Analysis of transforming growth factor beta 1 messenger RNA degradation by the transcript-selective, 12-O-tetradecanoylphorbol-13-acetate-regulated ribonuclease system from U937 promonocytes.

Transforming growth factor (TGF) beta 1 mRNA is selectively stabilized during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced differentiation of U937 promonocytes. In previous studies (R. Wager and R. Assoian, Mol. Cell. Biol., 10: 5983-5990, 1990), we showed that this phenotype results from the action of a RNase system that (a) recognizes the transcript selectively and (b) is inhibited upon exposure of cells to TPA. The studies reported here were designed to localize domains of TGF-beta 1 mRNA required for recognition by this TPA-regulated, transcript-selective RNase system. By examining the degradation of several truncated TGF-beta 1 in vitro transcripts with U937 cell extracts, we show that the coding domain is sufficient to allow selective degradation of the mRNA and that this process is enhanced by either the 5' or 3' untranslated regions. The 5' and 3' untranslated regions of TGF-beta 1 mRNA are also required for TPA-mediated inhibition of the transcript-selective RNase system. In contrast, an analysis of the half-lives of the 2.1- and 1.8-kilobase TGF-beta 1 mRNAs showed that the first 270 bases, unique to the larger TGF-beta 1 mRNA, minimally affect degradation of the transcript. Finally, a survey of several transcripts showed that gamma-actin mRNA levels are also controlled by the TPA-regulated RNase system. The regulated decay of TGF-beta 1 mRNA may reflect the behavior of a class of transcripts subject to similar posttranscriptional controls on overall gene expression.

Transforming growth factor-beta 1 inhibits scavenger receptor activity in THP-1 human macrophages.

The macrophage scavenger receptor, a 220-kDa trimeric membrane glycoprotein, mediates the internalization of modified forms of low density lipoprotein (LDL) such as acetyl-LDL and oxidized-LDL and thus is likely to play a key role in atheroma macrophage foam cell formation. In addition, recent evidence suggests that the scavenger receptor may be an important macrophage binding site for lipopolysaccharide involved in lipopolysaccharide scavenging by macrophages. However, little is known about the regulation of this important receptor. We now report that the induction of scavenger receptor activity (as measured by acetyl-LDL stimulation of intracellular cholesterol esterification) seen in phorbol ester-differentiated THP-1 human macrophages was completely suppressed to the level seen in undifferentiated THP-1 monocytes by picomolar concentrations of transforming growth factor-beta 1 (TGF-beta 1). 125I-Acetyl-LDL degradation was inhibited in a dose-dependent manner by TGF-beta 1, with maximal inhibition (approximately 70%) occurring at 24 pM TGF-beta 1. Scatchard analysis revealed that TGF-beta 1 treatment resulted in a approximately 2-fold decrease in receptor number, and Northern blot analysis of RNA isolated from differentiated THP-1 macrophages demonstrated approximately 2-fold less scavenger receptor mRNA in TGF-beta 1-treated cells compared with that in macrophages not treated with TGF-beta 1. Since TGF-beta 1 is thought to be present in both atherosclerotic and inflammatory lesions, the above findings may have physiological relevance regarding the regulation of atheroma foam cell formation and/or the regulation of lipopolysaccharide clearance by macrophages.

A phorbol ester-regulated ribonuclease system controlling transforming growth factor beta 1 gene expression in hematopoietic cells.

12-Tetradecanoylphorbol-13-acetate (TPA)-induced differentiation of U937 promonocytes leads to a 30-fold increase in transforming growth factor beta 1 (TGF-beta 1) gene expression, and this effect results from a stabilized mRNA. Similar up-regulation was detected in TPA-treated K562 erythroblasts but was absent from cell lines that do not differentiate in response to TPA. Related studies in vitro showed that postnuclear extracts of U937 promonocytes contain a ribonuclease system that degrades TGF-beta 1 mRNA selectively and that this system is completely blocked by prior treatment of the cells with TPA. These data identify a new mechanism for regulating TGF-beta 1 mRNA levels and allow us to establish the overall basis for control of TGF-beta 1 gene expression by activation of protein kinase C. Our results also provide a new basis for understanding the long-term up-regulation of TGF-beta 1 gene expression that can accompany hematopoietic cell differentiation.

Type beta 1 transforming growth factor gene expression. A corrected mRNA structure reveals a downstream phorbol ester responsive element in human cells.

