Enhanced Immunotherapy Based on Combining the Pro-phagocytosis and Anti-phagocytosis Checkpoint Blockade for Tumor Eradication.

Compared to the activation of acquired immunity by the immune checkpoint blockade, the activation of innate immunity via anti-phagocytosis checkpoint blockade could significantly increase the beneficiary population of immunotherapy. However, the activation of innate immunity and the occurrence of phagocytosis are only accomplished when the interaction between pro-phagocytosis signals and anti-phagocytosis signals is realized. Herein, a versatile nanoplatform (DHMR) based on mesoporous silicon nanoparticles (MSNPs) has been constructed. Two drugs, doxorubicin, a chemotherapeutic drug which could initiate tumor cells to release pro-phagocytosis signals, and RRx-001, an immunoadjuvant that could effectively implement the anti-phagocytosis checkpoint blockade, were loaded in MSNPs. Further decoration of hyaluronic acid encapsulation endows DHMR with the function of tumor targeting and long circulation. Ultimately, the DHMR system could efficiently and accurately target tumor tissue, release the drugs in the tumor microenvironment, achieve the activation of innate immunity, and finally dramatically inhibit the growth and metastasis of tumor cells.

Activating Innate Immunity by a STING Signal Amplifier for Local and Systemic Immunotherapy.

The number of patients who benefit from acquired immunotherapy is limited. Stimulator of interferon genes (STING) signal activation is a significant component to enhance innate immunity, which has been used to realize broad-spectrum immunotherapy. Here, M@P@HA nanoparticles, as a STING signal amplifier, are constructed to enhance innate immunotherapy. Briefly, when M@P@HA was targeted into tumor cells, the nanoparticles decomposed with Mn2+ and activated the release of protoporphyrin (PpIX). Under light irradiation, the generated reactive oxygen species disrupt the cellular redox homeostasis to lead cytoplasm leakage of damaged mitochondrial double-stranded (ds) DNA, which is the initiator of the STING signal. Simultaneously, Mn2+ as the immunoregulator could significantly increase the activity of related protein of a STING signal, such as cyclic GMP-AMP synthase (cGAS) and STING, to further amplify the STING signal of tumor cells. Subsequently, the STING signal of tumor-associated macrophages (TAM) is also activated by capturing dsDNA and Mn2+ that escaped from tumor cells, so as to enhance innate immunity. It is found that, by amplifying the STING signal of tumor tissue, M@P@HA could not only activate innate immunity but also cascade to activate CD8+ T cell infiltration even in a tumor with low immunogenicity.

Aptamer-Gold Nanocage Composite for Photoactivated Immunotherapy.

Immune checkpoint blockade (ICB) has been hailed as the hope for conquering cancer as ICB could produce a significant and durable response to tumor cells. However, the high cost and severe side effects of ICB drugs limited their application for further anticancer therapy. Here, we developed a photoactivated immunotherapy nanoplatform (Apt@AuNC). This nanoplatform could target tumor tissues via enhanced penetration retention (EPR) effect and the aptamer (Apt) could be released from Apt@AuNC in tumor sites via illumination. The immune system in the tumor area was then activated after the combination of Apt and PD-1 protein. The heat generated from AuNC was able to continue killing tumor cells. This nanoplatform could not only achieve the precise immunotherapy but also significantly facilitate the anticancer efficacy.

A Versatile Nanoplatform for Broad-Spectrum Immunotherapy by Reversing the Tumor Microenvironment.

Immunotherapy is currently an important adjuvant therapy for malignant tumors besides surgical treatment. However, the heterogeneity and low immunogenicity of the tumor are two main challenges of the immunotherapy. Here, we have constructed a nanoplatform (CP@mRBC-PpIX) to realize reversion of the tumor acidosis and hypoxia through alkali and oxygen generation triggered by tumor acidosis. By targeting tumor universal features other than endogenous biomarkers, it was found that CP@mRBC-PpIX could polarize tumor-associated macrophages to anti-tumor M1 phenotype macrophages to enhance tumor immune response. Furthermore, under regional light irradiation, the reactive oxygen species produced by photosensitizers located in CP@mRBC-PpIX could increase the immunogenicity of tumors, so that tumor changes from an immunosuppressive "cold tumor" to an immunogenic "hot tumor," thereby increasing the infiltration and response of T cells, further amplifying the effect of immunotherapy. This strategy circumvented the problem of tumor heterogeneity to realize a kind of broad-spectrum immunotherapy, which could effectively prevent tumor metastasis and recurrence.

