Circulating Cysteine Rich Protein 61 (Cyr61, CCN1) in Chinese Adults: Distribution and Reference Intervals.

BACKGROUND: The circulating levels of Cyr61 (also known as CCN1) may prove to have great clinical value in the diagnosis, monitoring and prognosis of many disorders in humans. However, the reference intervals (RIs) for this analyte in human subjects have not previously been well established. Therefore, establishing RIs and determining the distribution of circulating Cyr61 levels are very important for future clinical studies and could provide an orientation value for exploring its clinical usefulness. METHODS: The Cyr61 levels in 2,514 healthy Chinese Han subjects (1,250 males and 1,264 females, aged 18 - 88 years, recruited from 4 hospitals in Shanghai and Fujian) were measured with a sandwich ELISA (R&D Systems, USA). The RIs were determined in a manner consistent with the Clinical and Laboratory Standards Institute guidelines. RESULTS: The levels of serum Cyr61 showed a non-Gaussian distribution. A statistically significant difference was observed between the males and females such that the median level of Cyr61 in the males was significantly higher than that in the females. Furthermore, the Cyr61 levels significantly increased with age in the female group whereas no difference was observed among the different age groups among the males. The RIs for serum Cyr61 were 3.3 - 184 pg/mL and 5.0 - 182 pg/mL in females aged 18 - 45 and 46 - 88 years, respectively. The RI for serum Cyr61 was 4.0 - 198 pg/mL in the males. CONCLUSIONS: The RIs for serum Cyr61 were established among Chinese Han individuals. The effects of age and gender on the distribution characteristics of serum Cyr61 were studied, revealing that the RIs were gender and, in females, age-specific, which may suggest that a female hormone, estrogen plays a role in the regulation of Cyr61 expression in vivo.

Retraction Note: Correlation of CD44v6 expression with ovarian cancer progression and recurrence.

The authors have retracted this original article [1] because a number of the figures exhibit irregularities.

CYR61, a potential biomarker of tumor inflammatory response in epithelial ovarian cancer microenvironment of tumor progress.

BACKGROUND: Recent studies have found that inflammatory response is involved in the pathogenesis of ovarian cancer. Advanced ovarian cancer is often presented with ascites that is rich in cytokines, inflammatory factors or cancer cells. Therefore, it is important to study the microenvironment of ascites in order to further clarify the occurrence and progression of ovarian cancer. As a pro-inflammatory factor, the Cyr61 expression patterns are inconsistent in human tumors. Although it has been reported that Cyr61 is related to the progression of ovarian cancer, its specific mechanism is not yet clear. This study sought to evaluate the Cyr61 levels of ascites, serum and different tissues of ovarian cancer to explore the potential association of Cyr61with the tumor-associated inflammatory microenvironment of EOC. METHODS: Tumor specimens were procured from patients with ovarian serous cystadenocarcinoma and ovarian serous cystadenoma. Cyr61 and IL-6 levels of serum or ascites were determined by ELISA (Enzyme-Linked ImmunoSorbent Assay), while Cyr61 expressions of different ovarian tumor tissues were evaluated by IHC (Immunohistochemistry). Then the correlation of Cyr61 level in ascites with clinicopathologic features was analyzed. And other laboratory data were obtained from medical records. RESULTS: Both in ascites and serum, significantly higher Cyr61 levels were found in ovarian serous cystadenocarcinoma. In malignant ascites, higher Cyr61 level of ovarian serous cystadenocarcinoma was more closely associated with FIGO stage, initial tumor size > 10 cm and the residual tumor size. And the increased IL-6 level was linearly related to Cyr61 level. Moreover, the serum levels of Cyr61, IL-6 and CRP in advanced stage of ovarian cancer were much higher than those in early stage. Lastly, the IHC data demonstrate that Cyr61 expression of ovarian serous adenocarcinoma was higher than that of ovarian serous cystadenoma, but it was lower than the paired metastatic lesions. CONCLUSIONS: As a pro-inflammatory factor, increased ascites Cyr61 level is associated with FIGO stage, initial tumor size > 10 cm and the residual tumor size. Moreover, serum Cyr61 may be used as a potential marker for EOC inflammatory response. Finally, Cyr61 may be involved in the process of tumor metastasis and progression by producing IL-6 and CRP in the EOC inflammatory microenvironment.

