Stimulation of prostaglandin biosynthesis by urine of the human fetus may serve as a trigger for parturition.

Urine of the human fetus stimulated prostaglandin biosynthesis in vitro by increasing the conversion of arachidonic acid into prostaglandins. The stimulatory activity in urine from fetuses delivered at term after labor of spontaneous onset was greater than that in urine from fetuses delivered by cesarean section at term before the onset of labor. Such stimulation of prostaglandin biosynthesis by the fetal membranes, by way of a substance released into the urine and thence into amniotic fluid, could serve as a signal for the initiation of parturition.

Metabolism of deoxycorticosterone and deoxycorticosterone sulfate in men and women.

In this investigation we found that little of intravenously infused [14C]deoxycorticosterone (DOC) was converted to [14C]DOC-SO4 that entered plasma. Moreover little of intravenously infused [3H]DOC-SO4 was metabolized by way of DOC except by intestinal bacterial enzymes. However evidence was obtained that plasma DOC is converted to DOC-SO4 in liver, but little of the DOC-SO4 formed in liver escapes into blood; rather the DOC-SO4 enters bile and in the intestine is converted, in part, to progesterone (or metabolites thereof) by the action of bacterial enzymes. The estimated intrahepatic fractional conversion of DOC to DOC-SO4 was significantly greater in premenopausal women (0.72 +/- 0.118, mean +/- SEM) than in men (0.28 +/- 0.036, P less than 0.005).

17 beta-Hydroxysteroid dehydrogenase type 2: chromosomal assignment and progestin regulation of gene expression in human endometrium.

The cDNAs for two separate human 17 beta-hydroxysteroid dehydrogenases (17 beta-HSD) have been isolated and sequenced. The well-studied human placental cytosolic 17 beta-HSD (also referred to as estradiol dehydrogenase) preferentially catalyzes the reduction of estrone to estradiol-17 beta and the reduction of the C-20-ketone of progesterone to 20 alpha-dihydroprogesterone. This isoform of the enzyme has been referred to as 17 beta-HSD type 1 and localized to chromosome 17. A second 17 beta-HSD isoform (referred to as type 2) is localized in the endoplasmic reticulum of human trophoblast and is characterized by the preferential oxidation of the C-17 beta-hydroxyl group of C18- and C19-steroids and the C-20 alpha-hydroxyl group of 20 alpha-dihydroprogesterone. In this study, we determined the chromosomal localization of human 17 beta-HSD type 2, the expression of this gene in human endometrium, and the tissue distribution of the mRNA. We found that the human 17 beta-HSD type 2 gene is localized on chromosome 16, 16q24. 17 beta-HSD type 2 mRNA (approximately 1.5 kb) was identified in human endometrial tissues by Northern analysis of total RNA (10 micrograms). The highest levels of 17 beta-HSD type 2 mRNA were found in endometrial tissues obtained during the mid- to late secretory phase of the ovarian cycle (i.e., during the time of high plasma levels of progesterone). 17 beta-HSD type 2 mRNA levels were much greater in glandular epithelium than in the stromal cells isolated from secretory phase endometrium. The levels of 17 beta-HSD type 2 mRNA in secretory phase endometrium were approximately one-tenth that in villous trophoblast tissue from human placenta. We did not detect 17 beta-HSD type 1 mRNA in endometrial tissue by Northern analysis of total (10 micrograms) RNA. These findings are consistent with the view that the progestin-regulated 17 beta-HSD of the glandular epithelium of the human endometrium is primarily, if not exclusively, the product of the 17 beta-HSD type 2 gene. 17 beta-HSD type 2 mRNA was present in human placenta, liver, and small intestine; much smaller amounts, barely detectable by Northern analysis of poly(A)+ RNA, were present in prostate, kidney, pancreas, and colon, but not in heart, brain, skeletal muscle, spleen, thymus, ovary, or testis.

