Pleiotropic drug resistance in cystic fibrosis fibroblasts: increased resistance to cyclic AMP.

In previous studies, cystic fibrosis (CF) fibroblasts were demonstrated to be resistant to the cytotoxic effects of ouabain, dexamethasone, and the sex hormones, dihydrotestosterone, 17beta-estradiol, and progesterone. We now show that CF fibroblasts also exhibit greatly increased resistance to the cytotoxic effects of exogenous dibutyryl cyclic AMP (cAMP), as well as to isoproterenol and theophylline, drugs which are known to increase endogenous levels of cAMP. CF cells were also shown to have normal amounts of (3H)cAMP binding to protein kinase as well as normal amounts of cAMP-stimulated protein kinase activity. Phosphodiesterase in CF cells was also found to be stimulated by cAMP to the same degree as in normal cells. These findings suggest that there is no detectable protein kinase deficiency in CF cells. cf cells thus appear to be unlike some cAMP-resistant mutants described by others which are defective in protein kinase activity and cAMP regulation of phosphodiesterase levels. The cross-resistance of CF fibroblasts to ouabain, steroid hormones, and cAMP may provide a unique opportunity to study the biochemical events involved in the metabolism of these drugs as well as the basic biochemical defect in a common human genetic disease.     

Inhibition of Tamm-Horsfall glycoprotein induction of alkaline phosphatase in cystic fibrosis fibroblasts by medium conditioned by normal cells.

It is confirmed that the level of alkaline phosphatase in fibroblasts derived from cystic fibrosis patients can be induced many-fold by growing the cells in the presence of Tamm-Horsfall glycoprotein. It is further shown that normal fibroblasts produce a "CF corrective factor" which markedly inhibits this phenomenon. These observations support a previous hypothesis on the nature of the metabolic defect in cystic fibrosis.     

Repression of CFTR activity in human MMNK-1 cholangiocytes induces sulfotransferase 1E1 expression in co-cultured HepG2 hepatocytes

Mouse models of cystic fibrosis (CF) indicate that sulfotransferase (SULT) 1E1 is significantly induced in livers of many mice lacking cystic fibrosis transmembrane receptor (CFTR) activity. Increased SULT1E1 activity results in the alteration of estrogen-regulated protein expression in the livers of these mice. In this study, human MMNK-1 cholangiocytes with repressed CFTR function were used to induce SULT1E1 expression in human HepG2 hepatocytes to investigate whether SULT1E1 can be increased in human CF liver. CFTR expression was inhibited in MMNK-1 cholangiocytes using CFTR-siRNA, then the MMNK-1 and HepG2 cells were co-cultured in a membrane-separated Transwell system. Expression of SULT1E1 and selected estrogen-regulated proteins were then assayed in the HepG2 cells. Results demonstrate that inhibition of CFTR expression in MMNK-1 cells results in the induction of SULT1E1 message and activity in HepG2 cells in the Transwell system. The expression of estrogen-regulated proteins including insulin-like growth factor (IGF)-1, glutathione-S-transferase (GST) P1 and carbonic anhydrase (CA) II expression are repressed in the HepG2 cells cultured with the CFTR-siRNA-MMNK-1 cells apparently in response to the increased sulfation of beta-estradiol. Thus, we have shown that co-culture of HepG2 hepatocytes with MMNK-1 cholangiocytes with siRNA repressed CFTR expression results in the selective induction of SULT1E1 in the HepG2 cells. Loss of CFTR function in cholangiocytes may have a paracrine regulatory effect on hepatocytes via the induction of SULT1E1 and the increased sulfation of beta-estradiol. Experiments are presently underway in our laboratory to elucidate the identity of these paracrine regulatory factors.     

Immunochemical studies of the plasma and cultured fibroblast media fractions containing the cystic fibrosis ciliary inhibitor.