[Collagen Peptides Improve Lymphocyte Distribution in Peripheral Blood and T Lymphocyte Proliferation in Spleen of Mice under the Condition of Simulated Weightlessness].

OBJECTIVE: To investigate whether collagen peptides can improve the immune functions of mice under the condition of simulated weightlessness. METHODS: Mouse tail-suspension model was used to simulate the effects of weightlessness. Tail-suspended mice were intraperitoneally injected with 600 mg collagen peptides per kilogram body weight once a day for 10 days. Then, the mice were killed, and white blood cells were counted and classified. Lymphocyte subsets and T lymphocyte proliferations in spleens were analyzed. RESULTS: Compared with normal control group, total and differential count of leukocytes, lymphocytes, T cells，CD4+ and CD8+ T cells, B cells and NK cells, and splenic T lymphocyte proliferation all decreased in the weightlessness simulated mice (P＜0.05). Except for NK cells, the above-mentioned parameters were increased after administration of collagen peptides, and some of the parameters were recovered to the levels of normal control mice (P＜0.05). CONCLUSION: Collagen peptides can effectively improve peripheral blood lymphocyte distributions and T lymphocyte proliferations of mice under the condition of simulated weightlessness. This study nay provid the experimental basis for improvement of immune functions of astronauts.

Recombinant adenovirus of SEA and CD80 genes driven by MMRE and mouse TERT promoter induce effective antitumor immune responses against different types of tumor cells in vitro and in vivo.

Staphylococcus enterotoxin A (SEA) is a powerful immunostimulant and can stimulate T cells bearing certain T-cell receptor ß-chain variable regions when bound to major histocompatibility complex II molecules. SEA is widely used in research of antitumor therapy. The low affinity T-cell receptor (TCR) interaction with SEA in the absence of MHC class II antigens is sufficient for the induction of cytotoxicity but requires additional CD28/B7 signaling to result in proliferation of resting T cells. In this study, we constructed recombinant adenovirus (named as Ad-MMRE-mTERT-BIS) carrying membrane-expressing SEA (named as SEAtm) and CD80 driven by Myc-Max response elements (MMRE) and mouse telomerase reverse transcriptase (mTERT) promoter to reduce toxicity and to improve safety and efficiency. We demonstrated that Ad-MMRE-mTERT-BIS could make SEAtm and CD80 to co-express highly on the surface of Hepa1-6 and B16 cells, at low level on the surface of CT26 cells, but not in NIH3T3. Hepa1-6 and B16 cells infected by the recombinant adenovirus induced proliferation of CD4+ and CD8+ T cells and increased cytokine [interleukin (IL)-2, tumor necrosis factor (TNF)-α, interferon (IFN)-γ] production in vitro. Intratumoral injection of Ad-MMRE-mTERT-BIS in hepatoma and melanoma mouse models induced tumor-specific cytotoxic T cells in the spleen. Moreover, hepatoma and melanoma xenografts were suppressed by treatment with Ad-MMRE-mTERT-BIS and the survival time of treated mice was prolonged. These findings suggest that recombinant adenovirus of SEA and CD80 genes driven by mTERT promoter could induce effective antitumor immune responses against different kinds of tumor cells in vitro and in vivo.

Vitamin D improves immune function in immunosuppressant mice induced by glucocorticoid.