Despite a massive increase in cortisol secretion in women during parturition, there is an equally massive increase in prostaglandin synthesis. A paradox?

In this investigation, we sought to resolve the apparent paradox that is posed by the fact that there is a simultaneous increase in the production of prostaglandin and cortisol in women during labor. A paradox obtains, since in most tissues, cortisol acts to inhibit prostaglandin formation. Using previously characterized model systems for the in vitro study of arachidonic acid metabolism in amnion, decidua, and myometrium, we found that prostaglandin production by amnion and endometrial stromal cells in monolayer culture was not decreased by glucocorticosteroid treatment. On the other hand, prostaglandin production by myometrial smooth muscle cells in culture was inhibited by greater than 90% in response to dexamethasone (10(-7) M) treatment. Importantly, the major prostaglandin produced by myometrium, as well as myometrial smooth muscle cells in culture, is prostacyclin, a prostaglandin that acts to cause uterine quiescence. We suggest that the immunity of amnion and decidua to the action of glucocorticosteroids may allow for the accelerated production of prostaglandins E2 and F2 alpha, which act to cause myometrial contractions; simultaneously, glucocorticosteroid produced in large quantities in women in labor may lead to decreased production of prostacyclin by myometrium, thereby reducing uterine quiescence. In this coordinated manner, the uterine contractions that culminate in delivery of the fetus may proceed uninterrupted in the face of increased cortisol production.

Contractile elements and myosin light chain phosphorylation in myometrial tissue from nonpregnant and pregnant women.

Smooth muscle contraction is initiated primarily by an increase in intracellular Ca2+, Ca(2+)-dependent activation of myosin light chain kinase, and phosphorylation of myosin light chain. In this investigation, we identified pregnancy-associated alterations in myosin light chain phosphorylation, force of contraction, and content of contractile proteins in human myometrium. Steady-state levels of myosin light chain phosphorylation and contractile stress were correlated positively in both tissues, but the myometrial strips from pregnant women developed more stress at any given level of myosin light chain phosphorylation. During spontaneous contractions and during conditions that favor maximal generation of stress, the rate and extent of myosin light chain phosphorylation were attenuated in myometrial strips from pregnant women. The content of myosin and actin per milligram of protein and per tissue cross-sectional area was similar between myometrium of nonpregnant and pregnant women. Although cell size was significantly increased in tissues obtained from pregnant women, the amounts of contractile proteins per cellular cross-sectional area were similar. In addition, myosin light chain kinase and phosphatase activities were similar in the two tissues. The content of caldesmon was significantly increased in myometrium of pregnant women, whereas that of calponin (a smooth muscle-specific protein associated with the thin filaments) was not different. We conclude that adaptations of human myometrium during pregnancy include (a) cellular mechanisms that preclude the development of high levels of myosin light chain phosphorylation during contraction and (b) an increase in the stress generating capacity for any given level of myosin light chain phosphorylation.

Prevention of autoantibody formation and prolonged survival in New Zealand black/New Zealand white F1 mice fed dehydroisoandrosterone.

Dehydroisoandrosterone, administered orally to New Zealand Black/New Zealand White F1 hybrid mice, prevented the formation of antibodies to double-stranded DNA and prolonged survival in this murine model of lupus erythematosus.

Mesenchyme-epithelial interactions in human endometrium. Prostaglandin synthesis in separated cell types.

Glandular epithelium and stromal cells of human endometrium were separated and maintained in monolayer culture. At the time the cells became confluent, cell suspensions were prepared and incubated with [14C]arachidonic acid. Radiolabeled prostaglandin E2 and, to a lesser extent, prostaglandin F2 alpha and metabolites of these prostaglandins, were formed principally in stromal cells. There was considerably less prostaglandin formation in endometrial glands either after maintenance in monolayer culture or in freshly separated glands. In stromal cells of endometrium prostaglandin formation was linear with time of incubation for 2.5 min and with [14C]arachidonic acid concentrations up to 8 microM. When stromal cells and epithelial cells were combined, all prostaglandin formation could be accounted for by that produced in stromal cells. Little or no prostaglandin formation was detected in stromal cells from human adipose tissue or in fibroblasts from human genital or abdominal skin or human fallopian tube.