Cyr61/CCN1 is involved in the pathogenesis of psoriasis vulgaris via promoting IL-8 production by keratinocytes in a JNK/NF-κB pathway.

PURPOSE: Interleukin-8 (IL-8) is an important factor in the pathogenesis of psoriasis vulgaris, which is characterized by proliferation of keratinocytes, neutrophil infiltration and angiogenesis. Cysteine-rich 61 (Cyr61/CCN1), a secreted extracellular matrix protein, is a novel proinflammatory factor. Whether Cyr61 is involved in the development of psoriasis vulgaris via IL-8 production remains unknown. In this study we explore the role of Cyr61 in IL-8 expression regulation in vivo and in vitro. METHODS: Skin samples from normal donors and psoriasis vulgaris patients were examined the profile of Cyr61 and IL-8 using immunohistochemistry, real-time PCR and Western blotting. HaCaT cells were treated with Cyr61 and IL-8 expression was analyzed by real-time PCR and ELISA. Signal transduction pathways in Cyr61-induced IL-8 production were investigated by real-time PCR, western blotting, luciferase reporter assay or chromatin immunoprecipitation (ChIP) assay. IMQ-induced psoriasis-like mice were treated with anti-Cyr61monoclonal antibodies (mAb), or IgG1 as a control. RESULTS: We found that Cyr61 was abundant in the epidermis of patients with psoriasis vulgaris and positively correlated with the pathogenesis of skin lesions. Cyr61 induced IL-8 production by keratinocytes in a dose dependent manner. This IL-8 synthesis occurred in an IL-1ß- and TNF-α- independent mode via PI3K, AKT and JNK activation, with binding of enhanced AP-1, C/EBPß and NF-κB to sites located in the IL-8 promoter region. Treatment with anti-Cyr61 mAb led to reduction of MIP-2 level, decreased neutrophil infiltration, and attenuated inflammation in vivo. CONCLUSIONS: Our results not only reveal a novel mechanism illustrating the role of Cyr61 in epidermis pathogenesis but also suggest that therapies targeting Cyr61 may represent a novel strategy in the treatment of psoriasis vulgaris.

Molecular basis for the recognition of CCN1 by monoclonal antibody 093G9.

CCN1, also named Cyr61 (cysteine-rich protein 61), is the first identified member of the CCN family that is composed of 6 secreted extracellular matrix-associated glycoproteins. CCN1 has been demonstrated to participate in pathogenesis of rheumatoid arthritis through various pathways. A monoclonal antibody, namely, 093G9, is effective to antagonize the effects of CCN1 and hence has potential therapeutic benefits against rheumatoid arthritis. Here, we show that the epitope recognized by 093G9 is mapped to residues 77 to 80 of CCN1, and a cyclic peptide encompassing residues 75 to 81 of CCN1 displays high binding affinity for 093G9. The crystal structure of the 093G9 Fab in complex with the cyclic peptide was determined at 2.7 Å resolution, which reveals the intensive interactions between CCN1 and 093G9. Particularly, residues Asn79 and Phe80 of CCN1 are inserted into cavities mainly formed by residues of complementarity-determining region loop L3 and framework region L2 and by residues of complementarity-determining region loops H2 and H3, respectively, which contribute most of the interactions and therefore are critical for the recognition by 093G9. Together, these findings not only identify the epitope of CCN1 for 093G9 but also reveal the molecular mechanism of recognition and binding of CCN1 by 093G9.

