CCN1 upregulates IL-36 via AKT/NF-κB and ERK/CEBP ß-mediated signaling pathways in psoriasis-like models.

Psoriasis is a chronic skin disorder characterized by epidermal keratinocyte hyperproliferation and inflammatory infiltration. CCN1 (also termed CYR61 or cysteine-rich angiogenic inducer 61) is an extracellular matrix-associated protein that is involved in multiple physiological functions. In psoriasis, we recently demonstrated that the overexpression of CCN1 promoted keratinocyte proliferation and activation. Furthermore, CCN1 was highly expressed in psoriatic skin lesions from psoriasis vulgaris patients. Here, we dissect the underlying molecular mechanism in imiquimod (IMQ) and interleukin (IL)-23-induced psoriasis-like models. Our results demonstrate that CCN1 can significantly upregulate IL-36 production in the murine skin of IMQ and IL-23-induced psoriasis-like models. Injection of CCN1-neutralizing antibody improved epidermal acanthosis and significantly reduced IL-36 production in vivo. These results suggest that CCN1 can be a critical upstream pro-inflammatory factor in psoriasis. In primary normal human epidermal keratinocytes, we demonstrated that CCN1 can selectively induced the production of IL-36α and IL-36γ through the activation of the protein kinase B (AKT)/nuclear factor kappa light chain enhancer of activated B cells (NF-κB) and extracellular-regulated kinase (ERK)/CCAAT/enhancer binding protein ß (CEBPß) signaling pathways via integrin receptor α6ß1 in vitro. Our results suggest that targeting CCN1 can be a potential therapeutic strategy for psoriasis.

Generation of a safe and efficacious llama single-domain antibody fragment (vHH) targeting the membrane-proximal region of 4-1BB for engineering therapeutic bispecific antibodies for cancer.

BACKGROUND: The discovery of checkpoint inhibitors towards cytotoxic T-lymphocyte protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) has been revolutionary for the treatment of cancers. These therapies have only offered an average of 20%-30% response rates across the tumor spectrum and the combination of agonists towards the tumor-necrosis superfamily members, such as 4-1BB and CD40, has shown potent efficacy in preclinical studies; however, these agonists have exhibited high degrees of toxicity with limited efficacy in human trials. In this study, we have generated a single-domain antibody towards a unique epitope of 4-1BB that limits its potential on-target toxicity while maintaining sufficient potency. This 4-1BB binder is ideal for use in the engineering of multispecific antibodies to localize 4-1BB activation within the tumor microenvironment, as shown here by a anti-PD-L1/4-1BB bispecific candidate (PM1003). METHODS: To determine the functional activity of the 4-1BB- and PD-L1-binding elements of PM1003, in vitro luciferase reporter and primary cell assays were used to test the potency of programmed cell death 1 ligand 1 (PD-L1) blockade and PD-L1-mediated 4-1BB activation via cross-bridging. X-ray crystallography was conducted to resolve the binding epitopes of the respective binding arms, and accurate binding kinetics were determined using standard affinity measurement techniques. Human 4-1BB and/or PD-L1 knock-in mice were used in cancer models for testing the in vivo antitumor efficacy of PM1003, and safety was evaluated further. RESULTS: PM1003 shows potent activation of 4-1BB and blockade of PD-L1 in cell-based assays. 4-1BB activation was exerted through the bridging of PD-L1 on target cells and 4-1BB on effector cells. No PD-L1-independent activation of 4-1BB was observed. Through X-ray crystallography, a unique binding epitope in the cysteine-rich domain 4 (CRD4) region was resolved that provides high potency and potentially low on-target toxicity as determined by primary immune cell assays and toxicity evaluation in vivo. CONCLUSIONS: A unique single-domain antibody was discovered that binds to the CRD4 domain of 4-1BB. When incorporated into a 4-1BB/PD-L1 bispecific (PM1003), we have shown the potent inhibition of PD-L1 activity with 4-1BB agonism upon cross-bridging with PD-L1 in vitro. Antitumor activity with minimal toxicity was found in vivo. Thus, PM1003 is a uniquely differentiating and next generation therapeutic agent for cancer therapy.

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.

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.

CCN1 promotes IL-1ß production in keratinocytes by activating p38 MAPK signaling in psoriasis.

CCN1, an extracellular protein also known as cysteine-rich protein 61 (Cyr61), is a novel pro-inflammatory factor involved in the pathogenesis of rheumatoid arthritis. As an inflammatory disease, psoriasis is characterized by keratinocyte activation-induced epidermal hyperplasia and cytokine-mediated inflammation. We demonstrated in our previous study that CCN1 promoted keratinocyte activation in psoriasis. However, the role of CCN1 in regulating inflammation in psoriasis is still unknown. Here, we showed that CCN1 increased inflammatory cytokine IL-1ß production in keratinocytes. Furthermore, endogenous ATP and caspase-1 were required for mature IL-1ß production stimulated by CCN1 in keratinocytes. After binding to the receptor of integrin α6ß1, CCN1 activated the downstream p38 MAPK signaling pathway, thus inducing the expression of IL-1ß. In addition, we inhibited CCN1 function in mouse models of psoriasis, and decreased IL-1ß production was observed in vivo. Overall, we showed that CCN1 increased IL-1ß production via p38 MAPK signaling, indicating a role for CCN1 protein in regulating inflammation in psoriasis.

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.

Paeoniflorin ameliorates symptoms of experimental Sjogren's syndrome associated with down-regulating Cyr61 expression.