Vitamin D is an essential fat-soluble vitamin with multiple functions. Vitamin D receptor has been shown to be expressed in several types of immune cells suggesting vitamin D may have immune regulatory roles. Vitamin D insufficiency has been suggested to increase the risk of autoimmune diseases. However, little is known regarding its immunomodulatory effects in the condition of immune suppression. The aim of the present study was to investigate the regulatory effects of vitamin D on immune function in immunosuppressant mice. An immunosuppressant mouse model was induced by intraperitoneal injection with glucocorticiod for 3 days. Immunosuppressant mice were intragastrically administered with 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3; 0,4, 6 or 10 IU/g body weight] for 7 days. On day 8, the mice were decapitated. The body weight and the weights of thymus and spleen were measured. Thymus and spleen indexes were calculated. The ratio of CD4+/CD8+ T lymphocytes in the peripheral blood, proliferation and interleukin-2 (IL-2) production of spleen T lymphocytes was detected. Compared with the mice in the control group, the body weight, thymus and spleen indexes, the ratios of CD4+/CD8+ in peripheral blood and IL-2 production and proliferation of spleen T lymphocytes were decreased in immunosuppressant mice induced by glucocorticiod. However, in vitamin D-treated mice, the thymus indexes, the ratios of CD4+/CD8+, secretion of IL-2 and the proliferation index of spleen T lymphocytes were significantly increased (P<0.05). Among the three doses of 1,25(OH)2D3, 6 IU/g was most effective in improving the immune function. These results indicate that vitamin D supplementation can improve immune recovery in immunosuppressant mice by stimulating T-cell proliferation and elevating IL-2 production.

The Associations of Serum Lipids with Vitamin D Status.

AIMS: Vitamin D deficiency has been associated with some disorders including cardiovascular diseases. Dyslipidemia is a major risk factor for cardiovascular diseases. However, data about the relationships between vitamin D and lipids are inconsistent. The relationship of vitamin D and Atherogenic Index of Plasma (AIP), as an excellent predictor of level of small and dense LDL, has not been reported. The objective of this study was to investigate the effects of vitamin D status on serum lipids in Chinese adults. METHODS: The study was carried out using 1475 participants from the Center for Physical Examination, 306 Hospital of PLA in Beijing, China. Fasting blood samples were collected and serum concentrations of 25(OH)D, total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) were measured. AIP was calculated based on the formula: log [TG/HDL-C]. Multiple linear regression analysis was used to estimate the associations between serum 25(OH)D and lipids. The association between the occurrences of dyslipidemias and vitamin D levels was assessed by multiple logistic regression analysis. Confounding factors, age and BMI, were used for the adjustment. RESULTS: The median of serum 25(OH)D concentration was 47 (27-92.25) nmol/L in all subjects. The overall percentage of 25(OH)D ≦ 50 nmol/L was 58.5% (males 54.4%, females 63.7%). The serum 25(OH)D levels were inversely associated with TG (ß coefficient = -0.24, p < 0.001) and LDL-C (ß coefficient = -0.34, p < 0.001) and positively associated with TC (ß coefficient = 0.35, p < 0.002) in men. The associations between serum 25(OH)D and LDL-C (ß coefficient = -0.25, p = 0.01) and TC (ß coefficient = 0.39, p = 0.001) also existed in women. The serum 25(OH)D concentrations were negatively associated with AIP in men (r = -0.111, p < 0.01) but not in women. In addition, vitamin D deficient men had higher AIP values than vitamin D sufficient men. Furthermore, the occurrences of dyslipidemias (reduced HDL-C, elevated TG and elevated AIP) correlated with lower 25(OH)D levels in men, whereas the higher TC and LDL-C associated with higher 25(OH)D levels in women. CONCLUSION: It seems that the serum 25(OH)D levels are closely associated with the serum lipids and AIP. Vitamin D deficiency may be associated with the increased risk of dyslipidemias, especially in men. The association between vitamin D status and serum lipids may differ by genders.

Successful treatment of recurrent Kimura's disease with radiotherapy: a case report.

Kimura's disease is a rare, chronic inflammatory disorder affecting the skin and subcutaneous tissue, predominantly in the head and neck region. It is benign but may be recurrent and difficult to eradicate. A case of recurrent Kimura disease in a 53-year-old man was reported. Radiation therapy was performed for recurrence after surgical excision twice. The prescribed radiation dose was 36 Gy. With a follow-up time of 68 months, the patient was free of the disease.

Vitamin D status in Chinese pregnant women and their newborns in Beijing and their relationships to birth size.

