Saturated Phosphatidylcholine Synthesis Research Articles

The inhibition of Aurora A kinase regulates phospholipid remodeling by upregulating LPCAT1 in glioblastoma.

Metabolic reprogramming is a common feature of glioblastoma (GBM) progression and metastasis. Altered lipid metabolism is one of the most prominent metabolic alterations in cancer. Understanding the links between phospholipid remodeling and GBM tumorigenesis may help develop new anticancer strategies and improve treatments to overcome drug resistance. We used metabolomic and transcriptomic analyses to systematically investigate metabolic and molecular changes in low-grade glioma (LGG) and GBM. We then re-established the reprogrammed metabolic flux and membrane lipid composition in GBM based on metabolomic and transcriptomic analyses. By inhibiting Aurora A kinase via RNA interference (RNAi) and inhibitor treatment, we investigated the effect of Aurora A kinase on phospholipid reprogramming LPCAT1 enzyme expression and GBM cell proliferation in vitro and in vivo. We found that GBM displayed aberrant glycerophospholipid and glycerolipid metabolism compared with LGG. Metabolic profiling indicated that fatty acid synthesis and uptake for phospholipid synthesis were significantly increased in GBM compared to LGG. The unsaturated phosphatidylcholine (PC) and phosphatidylethanolamine (PE) levels were significantly decreased in GBM compared to LGG. The expression level of LPCAT1, which is required for the synthesis of saturated PC and PE, was upregulated in GBM, and the expression of LPCAT4, which is required for the synthesis of unsaturated PC and PE, was downregulated in GBM. Notably, the inhibition of Aurora A kinase by shRNA knockdown and treatment with Aurora A kinase inhibitors such as Alisertib, AMG900, or AT9283 upregulated LPCAT1 mRNA and protein expression in vitro. In vivo, the inhibition of Aurora A kinase with Alisertib increased LPCAT1 protein expression. Phospholipid remodeling and a reduction in unsaturated membrane lipid components were found in GBM. Aurora A kinase inhibition increased LPCAT1 expression and suppressed GBM cell proliferation. The combination of Aurora kinase inhibition with LPCAT1 inhibition may exert promising synergistic effects on GBM.

Orphan G Protein–Coupled Receptor GPR116 Regulates Pulmonary Surfactant Pool Size

Pulmonary surfactant levels within the alveoli are tightly regulated to maintain lung volumes and promote efficient gas exchange across the air/blood barrier. Quantitative and qualitative abnormalities in surfactant are associated with severe lung diseases in children and adults. Although the cellular and molecular mechanisms that control surfactant metabolism have been studied intensively, the critical molecular pathways that sense and regulate endogenous surfactant levels within the alveolus have not been identified and constitute a fundamental knowledge gap in the field. In this study, we demonstrate that expression of an orphan G protein-coupled receptor, GPR116, in the murine lung is developmentally regulated, reaching maximal levels 1 day after birth, and is highly expressed on the apical surface of alveolar type I and type II epithelial cells. To define the physiological role of GPR116 in vivo, mice with a targeted mutation of the Gpr116 locus, Gpr116(Δexon17), were generated. Gpr116(Δexon17) mice developed a profound accumulation of alveolar surfactant phospholipids at 4 weeks of age (12-fold) that was further increased at 20 weeks of age (30-fold). Surfactant accumulation in Gpr116(Δexon17) mice was associated with increased saturated phosphatidylcholine synthesis at 4 weeks and the presence of enlarged, lipid-laden macrophages, neutrophilia, and alveolar destruction at 20 weeks. mRNA microarray analyses indicated that P2RY2, a purinergic receptor known to mediate surfactant secretion, was induced in Gpr116(Δexon17) type II cells. Collectively, these data support the concept that GPR116 functions as a molecular sensor of alveolar surfactant lipid pool sizes by regulating surfactant secretion.

