Effect of a reconstituted basement membrane on expression of surfactant apoproteins in cultured adult rat alveolar type II cells.
Pulmonary surfactant, which is composed of phospholipids and three lung-specific apoproteins, is synthesized and secreted by alveolar type II cells. Previous work from this laboratory (Biochim. Biophys. Acta 1987; 931:143-156) has shown that cell-extracellular matrix interactions and cuboidal cell shape affect both the ultrastructural appearance and pattern of phospholipids synthesized by cultured rat type II cells. In the present study, we have examined the effects of cell-matrix interactions and cell shape on the ability of adult rat type II cells to express the surfactant apoproteins in culture. Isolated adult rat type II cells were cultured for 2, 4, and 8 days on either tissue culture plastic, on an extract of the Engelbreth-Holm-Swarm (EHS) tumor, or on laminin-coated plastic dishes. Expression of surfactant proteins A, B, and C (SP-A, SP-B, and SP-C) was evaluated by Northern analysis using specific rat cDNA probes for these mRNAs. SP-A content was determined by enzyme-linked immunosorbent assay using a polyclonal antibody raised against rat SP-A purified from lavage. Type II cells cultured on plastic dishes assumed an attenuated morphology soon after being placed in culture. Except for an occasional positive signal on day 2 of culture, these cells were uniformly negative for the presence of mRNA for SP-A, SP-B, or SP-C. Type II cells cultured on plastic did not contain SP-A. In contrast, type II cells cultured on EHS gels formed three-dimensional aggregates on the surface of the substratum; these aggregates were composed of polarized cells that had their apical surfaces directed inward. Type II cells cultured on this substratum showed a positive signal for mRNA for all three surfactant proteins; the abundance of these mRNAs, however, was significantly below that seen in freshly isolated type II cells. While the abundance of mRNA for SP-A and SP-B steadily increased with time in culture under these conditions, the abundance of SP-C mRNA decreased, suggesting that SP-C is regulated independently of SP-A and SP-B. These cultures were also positive for SP-A content, which increased with increasing time in culture. Type II cells cultured on laminin-coated dishes initially spread more slowly across the culture surface than cells on plastic, but were extremely attenuated by day 8 in culture. These cells contained neither SP-A, nor mRNA for any of the three surfactant proteins.(ABSTRACT TRUNCATED AT 400 WORDS)
Shannon
J M
JM
Department of Medicine, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO 80206.
Emrie
P A
PA
Fisher
J H
JH
Kuroki
Y
Y
Jennings
S D
SD
Mason
R J
RJ
D000818:Animals; D001059:Apoproteins / Q000032:analysis / Q000096:biosynthesis*; D001485:Basement Membrane / Q000502:physiology; D015152:Blotting, Northern; D002454:Cell Differentiation; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005109:Extracellular Matrix / Q000502:physiology*; D008854:Microscopy, Electron; D010641:Phenotype; D011510:Proteolipids / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D011650:Pulmonary Alveoli / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats
D001059:Apoproteins; D011510:Proteolipids; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D012333:RNA, Messenger; C046616:pulmonary surfactant apoprotein