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.

Aggrecan is a complex multidomain macromolecule that undergoes extensive processing and post-translational modification. A thorough understanding of the events and signals that promote translocation of aggrecan through the secretory pathway is lacking. To investigate which features of the C-terminal G3 region are necessary for successful translocation of the core protein, a number of deletion constructs based on the chick aggrecan cDNA sequence were prepared and transiently expressed in COS-1 cells and the natural host, embryonic chick chondrocytes; stable cell lines were established as well. The present results clearly establish a precise requirement for that portion of the G3 C-lectin domain encoded by exon 15 for: (i) translocation from the endoplasmic reticulum (ER) to the Golgi, (ii) secretion from the cell, (iii) galactosylation of chondroitin sulfate (CS) chains, (iv) generation of Ca ؉2 -dependent galactose binding ability. Furthermore, in the absence of this subdomain there is excess accumulation in the ER of expression products leading to a stress-related response involving the chaperones Grp78 and protein disulfide isomerase, followed by degradation via a ubiquitin-proteosome pathway. All of these events in the model system faithfully mimic the naturally occurring nanomelic mutant, which also elicits a ubiquitin-mediated degradation response due to the accumulation of the truncated core protein precursor. This study represents the first report of the mode of degradation of overexpressed or misfolded proteoglycans and suggests that, although proteoglycans follow different glycosylation pathways from other glycoproteins, they are monitored by an ER surveillance system similar to that which detects other misfolded proteins.Aggrecan, the major proteoglycan of the cartilage extracellular matrix, contains two globular domains in the N-terminal portion, G1 and G2, and one C-terminal globular domain, G3. Between the G2 and G3 globular domains, glycosaminoglycan chains (GAGs) 1 are covalently attached to an extended core protein. Functionally, it is well established that the G1 domain is responsible for interaction with hyaluronan in the extracellular matrix. The C-terminal G3 domain has been implicated in synthesis and maturation of aggrecan, because its absence is associated with the avian chondrodystrophy, nanomelia. The nanomelic chick bears a mutation in the aggrecan gene that introduces an early stop codon into the translated sequence, resulting in synthesis of a truncated core protein, which is neither glycosylated nor secreted by chondrocytes (1). The mutant precursor is modified by addition of N-linked oligosaccharides and the chondroitin sulfate chain initiating xylose, but does not acquire mature CS chains, consistent with the conclusion that it progresses no further than the endoplasmic reticulum (ER) in the secretory pathway (2). These studies suggested a previously unrecognized role for the C-terminal globular domain: that it might contain recognition or retention signals or that proper folding o...

No abstract

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. Developmental Dynamics 226:42-50, 2003.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.