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A peptidyl-glucosamine derivative affects IKKalpha kinase activity in human chondrocytes.

Scotto d'Abusco A, Politi L, Giordano C, Scandurra R - Arthritis Res. Ther. (2010)

Bottom Line: We investigated the possibility that GlcN and NAPA inhibit IKK kinase activity and found that NAPA inhibits the IKKalpha kinase activity, whereas GlcN does not.Our results demonstrate that glucosamine and its peptidyl derivative can interfere with NF-kappaB signaling pathway by inhibiting IKKalpha activity in human chondrocytes.While NAPA can both specifically inhibit the IKKalpha kinase activity and IKKalpha nuclear re-localization, GlcN only acts on IKKalpha nuclear re-localization.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemical Sciences, Sapienza University of Roma, P,le Aldo Moro, 5, 00185 Roma, Italy. anna.scottodabusco@uniroma1.it

ABSTRACT

Introduction: Nuclear factor-kappaB (NF-kappaB) transcription factor regulates several cell signaling pathways, such as differentiation and inflammation, which are both altered in osteoarthritis. Inhibitor kappaB kinase (IKK)alpha and IKKbeta are kinases involved in the activation of the NF-kappaB transcription factor. The aim of the present study was to determine the effects of glucosamine (GlcN), which is administered in the treatment of osteoarthritis, and of its 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-beta-D-glucose (NAPA) derivative on IKK kinases and, consequently, on NF-kappaB activation in human chondrocytes.

Methods: The human chondrosarcoma cell line HTB-94 and human primary chondrocytes were stimulated with tumor necrosis factor (TNF)alpha after pre-treatment with GlcN or NAPA. Gene mRNA expression level was evaluated by real-time PCR. Inhibitor kappaB protein (IkappaB)alpha phosphorylation and p65 nuclear re-localization were analyzed by Western blotting; IKKalpha nuclear re-localization was also investigated by immunocytochemistry and Western blotting. IKK kinase activity was studied by in vitro kinase assay.

Results: After TNFalpha stimulation, the mRNA expression level of some of the genes under NF-kappaB control, such as interleukin (IL)-6 and IL-8, increased, while treatment with GlcN and NAPA reverted the effect. We investigated the possibility that GlcN and NAPA inhibit IKK kinase activity and found that NAPA inhibits the IKKalpha kinase activity, whereas GlcN does not. Interestingly, both GlcN and NAPA inhibit IKKalpha nuclear re-localization.

Conclusions: Our results demonstrate that glucosamine and its peptidyl derivative can interfere with NF-kappaB signaling pathway by inhibiting IKKalpha activity in human chondrocytes. However, the mechanism of action of the two molecules is not completely overlapping. While NAPA can both specifically inhibit the IKKalpha kinase activity and IKKalpha nuclear re-localization, GlcN only acts on IKKalpha nuclear re-localization.

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Effect of glucosamine (GlcN) and NAPA on inhibitor κB kinase alpha (IKKα) nuclear translocation in human primary chondrocytes. The analysis was performed by Western blot. Cells were untreated (CTL), treated with tumor necrosis factor-alpha (TNFα) or pre-treated with 10 mM GlcN (G10) or NAPA (N10) and then stimulated with TNFα for 1 hour. (a) Cytosolic extract probed with antibodies against IKKα and β-actin. (b) Nuclear extract probed with antibodies against IKKα and fibrillarin. *P ≤ 0.05. Results are expressed as fold change with respect to control. A.U., arbitrary units; NAPA, 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-β-D-glucose.
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Figure 5: Effect of glucosamine (GlcN) and NAPA on inhibitor κB kinase alpha (IKKα) nuclear translocation in human primary chondrocytes. The analysis was performed by Western blot. Cells were untreated (CTL), treated with tumor necrosis factor-alpha (TNFα) or pre-treated with 10 mM GlcN (G10) or NAPA (N10) and then stimulated with TNFα for 1 hour. (a) Cytosolic extract probed with antibodies against IKKα and β-actin. (b) Nuclear extract probed with antibodies against IKKα and fibrillarin. *P ≤ 0.05. Results are expressed as fold change with respect to control. A.U., arbitrary units; NAPA, 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-β-D-glucose.

Mentions: IKKβ activates the canonical NF-κB pathway by phosphorylation of IκBα, whereas IKKα is not required to phosphorylate IκBα, but it plays an important role by localizing into the nucleus of activated cells and inducing the transcription of NF-κB-dependent genes. To determine whether GlcN and NAPA could inhibit the IKKα nuclear translocation, we analyzed its subcellular localization by immunocytochemistry. Detection of IKKα revealed that this protein is mainly cytoplasmic in unstimulated cells, while it accumulates in the nucleus of cells stimulated with TNFα. Cells pre-treated with GlcN and NAPA and subsequently stimulated with TNFα showed a prevalent cytoplasmic IKKα localization (Figure 4). This result was confirmed in human primary chondrocytes by Western blot analysis in which both GlcN and NAPA were able to inhibit the re-localization of IKKα into nuclei (Figure 5a, b).


