| Description |
The phenotypic expression of tropoelastin (TE) by cultured human skin fibroblasts was studied utilizing an enzyme-linked immunosorbant assay (ELISA). Tropoelastin production, on a per cell basis, was found to be serum and density dependent, linear for at least 72h, and stable over approximately 28 population doublings. Rates of TE accumulation in culture medium were not significantly altered by cell-associated degradation. Standardized culture conditions were used to establish the normal production values for human skin fibroblasts at 25-69 x 10('3) molecules per cell per hour. A decline in TE production was shown to accompany the aging of cells in vitro and TE production appeared to decline in cells from donors aged beyond the sixth decade. Developmental initiation of TE production by human fetal fibroblast strains was shown to initiate at the 19th week of gestation. The induction was pretranslationally controlled, as evidenced by the simultaneous appearance of elastin specific transcripts. Relative total collagen synthesis and type I collagen (alpha 2 chain) mRNA expression were invariant over the same period, implying that these two genes are under different developmental control. The premature aging syndrome, Hutchinson-Gilford progeria, results from a fatal genetic defect which affects primarily tissues of mesodermal origin. TE produced by three progeroid fibroblast strains, which was shown to migrate with TE peptides secreted by normal cells, was increased 5- to 10-fold, while total protein synthesis was found to be normal. Expression of elastin specific transcripts, measured by dot blot analysis, was shown to be similarly increased, suggesting modified control at the pretranslational level. Finally, six cell strains from donors with apparent autosomal recessive cutis laxa were examined for defects in elastin metabolism. Absolute TE production by three cutis laxa cell strains was significantly decreased when compared to an age-matched normal average. Two strains exhibited significantly reduced relative TE production, which was also associated with reduced expression of TE transcripts. These studies represent the first systematic characterization of elastin synthesis by a human cell strain. They also advance the first biochemical evidence for initiation of elastin synthesis during human development and its altered expression in two heritable human developmental diseases. |
