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. 2007 Oct;56(10):3284-96.
doi: 10.1002/art.22933.

Biomechanical signals inhibit IKK activity to attenuate NF-kappaB transcription activity in inflamed chondrocytes

Anar Dossumbekova  1 , Mirela Anghelina, Shashi Madhavan, Lingli He, Ning Quan, Thomas Knobloch, Sudha Agarwal
Affiliations

Biomechanical signals inhibit IKK activity to attenuate NF-kappaB transcription activity in inflamed chondrocytes

Anar Dossumbekova et al. Arthritis Rheum. 2007 Oct.

Abstract

Objective: While the effects of biomechanical signals in the form of joint movement and exercise are known to be beneficial to inflamed joints, limited information is available regarding the intracellular mechanisms of their actions. This study was undertaken to examine the intracellular mechanisms by which biomechanical signals suppress proinflammatory gene induction by the interleukin-1-beta (IL-1beta)-induced NF-kappaB signaling cascade in articular chondrocytes.

Methods: Primary rat articular chondrocytes were exposed to biomechanical signals in the form of cyclic tensile strain, and the effects on the NF-kappaB signaling cascade were examined by Western blot analysis, real-time polymerase chain reaction, and immunofluorescence.

Results: Cyclic tensile strain rapidly inhibited the IL-1beta-induced nuclear translocation of NF-kappaB, but not its IL-1beta-induced phosphorylation at serine 276 and serine 536, which are necessary for its transactivation and transcriptional efficacy, respectively. Examination of upstream events revealed that cyclic tensile strain also inhibited the cytoplasmic protein degradation of IkappaBbeta and IkappaBalpha, as well as repressed their gene transcription. Additionally, cyclic tensile strain induced a rapid nuclear translocation of IkappaBalpha to potentially prevent NF-kappaB binding to DNA. Furthermore, the inhibition of IL-1beta-induced degradation of IkappaB by cyclic tensile strain was mediated by down-regulation of IkappaB kinase activity.

Conclusion: These results indicate that the signals generated by cyclic tensile strain act at multiple sites within the NF-kappaB signaling cascade to inhibit IL-1beta-induced proinflammatory gene induction. Taken together, these findings provide insight into how biomechanical signals regulate and reduce inflammation, and underscore their potential in enhancing the ability of chondrocytes to curb inflammation in diseased joints.

