Abstract
The reverse transcription-polymerase chain reaction was used to examine alternative splicing at each of the three fibronectin exons known to undergo alternative splicing, i.e. extra domain A (ED-A), extra domain B (ED-B), and type III connecting sequence (IIICS). Ratios of fibronectin mRNAs with or without a given exon were determined in several rat tissues and human cell lines during aging in vivo and cellular senescence in vitro. We demonstrate that statistically significant shifts in the alternative splicing of fibronectin occur during aging in vivo and in vitro. Since all three alternatively spliced exons are spliced out at a higher frequency in aging tissues and cells, the fibronectin protein produced by old cells should be slightly smaller than that obtained from young cells. The reverse transcription-polymerase chain reaction demonstrates tissue-specific patterns of alternative splicing in several tissues. Whereas fibronectin mRNAs from adult rat tissues were found to range from 0 to 25% ED-A+ and from 0 to 10% ED-B+, fibronectin mRNAs from cultured cell lines were found to be approximately 50-60% ED-A+ and 15-25% ED-B+. We observed similarity in splicing of fibronectin RNA by the different cultured cell lines obtained from many tissues and attribute this observation to the effect of growth factors. We demonstrate that serum deprivation; placement of cells into primary culture; and growth factors such as transforming growth factor beta 1, retinoic acid, and 1,25-dihydroxyvitamin D3 can all change the alternative splicing of fibronectin pre-mRNA in the ED-A, ED-B, and type III connecting sequence exons. Possible mechanisms for the regulation of the alternative splicing of fibronectin RNA by growth factors are discussed.
Highlights
A decrease in the biological activity of fibronectin has been observed during senescence in vitro; for example, fibronectin isolated from late passage cells is less able to promote cell adhesion than fibronectin obtained from young cells [28, 43]
Late passage fibroblasts show a decrease in the amountof cell-surface fibronectin; and as in the case of transformed cells, fibronectin is present in a diffuse rather than fibrillar pattern [44,45]
The apparent molecular size of fibronectin is increased in aging E l l foreskin fibroblasts [29], and monoclonal antibodies have been isolated that recognize an antigenic determinant on aging cell-derived fibronectin not found on young cells [27]
Summary
Pg of total RNA was used (0.5-1 pg for cell lines and 2-5pg for rat tissues) to obtain fibronectin cDNAsfor use in PCR amplification. In vitro aging studies used early and latepassage IMR-90 human fibroblasts obtained from theCoriel Institute (Camden, NJ) at population doubling levels (PDL) 12 and 40 and grown cDNAs were generated using a fibronectin RNA-specific antisense oligonucleotide primer to primreeverse transcription in thefollowing 20.~1 reactionsolution: 40 mM KCl, 50 mM Tris-HC1 (pH8.3), 8 mM MgCl,, 0.5 mM each dNTP (2mM total dNTPs), 200 ng of antisense primer, 10 units of avian myeloblastosis virus reverse transcriptase, t o PDLs 16 and 50, respectively.
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