Parathyroid hormone (PTH) and PTH-related protein stimulate surfactant phospholipid synthesis in rat fetal lung, apparently by a mesenchymal-epithelial mechanism

We examined the effects of parathyroid hormone (PTH) and PTH-related protein (PTHrP) on rat fetal lung fibroblast and pneumocyte cell signalling. We also studied the effects of PTH and PTHrP on surfactant phospholipid synthesis to determine whether these peptides can modulate pulmonary maturation. Exposure of fibroblasts (gestational days 18–21) to PTH(1–34) or PTHrP(1–34) produced time- and dose-dependent stimulations of cAMP and inositol phosphate accumulation. Maximal stimulation of cAMP accumulation occurred with 1 · 10 −8 M of either peptide. These effects upon cAMP accumulation were competitively inhibited by the PTH antagonist, [Nle 8, Nle 18, Tyr 34]bPTH(3–34)amide. Maximal stimulation of fibroblast inositol phosphates was reached at 1 · 10 −7 M of either peptide. In contrast, PTH and PTHrP at these concentrations produced no changes in cAMP or inositol phosphate metabolism in isolated type II pneumocytes. When pneumocytes were exposed to PTH or PTHrP and pulse-labelled with [ methyl- 3H]choline chloride, no hormone-stimulated changes in saturated phosphatidylcholine (PC) synthesis were detected. However, PTH and PTHrP stimulated saturated PC synthesis in rat fetal lung explants (gestational day 19–20) by 46% and 106%, respectively. When fibroblasts and pneumocytes were co-cultured, PTH and PTHrP again stimulated saturated PC synthesis by 45% and 73%, respectively. Taken together, these findings suggest that PTH and PTHrP may be endocrine and/or paracrine regulators of fetal lung development.

Androgens Delay Human Fetal Lung Maturationin Vitro*

The mature lung produces pulmonary surfactant, a lipid-protein complex that prevents lung alveoli from becoming atelectatic. The prenatal surge of surfactant production in preparation for birth occurs between 28-34 weeks gestation and is triggered by glucocorticoids, which stimulate surfactant synthesis by alveolar epithelial cells through a series of biochemical steps mediated by mesenchymal-epithelial interactions. In contrast to this, when explanted midgestation human fetal lung tissue is maintained in serum-free medium, there is a spontaneous increase (40%) in de novo saturated phosphatidylcholine (SPC) synthesis on the fifth day in culture. Addition of dexamethasone (DEX; 1 x 10(-8) M) to the culture medium causes the increased synthesis in SPC to occur earlier (day 4) and to a greater extent (87%). Addition of an equimolar concentration of dihydrotestosterone (DHT) to the medium delays both the spontaneous and DEX-stimulated increases in SPC synthesis by 24 h. Weak fetal adrenal androgens are also able to block both spontaneous and DEX-stimulated SPC synthesis. Addition of DHT to the explant cultures at daily intervals reveals that inhibition of the DEX effect occurs within the first 24 h after exposure. The antiandrogen flutamide neutralizes the effect of DHT, indicating that it acts through the androgen receptor to block the glucocorticoid. Therefore, the human fetal adrenal cortex may time lung development through both inhibitory and stimulatory mechanisms.

Effects of two extracellular matrices on morphologic and biochemical properties of human type II cells in vitro.

Previous observations have suggested that differentiated functions of adult rat type II alveolar cells are affected in part by cell-matrix interactions. We examined several aspects of differentiated adult human type II cells cultured on either bovine corneal endothelial cell extracellular matrix (BCECM) or matrix derived from the Englebreth-Holm-Swarm tumor (EHS). Compared to cells cultured on BCECM, adult human type II cells grown on EHS assumed a more cuboidal shape, had a more defined apical-basal polarity, and appeared to contain a greater number of lamellar bodies and neutral lipid inclusions. These cells also incorporated a greater percentage of [14C]acetate into saturated phosphatidylcholine (SPC) than did their counterparts grown on BCECM. In contrast, the relative incorporation of [14C]acetate into phosphatidylglycerol (PG) was lower in cells grown on EHS than cells cultured on BCECM. The histochemical stain for alkaline phosphatase was useful in identification of human type II cells. Alkaline phosphatase expression was elevated in cells cultured on EHS compared to those cultured on BCECM. These results suggest that maintenance of a differentiated morphology, lipid synthesis, and expression of alkaline phosphatase activity by primary cultures of adult human type II cells are also influenced by cell-matrix interactions. All markers of differentiated function of type II cells except synthesis of PG are better maintained on EHS than on BCECM. Under the conditions of these experiments, synthesis of SPC and PG appears to be independently regulated.

