OBJECTIVETo investigate the relationship between connective tissue growth factor (CCN5) and hepatic stellate cell (HSC) activation as well as the mechanism of action.

METHODSAs the research object, LX-2 cells were stimulated with transforming growth factor-beta1 ( TGF-(beta1), and the protein expression levels of CCN5 and CCN2 were determined by Western blot; Hepatocyte high expression system of CCN5 was constructed and transfected hepatic stellate cells (HSC) to make CCN5 overexpression; The expression levels of alpha-smooth muscle actin (alpha-SMA) and collagen I were determined by RT-PCR and Western blot. To further study its mechanism of action, Smad2 and phosphorylation level of Smad2 were determined by RT-PCR and Western blot.

RESULTSUnder normal circumstances, CCN2 expression levels were much higher than CCN5 in LX-2 cells, while CCN2 expression was significantly higher than CCN5 if LX-2 cells were stimulated by TGF-beta1. However, there was no change for CCN5. Compared with the control group and the vector group, CCN5 was successfully overexpressed in the transfection group, and mRNA and protein levels of alpha-SMA and collagen I were significantly decreased (P < 0.01). Meanwhile, phosphorylation level of Smad2 was also significantly decreased (P < 0.01).

CONCLUSIONCCN5, which has the function that inhibits HSC activation, has the opposite role compared with CCN2, therefore, a new idea was proposed for the prevention and treatment of liver fibrosis.

Actins ; metabolism ; CCN Intercellular Signaling Proteins ; metabolism ; Cell Line ; Collagen Type I ; metabolism ; Connective Tissue Growth Factor ; metabolism ; Hepatic Stellate Cells ; metabolism ; Humans ; Liver Cirrhosis ; metabolism ; pathology ; Repressor Proteins ; metabolism ; Smad2 Protein ; metabolism

OBJECTIVES: 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. METHODS: 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 (=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. RESULTS: Compared with the sham group, the articular cartilage was severely damaged, the Mankin score was increased significantly (<0. 05), the mRNA and protein expression levels of DKK1 in cartilaginous tissue were markedly decreased(<0.05), while those of WISP, Wnt1, LRP5 and beta-catenin were increased significantly in model group(<0.05). Compared with model group, the articular cartilage lesions was light (<0.05), the Mankin Score was decreased significantly(<0.05), and the mRNA and protein levels of DKK1 in cartilaginous tissue were increased(<0.05), while those of WISP, Wnt1, LRP5 and beta-catenin were decreased in Yougui pill high-dose group and glucosamine sulfate group (<0.05). CONCLUSIONS 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.
Animals ; CCN Intercellular Signaling Proteins ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Glucosamine ; pharmacology ; Intercellular Signaling Peptides and Proteins ; metabolism ; Osteoarthritis, Knee ; drug therapy ; Proto-Oncogene Proteins ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Wnt Signaling Pathway ; Wnt1 Protein ; metabolism ; beta Catenin ; metabolism

OBJECTIVEProgressive pseudorheumatoid dysplasia (PPD) (MIM#208230) is a rare autosomal recessive disease of cartilage homeostasis characterized by axial and peripheral skeletal dysplasia. Analysis of WISP3 (Wnt1-inducible signaling pathway protein 3, MIM#603400) gene mutation can confirm the clinical and radiographic diagnosis for PPD. This study aimed to recognize PPD based on clinical manifestations and imaging characteristics of bones, and to investigate the mutations of WISP3 gene in three patients with PPD.

METHODThree male patients (9 - 16 years old) from three unrelated Chinese families, who presented with joint pain, swelling, deformities and motion limitation, were referred to this study. PPD was diagnosed on the basis of the clinical manifestations, imaging characteristics of bones and laboratory evaluation. All five exons and their exon/intron boundaries of the WISP3 gene were amplified by polymerase chain reaction (PCR) from the peripheral blood DNA of three PPD family members, and mutation analysis was performed by bidirectional DNA sequencing.

