Dose- and time-dependent synthesis of 20-hydroxyecdysone modulated polypeptides in the epidermis of Tenebrio molitor

Cellular (non-cuticular) polypeptides were isolated from mid-instar larval epidermis incubated in the presence or absence of 20-hydroxyecdysone (20HE) for 14-16 h in vitro. Polypeptides synthesized during the last few hours of incubation were labelled with [³⁵S]methionine, separated in cell lysates by two-dimensional polyacrylamide gel electrophoresis and detected fluorographically. Cells incubated with hormone incorporated 28% more label into polypeptide. The synthesis of over 250 polypeptides was detected in total cell lysates. Of these, 54 showed an altered level of synthesis in response to 20HE treatment. The synthesis of most (33) was depressed. The relative synthetic rate of most "down-regulated" 20HE-sensitive polypeptides began to drop at 10^-3 μg/ml whereas that of most "up-regulated" polypeptides increased only at concentrations above 10^-1 μg/ml. An early response to 20HE, involving the de novo synthesis of a 43 kDa polypeptide, was first detectable after 2 h of exposure to hormone, and peaked after 4 h. The synthesis of this 43 kDa polypeptide was selectively enhanced by the addition of 10^-2 μg/ml cycloheximide to medium containing 20HE. The long-term effect (12-16 h) of 20HE on polypeptide synthesis in subcellular fractions of the epidermis was also examined. Polypeptide synthesis found in the nuclear, mitochondrial-lysosomal, microsomal and soluble fractions changed in response to 20HE. It appears that 20HE influences epidermal behaviour predominantly through its ability to modulate the synthetic rate of many cellular polypeptides, rather than by turning off the synthesis of a few pre-existing ones and switching on that of a few new ones.