Functional differentiation of alveolar type II epithelial cells in vitro: Effects of cell shape, cell-matrix interactions and cell-cell interactions

Alveolar type II epithelial cells rapidly lose characteristics of differentiated function when cultured on plastic dishes. We have attempted to circumvent this problem by culturing type II cells under conditions that might better reproduce their environment in vivo. Cell-matrix interactions were studied by culturing isolated adult rat type II cells on Engelbreth-Holm-Swarm (EHS) tumor basement membrane. Aggregates of type II cells formed on the surface of the matrix during 4 days in culture. Microscopic examination of these aggregates revealed cuboidal cells that retained more characteristics of differentiated type II cells than did cells cultured on plastic. Type II cells cultured on EHS matrix incorporated a higher percentage of acetate into phosphatidylcholine (PC) than did cells on plastic, and a higher percentage of this PC was saturated. Phosphatidylglycerol (PG) synthesis by these cells was no different from that seen in cells on plastic. The effects of cell-cell interactions and cell shape were evaluated by culturing type II cells on feeder layers that in turn were grown on collagen gels. The feeder layer cells included fetal rat lung fibroblasts, adult rat lung fibroblasts, fetal rat skin fibroblasts, bovine aortic endothelial cells, and rat mammary tumor epithelial cells. One-half of the gels remained attached to the culture dish and one-half of the gels were detached after 24 h and allowed to float free in the medium. Type II cells grown in association with any of the attached feeder layers became flattened and lost their differentiated phenotype. These cells incorporated no greater percentage of acetate into PC than did cells on plastic. Saturated PC synthesis was modestly increased. PG synthesis declined in parallel with that seen in cells cultured on plastic. Type II cells cultured on feeder layers that were detached assumed their native cuboidal shape and also exhibited many morphological characteristics of differentiated function. These cells incorporated a significantly greater percentage of acetate into PC compared to cells on either plastic or attached feeder layers. Saturated PC synthesis also increased markedly. These cells, however, incorporated no greater percentage of acetate into PG than did cells on plastic or attached feeder layers. These data suggest an important role for cell shape and cell-matrix interactions and maintenance of type II cell differentiation. The effects of cell-cell interactions, while beneficial, appear to be non-specific.

Temporal linkage of glycogen and saturated phosphatidylcholine in fetal lung type II cells.

The developmental profiles of glycogen and surfactant-associated saturated phosphatidylcholine were investigated in type II cells isolated from fetal rat lung. Incorporation of radiolabeled glucose into glycogen and type II cell unlabeled glycogen content decreased as a function of gestational age. Conversely, an increase was noted in radioactive choline incorporation into saturated phosphatidylcholine and in the content of unlabeled saturated phosphatidylcholine as a function of gestational age. Type II cells from days 19 and 21 of gestation were also studied by electron microscopy. Temporal relationships similar to those noted biochemically were observed by morphometric analysis. A decrease in glycogen content and an increase in lamellar bodies (the storage organelles for the pulmonary surfactant) were noted as gestation progressed. These studies biochemically and morphologically demonstrate a temporal relationship between glycogen degradation and saturated phosphatidylcholine synthesis in type II cells isolated from fetal rat lung. These findings provide further support for the use of such type II cell preparations for studies of development at the cellular level.

SURFACTANT SYNTHESIS IN ISOLATED LUNG CELLS

Explant culture and hormone treatment increase synthesis of saturated phosphatidylcholine (PC) and surfactant apoprotein SP 28-36 in fetal type II cells. We asked whether these effects were maintained during cell monolayer culture. Human fetal lung (18-22 wk gestation) was cultured as explants for 5 d with 10 nM dexamethasone plus 2 nM T3; then type II cells (85% pure) or mixed cells (54% type II) were prepared and maintained as monolayers in MEM+ 10% fetal calf serum for 1-5 d. During culture the distribution of acetate changed (decrease into PC and lyso PC and increase into most other phospholipids) and the number of lamellar bodies in type II cells decreased progressively. The changes were similar in type II and mixed cell populations and were not prevented by the presence of T3 plus dexamethasone. In fibroblasts (87% pure) choline incorporation increased from d 1 to d 5 (17.1±6.1 to 39.3 ± 7.7 nmol/mg DNA/4 h) with no change in saturation (25.5 ± 0.8 to 22.6 ± 2.5%) consistent with membrane PC synthesis. We conclude that synthesis of both lipids and protein of surfactant rapidly ceases (dedifferentiation) when fetal type II cells are cultured as monolayers.

