The first intron of the human Pro alpha 1(I) collagen gene contains an orientation-dependent enhancer composed of both positive and negative cis-acting elements involved in the transcriptional regulation of this gene. Deletion of a 360 bp Sau 3A intronic fragment spanning nucleotide +494 to +854 (S360) resulted in dramatic down-regulation of pCOL-KT (Thompson et al., J Biol Chem 266: 2549-2556, 1991). Using a DNaseI protection assay, we demonstrate a single footprint located at +590 to +615 in the S360 fragment; nuclear extracts prepared from mesenchymal and nonmesenchymal cells exhibited similar binding characteristics. A double stranded oligonucleotide representing a consensus Ap-1 binding sequence competed with S360 for binding. In contrast to what occurred in response to S360 deletion which was always accompanied by reduced expression, the deletion of the Ap-1 binding site (+598 to +off) caused either increased or decreased expression of the reporter gene depending on the target cell. Site-directed mutations in the Ap-1-like cis-element of Pro alpha 1(I) were also tested in transient expression assays. Consistent with the paradoxical results of Ap-1 deletion, we observed that the functional consequences of mutations in the Ap-1 site also varied in different cells. In A204 cells, one point mutation, which resulted in the loss of protein binding to S360, led to increased CAT activity while another point mutant, which retained binding of the Ap-1 like trans-acting factor(s), showed decreased CAT expression. The effects of these two mutations in the HFL-1 cells were exactly opposite of what was seen for A204 cells. Based on these observations, we postulate that the Ap-1 site plays a critical role in the transcriptional activity of the human Pro alpha 1(I) gene. The implications of an apparently dual mode of regulation through a single cis-regulatory element are discussed.
Macrophages express a receptor on the cell surface that functions to clear glycoproteins from the extracellular milieu. The activity of this receptor is sensitive to treatment with trypsin. In inflammatory situations, macrophages are activated and exposed to increased levels of extracellular proteases. Under these conditions, mannose receptor activity on the macrophages is diminished. We therefore decided to study the effects of trypsin treatment on the structure and activity of cell-associated and purified receptor that might contribute to the activation-associated receptor down-regulation. Trypsin treatment (1 microgram/ml for 3 h) resulted in the production of a 140 kDa, trypsin-resistant fragment from both intact cells and isolated receptor. This fragment was no longer able to bind ligand. The remaining 35 kDa fragment apparently is further degraded into smaller fragments, since no evidence of this domain was found on Coomassie Blue-stained gels. The 140 kDa fragment retained immunoreactivity and contained at least a portion of the iodinated tyrosine residues following surface labelling with Na125I. Neither calcium nor ligand protected the receptor from proteolysis. In addition, prior treatment with oxidants did not increase the susceptibility of the receptor to trypsin digestion. We conclude from these results that the macrophage mannose receptor is clipped by the serine protease trypsin at the cell surface, resulting in the release and further degradation of the binding domain, and the production of a membrane-associated 140 kDa fragment. This trypsin-mediated down-regulation of receptor activity might be important in controlling glycoprotein clearance during inflammation.
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