Cyr61 participates in the pathogenesis of rheumatoid arthritis via promoting MMP-3 expression by fibroblast-like synoviocytes.

OBJECTIVES: The aim of this study was to investigate the effect and potential mechanism of Cysteine-rich 61 (Cyr61) on stimulating MMP-3 expression by fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients. METHODS: Primarily cultured RA FLS were treated with exogenous Cyr61 protein or Cyr61-siRNA, then, MMP-3 expression was analyzed by real-time PCR, western blotting and ELISA. Signal transduction pathways in Cyr61-induced MMP-3 production were examined by real-time PCR, western blotting, confocal microscopy, luciferase reporter assay. Mice with collagen-induced arthritis (CIA) were treated with anti-Cyr61 monoclonal antibodies (mAb), or IgG1 as control and MMP-3 in the joint was detected by IHC, real-time PCR and western blotting. RESULTS: High expressed MMP-3 and Cyr61 were positively correlated in RA ST; Cyr61 stimulated MMP-3 production in FLS of RA patients in an IL-1ß and TNF-α independent manner. Cyr61 induced MMP-3 could further enhance the invasive ability of RA FLS. Mechanistically, we found that Cyr61 promoted MMP-3 production via the P38, JNK-dependent AP-1 signaling pathway. Blockage of Cyr61 function with monoclonal antibody could decrease MMP-3 expression in the joints of CIA mice. CONCLUSION: This study provides new evidence that Cyr61 participates in RA pathogenesis not only as a pro-inflammatory factor but also plays a key role in bone erosion via promoting MMP-3 expression. We suggest that targeting of Cyr61 may represent a potential strategy in RA treatment.

Unique immunomodulatory effect of paeoniflorin on type I and II macrophages activities.

It has been widely accepted that macrophages are divided into M1 "pro-inflammatory" macrophages and M2 "anti-inflammatory" macrophages and an uncontrolled macrophage polarization plays an important role in the pathogenesis of different diseases. As the main substance of total glucosides of peony, paeoniflorin (PF), has been widely used to treat autoimmune and autoinflammatory diseases for years. Mechanistically, PF has been found to alter activities of many immune cells, which could further reduce inflammation and tissue damage. However, whether and how PF affects macrophages activities in vitro remains unknown. In current study, using M1 and M2 cells generated from mouse bone marrow precursors, we explored the role of PF in regulating M1/M2 cells activity in vitro. The results showed that PF inhibited LPS-induced M1 activity by reducing iNOS expression and NO production via decreasing LPS/NF-κB signaling pathway; whereas, PF enhanced IL-4-provoked M2 function by up-regulating Arg-1 production and activity via increasing IL-4/STAT6 signaling pathway. Our new finding indicates that PF can suppress M1 cells activity and enhance M2 cells function simultaneously, which could help to ameliorate autoimmune and autoinflammatory diseases in clinical treatment.

Cyr61 participates in the pathogenesis of rheumatoid arthritis by promoting proIL-1ß production by fibroblast-like synoviocytes through an AKT-dependent NF-κB signaling pathway.

IL-1ß plays a major role in the development of rheumatoid arthritis (RA). We previously showed that Cyr61 participates in RA pathogenesis as a proinflammatory factor. Here, we found that the levels of IL-1ß and Cyr61 were higher in RA SF than in osteoarthritis (OA) SF. IL-1ß mRNA and proIL-1ß protein levels were remarkably increased in Cyr61-stimulated FLS; however, IL-1ß was hardly detectable in the supernatant. We also found that the level of adenosine triphosphate (ATP) in SF and ST was significantly increased in RA patients and that the level of IL-1ß in supernatants from Cyr61-activated FLS increased significantly when we added exogenous ATP to the culture. Mechanistically, Cyr61 induced proIL-1ß production in FLS via the AKT-dependent NF-κB signaling pathway, and ATP caused Cyr61-induced proIL-1ß to generate IL-1ß in a caspase-1-dependent manner. Our results reveal a novel role of Cyr61 in RA that involves the promotion of proIL-1ß production in FLS.

