HORMONAL REQUIREMENTS FOR BASEMENT-MEMBRANE COLLAGEN DEPOSITION BY CULTURED RAT MAMMARY EPITHELIUM

Abstract

Alveoli and ducts isolated from virgin rat mammary glands synthesize basement membrane collagen (type IV) in primary culture. Using purified antibodies to type IV collagen, prominent intracellular and extracellular fluorescence is observed in the epithelium. No fluorescence is observed with antibodies to collagen type I and III. From quantitation of the incorporation of labeled proline into hydroxyproline and the estimation of the fraction of collagenase-sensitive [14C]Pro-labeled proteins, 1.5-2.5% of the newly synthesized proteins are collagen. Type IV collagen from these cultures was biochemically identified on the basis of the high ratio of labeled 3-hydroxyproline to 4-hydroxyproline (1:10), the gel electrophoretic pattern of the collagenase-sensitive proteins precipitated with 1.7 M NaCl, the failure of the collagen to bind to diethylaminoethyl-cellulose and the immunologic cross-reactivity with mouse tumor type IV collagen. The apparent MW of the mammary type IV collagen chain doublet (175,000) is identical with that of type IV collagen from other sources. When the supportive hormones, insulin, prolactin, hydrocortisone, progesterone and estradiol are removed from the cultures, there is a 90% reduction in the amount of [3H]Pro recovered in collagen compared to cultures in which hormonal support is continued. This 90% decrease in collagen synthesis coincides with only a 30% drop in the growth rate and a 20% drop in total protein synthesis of the cells over the 24-h period without hormones. Pulse-chase experiments revealed an enhanced turnover of collagen following hormone withdrawal. This system may be an in vitro model of collagen turnover in mammary gland in involution.