Browsing by Subject "Chondrocyte"

Now showing 1 - 3 of 3
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    CAMKK2 is Upregulated in Primary Human Osteoarthritis and its Inhibition Protects Against Chondrocyte Apoptosis
    (Elsevier, 2023) Dilley, Julian E.; Seetharam, Abhijit; Ding, Xinchun; Bello, Margaret A.; Shutter, Jennifer; Burr, David B.; Natoli, Roman M.; McKinley, Todd O.; Sankar, Uma; Anatomy, Cell Biology and Physiology, School of Medicine
    Objective: To investigate the role of calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) in human osteoarthritis. Materials and methods: Paired osteochondral plugs and articular chondrocytes were isolated from the relatively healthier (intact) and damaged portions of human femoral heads collected from patients undergoing total hip arthroplasty for primary osteoarthritis (OA). Cartilage from femoral plugs were either flash frozen for gene expression analysis or histology and immunohistochemistry. Chondrocyte apoptosis in the presence or absence of CAMKK2 inhibition was measured using flow cytometry. CAMKK2 overexpression and knockdown in articular chondrocytes were achieved via Lentivirus- and siRNA-mediated approaches respectively, and their effect on pro-apoptotic and cartilage catabolic mechanisms was assessed by immunoblotting. Results: CAMKK2 mRNA and protein levels were elevated in articular chondrocytes from human OA cartilage compared to paired healthier intact samples. This increase was associated with elevated catabolic marker matrix metalloproteinase 13 (MMP-13), and diminished anabolic markers aggrecan (ACAN) and type II collagen (COL2A1) levels. OA chondrocytes displayed enhanced apoptosis, which was suppressed following pharmacological inhibition of CAMKK2. Levels of MMP13, pSTAT3, and the pro-apoptotic marker BAX became elevated when CAMKK2, but not its kinase-defective mutant was overexpressed, whereas knockdown of the kinase decreased the levels of these proteins. Conclusions: CAMKK2 is upregulated in human OA cartilage and is associated with elevated levels of pro-apoptotic and catabolic proteins. Inhibition or knockdown of CAMKK2 led to decreased chondrocyte apoptosis and catabolic protein levels, whereas its overexpression elevated them. CAMKK2 may be a therapeutic target to prevent or mitigate human OA.
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    Knee loading reduces MMP13 activity in the mouse cartilage
    (Springer Nature, 2013-11-01) Hamamura, Kazunori; Zhang, Ping; Zhao, Liming; Shim, Joon W.; Chen, Andy; Dodge, Todd R.; Wan, Qiaoqiao; Shih, Han; Na, Sungsoo; Lin, Chien-Chi; Sun, Hui Bin; Yokota, Hiroki; Anatomy, Cell Biology and Physiology, School of Medicine
    Background: Moderate loads with knee loading enhance bone formation, but its effects on the maintenance of the knee are not well understood. In this study, we examined the effects of knee loading on the activity of matrix metalloproteinase13 (MMP13) and evaluated the role of p38 MAPK and Rac1 GTPase in the regulation of MMP13. Methods: Knee loading (0.5-3 N for 5 min) was applied to the right knee of surgically-induced osteoarthritis (OA) mice as well as normal (non-OA) mice, and MMP13 activity in the femoral cartilage was examined. The sham-loaded knee was used as a non-loading control. We also employed primary non-OA and OA human chondrocytes as well as C28/I2 chondrocyte cells, and examined MMP13 activity and molecular signaling in response to shear at 2-20 dyn/cm². Results: Daily knee loading at 1 N for 2 weeks suppressed cartilage destruction in the knee of OA mice. Induction of OA elevated MMP13 activity and knee loading at 1 N suppressed this elevation. MMP13 activity was also increased in primary OA chondrocytes, and this increase was attenuated by applying shear at 10 dyn/cm². Load-driven reduction in MMP13 was associated with a decrease in the phosphorylation level of p38 MAPK (p-p38) and NFκB (p-NFκB). Molecular imaging using a fluorescence resonance energy transfer (FRET) technique showed that Rac1 activity was reduced by shear at 10 dyn/cm² and elevated by it at 20 dyn/cm². Silencing Rac1 GTPase significantly reduced MMP13 expression and p-p38 but not p-NFκB. Transfection of a constitutively active Rac1 GTPase mutant increased MMP13 activity, while a dominant negative mutant decreased it. Conclusions: Knee loading reduces MMP13 activity at least in part through Rac1-mediated p38 MAPK signaling. This study suggests the possibility of knee loading as a therapy not only for strengthening bone but also preventing tissue degradation of the femoral cartilage.
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    Rac1 Mediates Load-Driven Attenuation of mRNA Expression of Nerve Growth Factor Beta in Cartilage and Chondrocytes
    (International Society of Musculoskeletal and Neuronal Interactions, 2013) Shim, Joon W.; Hamamura, Kazunori; Chen, Andy; Wan, Qiaoqiao; Na, Sungsoo; Yokota, Hiroki; Anatomy, Cell Biology and Physiology, School of Medicine
    Objectives: To determine effect of gentle loads applied to the knee on mRNA expression of nerve growth factor, particularly, the active beta subunit (NGFβ) in cartilage and chondrocyte. Methods: Cyclic compressive loads in vivo and fluid flow in vitro were used to determine the mRNA levels. Alteration of Rac1 GTPase as well as effect of salubrinal, a specific inhibitor of eIF2α phosphatase was assessed using fluorescence resonance energy transfer (FRET)-based Rac1 biosensor. Results: Knee loading at 1 N reduced mRNA levels of NGFβ and its low affinity receptor, p75 in cartilage and subchondral bone. In cartilage, knee loading at 1 N reduced the phosphorylation level of p38 MAPK (p38-p) and activity of Rac1 GTPase. Consistent with in vivo results, fluid flow at 5 and 10 dyn/cm(2) reduced mRNA levels of NGFβ and p75 in C28/I2 human chondrocytes. SB203580, which decreases p38-p, reduced the mRNA levels of NGFβ and p75. Silencing Rac1 by siRNA decreased the levels of p38-p and NGFβ mRNA but not p75. Furthermore, administration of salubrinal reduced FRET-based activity of Rac1 as well as the mRNA levels of NGFβ and p75. Conclusions: These results provide evidence that mechanical stimulation and salubrinal may attenuate pain perception-linked NGFβ signaling through Rac1-mediated p38 MAPK.