Regulatory T cells in rheumatoid arthritis showed increased plasticity toward Th17 but retained suppressive function in peripheral blood.

OBJECTIVE: Regulatory T cells (Tregs) with the plasticity of producing proinflammatory cytokine IL-17 have been demonstrated under normal and pathogenic conditions. However, it remains unclear whether IL-17-producing Tregs lose their suppressive functions because of their plasticity toward Th17 in autoimmunity. The aim of this study was to investigate IL-17-producing Tregs from patients with rheumatoid arthritis (RA), and characterise their regulatory capacity and clinical significance. METHODS: Foxp3 and IL-17 coexpression were evaluated in CD4 T lymphocytes from RA patients. An in vitro T cell polarisation assay was performed to investigate the role of proinflammatory cytokines in IL-17-producing Treg polarisation. The suppressive function of IL-17-producing Tregs in RA was assessed by an in vitro suppression assay. The relationship between this Treg subset and clinical features in RA patients was analysed using Spearman's rank correlation test. RESULTS: A higher frequency of IL-17-producing Tregs was present in the peripheral blood of RA patients compared with healthy subjects. These cells from peripheral blood showed phenotypic characteristics of Th17 and Treg cells, and suppressed T cell proliferation in vitro. Tregs in RA synovial fluid lost suppressive function. The Th17 plasticity of Tregs could be induced by IL-6 and IL-23. An increased ratio of this Treg subset was associated with decreased levels of inflammatory markers, including the erythrocyte sedimentation rate and C-reactive protein level, in patients with RA. CONCLUSIONS: Increased levels of IL-17-producing Tregs were identified in RA patients. This Treg subset with Th17 plasticity in peripheral blood retained suppressive functions and was associated with milder inflammatory conditions, suggesting that this Treg population works as a negative regulator in RA, but in RA synovial site it may be pathogenic.

Paeoniflorin selectively inhibits LPS-provoked B-cell function.

B cells are important in the development of autoimmune disorders through mechanisms involving dysregulated polyclonal B-cell activation, production of pathogenic antibodies, and targeting which reduces inflammation and tissue damage effectively but often leads to patients suffering from secondary infection. Paeoniflorin (PF) is the main substance of the Total glucosides of peony and has been widely used to treat autoimmune diseases for years. However, whether PF affects B cell activity remains unknown. In this study, using purified murine spleen B cells, we analyzed the effects of PF on B-cell function in vitro. We found that PF inhibited the expression of CD69/CD86 and the proliferation of B cells stimulated by LPS. In addition, PF reduced the B-cell differentiation and immunoglobulin production that was stimulated by LPS. Interestingly, PF did not alter B-cell activation and proliferation provoked by anti-CD40 or IL-4. These results indicated for the first time that PF inhibits B-cell activation, proliferation and differentiation by selectively blocking the LPS/TLR4 signaling pathway. Furthermore, our data suggest that PF selectively inhibits inflammation and tissue damage mediated by LPS-activated B cells but does not alter CD40/CD40L- or IL-4-provoked B-cell function in autoimmune diseases treatment, which might aid in protecting patients from secondary infection.

Cyr61 induces IL-6 production by fibroblast-like synoviocytes promoting Th17 differentiation in rheumatoid arthritis.