The cystic fibrosis ciliary inhibitor (CFCI) has been fractionated from plasma of cystic fibrosis (CF) homozygotes and from the media of cultured fibroblasts derived from CF homozygotes. Plasma and fibroblast media from normal controls have been fractionated in an identical manner. Fractions from plasma and fibroblast culture media that demonstrate ciliary inhibitory activity contain several proteins in a molecular weight range of approximately 5,000-11,000. These proteins have been partially characterized by immunochemical analysis with antisera to 33 human serum proteins. Immunological determinants of albumin, C3 (but not C3a), C4, C5, alpha1-lipoprotein, beta-lipoprotein, beta2-microglobulin and immunoglobulin light chains have been detected by hemagglutination in fractions of CF plasma that inhibited ciliary activity and in analogous fractions from normal sera. None of the proteins were detected in media of cultured fibroblasts from either genotype. Since the same proteins and protein fragments were identified in both CF and normal plasma fractions, and were not detected in CF fibroblast media, it appears that none of these proteins can be identified as the CFCI. Identification of these proteins will permit further purification of the CFCI by immunochemical methods.     

Estrogen regulates the IFN-gamma promoter

The greater immune reactivity of females has been attributed in part to the influence of sex steroid hormones, but the underlying mechanisms are unknown. Here we report evidence that expression of the IFN-gamma gene may be subject to direct hormonal control. In a transient expression assay, the sex steroid 17 beta-estradiol markedly increases activity of the IFN-gamma promoter in lymphoid cells that express the appropriate hormone receptor. This effect is mediated by sequences in the 5'-flanking region of the gene, and can augment the effect of T cell-activating agents. Short term exposure to estradiol also increases IFN-gamma mRNA expression in Con A-treated murine spleen cells. Hormonal regulation of this pleiotropic cytokine may account in part for the ability of estrogen to potentiate many types of immune responses, and for the disproportionate susceptibility of females to autoimmune disease.     

Characterization of the cell kinetics and growth properties of cystic fibrosis diploid fibroblasts in vitro.

The population growth kinetics of human diploid skin fibroblasts derived from cystic fibrosis homozygotes and heterozygotes and from normal subjects were investigated. Our data suggest the following: 1. Population doubling times increase with time in culture, with no significant differences observed among the three genotypes tested, when data were compared at the same subculture times or phases of growth. 2. The fraction of dividing cells in a population decreased with the duration in which the cells were in culture. No significant differences were obtained for cells derived from the three genotypes. 3. Cell cycle times were very similar (18-20 hr) when comparing the normal and cystic fibrosis lines or when comparing cystic fibrosis lines in phases 1 and 2 of growth. 4. No significant variations in population doubling times or growth fractions could be attributed to age or sex of the biopsy donor. 5. Variability in growth fractions and doubling times was minimal through the eighth subculture period but was very great in older cultures (tenth subculture). 6. Changes in growth fractions and doubling times appear to be due to the possibility of "aging" of human diploid fibroblasts in culture rather than to the presence or absence of genes for cystic fibrosis. 7. It is strongly indicated that differences in cell kinetic parameters cannot be used as the basis for differentiation between cells derived from normal or cystic fibrosis genotypes.     

Membrane function in cystic fibrosis. I. Putrescine transport in normal and cystic fibrosis fibroblasts

Putrescine transport was examined in normal and cystic fibrosis fibroblasts. No differences were observed in accumulation pattern, kinetics of uptake, or efflux between CF and normal cells. In both growing and growth-arrested CF and normal fibroblasts, exogenously supplied putrescine remained unchanged for at least 60 min. Some differences were observed in the response of CF and normal cells to environmental (media) changes.This research was supported by a grant from the Cystic Fibrosis Foundation and by a grant from the National Institutes of Health, Training Grant (GM01316 11 GNC).     

Glycosyl acceptors in intact and permeabilized normal and cystic fibrosis fibroblasts

