Wnt-induced Secreted Protein-1 Research Articles

Unraveling the mechanism in l-Caldesmon regulating the osteogenic differentiation of PDLSCs: An innovative perspective

Maxillofacial bone defect is one of the common symptoms in maxillofacial, which affects the function and aesthetics of maxillofacial region. Periodontal ligament stem cells (PDLSCs) are extensively used in bone tissue engineering. The mechanism that regulates the osteogenic differentiation of PDLSCs remains not fully elucidated. Previous studies demonstrated that l-Caldesmon (l-CALD, or CALD1) might be involved in the osteogenic differentiation of PDLSCs. Here, the mechanism by which CALD1 regulates the osteogenic differentiation of PDLSCs is investigated. The osteogenic differentiation of PDLSCs is enhanced with Cald1 knockdown. Whole transcriptome sequencing (RNA-seq) analysis shows that bone morphogenetic proteins (BMP) signaling pathway and Wingless type (Wnt) pathway have significant change with Cald1 knockdown, and the expressions of Wnt-induced secreted protein 1 (WISP1), BMP2, Smad1/5/9, and p-Smad1/5/9 are significantly upregulated, while Glycogen synthase kinase 3β (GSK3β) and p-GSK3β are downregulated. In addition, subcutaneous implantation in nude mice shows that knockdown of Cald1 enhances the osteogenic differentiation of PDLSCs in vivo. Taken together, this study demonstrates that knockdown of Cald1 enhances the osteogenic differentiation of PDLSCs by BMP and Wnt signaling pathways, and provides a novel approach for subsequent clinical treatment.

Analysis of CCN4/WISP1 Effects on Joint Tissues Using Gain- and Loss-of-Function Approaches.

The matricellular protein Wnt-induced secreted protein 1 (WISP1) is the fourth member of the CCN family of proteins, which has been shown to affect tissues of the musculoskeletal system. In the context of the musculoskeletal disorder osteoarthritis, our lab studied the function of CCN4/WISP1 in joint tissues, including synovium and cartilage, using both gain- and loss-of-function approaches. In mice, this was done by genetic engineering and recombination to generate mice deficient in CCN4/WISP1 protein. Various experimental models of osteoarthritis with different characteristics were induced in these mice. Moreover, CCN4/WISP1 levels in joints were experimentally increased by adenoviral transfections. Osteoarthritis pathology was determined using histology, and the effect of different CCN4/WISP1 levels on gene expression was evaluated in individual tissues. Effects of high levels of CCN4/WISP1 on chondrocytes were studied with an in vitro chondrocyte pellet model. In this chapter, we describe the procedures to conduct these experiments.

Application of Hyaluronic Acid as a Biopolymer Material in Reconstruction of Interdental Papilla in Rats

Applying hyaluronic acid, a biopolymer material, in the treatment of interdental papilla reconstruction has become a trend. The main objective of this research is to investigate the histologic effect of hyaluronic acid on interdental papilla over time. Deficient interdental papilla models were surgically created in sixty-two Sprague-Dawley (SD) rats and were randomly treated with the injection of hyaluronic acid (HA group) or phosphate-buffered saline (sham control group) or left untreated (control group). After 2, 4, and 8 weeks, the rats were sacrificed in batches to observe the histological changes. A fluorochrome label was used to monitor bone formation in 8 weeks. Immunohistochemical analyses were performed to evaluate the expression of potentially relevant cytokines, vascular endothelial growth factor (VEGF), bone morphogenetic protein-2 (BMP-2), and Wnt-induced secreted protein 1 (WISP1) in the gingival tissue in 8 weeks. A preliminary study of HA degradation after 24 weeks was performed in two rats. Following the HA injection, no inflammation or granulomatous foreign body reaction was observed. HA was able to promote collagen fiber and alveolar bone regular formation in the reconstruction site. HA also enhanced VEGF, BMP-2, and WISP-1 expression in gingival tissue (p＜0.05). After 24 weeks, there was no HA filler observed in the interdental papilla. In conclusion, our study suggested that HA is an effective way to reconstruct deficient interdental papilla.

