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

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-PA⅐PAI-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 125 I-labeled t-PA or t-PA⅐PAI-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 125 I-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.Fibrinolysis is a complex process that requires precise regulation in vivo to ensure that it is neither deficient nor excessive. The plasminogen activator system is critical for thrombolysis, as well as other physiological processes including cell migration and tissue remodeling (1, 2). Human tissue-type plasminogen activator (t-PA) 1 (M r 68,000), a key serine protease in this system, is of particular pharmacological interest because of its value in the treatment of thromboembolic disorders. Regulation of the plasminogen activator system involves modulation by cofactors and inhibitors as well as controlled synthesis of the key components. It is well documented that t-PA expression is influenced by a variety of exogenous factors such as hormones, growth factors, and cytokines (3-6). Regulation by these factors appears to be receptor-mediated and is imposed at the transcriptional level (7-10). However, information on the role of t-PA in the local regulation of its own biosynthesis, release, and clearance is limited. Kadouri and Bohak (11) suggested a negative feedback-type control for t-PA expression in human lung fibroblasts but did not identify the mechanism by which expression was regulated. Although t-PA has been reported to undergo receptor-mediated binding to a variety of cultured cells (12-15), the biological significance of this phenomenon remains uncertain. Reports on the internalization and lysosomal degradation of t-PA have been largely confined to studies on hepatocytes, which have a known clearance function (for reviews, see Refs. 16,17)....

Nerve Growth Factor Induces Rapid Increases in Functional Cell Surface Low Density Lipoprotein Receptor-related Protein

“…For example, our recent data suggest that negative feedback regulation of tissue plasminogen activator expression in colon fibroblasts is mediated via cell surface LRP (63). These observations suggest that at least one LRP ligand may modulate its own synthesis through communication between LRP and the transcriptional machinery of the cell.…”

Role of Tissue Plasminogen Activator Receptor LRP in Hippocampal Long-Term Potentiation

“…First, several studies have shown that apoE3, a ligand of LRP, increases neurite extension via LRP (Bellosta et al, 1995;Holtzman et al, 1995;Narita et al, 1997). Second, our previous studies have shown that negative feedback regulation of tPA gene expression in colon fibroblasts is mediated via tPA interaction with cell surface LRP (Hardy et al, 1997). Third, recent studies have shown that the LRP tail interacts with a heterotrimeric GTP-binding protein (Goretzki and Mueller, 1998) and two cytoplasmic adaptor proteins, FE65 and mammalian Disabled (Trommsdorff et al, 1998).…”

Identification of a Major Cyclic AMP-Dependent Protein Kinase A Phosphorylation Site within the Cytoplasmic Tail of the Low-Density Lipoprotein Receptor-Related Protein: Implication for Receptor-Mediated Endocytosis

“…First, several studies have shown that apoE3, a ligand of LRP, increases neurite extension via LRP (1,11,23,37). Second, our previous studies have shown that negative feedback regulation of tissue plasminogen activator gene expression in colon fibroblasts is mediated via cell surface LRP (18). Third, studies have shown that the LRP tail interacts with a GTP-binding protein induces cAMP-dependent protein kinase activity (16).…”