OBJECTIVE: To evaluate the prevalence of vitamin D deficiency in pregnant women and their newborns in Beijing, China and the influence of vitamin D deficiency on birth size. DESIGN: A cross-sectional study. SETTING: Data were collected from pregnant women who delivered during April to May 2010 at 306 Hospital of PLA in Beijing, China. SUBJECTS: Participants in the study were seventy healthy nulliparous pregnant women with singleton pregnancies who delivered healthy babies at full term and their newborns. RESULTS: Severe vitamin D deficiency (25-hydroxyvitamin D (25(OH)D) < 25 nmol/l) was detected in 54·5 % of mothers and 46·6 % of newborns. Neither mothers nor newborns had serum 25(OH)D concentrations that reached the normal level (>75 nmol/l). The concentration of 25(OH)D in mothers was positively correlated with that in cord blood (r = 0·89, P < 0·001). Newborns of mothers with severe vitamin D deficiency had lower birth length and birth weight. The head circumference and birth weight were lower in vitamin D-deficient newborns. CONCLUSIONS: The study indicates that pregnant women and neonates residing in Beijing are at high risk of vitamin D deficiency. Neonatal 25(OH)D concentrations are dependently related to maternal 25(OH)D levels. Maternal and neonatal vitamin D status influences newborn size.

The high prevalence of vitamin D deficiency and its related maternal factors in pregnant women in Beijing.

Maternal vitamin D deficiency has been suggested to influence fetal and neonatal health. Little is known about vitamin D status in Chinese pregnant women. The purpose of this study was to assess the vitamin D status of pregnant women residing in Beijing in winter and evaluate the impact of maternal factors on serum 25-hydroxyvitamin D [25(OH)D] levels. The study was conducted on 125 healthy pregnant women. For each individual, data concerning pre-pregnancy weight, educational status, use of multivitamins and behavioral factors such as daily duration of computer use, walking and sun exposure were obtained. Serum concentrations of 25(OH)D were measured by enzyme-linked immunosorbent assay. The prevalence of vitamin D deficiency (25(OH)D < 50 nmol/L) was 96.8% and almost half (44.8%) of women were severely vitamin D deficiency (25(OH)D < 25 nmol/L). The concentration of 25(OH)D was lower in women with shorter duration of sun exposure (≤ 0.5 h/day, 25.3 ± 8.9 nmol/L) than that in women with longer duration of sun exposure (> 0.5 h/day; 30.3 ± 9.5 nmol/L; P = 0.003). Thirty six women (28.8%) had sun exposure duration ≥ 1.5h/day. The 25(OH)D concentration in these women was 31.5 ± 9.4 nmol/L which was also much lower than the normal level. Women who reported taking a multivitamin supplement had significantly higher 25(OH)D concentrations (32.3 ± 9.5 nmol/L) when compared with non-users (24.9 ± 8.2 nmol/L; P < 0.001). Pregnant women in Beijing are at very high risk of vitamin D deficiency in winter. Duration of Sun exposure and the use of multivitamin were the most important determinants for vitamin D status. However, neither prolonging the time of sunlight exposure nor multivitamin supplements can effectively prevent pregnant women from vitamin D deficiency. Other measures might have to be taken for pregnant women to improve their vitamin D status in winter.

[Construction of recombinant adenovirus of SEA and CD80 genes co-expression regulated by mouse TERT promoter and identification of its expression in hepatoma cells].

AIM: To construct recombinant co-expression adenovirus vector of SEA and CD80 genes regulated by mouse TERT(telomerase reverse transcriptase, TERT) promoter and to observe the expression of SEA and CD80 in the Hepa1-6 cells mediated by it. METHODS: Using AdEasy adenovirus system, the core promoter region of mTERT was subcloned to shuttle plasmid pShuttle2 and Myc-Max response element was inserted upstream of it to regulate the expression of SEA and CD80. The recombinant co-expression adenovirus vector of SEA and CD80 genes was constructed and named as Ad-MMRE-mTERT-BIS. Hepatoma cell line Hepa1-6 and fibroblast cell line NIH3T3 were infected by recombinant adenovirus at MOI(multiplicity of infection)of 100, the expression of SEA and CD80 on the surface of cells was detected by indirect immunofluorescent staining. RESULTS: SEA and CD80 was specifically co-expressed on the surface of infected Hepa1-6 cells but not on NIH3T3 cells. CONCLUSION: The recombinant co-expression adenovirus vector of SEA and CD80 gene regulated by mTERT promoter was sucessfully constructed and make targeting-expression of SEA and CD80 on the surface of hepatoma cells, which lays the foundation for further research on application of SEA and CD80 in targeted genetherapy for hepatoma.

Cloning of mouse telomerase reverse transcriptase gene promoter and identification of proximal core promoter sequences essential for the expression of transgenes in cancer cells.