The effect of substratum and serum on the lipid synthesis and morphology of alveolar type II cells in vitro.

To determine the effect of various culture conditions on the maintenance of lipid synthesis and morphology in alveolar type II cells, we cultured isolated adult rat alveolar type II cells on either plastic or denuded human amnionic basement membrane (ABM) in medium supplemented with either fetal bovine, porcine, horse, rat, or human serum. Lipid synthesis was assessed by incubation with [1-14C]acetate and determination of the distribution of radiolabel into individual lipid classes. Cells cultured on ABM incorporated significantly higher percentages of acetate into either phosphatidylcholine (PC) or phosphatidylglycerol (PG), and retained lamellar inclusions and a more characteristic cuboidal shape for longer periods than did cells cultured on plastic. Compared to other sera, cells cultured in the presence of rat serum incorporated the highest percentages of acetate into PC and saturated PC, had the best preservation of lamellar-body ultrastructure, and also appeared to contain more multivesicular bodies. The percent composition of linoleic acid, an essential fatty acid, was found to vary widely among the different sera. Supplementing media with linoleic acid resulted in a marked increase in acetate incorporation into saturated PC and a decreased incorporation into PG. We conclude that for maintenance of differentiated function of adult rat alveolar type II cells in primary culture (1) ABM is preferable to plastic as a culture substratum, (2) rat serum is preferable to fetal bovine serum as a serum supplement, and (3) the regulation of lipid synthesis by linoleic acid causes disparate effects on PG and saturated PC synthesis.

The cellular mechanism of glucocorticoid acceleration of fetal lung maturation. Fibroblast-pneumonocyte factor stimulates choline-phosphate cytidylyltransferase activity.

The cellular mechanism by which glucocorticoids stimulate phosphatidylcholine biosynthesis has been studied in the fetal rat lung in vivo and in cultured fetal rat lung cells of varying levels of complexity. Administration of dexamethasone to pregnant rats at 18 days gestation resulted in a significant increase in saturated phosphatidylcholine content in fetal lung 24 h after injection. Dexamethasone administration increased the activity of fetal lung choline-phosphate cytidylyltransferase by 34%. It had no effect on the activities of fetal lung choline kinase and choline phosphotransferase. Exposure of fetal lung type II cells in organotypic cultures (which contain both type II cells and fibroblasts) to cortisol resulted in a 1.6-fold increase in the incorporation of [Me-3H]choline into saturated phosphatidylcholine. The activities of the enzymes in the choline pathway for the de novo biosynthesis of phosphatidylcholine were not significantly altered except for a 105% increase in choline-phosphate cytidylyltransferase activity. Treatment of monolayer cultures of fetal type II cells with cortisol-conditioned medium from fetal lung fibroblasts resulted in a 1.5-fold increase in saturated phosphatidylcholine production. This effect correlated with a doubling of choline-phosphate cytidylyltransferase activity. Additional evidence that this stimulatory action is mediated by fibroblast-pneumonocyte factor, produced by fetal lung fibroblasts in response to cortisol, was obtained. The factor was partially purified from cortisol-conditioned medium of fetal lung fibroblasts by gel filtration and affinity chromatography. Based on biological activity, a 3000-fold purification was obtained. Stimulation of saturated phosphatidylcholine synthesis in type II cells by fibroblast-pneumonocyte factor was maximal within 60 min of incubation. Pulse-chase experiments indicated that the stimulatory effect was correlated with an increased conversion of choline phosphate into CDP choline. Moreover, the enhanced phosphatidylcholine formation by fetal type II cells in response to fibroblast-pneumonocyte factor was accompanied by decreased levels of cellular choline phosphate. These findings further support the concept that glucocorticoid action on surfactant-associated phosphatidylcholine synthesis occurs ultimately at the level of the alveolar type II cell and involves fibroblast-pneumonocyte factor which stimulates the activity of choline-phosphate cytidylyltransferase.

Compartmentalization of 11-oxidoreductase within fetal lung alveolus.

