β-Adrenoceptors regulate matrix metalloproteinase expression in human urothelial cells under hydrostatic pressure

The bladder wall is constantly subjected to intravesical pressure during the filling and voiding cycles. An imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) under elevated intravesical pressure contributes to pathological changes in the bladder. To investigate the changes in human urothelial cells (HUCs) under elevated intravesical pressure, this study analyzed the effect of β-adrenoceptor signaling on the expression of MMPs and TIMPs in HUCs exposed to pathological hydrostatic pressure (HP) (70 cm H₂O) for 6 hours. Quantitative polymerase chain reaction, Western blot analysis, and cell fluorescence staining were used to explore the effect of β-adrenoceptor signaling on the expression of MMPs and TIMPs in HUCs after agonist and/or antagonist treatment. The expression levels of β₂- and β₃-adrenoceptor, MMP1, and MMP2 were greatly downregulated, while the expression of TIMP1 was greatly upregulated. Formoterol and BRL 37344, which are agonists of β₂- and β₃-adrenoceptor, respectively, significantly increased MMP1 and MMP2 expression under 70 cm H₂O. As a classic downstream pathway of β₂- and β₃-adrenoceptor, protein kinase A (PKA) signaling inhibited MMP1 and MMP2 expression by regulating cAMP response element binding protein (CREB) activity. MMP1 and MMP2 expression in HUCs under 70 cm H₂O was modified by β₂- and β₃-adrenoceptor via the PKA/CREB pathway. This outcome suggests that MMPs likely participate in the pathological effects of elevated intravesical pressure. The underlying mechanism of β₂- and β₃-adrenoceptor in elevated intravesical pressure was also revealed; this mechanism constitutes a new potential therapeutic target for partial bladder outlet obstruction.     

High-frequency near-infrared diode laser irradiation suppresses IL-1β-induced inflammatory cytokine expression and NF-κB signaling pathways in human primary chondrocytes

Lasers in Medical Science - Osteoarthritis (OA) and rheumatoid arthritis (RA) are common inflammation-associated cartilage degenerative diseases. Recent studies have shown that low-level diode...     

Cordycepin prevented IL-β-induced expression of inflammatory mediators in human osteoarthritis chondrocytes

Purpose

Cordycepin, a nucleoside derivative isolated from Cordyceps, has been reported to exert anti-inflammatory, antitumor, antidiabetic and renoprotective effects. Osteoarthritis (OA) is a degenerative joint disease with an inflammatory component that drives the degradation of cartilage extracellular matrix. This study aimed to assess the effects of cordycepin on human OA chondrocytes.

Methods

In this study, human OA chondrocytes were pretreated with cordycepin at 10, 50 or 100 μM and subsequently stimulated with interleukin-1β (IL-1β) (5 ng/ml) for 24 h. Production of prostaglandin E₂ (PGE2) and nitric oxide (NO) were evaluated by the Griess reaction and an enzyme-linked immunosorbent assay (ELISA). Gene expression of matrix metalloproteinase (MMP)-13, IL-6, inducible nitric oxide synthase (iNOS) and cyclo-oxygenase (COX-2) was measured by real-time polymerase chain reaction (PCR). MMP-13 and IL-6 proteins in culture medium were determined using cytokine-specific ELISA. Western immunoblotting was used to analyse the iNOS and COX-2 protein production in culture medium. Nuclear factor kappa-B (NF-κB) activity regulation was explored using Western immunoblotting.

Results

Pretreatment with cordycepin significantly inhibited the production of PGE2 and NO induced by IL-1β. Cordycepin also significantly decreased the IL-1β-stimulated gene expression and production of MMP-13, IL-6, iNOS and COX-2 in OA chondrocytes. Pretreatment with cordycepin attenuated IL-1β-induced activation of NF-κB by suppressing degradation of its inhibitory protein nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκB-α) in the cytoplasm.

Conclusions

We show for the first time the anti-inflammatory activity of cordycepin in human OA chondrocytes. Thus, with this unique profile of actions, cordycepin may prove to be a potentially attractive and new therapeutic/preventive agent for OA.     

