Miillerian

Inhibiting Substance Blocks Epidermal Growth Factor Phosphorylation in Fetal Rat Lung Membranes

Receptor

Elizabeth A. Catlin, Neal D. Uitvlugt, Patricia K. Donahoe, David M. Powell, Masando Hayashi, and David T. MacLaughlin Neonatal males develop respiratory distress syndrome more frequently than females for unknown reasons. The fetal testis secretes testosterone and mfillerian 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 lg.5-day fetal rats and phosphorylation assays performed with 3 to 4 pg of membrane protein, with or without EGF (26 nmol/L), 0.025 mCi AFP (0.136 pmol/L), and either recombinant human MIS (rhMIS, 30 pmol) from media of Chinese hamster ovary (CHO) cells, rhMlS dialysis buffer, or wild-type CHO media. The 170,006 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 < 0.001; rhMl&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. lmmunoabsorption experiments using coincubation of rhMlS with anti-rhMIS IgG polyclonal antibodies or equiprotein normal IgG demonstrated MIS antibody-specific reversal of rhMlS activity in membrane phosphorylation. These observations in fetal rat lung membranes extend the previous data showing inhibitory effects by MIS in fetal lung, confirm findings in natural tissue previously confined to tumor cells (A431). and suggest that rhMlS signal transduction could be mediated via inhibition of EGF receptor autophosphorylation. Copyright 6 1991 by W.B. Saundars Company

R

ESPIRATORY distress syndrome (RDS) remains a major cause of death in premature neonates, despite recent advances in its treatment, such as the use of exogenous surfactants. Neonatal males develop RDS more frequently and with increased severity than females,112and while the etiology of this male disadvantage is unclear, it may be due to a male-specific factor. Beginning early in embryogenesis, the testis secretes two principle hormones directing male genital development, namely, testosterone and mtillerian inhibiting substance (MIS), a 140,000 molecular weight glycoprotein. MIS acts locally to cause involution of the embryonic epithelial-mesenchymal miillerian duct and, in addition, MIS is now known to be a circulating hormone with levels as high as 70 ng/mL in human neonatal males.3 Inhibitory effects on fetal lung maturation have been demonstrated with dihydrotestosterone exposure,“a5 thus, androgens may mediate the sexual dimorphism in pulmonary surfactant production. However, plasma levels of testosterone in the male and female rat fetus in the last several days of gestation, the window of male disadvantage in surfactant production in the fetal rat, are not significantly

From the Pediatric Surgical Research Laboratory and the Division of Neonatal and Pediatric Intensive Care, Massachusetts General Hospital, and the Departments of Pediatrics and Surgery, Harvard Medical School, Boston, MA; and the Department of Pathology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ. Supported in part by a Charles H. Hood Foundation Research Grant to EA. C. and by grants from the National Institutes of Health (No. CA17393) and American Cancer Society (No. PDT360) to P.K.D. Address reprint requests to Elizabeth A. Catlin, MD, Neonatal Intensive Care Unit, Massachusetts General Hospital, Boston, MA 02114. Copyright 0 1991 by WB. Saunders Company 0026~0495/91/4011-0011$03.00/0 117%