Serum WNT-induced secreted protein 1 level as a potential biomarker for thyroid nodules

Background. Thyroid nodule (TN) is a common thyroid disease worldwide, and it has increased significantly last decades. Most TNs are usually incidental findings of asymptomatic, benign lesions discovered by imaging modalities performed for reasons unrelated to thyroid diseases. The purpose of this study was to investigate the value of serum WNT-induced secreted protein 1 (WISP1) level as a supporting biomarker to perform differential diagnosis of benign and non-benign thyroid nodules. Materials and methods. The study was completed with the 89 patients undergone fine needle aspiration biopsy and 43 controls. The patients were composed of 96 (72.7%) females and 36 (27.3%) males. And they were divided into 2 group according to the Bethesda cytological evaluation as Benign (Bethesda 2) and Non-Benign (Bethesda 3–6) groups. Their serum WISP1 levels were measured by an ELISA method. Results. There were 58 (43.9%) patients in Benign (Bethesda 2) and 31 (23.5%) in non-Benign (Bethesda 3–6) groups. In the contrary nodule size was bigger in the Non-benign group than that benign group (p=0.006). The serum WISP1 level in the Benign (Bethesda 2) group was significantly higher than that in the and Non-Benign (Bethesda 3–6) group, and controls (p<0). The difference between benign and non-benign group accordingly to their echogenicitiy was significant (p<0.05). In benign group there was 76.9% mixed echoic nodules, 76.7% isoechoic nodules 68.4% isohypoechoic nodules and 35.7% hypoechoic nodules. In the non-benign group, the highest hypoechoic echo (64.3%), the least mixed echo (23.1%), while in the benign group, the most mixed echo (76.9%), the least hypoechoic echo (35.7%) was present. There was no relation between WISP1 levels and echogenicity with Kruskal-Wallis H test. Conclusions. According to the preliminary results of current study, addition of serum WISP1 measurement to the differential diagnostic work-up of thyroid nodules patients may provide supportive information. Inthyroid nodules patients with Benign (Bethesda 2) category of cytological evaluation, a higher level of serum WISP1 may support cytological diagnosis.

Knockdown of long non-coding RNA CCAT2 suppresses growth and metastasis of esophageal squamous cell carcinoma by inhibiting the β-catenin/WISP1 signaling pathway.

ObjectiveLong non-coding RNA (lncRNA) colon cancer-associated transcript 2 (CCAT2) plays oncogenic roles in several cancers, including esophageal squamous cell carcinoma (ESCC). However, the specific mechanism of how CCAT2 influences ESCC tumorigenesis is still unknown.MethodsUsing RT-qPCR, the mRNA expression levels of CCAT2 in 33 paired ESCC and adjacent non-cancer tissues and cell lines were measured. Lentiviral vector sh-CCAT2 was designed and transfected into TE10 cells. CCK-8 and transwell assays were employed to detect the effects of CCAT2 knockdown on cell proliferation and invasion, respectively. RT-qPCR and western blots were used to detect the effects of CCAT2 knockdown.ResultsCCAT2 was overexpressed in ESCC tissues compared with corresponding adjacent tissues. CCAT2 knockdown could suppress cell proliferation and invasion in vitro. Furthermore, knockdown of CCAT2 could suppress the mRNA and protein levels of β-catenin and Wnt-induced-secreted-protein-1 (WISP1), as well as the mRNA levels of their downstream targets VEGF-A, MMP2, and ICAM-1. High expression of CCAT2 and WISP1 were associated with poor prognosis of ESCC patients.ConclusionsIn conclusion, a novel CCAT2/β-catenin/WISP1 axis was revealed in ESCC progression and may provide a promising therapeutic target against ESCC. CCAT2 and WISP1 are potential molecular biomarkers for predicting prognosis of ESCC.

Wingless/int-1induced secreted protein-1: a new biomarker for renal fibrosis.

This study aimed to detect the expression of Wnt-induced secreted protein-1 (WISP-1) in renal fibrosis (RF) and to clarify the underlying mechanism. An in vivo mousee model of unilateral ureteral obstruction (UUO) and in vitro model of fibrosis on renal tubular epithelial NRK52E cells after transforming growth factor-β1 (TGF-β1) stimulation were used. Quantitative real-time PCR (qRT-PCR), Western blot (WB), and immunohistochemistry were used to detect WISP-1 and fibrosis markers, including the expression of fibronectin (FN), collagen I (Col I), collagen IV (Col IV), and α-smooth muscle actin (α-SMA). In vitro experiments showed that the expression of WISP-1 and fibrosis markers FN, Col I, Col IV, and α-SMA in rat renal tubular epithelial cells were significantly higher than that in the control group after 48 h of TGF-β1 stimulation. In vivo experiments showed that the expressions of WISP-1 and fibrosis markers FN, Col I, Col IV, and α-SMA in the obstructed kidney of UUO animal models were significantly increased in mRNA and protein levels compared to normal mice. This study showed that WISP-1 may be an essential cytokine that promotes renal fibrosis, being involved in the development of renal fibrosis.

