exacerbated the inflammation-induced decrease in food intake. Correspondingly, estradiol potentiated the DNA-binding activity of NF-κB and the transcription of its target genes in the synovial membrane. Furthermore, the estrogen receptor antagonist ICI 182780 or the NF-κB inhibitor pyrrolidine dithiocarbamate partially blocked the effects of estradiol on TMJ inflammation and pain and the NF-κB pathway. CONCLUSION: These results suggest that estradiol aggravates TMJ inflammation through the NF-κB pathway, leading to the induction of proinflammatory cytokines.

17-Beta-estradiol enhanced allodynia of inflammatory temporomandibular joint through upregulation of hippocampal TRPV1 in ovariectomized rats.

Temporomandibular disorders (TMDs) predominantly affect reproductive female patients, with pain the most frequent complaint. Although estrogens are believed to play important roles in TMD pain, the mechanism underlying modulation of TMD pain by estrogens remains largely unknown. Accumulating evidence implies that the hippocampus is involved in sexual dimorphism of pain sensitivity. In this study, we investigated the hippocampal TRPV1 (transient receptor potential vanilloid 1) expression in ovariectomized rats that received 17-beta-estradiol substitution and found that 17-beta-estradiol enhanced the mechanical allodynia of inflamed temporomandibular joint (TMJ) induced by complete Freund's adjuvant. Real-time PCR and immunoblotting demonstrated that TMJ inflammation significantly induced hippocampal TRPV1 expression compared with the control group but failed to induce it in the ovariectomized rats that received no estradiol replacement. In addition, estradiol potentiated TMJ inflammation-induced hippocampal TRPV1 expression in a dose-dependent manner in the ovariectomized rats. In contrast, TRPV1 transcription in amygdala, prefrontal cortex, and thalamus was not affected by TMJ inflammation and estradiol. Immunostaining showed TRPV1 localized in the processes and cytoplasm of pyramidal neurons in CA1-CA3 regions of the hippocampus. Moreover, intrahippocampal injection of TRPV1 antagonists capsazepine and 5'-iodo-resiniferatoxin into the CA1 region of the hippocampus significantly attenuated allodynia of inflamed TMJ in both nonovariectomized and ovariectomized rats that received estradiol replacement. Our results suggested that hippocampal TRPV1 can modulate central pain processing and estradiol may contribute to the sexual dimorphism of TMD pain sensitivity through upregulation of TRPV1 expression in the hippocampus.