Hepsin was previously identified as a putative cell-surface serine protease. When hepatoma cells were treated with anti-hepsin antibodies, their growth was substantially arrested, suggesting the requirement of hepsin molecules present at the cell surface for normal cell growth. This was further supported by a gross inhibition of cell growth with hepsinspecific antisense oligonucleotides. Upon treatment of cells with antisense oligonucleotides, rapid reduction in cellular hepsin was observed. This reduction in cellular hepsin levels was accompanied by drastic morphological changes. Various tissues in the developing mouse embryo showed greatly elevated hepsin levels in regions of active proliferation. These results indicate that hepsin plays an essential role in cell growth and maintenance of cell morphology.Cell-surface serine proteases have been known to play important roles in various cell functions (1). Our current understanding of this class of plasma membrane proteins, however, is significantly limited. Hepsin is a putative membrane-associated serine protease of 51 kDa (2). It is synthesized as a single polypeptide chain of 417 amino acid residues with a 27-residue-long internal hydrophobic sequence (2, 3). Hepsin is located primarily in the plasma membrane with its trypsin-type protease module (the C-terminal half) at the external surface of cells. This molecular orientation makes hepsin particularly interesting since very little is known about the biological roles of such serine proteases in spite of their predicted importance in cell growth. Hepsin is present at significant levels in many different types of mammalian cells such as human hepatoma cells (HepG2 and PLC/PRF/5 cells), mammary cancer cells (MCF784 and T470), peripheral nerve cells (PC12), and baby hamster kidney cells, but at undetectable levels in some types of cells such as human umbilical cord as well as rat capillary endothelial cells. Hepsin is produced in most tissues but at a particularly high level in the liver (2).In the present report, we describe the importance of hepsin for mammalian cell growth. Experimental evidence indicates that the expression of hepsin is necessary for normal cell growth and morphology. MATERIALS AND METHODSEffects of Anti-Hepsin Antibodies and Antisense Oligonucleotides on the Growth of PLC/PRF/5 Cells. To test the importance of hepsin for cell growth, a set of antisense oligonucleotides and their thioate derivatives were prepared. Synthetic phosphodiester oligonucleotides including sense strand (SS-pd-oligo237, 5'-GGCAGTGACATGGCGCA-GAAG-3') and antisense strand (AS-pd-oligo237, 5'-CTTCTGCGCCATGTCACTGCC-3') of hepsin, which areThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. purified on reverse-phase high-performance liquid chromatography, corresponded to the.5' end region of hepsin cDNA (nt 237-257) with the first in-frame ATG at the center (3...
Overproduction of Apo CIII causes elevated plasma triglyceride levels in transgenic animals and is associated with hypertriglyceridemia in humans. The regulation of apo CIII production is likely to play an important role in controlling plasma triglyceride levels. As an initial step in determining the role of transcriptional regulation in the production of apo CIII and in triglyceride metabolism, we have begun to characterize the activity of specific transcriptional regulatory elements in the CIII promoter. In the current study, we have identified and characterized an NF-kappa B regulatory element located 150 nucleotides upstream from the transcriptional start site of the apo CIII gene. Purified NF-kappa B, as well as an NF-kappa B protein in HepG2 cell nuclear extracts, bound specifically to this sequence element. The hepatic protein was induced by phorbol ester (PMA), and reacted with antibodies to the p50 and p65 subunits of NF-kappa B. The NF-kappa B element conferred PMA and IL1-beta inducible transcriptional activity to a heterologous promoter/reporter construct when transfected into HepG2 cells. Analysis of the full length CIII promoter demonstrated that the inducible activity of the NF-kappa B element was suppressed by sequences in the apo CIII enhancer element located approximately 500 nucleotides upstream of the NF-kappa B binding site. A deletion removing the enhancer restored the PMA inducible activity of the NF-kappa B binding site. These results indicate that apo CIII gene expression is regulated by NF-kappa B, and suggest that apo CIII production may be modulated by cellular signals, like inflammatory cytokines, that activate NF-kB.
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Nuclear protein factor type 1 (NPF-1) thatsimulates IMR-32 primase-associated DNA polymerase a, and a2 activities has been purified from a high-salt extract of liver chromatin from 6-month-old rats. The final purified factor lacks DNA polymerase a, RNA polymerase, and DNA-unwinding or topoisomerase type I activities. The stimulatory activity is destroyed by trypsin (60 min at 37C), DNase II (60 min at 370C), and heat treatment (2 min at 68C). The '2I-labeled NPF-1 does not bind to activated calf thymus DNA or poly(dC). However, it forms a ternary complex with DNA in the presence of DNA polymerase a-primase complex (a, and a2). The ternary complex sediments on sucrose density gradient as a heavier band (11S). The NPF-1 also stimulates (2.5-fold) primase-catalyzed incorporation of GMP and dGMP from the corresponding triphosphates on poly(dC) template even in the presence of a high concentration of a-amanitin (400 ,tg/ml). 8.0/1 mM EDTA/6 mM KCI/2 mM MgCl2/1 mM 2-mercaptoethanol/0.1 mM phenylmethylsulfonyl fluoride/0.1% polyethylene glycol (PEG; 8 kDa) using a glass-Teflon homogenizer. The homogenate was centrifuged at 100,000 x g for 1 hr. The supernatant (1.5 ml) was applied to a DE 23 column (1.5 x 10 cm) equilibrated with 50 mM potassium phosphate, pH 7.6/1 mM MgCl2/1 mM 2-mercaptoethanol/0.1% PEG/ 0.1 mM phenylmethylsulfonyl fluoride (buffer C). After
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