Changing Expression Profiles of lncRNAs During Ischemia-Reperfusion-Induced Renal Fibrosis

The pathological physiology of end-stage renal disease is characterized by renal fibrosis, for which suitable therapeutics have not yet been developed. Early tubular damage in ischemia-reperfusion (IR) injury induced acute kidney injury (AKI) is highly relevant to later fibrosis, but the mechanism remains unclear. Moreover, more and more evidence has proven that long non-coding ribonucleic acids (lncRNA) regulate gene expression associated with renal fibrosis. Therefore, we analyzed lncRNA profiles in the kidneys of mice using RNA sequencing during the pathogenesis of IR-induced renal fibrosis. Our results showed that expression levels of 43 lncRNAs and 141 lncRNAs were respectively significantly changed 7 days and 2 weeks after IR treatment. We predicted the functions of the differentially expressed (DE) lncRNAs and target genes with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Interaction networks of lncRNAs, microRNAs (miRNAs) and mRNA were constructed based on the correlation analysis of the DE genes. Based on these informations, we further verified lncRNA (Gm12840) play competing endogenous RNA (ceRNA) mechanism with miR-677-5p to meidiate TGF-β1 induced fibroblast activation via Wnt-induced secreted protein-1 (WISP1)/ protein kinase B (Akt) signaling pathway. Our findings identify that lncRNAs are involved in IR-induced late fibrosis of kidney and may represent a systematic perspective on the important function of IR-induced renal fibrosis as well as new therapeutic targets. Funding Statement: This work was supported by grants from National Natural Science Foundation of China (81860130), Natural Science Foundation of Guangdong province (2016A030313376), Guangdong Province Science and Technology Planning Project (2017B020247035, 2014A020212612). Declaration of Interests: All authors declare that they have no competing interests. Ethics Approval Statement: All experiments using animals were approved by the Institutional Animal Care and Use Committee of the First People hospital of Foshan and breed as approved by the Institutional Animal Care protocol.

Non‑canonical Wnt signaling contributes to ventilator‑induced lung injury through upregulation of WISP1 expression.

Mechanical ventilation may cause ventilator-induced lung injury (VILI). Canonical Wnt signaling has been reported to serve an important role in the pathogenesis of VILI. Bioinformatics analysis revealed that canonical and non-canonical Wnt signaling pathways were activated in VILI. However, the role of non-canonical Wnt signaling in the pathogenesis of VILI remains unclear. The present study aimed to analyze the potential role of non-canonical Wnt signaling in VILI pathogenesis. Lung injury was assessed via Evans blue albumin permeability and histological scoring, as well as by inflammatory cytokine expression and total protein concentration in bronchoalveolar lavage fluid. The relative protein expression of canonical and non-canonical Wnt signaling pathway components were examined via western blotting and immunohistochemistry. The results demonstrated that 6 h of mechanical ventilation at low tidal volume (LTV; 6 ml/kg) or moderate tidal volume (MTV; 12 ml/kg) induced lung injury in sensitive A/J mice. Ventilation with MTV increased the protein levels of Wnt-induced secreted protein 1 (WISP1), Rho-associated protein kinase 1 (ROCK1), phosphorylated (p)-Ras homolog gene family, member A and p-C-Jun N-terminal kinase (JNK). Inhibition of ROCK1 by Y27632 and JNK by SP600125 attenuated MTV-induced lung injury and decreased the expression of proteins involved in non-canonical Wnt signaling, including WISP1. In conclusion, non-canonical Wnt signaling participates in VILI by modulating WISP1 expression, which has been previously noted as critical for VILI development. Therefore, the non-canonical Wnt signaling pathway may provide a preventive and therapeutic target in VILI.

MicroRNA-384 regulates cell proliferation and apoptosis through directly targeting WISP1 in laryngeal cancer.

