Objective A reduced response of articular chondrocytes to growth factors with aging could contribute to the development of osteoarthritis. The purpose of this study was to determine the effects of aging and oxidative stress on the response of human articular chondrocytes to insulin-like growth factor-1 (IGF-1) and osteogenic protein-1 (OP-1). Methods Chondrocytes isolated from normal human articular cartilage obtained from tissue donors were cultured in alginate beads or monolayer. Cells were stimulated with 50–100 ng/ml of IGF-1, OP-1, or both. Oxidative stress was induced using tert-butyl-hydroperoxide. Sulfate incorporation was used to measure proteoglycan synthesis and cell lysates to evaluate signaling proteins by immunoblotting. Confocal microscopy was used to measure nuclear translocation of Smad4. Results Chondrocytes isolated from tissue donors ranging in age from 24 to 81 years demonstrated an age-related decline in IGF-1 and IGF-1+OP-1 stimulated proteoglycan synthesis. Induction of oxidative stress inhibited both IGF-1 and OP-1 stimulated proteoglycan synthesis. Signaling studies revealed oxidative stress inhibited IGF-1 stimulated Akt phosphorylation while increasing phosphorylation of ERK and these effects were greater in cells from older donors. Oxidative stress also increased p38 phosphorylation which resulted in phosphorylation of Smad1 at the Ser206 inhibitory site and reduced Smad1 nuclear accumulation. Oxidative stress also modestly reduced OP-1 stimulated nuclear translocation of Smad4. Conclusion These results demonstrate an age-related reduction in response of human chondrocytes to IGF-1 and OP-1, two important anabolic factors in cartilage, and suggest oxidative stress may be a contributing factor by altering IGF-1 and OP-1 signaling.
Previously we reported that the G protein-coupled receptor (GPCR) agonist thrombin potentiated the mitogenic effect of epidermal growth factor (EGF) on human airway smooth muscle (ASM) by promoting sustained late-phase activation of PI3K and p70S6K via a pathway dependent on Gbetagamma subunits of heterotrimeric G proteins. Here, we provide additional mechanistic insight and reveal the robustness of this phenomenon by demonstrating that H1 histamine and thromboxane receptors utilize the same mechanism to augment ASM growth via specific activation of the heterotrimeric G protein G(q/11). Thrombin, histamine, and U46619 all enhanced EGF-stimulated [3H]-thymidine incorporation as well as late-phase Akt and p70S6K phosphorylation in ASM cultures. Heterologous expression of Gbetagamma sequestrants (GRK2CT-GFP or Galpha(i)G203A), as well as GRK2NT-GFP (an RGS protein for G(q/11)) but neither p115RhoGEFRGS-GFP (an RGS for G(12/13)) nor pertussis toxin pretreatment (inactivating G(i/o)), attenuated the effects on both signaling and growth. Inhibition of Rho, Rho kinase, or Src, or modulation of arrestin expression did not significantly affect the cooperative signaling by EGF and any of the GPCR agonists. Thus, G(q/11)-coupled receptors are the principal GPCR subfamily mediating cooperative mitogenic signaling in ASM, acting through Gbetagamma-dependent, and Src/arrestin-independent activation of PI3K and p70S6K.
Although G protein-coupled receptor (GPCR) kinases (GRKs) have been shown to mediate desensitization of numerous GPCRs in studies using cellular expression systems, their function under physiological conditions is less well understood. In the current study, we employed various strategies to assess the effect of inhibiting endogenous GRK2/3 on signaling and function of endogenously expressed Gs-coupled receptors in human airway smooth muscle (ASM) cells. GRK2/3 inhibition by expression of a Gβγ sequestrant, a GRK2/3 dominant-negative mutant, or siRNA-mediated knockdown increased intracellular cAMP accumulation mediated via β-agonist stimulation of the beta-2-adrenergic receptor (β2AR). Conversely, neither 5′-(N-ethylcarboxamido)-adenosine (NECA; activating the A2b adenosine receptor) nor prostaglandin E2 (PGE2; activating EP2 or EP4 receptors)-stimulated cAMP was significantly increased by GRK2/3 inhibition. Selective knockdown using siRNA suggested the majority of PGE2-stimulated cAMP in ASM was mediated by the EP2 receptor. Although a minor role for EP3 receptors in influencing PGE2-mediated cAMP was determined, the GRK2/3-resistant nature of EP2 receptor signaling in ASM was confirmed using the EP2-selective agonist butaprost. Somewhat surprisingly, GRK2/3 inhibition did not augment the inhibitory effect of the β-agonist on mitogen-stimulated increases in ASM growth. These findings demonstrate that with respect to Gs-coupled receptors in ASM, GRK2/3 selectively attenuates β2AR signaling, yet relief of GRK2/3-dependent β2AR desensitization does not influence at least one important physiological function of the receptor.
This study presents a simple voltage oriented vector control scheme to regulate active and reactive power in a grid connected variable speed wind electrical system that consists of permanent magnet synchronous generator and matrix converter, which enables the maximum power tracking in wind electrical systems. The maximum extractable power from wind is derived from power curves and set as reference active power. Reactive power reference is chosen as zero. The deviations in powers are processed by proportional and integral (PI) controllers which vary the voltage gain of MC through which the desired regulation of powers is carried out. Since the relationship between wind speed and reference active power is nonlinear, single PI controller is insufficient, hence gain scheduling is required to adjust the controller parameters in response to changes in wind speeds. The controller parameter change is abrupt in conventional gain scheduling which leads to unstable performance. To avoid this, neural network-based PI (NN-PI) is designed which is more robust. In the presented work, both conventional gain scheduled PI and NN-PI are applied to validate the suggested voltage oriented control for power regulation. Simulation and experimental studies are presented to confirm the effective functionality of the system.
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