1 Induction of lipocortin 1 secretion by dexamethasone has been demonstrated, although the secretory mechanism is still unknown. We have studied the effects of 12-tetradecanoyl phorbol 13-acetate (TPA) and/or dexamethasone on the expression, translocation, and secretion of lipocortin 1 in U937 cells. 2 The expression of lipocortin 1 and its mRNA increased during TPA-induced differentiation of U937 cells to a maximum of 1.9 fold and 8.2 fold, respectively, after 48 h. Both the protein and the mRNA levels decreased after 48 h. 3 TPA caused the translocation of lipocortin 1 from the cytosol to the membrane of U937 cells in a time-dependent manner, as determined by Western blot analysis. The translocation was concurrent with the differentiation of the cells. After 48 h of TPA treatment, 82.6+6.5% of lipocortin 1 was present in the membrane fraction compared to 41.6+1.7% in untreated cells. 4 The amount of lipocortin 1 that was externally bound (associated) with the membrane increased to 3.2 fold as the cytosol to membrane translocation of lipocortin 1 increased. 5 Dexamethasone decreased the externally bound lipocortin 1, but had no effect on the cytosol to membrane translocation. 6 This offers a model system with which the function and the secretion mechanism of lipocortin 1 can be studied. Our data is consistent with the hypothesis that the secretory mechanism is through an unknown pathway, involving the translocation of lipocortin 1 from the cytosol to the internal membranes, and then, its secretion to the external membrane.

Hyperlipidemia, especially hypercholesterolemia, may contribute to glomerulosclerosis as it does to atherosclerosis. Low density lipoprotein (LDL) stimulates the production of extracellular matrix by mesangial cells in culture as well as the proliferation of mesangial cells. This study was carried out to examine the effects of LDL on the type IV collagen (CIV) production by cultured rat mesangial cells (CRMC). Subconfluent CRMC monolayers which were grown in RPMI with 20% lipid-free fetal calf serum for 48 h were challenged with LDL (0, 50,100,150 and 200 μg/ml) for another 48 h. LDL was prepared from normal human plasma. Mesangial cell proliferation was examined by [³H]-thymidine uptake. Production of CIV was evaluated as the expression of CIV on the cell surface by flow-cytometric analysis. The collagen synthesis was measured by the [³H]-proline uptake. Total RNA was extracted from CRMC at 6 and 24 h of incubation with 150 μg/ml LDL, and Northern blotting and hybridization was performed with cDNAs for α_rCIV, for 72-kD collagenase and for tissue inhibitor of metalloproteinase (TIMP)-2. The amount of total mRNA was corrected with β-actin mRNA. Mesangial cell proliferation increased in all concentrations studied and had a peak value of 221% with 150 μg/ml of LDL. Expression of CIV increased by 30-60% in 100-200 μg/ml of LDL. Collagen synthesis also increased by 50-70% in 150-200 μg/ml of LDL. The mRNA ratio (procollagen α₁(IV)/β-actin) increased to 133% at 24 h. The mRNA ratio (TIMP-2/β-actin) increased to 137% at 24 h. mRNA ratios at 6 h showed no change. LDL had little effect on collagenase mRNA expression. These results show that LDL stimulates, in addition to stimulated mesangial cell proliferation, collagen production through a combination of increased collagen synthesis and possibly decreased collagen degradation. Hyperlipidemia may contribute to the pathogenesis of glomerulosclerosis through the direct effect of LDL on mesangial cells.