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. 2005 Sep;204(3):956-63.
doi: 10.1002/jcp.20368.

Polyamine depletion inhibits NF-kappaB binding to DNA and interleukin-8 production in human chondrocytes stimulated by tumor necrosis factor-alpha

Annalisa Facchini  1 , Rosa Maria Borzí, Kenneth B Marcu, Claudio Stefanelli, Eleonora Olivotto, Mary B Goldring, Andrea Facchini, Flavio Flamigni
Affiliations

Polyamine depletion inhibits NF-kappaB binding to DNA and interleukin-8 production in human chondrocytes stimulated by tumor necrosis factor-alpha

Annalisa Facchini et al. J Cell Physiol. 2005 Sep.

Abstract

The activation of the NF-kappaB pathway by pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNFalpha), can be an important contributor for the re-programming of chondrocyte gene expression, thereby making it a therapeutic target in articular diseases. To search for new approaches to limit cartilage damage, we investigated the requirement of polyamines for NF-kappaB activation by TNFalpha in human C-28/I2 chondrocytes, using alpha-difluoromethylornithine (DFMO), a specific polyamine biosynthesis inhibitor. The NF-kappaB pathway was dissected by using pharmacological inhibitors or by expressing a transdominant IkappaBalpha super repressor. Treatment of C-28/I2 chondrocytes with TNFalpha resulted in a rapid enhancement of nuclear localization and DNA binding activity of the p65 NF-kappaB subunit. TNFalpha also increased the level and extracellular release of interleukin-8 (IL-8), a CXC chemokine that can have a role in arthritis, in an NF-kappaB-dependent manner. Pre-treatment of chondrocytes with DFMO, while causing polyamine depletion, significantly reduced NF-kappaB DNA binding activity. Moreover, DFMO also decreased IL-8 production without affecting cellular viability. Restoration of polyamine levels by the co-addition of putrescine circumvented the inhibitory effects of DFMO. Our results show that the intracellular depletion of polyamines inhibits the response of chondrocytes to TNFalpha by interfering with the DNA binding activity of NF-kappaB. This suggests that a pharmacological and/or genetic approach to deplete the polyamine pool in chondrocytes may represent a useful way to reduce NF-kappaB activation by inflammatory cytokines in arthritis without provoking chondrocyte apoptosis.

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Figures

Fig. 1
Fig. 1
Effect of DFMO on polyamine content and cell number of C-28/I2 chondrocytes. Cells were grown for 3 days after seeding without any addition (Control), or in the presence of 4 mM DFMO, or in the presence of 4 mM DFMO plus 100 μM putrescine (Put). Then cells were harvested and assayed for polyamine content (A) or for cell number by the PicoGreen dsDNA method (B). * p< 0.05 vs. control cells. Data represent means ± SEM of three (A) or five (B) determinations. Spd, spermidine; Spm, spermine.
Fig. 2
Fig. 2
Effect of NF-κB pathway inhibitors on TNFα-stimulated NF-κB DNA binding activity in C-28/I2 chondrocytes. (A) Cells, grown for 3 days after seeding, were pre-treated with 10 μM Bay 11-7082 for 1 h or 5 μM MG-132 for 30 min and then incubated in the presence or absence of TNFα for 30 min. (B) Cells infected with IκBαSR or the empty vector were treated with TNFα as previously described. Then cells were analysed for p65 NF-κB DNA binding activity. Data are means ± SEM (N=4); * p<0.05 vs. TNFα-treated cells (A) or vs. TNFα-treated cells infected with empty vector (B).
Fig. 3
Fig. 3
Effect of polyamine depletion on TNFα-stimulated NF-κB activation in C-28/I2 chondrocytes. (A) Cells were grown for 3 days after seeding without any addition (Control), or in the presence of 4 mM DFMO, or in the presence of 4 mM DFMO plus 100 μM putrescine, and then treated with TNFα for 30 min. Cells were collected and nuclear extracts were analyzed for p65 NF-κB DNA binding activity. Data are means ± SEM (N=3); * p<0.05 vs. TNFα-treated cells. (B) The cells, treated as indicated in the legend of panel A, were collected and analysed for the amount of nuclear p65 NF-κB or the level of phosphorylated p65 NF-κB by Western blot.
Fig. 4
Fig. 4
Effect of TNFα and NF-κB pathway inhibitors on IL-8 production by C-28/I2 chondrocytes. (A,B) Cells, grown for 3 days after seeding, were pre-treated with 10 μM Bay 11-7082 for 1 h or 5 μM MG-132 for 30 min and then incubated with TNFα for 24 h. (C,D) Cells infected with IκBαSR or the empty vector were treated with TNFα as previously described. Then cells were analysed for IL-8 content in cell lysates (A,C) or in cell medium (B,D). Data are means ± SEM (N=3); * p<0.05 vs. TNFα-treated cells (A) or vs. TNFα-treated cells infected with empty vector (B).
Fig. 5
Fig. 5
Effect of polyamine depletion on TNFα-stimulated IL-8 production by C-28/I2 chondrocytes. Cells were grown for 3 days after seeding without any addition (Control), or in the presence of 4 mM DFMO, or in the presence of 4 mM DFMO plus 100 μM putrescine, and then treated with TNFα for 24 h. Cells were analyzed for IL-8 content in cell lysates (A) or in cell medium (B). Data are means ± SEM (N=3 for A and N=5 for B); * p<0.05 vs. TNFα-treated cells.
Fig. 6
Fig. 6
Effect of TNFα and polyamine depletion on caspase activity and cell viability of C-28/I2 chondrocytes. Cells were grown for 3 days after seeding without any addition (Control), or in the presence of 4 mM DFMO, or in the presence of 4 mM DFMO plus 100 μM putrescine, and then treated with TNFα. After 20h incubation cells were collected and assayed for caspase activity (A). Alternatively, cells viability (B) was evaluated by trypan blue exclusion at the end of a 24h incubation with TNFα. Data are means ± SEM (N=3 for A and N=6 for B); * p<0.05 vs. control cells.
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