scholarly journals The β-catenin/TCF-4 pathway regulates the expression of OPN in human osteoarthritic chondrocytes

2020 ◽  
Author(s):  
Jian Tian ◽  
Shu-Guang Gao ◽  
Yu-Sheng Li ◽  
Chao Cheng ◽  
Zhen-Han Deng ◽  
...  
Keyword(s):  
Cartilage Destruction ◽  
Transient Transfection ◽  
Human Chondrocytes ◽  
Mrna Levels ◽  
Osteoarthritic Chondrocytes ◽  
Mrna And Protein Expression ◽  
Oa Chondrocytes ◽  
Canonical Wnt ◽  
Dickkopf 1

Abstract Background: Cartilage destruction is the main characteristic of osteoarthritis (OA), and osteopontin (OPN) is elevated in OA articular cartilage; however, the reason for the increased OPN level is not determined. In addition, Wnt/β-catenin signaling participates in the progression of OA. The aim of the present study was to evaluate whether canonical Wnt signaling could regulate the expression of OPN in human chondrocytes in vitro.Methods: Human chondrocytes were cultured in vitro, and we first assayed the mRNA levels of OPN and β-catenin in chondrocytes. Next, we performed transient transfection of TCF 4 shRNA into chondrocytes to inhibit TCF 4 expression and explore changes in the OPN level. Then, the Wnt/β-catenin signaling inhibitor Dickkopf-1 (Dkk-1) was incubated with chondrocytes, and we assayed the changes in β-catenin and OPN.Results: Our results showed that the expression of both β-catenin and OPN was increased in OA chondrocytes, but there were no correlations between β-catenin and OPN expression. TCF4 shRNA downregulated the expression of TCF 4 and OPN in chondrocytes, while after treatment with rDKK-1 at a concentration of 400 ng/ml for 24 h, the mRNA and protein expression of both β-catenin and OPN was significantly decreased in chondrocytes.Conclusions: Elevated OPN expression might be regulated by the β-catenin/TCF-4 pathway, and the Wnt/β-catenin inhibitor DKK1 could inhibit the expression of β-catenin and OPN in OA chondrocytes.

scholarly journals The β-catenin/TCF-4 pathway regulates the expression of OPN in human osteoarthritic chondrocytes

2020 ◽  
Author(s):  
Jian Tian ◽  
Shu-Guang Gao ◽  
Yu-Sheng Li ◽  
Chao Cheng ◽  
Zhen-Han Deng ◽  
...  
Keyword(s):  
Cartilage Destruction ◽  
Transient Transfection ◽  
Human Chondrocytes ◽  
Mrna Levels ◽  
In Vitro Methods ◽  
Osteoarthritic Chondrocytes ◽  
Mrna And Protein Expression ◽  
Oa Chondrocytes ◽  
Canonical Wnt

Abstract Background Cartilage destruction is the main characteristic of osteoarthritis (OA), and osteopontin (OPN) is elevated in OA articular cartilage; however, the reason for the increased OPN level is not determined. In addition, Wnt/β-catenin signaling participates in the progression of OA. The aim of the present study was to evaluate whether canonical Wnt signaling could regulate the expression of OPN in human chondrocytes in vitro. Methods Human chondrocytes were cultured in vitro, and we first assayed the mRNA levels of OPN and β-catenin in chondrocytes. Next, we performed transient transfection of TCF 4 shRNA into chondrocytes to inhibit TCF 4 expression and explore changes in the OPN level. Then, the Wnt/β-catenin signaling inhibitor dinkkopf-1 (Dkk-1) was incubated with chondrocytes, and we assayed the changes in β-catenin and OPN. Results Our results showed that the expression of both β-catenin and OPN was increased in OA chondrocytes, but there were no correlations between β-catenin and OPN expression. TCF4 shRNA downregulated the expression of TCF 4 and OPN in chondrocytes, while after treatment with rDKK-1 at a concentration of 400 ng/ml for 24 h, the mRNA and protein expression of both β-catenin and OPN was significantly decreased in chondrocytes. Conclusions Elevated OPN expression might be regulated by the β-catenin/TCF-4 pathway, and the Wnt/β-catenin inhibitor DKK1 could inhibit the expression of β-catenin and OPN in OA chondrocytes.

