A peptidyl-glucosamine derivative affects IKKα kinase activity in human chondrocytes

Abstract

IntroductionNuclear factor-κB (NF-κB) transcription factor regulates several cell signaling pathways, such as differentiation and inflammation, which are both altered in osteoarthritis. Inhibitor κB kinase (IKK)α and IKKβ are kinases involved in the activation of the NF-κB 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-β-D-glucose (NAPA) derivative on IKK kinases and, consequently, on NF-κB activation in human chondrocytes.MethodsThe human chondrosarcoma cell line HTB-94 and human primary chondrocytes were stimulated with tumor necrosis factor (TNF)α after pre-treatment with GlcN or NAPA. Gene mRNA expression level was evaluated by real-time PCR. Inhibitor κB protein (IκB)α phosphorylation and p65 nuclear re-localization were analyzed by Western blotting; IKKα nuclear re-localization was also investigated by immunocytochemistry and Western blotting. IKK kinase activity was studied by in vitro kinase assay.ResultsAfter TNFα stimulation, the mRNA expression level of some of the genes under NF-κB 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 IKKα kinase activity, whereas GlcN does not. Interestingly, both GlcN and NAPA inhibit IKKα nuclear re-localization.ConclusionsOur results demonstrate that glucosamine and its peptidyl derivative can interfere with NF-κB signaling pathway by inhibiting IKKα activity in human chondrocytes. However, the mechanism of action of the two molecules is not completely overlapping. While NAPA can both specifically inhibit the IKKα kinase activity and IKKα nuclear re-localization, GlcN only acts on IKKα nuclear re-localization.

A pharmacoproteomic study confirms the synergistic effect of chondroitin sulfate and glucosamine

“…Proteins can be modified reversibly at serine or threonine residues by attachment of a GlcNAc molecule. As suggested by Scotto D'Abusco et al the effects of GlcN could derive, at least in part, from its capacity to O-GlcNAcylate proteins, primarily kinases (JNK, p38 and IKKα)5455. In the case of chondrocytes treated with GlcN, this protein modification competes with phosphorylation by utilizing the same amino acidic sites and, as a consequence, GlcN reduces NF-κB nuclear translocation and abrogates the transcription of proteolytic and pro-inflammatory target genes.…”

The induction of Maspin expression by a glucosamine-derivative has an antiproliferative activity in prostate cancer cell lines

“…Previously, in our laboratory, we studied glucosamine (GlcN) and a glucosamine-derivative, NAPA, which showed a good anti-inflammatory activity inhibiting p38 and JNK kinases, and in turn interfere with AP-1 pathway [29,30]. GlcN and mainly NAPA were also able to inhibit NF-κB pathway, particularly by inhibiting IKKα migration into the nucleus and its kinase activity [16]. IKKα belongs to IKK complex, which controls the activation of NF-κB pathway, and considering that recently, the nuclear role of IKKα has been associated to cancer progression through repression of Maspin expression [14], we decided to study whether our molecules could be able to interfere with Maspin production.…”

“…We found that some of these glucosamine derivatives, i.e. 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-β-D-glucose (NAPA) and 2-(N-Carbobenzyloxy)L-phenylalanylamido-2-deoxy-β-D-glucose (NCPA), were able to inhibit the migration into the nucleus of IKKα and NAPA was also able to partially inhibit IKKα kinase activity [16]. Taking into account the role of nuclear IKKα in cancer progression through repression of Maspin expression, we investigated whether these molecules were able to stimulate the Maspin expression by inhibiting IKKα nuclear migration in 143B osteosarcoma cells, demonstrating that NCPA was strongly able to interfere with this event [17].…”

Effect of Glucosamine-Derivate Molecules on the Extracellular Matrix Remodeling In Human Dermal Fibroblast

“…Starting from glucosamine, we designed a new and more selective molecule, 2-(N-Carbobenzyloxy)-L-phenylalanylamido-2-deoxy-β-Dglucose (NCPA) that worked at lower concentration than glucosamine because more hydrophobic, and were able to inhibit IKKα nuclear translocation and its activity, but did not affect IKKß activity. Furthermore, NAPA and GlcN were able to interfere with AP-1 pathway by inhibiting p38 and JNK kinases and consequently by inhibiting MMP-1, -3 and -13 production [12][13][14]. The successful results have prompting us to analyze if this molecules could be able to induce ECM production and to inhibit IKKα activation followed by UVB irradiation also in other connective tissue, as skin, to exert an anti-aging effect and prevention from photodamage.…”