The role of 11-oxidoreductase in the cellular process of fetal lung surfactant production and its localization within the alveolar domain have been investigated. In organotypic cultures of fetal rat lung, cortisol and cortisone markedly stimulate saturated phosphatidylcholine synthesis by the alveolar type II cell; a 10-fold excess of 11-ketoprogesterone blocks the bioactivity of cortisone. Both cortisol and cortisone also stimulate fibroblast-pneumonocyte factor production, whereas 11-ketoprogesterone blocks the effect of cortisone, but not of cortisol, suggesting that cortisone stimulation of fibroblast-pneumonocyte factor production depends on its conversion to cortisol by 11-oxidoreductase. A survey of the cells that are present in the alveolar domain revealed that 11-oxidoreductase activity is only present in the fibroblast. Localization of both 11-oxidoreductase and fibroblast-pneumonocyte factor production within the same cell emphasizes the significance of 11-oxidoreductase in the regulation of fetal lung surfactant production.

Dihydrotestosterone inhibits fibroblast-pneumonocyte factor-mediated synthesis of saturated phosphatidylcholine by fetal rat lung cells.

Dihydrotestosterone has previously been found to inhibit pulmonary surfactant production in vivo. In the present study, we have investigated the effects of this androgen on the fetal lung fibroblast and type II cell in culture. Addition of 1 X 10(-8) M cortisol to organotype cultures stimulates the synthesis of radiolabelled saturated phosphatidylcholine; however, preincubation of such cultures with 1 X 10(-8) M dihydrotestosterone completely blocks cortisol stimulation of saturated phosphatidylcholine synthesis without affecting basal activity. Cortisol treatment of fetal lung fibroblasts stimulates the production of fibroblast-pneumonocyte factor; pre-treatment with dihydrotestosterone blocks this stimulation. Fibroblast-pneumonocyte factor stimulates the synthesis of saturated phosphatidylcholine by type II pneumonocytes; this effect is also blocked by dihydrotestosterone pre-treatment. I conclude that dihydrotestosterone blocks cortisol-inducible saturated phosphatidylcholine synthesis by inhibiting the production and activity of fibroblast-pneumonocyte factor in vitro.

321 Dihydrotestosterone Antagonizes Glucocorticoid-de-Pendent Surfactant Synthesis in Explant Culture

Male fetuses produce less pulmonary surfactant than female fetuses during the active phase of lung maturation characterized by increased synthesis of saturated phosphatidylcholine (SPC). A previous study has shown that Dihydrotestosterone (DHT) can inhibit SPC production by the fetal rabbit lung in vivo (Nielsen et al JCI 69; 611, 1982). The present study was designed to test whether DHT antagonizes glucocorticoid-stimulated surfactant synthesis by fetal rabbit lung explants, a system in which active SPC synthesis is glucocorticoid-dependent. Day 19 fetal lung explants were cultured in Waymouth's medium plus 10% charcoal-stripped fetal calf serum (FCS-) and 10−7M DHT, 10−7M cortisol or 10−7M DHT+10−7M cortisol for 9 days (=day 28 in utero). Ex-plants maintained in FCS- showed no increase in 3H-choline incorporation into 3H-SPC; cortisol caused a 4-fold increase in SPC synthesis; DHT had no effect on baseline SPC synthesis - however DHT-treated explants did not respond to a cortisol challenge. Furthermore, sex-specific explants showed no difference in either baseline or cortisol-challenged SPC synthesis. These data support the hypothesis that the sex difference in pulmonary surfactant production is due to androgen antagonism of cortisol-stimulated surfactant phospholipid synthesis. There is extensive evidence that this increase in SPC synthesis is due to glucocorticoid stimulation, and that the delay in male surfactant production is due to androgen inhibition. This study was supported by NIH grant #28315.

Insulin acts on the fibroblast to inhibit glucocorticoid stimulation of lung maturation.

The effects of insulin and cortisol on saturated phosphatidylcholine synthesis are examined in fetal type II cell cultures and in mixed cell cultures containing type II cells and fibroblasts. In 19-day fetal rat lung type II cell cultures, 100 nM cortisol and 2 nM insulin have no significant effect. Fibroblast-pneumonocyte factor results in enhanced saturated phosphatidylcholine synthesis by fetal type II cells. The significant stimulatory effect of cortisol in mixed-cell cultures is abolished in the presence of insulin or of monoclonal antibodies to fibroblast-pneumonocyte factor. Incubation of type II cells with conditioned media from fibroblasts exposed to cortisol results in increased saturated phosphatidylcholine synthesis. This process is not stimulated when type II cells are incubated with conditioned media from fibroblasts exposed to insulin and cortisol (or to insulin alone). These observations demonstrate that insulin inhibits cortisol induction of lung maturation and suggest that this antagonism results from an inhibitory effect of insulin on the elaboration of fibroblast-pneumonocyte factor by fetal lung fibroblasts.

