Am. J. Respir. Cell Mol. Biol.,
Volume 20, Number 2, February, 1999 327-331
Isolation of a Gene Product Expressed by a Subpopulation of Human
Lung Fibroblasts by Differential Display
J.
Lurton,
T. M.
Rose,
G.
Raghu,
and
A. S.
Narayanan
Departments of Medicine and Pathology, School of Medicine; and Department of Pathobiology, School of Public
Health and Community Medicine, University of Washington, Seattle, Washington
Fibroblasts are the major cell type responsible for synthesizing matrix constituents in lung and other connective tissues. Evidence indicates that fibroblasts are heterogeneous, and that subpopulations with some
distinct properties are clonally selected and expanded in fibrotic diseases. However, few distinct markers capable of demonstrating the presence of fibroblast subpopulations in tissues have been isolated so far.
With the objective of identifying proteins that could detect fibroblast subpopulations, we compared the
messenger RNA (mRNA) expression of two cultured human lung fibroblast subpopulations by differential
display. Total RNA was obtained, complementary DNA (cDNA) was synthesized, and the polymerase chain reaction (PCR) products obtained with several primer pairs were compared. One 724-bp product,
which was strongly expressed by one human lung fibroblast subpopulation, was identified and cloned.
This product was poorly expressed by the other lung fibroblast subpopulation. The mRNA for the gene encoding this product was not detectable in human smooth-muscle cells, endothelial cells, or epithelial cells,
although it was present in dermal fibroblasts. The mRNA was detected in normal and fibrotic human
lungs. Search of the National Center for Biotechnology (NCBI) GenBank DNA database with the sequence obtained from this clone revealed no significant matches. However, a search of the NCBI database
of expressed sequence tags (dBEST) revealed five different human expressed sequence tag (EST) clones
corresponding to the LR8 cDNA sequence. Six additional mouse and one pig EST clones were identified
that showed significant similarity to the human fibroblast cDNA. Composites of the entire coding sequences for the human fibroblast gene product and the mouse homologue were assembled from the respective overlapping EST sequences. The open reading frame identified for each composite sequence predicted
protein products of 270 and 263 amino acids for the human and mouse sequences, respectively, which
were 52% identical, with three gaps. At the amino acid level, no significant sequence similarity was detected with any other sequences in exhaustive searches of the NCBI DNA and protein databases or the
Blocks databases. A PCR product with predicted length and sequence was obtained by using a sense
primer upstream to LR8 and an antisense primer within LR8. Our results indicate that this differentially
displayed product represents a previously undescribed protein that could be useful for distinguishing fibroblasts, and possibly fibroblast subpopulations, from other cell types in lungs and other tissues.