Telomerase is a ribonucleoprotein complex, whose function is to add motif-specific nucleotides to the end of chromosomes. Telomerase consists of three major subunits, the telomerase RNA template (hTR), the telomerase-associated protein (TEP1) and telomerase reverse transcriptase (TERT). TERT is the most important component responsible for the catalytic activity of telomerase and a rate-limiting determinant of the activity. Telomerase activities were at high levels in approximately 90% of mouse cancers or tumor-derived cell lines through TERT transcriptional up-regulation. Unlike human telomerase, telomerase activity exists in colon, liver, ovary and testis but not in brain, heart, stomach and muscle in normal mouse tissues. In this study, we prepared 5' truncations of 1086 bp fragments upstream of the initiating ATG codon of the mTERT gene to construct luciferase reporter gene plasmids, and transfected these plasmids into a normal mouse cell line and several cancer lines to identify the core promoter region essential for transcriptional activation in cancer cells by a luciferase assay. We constructed a eukaryotic expression vector of membrane-expressing staphylococcal endotoxin A (SEA) gene driven by the core promoter region of the mTERT gene and observed if the core promoter region could express the SEA gene in these cancer cells, but not in normal cells following transfection with the construct. The results showed that the transcriptional activities of each fragment of the mTERT gene promoter in the cancer cell lines Hepa1-6, B16 and CT26 were higher than those in NIH3T3 cells, and the proximal 333-bp fragment was the core promoter of the mTERT gene in the cancer cells. The proximal 333-bp fragment was able to make the SEA express on the surface of the cancer cells, but not in NIH3T3 cells. It provides a foundation for cancer targeting gene therapy by using the mTERT gene promoter.

Thrombin-stimulated growth factor and cytokine expression in osteoblasts is mediated by protease-activated receptor-1 and prostanoids.

Thrombin exerts multiple effects upon osteoblasts including stimulating proliferation, and inhibiting osteoblast differentiation and apoptosis. Some of these effects are believed to be mediated by the synthesis and secretion of autocrine factors such as growth factors and cytokines. Many but not all cellular responses to thrombin are mediated by members of the protease-activated receptor (PAR) family of G protein-coupled receptors. The current study was undertaken to investigate the nature of thrombin's induction of autocrine factors by analysing the expression of twelve candidate genes in thrombin-stimulated primary mouse osteoblasts. Analysis by quantitative reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that thrombin induced transforming growth factor beta, cyclooxygenase-2, tenascin C, fibroblast growth factor-1 and -2, connective tissue growth factor and interleukin-6 expression in wild type osteoblasts, but not PAR-1 null mouse osteoblasts. Induction of all the thrombin-responsive genes was blocked by the presence of the non-selective cyclooxygenase inhibitor indomethacin. Further studies were conducted on interleukin-6, which was the gene that showed the greatest increase in expression following stimulation of osteoblast-like cells with thrombin. A PAR-1-specific activating peptide, but neither a PAR-4-activating peptide nor catalytically inactive thrombin induced release of interleukin-6 by osteoblasts. Furthermore, in the presence of the selective cyclooxygenase-1 and -2 inhibitors SC-560 and NS-398 thrombin-induced interleukin-6 release was prevented. Levels of both prostaglandin E(2) and interleukin-6 in medium conditioned by thrombin-stimulated osteoblast-like cells were found to be significantly increased compared to medium conditioned by non-stimulated cells, however release of prostaglandin E(2) was found to precede release of interleukin-6. Treatment of isolated osteoblast-like cells with a number of synthetic prostanoids stimulated secretion of interleukin-6 with differing potencies. These studies suggest that activation of PAR-1 on osteoblasts by thrombin induces cyclooxygenase activity, which in turn results in the increased expression of multiple secreted factors. The induction of these secreted factors may act in an autocrine fashion to alter osteoblast function, allowing these cells to participate in the earliest stages of bone healing by both autocrine and paracrine mechanisms.

Heparan sulfate mediates the proliferation and differentiation of rat mesenchymal stem cells.