Effects of interleukin-1, tumor necrosis factor-β, and forskolin on tissue plasminogen activator activity in human osteoblastic osteosarcoma cells

Summary The effects of interleukin-1 (IL-1), forskolin, and tumor necrosis factor (TNF-) on tissue plasminogen activator (t-PA) activity were studied in the human osteoblastic osteosarcoma cell line, G292. t-PA activity was measured in the cell media using the chromogenic substrate, S-2251. After a 24 hour incubation period, IL-1 increased t-PA in a dose-dependent manner. The effect of IL-1 at 10.0 U/ml was partially inhibited in the presence of indomethacin. Forskolin (1.0 M) increased t-PA activity after 24 hours with the effects of combined treatment of IL-1 (1.0 U/ml, 10.0 U/ml) and forskolin being apparently additive in nature. TNF- (10^-8–10^-7 M) also produced increased t-PA activity in the cell medial after a 24 hour incubation period. These results suggest that the cytokines, IL-1 and TNF-, can increase t-PA activity in G292 cells and that there is both a cAMP-dependent as well as a cAMP-independent pathway involved in the regulation of this osteoblastic cell function.     

Mechanotransduction pathways in articular chondrocytes and the emerging role of estrogen receptor-α

<正>In the synovial joint, mechanical force creates an important signal that influences chondrocyte behavior. The conversion of mechanical signals into biochemical cues relies on different elements in mechanotransduction pathways and culminates in changes in chondrocyte phenotype and extracellular matrix composition/structure. Recently, several mechanosensors, the first responders to mechanical force, have been discovered. However, we still have limited knowledge about the downstream molecu...     

Green fluorescent protein as marker in chondrocytes overexpressing human insulin-like growth factor-1 for repair of articular cartilage defects in rabbits

Objective:To label the primary articular chondrocytes overexpressing human insulin-like growth factor (hIGF-1) with green fluorescent protein (GFP) for repair of articular cartilage defects in rabbits. Methods: GFP cDNA was inserted into pcDNA3. 1-hIGF-1 to label the expression vector. The recombinant vector, pcGI, a mammalian expression vector with multiple cloning sites under two respective cytomegalovirus promoters/enhancers, was transfected into the primary articular chondrocytes with the help of lipofectamine. After the positive cell clones were selected by G418, G418-resistant chondrocytes were cultured in medium for 4 weeks. The stable expression of hIGF-1 in the articular chondrocytes was determined by in situ hybridization and immunocytochemical analysis and the GFP was confirmed under a fluorescence microscope. Methyl thiazolyl tetrazolium ( MTT) and flow cytometer methods were employed to determine the effect of transfection on proliferation of chondrocytes. Gray value was used to analyze quantitatively the expression of type II collagen. Results: The expression of hIGF-1 and GFP was confirmed in transfected chondrocytes by in situ hybridization, immunocytochemical analysis and fluorescence microscope observation. Green articular chondrocytes overexpressing hIGF-1 could expand and maintain their chondrogenic phenotypes for more than 4 weeks. After the transfection of IGF-1, the proliferation of chondrocytes was enhanced and the chondrocytes could effectively maintain the expression of typeⅡcollagen. Conclusions: The hIGF-1 eukaryotic expression vector containing GFP marker gene has been successfully constructed. GFP, which can be visualized in real time and in situ, is stably expressed in articular chondrocytes overexpressing hIGF-1. The labeled articular chondrocytes overexpressing hIGF-1 can be applied in cell-mediated gene therapy as well as for other biomedical purposes of transgenic chondrocytes.     

Inhibition of TGF-β receptor I by siRNA suppresses the motility and invasiveness of T24 bladder cancer cells via modulation of integrins and matrix metalloproteinase

Background  

Urinary bladder transitional-cell carcinoma is still challenging because the mechanisms underlying the tumor progression are still largely unknown. Transforming growth factor β1 (TGF-β1) is considered a crucial molecule in the tumorigenesis of urinary bladder carcinoma. Many studies have indicated that it is also associated with epithelial–mesenchymal transition, angiogenesis, migration and metastases in many types of malignant tumors.