RESULT(1) Three patients were diagnosed as PPD by characteristic evidences: all patients presented with non-inflammatory multiple joints swelling and stiffness including joints in hand and feet as they age. Radiographs showed platyspondyly, ovoid or wedged anterior end-plate of vertebral bodies, coxa vara, widened epiphyses or metaphyses including capital femoral, metacarpophalangeal, interphalangeal joints and metatarsals. Normal laboratory values were found for the erythrocyte sedimentation rate and C-reactive protein, rheumatoid factors, antinuclear antibodies etc. (2) The three different mutations of WISP3 gene were identified in three patients with PPD, including two small insert mutations (c.624_625insA, c.866_867insA), one was deletion mutation (c.729_735delGAGAAAA). The types of mutation of two alleles in three patients were c.624_625insA/c.729_735delGAGAAAA, c.624_625insA/c.866_867insA and c.866_867 insA/c.866_867insA, respectively. These mutations were found in exon 4 and exon 5 of WISP3 gene, accounting for 50%(3/6) respectively. All three different mutations were novel variations, and none of 3 novel variations was found in the 50 controls.

CONCLUSIONThe characteristic evidences of PPD were non-inflammatory multiple enlarged joints (including hand and feet), limited movement, normal laboratory values such as rheumatoid factors. It is essential for making diagnosis to carefully examine the entire skeleton including spine. The characteristics of bone imaging are platyspondyly, widened epiphyses or metaphyses including large and small joints and narrow joint spaces. Three different novel variations of WISP3 gene were identified in three PPD patients, they are c.624_625insA, c.866_867insA and c.729_735delGAGAAAA. Each of novel mutations is insert or deletion mutation.

Adolescent ; Arthropathy, Neurogenic ; diagnosis ; genetics ; CCN Intercellular Signaling Proteins ; Child ; Humans ; INDEL Mutation ; Insulin-Like Growth Factor Binding Proteins ; genetics ; Joint Diseases ; congenital ; Male ; Molecular Sequence Data

OBJECTIVE: To construct two types of Wnt-inducible secreted protein 3(WISP3) gene's mutants(1000T/C,840delT) found in spondyloepiphyseal dysplasia tarda with progressive anthopathy (SEDT-PA) patients, and to observe their expression in COS-7 cells. METHODS: Full-length cDNA of wild type WISP3 gene(WT-WISP3) was amplified from human chondrocytes by RT-PCR, and site-directed mutagenesis was used to obtain full-length cDNAs of the mutated WISP3 genes(MUT1000T/C and MUT840delT). The recombined plasmids WT-WISP3/pcDNA3.1(+), MUT1000T/C/pcDNA3.1(+) and MUT840delT/pcDNA3.1(+) were transfected transiently into COS-7 cells by liposome-mediated method, and pcDNA3.1(+) vector was used as a control. The total RNA and protein of the transfected COS-7 cells were extracted after 48 hours of transfection. The expression of WISP3 gene in the transfected COS-7 cells was detected by semi-quantitative RT-PCR and Western blot. RESULTS: By restriction endonuclease analysis and sequencing, the sequence of MUT1000T/C and MUT840delT were consistent with that mutated in SEDT-PA, and the open reading frames matched with the vector sequence. Semi-quantitative RT-PCR and Western blot showed that the recombined plasmids were highly expressed in COS-7 cells. CONCLUSION WISP3 gene's mutants of SEDT-PA are successfully constructed by genetic recombination, and expressed in COS-7 cells, which lays the foundation for the further study on its molecular functions in SEDT-PA.
Animals ; Base Sequence ; CCN Intercellular Signaling Proteins ; COS Cells ; metabolism ; Chlorocebus aethiops ; Humans ; Insulin-Like Growth Factor Binding Proteins ; biosynthesis ; genetics ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Mutation ; Osteochondrodysplasias ; genetics ; metabolism ; Transfection

OBJECTIVETo investigate the effect of irradiated human lung fibroblasts (HLFs) on the canonical Wnt/β-catenin signaling pathway in human umbilical cord mesenchymal stem cells (HUMSCs).

METHODSHUMSCs were cultured alone (single group) or co-cultured with HLFs exposed to 5Gy X-rays (co-culture group). The protein levels of GSK-3β, p-GSK-3β, FRAT1 and β-catenin in HUMSCs were examined by Western blotting 3 days after culture or co-culture. WISP-1 protein levels in conditioned medium were examined by ELISA.

RESULTSThe levels of p-GSK3β/GSK3β (0.15 ± 0.05), FRAT1 (0.48 ± 0.07) and β-catenin (0.50 ± 0.07) in co-cultured HUMSCs significantly decreased compared to those in single group (0.55 ± 0.05, 1.16 ± 0.13 and 2.39 ± 0.15, all P<0.05). The supernatant level of WISP-1 in co-culture group was significantly decreased [(602.23 ± 161.47) ng/mL], compared to the single group [(977.77 ± 110.56) ng/mL, P<0.05].