Cachectin/tumor necrosis factor-alpha formation in human decidua. Potential role of cytokines in infection-induced preterm labor.

This study was conducted as part of an investigation to evaluate the hypothesis that bacterial toxins (LPS or lipoteichoic acid), acting on macrophage-like uterine decidua to cause increased formation of cytokines, may be involved in the pathogenesis of infection-associated preterm labor. We found that cachectin/tumor necrosis factor-alpha (TNF-alpha) was synthesized and secreted into the culture medium by human decidual cells and explants in response to treatment with LPS. LPS treatment also caused an increase in PGF2 alpha production by decidual cells and explants. In amnion cells in monolayer culture, TNF-alpha stimulated PGE2 formation, and TNF-alpha was cytostatic (inhibited [3H]thymidine incorporation into DNA) but not cytolytic in amnion cells. TNF-alpha was not detectable (less than 0.34 ng/ml) in the amniotic fluid of normal pregnancies at midtrimester or at term before or after the onset of labor (n = 44); but TNF-alpha was present at concentrations between 2.8 and 22.3 ng/ml in amniotic fluids of 4 of 20 pregnancies with intact membranes complicated by preterm labor (less than 34 wk gestational age). LPS was present in 10 of the 20 amniotic fluids of preterm labor pregnancies, including all four in which TNF-alpha was present. Bacteria were identified in only one of the four LPS-positive, TNF-alpha-positive fluids. Cytokine formation in macrophage-like decidua may serve a fundamental role in the pathogenesis of preterm labor, including increased prostaglandin formation and premature rupture of the membranes.

Origin of urinary nonconjugated 19-nor-deoxycorticosterone and metabolism of infused radiolabeled 19-nor-deoxycorticosterone in men and women.

It is known that 19-nor-deoxycorticosterone (19-nor-DOC) is a potent mineralocorticosteroid that is present in urine of rats and humans in a free, i.e., nonconjugated, form. In some forms of hypertension in rats, the levels of free 19-nor-DOC in urine are increased compared with those in urine of normotensive animals. Yet, despite the potential importance of this mineralocorticosteroid in the pathogenesis of certain forms of hypertension, little is known of its site of origin or metabolism. In the present investigation, we evaluated the metabolism of intravenously infused [3H]19-nor-DOC and the possibility that 19-nor-DOC was formed from plasma DOC. We found that the metabolism of [3H]19-nor-DOC infused intravenously in men and women was similar to that of DOC with important exceptions. The majority of the radiolabeled urinary metabolites of intravenously infused [3H]19-nor-DOC were excreted in urine as glucuronosides. Little radioactivity, infused as [3H]19-nor-DOC, was recovered in urine as nonconjugated or sulfoconjugated steroids. There was no free radiolabeled 19-nor-DOC in urine after the simultaneous infusion of [3H]19-nor-DOC and [14C]DOC. A major metabolite of [3H]19-nor-DOC in urine was 19-nor-DOC-21-glucuronoside, whereas little or no intravenously infused radiolabeled DOC was excreted as radiolabeled DOC-glucuronoside. We also found that intravenously infused [14C]DOC was not converted to urinary [14C]19-nor-DOC (glucuronoside) and that other tritium-labeled metabolites of infused [3H]19-nor-DOC contained no carbon-14. The production rate of 19-nor-DOC, computed from the specific activity of urinary 19-nor-DOC (glucuronoside), in one normal man was 16 micrograms/d and in the two women of this study, it was 10 micrograms/d. These findings are supportive of the proposition that free urinary 19-nor-DOC is not formed from plasma DOC; it may be formed in kidney from a precursor other than DOC or it may be formed nonenzymatically in kidney or urine from a precursor such as 19-oic-DOC.

