Progestin and estrogen control of cathepsin D expression and processing in rat uterine luminal epithelium and stroma-myometrium.
Progestins increase the activity and rate of synthesis of cathepsin D, a lysosomal aspartyl protease, in the uterine luminal epithelium in ovariectomized rats. Western blot analysis of luminal epithelial proteins determined that the progestin, medroxyprogesterone acetate (MPA) increased the 43-kDa form of cathepsin D by 7-fold in 24 hr, whereas estradiol increased the amount of the same form by only 2-fold. To examine the precursor-product relationship between cathepsin D proteins in the luminal epithelium and stroma-myometrium after progestin or estradiol treatment, uterine proteins were prelabeled by incubation with [35S]methionine in vitro, cathepsin D was isolated by immunoprecipitation, and equal amounts of labeled cathepsin D were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After each hormonal treatment in each uterine tissue, a 48-kDa precursor was processed into a 44-kDa cathepsin D product. Endoglycosidase H digestion of [35S]methionine-labeled cathepsin D from the luminal epithelium and stroma-myometrium of medroxyprogesterone-treated rats shifted the molecular masses of the cathepsin D proteins by approximately 5.7 kDa. To examine the contribution of increased mRNA to increased rates of cathepsin D synthesis, we measured levels of cathepsin D mRNA in uterine tissues after progestin and estrogen treatment. Total RNA was isolated from the uterine luminal epithelium and from the stroma-myometrium. Northern blot analysis identified a single 2.2-kb RNA band corresponding to the size expected for cathepsin D mRNA. Medroxyprogesterone increased levels of cathepsin D mRNA in the luminal epithelium (greater than 17-fold) and in the stroma myometrium (3-fold), with maximum increases at 9 hr after treatment. Estradiol also increased cathepsin D mRNA levels in both uterine tissues, but by only 2-fold. No hormonal effects on liver cathepsin D mRNA were observed. Increases in cathepsin D synthesis and activity in uterine tissues in response to progestin and estrogen appear to depend in part upon increased levels of mRNA.
Moulton
B C
BC
Department of Obstetrics and Gynecology, University of Cincinnati, College of Medicine, Ohio 45267.
Khan
S A
SA
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002402:Cathepsin D / Q000096:biosynthesis*; D004848:Epithelium / Q000378:metabolism; D004967:Estrogens / Q000494:pharmacology*; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D006031:Glycosylation / Q000187:drug effects; D006596:Hexosaminidases / Q000494:pharmacology; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D008525:Medroxyprogesterone / Q000031:analogs & derivatives* / Q000494:pharmacology; D017258:Medroxyprogesterone Acetate; D009215:Myometrium / Q000378:metabolism; D010052:Ovariectomy; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D011919:Rats, Inbred Strains; D014599:Uterus / Q000378:metabolism*
D004967:Estrogens; D007166:Immunosuppressive Agents; D012333:RNA, Messenger; D017258:Medroxyprogesterone Acetate; D006596:Hexosaminidases; D002402:Cathepsin D; D008525:Medroxyprogesterone