Procollagen messenger RNA levels and activity and collagen synthesis during the fetal development of sheep lung, tendon, and skin.

P Tolstoshev,R Haber,B.C Trapnell,R.G Crystal

doi:10.1016/s0021-9258(19)68815-0

Abstract

The rates of type I collagen synthesis in sheep lung, tendon, and skin were evaluated during the latter half of fetal development and compared with the levels of type I procollagen mRNA, quantified by molecular hybridization with a type I procollagen specific complementary DNA, and with the activity of total procollagen mRNA measured by in vitro cell-free translation. In the lung and tendon, the levels of type I procollagen mRNA and activity of total procollagen mRNA parallel collagen synthesis during development. In the skin, however, type I collagen synthesis declines sharply during fetal development, but both type I procollagen mRNA levels and total procollagen mRNA activity remain at the high levels of early development. These observations suggest that in developing lung and tendon, type I procollagen mRNA levels are likely the major determinants of the levels of type I collagen synthesis. In contrast, the dichotomy between type I procollagen mRNA levels and rates of type I collagen synthesis in the developing sheep skin suggest the skin utilizes mechanisms in addition to mRNA levels to modulate expression of the type I collagen gene.

Highlights

For explant incucontrast,thedichotomy between type I procollagen bations, tissue was cut intosmall pieces and washed with PBS.' Prior mRNA levels and rates of type I collagen synthesis in to labeling, the tissue was incubated in 100-mgportions for 30 min at thedeveloping sheep skin suggest the skin utilizes mechanisms in addition to mRNA levels to modulate expression of thetype I collagen gene
Densitometric quantitation showed that the translatiotnyaple I collagen synthesis declined 5.7-fold over the developactivity of procollagen mRNA did not change significantly in ment period studied, typeI11collagen declined23.4-fold.total RNA from the skinat the three developmental stages, it is quite clear that, irrespectiveof whether type 111procoland that thecollagenase-sensitive cell-free product remained lagen mRNA was able to cross-react with the typeI procolconstant ( p> 0.1, all comparisons) (Fig. 9B)
Remained at the levels characteristic of the earliest develop- Mechanisms Controlling Type I Procollagen mRNA Leumental age studied, itis clear that mechanisms other than theels-In the developing fetal sheeplung and tendon, thelevels levels of procollagen mRNA must beinvolved in determining of procollagen mRNA appear to be major determinoafntthse the rate of collagen synthesis in thedeveloping sheep skin. levels of collagen synthesis.Thereare a number of other examples of collagen-producing cells in whichprocollagen mRNA levels appear to determine the number of collagen

Summary

Preparation of the Purified Type I Procollagen

Tendon mRNA was translated at a concentration of 2Opg/ml in the nuclease-treated reticulocyte lysate cell-free system with [“Hlproline as tracer. Ethylmaleimide and CaCIZ were each added to a 12.5-pl aliquot of the cell-free translation system to final concentrations of 2.5 mM. This mixture was incubated in a final volume of 25 ~1 at 37 “C for 1 h in the presence or absence of 5 units of clostridial collagenase. %labeled procollagen produced by human fetal lung fibroblasts (HFL-1) (20); lane 2, translation products of the nuclease-treated lysate with no added RNA; lane 3, translation products of purified sheep tendon type I procollagen mRNA; lane 4, as for lane 3, but incubated with collagenase; lane 5, translation products directed by. Since rabbit reticulocytes do not produce collagen, this demonstrates the specificity of the collagenase used

RNAIcDNA Ratio

Proportion of collagen synthesis represented by type

DISCUSSION