Differential use of chondroitin sulfate to regulate hyaluronan binding by receptor CD44 in Inflammatory and Interleukin 4-activated Macrophages.

CD44 is a cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan and is involved in processes ranging from leukocyte recruitment to wound healing. In the immune system, the binding of hyaluronan to CD44 is tightly regulated, and exposure of human peripheral blood monocytes to inflammatory stimuli increases CD44 expression and induces hyaluronan binding. Here we sought to understand how mouse macrophages regulate hyaluronan binding upon inflammatory and anti-inflammatory stimuli. Mouse bone marrow-derived macrophages stimulated with tumor necrosis factor α or lipopolysaccharide and interferon-γ (LPS/IFNγ) induced hyaluronan binding by up-regulating CD44 and down-regulating chondroitin sulfation on CD44. Hyaluronan binding was induced to a lesser extent in interleukin-4 (IL-4)-activated macrophages despite increased CD44 expression, and this was attributable to increased chondroitin sulfation on CD44, as treatment with ß-d-xyloside to prevent chondroitin sulfate addition significantly enhanced hyaluronan binding. These changes in the chondroitin sulfation of CD44 were associated with changes in mRNA expression of two chondroitin sulfotransferases, CHST3 and CHST7, which were decreased in LPS/IFNγ-stimulated macrophages and increased in IL-4-stimulated macrophages. Thus, inflammatory and anti-inflammatory stimuli differentially regulate the chondroitin sulfation of CD44, which is a dynamic physiological regulator of hyaluronan binding by CD44 in mouse macrophages.

Expression of N-acetylglucosamine 6-O-sulfotransferases (GlcNAc6STs)-1 and -4 in human monocytes: GlcNAc6ST-1 is implicated in the generation of the 6-sulfo N-acetyllactosamine/Lewis x epitope on CD44 and is induced by TNF-alpha.

Sulfation at the 6-O position of N-acetylglucosamine (GlcNAc) in the context of sialyl 6-sulfo Lewis x occurs constitutively on specific glycoproteins present on high-walled endothelial venules (HEV) and is important for L-selectin dependent homing of lymphocytes. Here, the proinflammatory cytokine, TNF-alpha, induced the expression of 6-sulfo N-acetyllactosamine (LacNAc)/Lewis x on human peripheral blood monocytes (PBM). This epitope was detected by monoclonal antibody (mAb) AG107 after neuraminidase treatment suggesting a sialylated epitope, which was present on the cell adhesion molecule, CD44. Treatment of human PBM with TNF-alpha up-regulated the expression of N-acetylglucosamine 6-O-sulfotransferase-1 (GlcNAc6ST-1) and GlcNAc6ST-4, as determined by reverse transcriptase polymerase chain reaction (RT-PCR). However, only GlcNAc6ST-1 was induced by TNF-alpha in the human SR91 cell line, which also up-regulated the AG107 epitope. In ECV304 cells, the expression of GlcNAc6ST-4 alone was insufficient to generate the AG107 epitope. However, the transfection of GlcNAc6ST-1 resulted in significant sulfate incorporation into CD44 and generated the 6-sulfo LacNAc/Lewis x epitope on CD44, which was present largely on N-linked glycans. This demonstrates the induction of GlcNAc6STs in human monocytes in response to TNF-alpha and implicates GlcNAc6ST-1 in the generation of the 6-sulfo LacNAc/Lewis x epitope on CD44.

Opposing roles of C/EBPbeta and AP-1 in the control of fibroblast proliferation and growth arrest-specific gene expression.

Chicken embryo fibroblasts (CEF) express several growth arrest-specific (GAS) gene products in G0. In contact-inhibited cells, the expression of the most abundant of these proteins, the p20K lipocalin, is activated at the transcriptional level by C/EBPbeta. In this report, we describe the role of C/EBPbeta in CEF proliferation. We show that the expression of a dominant negative mutant of C/EBPbeta (designated Delta184-C/EBPbeta) completely inhibited p20K expression at confluence and stimulated the proliferation of CEF without inducing transformation. Mouse embryo fibroblasts nullizygous for C/EBPbeta had a proliferative advantage over cells with one or two functional copies of this gene. C/EBP inhibition enhanced the expression of the three major components of AP-1 in cycling CEF, namely c-Jun, JunD, and Fra-2, and stimulated AP-1 activity. In contrast, the over-expression of C/EBPbeta caused a dramatic reduction in the levels of AP-1 proteins. Therefore, C/EBPbeta is a negative regulator of AP-1 expression and activity in CEF. The expression of cyclin D1 and cell proliferation were stimulated by the dominant negative mutant of C/EBPbeta but not in the presence of TAM67, a dominant negative mutant of c-Jun and AP-1. CEF over-expressing c-Jun, and to a lesser extent JunD and Fra-2, did not growth arrest at high cell density and did not express p20K. Therefore, AP-1 interfered with the action of C/EBPbeta at high cell density, indicating that these factors play opposing roles in the control of GAS gene expression and CEF proliferation.