CONCLUSIONIrradiated HLFs attenuate the activation of canonical Wnt/β-catenin signaling pathway in HUMSCs in vitro.

CCN Intercellular Signaling Proteins ; metabolism ; Cells, Cultured ; Coculture Techniques ; Fibroblasts ; cytology ; radiation effects ; Gamma Rays ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Humans ; Intracellular Signaling Peptides and Proteins ; metabolism ; Mesenchymal Stromal Cells ; metabolism ; Proto-Oncogene Proteins ; metabolism ; Umbilical Cord ; cytology ; Wnt Signaling Pathway ; X-Rays ; beta Catenin ; metabolism

This study was aimed to investigate the mRNA and protein expression of CTGF, CYR61, VEGF-C and VEGFR-2 in bone marrow of patients with leukemia, and to analyze the role and clinical significance of these 4 factors in genesis and development of leukemia, infiltration and metastasis of leukemic cells. A total of 100 cases of newly diagnosed leukemia, 26 cases of acute leukemia in complete remission and 30 controls were enrolled in this study. The mononuclear cells of bone marrow were collected, the mRNA and protein expression levels of CTGF, CYR61, VEGF-C, VEGFR-2 in leukemia patients and controls were detected by real time PCR and Western blot, respectively. The results showed that the mRNA and protein expression levels of above mentioned 4 factors were significantly higher than those in control (P < 0.05), only CTGF mRNA expression in AL patients after complete remission showed statistical difference as compared with control (P < 0.05), but the expression of CTGF mRNA showed statistical significance in different bone marrow hyperplasia of acute leukemia (P < 0.05). The expression level of CTGF protein showed difference in different chromosome karyotypes of leukemia (P < 0.05). The expression levels of CYR61 and VEGF-C proteins showed statistical difference in different bone marrow hyperplasia of acute leukemia (P < 0.05). The expression level of CTGF, CYR61, VEGF-C mRNA and protein in CML group were higher than that in control group. The expression levels of CTGF and CYR61 protein were higher than that in control. The mRNA and protein expression levels of above-mentioned 4 factors in sex and infiltration lf leukemic cells did not show statistical significance(P < 0.05). In correlative analysis, the mRNA expressions of above mentioned 4 factors were positively correlated with bone marrow blast count(P < 0.05), the protein expression of CTGF, CYR61 and VEGF-C were positively correlated with bone marrow blast count. It is concluded that the CTGF, CYR61, VEGF-C and VEGFR-2 mRNA and protein play a role in acute leukemia. In acute leukemia (AML/ALL), the expression of above mentioned factor was high, but except VEGFR-2. Most of them were positively correlated with bone marrow blast count. Joint block of these angiogenesis-related factors is likely to play an important role in targeting treatment of leukemia.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Bone Marrow ; metabolism ; pathology ; Case-Control Studies ; Child ; Child, Preschool ; Connective Tissue Growth Factor ; metabolism ; Cysteine-Rich Protein 61 ; metabolism ; Female ; Humans ; Leukemia ; metabolism ; pathology ; Male ; Middle Aged ; RNA, Messenger ; genetics ; Vascular Endothelial Growth Factor C ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism ; Young Adult

The study was aimed to detect the levels of CYR61, CTGF, VEGF-C, VEGFR-2 mRNA in bone marrow (BM) of leukemia patients and investigate the interaction of CYR61, CTGF, VEGF-C, VEGFR-2 proteins in occurrence, development, infiltration and metastasis of leukemia and its clinical significance, to find a new tumor marker for diagnosis and treatment of leukemia with some new directions. 74 patients with leukemia were enrolled in this study, 38 out of them were males and 36 were females, aged from 6 to 77 years old with the median age of 45 years old. In the control group, 7 males and 5 females, aged from 16 to 78 years old with the median age of 46. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to detect the levels of CYR61, CTGF, VEGF-C, VEGFR-2 mRNA. The results showed that the levels of CYR61, CTGF, VEGF-C, VEGFR-2 mRNA in BM of newly diagnosed patients with acute and chronic leukemia of each group were significantly higher as compared with the control group (p < 0.05). The levels of CYR61, CTGF mRNA in acute leukemia remission group were significantly higher than those in control group (p = 0.039, 0.025). The level of CTGF mRNA was highest in B-ALL group, and was higher than that in AML, CML, CLL, T-ALL groups (p = 0.002, 0.034, 0.002, 0.010). In AML group, mRNA expressions of CYR61 and CTGF, CYR61 and VEGF-C, CTGF and VEGFR-2 were positively correlated (r = 0.452, 0.466, 0.464; p = 0.045, 0.038, 0.039), and in CML group mRNA expression of CYR61 and VEGF-C was positively correlated (r = 0.882, p = 0.000). The expression levels of VEGF-C, VEGFR-2 mRNA in acute leukemia patients with extramedullary infiltration were higher than those in acute leukemia patients without extramedullary infiltration (p = 0.028, 0.047). VEGF-C mRNA expression and the original cell counts in AML group were positively correlated (r = 0.418, p = 0.034). It is concluded that CYR61, CTGF, VEGF-C and VEGFR-2 interact each other in the pathogenesis of leukemia, promote the development, metastasis and infiltration of leukemia; and these factors in different types of leukemia and extramedullary infiltration are different, which may become tumor markers of leukemia; and blocking VEGF-C and VEGFR-2 may block tumor growth and metastasis.
Adolescent ; Adult ; Aged ; Bone Marrow ; metabolism ; Case-Control Studies ; Child ; Connective Tissue Growth Factor ; metabolism ; Cysteine-Rich Protein 61 ; metabolism ; Female ; Humans ; Leukemia ; metabolism ; pathology ; Male ; Middle Aged ; RNA, Messenger ; genetics ; Vascular Endothelial Growth Factor C ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism ; Young Adult