Cysteine-rich protein 61 (Cyr61)/CCN1 is a product of an immediate early gene and functions in mediating cell adhesion and inducing cell migration. We previously showed that increased production of Cyr61 by fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) promotes FLS proliferation and participates in RA pathogenesis with the IL-17-dependent pathway. However, whether Cyr61 in turn regulates Th17 cell differentiation and further enhances inflammation of RA remained unknown. In the current study, we explored the potential role of Cyr61 as a proinflammatory factor in RA pathogenesis. We found that Cyr61 treatment dramatically induced IL-6 production in FLS isolated from RA patients. Moreover, IL-6 production was attenuated by Cyr61 knockdown in FLS. Mechanistically, we showed that Cyr61 activated IL-6 production via the αvß5/Akt/NF-κB signaling pathway. Further, using a coculture system consisting of purified CD4(+) T cells and RA FLS, we found that RA FLS stimulated Th17 differentiation, and the pro-Th17 differentiation effect of RA FLS can be attenuated or stimulated by Cyr61 RNA interference or addition of exogenous Cyr61, respectively. Finally, using the collagen-induced arthritis animal model, we showed that treatment with the anti-Cyr61 mAb led to reduction of IL-6 levels, decrease of Th17 response, and attenuation of inflammation and disease progression in vivo. Taken together, our results reveal a novel role of Cyr61 in promoting Th17 development in RA via upregulation of IL-6 production by FLS, thus adding a new layer into the functional interplay between FLS and Th17 in RA pathogenesis. Our study also suggests that targeting of Cyr61 may represent a novel strategy in RA treatment.

Total Flavonoids Isolated from the Leaves of Eucommia ulmoides Augment Peak Bone Mass in Female Rats and Show no Side Effects in Other Organs.

INTRODUCTION: Eucommia ulmoides is a unique monophyletic and tertiary relict in China and is listed as a national second-class precious protected tree species. Eucommia ulmoides, recognized as a traditional Chinese medicine, can tonify the liver and kidneys and strengthen bones and muscles. Modern pharmacological research has proved that Eucommia ulmoides has multiple osteoprotective effects, including prohibiting the occurrence of osteoporosis and arthritis and enhancing the healing of bone fractures and bone defects. AIM: To check its osteotropic effects, which may provide ideas for its potential use for the development of novel drugs to treat osteoporosis, this study evaluated the effect of total flavonoids from Eucommia ulmoides leaves (TFEL) on the acquisition of Peak Bone Mass (PBM) in young female rats. MATERIALS AND METHODS: TFEL was isolated, and its purity was confirmed by using a UV spectrophotometer. TFEL with a purity of 85.09% was administered to 6-week-old female rats by oral gavage at a low (50), mid (100), or high (200 mg/kg/d) dose, and the control group was administrated only with the same volume of water. After 13 weeks of treatment, the rats were sacrificed, and serum, different organs, and limb bones (femurs and tibias) were harvested, and the bone turnover markers, organ index, Bone Mineral Density (BMD), biomechanical property, and microstructure parameters were assayed. Furthermore, molecular targets were screened, and network pharmacology analyses were conducted to reveal the potential mechanisms of action of TFEL. RESULTS: Oral administration of TFEL for 13 weeks decreased the serum level of bone resorption marker TRACP-5b. As revealed by micro-computer tomography analysis, it elevated BMD even at a low dose (50 mg/kg/d) and improved the microstructural parameters, which were also confirmed by H&E histological staining. However, TFEL showed no effects on body weights, organ index, and micromorphology in the uterus. In our network pharmacology study, an intersection analysis screened out 64 shared targets, with quercetin, kaempferol, naringenin, and apigenin regulating the greatest number of targets associated with osteoporosis. Flavonoids in Eucommia ulmoides inhibited the occurrence of osteoporosis potentially through targeting signaling pathways for calcium, VEGF, IL-17, and NF-κB. Furthermore, AKT1, EGFR, PTGS2, VEGFA, and CALM were found to be potentially important target genes for the osteoprotective effects of flavonoids in Eucommia ulmoides. CONCLUSION: The above results suggested that TFEL can be used to elevate the peak bone mass in adolescence in female individuals, which may prevent the occurrence of postmenopausal osteoporosis, and the good safety of TFEL also suggests that it can be used as a food additive for daily life to improve the bone health.

Correlation of CD44v6 expression with ovarian cancer progression and recurrence.