Regulation of extraadrenal steroid 21-hydroxylase activity. Increased conversion of plasma progesterone to deoxycorticosterone during estrogen treatment of women pregnant with a dead fetus.

We measured deoxycorticosterone (DOC) and progesterone (P) in plasma of 47 women pregnant with a dead fetus and sequentially throughout gestation in 35 women pregnant with a live fetus. When P levels in plasma were low, the plasma levels of DOC in women pregnant with a dead fetus varied but usually were similar to those in women pregnant with a live fetus. However, when P levels were high, the levels of DOC in some women pregnant with a dead fetus were considerably lower than those in women pregnant with a live fetus. To test whether this finding was due to loss of transfer of DOC from fetus to mother or else loss of extraadrenal steroid 21-hydroxylase activity in the mother after death of the fetus, we conducted several studies. The levels of P and DOC in plasma of one woman remained constant from 30 min after fetal death until delivery occurred 13 h later. Estrogen treatment of four women pregnant with a dead fetus brought about an increase in plasma levels of DOC in three of the women. In one woman the ratio of plasma DOC to P was 0.015, a value similar to that found before fetal death, but was 0.003 after fetal death but before estrogen treatment. In two women pregnant with a dead fetus the transfer constants of conversion of plasma P to DOC were 0.011 and 0.005 before, and 0.024 and 0.013, respectively, during estrogen treatment. In one woman pregnant with a deformed fetus with adrenal agenesis, the metabolic clearance rates of DOC before and during estrogen treatment were similar, whereas the plasma production rates of DOC were 2.75 before and 4.31 mg/24 h during estrogen treatment. We suggest that (a) the DOC in plasma of near-term pregnant women arises in part by extraadrenal 21-hydroxylation of plasma P and (b) estrogen stimulates steroid 21-hydroxylase activity in extraadrenal tissues.

Tissue distribution and ontogeny of steroid 5 alpha-reductase isozyme expression.

The synthesis of dihydrotestosterone is catalyzed by steroid 5 alpha-reductase isozymes, designated types 1 and 2. Mutation of type 2 results in male pseudohermaphroditism, in which the external genitalia are phenotypically female at birth. Two striking and unexplained features of this disorder are that external genitalia of affected males undergo virilization during puberty and that these individuals have less temporal hair regression. The tissue-specific and developmental expression patterns of the 5 alpha-reductase isozymes were investigated by immunoblotting. The type 1 isozyme is not detectable in the fetus, is transiently expressed in newborn skin and scalp, and permanently expressed in skin from the time of puberty. There was no qualitative difference in 5 alpha-reductase type 1 expression between adult balding vs. nonbalding scalp. The type 2 isozyme is transiently expressed in skin and scalp of newborns. Type 2 is the predominant isozyme detectable in fetal genital skin, male accessory sex glands, and in the prostate, including benign prostatic hyperplasia and prostate adenocarcinoma tissues. Both isozymes are expressed in the liver, but only after birth. These results are consistent with 5 alpha-reductase type 1 being responsible for virilization in type 2-deficient subjects during puberty, and suggest that the type 2 isozyme may be an initiating factor in development of male pattern baldness.

Endothelin-1 gene expression and biosynthesis in human endometrial HEC-1A cancer cells.

In this study, evidence was obtained that endothelin-1 (ET-1) is produced by an established endometrial cancer (HEC-1A) cell line. PreproET-1 mRNA is present in HEC-1A cells, and immunoreactive endothelin is secreted into the medium of these cells maintained in culture. Cycloheximide treatment of these cells caused superinduction of preproET-1 mRNA. Transforming growth factor-beta acts in these cells to increase the levels of preproET-1 mRNA. This effect of transforming growth factor-beta on preproET-1 mRNA accumulation was accompanied by an increase in the amount of immunoreactive endothelin secreted into the culture medium. ET-1, added to the culture medium, did not act as a mitogen in HEC-1A cells. We speculate that ET-1 (which is known to stimulate fibroblast proliferation) produced by endometrial adenocarcinoma cells may participate in the angiogenic process that occurs during the establishment of this carcinoma in vivo.