A combined approach of cDNA cloning and direct oligonucleotide mapping of TGF-beta 1 mRNA from several human cell lines has revealed that the major human TGF-beta 1 transcript is 381 bases shorter than originally reported, and that the reduced mRNA size is due to polyadenylation from an ATTAAA signal at position 2136 rather than use of the expected AATAAA signal at position 2517. Moreover, there is no evidence for a significant amount of structural heterogeneity, as a result of alternative polyadenylation, in the human TGF-beta 1 transcripts. Considering that the 381-base domain is not part of the major human TGF-beta 1 mRNA, we analyzed this sequence for potential transcriptional regulatory elements. We have identified a 16-base pair domain which contains three putative phorbol ester responsive elements (TREs) based on homology to the TRE consensus sequence. We also show that this 16-base pair fragment confers phorbol ester responsiveness to the chloramphenicol acetyltransferase gene after transient transfection of the heterologous construct in NIH-3T3 cells. The identification of a TRE immediately downstream of the last TGF-beta 1 exon suggests that a 3' enhancer may play an important role in human TGF-beta 1 gene transcription, and suggests a basis for growth factor-mediated regulation of TGF-beta 1 expression by activation of protein kinase C.

Major and minor forms of the rat liver asialoglycoprotein receptor are independent galactose-binding proteins. Primary structure and glycosylation heterogeneity of minor receptor forms.

Preparations of the rat liver asialoglycoprotein receptor (rat hepatic lectin, RHL), which is responsible for the selective uptake of partially deglycosylated serum glycoproteins, have been found to contain multiple polypeptide species. A method has been developed for separating the predominant species (RHL-1) from the minor species (RHL-2/3) using conditions in which RHL-1 retains its galactose-binding activity. Endoglycosidase digestion and lectin blotting have been utilized to demonstrate that RHL-2 and RHL-3 differ by the presence of different carbohydrate structures attached to a common peptide backbone. Antisera specific for RHL-1 and RHL-2/3 have been prepared and utilized to analyze the results of cross-linking experiments performed on purified receptor and hepatocyte membranes. The results show that RHL-1 and RHL-2/3 polypeptides are each associated into homooligomers but are physically unlinked to each other. The structure of the RHL-2/3 polypeptide has been established by protein and cDNA sequence analysis, which reveals that this protein is homologous to RHL-1 throughout its length but contains one major insertion of 18 amino acids near its NH2 terminus. The COOH-terminal portion of the RHL-2/3 polypeptide has been demonstrated to contain a galactose-recognition domain by expression in an in vitro transcription/translation system. The results of these experiments indicate that RHL-1 and RHL-2/3 polypeptides are self-associated into two distinct molecules, each of which has galactose-binding activity.

Surfactant apoprotein Mr = 26,000-36,000 enhances uptake of liposomes by type II cells.

The alveolar type II cell which synthesizes and secretes surfactant also plays a major role in the reuptake of surfactant lipids. In a recent in vivo study we found that the subfractions of natural surfactant that contained the surfactant protein with molecular weights of 26,000-36,000 (SP-26-36) were preferentially taken up into lamellar bodies of type II cells to a greater extent than were fractions that did not contain SP-26-36. Because the subfractions of natural surfactant in that study differed in other properties than the presence or absence of SP-26-36, the current study was undertaken to determine whether purified SP-26-36 enhanced the uptake of surfactant-like lipids by freshly isolated type II cells. SP-26-36 increased the uptake of label in radioactive surfactant-like lipids by up to 10-fold, and the effect of SP-26-36 was dependent on time, protein concentration, and temperature. The enhancement was inhibited by heat-treating the protein, by a polyclonal antibody against SP-26-36, and by metabolic inhibitors. The distribution of radioactivity in cell-associated phospholipids differed if cells were incubated with or without SP-26-36. If SP-26-36 was present during the incubation, greater than 96% of the radioactivity remained associated with phosphatidylcholine. In the absence of SP-26-36, only 85% of the radioactivity remained associated with phosphatidylcholine and 7% of the label appeared in phosphatidylglycerol. We hypothesize that SP-26-36 may act as a ligand to direct surfactant lipids to type II cells, perhaps to different metabolic pathways, and to regulate recycling and surfactant homeostasis.

Uptake of lung surfactant subfractions into lamellar bodies of adult rabbit lungs.

The goals of this investigation were to determine whether subfractions of alveolar surfactant that have different physical and biochemical properties are preferentially taken up from the alveolar air space into lamellar bodies and to correlate the magnitude of the uptake with the properties of the fractions. Radiolabeled subfractions were obtained by differential centrifugation of lavage fluid from rabbits that had been intravenously injected with radioactive palmitate. The subfractions were P (pellet) 3 (1,000 g, 20 min), P4 (60,000 g, 60 min), P5 (100,000 g, 16 h). Subfractions were instilled into the lungs of anesthetized spontaneously breathing adult rabbits, and lavage and lamellar body fractions were isolated at later times. P3 and P4 were taken up to a larger extent than was P5 or liposomes prepared from a P4 lipid extract. The fractions that were preferentially taken up (P3 and P4) contained surfactant apoprotein (APO) 36, tubular myelin, multilamellar vesicles, and were rapidly adsorbed to an air-water interface. P3 also contained APO 10. These results demonstrate that different forms of surfactant are recycled at different rates and suggest that there is specificity in the recycling process.