different,b and human fetal male testosterone levels decrease sharply in the last trimester of gestation so that male levels are only slightly higher than females at full term7.* when the male disadvantage in RDS compared with females increases significantly to 5:l. In contrast, MIS levels in male fetuses do not decrease late in pregnancy and, in fact, MIS at high levels is found for several years postnatally.3 Therefore, the effect of bovine MIS and recombinant human MIS (rhMIS) on fetal lung development were studied in cultured fetal rat lungs and were found, in picomolar to nanomolar concentrations, to inhibit lung maturation measured as disaturated phosphatidylcholine (DSPC) accumulation.’ More recently, rhMIS was shown to have significant inhibitory effects on the fetal lung in vivo,‘” similar to that measured in vitro. The present study was undertaken to investigate a potential mechanism whereby MIS might inhibit fetal lung development. Membrane phosphorylation has been proposed by virtue of the temporal relationship between increased phosphorylation and the observed effects as a key event in control of cellular growth, differentiation, and transformation by viruses.” Signal transductions by serine and threonine kinases are common events, whereas signaling via tyrosine kinases are less commonly observed.” Epidermal growth factor (EGF) is a 6,000 molecular weight polypeptide with membrane receptors on most mammalian cells,‘3 including the fetal lung.14 The EGF receptor has an external EGFbinding domain, a transmembrane domain, and a cytosolic tyrosine-specific kinase region. Autophosphorylation of the EGF receptor at specific tyrosine residues can be inhibited by bovine and rhMIS in a human cell line, A431, that over expresses EGF receptors with respect to normal cells.“~‘” While the role of EGF in lung development has not yet been clearly defined, in certain studies EGF can induce functional and morphologic maturation of fetal Iung’7-20; Metabolism, Vol40, No 11 (November), 1991:

pp 1178-l

184

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MIS AND FETAL LUNG

since MIS and EGF have been found to have antagonist

activities in the miillerian duct,” on oocyte meiosis,= and on cell and colony growth of reproductive tumor lines such as A431,” we hypothesized that the inhibitory effect of MIS on lung maturation might be correlated with inhibition of membrane phosphorylation of EGF receptors, and have provided an ultrastructural analysis of MIS-exposed fetal lung during these events to assess the effect of MIS on tissue morphology. MATERIALS

AND METHODS

Sprague-Dawley, Holtzman strain, timed-pregnant rat dams were obtained from Holtzman Laboratories, Madison, WI. Fetal gestational age was defined using the approximate time of maternal coitus during overnight caging as time 0; thus, when sperm was present in the morning vaginal smear this was considered day 0.5 of a total gestation time of 22.5 days. The experimental protocol was approved by our institutional Subcommittee on Animal Care. rhMIS was produced and purified in this laboratory (see below). Receptor-grade EGF was purchased from Collaborative Research, Lexington, MA. Normal rabbit IgG was obtained from Dakopatts, Denmark, and IgG-free horse serum from GIBCO, Grand Island, NY. Analytical-grade buffer reagents were obtained from Sigma Chemical, St Louis, MO, as was protein A Sepharose and electron microscopy (EM)-grade glutaraldehyde. [y’“P]ATP was purchased from the New England Nuclear Corporation, Billerica, MA. Kodak XRP-5 film (Eastman Kodak, Rochester, NY) was used in all autoradiograms. Molecular weight markers were obtained from Bio-Rad, Richmond, CA. Epon was purchased from Polysciences, Warrington, PA, and araldite from Ladd Research Industries, Burlington, VT. MIS Preparations rhMIS was purified from conditioned media of Chinese hamster ovary (CHO) cells transfected with a linear construct of the human MIS gene.= rhMIS, in media concentrated 20-fold, was isolated using ion exchange and dye-ligand affinity chromatography (called DG3 rhMIS),” to produce bioactive preparations of rhMIS with average enzyme-linked immunosorbent assay (ELISA)’ values of 161 kg/mL. rhMIS was also purified by immunaffinity chromatography (termed IAP rhMIS) using a mouse monoclonal antibody.‘” The conditioned media from wild-type nontransfected CHO cells were purified in an identical manner to produce control material lacking rhMIS. Biologic potency in each rhMIS preparation was verified using an established organ culture assay, which grades regression of the 14.5-day gestation rat urogenital ridge,% and by quantitation with an ELISA specific for human MIS.’ MIS Antibody Preparation and Preabsorption To generate polyclonal antisera in female New Zealand white rabbits, sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE)purified 140,000 molecular weight dimeric rhMIS (25 pg) was injected into popliteal lymph nodes with complete adjuvant and boosted 1 month later with incomplete Freund’s adjuvant (1:l). The IgG fraction (MGH-1) was purified from serum by (NH&SO, precipitation, dialyzed against phosphate-buffered saline, pH 8.2, and eluted from protein A Sepharose.3 Polyclonal antibodies were also raised against two peptides (MIS N-l and MIS C-l) synthesized by solid-phase peptide synthesis techniques and corresponding to amino acids no. 411-422 and 471-482 of intact human MIS, respectively, and purified as described above. These regions of MIS were chosen for antibody production as they are either amino terminal or carboxy terminal to a known monobasic cleavage site