Nicotinamide adenine dinucleotide-dependent 15-hydroxyprostaglandin dehydrogenase activity in hydatidiform mole tissue and choriocarcinoma cells.

Prostaglandins may occupy an important role in viral and chemical carcinogen-induced neoplasia. To evaluate the possible role of prostaglandin catabolism in neoplastic cells, we measured nicotinamide adenine dinucleotide-dependent 15-hydroxyprostaglandin dehydrogenase activity in hydatidiform mole tissue and in choriocarcinoma cells maintained in monolayer culture. The specific activity of nicotinamide adenine dinucleotide-dependent 15-hydroxyprostaglandin dehydrogenase in hydatidiform mole tissue (0 to 1.2 nmol 15-ketoprostaglandin E2 formed x min-1 x mg-1 cytosolic protein) and in choriocarcinoma cells (1.0 nmol 15-ketoprostaglandin E2 x min-1 x mg-1 protein) was strikingly less than that found in normal placental tissue [11.4 +/- 2.3 (S.E.) nmol 15-ketoprostaglandin x min-1 x mg-1 protein].

Metabolism of arachidonic acid and prostanoids in human endometrial stromal cells in monolayer culture.

In the present investigation, we compared the metabolism of arachidonic acid in human endometrial stromal cells maintained in monolayer culture with that in human decidual tissues. By gas-chromatographic analysis, the distribution of arachidonic acid in glycerophospholipids and in the neutral lipids of decidual tissues and stromal cells in culture was similar. After the addition of [14C]arachidonic acid to the culture medium, steady-state conditions with respect to radioactive labeling of the lipids of the cells were attained after 24 h, except for phosphatidylethanolamine and neutral lipids. The percentage distribution of [14C]arachidonic acid in the lipids of the cells in culture was as follows: phosphatidylcholine, 41%; phosphatidylserine, 5%; phosphatidylinositol, 19%; phosphatidylethanolamine, 22%; neutral lipids, 11%. This distribution of arachidonic acid among the lipids is similar to that in decidual tissue, except for that in phosphatidylethanolamine. The amount of radioactivity in phosphatidylethanolamine continued to increase up to 72 h whereas that in neutral lipids declined after a maximum amount was present at 4 h. In the cells in monolayer culture, [14C]prostaglandin E2 and [14C]prostaglandin F2 alpha were produced from [14C]arachidonic acid, as is true in superfused decidual tissue. The similarities in arachidonic acid metabolism in these cells to that in decidual tissue are supportive of the proposition that endometrial stromal cells in monolayer culture are an appropriate model for the study of the regulation of arachidonic acid release and prostaglandin formation by endometrium and decidua vera.

The accumulation of prostaglandins (PG) in amniotic fluid is an aftereffect of labor and not indicative of a role for PGE2 or PGF2 alpha in the initiation of human parturition.