Using cell permeabilization, a technique which allows addition of exogenously supplied radiolabeled sugar nucleotides to serve as direct glycosyl donors, oligosaccharide biosynthesis was examined in fibroblasts obtained from normal and cystic fibrosis (CF) subjects. Incubation of logarithmically growing cells with either radiolabeled leucine or xylose has indicated that there was a difference in the synthetic rate between the cell types. Protein synthesis in normal cells made permeable with 50 m?g/ml lysolecithin (LL) was demonstrated to be absent, and could not be induced to take place by adding exogenous components, including energy sources and amino acids, normally required for protein synthesis. Thus radiolabeled sugars were being added to peptide acceptors which were already present at the time of LL addition. Both permeable and intact fibroblasts were exposed to labeled UDP-xylose, UDP-galactose, and UDP-glucuronic acid, all donors of mucopolysaccharide precursors. The uptake of xylose into protein was the same for both normal and CF cells, but permeable CF fibroblasts incorporated statistically greater amounts of sugar from UDP-galactose and UDP-glucuronic acid. Intact CF cells were also labeled using these two sugar nucleotides. Trypan blue exclusion indicated CF and normal fibroblasts were equally intact. This and the fact that preincubation of CF cells with the appropriate cold sugar nucleotide eliminated the differences in incorporation between the normal and CF cells suggested that CF fibroblasts had more cell surface acceptor than the normal cells.     

Estrogen-mediated regulation of CYP7B1: a possible role for controlling DHEA levels in human tissues

The current study examines regulation of CYP7B1, a DHEA 7alpha-hydroxylase, by sex hormones. Transfection with estrogen receptor alpha and treatment with 17beta-estradiol in human embryonic kidney 293 cells significantly increased CYP7B1 catalytic activity and mRNA, and stimulated a human CYP7B1 reporter gene. Transfection with estrogen receptor beta showed similar but less significant effects. In the absence of receptors, 17beta-estradiol suppressed CYP7B1 activity, suggesting that estrogenic effects may be different in cells not expressing receptors. Quantitation of CYP7B1 mRNA in adult and fetal human tissues showed markedly higher CYP7B1 mRNA levels in fetal tissues compared with the corresponding adult ones, except in the liver. This indicates a tissue-specific, developmental regulation of CYP7B1 and suggests an important function for this enzyme in fetal life. DHEA secreted by fetal adrenals is an essential precursor for placental estrogen formation. Since CYP7B1 diverts DHEA from the sex hormone biosynthetic pathway, estrogen receptor-mediated up-regulation of CYP7B1 should lead to less DHEA available for sex hormone synthesis and may help to maintain normal levels of estrogens and androgens in human tissues, especially during fetal development. Regulation by estrogens may also be of importance in other processes where CYP7B1 is involved, including cholesterol homeostasis, cellular proliferation, and CNS function.     

Sex steroid effects at target tissues: mechanisms of action

Our understanding of the mechanisms of sex hormone action has changed dramatically over the last 10 years. Estrogens, progestins, and androgens are the steroid hormones that modulate reproductive function. Recent data have shown that many other tissues are targets of sex hormones in addition to classical reproductive organs. This review outlines new advances in our understanding of the spectrum of steroid hormone ligands, newly recognized target tissues, structure-function relationships of steroid receptors, and, finally, their genomic and nongenomic actions. Sex-based specific effects are often related to the different steroid hormone mileu in men compared with women. Understanding the mechanisms of sex steroid action gives insight into the differences in normal physiology and disease states.     

X-ray microanalysis of cAMP-induced ion transport in cystic fibrosis fibroblasts.

cAMP-induced ion transport in normal and cystic fibrosis (CF) fibroblasts was investigated by X-ray microanalysis. Stimulation with cAMP causes an increase in cellular Na content and a decrease in cellular Cl and K content. No significant difference in response between CF and normal cells was noted. In this respect, fibroblasts differ from epithelial cells, where cAMP-induced Cl- efflux blocked in CF patients. Isoproterenol produced similar changes in Na and K content as cAMP, but did not effect Cl content.     

Identification of a defective cAMP-stimulated Cl- channel in cystic fibrosis fibroblasts

Cl- efflux from normal human fibroblasts is stimulated by elevation of cAMP and by elevation of intracellular free Ca2+. In both cases the stimulated Cl- transport occurs via electrically conductive pathways. In six lines of normal human fibroblasts, dibutyryl cAMP increased total Cl- efflux by an average of 13%. In six lines of fibroblasts from patients with cystic fibrosis, dibutyryl cAMP was without effect. The electrically conductive component of Cl- transport was increased an average of 30% by dibutyryl cAMP in normal cells and was unaffected by dibutyryl cAMP in cystic fibrosis cells. Stimulation of the Ca2+-sensitive Cl- channel by addition of A23187 increased Cl- efflux by an average of 30% in normal and 30% in cystic fibrosis fibroblasts. The data indicate that there is a defect in a cAMP-activated Cl- channel in cystic fibrosis fibroblasts.     