Laryngeal cancer (LC) is an increasingly common malignant tumors of head and neck cancer. Aberrant expression of microRNA (miRNA) is closely related with LC development. In the current study, we investigated the biological function and underlying molecular mechanism of miR-384 in LC. The results showed that the miR-384 expression was markedly downregulated in LC tissue and cell lines (TU212 and TU686) as compared with that of adjacent nontumor tissues and a normal human bronchial epithelial cell line. Next, we performed gain-of-function and loss-of-function experiments in the TU212 and TU686 cells by transfecting the cells with miR-384 mimics, miR-384 inhibitor, or miRNA control. Moreover, results showed that miR-384 mimic remarkably inhibited LC cell proliferation, which was notably decreased by miR-384 inhibitor. Furthermore, miR-384 mimics notably increased the amounts of DNA fragmentation from the apoptotic cells (a hallmark of apoptosis) and the caspase-3 activity, whereas miR-384 inhibitor resulted in a decline of DNA fragmentation and the caspase-3 activity compared with its control. In addition, a dual-luciferase reporter assay confirmed that Wnt-induced secreted protein-1 (WISP1) gene was a direct target of miR-384. MiR-384 mimic remarkably inhibited the messenger RNA and protein expression of WISP1, which was upregulated by miR-384 inhibitor as compared to its control. WISP1 knockdown by small interfering RNA inhibited LC cell proliferation and promoted cell apoptosis. WISP1 overexpression partly abrogates the effect of miR-384 overexpression. Taken together, these data indicate that miR-384 regulates LC cell proliferation and apoptosis through targeting WISP1 signaling pathway, providing a novel insight into the LC treatment.

Human Leukocyte Antigen F Locus Adjacent Transcript 10 Overexpression Disturbs WISP1 Protein and mRNA Expression to Promote Hepatocellular Carcinoma Progression.

Recently, studies on transcriptome-proteome relationships have revealed mRNA/protein expression discordance for certain genes and speculated that protein posttranslational modification (PTM) may be involved. However, there is currently no evidence to support this hypothesis. Wnt-induced secreted protein-1 (WISP1) is the downstream target gene of β-catenin and plays an important role in tumorigenesis and progression, but the expression and role of WISP1 in different tumor types are controversial. Here, we first confirmed that WISP1 protein expression was significantly down-regulated in hepatocellular carcinoma (HCC) tissue and could be an independent predictor of poor prognosis for patients with HCC. In vivo and in vitro evidence was provided that WISP1 can suppress HCC cell proliferation. Further studies have found that low WISP1 protein expression was related to expression of human leukocyte antigen F locus adjacent transcript 10 (FAT10), a specific ubiquitin-like protein with both degradation and stabilization functions, which plays an important role in PTM. FAT10 overexpression facilitated WISP1 degradation by FAT10ylation to decrease WISP1 protein expression, thus promoting HCC proliferation. Interestingly, we found and demonstrated that FAT10 overexpression could result in WISP1 protein/mRNA expression discordance, with protein expression decreasing while mRNA expression increased. The underlying mechanism is that FAT10 exerts substrate stabilization and degradation functions simultaneously, while FAT10 overexpression promotes WISP1 mRNA expression by stabilizing β-catenin and directly degrades WISP1 protein. Conclusion: Our study demonstrated that overexpression of FAT10 results in expression discordance between WISP1 protein and mRNA, thereby promoting HCC progression by down-regulating WISP1 protein expression.

Identification of Senescence‐associated Genes in Rhesus Monkey Bone Marrow‐Derived Mesenchymal Stem Cells Cultured in A Defined Serum‐free Media

BackgroundMesenchymal stem cells (MSCs) are multipotent cells that are currently being investigated in a wide variety of clinical trials for their anti‐inflammatory and immunomodulatory properties. The purity and properties for the resulting populations can be affected by the conditions under which MSCs are cultured. Serum‐free medium would greatly facilitate the development of robust, clinically acceptable bioprocesses for reproducibly generating quality‐assured cells. Clinical trials as well as animal studies have shown that the regeneration potential of bone and other tissues declines with age due to a decline in the number or frequency of stem cells present in adult organs; these factors may contribute to human aging and age‐related disease.MethodsOur in‐house serum free medium, consists of α‐MEM supplemented with PDGF, TGF‐β1, bFGF, IGF, Sodium Bicarbonate, L‐Glutamine, Hepes, Putrescine, progesterone, hydrocortisone, defined lipid concentrate, Fetuin, ascorbic acid, Insulin‐transferrin‐selenium, human albumin, serotonin and Folic acid. MSCs were first cultured under both our defined serum‐free medium and 10% FBS medium conditions. Immunophenotype analysis was performed with CD90, CD29, CD105, HL‐ADR, CD44 and CD34. And cells were treated with CCK‐8 for 24 h, 48 h, 96 h, 120 h,144 h,168 h and 192 h, respectively. Then, this study compared rBMSCs in three age groups: young (< 5 years), middle (8–10 years), and old (>15years). Using NOISeq method to filter 1909 differentially expressed genes (DEGs) out of 24,204 total genes analyzed, we identified 224 highly senescence‐related DEGs.ResultsThe expansion levels of MSCs in serum‐free culture exhibited better cell morphology and immunophenotype, positive for CD29 (99.9%) and CD105 (98.2%), and negative for CD34 (2.4%) and HLA‐DR (0.2%)(Figure. 1–2). And CCK‐8 assay showed that MSCs cultured in serum‐containing medium exhibited better cell proliferation activity. Also, the serum‐free culture cells showed better differentiation capacity. And then, GO term categorization revealed that the identified genes are strongly related to known biological process, such as angiogenesis and immune responses (Figure 3–5). Notably, many of the SASP components over‐secreted by senescent rBMSCs are to the immune system process. And we found a robust increase of several inflammatory cytokine genes in senescent rBMSCs, including IL‐6, IL‐8, CCL‐2, GM‐CSF, MMP3 and ICAM. Meanwhile, we found that the senescence of MSCs are strongly related to the angiogenesis genes, such as PDGFR, WISP1, SDF‐1 and TGF‐β1(Figure 6). And WNT‐induced secreted protein 1 (WISP1), also a downstream factor of the canonical WNT signaling pathway, is known to affect proliferation and differentiation of MSCs.ConclusionsOur results indicate that the use of SFM in MSC culture can give better cell quality than serum‐containing medium. And SASP increases the complexity of paracrine communication among rBMSCs and their physiological/pathological microenvironment. The genes identified using RNA‐seq will facilitate future studies of the mechanisms underlying the cellular senescence of MSCs.Support or Funding InformationFounding: Chengdu Giant Panda Breeding Research Foundation (2016cdspt001) Project Name: Rhesus Monkey Mesenchymal Stem Cells Biological Study and Resource Pool EstablishmentThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The therapeutic effects of Yougui pill on knee osteoarthritis and the expression of Wnt signal pathway related factors in rats