BACKGROUND: Previously some groups demonstrated that CD44 variant 6 (CD44v6) is correlated with progression and metastasis of ovarian cancer. However, a number of other groups failed to find such an association. Moreover, epithelial ovarian cancer is known to easily metastasize to distinct sites such as the pelvic and abdominal cavities, but the potential association of CD44v6 expression with site-specific metastasis of ovarian cancer has not been explored. This study sought to evaluate the expression of CD44 standard (CD44s) and CD44v6 in primary, metastatic and recurrent epithelial ovarian cancer to explore the potential association of CD44s and CD44v6 with tumor progression and recurrence. METHODS: Tumor specimens were procured from patients with advanced (FIGO III, G3) and recurrent ovarian serous adenocarcinoma. CD44s and CD44v6 expression in the tumor tissues was evaluated by real-time RT-PCR and Western blot. Moreover, serum soluble CD44s or CD44v6 concentrations of early stage (FIGO I, G1), advanced (FIGO III, G3) and recurrent ovarian serous adenocarcinoma patients were determined by enzyme-linked immunosorbent assays (ELISA). CD44v6 expression in a different set of tumor samples on an ovarian cancer tissue chip was evaluated by immunohistochemistry (IHC) and the correlation of CD44v6 expression with clinicopathologic features was analyzed. Finally, the effects of knockdown of CD44v6 in SKOV3 cells on cell adhesion, invasion and migration were assessed. RESULTS: The expression of CD44v6, but not CD44s, is up-regulated in recurrent ovarian serous cancer compared to advanced primary tumor. CD44v6 expression is also preferentially increased in the tumor at the abdominal cavity metastasis site of advanced diseases. Consistently, serum soluble CD44v6 levels of recurrent ovarian cancer were higher than those of early stage and advanced primary diseases. The IHC data demonstrate that CD44v6 expression is correlated with clinicopathologic features and tumor progression. Lastly, knockdown of CD44v6 decreases the adhesion and migration but not invasion capacities of SKOV3 cells. CONCLUSIONS: CD44v6 expression levels are associated with epithelial ovarian cancer progression, metastasis and relapse. Moreover, serum soluble CD44v6 may be used as a potential marker for identifying tumor relapse. Finally, CD44v6 may play a role in ovarian cancer metastasis by mediating tumor cell adhesion and migration.

A novel anti-Cyr61 antibody inhibits breast cancer growth and metastasis in vivo.

Cysteine-rich protein 61(CCN1/Cyr61) has been implicated as an important mediator in proliferation and metastasis of breast cancer, which indicated that blockage of Cyr61 might be a potent target for breast cancer treatment. However, the antitumor effect of anti-Cyr61 antibodies on breast cancer in vivo has not been reported so far. In this study, we reported the effect and likely mechanism of generated anti-human Cyr61 monoclonal antibodies (mAb) on Cyr61 high expression line MDA-MB-231, known as a highly malignant and invasive human breast cancer cell line, at aspects of proliferation and migration in vitro and in vivo. We found the mAb, denoted as 093G9, revealed inhibitory effects on MDA-MB-231 cell proliferation, migration, and invasion through downregulation of both AKT and ERK phosphorylation in vitro compared with its isotype control. 093G9 also showed significant efficacy on suppressing primary tumor growth and spontaneous lymph node metastasis in in vivo mouse model. The specific epitope recognized by 093G9 was identified to be (140)LPNLGCP(146), adjacent to the VWC domain of Cyr61 by Ph.D.-C7C phage library display system. Our study provides direct evidence that Cyr61 can be a potent therapeutic target for patients who bear high Cyr61 expression breast cancer. Furthermore, the mAb, 093G9 developed in our laboratory, has shown a promising therapeutic characteristic in breast cancer.

Total glucosides of paeony inhibits Th1/Th17 cells via decreasing dendritic cells activation in rheumatoid arthritis.

