Characterization of a serum factor that decreases albumin mRNA in cultured hepatocytes.
When primary cultures of hepatocytes are exposed to media containing fetal bovine serum (FBS) there is a rapid decrease in levels of tissue-specific mRNAs such as albumin mRNA. We used Northern blot analysis to examine mRNA levels in cultured hepatocytes, and characterized the factor in FBS that significantly reduces the steady state albumin mRNA level. Neonatal bovine serum or serum derived from platelet-poor calf plasma proved as potent as did FBS, but commercial bovine serum albumin did not exhibit this inhibitory activity. Inhibitory activity of FBS was not removed by moderate heat treatment, dialysis, or extraction with organic solvents. However, incubation of FBS with a highly anionic detergent such as 0.1% sodium dodecyl sulfate or N-lauroyl sarcosine, followed by extensive dialysis, resulted in sera that did not inhibit expression of albumin mRNA. These sera supported cell attachment and seemed non-toxic toward the cells. Ammonium sulfate fractionation of FBS showed the activity was present in the 45 to 70% fraction, and trypsin digestion destroyed the inhibitory activity. Gel exclusion chromatography gave a molecular weight of 60,000 to 70,000. Fractionation of serum proteins by DEAE-Sephacel or Cibacron blue-agarose showed enrichment for albumin in the most active fractions. Interestingly, metabolic labeling of secreted and cellular proteins with 35S-methionine and cysteine showed no significant difference between hepatocytes maintained for 2 days beforehand in serum-free or serum-supplemented media, and no difference between detergent-treated FBS and control FBS.(ABSTRACT TRUNCATED AT 250 WORDS)
Johnston
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DE
Department of Medicine, New England Medical Center, Boston, Massachusetts 02111.
Jefferson
D M
DM
D000818:Animals; D001685:Biological Factors / Q000097:blood* / Q000302:isolation & purification / Q000494:pharmacology; D002478:Cells, Cultured; D003902:Detergents; D005786:Gene Expression Regulation / Q000187:drug effects*; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D010949:Plasma; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012521:Sarcosine / Q000031:analogs & derivatives; D012709:Serum Albumin / Q000235:genetics*; D012967:Sodium Dodecyl Sulfate
D001685:Biological Factors; D003902:Detergents; D012333:RNA, Messenger; D012709:Serum Albumin; D012967:Sodium Dodecyl Sulfate; C025231:sarkosyl; D012521:Sarcosine