Regulation of alpha-smooth muscle actin and other polypeptides in proliferating and density-arrested vascular smooth muscle cells.
We have examined alpha-smooth muscle actin (alpha-SM actin) protein and mRNA levels in proliferating and density-arrested rabbit vascular smooth muscle cells (SMC) and also studied overall polypeptide synthesis in these cells by two-dimensional (2-D) gel electrophoresis. Of the approximately 1,000 cellular polypeptides resolved by 2-D gel analysis, we consistently detected increased expression of 12 polypeptides in growth-arrested SMC. These polypeptides, with apparent molecular weights of 24,000 to 55,000 exhibited relative increases of between fourfold to greater than tenfold. Three of these polypeptides were expressed at undetectable levels in proliferating SMC. We also detected 12 secreted polypeptides that were expressed at higher levels in growth-arrested SMC. More changes were associated with the secreted polypeptides, since they represented approximately 4% of the total resolved secreted polypeptides, while only 1% of the cellular polypeptides were increased in high-density growth-arrested cells. Under these conditions we observed no change in relative alpha-SM actin protein content as determined by 2-D gel analysis and Western blots. This was corroborated by high levels of alpha-SM actin mRNA levels in both proliferating and high-density growth-arrested SMC. These results indicate rabbit vascular SMC maintain a high level of expression of a smooth muscle differentiation marker (alpha-SM actin) in a proliferation- and density-independent manner. We also examined polypeptide synthesis in SMC isolated by enzymatic digestion of the aorta vs. cells isolated by the explant method. We found that although overall protein patterns were remarkably similar, several differences were observed. These differences were not due to increased contamination by fibroblasts, since both enzymatically- and explant-derived SMC contained high levels of alpha-SM actin as determined by immunofluorescence and by Northern analysis.
Liau
G
G
American Red Cross, Jerome H. Holland Laboratory for Biomedical Sciences, Rockville, Maryland 20855.
Janat
M F
MF
Wirth
P J
PJ
D000199:Actins / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D001013:Aorta, Thoracic; D015153:Blotting, Western; D002452:Cell Count; D002455:Cell Division; D046508:Culture Techniques; D015180:Electrophoresis, Gel, Two-Dimensional; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D003012:Microbial Collagenase; D009693:Nucleic Acid Hybridization; D010196:Pancreatic Elastase; D010452:Peptide Biosynthesis; D010455:Peptides / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D011817:Rabbits
D000199:Actins; D010455:Peptides; D012333:RNA, Messenger; D010196:Pancreatic Elastase; D003012:Microbial Collagenase