Total glucoside of paeony (TGP), an active compound extracted from paeony root, has been used in therapy for rheumatoid arthritis (RA). Th1 and Th17 cells are now believed to play crucial roles in the lesions of RA. However, the molecular mechanism of TGP in inhibition of Th1 and Th17 cells remains unclear. In this study, we found that TGP treatment significantly decreased percentage and number of Th1 and Th17 cells in collagen induced arthritis (CIA) mice. Consistently, treatment with TGP decreased expression of T-bet and RORγt as well as phosphorylation of STAT1 and STAT3. In particular, TGP treatment inhibited dendritic cells (DCs) maturation and reduced production of IL-12 and IL-6. Moreover, TGP-treatment RA patients showed shank population of matured DCs and IFN-γ-, IL-17-producing cells. Taken together, our results demonstrated that TGP inhibited maturation and activation of DCs, which led to impaired Th1 and Th17 differentiation in vivo.

[Effects of Drynaria total flavonoids on apoptosis of osteoblasts mediated by tumor necrosis factor-α].

OBJECTIVE: To investigate the influence of Drynaria total flavonoids on proliferation and apoptosis of osteoblasts in tumor necrosis factor-α (TNF-α)- mediated medium, so as to explore the mechanism of Drynaria total flavonoids in preventing and treating osteoporosis of rheumatoid arthritis. METHODS: Twenty Wistar rats with average weight of (200±20) g were randomly divided into two groups: blank control group and Qianggu capsule (Drynaria total flavonoids) group. Rats in Qianggu capsule group were fed with 75 mg Qianggu capsule daily for continuous 3 d. One hour after the last feed, blood samples were collected. The in vitro experiment of four groups was designed: blank control serum group, Drynaria total flavonoids-containing serum group, blank control serum plus TNF-α group and Drynaria total flavonoids-containing serum plus TNF-α group. Methyl thiazolyl tetrazolium method was used to detect the proliferation of osteoblasts. Flow cytometry was used to detect the apoptosis of osteoblasts and real-time fluorescent quantitative polymerase chain reaction to detect the expressions of Bcl-2 and Bax mRNAs in osteoblasts. RESULTS: Compared with the control serum, Drynaria total flavonoids-containing serum promoted the proliferation and decreased the apoptosis of osteoblasts in TNF-α-mediated inflammatory environment (P<0.05), and increased the ratio of Bcl-2 mRNA to Bax mRNA. CONCLUSION: In TNF-α-mediated inflammatory environment, Drynaria total flavonoids can promote the proliferation and decrease the apoptosis of osteoblasts by improving the ratio of Bcl-2 mRNA to Bax mRNA, which may be one of the mechanisms of Drynaria total flavonoids in preventing and treating osteoporosis of rheumatoid arthritis.

Hypoxia-Induced Cyr61/CCN1 Production in Infantile Hemangioma.

BACKGROUND: Hypoxia may play a role in the pathogenesis of infantile hemangioma. Cysteine-rich angiogenic inducer 61 (Cyr61), or CCN1, can be induced under hypoxic conditions in several types of cells. However, whether CCN1 has any impact on infantile hemangioma remains unknown. This study aims to explore the expression of CCN1 in infantile hemangioma and to investigate the effect of hypoxia on CCN1 and vascular endothelial growth factor-A (VEGF-A) production. METHODS: Hemangioma-derived endothelial cells and hemangioma-derived stem cells were isolated from surgical specimens of proliferative infantile hemangioma. RNA extracted from infantile hemangioma tissue, hemangioma-derived endothelial cells, and hemangioma-derived stem cells was used to analyze gene expression by real-time polymerase chain reaction. The effects of CCN1 blockade were examined in hemangioma-derived stem cells. Immunostaining, immunoblotting, and enzyme-linked immunosorbent assays were used to assess protein expression. RESULTS: By double-label immunofluorescence staining, the authors first identified that CCN1 was abundant in proliferative infantile hemangioma lesions and colocalized well with immature microvessels. The authors found that the mRNA level of CCN1 in proliferative infantile hemangioma was significantly higher than in healthy controls, as was involuting infantile hemangioma. Treatment with the hypoxia inducer cobalt chloride dramatically increased CCN1 production in hemangioma-derived endothelial cells in a time-dependent manner. Furthermore, blocking or knockdown of CCN1 expression reduced the expression of VEGF-A in hemangioma-derived stem cells. Lastly, the signaling pathway study showed that CCN1 up-regulation of VEGF-A synthesis in hemangioma-derived stem cells depends on nuclear factor-κB and JNK activation. CONCLUSIONS: These findings provide new evidence that CCN1 participates in the crosstalk between hemangioma-derived endothelial cells and hemangioma-derived stem cells through promoting VEGF-A expression in the hypoxic environment of infantile hemangioma angiogenesis and vasculogenesis. Targeting of CCN1 might be a novel therapeutic strategy for infantile hemangioma.