scholarly journals The β-catenin/TCF-4 pathway regulates the expression of OPN in human osteoarthritic chondrocytes

2020 ◽  
Author(s):  
Jian Tian ◽  
Shu-Guang Gao ◽  
Yu-Sheng Li ◽  
Chao Cheng ◽  
Zhen-Han Deng ◽  
...  
Keyword(s):  
Cartilage Destruction ◽  
Human Chondrocytes ◽  
Mrna Levels ◽  
In Vitro Methods ◽  
Osteoarthritic Chondrocytes ◽  
Mrna And Protein Expression ◽  
Oa Chondrocytes ◽  
Canonical Wnt ◽  
Dickkopf 1

Abstract Background Cartilage destruction is the main characteristic of osteoarthritis (OA), and osteopontin (OPN) is elevated in OA articular cartilage; however, the reason for the increased OPN level is not determined. In addition, Wnt/β-catenin signaling participates in the progression of OA. The aim of the present study was to evaluate whether canonical Wnt signaling could regulate the expression of OPN in human chondrocytes in vitro. Methods Human chondrocytes were cultured in vitro, and we first assayed the mRNA levels of OPN and β-catenin in chondrocytes. Next, we performed transient transfection of TCF 4 shRNA into chondrocytes to inhibit TCF 4 expression and explore changes in the OPN level. Then, the Wnt/β-catenin signaling inhibitor Dickkopf-1 (Dkk-1) was incubated with chondrocytes, and we assayed the changes in β-catenin and OPN. Results Our results showed that the expression of both β-catenin and OPN was increased in OA chondrocytes, but there were no correlations between β-catenin and OPN expression. TCF4 shRNA downregulated the expression of TCF 4 and OPN in chondrocytes, while after treatment with rDKK-1 at a concentration of 400 ng/ml for 24 h, the mRNA and protein expression of both β-catenin and OPN was significantly decreased in chondrocytes. Conclusions Elevated OPN expression might be regulated by the β-catenin/TCF-4 pathway, and the Wnt/β-catenin inhibitor DKK1 could inhibit the expression of β-catenin and OPN in OA chondrocytes.

scholarly journals Pleiotropic Roles of NOTCH1 Signaling in the Loss of Maturational Arrest of Human Osteoarthritic Chondrocytes

2021 ◽  
Vol 22 (21) ◽  
pp. 12012
Author(s):  
Manuela Minguzzi ◽  
Veronica Panichi ◽  
Stefania D’Adamo ◽  
Silvia Cetrullo ◽  
Luca Cattini ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Terminal Differentiation ◽  
Loss Of Function ◽  
Notch Receptors ◽  
Osteoarthritic Chondrocytes ◽  
Gene And Protein Expression ◽  
Luminescence Assay ◽  
Culture Models ◽  
Oa Chondrocytes

Notch signaling has been identified as a critical regulator of cartilage development and homeostasis. Its pivotal role was established by both several joint specific Notch signaling loss of function mouse models and transient or sustained overexpression. NOTCH1 is the most abundantly expressed NOTCH receptors in normal cartilage and its expression increases in osteoarthritis (OA), when chondrocytes exit from their healthy “maturation arrested state” and resume their natural route of proliferation, hypertrophy, and terminal differentiation. The latter are hallmarks of OA that are easily evaluated in vitro in 2-D or 3-D culture models. The aim of our study was to investigate the effect of NOTCH1 knockdown on proliferation (cell count and Picogreen mediated DNA quantification), cell cycle (flow cytometry), hypertrophy (gene and protein expression of key markers such as RUNX2 and MMP-13), and terminal differentiation (viability measured in 3-D cultures by luminescence assay) of human OA chondrocytes. NOTCH1 silencing of OA chondrocytes yielded a healthier phenotype in both 2-D (reduced proliferation) and 3-D with evidence of decreased hypertrophy (reduced expression of RUNX2 and MMP-13) and terminal differentiation (increased viability). This demonstrates that NOTCH1 is a convenient therapeutic target to attenuate OA progression.