Paeoniflorin (PF), an active compound extracted from Paeony root, has been used in therapy of autoimmune diseases with effective clinical efficiency and higher safety. Sjogren's syndrome (SS) is a chronic, systemic, immune-mediated inflammatory disease. In this study, we demonstrated that novel pro-inflammatory factor Cyr61/CCN1 was up-regulated in epithelial cells of salivary glands of primary SS patients and submandibular gland autoantigen-induced experimental SS mice. Blocking Cyr61 expression with special monoclonal antibody improved saliva secretion by ameliorating inflammatory infiltration and cytokines production in vivo. Furthermore, we showed that PF could alleviate inflammation by down-regulating Cyr61 expression in experimental SS mice. In conclusion, our new findings revealed for the first time that Cyr61 involves the pathogenesis of primary SS and PF alleviates SS-like symptoms associated with inhibiting Cyr61 expression, providing new insights into the potential molecular mechanism of PF in primary SS treatment.

CCN1, a Pro-Inflammatory Factor, Aggravates Psoriasis Skin Lesions by Promoting Keratinocyte Activation.

Psoriasis is a common chronic skin disease characterized by epidermal hyperplasia and inflammation. The pathogenesis of psoriasis is multifactorial and is not fully understood. Here we demonstrate that CCN1 (also called Cyr61, which is short for cysteine-rich 61), an extracellular matrix protein that is also considered a pro-inflammatory factor, is highly expressed in the lesional skin of psoriasis patients, as well as in that of imiquimod (IMQ)- and IL-23-treated psoriasis-like mice. Then we show that blocking CCN1 function in vivo attenuates epidermal hyperplasia and inflammation in psoriasis-like mice. Further, in primary cultured normal human keratinocytes and HaCaT (human keratinocyte cell line) cells, CCN1 promotes keratinocyte activation, including the proliferation and expression of immune-related molecules. Finally, we observe that integrin α6ß1 is the receptor of CCN1 in keratinocytes, and CCN1 stimulation activates the downstream phosphoinositide-3 kinase/Akt/NF-κB signaling pathway. Taken together, our findings reveal that CCN1 has a critical role in psoriasis pathogenesis. Moreover, as CCN1 is a secreted extracellular matrix (ECM) protein, our study also provides evidence that ECM, which is involved in psoriatic pathogenesis, could be a potent target for psoriasis 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.

Paeoniflorin inhibits skin lesions in imiquimod-induced psoriasis-like mice by downregulating inflammation.

Psoriasis is a common chronic immune-mediated inflammatory disease. It is well known that macrophages, neutrophils and T-helper 1 (Th1)/T-helper 17 (Th17) cells play important roles in skin lesions by provoking inflammation. Paeoniflorin (PF) is the major effective component extracted from the root of Paeonia lactiflora, which has been widely used in China to treat inflammatory and autoimmune diseases, including psoriasis. Although PF shows a clinical therapeutic effect on psoriasis patients, how PF affects infiltrated immune cells in psoriasis skin lesions is still unknown. In this study, using a generated imiquimod (IMQ)-induced psoriasis-like mouse model, we found that PF ameliorates inflammation and skin lesions. Subsequent analyses showed that PF decreases the number of F4/80(+)CD68(+) macrophages and their related cytokine production (TNF-α, IL-1ß, IL-6, IL-12 and inducible nitric oxide synthase (iNOS)) in the skin of IMQ-challenged mice. Moreover, PF suppresses the number of CD11b(+)Gr-1(+) neutrophils and the expression of macrophage inflammatory protein-2 (MIP-2; a counterpart of human IL-8, which is responsible for the recruitment of neutrophils in mice). Finally, PF also down-regulates Th1- and Th17-related cytokine expression. Therefore, our new findings reveal that PF alleviates psoriatic skin lesions by inhibiting inflammation, which provides new insights into the immunomodulatory effect of PF in psoriasis treatment.

CD133 expression in renal cell carcinoma (RCC) is correlated with nuclear hypoxia-inducing factor 1α (HIF-1α).

PURPOSE: In our previous study, we have found that the hypoxia-inducible factor 1α (HIF-1α) is upregulated in renal cell carcinoma (RCC) tissues compared with para-cancer normal tissues by 2-dimensional polyacrylamide gel electrophoresis. It was reported that hypoxic conditions were correlated with cancer stem cell generation and HIF-1α acted as a transcription regulator in nuclear HIF-1α expression. Therefore, in this study we investigate the relation between CD133 and nuclear HIF-1α expression levels in RCC tissues. METHODS: In this study 61 RCC tissues from the patients that treated with radical nephrectomy were collected. Then, we investigated the expression of CD133 and nuclear HIF-1α expression by immunohistochemistry. To verify the relation between CD133 and nuclear HIF-1α expression, we treated 786-O cells with cobalt chloride. The expression of CD133 on 786-O cells was analyzed by flowcytometry. RESULTS: The immunohistochemical study showed that CD133 was correlated with tumor stage and metastatic stage, whereas nuclear HIF-1α had no association with clinicopathological parameters. However, the expression of nuclear HIF-1α was correlated with CD133. The CD133 expression in 786-O cells was enhanced by cobalt chloride, which meant that CD133 expression was affected by hypoxia. CONCLUSIONS: Our study showed that in RCC, CD133 expression was strongly related to nuclear HIF-1α and the expression of CD133 might be upregulated under hypoxia environment.