K dependence of the Na-K pump is abnormal in erythrocytes from patients with cystic fibrosis and obligate heterozygotes

The affinity of the Na-K pump for K was significantly (P less than .001) lower in erythrocytes from patients with cystic fibrosis (Km 4.6 +/- 0.35 mM; n = 26) or from heterozygotes (Km 3.9 +/- 0.57 mM; n = 12) than in controls (Km 2.2 +/- 0.10 mM; n = 20). The affinity of the Na-K pump for K was lower in normal erythrocytes than in normal fibroblasts which may explain the variability in the severity of involvement of different organs in cystic fibrosis. We have now shown in human skin fibroblasts and erythrocytes, that the K affinity of the Na-K pump is lower in patients with cystic fibrosis than in controls. Since the abnormality is also present in erythrocytes from heterozygotes who are clinically normal, it is likely that this abnormality is closely related to the genetic defect in cystic fibrosis.     

BMP-6-induced osteogenic differentiation of mesenchymal cell lines is not modulated by sex steroids and resveratrol

Bone morphogenetic protein-6 (BMP-6) is a potent inducer of osteogenic differentiation and its expression is stimulated by 17beta-estradiol. The existence of a regulatory loop between sex steroids and BMP-6 is therefore reasonable to hypothesize. Here we determined whether the sex steroids 17beta-estradiol and dihydrotestosterone, and the phytoestrogen resveratrol can modulate BMP-6-induced alkaline phosphatase activity and osteocalcin expression. Mesenchymal cells of murine (osteoblastic MC3T3-E1 cells, preadipogenic ST2 cells, prechondrogenic ATDC5 cell) and human origin (osteosarcoma SaOS and HOS cells, primary bone marrow stromal cells) were cultured in the presence of recombinant BMP-6 under serum-free conditions. BMP-6 dose-, and time-dependently increased alkaline phosphatase activity in murine cell lines, but not in human cells. Osteocalcin expression was also increased upon stimulation with BMP-6. The presence of 17beta-estradiol, dihydrotestosterone, and resveratrol had no effect on BMP-6-induced alkaline phosphatase activity and osteocalcin expression. These data suggest that osteogenic differentiation in response to BMP-6 occurs independent of steroid hormones and resveratrol in mesenchymal cells that express basal receptor levels.     

Sex steroids and growth factors in the regulation of mammary gland proliferation, differentiation, and involution

The mammary gland is subjected to major morphological and biochemical changes during the lactation cycle. It is therefore not surprising that this dynamic process is strictly controlled. The importance of the sex steroid hormones 17beta-estradiol and progesterone for normal development of the mammary gland was recognized several decades ago and has been unequivocally confirmed since. Furthermore, it is now also established that the influence of sex steroids is not restricted to mammogenesis, but that these hormones also control involution. Another important regulatory role is played by growth factors that have been shown to modulate survival (epidermal growth factor, amphiregulin, transforming growth factor alpha, insulin like growth factor, and tumor necrosis factor alpha) or apoptosis (tumor necrosis factor alpha, transforming growth factor beta) of mammary cells. However, the molecular mechanism underlying the influence of sex steroid hormones and/or growth factors on the development and function of the mammary gland remains largely unknown to date. Also scarce is information on the interaction between both groups of modulators. Nevertheless, based on the current indications compiled in this review, an important functional role for sex steroid hormones in the lactation cycle in co-operation with growth factors can be suggested.     

Differences in the incorporation of thymidine into DNA of normal and cystic fibrosis fibroblasts in vitro.