To observe the effects of Yougui pill (Traditional Chinese Medicine) on the related factors of Wnt signal pathway of rats with knee osteoarthritis (KOA), and explore its protective mechanism. Sixty SPF SD rats were randomly divided into the sham-operative group, model group, glucosamine sulfate group, high-dose, middle-dose, low-dose of Yougui pill treated group (n=10). KOA model was established by modified Hulth method for six weeks. The rats in the high, middle and low-dose of Yougui pill group were treated with Yougui pills at the doses of 20,10 and 5 g/kg respectively by gastrogavage once a day for 8 weeks, while equal volume of normal saline was given to those in the sham and model control group and an equal volume of glucosamine sulfate (1.7 g/kg·d) was given to those in glucosamine sulfate group for 8 weeks. The knee joint was removed after the last dose of drug. The pathological changes of cartilaginous tissues were observed under a microscope. The mRNA levels of Dickkopf homolog 1(DKK1), Wnt induced secreted protein 1(WISP1), Wnt1, low density lipoprotein receptor related protein 5(LRP5) and beta -catenin in rats cartilaginous tissues were analyzed by using RT-PCR method, and the protein contents of DKK1, WISP1, Wnt1, LRP5 and beta-catenin in cartilaginous tissues were detected by Western blot. Compared with the sham group, the articular cartilage was severely damaged, the Mankin score was increased significantly (P<0. 05), the mRNA and protein expression levels of DKK1 in cartilaginous tissue were markedly decreased(P<0.05), while those of WISP, Wnt1, LRP5 and beta-catenin were increased significantly in model group(P<0.05). Compared with model group, the articular cartilage lesions was light (P<0.05), the Mankin Score was decreased significantly(P<0.05), and the mRNA and protein levels of DKK1 in cartilaginous tissue were increased(P<0.05), while those of WISP, Wnt1, LRP5 and beta-catenin were decreased in Yougui pill high-dose group and glucosamine sulfate group (P<0.05). Yougui pill has protective effects on the KOA by inhibiting the expressions of WISP, Wnt1, LRP5, beta-catenin and increasing the expression of DKK1 cytokine in the Wnt signaling pathway.

Abstract 4938: A vicious cycle between osteoblasts-derived WISP-1 and mesenchymal-like cancer cells is essential to prostate cancer metastasis

