The ductus arteriosus migratory smooth muscle cell phenotype processes tropoelastin to a 52-kDa product associated with impaired assembly of elastic laminae.
We established the identity of a 52-kDa protein secreted by fetal lamb ductus arteriosus (DA) smooth muscle cells (SMC) and suggest how it might be related to structural changes unique to DA development, i.e. reduced assembly of elastic laminae and associated formation of intimal cushions. We produced a monoclonal antibody (HI-20) to the 52-kDa protein and observed, by electron microscopy, immunogold labeling of elastin in both DA and aorta vessel walls. Western immunoblotting showed that HI-20, as well as antibodies to tropoelastin, reacted with the 52-kDa protein secreted by DA SMC, as well as with 68-kDa tropoelastin. The highly specific antibody to the carboxyl-terminal sequence of tropoelastin failed, however, to recognize the 52-kDa protein, although it reacted well with the 68-kDa tropoelastin. Amino acid analysis and sequencing data confirmed the identity of the affinity-purified 52-kDa protein as truncated tropoelastin with an intact amino terminus. Cell-free translation of mRNA extracted from DA and aorta SMC produced a 68-kDa, but not a 52-kDa, immunoprecipitated tropoelastin. When DA and aorta SMC were pulsed with [14C]valine, we immunoprecipitated, after only a 15-min chase, both 68-kDa and 52-kDa tropoelastin from cell extracts of DA SMC, but only the 68-kDa tropoelastin was present in aorta SMC. There was no evidence of proteolytic degradation of radiolabeled aorta 68-kDa tropoelastin to a 52-kDa species when mixed with DA SMC conditioned medium. This suggests that the 52-kDa tropoelastin is the result of cell-associated processing or degradation of an original 68-kDa product of translation. Furthermore, pulse-chase experiments showed initial secretion of equivalent amounts of 68-kDa and 52-kDa tropoelastins by cultured DA SMC with increasing accumulation of the 52-kDa species, suggesting its impaired insolubilization. The production, in high concentration, of a 52-kDa tropoelastin product that lacks the carboxyl terminus, may prevent its alignment on the microfibrillar scaffold, resulting in abnormal assembly of elastic laminae in the DA. The accumulation of this soluble tropoelastin may be associated with the previously described property of chemotaxis resulting in the increased SMC migration into the subendothelium associated with DA intimal thickening.
Hinek
A
A
Department of Pediatrics, University of Toronto, Ontario, Canada.
Rabinovitch
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000378:metabolism*; D015153:Blotting, Western; D002474:Cell-Free System; D004373:Ductus Arteriosus / Q000378:metabolism*; D004549:Elastin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D005865:Gestational Age; D007700:Kinetics; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D010641:Phenotype; D014176:Protein Biosynthesis; D012756:Sheep; D014333:Tropoelastin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D010446:Peptide Fragments; D014333:Tropoelastin; D004549:Elastin