Although similar fractions of cells were in the S phase of the cell cycle, normal human skin fibroblasts were shown to incorporate more than twice the 3HTdR into their DNA in vitro than did cells obtained from individuals with cystic fibrosis (CF). Obligate heterozygotes incorporated an intermediate amount of the DNA precursor. Studied were initiated to determine the basis of the differential incorporation of 3HTdR among the genotypes. An analog of thymidine, BUdR, produced varied effects on the growth kinetics of the three genotypes. The growth of cells in BUdR resulted in a 50% increase in the population doubling times of all three genotypes, and caused the cell morphology to change from a spindle shape to one in which the cells became broadened and flat, with numerous cytoplasmic projections extending for distances of several cell diameters. The activities of thymidine kinase and the participation of the exogenous and de novo pathways in the synthesis of TMP were found to be approximately the same in all three genotypes. The data suggest that an alteration in the transport of thymidine into the cells may account for the differences in TdR incorporation into DNA, and this may be associated with other changes in cystic fibrosis that are apparently membrane associated.     

Depot differences in steroid receptor expression in adipose tissue: possible role of the local steroid milieu

Sex hormones play an important role in adipose tissue metabolism by activating specific receptors that alter several steps of the lipolytic and lipogenic signal cascade in depot- and sex-dependent manners. However, studies focusing on steroid receptor status in adipose tissue are scarce. In the present study, we analyzed steroid content [testosterone (T), 17beta-estradiol (17beta-E2), and progesterone (P4)] and steroid receptor mRNA levels in different rat adipose tissue depots. As expected, T levels were higher in males than in females (P = 0.031), whereas the reverse trend was observed for P4 (P < 0.001). It is noteworthy that 17beta-E2 adipose tissue levels were higher in inguinal than in the rest of adipose tissues for both sexes, where no sex differences in 17beta-E2 tissue levels were noted (P = 0.010 for retroperitoneal, P = 0.005 for gonadal, P = 0.018 for mesenteric). Regarding steroid receptor levels, androgen (AR) and estrogen receptor (ER)alpha and ERbeta densities were more clearly dependent on adipose depot location than on sex, with visceral depots showing overall higher mRNA densities than their subcutaneous counterparts. Besides, expression of ERalpha predominated over ERbeta expression, and progesterone receptor (PR-B form and PR-A+B form) mRNAs were identically expressed regardless of anatomic depot and sex. In vitro studies in 3T3-L1 cells showed that 17beta-E2 increased ERalpha (P = 0.001) and AR expression (P = 0.001), indicating that estrogen can alter estrogenic and androgenic signaling in adipose tissue. The results highlighted in this study demonstrate important depot-dependent differences in the sensitivity of adipose tissues to sex hormones between visceral and subcutaneous depots that could be related to metabolic situations observed in response to sex hormones.     

Membrane function in cystic fibrosis. II. Methionine transport in normal and cystic fibrosis fibroblasts

Initial rate kinetics of methionine transport, time course of accumulation of methionine, and efflux of accumulated methionine were studied in three normal and four CF human diploid fibroblast strains. The range of apparent Km's was 12.7-32.1 micrometer for the CF strains and 18.3-39.2 micrometer for the normal strains. The range of apparent Vmax's was 6.69-9.22 nmole mg-1 min-1 for the CF strains and 5.59-7.87 nmole mg-1 min-1 for the normal strains. The patterns of accumulation and efflux are quite similar in all the strains studied except for WI-38, which showed somewhat higher efflux and lower accumulation than for others. There was no significant difference in the kinetic parameters of methionine transport between CF and normal skin fibroblasts, and methionine transport will not serve as a marker for cystic fibrosis in cultured fibroblasts.     

Taurine uptake by normal and cystic fibrosis fibroblasts

Taurine deficiency recently has been proposed to be clinically significant in cystic fibrosis (CF). Uptake of [14C]taurine by four cystic fibrosis (CF) and three control fibroblast lines was examined to determine whether a generalized defect in taurine transport could contribute to the deficiency. The time course of uptake was linear up to 20 h and was similar in both CF and control fibroblasts. Taurine was avidly retained after uptake, and the effect of metabolic (chlorpromazine) and competitive (hypotaurine, L-leucine) inhibitors was similar in both CF and control cells. In contrast, while taurine uptake in a calcium-free medium was impaired in both CF and control fibroblasts, the impairment was significantly less in CF cells. The findings suggest that a generalized abnormality in taurine transport is unlikely to be responsible for the taurine deficiency in CF.