(no. 427) in holo MIS, and highly conserved in bovine and human MIS.“To test that the measured effects on EGF receptor phosphorylation produced by rhMIS were specifically reversed by antibodies to rhMIS, rhMIS was incubated with a mixture of these antibodies or with normal rabbit IgG at 4°C for 24 hours (1:4 mole ratio of rhMIS:antibody). The antibodies were dialyzed overnight against 4 L of rhMIS buffer (20 mmol/L HEPES [4-(2-hydroxyethyl)-1-(piperazine ethane sulfonic acid)], 0.01% NP-40, pH 7.4) to a final protein concentration of 0.915 mg/mL prior to use. Antigen-antibody complexes were precipitated by the addition of protein A Sepharose beads, prewashed with ice-cold 20 mmol/L HEPES, 10% IgG-free horse serum in 20 mmol/L HEPES, and again with 20 mmol/L HEPES, pH 7.4. Following centrifugation of the protein A-antigen-antibody mixture, the supernatants were added to the usual fetal lung membrane phosphorylation assay. Membrane Preparation Cell plasma membranes were prepared from fresh lung tissue using a modification of the method described by Thorn et al.” Fetuses of 19.5 days’ gestation, from three to four litters per membrane preparation, were surgically delivered, fetal sex determined by direct inspection of gonads, and ltigs, trimmed of major vessels and connective tissue, were immediately placed in ice-cold calcium and magnesium-free harvesting solution (150 mmol/L NaCl, 20 mmol/L HEPES pH 7.4). All subsequent steps of the preparation were performed on ice or at 4°C. Pooled male or female lungs were weighed then homogenized in 40 vol (wt/vol) of fresh harvesting solulion using a brief (< 10 seconds) low-speed processing by a Polytron homogenizer with a model PTA 10 generator (Brinkmann Instruments, Westbury, NY). Homogenized lungs were pelleted at 500 x g for 5 minutes (1500 rpm, Damon/ IEC) and resuspended in 2 vol of harvesting solution. This resuspended pellet was added dropwise to 100 vol of extraction buffer (20 mmol/L boric acid, 0.2 mmol/L EDTA, pH 10.2) and stirred for 10 minutes. Following this hypotonic lysis of cells, cytoplasmic proteins were coagulated by the addition of 8 vol of borate solution (0.5 mol/L boric acid, pH 10.2) and 5 minutes of additional stirring. This solution was filtered through four layers of l-mm nylon mesh and cleared of particulate debris by centrifugation at 2,200 ‘pm for 5 minutes (Beckman 52-21). Crude membranes were collected by centrifuging the supernatant at 11,000 ‘pm for 30 minutes (Beckman 52-21). This pellet was suspended in 3 to 6 mL 20 mmol/L HEPES, pH 7.4, and gently layered onto 35% sucrose in Beckman polyallomer 14 x 89-mm centrifuge tubes. After a 60-minute centrifugation at 15,000 rpm (SW 41 rotor, Beckman L&55), the membrane-rich interface and upper HEPES layer were collected and recentrifuged at 15,000 ‘pm for 10 minutes (50.2 Ti rotor, Beckman U-55). The resulting pellet was suspended in 0.5 to 1.0 mL HEPES, pH 7.4, to give final membrane protein concentrations of 100 to 200 pg/mL, and stored at -70°C in lOO-p,L aliquots until use. Protein concentrations were measured using the method of Bradford.*’ Membrane Phosphovlation Assay EGF receptor autophosphorylation assays were performed using a method identical to one described for A431 membranes3’-” and used by our Laboratoty.‘S.‘6Lung membranes (3 to 4 pg of protein) were incubated in an ice bath with or without EGF (26 nmol/L) in a calcium-free solution containing 20 mmol/L HEPES, 0.75 mmol/L manganese chloride, and 0.0125% bovine serum albumin. After a lo-minute incubation, the phosphorylation reaction was initiated by the addition of 0.025 mCi [y’*P]ATP at a concentration of 0.136 kmol/L. rhMIS (30 pmol) was added before the AT-‘*Pto test its ability to inhibit the EGF-stimulated autophosphorylation of the