Circ-GLI1 promotes metastasis in melanoma through interacting with p70S6K2 to activate Hedgehog/GLI1 and Wnt/ß-catenin pathways and upregulate Cyr61.

Circular RNAs (circRNAs) are emerging regulators in the development of human cancers. However, the role of circRNAs in melanoma is poorly understood. Microarray analysis and qRT-PCR was applied to screen out circRNAs that were differentially expressed in melanoma cells compared to normal cells. Currently, we first proved that inhibition of CYR61, an angiogenesis factor with controversial functions in melanoma, restrained cell migration, invasion and angiogenesis in melanoma. Thereafter, a novel circRNA hsa_circ_0027247 derived from GLI1 (circ-GLI1) was identified to positively modulate CYR61 expression in melanoma cell lines. Besides, silencing circ-GLI1 hindered melanoma cell metastasis as well. Interestingly, we unveiled that circ-GLI1 enhanced CYR61 transcription by an indirect manner. Meanwhile, circ-GLI1 activated Hedgehog/GLI1 and Wnt/ß-catenin pathways by affecting the degradation of GLI1 and ß-catenin. Moreover, we found that circ-GLI1 interacted with p70S6K2 to induce GSK3ß phosphorylation at Ser9, and therefore blocked the binding of GSK3ß with GLI1 and ß-catenin so as to elevate their protein expression. Of note, CYR61 was transcriptionally activated by MYC, a well-recognized downstream target of both GLI1 and ß-catenin. In conclusion, circ-GLI1 exacerbates the metastasis and angiogenesis of melanoma by upregulating Cyr61 via p70S6K2-dependent activation of Hedgehog/GLI1 and Wnt/ß-catenin pathways.

Role of IFN-gamma in induction of Foxp3 and conversion of CD4+ CD25- T cells to CD4+ Tregs.

IFN-gamma is an important Th1 proinflammatory cytokine and has a paradoxical effect on EAE in which disease susceptibility is unexpectedly heightened in IFN-gamma-deficient mice. In this study, we provide what we believe is new evidence indicating that IFN-gamma is critically required for the conversion of CD4+ CD25- T cells to CD4+ Tregs during EAE. In our study, the added severity of EAE in IFN-gamma knockout mice was directly associated with altered encephalitogenic T cell responses, which correlated with reduced frequency and function of CD4+ CD25+ Foxp3+ Tregs when compared with those of WT mice. It was demonstrated in both human and mouse systems that in vitro IFN-gamma treatment of CD4+ CD25- T cells led to conversion of CD4+ Tregs as characterized by increased expression of Foxp3 and enhanced regulatory function. Mouse CD4+ CD25- T cells, when treated in vitro with IFN-gamma, acquired marked regulatory properties as evidenced by suppression of EAE by adoptive transfer. These findings have important implications for the understanding of the complex role of IFN-gamma in both induction and self regulation of inflammatory processes.