Abstract Prostate cancer (PCa) is the most commonly diagnosed malignancy in the United States and other Western countries. It is well established that tumor microenvironment plays an important role in cancer progression and metastasis. Wnt-induced secreted protein-1 (WISP-1) belongs to the CCN family (CTGF/CYR61/NOV) that plays a key role in osteogenesis and tumor growth. We found that osteoblast-conditioned media (OBCM) stimulated migration and invasion in human PCa cells. Furthermore, the medium was collected 2 days after treatment of osteoblasts with PCa conditioned media which were designed as PCa-OBCM. The results showed that treatment of PCa cells with PCa-OBCM further increased the migration and invasion activity, as well as epithelial-mesenchymal transition (EMT), including the epithelial marker (E-cadherin) down-regulation and mesenchymal marker (N-cadherin and twist) up-regulation. Moreover, human cytokine array was used to measure the secretion of 32 Cytokines into PCa-OBCM. The data showed that WISP-1 is highly up-regulated with PCa-OBCM treatment compared to OBCM. Stimulation of osteoblasts with PCa conditioned media induced WISP-1 expression. In contrast, knockdown WISP-1 in osteoblasts impacted PCa-OBCM-mediated cancer migration, invasion, and EMT. In addition, conditioned media from PCa cells was incubated with another cytokine array membranes. The data showed that ADAM-9 and cathepsinL were overexpressed in PCa conditioned media but only ADAM-9 could regulate WISP-1 expression in osteoblasts. Taken together, these results demonstrated that primary PCa-secreted ADAM-9 promotes osteoblasts-derived WISP-1 up-regulation, which in turn enhances primary PCa cells migration, invasion, and EMT, resulting in cancer metastasis. Our study suggests that inhibition of osteoblasts-derived WISP-1 is a new therapeutic target for treating metastasis in human PCa patients. Citation Format: An-Chen Chang, Show-Mei Chuang, Chih-Hsin Tang. A vicious cycle between osteoblasts-derived WISP-1 and mesenchymal-like cancer cells is essential to prostate cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4938. doi:10.1158/1538-7445.AM2017-4938

Expressions of Wingless and Int1 (Wnt)-induced secreted protein 1 in paraquat-poisoned patients

Purpose: To study the expression of Wingless &amp; Int1 (Wnt)-induced secreted protein 1 (WISP1) in paraquat (PQ)-poisoned patients.Methods: A total of 37 PQ-poisoned patients were enrolled in the study, and divided into non-survivor group (NS) and survival group (S) based on the final therapeutic outcome. Besides, another normal control group (NC) comprised of normal healthy people. Serum PQ concentration was determined by high performance liquid chromatography (HPLC), while reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to evaluate WISP1 in the serum of PQ poisoned patients.Results: PQ intake in NS and S groups were 23.58 ± 26.23 and 143.18 ± 263.04 mL, respectively, while serum PQ concentration was 2.07 ± 0.67 and 4.12 ± 1.74 mg/L, respectively. Significant correlation was found between the outcome of patients and serum PQ concentration (OR = 1.434, p &lt; 0.01). Serum PQ concentration was closely correlated with WISP1 gene expression levels (OR = 0.621, p &lt; 0.01) and serum WISP1 protein levels (OR = 0.596, p &lt; 0.01) on the first day after poisoning. Furthermore, a correlation between serum PQ concentration and WISP1 levels was found on the third after poisoning (OR = 0.447, p &lt; 0.01).Conclusion: WISP1 is over-expressed in PQ-poisoned patients, and serum PQ concentration may be a useful index for the prognosis of PQ poisoned patients.Keywords: Wingless &amp; Int1 (Wnt)-induced secreted protein 1, Poison, Paraquat, Prognosis, Correlation

Notch1-WISP-1 axis determines the regulatory role of mesenchymal stem cell-derived stromal fibroblasts in melanoma metastasis.

Mesenchymal stem cells-derived fibroblasts (MSC-DF) constitute a significant portion of stromal fibroblasts in the tumor microenvironment (TME) and are key modulators of tumor progression. However, the molecular mechanisms that determine their tumor-regulatory function are poorly understood. Here, we uncover the Notch1 pathway as a molecular determinant that selectively controls the regulatory role of MSC-DF in melanoma metastasis. We demonstrate that the Notch1 pathway's activity is inversely correlated with the metastasis-regulating function of fibroblasts and can determine the metastasis-promoting or -suppressing phenotype of MSC-DF. When co-grafted with melanoma cells, MSC-DFNotch1−/− selectively promote, while MSC-DFN1IC+/+ preferentially suppress melanoma metastasis, but not growth, in mouse models. Consistently, conditioned media (CM) from MSC-DFNotch1−/− and MSC-DFN1IC+/+ oppositely, yet selectively regulates migration, but not growth of melanoma cells in vitro. Additionally, when co-cultured with metastatic melanoma cells in vitro, MSC-DFNotch1−/− support, while MSC-DFN1IC+/+ inhibit melanoma cells in the formation of spheroids. These findings expand the repertoire of Notch1 signaling as a molecular switch in determining the tumor metastasis-regulating function of MSC-DF. We also identified Wnt-induced secreted protein-1 (WISP-1) as a key downstream secretory mediator of Notch1 signaling to execute the influential role of MSC-DF on melanoma metastasis. These findings reveal the Notch1—WISP-1 axis as a crucial molecular determinant in governing stromal regulation of melanoma metastasis; thus, establishing this axis as a potential therapeutic target for melanoma metastasis.