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Figures

Figure 1
Figure 1
Inhibition of interleukin-1β (IL-1β)–induced NF-κB DNA binding, nuclear translocation, and transcription by cyclic tensile strain (CTS). Rat chondrocytes cultured on Bioflex plates were either untreated (control) or exposed to IL-1β (1.0 ng/ml) alone, cyclic tensile strain (3% and 0.05 Hz) and IL-1β, or cyclic tensile strain alone for 10, 30, 60, or 90 minutes, as indicated. A, Results of electrophoretic mobility shift assay (EMSA) of nuclear extracts, using IR-dye–labeled NF-κB consensus sequences. Lane 1, Untreated control cells; lanes 2, 4, 6, and 8, cells treated with IL-1β; lanes 3, 5, 7, and 9, cells treated with CTS and IL-1β; lane 10, IL-1β–treated cells incubated with a 100-fold excess of unlabeled probe (ExL); lane 11, cells treated with CTS; lanes 12 and 13, results of super-shift EMSA, demonstrating NF-κB subunit p65. B, Localization of cytoplasmic NF-κB p65 (green arrows) and nuclear NF-κB p65 (white arrows) following treatment with IL-1β in the presence and absence of cyclic tensile strain, determined by immunofluorescence staining. NF-κB p65 was stained red, and β-actin was stained green. Bar = 20 μm. C and D, Semiquantitative analysis of nuclear NF-κB (C) and cytoplasmic NF-κB (D) in chondrocytes exposed to IL-1β alone or to IL-1β and cyclic tensile strain. Values are the mean and SEM from 1 of 3 separate experiments with similar results. E, Phosphorylation of NF-κB p65 (p-p65) at serine 276 (ser 276) and serine 536 in whole cell extracts, determined by Western blot analysis and densitometric analysis of the bands. F, Inducible nitric oxide synthase (iNOS) mRNA expression measured by real-time polymerase chain reaction, using GAPDH mRNA expression as an internal control. Values are the mean and SEM from 3 separate experiments. Color figure can be viewed in the online issue, which is available at http://www.arthritisrheum.org.
Figure 2
Figure 2
Inhibition of interleukin-1β (IL-1β)–induced degradation and synthesis of IκBβ by cyclic tensile strain (CTS). Chondrocytes were treated as described in Materials and Methods. A, Inhibition of IL-1β–induced IκBβ degradation by cyclic tensile strain, determined by Western blot analysis. The cytoplasmic extracts of articular chondrocytes were probed with rabbit anti-IκBβ, and the membrane was stripped and reprobed for β-actin as an internal control. B, Results of immunofluorescence analysis, demonstrating inhibition of IκBβ degradation by cyclic tensile strain. Arrows show IκBβ. In A and B, results are representative of 1 of 3 separate experiments with similar results. C, Semiquantitative analysis of the inhibition of IκBβ degradation by cyclic tensile strain. Values are the mean and SEM from 1 of 3 separate experiments with similar results. D, Expression of IκBβ mRNA in chondrocytes over a period of 10–90 minutes, determined by real-time polymerase chain reaction. Values are the mean and SEM from 3 separate experiments. * = P ≤ 0.05 versus cells treated with IL-1β alone. Color figure can be viewed in the online issue, which is available at http://www.arthritisrheum.org.
Figure 3
Figure 3
Down-regulation of interleukin-1β (IL-1β)–induced degradation and synthesis of IκBα by cyclic tensile strain (CTS). Articular chondrocytes were either untreated or treated with IL-1β in the presence or absence of cyclic tensile strain for the indicated time periods. A, Degradation and synthesis of IκBα, determined by Western blot analysis. The membrane was probed with anti-IκBα antibody, stripped, and reprobed for β-actin as an internal control. B, Localization of cytoplasmic IκBα (white arrows) and nuclear IκBα (green arrows) and IL-1β–induced degradation of IκBα, determined by immunofluorescence staining using rabbit anti-IκBα IgG. Bar = 20 μm. C and D, Semiquantitative analysis of cytoplasmic IκBα (C) and nuclear IκBα (D) in chondrocytes exposed to IL-1β alone or to IL-1β and cyclic tensile strain. In A–D, results are representative of 1 of 3 separate experiments with similar results. E, Expression of IκBα mRNA in chondrocytes over a period of 10–90 minutes, determined by real-time polymerase chain reaction. Values are the mean and SEM from 3 separate experiments. * = P ≤ 0.05. Color figure can be viewed in the online issue, which is available at http://www.arthritisrheum.org.
Figure 4
Figure 4
Suppression of interleukin-1β (IL-1β)–induced IKK activity by cyclic tensile strain (CTS). Chondrocytes were treated with IL-1β in the presence or absence of cyclic tensile strain for the indicated time periods. A, Phosphorylation of IκB substrate, determined by Western blot analysis. IKK complexes immunoprecipitated from cell lysates with anti–IKKγ/NF-κB–essential modulator antibodies were incubated with glutathione S-transferase (GST)–IκB and ATP, as described in Materials and Methods. Phosphorylation of IκB substrate was then analyzed using p-IκBα mouse monoclonal antibody and N-terminal anti-IκB–GST peptide IgG as IκB loading control. The total amount of IKK immunoprecipitated from chondrocytes was assessed by probing the blots with rabbit anti-IKKγ IgG. Secondary antibodies labeled with IR-dye 680 or 800 were used to obtain densitometric measurements of blots with the Odyssey Infrared Imaging System. Results are representative of 1 of 4 separate experiments with similar results. The y-axis shows the fold increase over untreated control cells. B, Inhibition of expression of mRNA for NF-κB–regulated genes in the presence of IL-1β. Cells treated with IL-1β in the presence or absence of cyclic tensile strain were examined for expression of mRNA for tumor necrosis factor receptor–associated factor 1 (TRAF1) and TRAF2, by real-time polymerase chain reaction. Values are the mean and SEM from 2 separate experiments performed in triplicate. * = P < 0.05. Color figure can be viewed in the online issue, which is available at http://www.arthritisrheum.org.
Figure 5
Figure 5
Schematic representation of the mechanisms of intracellular action of cyclic tensile strain (CTS). Cyclic tensile strain suppresses interleukin-1β (IL-1β)–induced proinflammatory gene induction by intercepting salient steps in the NF-κB signaling cascade to inhibit transcription activity. Cyclic tensile strain suppresses IL-1β–induced IKK activation, and thus phosphorylation and proteosomal degradation of IκBα and IκBβ. This leads to the inhibition of nuclear translocation of NF-κB. During the initial stages of IL-1β–mediated activation of cells, cyclic tensile strain up-regulates IκBα nuclear translocation to prevent NF-κB binding to DNA and facilitate export of nuclear NF-κB, which may enter the nucleus. Cyclic tensile strain represses IL-1β–induced IκBα and IκBβ mRNA expression. Collectively, these actions of cyclic tensile strain inhibit proinflammatory gene induction as well as expression of multiple molecules involved in the regulation of the NF-κB signaling cascade to suppress IL-1β–induced inflammation. IL-1R = IL-1 receptor; TRAF1 = tumor necrosis factor receptor–associated factor 1; iNOS = inducible nitric oxide synthase; COX-2 = cyclooxygenase 2; MMPs = matrix metalloproteinases; TNF = tumor necrosis factor. Color figure can be viewed in the online issue, which is available at http://www.arthritisrheum.org.
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