Cell, Vol. 32, 1181-l 190, April 1983, Copyright 0 1983 by MIT 0092-8674/83/041181-l 0 $02.00/O Hormonal Regulation of Plasminogen Activator mRNA Production in Porcine Kidney Cells Yoshikuni Nagamine, Marius Sudol and E. Reich The Rockefeller University 1230 York Avenue New York, New York 10021 Summary Plasminogen activator (PA) production in LLC-PK, pig kidney cell culture is induced to high levels by calcitonin and vasopressin, both of which stimulate adenylate cyclase, or by other compounds that also raise intracellular CAMP levels. Enzyme induction is transiently sensitive to inhibition by actinomycin D, suggesting that increased concentrations of CAMP mediate the inducing effects of the hormones by enhancing the transcription of PA-mRNA se- quences. We tested this hypothesis by measuring PA-mRNA sequences in the Xenopus oocyte trans- lation system which showed a 15-2D-fold enhanced PA-synthesizing capacity when supplied with poly(A)+RNA from induced cells, above that ob- tained from uninduced cell RNA. Changes in PA- mRNA levels measured by Northern hybridization using cloned PA-specific cDNA gave results that agreed well with those obtained from translation assays. Pretreatment with high concentrations of cycloheximide did not block calcitonin-induced PA- mRNA synthesis, indicating that PA gene activation was a primary transcriptional result of calcitonin stimulation and did not require new protein synthe- sis. Introduction Because hormones profoundly influence the pheno- type of vertebrate cells, the elucidation of the bio- chemical and molecular mechanisms that underlie their effects is of general interest. A good deal is known about the way in which some hormones act. For example, those whose effects are mediated by stimulating adenylate cyclase ultimately regulate met- abolic programs by controlling the activity of enzymes that are rate-determining for specific pathways; this form of regulation is achieved by a particular cycle of postsynthetic modifications, namely, protein phospho- rylation and dephosphorylation (Sutherland, 1972; Greengard, 1978). Another facet of hormone action involves the regulation of gene expression. It is ap- parent from many lines of observation that hormones can induce and repress the formation of specific gene products, but it remains unclear how this is accom- plished. The sequence of reactions intervening be- tween the hormone-receptor complex and the modu- lation of gene expression is not yet completely de- fined; and the precise level in the pathway of gene expression at which a given hormone exerts its regu- latory effect(s) is in most cases uncertain, although promising studies of mammary tumor virus are clari- fying some mechanisms of glucocorticoid action (Hynes et al., 1981; Buetti and Diggelmann, 1981). Plasminogen activators (PA) pose challenging ques- tions concerning the hormonal control of gene expres- sion. These enzymes are secreted by most vertebrate cell types, in nearly all of which PA production is governed by cell- and tissue-specific patterns of re- sponse to hormones. Depending on cell type, changes in the rate of PA synthesis occur promptly after ex- posure to physiological concentrations of adrenocor- tical (Vassalli et al., 1976) or sex steroids (Mak et al., 1976), to one of many polypeptide hormones (Beers et al., 1975; Strickland and Beers, 1976; Dayer et al., 1981) or to some combination of these. Enzyme formation is also modulated by low concentrations of hormone-like stimuli such as tumor promoters (Wigler and Weinstein, 1976; Wilson and Reich, 1979) reti- noids (Miskin et al., 1978; Wilson and Reich, 1978) prostaglandins (Strickland and Beers, 1976) or trans- forming viral genes (Unkeless et al., 1973; Ossowski et al., 1973). Furthermore, changes in PA production are of more biological interest because they have been correlated in particular tissues with macroscopic phenomena such as ovulation (Beers et al., 1975) mammary involution (Ossowski et al., 1979), tissue remodeling (Strickland et al., 1976; Valinsky et al., (1981), and oncogenic transformation (Unkeless et al., 1973; Ossowski et al., 1973) among others. These background facts made it of interest to explore the mechanisms that could mediate such a diversity of regulatory patterns. As a first step, we elected to study PA synthesis in LLC-PK, cells, a permanent line isolated from pig kidney that produces a single plasminogen activator of the urokinase type (Dayer et al., 1981; Sudol and Reich, unpublished). In a particular cloned derivative of this line, PA production is very low under basal conditions, but may be stimulated up to several hundred-fold by addition of calcitonin or vasopressin. In these cells, both hormones enhance the activity of adenylate cyclase, thereby raising the intracellular level of CAMP, and there is no doubt that hormonal induction of PA is mediated by cyclic nucleotides because it also occurs in response to other stimuli that increase CAMP such as cholera toxin, the phos- phodiesterase inhibitor isobutylmethylxanthine, and CAMP itself (Dayer et al., 1981). The present experi- ments were undertaken to establish whether hormonal induction occurred predominantly at the level either of genetic transcription or translation, and, more specif- ically, whether changes in the rates of enzyme syn- thesis were correlated with changes in PA-mRNA levels. CAMP-induced changes in levels of synthesis of mRNA have been demonstrated indirectly for a num- ber of enzymes. Actinomycin D-sensitive stimulation of enzyme activity has been reported for PA of rat granulosa cells (Strickland and Beers, 1976) and LLC-