scholarly journals Reduction of Matrix Metallopeptidase 13 and Promotion of Chondrogenesis by Zeel T in Primary Human Osteoarthritic Chondrocytes

2021 ◽  
Vol 12 ◽  
Author(s):  
Christelle Sanchez ◽  
Kathrin Hemmer ◽  
Natascha Krömmelbein ◽  
Bernd Seilheimer ◽  
Jean-Emile Dubuc ◽  
...  
Keyword(s):  
Differential Expression Analysis ◽  
Type Ii Collagen ◽  
Cartilage Degradation ◽  
Alginate Beads ◽  
Protein Quantification ◽  
Type Ii ◽  
Osteoarthritic Chondrocytes ◽  
Matrix Metallopeptidase ◽  
Oa Chondrocytes

Objectives: Zeel T (Ze14) is a multicomponent medicinal product. Initial preclinical data suggested a preventive effect on cartilage degradation. Clinical observational studies demonstrated that Ze14 reduced symptoms of osteoarthritis (OA), including stiffness and pain. This study aimed to explore these effects further to better understand the mode of action of Ze14 on human OA chondrocytes in vitro.Methods: Primary chondrocytes were obtained from the knees of 19 OA patients and cultured either as monolayers or in alginate beads. The cultures were treated with 20% or 10% (v/v) Ze14 or placebo. For RNA-seq, reads were generated with Illumina NextSeq5000 sequencer and aligned to the human reference genome (UCSC hg19). Differential expression analysis between Ze14 and placebo was performed in R using the DESeq2 package. Protein quantification by ELISA was performed on selected genes from the culture medium and/or the cellular fractions of primary human OA chondrocyte cultures.Results: In monolayer cultures, Ze14 20% (v/v) significantly modified the expression of 13 genes in OA chondrocytes by at least 10% with an adjusted p-value < 0.05: EGR1, FOS, NR4A1, DUSP1, ZFP36, ZFP36L1, NFKBIZ, and CCN1 were upregulated and ATF7IP, TXNIP, DEPP1, CLEC3A, and MMP13 were downregulated after 24 h Ze14 treatment. Ze14 significantly increased (mean 2.3-fold after 24 h, p = 0.0444 and 72 h, p = 0.0239) the CCN1 protein production in human OA chondrocytes. After 72 h, Ze14 significantly increased type II collagen pro-peptide production by mean 27% (p = 0.0147). For both time points CCN1 production by OA chondrocytes was correlated with aggrecan (r = 0.66, p = 0.0004) and type II collagen pro-peptide (r = 0.64, p = 0.0008) production. In alginate beads cultures, pro-MMP-13 was decreased by Ze14 from day 7–14 (from −16 to −25%, p < 0.05) and from day 17–21 (−22%, p = 0.0331) in comparison to controls.Conclusion: Ze14 significantly modified the expression of DUSP1, DEPP1, ZFP36/ZFP36L1, and CLEC3A, which may reduce MMP13 expression and activation. Protein analysis confirmed that Ze14 significantly reduced the production of pro-MMP-13. As MMP-13 is involved in type II collagen degradation, Ze14 may limit cartilage degradation. Ze14 also promoted extracellular matrix formation arguably through CCN1 production, a growth factor well correlated with type II collagen and aggrecan production.

scholarly journals DKK1 activates noncanonical NF-κB signaling via IL-6–induced CKAP4 receptor in multiple myeloma

2021 ◽  
Vol 5 (18) ◽  
pp. 3656-3667
Author(s):  
Xin Li ◽  
Jingjing Wang ◽  
Shuai Zhu ◽  
Jinxin Zheng ◽  
Ying Xie ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Wnt Signaling ◽  
Proteasome Inhibitors ◽  
Bone Destruction ◽  
Bortezomib Treatment ◽  
Canonical Wnt ◽  
Dickkopf 1