A peptidyl-glucosamine derivative affects IKKalpha kinase activity in human chondrocytes.

Scotto d'Abusco A, Politi L, Giordano C, Scandurra R - Arthritis Res. Ther. (2010)

Effect of glucosamine (GlcN) and NAPA on inhibitor κB kinase alpha (IKKα) nuclear translocation in human primary chondrocytes. The analysis was performed by Western blot. Cells were untreated (CTL), treated with tumor necrosis factor-alpha (TNFα) or pre-treated with 10 mM GlcN (G10) or NAPA (N10) and then stimulated with TNFα for 1 hour. (a) Cytosolic extract probed with antibodies against IKKα and β-actin. (b) Nuclear extract probed with antibodies against IKKα and fibrillarin. *P ≤ 0.05. Results are expressed as fold change with respect to control. A.U., arbitrary units; NAPA, 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-β-D-glucose.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875647&req=5

Figure 5: Effect of glucosamine (GlcN) and NAPA on inhibitor κB kinase alpha (IKKα) nuclear translocation in human primary chondrocytes. The analysis was performed by Western blot. Cells were untreated (CTL), treated with tumor necrosis factor-alpha (TNFα) or pre-treated with 10 mM GlcN (G10) or NAPA (N10) and then stimulated with TNFα for 1 hour. (a) Cytosolic extract probed with antibodies against IKKα and β-actin. (b) Nuclear extract probed with antibodies against IKKα and fibrillarin. *P ≤ 0.05. Results are expressed as fold change with respect to control. A.U., arbitrary units; NAPA, 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-β-D-glucose.
Mentions: IKKβ activates the canonical NF-κB pathway by phosphorylation of IκBα, whereas IKKα is not required to phosphorylate IκBα, but it plays an important role by localizing into the nucleus of activated cells and inducing the transcription of NF-κB-dependent genes. To determine whether GlcN and NAPA could inhibit the IKKα nuclear translocation, we analyzed its subcellular localization by immunocytochemistry. Detection of IKKα revealed that this protein is mainly cytoplasmic in unstimulated cells, while it accumulates in the nucleus of cells stimulated with TNFα. Cells pre-treated with GlcN and NAPA and subsequently stimulated with TNFα showed a prevalent cytoplasmic IKKα localization (Figure 4). This result was confirmed in human primary chondrocytes by Western blot analysis in which both GlcN and NAPA were able to inhibit the re-localization of IKKα into nuclei (Figure 5a, b).

Bottom Line: We investigated the possibility that GlcN and NAPA inhibit IKK kinase activity and found that NAPA inhibits the IKKalpha kinase activity, whereas GlcN does not.Our results demonstrate that glucosamine and its peptidyl derivative can interfere with NF-kappaB signaling pathway by inhibiting IKKalpha activity in human chondrocytes.While NAPA can both specifically inhibit the IKKalpha kinase activity and IKKalpha nuclear re-localization, GlcN only acts on IKKalpha nuclear re-localization.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemical Sciences, Sapienza University of Roma, P,le Aldo Moro, 5, 00185 Roma, Italy. anna.scottodabusco@uniroma1.it

ABSTRACT

Introduction: Nuclear factor-kappaB (NF-kappaB) transcription factor regulates several cell signaling pathways, such as differentiation and inflammation, which are both altered in osteoarthritis. Inhibitor kappaB kinase (IKK)alpha and IKKbeta are kinases involved in the activation of the NF-kappaB transcription factor. The aim of the present study was to determine the effects of glucosamine (GlcN), which is administered in the treatment of osteoarthritis, and of its 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-beta-D-glucose (NAPA) derivative on IKK kinases and, consequently, on NF-kappaB activation in human chondrocytes.

Methods: The human chondrosarcoma cell line HTB-94 and human primary chondrocytes were stimulated with tumor necrosis factor (TNF)alpha after pre-treatment with GlcN or NAPA. Gene mRNA expression level was evaluated by real-time PCR. Inhibitor kappaB protein (IkappaB)alpha phosphorylation and p65 nuclear re-localization were analyzed by Western blotting; IKKalpha nuclear re-localization was also investigated by immunocytochemistry and Western blotting. IKK kinase activity was studied by in vitro kinase assay.

Results: After TNFalpha stimulation, the mRNA expression level of some of the genes under NF-kappaB control, such as interleukin (IL)-6 and IL-8, increased, while treatment with GlcN and NAPA reverted the effect. We investigated the possibility that GlcN and NAPA inhibit IKK kinase activity and found that NAPA inhibits the IKKalpha kinase activity, whereas GlcN does not. Interestingly, both GlcN and NAPA inhibit IKKalpha nuclear re-localization.

Conclusions: Our results demonstrate that glucosamine and its peptidyl derivative can interfere with NF-kappaB signaling pathway by inhibiting IKKalpha activity in human chondrocytes. However, the mechanism of action of the two molecules is not completely overlapping. While NAPA can both specifically inhibit the IKKalpha kinase activity and IKKalpha nuclear re-localization, GlcN only acts on IKKalpha nuclear re-localization.

Show MeSH
Related in: MedlinePlus