Expression of porcine complement cytolysis inhibitor mRNA in cultured aortic smooth muscle cells. Changes during differentiation in vitro.
Porcine smooth muscle cells (SMC) grown to a high density monolayer culture undergo a morphological transition in which the cells draw away from the substrate and form multicellular nodules. The cells within the nodule resemble SMC in the aortic media and in some atherosclerotic plaques. The process of nodule formation is associated with the enhanced production of a secreted 38-kDa glycoprotein. To characterize the 38-kDa protein and its expression, a cDNA clone (pc38K) was isolated by immunological screening of an expression library. The 1646-base pair cDNA contains a single open reading frame encoding 446 amino acids. This sequence shows 72% homology with the human complement cytolysis inhibitor (CLI), also called serum protein-40,40, and 68% identity with rat sulfated glycoprotein-2. Based on this homology, we refer to the protein encoded by pc38K as CLI. This polypeptide includes a potential signal sequence, seven glycosylation sites and 10 cysteines in two clusters of five each. Southern blot analysis reveals that a single copy gene encoding CLI is present in mammals and chicken. In Northern blot analysis of SMC RNA, pc38K hybridizes to a mRNA of about 1.9 kilobases that is preferentially expressed in nodular SMC. The steady state level of this mRNA increases as the cultures begin to form multilayered regions. High levels of the mRNA persist after the cells are trypsin-dissociated. Culture medium conditioned by nodular SMC also induces an increase of CLI mRNA. Analysis of RNA extracted from porcine tissues show the highest levels of CLI mRNA in brain and liver; lower levels are detected in other tissues, including the aorta. Possible functions for the CLI are discussed.
Diemer
V
V
Department of Biological Sciences, University at Albany, State University of New York 12222.
Hoyle
M
M
Baglioni
C
C
Millis
A J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology / Q000502:physiology*; D001483:Base Sequence; D001798:Blood Proteins / Q000235:genetics*; D002454:Cell Differentiation; D002478:Cells, Cultured; D003001:Cloning, Molecular; D051152:Clusterin; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000502:physiology*; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D013552:Swine
D001798:Blood Proteins; C495242:CLU protein, human; D051152:Clusterin; D011506:Proteins; D012333:RNA, Messenger; D004247:DNA