Reduction of epidermal growth factor receptor phosphorylation by activated Mullerian inhibiting substance is vanadate-sensitive.

The carboxy-terminal domain of recombinant human Mullerian inhibiting substance (MIS) inhibits cellular proliferation in vitro and decreases epidermal growth factor (EGF)-dependent phosphorylation of the EGF receptor. Proteolytically cleaved and undissociated MIS is more potent than carboxy-terminal MIS alone, supporting a functional role for the amino-terminal region of the molecule. MIS does not block EGF binding to the EGF receptor, thus, MIS reduction of EGF receptor phosphorylation must occur distal to receptor ligand binding. The effect of proteolytically cleaved MIS on reduction of EGF receptor phosphorylation in membrane preparations is decreased by a specific phosphatase inhibitor, vanadate, thus implicating a membrane phosphatase in this MIS action at the EGF receptor.

Identification of a receptor for human müllerian inhibiting substance.

Müllerian inhibiting substance (MIS), a Sertoli cell-derived glycoprotein and member of the transforming growth factor-beta supergene family, plays a key down-stream role in mammalian sex determination. Identification of a receptor for MIS has now been achieved in a MIS-responsive human vulvar carcinoma cell line, A431, using fluorescein isothiocyanate labeling of recombinant human MIS (FITC-rhMIS) and RRAs with iodinated carboxy-terminal rhMIS. Confocal fluorescence microscopy of A431 cells incubated on ice with 30-nM concentrations of covalently bound FITC-rhMIS reveals specific punctate cell surface fluorescent signal. Cytosolic fluorescent signal is seen after incubation at 37 C for 1 h as well as occasional apparent perinuclear accumulation. FITC-rhMIS coincubated with molar excesses of unlabeled rhMIS in A431 cells eliminates cell surface and cytosolic fluorescent uptake. Double label experiments with FITC-rhMIS and tetramethyl rhodamine isothiocyanate epidermal growth factor establish separate binding of each ligand, displaceable, respectively, by large molar excesses of unlabeled rhMIS or epidermal growth factor. RRAs reveal a single, high affinity (Kd, 5.8 nM), saturable, low abundance binding species for carboxy-terminal rhMIS. Solubilized supernatants of A431 whole cells cross-linked with 125I-carboxy-terminal rhMIS identify a band with a mol wt of 88,000 on electrophoresis and autoradiography. This identification of a MIS receptor in A431 cells now permits the design of affinity purification protocols using rhMIS, followed by direct protein microsequencing.

Müllerian inhibiting substance: new perspectives and future directions.

MIS, as a differentiate and antiproliferative agent, is precisely regulated, for example, at the transcriptional level by such transacting factors as SRY, and posttranslationally by testosterone. Processing of MIS most likely requires an as yet unknown in vivo protease which probably serves to control cleavage of MIS and hence its activation at specific sites wherein a localized program of cell death is initiated via a receptor mediated event. Progress has been made in understanding the molecular domains of MIS; current efforts are focused on characterizing the wild type MIS receptor as well as cloning and expressing the MIS receptor. We need now to understand how to target and efficiently activate MIS at its projected site of action. We must focus, after structural analysis of its receptor, on elucidating the MIS initiated intracellular signals which result in localized cell inhibition. Understanding of these mechanisms will permit design of antitumor agents and therapeutic strategies. Similarly, understanding regulation of MIS expression may lead to therapeutic induction of expression in those states where depressed expression is associated with tumorigenesis, sexual ambiguity, or infertility.

Biochemical immaturity of lungs in congenital diaphragmatic hernia.