The accumulation of prostaglandins (PGs) in amniotic fluid (AF) during labor is cited frequently as one line of evidence in support of a role for these eicosanoids in the initiation of human parturition. In this study, we evaluated an alternate possibility, viz. that PGs entering AF at parturition are produced as a sequela of labor-associated processes. During labor, the AF normally becomes separated into two compartments, viz. the forebag and the upper compartment, by the obstruction produced as the descending fetal presenting part is engaged in the maternal pelvis. We theorized that the PGs that enter AF are produced in traumatized tissues lining the forebag, which is formed as the result of labor-driven cervical dilatation. In addition, these traumatized tissues are exposed to and bathed by the vaginal fluid, which contains many potent stimuli of PG formation, viz. large numbers of microorganisms and bacterial toxins. AF was collected at term before labor (n = 50) and from the upper compartment during labor (n = 47) by transuterine amniocentesis, and AF was collected by direct needle aspiration of the forebag during labor (n = 143). PGF2 alpha, 13,14-dihydro-15-keto-PGF2 alpha (PGFM), and PGE2 were quantified by RIA. The concentrations (nanomoles per L mean +/- SEM) of PGs in AF of the forebag (PGF2 alpha, 85.6 +/- 10.6; PGFM, 20.8 +/- 2.58; PGE2, 26.9 +/- 2.73) were much greater than those in the AF before labor (PGF2 alpha, 0.56 +/- 0.05; PGFM, 0.9 +/- 0.08; PGE2, 5.89 +/- 1.13) or in AF of the upper compartment during labor (PGF2 alpha, 7.14 +/- 1.64; PGFM, 5.11 +/- 0.82; PGE2, 8.74 +/- 1.71). The concentrations of PGs in AF of the upper compartment during early labor (< or = 2.5-cm cervical dilatation) were no greater than those in AF before labor began. The concentration and total content of PGs in AF of the forebag increased as a function of cervical dilatation until delivery. At 3- to 5-cm cervical dilatation, the levels of PGs in AF of the upper compartment were greater than those before labor, but significantly less than those in AF of the forebag at the same stage of labor progress. After 3-5 cm, the levels of PGs in the upper compartment did not increase further. These findings indicate that PGF2 alpha, PGFM, and PGE2, which enter AF in increased amounts during parturition, are produced during, not before, labor in tissues (principally decidua) lining for forebag.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor-beta inhibits progesterone-induced enkephalinase expression in human endometrial stromal cells.

The specific activity of enkephalinase in endometrial tissue of nonpregnant ovulatory women is correlated in a highly significant, positive manner with the plasma level of progesterone. The specific activity and levels of enkephalinase messenger ribonucleic acid and immunoreactive protein also are increased in human endometrial stromal cells in culture by treatment with a synthetic progestin, medroxyprogesterone acetate (MPA), in a time- and dose-dependent manner. From an analysis of the temporal relationship between the specific activity and half-life of enkephalinase in endometrial tissue and the level of progesterone in plasma, it appeared highly likely that some mechanism, in addition to progesterone withdrawal, was operative to reduce enkephalinase activity in endometrium during the late luteal phase of the ovarian cycle before progesterone levels had declined below those known to be effective for progesterone action. In stromal cells previously (and concurrently) treated with MPA (10(-9) mol/L), the addition of transforming growth factor-beta 1 (TGF beta 1) or TGF beta 2 (1 ng/mL) to the medium caused a decrease in enkephalinase specific activity despite the continued presence of MPA. The half-life of enkephalinase (activity) in stromal cells treated with MPA plus TGF beta 1 was 2.8 days, which is similar to the computed half-life for enkephalinase in endometrial tissue during the mid- to late secretory phase of the endometrial cycle (2.5 days). Simultaneous treatment of endometrial stromal cells with MPA (10(-9) mol/L) and TGF beta 1 (1 ng/ mL) prevented the progestin-induced increase in enkephalinase specific activity and immunoreactive enkephalinase protein. Thus, TGF beta acts to oppose the progesterone-induced increase in enkephalinase expression in endometrial stromal cells, even in the continued presence of MPA.

Lysyl oxidase (ras recision gene) expression in human amnion: ontogeny and cellular localization.

The tensile strength of human fetal membranes is attributable to interstitial collagens of the zona compacta of the avascular amnion. Collagen fiber strength and proteolytic resistance is provided by inter and intramolecular cross-links of collagen fibrils, which are formed in a series of reactions initiated by lysyl oxidase. Lysyl oxidase activity in amnion tissues varied by more than 400-fold in a highly significant inverse manner as a function of gestational age (12-43 weeks). At 12-14 weeks gestation, the levels of lysyl oxidase messenger ribonucleic acid, protein, and activity in amnion are very high. During the second trimester of pregnancy, however, these decline abruptly, and a nadir is reached at about 20-24 weeks gestation, which persists to term. The level of lysyl oxidase messenger ribonucleic acid was greater in amnion mesenchymal cells than in amnion epithelial cells. The decline in lysyl oxidase in amnion may be attributable to a correspondent decline in the density of amnion mesenchymal cells with fetal development.