Abstract Proteasome inhibitors, such as bortezomib (BTZ), represent the key elements in chemotherapy regimens for multiple myeloma (MM), whereas acquired chemoresistance and ultimately relapse remain a major obstacle. In the current study, we screened differently expressed cytokines in bortezomib-resistant MM cells and found that Dickkopf-1 (DKK1) level was remarkably augmented, whereas CD138 level was significantly suppressed. DKK1 in vitro specifically enhanced the resistance of myeloma cells to bortezomib treatment, and excessive DKK1 drove CD138 downregulation via inhibition of canonical Wnt signaling. Notably, DKK1 mainly induced drug resistance in MM cells via the receptor of CKAP4. Mechanistically, CKAP4 transduced DKK1 signal and evoked NF-κB pathway through recruiting and preventing cullin associated and neddylation dissociated 1 from hampering the assembly of E3 ligase-mediated ubiquitination of IκBα. In addition, we found that interleukin-6 (IL-6) stimulated CKAP4 expression to generate drug resistance, and disturbance of DKK1-CKAP4 axis improved sensitivity to BTZ treatment of MM and attenuated bone destruction in a mouse model. Collectively, our study revealed the previously unidentified role of DKK1 in myeloma drug resistance via Wnt signaling dependent and independent manners, and clarified the importance of antagonism of DKK1-IL-6 loop in bone marrow microenvironment.

The Expression and Biological Function of DKK1 in Oral Squamous Cell Carcinomas by Bioinformatics Analysis

2020 ◽  
Author(s):  
Huijie Yu ◽  
Tianhua Li ◽  
Xuemei Mao
Keyword(s):  
Squamous Cell ◽  
Bioinformatics Analysis ◽  
Prognostic Significance ◽  
Squamous Cell Carcinomas ◽  
Oral Squamous Cell Carcinomas ◽  
Dkk1 Expression ◽  
Mrna And Protein Expression ◽  
Proliferation Ability ◽  
Dickkopf 1

Abstract Background To investigate the expression of transcription factor Dickkopf-1 (DKK1) in oral squamous cell carcinomas (OSCC) by bioinformatics analysis and to clarify the connection between expression of DKK1 and clinicopathological features of OSCC, so as to elucidate the early diagnosis and prognostic significance of OSCC by DKK1. Methods This study used the GEPIA database in conjunction with the TCGA database to analyze the expression level of DKK1 in OSCC tissues and then verify it by QRTPCR and Western-blot analysis in vitro. The LinkedOmics database was used to describe the correlation between DKK1 expression and clinical pathological parameters of OSCC and its impact on prognosis. DKK1 was knocked down by RNA interference approach in SCC-4 and SCC-25 OSCC cell lines. In addition, the proliferation ability was assessed by MTT assay. Results DKK1 was highly expressed in OSCC and positively correlated with OSCC pathological grade and T stage. The results of TCGA showed that high DKK1 mRNA expression was associated with overall survival in OSCC. Besides, both DKK1 mRNA and protein expression was confirmed increased significantly in oral squamous cell carcinomas SCC-25 and SCC-4. Furthermore, MTT analysis investigated that knockdown of DKK1 caused reduced proliferation ability of OSCC cells. Conclusions The TCGA database analysis found that DKK1 is highly expressed in OSCC and is associated with multiple pathological indicators of OSCC, which will provide important theoretical guidance for subsequent oral squamous cell carcinoma research.

scholarly journals GRO-alpha in normal and pathological thyroid tissues and its regulation in thyroid-derived cells

2001 ◽  
Vol 170 (3) ◽  
pp. 513-520 ◽  
Author(s):  
G Aust ◽  
M Steinert ◽  
C Boltze ◽  
S Kiessling ◽  
C Simchen
Keyword(s):  
T Cells ◽  
Mrna Level ◽  
Thyroid Tissue ◽  
Thyroid Peroxidase ◽  
Mrna Levels ◽  
Carcinoma Cell Lines ◽  
Leukocytic Infiltration ◽  
Cellular Source ◽  
Mrna And Protein Expression

