Low Density Lipoprotein Receptor-related Protein Modulates the Expression of Tissue-type Plasminogen Activator in Human Colon Fibroblasts

Medora M Hardy,Joseph Feder,Richard A Wolfe,Guojun Bu

doi:10.1074/jbc.272.10.6812

Abstract

Human colon fibroblasts (HCF) produce tissue-type plasminogen activator (t-PA) in culture, but after 24-48 h, t-PA ceases to accumulate in the medium. Here, we report negative feedback regulation of t-PA expression, exerted by t-PA or complexes of t-PA with its physiological inhibitor, plasminogen activator inhibitor type 1 (PAI-1). Inhibition of t-PA expression could be induced by addition of exogenous t-PA or t-PA.PAI-1 complexes and reversed by monoclonal antibody directed against the active site of t-PA. Analysis of metabolically radiolabeled protein and cellular mRNA showed that both t-PA protein and mRNA levels declined considerably after 24 h. When 125I-labeled t-PA or t-PA.PAI-1 complexes were incubated with HCF, monensin-inhibitable endocytosis and catabolism were observed. The low density lipoprotein receptor-related protein (LRP) was found to be expressed by HCF and to mediate these events. Addition of the 39-kDa receptor-associated protein (RAP), an antagonist for ligand interactions with LRP, removed the block to t-PA expression and restored its accumulation in the medium. Moreover, RAP completely prevented the degradation of exogenous 125I-labeled t-PA by HCF, suggesting that LRP is the endocytic receptor for t-PA in these cells. These results demonstrate that cellular modulation of t-PA expression in HCF involves LRP receptor-mediated clearance of t-PA. This LRP receptor-mediated event results in down-regulation of t-PA expression at the mRNA level.

Highlights

Fibrinolysis is a complex process that requires precise regulation in vivo to ensure that it is neither deficient nor excessive
We report that lipoprotein receptor-related protein (LRP) mediates binding and endocytosis of type plasminogen activator (t-PA) and t-PA1⁄7PAI-1 complexes in human colon fibroblasts
Production of Plasminogen Activators by Human Colon Fibroblasts—When confluent cultures of Human colon fibroblasts (HCF) cells were assayed for t-PA production, it was found that accumulation of t-PA in the medium ceased after the first 24 – 48 h at concentrations ranging from ϳ100 –300 ng/ml (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—The normal human colon fibroblast strain CCD-18CO was obtained from American Type Culture Collection (ATCC CRL 1459). After the 2 h labeling period, the cells were washed and scraped into 0.5 ml of 3D detergent lysis buffer containing phenylmethylsulfonyl fluoride, as described previously [27]. Immunoprecipitation of radiolabeled t-PA from the medium and cell lysates was performed using monoclonal antibody 79 –7 that had been conjugated to cyanogen bromide-activated Sepharose 4B according to the manufacturer directions (Pharmacia). 125I-t-PA1⁄7PAI-1 complexes were separated from free t-PA by gel filtration on a Zorbax GF-250 column (DuPont Co.). SDS-PAGE analysis, followed by silver staining, revealed a single band migrating with Mr ϳ120,000, indicative of t-PA1⁄7PAI-1 complexes. Slot Blot Analysis of RNA—The isolation of total cytoplasmic RNA from cultured cells was performed by guanidinium thiocyanate/phenol/ chloroform extraction as described previously [29]. Nonspecific degradation was determined in the presence of 300 nM unlabeled t-PA and was subtracted from the experimental values

RESULTS

Percent of total radioactivity released by the cells

DISCUSSION