Developmental expression of the HNK-1 carbohydrate epitope on aggrecan during chondrogenesis.

Previously, we showed that the HNK-1 carbohydrate epitope is expressed on aggrecan synthesized in the notochord but not in mature cartilage. In the present study, we demonstrate that in immature cartilage (embryonic day 6) the HNK-1 epitope is also expressed predominantly on aggrecan proteoglycan molecules. This finding was verified by using an aggrecan-deficient mutant, the nanomelic chick, which lacks HNK-1 immunostaining in the extracellular matrix of dividing and hypertrophic chondrocytes as late as embryonic day 12. By using both biochemical and immunologic approaches, the initially prominent expression of the HNK-1 epitope is down-regulated as development of limb and vertebral cartilage proceeds, so that by embryonic day 14 no HNK-1 is detectable. Localization changes with development and the HNK-1-aggrecan matrix becomes restricted to dividing and hypertrophic chondrocytes and is particularly concentrated in the intraterritorial matrix. Concomitant with the temporal and spatial decreases in HNK-1, there is a significant increase in keratan-sulfate content and the aggrecan-borne HNK-1 epitope is closely associated with proteolytic peptides that contain keratan sulfate chains, rather than chondroitin sulfate chains or carbohydrate-free domains. Lastly, the diminution in HNK-1 expression is consistent with a reduction in mRNA transcripts specific for at least one of the key enzymes in HNK-1 oligosaccharide biosynthesis, the HNK-1 sulfotransferase. These findings indicate that the HNK-1 carbohydrate may be a common modifier of several proteoglycans (such as aggrecan) that are usually expressed early in development, and that HNK-1 addition to these molecules may be regulated by tissue- and temporal-specific expression of requisite sulfotransferases and glycosyltransferases.

NF-kappa B inhibition by omega -3 fatty acids modulates LPS-stimulated macrophage TNF-alpha transcription.

Omega-3 fatty acid (FA) emulsions reduce LPS-stimulated murine macrophage TNF-alpha production, but the exact mechanism has yet to be defined. The purpose of this study was to determine the mechanism for omega-3 FA inhibition of macrophage TNF-alpha production following LPS stimulation. RAW 264.7 cells were pretreated with isocaloric emulsions of omega-3 FA (Omegaven), omega-6 FA (Lipovenos), or DMEM and subsequently exposed to LPS. IkappaB-alpha and phospho-IkappaB-alpha were determined by Western blotting. NF-kappaB binding was assessed using the electromobility shift assay, and activity was measured using a luciferase reporter vector. RT-PCR and ELISA quantified TNF-alpha mRNA and protein levels, respectively. Pretreatment with omega-3 FA inhibited IkappaB phosphorylation and significantly decreased NF-kappaB activity. Moreover, omega-3-treated cells demonstrated significant decreases in both TNF-alpha mRNA and protein expression by 47 and 46%, respectively. These experiments demonstrate that a mechanism for proinflammatory cytokine inhibition in murine macrophages by omega-3 FA is mediated, in part, through inactivation of the NF-kappaB signal transduction pathway secondary to inhibition of IkappaB phosphorylation.

Modulation of lipopolysaccharide-stimulated macrophage tumor necrosis factor-alpha production by omega-3 fatty acid is associated with differential cyclooxygenase-2 protein expression and is independent of interleukin-10.

BACKGROUND: The role of omega-3 fatty acids (FA) as anti-inflammatory agents involves the inhibition of macrophage (Mphi) cytokine production, but the mechanisms involved are not well defined. The effects of omega-3 FA on the transcription and translation of cyclooxygenase-2 (COX-2), the production of prostaglandin E(2) (PGE(2)), and the production of interleukin-10 (IL-10) were investigated as potential mechanisms for the down-regulation of lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha production. METHODS: RAW 264.7 Mphi were incubated with Omegaven (10 mg% omega-3 FA), Lipovenos (10 mg% omega-6 FA), or DMEM for 4 h of pretreatment. The cells were then exposed to LPS (1 microg/ml) or medium alone for 3 h. COX-2 mRNA levels were determined by semi-quantitative reverse transcriptase polymerase chain reaction, and COX-2 protein levels were determined by Western blotting. The levels of PGE(2) and IL-10 proteins secreted into the medium were quantified using enzyme-linked immunosorbent assays. RESULTS: Pretreatment with omega-3 FA increased Mphi COX-2 protein expression levels without altering the levels of COX-2 mRNA in response to LPS stimulation. In addition, pretreatment with omega-3 FA dramatically decreased the PGE(2) and IL-10 production induced by LPS, whereas pretreatment with an equivalent dose of omega-6 FA only resulted in a modest increase in PGE(2) and a slight decrease in IL-10 production compared to controls. CONCLUSION: As COX-2 protein levels were increased without a change in COX-2 mRNA levels with omega-3 FA pretreatment, this suggested that omega-3 FA did not upregulate COX-2 at the transcriptional level. The omega-3 FA may instead posttranscriptionally stabilize existing COX-2 mRNA. The increased COX-2 expression may thus be explained by increased translation of COX-2 and/or decreased COX-2 degradation. The decreased PGE(2) production could be attributed to the replacement of Mphi membrane omega-6 FA substrates by omega-3 FA and the competitive inhibition of COX-2 enzyme by omega-3 FA. The reduction of active COX-2 product associated with an increase in COX-2 enzyme implies the existence of a negative feedback mechanism. Surprisingly, IL-10 production was decreased by omega-3 FA pretreatment, indicating that the reduced IL-10 inhibition of Mphi cytokine production was superceded by the other actions of omega-3 FA.