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CATLIN ET AL

EGF receptor. Controls consisted of final dialysis buffer in which the rhMIS was prepared (20 mmol/L HEPES, 0.01% NP-40, pH 7.4), or an equal amount of protein purified from wild-type non-MIS transfected CHO cell media. All reactions had a final volume of 62.5 pL. The phosphorylation reaction was terminated exactly 10 minutes after the radiolabeled ATP addition with 20 PL of Laemmli sample buffer?’ Samples were heated to 90°C for 10 minutes, and labeled proteins separated on 7.5% polyacrylamide gels by SDS gel electrophoresis under disulfide bond reducing conditions.3* The phosphorylated EGF receptor was detected by autoradiography showing a characteristic labeled band at 170,ooO molecular weight and was quantitated by Cerenkov counting in a beta scintillation counter after cutting the 170,000 molecular weight receptor band from dried gels. Electron Microscopy Fresh 21.5day gestation fetal rat control or rhMIS-treated lung tissue (left upper lobe) was first fixed in situ for 5 minute? then diced into l- to Z-mm cubes and fixed in 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer, pH 7.4, after 48 hours of in vivo exposure to lo-* mol/L rhMIS or vehicle buffer. The technique of transuterine subcutaneous fetal rat injection at 19.5 days’ gestation” used to treat the fetuses was recently validated,” and produces serum levels, as quantitated by ELISA, of rhMIS in injected fetuses of approximately 100% of injected hormone. Following 1 hour of fixation at room temperature, specimens were rinsed in three changes of 0.1 mol/L cacodylate buffer and post-fixed overnight in 1% osmium tetroxide in 0.1 mol/L cacodylate buffer. The following day, specimens were rinsed in two changes of 0.1 mol/L cacodylate buffer, pH 7.4, at 4°C. and two changes of 0.05 mol/L maleate, pH 7.4, then stained with 1.5% uranyl acetate in 0.05 mol/L maleate, pH 5.2, for 90 minutes at 4°C. Specimens were next rinsed three times in 0.05 mol/L maleate, pH 7.4, then dehydrated in a graded series of alcohols at 4°C and transferred to propylene oxide for 10 minutes. Sections were transferred to fresh Epon mixed with propylene oxide and dimethylaminomethyl phenol (DMP-30) for 2 hours, then placed in fresh Epon mixtures three more times before embedding at 60°C overnight in Epon with DMP-30. Thin sections were cut with a diamond knife on a Porter-Blum MT-2 ultramicrotome and photographed in a Philips 420 transmission electron microscope.

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Fig 1. inhibition of EGF receptor phosphorylation by rhMIS. Autoradiogram (4-hour exposure) of a 7.5% polyacrylamide gel showing stimulation of phosphorylatlon by EGF and inhibition by rhMlS in male lg.5-day fetal rat lung membranes. Lane 1: lung membrane without EGF or rhMlS (basal phosphorylation) with CHO wild-type medium; lane 2: lung membrane plus EGF (EGF-stimulated phosphorylatfon) and CHO wild-type medium; lane 3: lung membrane with EGF and rhMlS (DG3) showing rhMlS inhibition of EGF-stimulated phosphorylation. Molecular weight marker position is shown on the left.

phosphorylation assay, the EGF-stimulated EGF receptor phosphorylation band equaled 822.2 cpm and the band of rhMIS inhibition of EGF-stimulated phosphorylation equaled 361.4 cpm. The identical levels of EGF-augmented l

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0.6

Statistical Analysis Statistical analysis was performed on a VAX/VMS mainframe computer using CLINFO software (BBN, Cambridge, MA). ANOVA was used to test the null hypotheses and multiple comparison tests were then performed. P values less than .05 were regarded as significant. SEM was used to express the variability around the mean in the descriptive statistics.