Thymosin β4 Prevents Angiotensin II-Induced Cardiomyocyte Growth by Regulating Wnt/WISP Signaling.

Thymosin beta-4 (Tβ4) is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. However, the role of Tβ4 in cardiomyocyte hypertrophy is currently unknown. The purpose of this study was to determine the cardio-protective effect of Tβ4 in angiotensin II (Ang II)-induced cardiomyocyte growth. Neonatal rat ventricular cardiomyocytes (NRVM) were pretreated with Tβ4 followed by Ang II stimulation. Cell size, hypertrophy marker gene expression and Wnt signaling components, β-catenin, and Wnt-induced secreted protein-1 (WISP-1) were evaluated by quantitative real-time PCR, Western blotting and fluorescent microscopy. Pre-treatment of Tβ4 resulted in reduction of cell size, hypertrophy marker genes and Wnt-associated gene expression, and protein levels; induced by Ang II in cardiomyocyte. WISP-1 was overexpressed in NRVM and, the effect of Tβ4 in Ang II-induced cardiomyocyte growth was evaluated. WISP-1 overexpression promoted cardiomyocytes growth and was reversed by pretreatment with Tβ4. This is the first report which demonstrates that Tβ4 targets Wnt/WISP-1 to protect Ang II-induced cardiomyocyte growth. J. Cell. Physiol. 231: 1737-1744, 2016. © 2015 Wiley Periodicals, Inc.

MECHANICAL VENTILATION AUGMENTS POLY(I:C)INDUCED LUNG INJURY VIA A WISP1-INTEGRIN β3 DEPENDENT PATHWAY IN MICE.

Mechanical ventilation can improve hypoxemia, but can also cause the so-called ventilator-induced lung injury (VILI). Polyinosinic-polycytidylic acid (poly(I:C)), an analogue of natural double strand RNA virus, can induce lung inflammation. The purpose of this study was to determine whether moderate tidal volume mechanical ventilation (MTV) augments Poly(I:C)-induced lung injury, and if so, the mechanism responsible for it. Poly(I:C) (2μg/g) were instilled intratracheally in C57BL/6J wide type (WT) mice. They were then randomized to MTV (10ml/kg tidal volume) or spontaneous breath. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected 4h later for various measurements. Our results showed that MTV did not cause significant injury in normal lungs, but augmented Poly(I:C)-induced lung injury. The expression level of WNT-induced secreted protein 1 (WISP1) was consistent with lung injury, and the amplification of lung injury by MTV can be alleviated by anti-WISP1 antibody treatment. MTV further increased Poly(I:C)-induced integrin β3 expression in the lung. And co-immunoprecipitation (Co-IP) results suggested there was an interaction between WISP1 and β3. WISP1 significantly increased Poly(I:C)-induced TNF-α production in macrophages isolated from WT mice but not in macrophages isolated from β3 knock-out mice. Co-treatment with WISP1 and Poly(I:C) markedly increased the phosphorylation of extracellular signal-related kinase (ERK) in macrophages. Pretreating macrophages with an ERK inhibitor, U0126, dose-dependently antagonized WISP's synergistic effect on Poly(I:C)-induced TNF-α release. In conclusion, MTV exaggerates Poly(I:C)-induced lung injury in a WISP1 and integrin β3 dependent manner, involving, at least part, the activation of the ERK pathway. The WISP1-integrin β3 pathway could be an important target for novel therapy.

Effects of WISP-1 on Matrix Metalloproteinase Expression and Wound Healing in Human Skin Fibroblasts via Down-Regulation of miR-519d

WISP-1 (Wnt-induced secreted protein-1), from the CCN gene family, also call CCN4, which is involved in many cellular activities such as growth, differentiation, cell motility, adhesion, division and repair. Wound healing is an intricate process whereby the skin repairs itself after injury. Here, we report that WISP-1 increased wound healing ability in human skin fibroblasts. Moreover, incubation of cells with WISP-1 increased the mRNA and protein expression of matrix metalloproteinase (MMP)-2 and MMP-3. Transfection of cells with siRNA against MMP-2 and MMP-3 reduced WISP-1-promoted wound healing ability. In addition, incubation of cells with WISP-1 diminished microRNA (miR)-519d expression. Co-transfection with miR-519d mimic reversed WISP-1-mediated MMP expression and wound healing ability. Our results suggest that WISP-1 increases MMP-2 and MMP-3 expression and promotes wound healing via inhibition of miR-519d expression in human skin fibroblasts.