Thyroid glands affected by Graves' disease (GD) show striking leukocytic infiltration, mainly by T-cells. The mechanisms by which the various leukocytes are maintained in the thyroid are unknown. Growth-regulated oncogene-alpha (GRO-alpha) in interaction with its receptor CXCR2 is a chemoattractant for both T-cells and neutrophils and may be one of the chemokines involved in the cell maintenance. GRO-alpha and CD18 mRNA as a marker of leukocytic infiltration were quantified in thyroid tissue using competitive RT-PCR. We found very high GRO-alpha mRNA levels in all thyroid tissues. In GD patients (n=16), the GRO-alpha mRNA did not correlate with the CD18 mRNA level or thyroid peroxidase and TSH-receptor antibodies in patients' sera. In thyroid autonomy (n=10), the GRO-alpha mRNA levels were significantly lower in autonomous single adenomas compared with the corresponding normal tissue. In order to define the cellular source of GRO-alpha mRNA and protein, we examined various thyroid-derived cells. Thyrocytes, thyroid-derived leukocytes and fibroblasts showed basal GRO-alpha mRNA and protein expression, which was remarkably upregulated by different stimuli in vitro. The expression of GRO-alpha by thyroid carcinoma cell lines confirms that thyrocytes may actually produce GRO-alpha. As shown by flow cytometry and immunohistology, CD68+ monocytes/macrophages are the only cell population strongly expressing CXCR2 in the thyroid.

scholarly journals Effects of 5-aza-2’-deoxycytidine on human osteoarthritic chondrocytes

2018 ◽  
Author(s):  
Shirin Kadler ◽  
Özlem Vural ◽  
Luzia Reiners-Schramm ◽  
Roland Lauster ◽  
Mark Rosowski
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Late Stage ◽  
Chondrogenic Differentiation ◽  
Disease Models ◽  
Mrna Levels ◽  
Differentiation Potential ◽  
Differentiation Capacity ◽  
Oa Chondrocytes

AbstractBackgroundGiven regenerative therapies, the utilization of primary human cells is desired and requested in the development of in vitro systems and disease models. After a few passages in vitro, all cells from the connective tissue end up in a similar fibroblastoid cell type marked by loss of the specific expression pattern. It is still under discussion whether different de-differentiated mesenchymal cells have similar or identical differentiation capacities in vitro.MethodsChondrocytes isolated from patients with late-stage osteoarthritis were cultured for several passages until de-differentiation was completed. The mRNA level of cartilage markers was investigated, and the adipogenic, osteogenic and chondrogenic differentiation capacity was examined. By adding 5-aza-2’-deoxycytidine (5-aza-dC) to the media, the influence of DNA methylation on the differentiation capacity was analyzed.ResultsThe chondrocytes used in this work were not affected by the loss of specific gene expression upon cell culture. The mRNA levels of SOX5, SOX6, SOX9, aggrecan, and proteoglycan-4 remained unchanged. The underlying mechanisms of cartilage marker maintenance in osteoarthritic (OA) chondrocytes were investigated with a focus on the epigenetic modification by DNA methylation. The treatment of de-differentiated chondrocytes with the DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-aza-dC) displayed no appreciable impact on the observed maintenance of marker gene expression, while the chondrogenic differentiation capacity was compromised. On the other hand, the pre-cultivation with 5-aza-dC improved the osteogenesis and adipogenesis of OA chondrocytes. Contradictory to these effects, the DNA methylation levels were not reduced after treatment with 1 μM 5-aza-dC for four weeks.ConclusionChondrocytes isolated from late-stage osteoarthritic patients represents a reliable cell source for in vitro studies as wells as disease models since the chondrogenic differentiation potential remains. 5-aza-2’-deoxycytidine could not further improve their chondrogenic potential.

scholarly journals N-Acetylcysteine Inhibits Lipids Production in Mature Adipocytes through the Inhibition of Peroxisome Proliferator-Activated Receptor 