The growth and differentiation of mesenchymal stem cells (MSCs) is controlled by various growth factors, the activities of which can be modulated by heparan sulfates (HSs). We have previously noted the necessity of sulfated glycosaminoglycans for the fibroblast growth factor type 2 (FGF-2)-stimulated differentiation of osteoprogenitor cells. Here we show that exogenous application of HS to cultures of primary rat MSCs stimulates their proliferation, leading to increased expression of osteogenic markers and enhanced bone nodule formation. FGF-2 can also increase the proliferation, and osteogenic differentiation of rat bone marrow stem cells (rMSCs) when applied exogenously during their linear growth. However, as opposed to exogenous HS, the continuous use of FGF-2 during in vitro differentiation completely blocked rMSC mineralization. We show that the effects of both FGF-2 and HS are mediated through FGF receptor 1 (FGFR1) and that inhibition of signaling through this receptor arrests cell growth, resulting in the cells being unable to reach the critical density necessary to induce differentiation. Blocking FGFR1 signaling in postconfluent osteogenic cultures significantly increased calcium deposition. Taken together our data suggest that FGFR1 signaling plays an important role during osteogenic differentiation, first by stimulating cell growth that is closely followed by an inhibitory effect once the cells have reached confluence. It also confirms the importance of HS as a coreceptor for the signaling of endogenous FGF-2 and suggests that purified glycosaminoglycans may be attractive alternatives to growth factors for improved ex vivo growth and differentiation of MSCs.

Regulated expression of syndecan-4 in rat calvaria osteoblasts induced by fibroblast growth factor-2.

Fibroblast growth factor-2 (FGF2) is a member of a prominent growth factor family that drives proliferation in a wide variety of cell types, including osteoblasts. The binding and signal transduction triggered by these mitogens is dependent on glycosaminoglycan (GAG) sugars, particularly of the heparan sulfate (HS) class. These are secreted in proteoglycan (PG) complexes, some of which become FGF co-receptors. The syndecans, the transmembrane forms of HSPG of which there are four members, act as multifunctional receptors for a variety of ligands involved in cell-extracellular matrix (ECM) adhesion as well as growth factor binding. To understand the role of syndecans in developing osteoblasts, the effects of exogenous FGF2 on syndecan expression were examined using primary rat calvarial osteoblasts. All four syndecan mRNAs were expressed in the osteoblasts, although only syndecan-4 was upregulated by FGF2 treatment in a dose-dependent manner. This upregulation could be abrogated by pretreatment with the protein synthesis inhibitor cycloheximide, suggesting that the upregulation of syndecan-4 by FGF2 is not a primary response. Osteoblast proliferation and mineralization were enhanced by exogenous FGF2 treatment, but could be specifically diminished by anti-syndecan-4 antibody pretreatment. This treatment also blocked FGF2-induced extracellular signal-regulated kinase activation, but not the expression of the bone-specific transcription factor Runx2. These results demonstrate that mitogen-triggered syndecan-4 expression is an intrinsic part of the pathways subtending osteoblast proliferation and mineralization.

The role of protease-activated receptor-1 in bone healing.

Protease-activated receptor (PAR)-1, a G-protein-coupled receptor activated by thrombin, mediates thrombin-induced proliferation of osteoblasts. The current study was undertaken to define the role of PAR-1 in bone repair. Holes were drilled transversely through the diaphysis of both tibiae of PAR-1-null and wild-type mice. Three days later, fewer cells had invaded the drill site from adjacent bone marrow in PAR-1-null mice than in wild-type mice, and a lower percentage of cells were labeled with [(3)H]thymidine in PAR-1-null drill sites. More osteoclasts were also observed in the drill site of PAR-1-null mice than in wild-type mice 7 days after drilling. New mineralized bone area was less in the drill site and on the adjacent periosteal surface in PAR-1-null mice than in wild-type mice at day 9. From day 14, no obvious differences could be seen between PAR-1-null and wild-type tibiae. In vitro thrombin caused a dose-dependent increase in proliferation of bone marrow stromal cells isolated from wild-type mice but not PAR-1-null mice. Thrombin stimulated survival of bone marrow stromal cells from both wild-type and PAR-1-null mice, but it did not affect bone marrow stromal cell migration in either wild-type or PAR-1-null cells. The results indicate that PAR-1 plays an early role in bone repair.

Activation of protease-activated receptor-2 leads to inhibition of osteoclast differentiation.