Synthesis of saturated phosphatidylcholine and phosphatidylglycerol by freshly isolated rat alveolar type II cells

Saturated phosphatidylcholine and phosphatidylglycerol are important components of pulmonary surface active material, but the relative contributions of different pathways for the synthesis of these two classes of phospholipids by alveolar type II cells are not established. We purified freshly isolated rat type II cells by centrifugal elutriation and incubated them with [1- 14C]palmitate as the sole exogenous fatty acid in one series of experiments or with [9,10- 3H]palmitate, mixed fatty acids (16:0, 18:1 and 18:2), and [U- 14C]glucose in another series of experiments. Type II cells readily incorporated [1- 14C]palmitate into saturated phosphatidic acid (55–59% of total phosphatidic acid), saturated diacylglycerol (82–87% of total diacylglycerol), saturated phosphatidylcholine (69–76% of total phosphatidylcholine), and saturated phosphatidylglycerol (55–59% of total phosphatidylglycerol). Saturated phosphatidic acid, diacylglycerol and phosphatidylglycerol were nearly equally labeled in the sn-1 and sn-2 positions, whereas saturated phosphatidylcholine was preferentially labeled in the sn-2 position. With [9,10- 3H]palmitate and [U- 14C]glucose, the labeling patterns of phosphatidic acid, diacylglycerol and phosphatidylglycerol were similar to each other but different from that of phosphatidylcholine. The glucose label was found predominantly in the unsaturated phosphatidylcholines at early times (3–10 min) and in the saturated phosphatidylcholines at later times (30–90 min). Similarly, the 3H/ 14C ratio was very high in saturated phosphatidylcholine and always above that in saturated diacylglycerol. We conclude that freshly isolated type II cells synthesize saturated phosphatidic acid, diacylglycerol, phosphatidylcholine and phosphatidylglycerol and that under our in vitro conditions the deacylation-reacylation pathway is important for the synthesis of saturated phosphatidylcholine but is less important for the synthesis of saturated phosphatidylglycerol. By the assumptions stated in the text during the pulse chase experiment de novo synthesis of saturated phosphatidylcholine from saturated diacylglycerol accounted for 25% of the total synthesis of saturated phosphatidylcholine.

THE CELLULAR SITE OF INSULIN INHIBITION OF GLUCOCORTICOID-INDUCED LUNG MATURATION

The infant of the diabetic mother shows delayed lung maturation and available evidence suggests that both hyperinsulinemia and hyperglycemia may mediate this delay. In vitro studies have shown that insulin blocks cortisol stimulation of surfactant-associated phospholipid synthesis. Cortisol induces the synthesis of fibroblast-pneumonocyte factor (FPF) which in turn stimulates surfactant synthesis by alveolar type II cells. In the present study, we have examined the effects of insulin, cortisol and FPF on saturated phosphatidylcholine (SPC) synthesis by fetal type II cells alone and in the presence of fetal lung fibroblasts and the effects of insulin and cortisol upon the production of FPF activity by fetal lung fibroblasts. In fibroblast/type II cell cultures, cortisol stimulates (3H)choline incorporation into SPC and this effect is blocked by insulin as well as by monoclonal antibodies directed against FPF. In type II cell cultures, cortisol is ineffective but SPC synthesis is stimulated by FPF. This stimulation is not inhibited by insulin. In contrast, preliminary studies suggest that insulin inhibits the elaboration of FPF activity by fetal lung fibroblasts exposed to cortisol. These observations confirm the ability of insulin to block cortisol induction of lung maturation and suggest that this action is exerted on the fetal lung mesenchyme. (Supported by NIH grant HL-25907)