Origin and production rates of deoxycorticosterone and deoxycorticosterone sulfate in men and nonpregnant women.

In the present investigation, we evaluated the origin of deoxycorticosterone sulfate (DOC-SO4) and the production rates of DOC and DOC-SO4 in men and women. Previously, we found that there was little or no interconverion of plasma DOC and DOC-SO4; this finding was reconfirmed in the present investigation. After the iv infusion of [3H]DOC-SO4 and [14C]DOC, urine was collected for 5 days, DOC-SO4 was isolated and purified as unconjugated DOC, and tetrahydro-DOC glucuronoside was isolated and purified as the unconjugated metabolite. The production rate of DOC in these subjects (mean +/- SEM, 66 +/- 9.8 micrograms/24 h) was computed from the specific activity of urinary [14C]tetrahydro-DOC (glucuronoside); the production rate of DOC-SO4 in these subjects (92 +/- 15.9 micrograms/24 h) was computed from the specific activity of urinary [3H]DOC-SO4. The production rates are expressed on the basis of the molecular weight of DOC. Since plasma DOC and DOC-SO4 are not interconverted, we conclude that both steroids are secretory products, presumably from the adrenal cortex.

Formation of deoxycorticosterone from progesterone in extraadrenal tissues: demonstration of steroid 21-hydroxylase activity in human aorta.

In the present investigation, we demonstrated the presence of steroid 21-hydroxylase activity in microsome-enriched fractions prepared from homogenates of aortal tissues of human abortuses and a prepubertal boy. The specific activities of this enzyme in microsomes prepared from aortal tissue of various abortuses varied but was similar to that found previously in microsomes prepared from human fetal kidney tissue. However, the specific activity of steroid 21-hydroxylase in microsomes prepared from smooth muscle tissue of the aorta of a prepubertal boy was extraordinarily high, viz., 705 pmol x h-1 x mg-1 protein. Thus the potential exists for the formation of deoxycorticosterone (DOC) in aorta, another tissue site of DOC action.

Keratinocyte growth factor expression in the mesenchymal cells of human amnion.

Amnion epithelial and mesenchymal cells were separated by differential protease treatment, and the separated cells were maintained in monolayer culture. Keratinocyte growth factor (KGF) messenger RNA (mRNA) was readily detected by Northern analysis of amnion mesenchymal cell total RNA (10 micrograms) but not in amnion epithelial cells. Treatment of the amnion mesenchymal cells in serum-free medium with tetradecanoyl phorbol acetate (1 nM) caused an increase in the level of KGF mRNA. Forskolin treatment also caused an increase in KGF mRNA but not to the levels attained with tetradecanoyl phorbol acetate treatment. Dexamethasone (1 nM) treatment of these cells effected a reduction in the level of KGF mRNA. Prolonged maintenance of mesenchymal cells in serum-free medium also was associated with an increase in the level of KGF mRNA. Treatment with a variety of other agents, viz., interleukin (IL)-1, IL-6 plus or minus IL-6 soluble receptor, IL-11, oncostatin M, epidermal growth factor (EGF), and transforming growth factor-beta and not modify the level of KGF mRNA. Treatment of amnion epithelial cells with KGF caused an increase in the rate of [3H]thymidine incorporation, but the rate of cell replication induced by KGF was less than that induced by treatment with EGF. Transforming growth factor-beta treatment inhibited basal and EGF- and KGF-stimulated amnion epithelial cell replication. The findings of this study are indicative the KGF is expressed in human amnion mesenchymal cells, and that KGF may act on the epithelial cells of this tissue.