Neonates with congenital diaphragmatic hernia (CDH) continue to have unacceptably high mortality rates. To better understand the associated pulmonary pathology we measured biochemical parameters of lung maturity in neonatal rats with or without congenital diaphragmatic hernia created by maternal feeding of a single dose of nitrofen on day 9.5 or day 11.5 of gestation. Lungs from neonatal rats with large CDH (n = 9, 5 right-sided, 4 left-sided) had a significantly lower lung weight (P = .0001), lung weight/body weight ratio (P = .0001), disaturated phosphatidylcholine (DSPC) per microgram DNA (P < .005), total DSPC (P = .0001), total DNA (P < .05), protein per microgram DNA (P < .05), and total protein content (P < .005) when compared with lungs from the litter mates without congenital diaphragmatic hernia (n = 10). The lungs of rats with hernia also had significantly higher DNA concentrations (P < .05) and glycogen concentrations (P < .05). These data demonstrate that lungs in neonatal rats with large CDH are biochemically immature. Treatment directed toward correcting the pulmonary biochemical immaturity of affected fetuses before birth may improve the prognosis for these babies.

Identification of müllerian inhibiting substance specific binding in human cell lines.

The receptor for Müllerian Inhibiting Substance (MIS), a gonadal glycoprotein hormone, has not been previously identified. Plasma membranes from MIS-sensitive human tumor cell lines (HTB-111, endometrial carcinoma; and A-431, vulvar squamous carcinoma) were detergent extracted and incubated with 125I-labeled MIS anti-idiotypic antibody, or radioiodinated human recombinant MIS (125I rhMIS), with and without unlabeled competitors. 125I anti-idiotypic MIS antibody bound to HTB-111 membrane extracts was displaceable by unlabeled anti-idiotypic antibody, but not by anti-isotypic antibody prior to cross-linking. Specific binding of the anti-idiotypic MIS antibody to endometrial carcinoma cells was verified using fluorescence activated cell analysis and fluoresceinated antibody. Furthermore, unlabeled anti-idiotypic MIS antibody competed for 125I rhMIS binding to A-431 vulvar carcinoma membranes. The labeled anti-idiotypic MIS antibody binding complex could be separated from 32P labeled EGF receptor in the A-431 membranes, indicating that EGF, a natural inhibitor of MIS activity, and MIS itself bind to different receptors. These studies demonstrate a specific, displaceable binder for MIS in the plasmalemmae of two human tumor lines. Purification of this cell surface receptor protein will be greatly aided by using the MIS anti-idiotypic antibody.

Müllerian inhibiting substance blocks epidermal growth factor receptor phosphorylation in fetal rat lung membranes.

Neonatal males develop respiratory distress syndrome more frequently than females for unknown reasons. The fetal testis secretes testosterone and müllerian inhibiting substance (MIS); MIS has been shown to inhibit fetal lung maturation in vitro and in vivo and to block phosphorylation of epidermal growth factor (EGF) receptors in A431 cells. We hypothesized that MIS would also inhibit membrane phosphorylation of EGF receptors in fetal lung, and that ultrastructural study of MIS-exposed lung might complement the biochemical data by assessing the effect of MIS on tissue morphology. Lung membranes were prepared from 19.5-day fetal rats and phosphorylation assays performed with 3 to 4 micrograms of membrane protein, with or without EGF (26 nmol/L), 0.025 mCi AT32P (0.136 mumol/L), and either recombinant human MIS (rhMIS, 30 pmol) from media of Chinese hamster ovary (CHO) cells, rhMIS dialysis buffer, or wild-type CHO media. The 170,000 molecular weight EGF receptor, visualized by autoradiography of polyacrylamide gels, was phosphorylated in both female and male membranes. rhMIS, when added to EGF-stimulated membranes, caused significant inhibition of EGF receptor phosphorylation (females: 32.42% +/- 11.5%; males: 32.3% +/- 19.1%, P less than 0.001; rhMIS-treated v EGF-stimulated state, P = NS, male v female, Cerenkov counting). Electron microscopy (EM) of rhMIS-exposed lung showed decreased lamellar bodies (LB) in both male alveolar spaces and female parenchyma, and, unexpectedly, increased numbers in female alveoli. Immunoabsorption experiments using coincubation of rhMIS with anti-rhMIS IgG polyclonal antibodies or equiprotein normal IgG demonstrated MIS antibody-specific reversal of rhMIS activity in membrane phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)