2020 ◽  
pp. 17-29
Author(s):  
Daniela Soto ◽  
Claudia Martini ◽  
Evelyn Frontera ◽  
Laura Montaldo ◽  
Maria C. Vila ◽  
...  
Keyword(s):  
In Vitro Study ◽  
Nutritional Supplement ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
Peroxisome Proliferator Activated Receptor ◽  
Mrna And Protein Expression ◽  
Species Production ◽  
Lipids Production

Aims: Reports regarding the effects of antioxidants in obesity have been contradictory. Antioxidant N-acetylcysteine is usually considered a nutritional supplement. Our aim is to evaluate bioactivity of N-acetylcysteine (NAC) on mature adipocytes, which is a close model to in vivo condition. Study Design: In vitro study. Place and Duration of Study: Department of Basic Science (Universidad Nacional de Lujan), Department of Chemical Biology (Universidad de Buenos Aires), CONICET – INEDES and CONICET – IQUIBICEN, between March 2017 and June 2019. Methodology: We evaluated the bioactivity of different concentrations of NAC for 5 days (0.01 mM to 5 mM) on fully differentiated 3T3-L1 cells (mature adipocytes). Results: We demonstrated that NAC treatment was not toxic to mature adipocytes. Only 5mM NAC inhibited reactive oxygen species production. 5 mM NAC treatment resulted in a 60% decrease in cellular triglycerides content and inhibited 70% cholesterol accumulation.  We also determined the mRNA and protein expression levels of Peroxisome Proliferator-Activated Receptor g as well as, mRNA levels of lipid protein Perilipin in NAC treated adipocytes; we observed that 5mM NAC treatment caused nearly 30% decrease in the expression of these parameters. Conclusion: These results suggest that NAC could avoid lipid accumulation in mature adipocytes; the antioxidant NAC could be beneficial in obesity treatment.

scholarly journals In vitro effects of nutraceutical treatment on human osteoarthritic chondrocytes of females of different age and weight groups

2021 ◽  
Vol 10 ◽  
Author(s):  
Mahmoud Amr ◽  
Alia Mallah ◽  
Haneen Abusharkh ◽  
Bernard Van Wie ◽  
Arda Gozen ◽  
...  
Keyword(s):  
Negative Control ◽  
Average Weight ◽  
Mrna Levels ◽  
Catechin Hydrate ◽  
Engineered Tissues ◽  
Osteoarthritic Chondrocytes ◽  
Mrna Gene ◽  
Group 2 ◽  
Group 1

Abstract The in vitro effects of four nutraceuticals, catechin hydrate, gallic acid, α-tocopherol and ascorbic acid, on the ability of human osteoarthritic chondrocytes of two female obese groups to form articular cartilage (AC) tissues and to reduce inflammation were investigated. Group 1 represented thirteen females in the 50–69 years old range, an average weight of 100 kg and an average body mass index (BMI) of 34⋅06 kg/m2. Group 2 was constituted of three females in the 70–80 years old range, an average weight of 75 kg and an average BMI of 31⋅43 kg/m2. The efficacy of nutraceuticals was assessed in monolayer cultures using histological, colorimetric and mRNA gene expression analyses. AC engineered tissues of group 1 produced less total collagen and COL2A1 (38-fold), and higher COL10A1 (2⋅7-fold), MMP13 (50-fold) and NOS2 (15-fold) mRNA levels than those of group 2. In comparison, engineered tissues of group 1 had a significant decrease in NO levels from day 1 to day 21 (2⋅6-fold), as well as higher mRNA levels of FOXO1 (2-fold) and TNFAIP6 (16-fold) compared to group 2. Catechin hydrate decreased NO levels significantly in group 1 (1⋅5-fold) while increasing NO levels significantly in group 2 (3⋅8-fold). No differences from the negative control were observed in the presence of other nutraceuticals for either group. In conclusion, engineered tissues of the younger but heavier patients responded better to nutraceuticals than those from the older but leaner study participants. Finally, cells of group 2 formed better AC tissues with less inflammation and better extracellular matrix than cells of group 1.

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