ldn-193189 and Osteoarthritis

Chondrocytes in injured cartilage tissue are susceptible to mechanical loading; mechanical overloading can induce cartilage degeneration. The aim of the present study was to investigate whether mechanical loading can regulate chondrocyte degeneration and angiogenesis via the tissue inhibitor of matrix metalloproteinase‑3 (TIMP3)/transforming growth factor (TGF)‑β1 axis. Primary human chondrocytes were obtained from knee articular cartilage of a healthy donor. Then, normal chondrocytes or TIMP3 lentivirus‑transfected (LV‑TIMP3) chondrocytes were subjected to mechanical loading (10 MPa compression). Then, chondrocytes were stimulated with 1 µg/ml lipopolysaccharide (LPS) or treated with LDN‑193189 (inhibitor of TGF‑β1 signaling pathway). In addition, human umbilical vein endothelial cells (HUVECs) were co‑cultured with chondrocytes or LV‑TIMP3 chondrocytes. The expression levels of collagen‑I, proteoglycan, TIMP3, TGF‑β1, Smad2 and Smad3 were detected by reverse transcription‑quantitative PCR and western blotting. Moreover, cell apoptosis and viability were determined using flow cytometry and MTT analysis, while cell migration was observed by Transwell assays. In addition, the vascular endothelial growth factor (VEGF)/VEGF receptor (R)2 binding rate in HUVECs was detected by a solid‑phase binding assay. It was demonstrated that mechanical loading significantly inhibited the expression levels of collagen‑I and proteoglycan in chondrocytes, as well as reducing cell proliferation and promoting cell apoptosis. In addition, the expression levels of TIMP3, TGF‑β1, phosphorylated (p)‑Smad2 and p‑Smad3 were significantly decreased in degenerated chondrocytes that were induced by LPS, as well as in chondrocytes treated with LDN‑193189. Furthermore, TIMP3 overexpression suppressed cell migration and reduced the VEGF/VEGFR2 binding rate in HUVECs. Mechanical loading significantly inhibited the expression levels of TIMP3, TGF‑β1, p‑Smad2 and p‑Smad3 in chondrocytes, and also increased cell migration of HUVECs; TGF‑β1 treatment or TIMP3 overexpression reversed these effects. Thus, the TIMP3/TGF‑β1 axis may be a vital signaling pathway in mechanical loading‑induced chondrocyte degeneration and angiogenesis.

Topics: Aged; Cell Survival; Cells, Cultured; Chondrocytes; Coculture Techniques; Gene Expression Regulation; Human Umbilical Vein Endothelial Cells; Humans; Lipopolysaccharides; Male; Mechanical Phenomena; Models, Biological; Osteoarthritis; Pyrazoles; Pyrimidines; Signal Transduction; Tissue Inhibitor of Metalloproteinase-3; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

Dysregulated transforming growth factor β (TGFβ) signaling is implicated in osteoarthritis development, making normalizing TGFβ signaling a possible therapy. Theoretically, this can be achieved with small molecule inhibitors specifically targeting the various TGFβ receptors and downstream mediators. In this study we explore in primary chondrocytes the use of small molecule inhibitors to target TGFβ-induced pSmad1/5/9-, pSmad2/3- and TGFβ-activated kinase 1 (TAK1)-dependent signaling.. Primary bovine chondrocytes and explants were isolated from metacarpophalangeal joints. To modulate TGFβ signaling the activin receptor-like kinase (ALK)1/2/3/6 inhibitor LDN-193189, the ALK4/5/7 inhibitor SB-505124 and the TAK1 inhibitor (5Z)-7-Oxozeaenol were used. pSmad1/5 and pSmad2 were measured using western blot analysis and TGFβ1-induced gene expression was measured using quantitative real time PCR (qPCR).. In chondrocytes, TGFβ1 strongly induced both pSmad1/5 and pSmad2. Remarkably, LDN-193189 did not inhibit TGFβ-induced pSmad1/5. In contrast, SB-505124 did inhibit both TGFβ-induced Smad2 and Smad1/5 phosphorylation. Furthermore, (5Z)-7-Oxozeaenol also profoundly inhibited TGFβ-induced pSmad2 and pSmad1/5. Importantly, both SB-505124 and (5Z)-7-Oxozeaenol did not significantly inhibit constitutively active ALK1, making an off-target effect unlikely. Additionally, LDN-193189 was able to potently inhibit BMP2/7/9-induced pSmad1/5, showing its functionality. On gene expression, LDN-193189 did not affect TGFβ1-induced regulation, whereas both SB-505124 and (5Z)-7-Oxozeaenol did. Similar results were obtained in cartilage explants, although pSmad1/5 was not strongly induced by addition of TGFβ1.. Our data suggest that ALK5 kinase activity plays a central role in both TGFβ-induced Smad1/5 and Smad2/3 phosphorylation, making it difficult to separate both pathways with the use of currently available small molecule inhibitors. Furthermore, our data regarding (5Z)-7-Oxozeaenol suggest that TAK1 facilitates Smad-dependent signaling.

Topics: Animals; Benzodioxoles; Cattle; Chondrocytes; Enzyme Inhibitors; Imidazoles; MAP Kinase Kinase Kinases; Osteoarthritis; Phosphorylation; Protein Serine-Threonine Kinases; Pyrazoles; Pyridines; Pyrimidines; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Smad Proteins; Transforming Growth Factor beta1; Zearalenone