0.4

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RESULTS

Female and male fetal rat lung membrane EGF receptor was consistently phosphorylated by EGF in vitro. The EGF-stimulated phosphorylation states (100% phosphorylation) in female membrane experiments, as well as in male membrane experiments, were significantly elevated above the nonstimulated states (P < .OOl for both male and female EGF-stimulated v basal phosphorylation, n = 4 and 5 experimental groups, respectively, v controls). There were no differences among male and female EGF-stimulated or basal membrane phosphorylation data (P = .42). rhMIS DG3 completely abolished the EGF-dependent EGF receptor autophosphorylation to levels not significantly different from basal, P < .OOl (Figs 1 and 2). In a representative

0 Female Fig 2. Phosphoylation of the 170,000 molacular weight EGF receptor in male and famale fetal rat lung membrane5 shown as fraction of EGF-stimulatad phosphorylation. The basal phosphorylation statas are shown as stlpplod bars, EGF stimulated male and female membrane5 are shown as black bars, and membranes incubated wfth rhMlS (DG3) addad in the presence of EGF are shown as white bars wfth SEM im. Each data point represents a minimum of four replications of the same sex-spscifk lung membrane preparations. lP < .OOl comparing basal phosphorylation states with EGF-stimulated states and comparing rhMfS treated with EGFtreated membranes; P = NS comparing male vemus female membrane effects of EGF and rhMIS.

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MIS AND FETAL LUNG

phosphotylation and rhMIS reduction of phosphorylation measured in the male and female lung plasma membranes permitted these to be combined for immunoabsorption experiments. The ability of rhMIS (DG3) to inhibit male and female lung membrane EGF receptor autophosphoiylation could in turn be specifically and reproducibly inhibited by rhMIS antibodies (Fig 3) compared with equiprotein normal rabbit IgG (n = 3). In a typical immunoabsorption experiment (Fig 4) normal IgG did not effect rhMIS inhibition of EGF receptor autophosphorylation, but equiprotein rhMIS antibody coincubated with rhMIS significantly reversed the rhMIS effect. When rhMIS was purified by immunoaffinity column purification, its inhibitory effect on fetal lung membrane phosphorylation was lost, duplicating the results of Cigarroa et al in A431 membranes.16 Electron micrographs of lung specimens from 21.5-day gestation male (n = 9) and female (n = 10) MIS-injected and vehicle buffer-injected rat fetuses showed lamellar bodies (LB) with characteristic morphology and similar tissue content of LB in all groups except for decreased tissue LB numbers in female MIS-exposed specimens. Secreted LB numbers in presumptive alveolar spaces were depressed in the male MIS specimens (Fig 5) and, in a different pattern, appeared to be increased in the alveolar spaces by MIS exposure in females. Other systematic effects associated with rhMIS exposure were not observed at magnifications of up to 12,500x, although only a small fraction of each whole lung was studied. DISCUSSION

The mechanisms directing cellular growth and differentiation in the fetus are far from understood. Our current understanding of fetal development, including the lung, supports complex levels of stimulatory, inhibitory, and The importance of inhibitory multifunctional contr01.5~35”8 factors in lung maturation is suggested by the precocious differentiation observed when human type II pneumocytes 100