The protein expression of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl(-) channel, in ovarian stimulated premature female rat ovary during a cycle of follicle development and corpus luteum formation was investigated. Animals were injected with 10 U pregnant Mare's serum gonadotropin (PMSG) and subsequently 10 U hCG 48 h later. Time-dependent immunohistochemistry and Western blotting experiments were performed before and 24, 48, 72 h after hCG treatment. The immunohistochemistry revealed that administration of PMSG stimulated the CFTR expression in thecal cell layer and granulosa cell layer of mature follicles 48 h post injection, coincident with the PMSG-induced peak in follicular estradiol. However, the expression of CFTR in the granulose lutein cell layer and thecal lutein cell layer was time-dependently reduced following hCG injection, in accordance with the gradually increased progestogen level during luteum corpus formation. Western blotting analysis demonstrated that rat ovarian tissue expressed the special CFTR band at 170 kD. It is concluded that cAMP-dependent Cl(-) channels are involved in regulation of follicle development and luteum formation.
Connective Tissue Growth Factor/genetics ; Connective Tissue Growth Factor/*metabolism ; Connective Tissue Growth Factor/*physiology ; Corpus Luteum/growth & development ; Ovarian Follicle/growth & development ; Ovary/*metabolism ; Rats, Wistar

Connective Tissue Growth Factor ; Humans ; Myocardial Infarction ; physiopathology ; Ventricular Remodeling

OBJECTIVE: To investigate the role of Cyr61 in angiotensin Ⅱ (AngⅡ)-induced functional changes in HEK293 cells and explore the mechanism. METHODS: Cyr61 knockdown in cultured HEK293T cells was achieved by transfection of the cells with CRISPR/Cas9 KO plasmid. The changes in apoptosis and expression levels of Cyr61 and Bcl-2 in the cells with or without Cyr61 knockdown in response to treatment with 10 mol/L AngⅡ for 48 h were analyzed using flow cytometry, qRT-PCR and Western blotting. RESULTS: The cells with Cyr61 knockdown showed significantly decreased expression of Cyr61 protein as compared with the control cells ( < 0.05). AngⅡ treatment for 48 h significantly increased the expression of Cyr61 and lowers the expression of Bcl-2 at both the protein and mRNA levels in HEK293T cells. In HEK293T cells with Cyr61 knockdown, AngⅡ treatment resulted in significantly increased expression of Bcl-2 in HEK293T cells as compared with that of the control group ( < 0.05). AngⅡ treatment caused significantly increased apoptotic rate in HEK293T cells as compared with the cells with Cyr61 knockdown [(26.94 ± 3.73)% (3.87 ± 0.83)%, < 0.05), and the apoptosis rate was significantly lowered to (15.76 ± 1.31)% in HEK293T cells with Cyr61 knockdown following AngⅡ treatment ( < 0.05). CONCLUSIONS The up-regulation of Cyr61 expression is related with AngⅡ-induced injury in HEK293T cells, and down-regulating Cyr61 expression can effectively protect HEK293T cells against AngⅡ-induced injury.
Angiotensin II ; Apoptosis ; Cysteine-Rich Protein 61 ; HEK293 Cells ; Humans ; Up-Regulation