UNLABELLED: PAR-2 is expressed by osteoblasts and activated by proteases present during inflammation. PAR-2 activation inhibited osteoclast differentiation induced by hormones and cytokines in mouse bone marrow cultures and may protect bone from uncontrolled resorption. INTRODUCTION: Protease-activated receptor-2 (PAR-2), which is expressed by osteoblasts, is activated specifically by a small number of proteases, including mast cell tryptase and factor Xa. PAR-2 is also activated by a peptide (RAP) that corresponds to the "tethered ligand" created by cleavage of the receptor's extracellular domain. The effect of activating PAR-2 on osteoclast differentiation was investigated. MATERIALS AND METHODS: Mouse bone marrow cultures have been used to investigate the effect of PAR-2 activation on osteoclast differentiation induced by parathyroid hormone (PTH), 1,25 dihydroxyvitamin D3 [1,25(OH)2D3], and interleukin-11 (IL-11). Expression of PAR-2 by mouse bone marrow, mouse bone marrow stromal cell-enriched cultures, and the RAW264.7 osteoclastogenic cell line was demonstrated by RT-PCR. RESULTS: RAP was shown to inhibit osteoclast differentiation induced by PTH, 1,25(OH)2D3, or IL-11. Semiquantitative RT-PCR was used to investigate expression of mediators of osteoclast differentiation induced by PTH, 1,25(OH)2D3, or IL-11 in mouse bone marrow cultures and primary calvarial osteoblast cultures treated simultaneously with RAP. In bone marrow and osteoblast cultures treated with PTH, 1,25(OH)2D3, or IL-11, RAP inhibited expression of RANKL and significantly suppressed the ratio of RANKL:osteoprotegerin expression. Activation of PAR-2 led to reduced expression of prostaglandin G/H synthase-2 in bone marrow cultures treated with PTH, 1,25(OH)2D3, or IL-11. RAP inhibited PTH- or 1,25(OH)2D3-induced expression of IL-6 in bone marrow cultures. RAP had no effect on osteoclast differentiation in RANKL-treated RAW264.7 cells. CONCLUSION: These observations indicate that PAR-2 activation inhibits osteoclast differentiation by acting on cells of the osteoblast lineage to modulate multiple mediators of the effects of PTH, 1,25(OH)2D3, and IL-11. Therefore, the role of PAR-2 in bone may be to protect it from uncontrolled resorption by limiting levels of osteoclast differentiation.

Inhibition of osteoblast apoptosis by thrombin.

The multifunctional serine protease thrombin has been shown to be a specific agonist for a variety of functional responses of cells including osteoblasts. The current study was conducted to determine if thrombin was capable of inhibiting apoptosis in osteoblasts, and if so, to examine the mechanism by which this occurred. Thrombin (20-100 nM) significantly inhibited apoptosis in serum-starved cultures of the human osteoblast-like Saos-2 cell line and cultures of primary osteoblasts isolated from mouse calvariae, as well as dexamethasone-treated primary mouse osteoblasts. Inhibition of serum deprivation-induced apoptosis was shown to require thrombin's specific proteolytic activity. Primary mouse osteoblasts were found to express two functional thrombin receptors, PAR-1 and PAR-4. Thrombin inhibited serum deprivation-induced apoptosis in osteoblasts isolated from PAR-1 null mice to the same degree as in osteoblasts isolated from wild-type mice. Treatment of serum-deprived osteoblasts, isolated from either PAR-1 null or wild-type mice, with a PAR-4-activating peptide failed to significantly inhibit apoptosis compared to the relevant control. Medium conditioned by thrombin-treated osteoblasts, in which thrombin had been inactivated, was able to inhibit serum deprivation-induced osteoblast apoptosis almost as well as thrombin itself. Blocking protein synthesis, by cycloheximide pretreatment of the conditioning cells, prevented this action. The ability of known osteoblast survival factors, such as transforming growth factor beta1, fibroblast growth factor-2, insulin-like growth factor-II, and interleukin-6, to inhibit serum deprivation-induced osteoblast apoptosis was also tested. None of these factors was able to inhibit serum deprivation-induced osteoblast apoptosis to the same extent as thrombin. The results presented here demonstrate that thrombin treatment of osteoblasts inhibits apoptosis induced either by dexamethasone or by serum deprivation. Furthermore, it does so independently of the known thrombin receptors by bringing about the synthesis and/or secretion of an unknown survival factor or factors, which then act in an autocrine fashion to inhibit apoptosis.