60

Fig 3. Immunoabsorption of rhMlS activity in lung membranes by anti-rhMIS antibodies (n = 3, CSEM) shown as percent of EGFstimulated phosphorylation. Bar 1: EGF-stimulated phosphorylation of the 170,000 molecular weight receptor; bar 2: rhMlS (170 nmol/L DG3) added alone to stimulated membranes; bar 3: rhMlS (170 nmol/L DG3) coincubated with normal rabbit IgG; bar 4: rhMlS (170 nmol/L DG3) coincubated with equiprotein anti-rhMIS IgG antibodies.

are explanted.39 Androgens depress pulmonary surfactant production in several model systems,45@ and MIS, a wellknown regressor for the embryonic female urogenital system, has recently been shown to significantly inhibit fetal lung maturation both in vitro’ and in viva.“’ It is also noteworthy that testosterone has previously been shown to enhance MIS effects.4’ The present study demonstrates that rhMIS can suppress membrane phosphorylation of the EGF receptor in the male and female fetal rat lung in vitro and that this inhibitory effect can be specifically reversed by antibodies to rhMIS, raising the possibility that the inhibitory effect of MIS on fetal lung surfactant accumulation may be correlated with this signal transduction event. The inability of IAP rhMIS, except at high concentrations, to inhibit autophosphorylation of EGF receptors in membrane preparations has previously been observed.16 Speculations on the mechanism of IAP rhMIS inactivation include macromolecular aggregation of rhMIS due to its hydrophobic character, depletion of a necessary cofactor for rhMIS activity, incomplete cleavage of the glycoprotein, or configurational changing of rhMIS by the acid or concentrated chaotropic salt used for the IAP column elution. All of these possibilities are being rigorously examined. Morphologic study of MIS-exposed male and female lung suggests that MIS treatment does not induce the structural changes in lung, such as degeneration and phagocytosis of epithelial and mesenchymal cells and loss of basement membrane, that it does in the urogenital ridge,42 nor did it appear to have toxic effects. The smaller number of LB in alveolar spaces of MIS-treated males implies an MISassociated inhibition of lung maturation. Previous in vivo studies in fetal male lung suggested an MIS-mediated decrease in biochemical maturation as measured by elevated lung glycogen content with a small decrease in DSPC accumulation”; female lungs exposed to lo-* to 10e9 mol/L rhMIS showed markedly decreased DSPC content,“’ and the current observation of lesser LB numbers in female MIS exposed tissue is consistent with this, but quantitative stereological studies were not performed. The high numbers of LB in female MIS-treated alveolar spaces are difficult to reconcile. One speculation is that MIS exposure in females accelerates LB exocytosis, resulting in decreased LB tissue content, and decreased DSPC content,” and increased alveolar space LB. Since male lung samples did not show this, possibly a different mechanism of MIS action occurs in males and females, especially androgenic augmentation of MIS action:l or sex-specific EGF lung effects,” or prior exposure of males to endogenous MIS in vivo might alter MIS receptor affinity and/or numbers. The initial observation of antagonism between EGF and MIS was made in the urogenital ridge bioassay” and subsequent experiments have shown EGF/MIS antagonism in other whole cells and membrane preparations.‘5~‘6~*Z Inhibition of EGF receptor phosphorylation in fetal lung membranes by MIS was similar in male and female membranes, as were basal phosphorylation states initially. Since activation of EGF receptor tyrosine kinase by growth regulators is transient,” once male fetal tissue was removed from its hormonal milieu (MIS, etc) and processed to

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1

2

obtain the plasma membrane fraction, it is not apparent that persistent inhibition of EGF receptor phosphorylation by endogenous MIS would occur, thus explaining the equivalent initial male and female phosphorylation values. While the specific inhibitory effect of MIS on phosphorylation of the EGF receptor first observed in vulvar squamous carcinoma cells (A431) has now been observed in fetal lung membranes, it is not known precisely how MIS blocks the EGF receptor kinase activity, or specifically how the action of EGF at its receptor relates to the observed subsequent metabolic events such as increased colony formation in soft agar, oocyte meiosis, or aspects of fetal lung development.l’-m One possibility is that MIS may block ATP binding to the ATP site on the EGF receptor, or, MIS may reduce the affinity constant of EGF for its receptor, although the MIS effect is not reversed by increasing the

