Identification of stretch-responsive genes in pulmonary artery smooth muscle cells by a two arbitrary primer-based mRNA differential display approach.
Physical forces induce profound changes in cell phenotype, shape and behavior. These changes can occur in vascular structures as a result of pressure overload and their effects can be seen in atherosclerotic vessels in which smooth muscle cells have undergone hyperplastic and hypertrophic changes. At the molecular level, mechanical stimuli are converted into chemical ones and lead to modulation of gene expression and/or the activation of a new repertoire of genes whose encoded proteins help the cells to adapt to their microenvironment. In this study, we have used a two primer-based mRNA differential display technique to identify candidate mechano-responsive genes in pulmonary artery smooth muscle cells. As compared to the original method described by Liang and Pardee, this technique uses two arbitrary primers instead of an anchored oligo(dt) plus an arbitrary primer in the polymerase chain reaction. The chief advantages of these modifications are an increase in the efficiency of the amplification and in the identification of differentially expressed clones. Using this approach, we compared the pattern of expressed genes in cells cultured under static conditions with those in cells that were mechanically stretched (1 Hz) for 24 h in a well-defined in vitro mechanical system. Three candidate genes that showed reproducible differences were chosen for further characterization and cloning. One clone was under expressed in stretched cells and had a DNA sequence with 90% homology to the human fibronectin gene. Two other clones were highly expressed in stretched cells and had a 92% and a 83% sequence homology with human platelet-activating factor (PAF) receptor and rat insulin-like growth factor-I (IGF-I) genes respectively. Northern blot analysis confirmed low levels of fibronectin mRNA transcripts in stretched cells. In contrast, accumulation of PAF receptor mRNA occurred 30 min after mechanical stretch was initiated whereas IGF-I mRNA levels peaked at 8 h. Both mRNA levels were sustained for up to 24 h of mechanical stretching. These results demonstrate the usefulness of the two primer-based mRNA differential display that enabled us to identify and characterize alterations at the level of gene expression among matrix proteins, G-protein coupled receptors and growth factors, each of whose response to mechanical strain is different. A more complete understanding of these responses will provide further insight into the pathologic processes associated with hypertension and atherosclerosis.
Chaqour
B
B
University of Pennsylvania, Department of Anatomy Histology, Philadelphia 19104, USA.
Howard
P S
PS
Macarak
E J
EJ
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D005353:Fibronectins / Q000235:genetics*; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000235:genetics*; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000502:physiology*; D010812:Physical Stimulation; D010980:Platelet Membrane Glycoproteins / Q000235:genetics*; D011651:Pulmonary Artery / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D012689:Sequence Homology, Nucleic Acid
D017931:DNA Primers; D018076:DNA, Complementary; D005353:Fibronectins; D010980:Platelet Membrane Glycoproteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C039351:platelet activating factor receptor; D007334:Insulin-Like Growth Factor I