Deletion of WISP1 leads to higher sensitivity to carbon tetrachloride-induced liver damage

WISP1 (Wnt-induced secreted protein-1) is a member of the CCN family of matricellular proteins. These highly conserved and secreted proteins can interact with integrins and modulate many biological processes like migration, proliferation, differentiation and apoptosis. CCN proteins have been show to play divergent roles in liver pathophysiology, in spite of having very similar structural domains. Importantly, little is known about WISP1 in this context. Gene array analysis of mouse liver after acute injury by intraperitoneal injection of CCl4 in C56BL/6N mice showed WISP1 mRNA upregulated during early injury phase, between 2h and 24h after CCl4 injection. These results were confirmed by real time PCR, showing a maximal induction of 40 fold over healthy liver at 24h. To establish the role of WISP1 in acute liver damage we used a knockout mouse model, in which WISP1 expression is disrupted by replacing Exon 2 with a resistance cassette and a stop codon. Heterozygous mice were bred to establish homozygous knockout and wild type lines which were validated by genotyping. Real time PCR analysis confirmed the absence of WISP1 mRNA expression in KO mice challenged with CCl4. Remarkably, deletion of WISP1 led to a significant increase of necrotic area at 24h. This was show by hematoxylin & eosin staining reaching 49% in KO vs. 27% in WT mice (p < 0.0001; n = 13 wild type, n = 11 KO) and serum transaminase activity (GPT: 8230 U/l in WT, 12300 U/l in KO). Importantly, immunofluorescence and real time PCR showed that WT and WISP1 KO mice have no significant differences in CYP2E1 expression, suggesting that the difference in tissue injury does not depend on alterations of CCl4 metabolism in KO mice. To further characterize this different phenotype, we performed a time course after CCl4 intoxication (460 mg/kg, i.p.) by collecting liver tissue at 30 min, 2h, 8h, 18h, and 24h. Interestingly, already at 18h KO mice showed a clear necrotic area around the central veins whereas WT mice showed ballooned hepatocytes with cytoplasmic translocation of high-mobility group protein B1 (HGMB1), indicating that WISP1 KO mice had a more advanced stage of liver injury at this time point than the WT counterparts. This phenotype was also observed upon intoxication with lower doses of CCl4 (132.4 mg/kg and 76 mg/kg). Analysis of cytokine expression by real time PCR showed a stronger induction of TNFα in WISP1 KO mice (7-fold v/s 10-fold at 18h, and 3-fold vs. 6-fold at 24h in WT vs. WISP1 KO respectively), and IL6 (5-fold vs. 10-fold at 18h, and no induction vs. 3-fold at 24h in WT v/s WISP1 KO respectively). Furthermore, western blot analysis showed marked alterations in signal transduction pathways in liver tissue of WISP1 KO mice, with stronger activations of p-JNK at 2h and p-STAT3 at 18h and 24h. Currently we are investigating the mechanism through which WISP1 exerts a protective role upon CCl4 stimulation in liver damage and in the subsequent regeneration process. In conclusion we have strong evidence that WISP1 plays an important role in acute liver damage.

Expression of CCN family members correlates with the clinical features of hepatocellular carcinoma.

Studies have reported that the CCN family of proteins plays an important role in stimulating tumorigenesis. However, the relationship between the CCN protein family members and the features of hepatocellular carcinoma (HCC) remains unclear. The objective of this study was to determine the relationship between the expression levels of CCN protein family members and the features of HCC. Expression levels of the CCN family of proteins in 80-paired primary HCC samples and 11 normal liver samples were determined by a quantitative real-time PCR assay. Enhanced expression of nephroblastoma overexpressed protein (NOV) and decreased expression of Wnt-induced secreted protein 1 (WISP1), cysteine-rich protein 61 (CYR61) and connective tissue growth factor (CTGF) were found in HCC samples when compared to levels in matched non-cancerous tissues. No significant difference in WISP2 was found between matched-pair samples; only a few samples showed WISP3 expression. Furthermore, the expression levels of NOV, WISP1 and CYR61 were closely correlated with certain clinical features, including venous invasion, cellular differentiation, pTNM stage, disease-free survival and overall survival. Our results suggest that HCC progression may be enhanced by NOV and suppressed by WISP1 and CYR61. Our statistical analysis suggests that these proteins may be valuable in determining the prognosis of this deadly disease and directs attention to modulating the levels of these proteins as a potential mode of therapy.