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4

Fig 4. EGF-stimulated phosphoryfetlon of tfte 170,ooO molecular welgftt EGF receptor in the presence of 30 nmd/L (lane 1) or 170 nmol/L rhBlfS (lsnee 2 to 4). end lntf-rhbBS antfbodles (lanes 1 and 4) or control rebblt IgG (lane 3). The anti-rhMfS antibodies block ths 170-nmol/L rfMS effect (lane 4) end do not alter the phosphoryletion of EGF receptor observed wlth a dose of rhMlS below the threshold of ectlvfty (lane 1). Control rabbit IgG did not reverse the rhMlS effect. Molecular weight marker position is shown on the left.

ATP concentration in vitro.16 Recent experiments crosslinking ‘zI-rhMIS to fetal rat lung plasma membranes (E.A. Catlin, in preparation) support that MIS and EGF act through different receptors and that molar excesses of EGF cannot displace 1251-rhMISfrom its high molecular weight binding species (molecular weight, - 240,000 when linked to reduced ‘“I-rhMIS). Evidence to date argues against MIS acting as a phosphatase itself or as an inducer of a phosphatase; however, these possibilities have not been completely ruled out and Grupusso et al have recently shown that transforming growth factor-l3 (TGF-S)-induced growth arrest in keratinocytes is associated with activation of two protein phosphatases.43 Protein kinase C is a serinelthreonine kinase that regulates function of EGF receptors, ie, EGF receptor autophosphorylation is decreased if the receptor is first

Flg 5. Elsctron micrographs of portions of lung from male fetal rats injected perenterellv with (A) vehicle buffer (controls), or (8) lo-* mol/L rhMIS. Orlginel megnlflcations: (A) x 8,250, bar = 2.42 pm, arrow lndketes LB In presumptive alveolar space; (B) x 12,250.

MIS AND FETAL LUNG

phosphorylated by protein kinase C (PKC).” We have taken steps to address the possibility that the MIS effect is mediated via PKC; since PKC is calcium-dependent, the lung membrane phosphorylation assays were performed with calcium-free reagents and the membranes prepared with calcium chelators. We have not been able to demonstrate a PKC effect with calcium stimulation and have seen no effect of phorbol esters. Phosphoamino analysis of both A431 whole cells and plasma membranes treated with EGF and MIS in this laboratory has shown inhibition of EGFstimulated tyrosine phosphorylation’6; thus, it seems unlikely but not impossible that the MIS depression of EGF phosphorylation could be mediated by PKC. Regulation of normal cellular proliferation and differentiation by the balancing of inhibitory, as well as stimulatory, growth factors is suggested by the existence of such factors and supported by demonstration of their growth-modifying capabilities in biologic systems. Examples of two such protein inhibitors include MIS, a natural inhibitor of EGF receptor tyrosine kinase,‘5,‘6 and a recently described phos-

phorylated glycoprotein product of rat hepatocytes, pp63, which in addition to suppressing hepatoma cell growth also inhibits insulin receptor tyrosine kinase.” The previous findings in fetal lung that physiologic concentrations of MIS can suppress biochemical maturation in vitro and in vivo the present ultrastructural findings, and the observation that MIS inhibitory activity in the fetal lung membrane can be measured at a level proximal to the receptor for epidermal growth factor, support the hypotheses that this fetal regressor might serve as a natural inhibitor of fetal lung development and that MIS signal transduction is potentially correlated with altered membrane and perhaps cytoplasmic or nuclear protein phosphorylation induced by EGF. Understanding the complete signal transduction pathway of MIS from extracellular lung to the cell nucleus, including complete characterization of the MIS receptor, may be key in understanding the human newborn male disadvantage in RDS. In addition, the lung provides a hitherto unavailable source for purification of nonneoplastic MIS receptor and cloning of its gene.

REFERENCES

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