DIHYDROTESTOSTERONE (DHT) INHIBITS SYNTHESIS OF SATURATED PHOSPHATIDYLCHONE

Male newborns are at a 2-fold greater risk of Respiratory Distress Syndrome than females. This risk is associated with delayed production of saturated phosphatidylcholine (SPC) in male human, rabbit, and rat fetuses. DHT administration to rabbit fetuses inhibits the production of SPC in an organ-specific manner and the anti-androgen Flutamide negates the native sex difference in SPC production. In the present study DHT was administered to pregnant rabbit does at 1 mg/kg from day 12 to day 27 of gestation daily and the fetuses were sacrificed on day 28. The fetal lungs were incubated with 3H-choline chloride (10μci) for 1 hour and subsequently analyzed for radiolabelled PC and SPC content.Male and female control lungs synthesized comparable amounts of both PC and SPC. DHT had no measurable effect on either PC or SPC synthesis by female lungs, or on PC synthesis by male lungs. However, DHT did have a significant inhibitory effect on SPC synthesis by male lungs. These data suggest that DHT inhibits the synthesis of pulmonary surfactant in a sex-specific fashion under these experimental conditions. The mechanism underlying this inhibition is currently under investigation. (Supported by NIH Grant #HL28315-02).

DIRECT PHARMACOLOGIC EFFECTS OF DHT ON THE ALVEOLAR TYPE II CELL IN VITRO

Previous studies from our laboratory have demonstrated that dihydrotestosterone (DHT) inhibits the production of pulmonary surfactant in vivo and its de novo synthesis in tissue culture (J. Cell Biology, 97, 86a, 1983). Studies in tissue culture suggest that DHT has no effect on the fibroblast-type II cell interaction which gives rise to surfactant, but blocks this mechanism in response to cortisol at very low doses (≤ 10−8M) comparable to the circulating level of androgens in the fetus and to the Kd of the androgen receptor. More recent studies suggest that 10−7M to 10−5M DHT stimulates the growth of the fetal type II cell by 40-100% while reducing the synthesis of saturated phosphatidylcholine in a reciprocal manner. These results may explain the observed in vivo effects of DHT initially on the female fetuses (10−8M) and at pharmacologic levels on both sexes at serum androgen levels at an order of magnitude greater. We conclude that the native sex difference in pulmonary surfactant production is due to the effects of androgen on mesenchymal-epithelial interactions, but that DHT may have direct, pharmacologic effects on type II cell maturation as well. (Supported by NIH Grant #HL28315-02).

Inhibition of lung maturation by monoclonal antibodies against fibroblast-pneumonocyte factor.

Fetal lung maturation, especially the onset of surfactant formation by alveolar type II cells, seems to be regulated by endogenous fetal glucocorticoids. Recent studies suggest that glucocorticoids do not act directly on the type II cell but rather on the lung mesenchyme. In response to glucocorticoids, the mesenchyme produces and secretes a polypeptide, fibroblast-pneumonocyte factor, which in turn stimulates surfactant synthesis by the alveolar type II cell. We report here the generation of hybridomas secreting monoclonal antibodies to rat lung fibroblast-pneumonocyte factor. Two monoclonal antibodies studied in detail reduced the cortisol-stimulated synthesis of saturated phosphatidylcholine in organotypic cultures of fetal rat lung cells and blocked the stimulatory effect of fibroblast-pneumonocyte factor in type II cells from these cultures. When embryonic chicks were injected on day 15 of incubation with either monoclonal antibody, they showed on days 20 and 21 biochemical evidence of delayed lung maturation as compared with controls. These effects were organospecific. Our observations support a physiological role for fibroblast-pneumonocyte factor in prenatal lung maturation.

Synergistic effect of triiodothyronine and dexamethasone on male and female fetal rat lung surfactant synthesis.

It has previously been reported that morphologic and functional indices of fetal lung maturation are delayed in male rabbit fetuses, and these differences cannot be overcome by glucocorticoid treatment. Since thyroid hormone also stimulates surfactant production we have studied the effects of triiodothyronine (T3) and dexamethasone on saturated phosphatidylcholine (SPC) synthesis by day 20 male and female rat lung. Control male lung slices incorporated significantly less 3H-choline into SPC than females; surgery on day 17 markedly stimulated 3H-choline incorporation into SPC by both sexes, but the sex difference persisted. The optimal dose of T3 inhibited SPC synthesis by both sexes while dexamethasone was stimulatory. When T3 and dexamethasone were given together they had a greater effect than that of dexamethasone or T3 alone. Based on these results we conclude that the male deficit in pulmonary surfactant production may be overcome by the synergistic effects of T3 and dexamethasone.