Altered patterns of proteoglycan deposition during maturation of the fetal mouse lung

83

Cell Differentiation and Development, 32 (1990) 83-96 Elsevier Scientific Publishers Ireland, Ltd.

CELDIF

00698

Altered patterns

Candyce

’ Department

of proteoglycan deposition of the fetal mouse lung

I. Smith ‘3*, Earl H. Webster 2, Mark A. Nathanson and S. Robert Hilfer ’ of Biology, Temple University, Philadelphia, PA and ’ Department Newark, NJ, U.S.A.

during maturation

2, Robert

L. Searls ’

oj Anatomy, New Jersey Medical School,

(Accepted 30 July 1990)

Previous studies have shown that g-xyloside inhibits maturation of the fetal mouse lung (Smith et al., Dev. Biol. 138, 42-52, 1990). Insofar as this drug inhibits proteoglycan deposition, the present studies were undertaken to examine the chemical composition and tissue distribution of proteoglycans in order to determine, more precisely, their role during lung morphogenesis. Autoradiography of labeled 16- and 19-day embryonic lungs demonstrated greater incorporation over the mesenchyme. Treatment with p-xyloside did not alter the autoradiographic appearance; however, fi-xyloside treatment followed by nitrous acid digestion, eliminated most silver grains. Isolation of proteoglycans from extracellular, membrane and intracellular pools over the 16 to 19-day interval demonstrated redistribution of heparan sulfate proteoglycan from an intracellular to a membrane location, while chondroitin sulfate proteoglycan redistributed from intracellular to extracellular. Only the synthesis of chondroitin sulfate proteoglycan was inhibited by p-xyloside. On the basis of these results we suggest that a chondroitin sulfate proteoglycan is required for lung maturation and that inhibition of its synthesis results in inhibition of septa formation and subsequent failure of morphogenesis and differentiation. Chondroitin sulfate proteoglycan; Fetal lung maturation;

Introduction The pattern of terminal branching and differentiation of the respiratory epithelium is influenced by the mesenchyme with which the epithelial component is combined (Hilfer et al., 1985). * Present address: Environmental Medicine Institute, University of Pennsylvania, Philadelphia, PA 19104, U.S.A. Correspondence address: Dr. Robert L. Searls, Department of Biology, Temple University, Philadelphia, PA 19122, U.S.A. 0922-3371/90/$03.50

fi-Xyloside; Cell interaction, lung

Mesenchyme has also been shown to play an important role during early morphogenetic stages of the pancreas (Grobstein, 1962; Rutter et al., 1964; Wessells, 1968) and salivary gland (Bernfield and Wessells, 1970; Bernfield et al., 1973). At least part of this mesenchymal influence is exerted through secretion of macromolecules of the extracellular matrix. Inhibition of collagen deposition interferes with branching in both the lung and salivary gland (Alescio, 1973; Spooner and Faubion, 1980), while collagen deposition appears

0 1990 Elsevier Scientific Publishers Ireland, Ltd.

84

to stabilize branching (Wessells, 1970; Nakanishi et al., 1986a, b; Fukuda et al., 1988). Inhibition of proteoglycan synthesis also interferes with salivary branching (Thompson and Spooner, 1982, 1983; Spooner et al., 1985) and kidney tubule formation (Platt et al., 1987), and it has been suggested that one role of mesenchyme is proteoglycan degradation at the basal lamina of salivary bud tips where branching will occur (Banerjee et al., 1977; Bernfield and Banerjee, 1978, 1982; Smith and Bernfield, 1982). Proteoglycan synthesis may play a role in differentiation as well, since suppression of proteoglycan synthesis by P-xyloside stimulates haematopoiesis in bone marrow cultures (Spooncer et al., 1983) and casein production by mammary epithelial cells in culture (Parry et al., 1988). Previous studies in this laboratory have shown that morphogenesis of the respiratory epithelium is dependent upon proteoglycan synthesis. Treatment of immature respiratory endings with pxyloside, a drug that acts as an acceptor for glycosaminoglycan (GAG) synthesis at the expense of proteoglycan synthesis (Schwartz, 1977), prevents maturation of alveolar endings, inhibits differentiation of type II pneumocytes, and depresses synthesis of surfactant phospholipid (Smith et al., 1990a). The effects of P-xyloside on lung maturation are very similar to the effects of heterologous mesenchyme (Hilfer et al., 1985) or to the effects of culture without corticosteroids (Smith et al., 1990b). In the present study, we have continued to investigate proteoglycan synthesis in order to answer the following questions: (a) which glycosaminoglycans are synthesized during maturation of the lung; (b) what is their normal distribution within the tissue; and (c) what are the effects of /3-xyloside on the types of GAG and their distribution? In combination with our previous observation that P-xyloside also inhibits maturation of the respiratory epithelium, the present data suggest that fl-xyloside inhibited synthesis of a chondroitin sulfate proteoglycan that was most likely in an extracellular location, synthesized by mesenchyme, and required for maturation of the lung. We hypothesize that agents which enhance morphogenesis of the fetal respiratory mesenchyme act to enhance the synthesis of a chondroitin sulfate proteoglycan.

Materials and Methods Swiss-Webster mice were purchased from the Institute for Cancer Research, Philadelphia, PA, and Taconic Farms, Germantown, NY. Tissue culture medium and supplemental components were from GIBCO, Grand Island, NY. Organ culture dishes were purchased from Falcon Plastics, Oxnard, CA. Polycarbonate membrane filters were from the Nuclepore Corp., Pleasanton, CA. Isotopes [6- 3H]glucosamine, 35S0,Na *, and fluorography spray (EN3HANCE) were purchased from New England Nuclear, Boston, MA. Guanidinium-HCl, Triton X-100, r-aminocaproic acid, benzamidine-HCl, phenylmethylsulfonyl fluoride, chondroitin sulfate (mixed isomers from whale and shark cartilage), ninhydrin reagent, and BSA (fraction VI) were purchased from the Sigma Chemical Co., St. Louis, MO. Trypsin was purchased from Difco, Detroit, MI. p-nitrophenyl-/_?-D-xylopyranoside and p-nitrophenyl-a-Dxylopyranoside were from Koch-Light Laboratories, Colnbrook, U.K. Pronase (protease from Streptomyces griesus), soybean trypsin inhibitor, and Zwittergent 3-12 were from Calbiochem, La Jolla, CA. DEAE-Sephacel was from Pharmacia, Uppsala, Sweden. Chondroitinases ABC and ACII, and purified disaccharide standards, were purchased from Seikagaku Kogyo Ltd. via ICN Immunobiologicals, Lisle, IL. Cellulose thin-layer chromatography plates (without indicator), XAR-5 X-ray film, NTB-3 autoradiography emulsion and photographic processing chemicals were from the Eastman Kodak Co., Rochester, NY, with the exception of Rodinal film developer from AgfaGaevart, Teterboro, NJ. Scintillation cocktail (Ecoscint) and tissue solubilizer (Solusol) were purchased from National Diagnostics, Manville, NJ. Deionized water was 18 MO/cm from a MilliQ system by Millipore, Corp., Boston, MA. All other components were reagent grade. Preparation of cultures Pregnant mice were killed by cervical dislocation. Fetuses at 16 and 19 days of gestation were removed under aseptic conditions and the lungs isolated into culture medium. The thin outer margins from all five lobes, which contained

85

terminal branches of the respiratory tree, were removed using sharpened tungsten needles as described previously (Smith et al., 1990b). Respiratory regions were prepared in the same manner from lungs of newborn and adult mice. Experimental and control cultures always consisted of equal numbers of tissue strips, containing approximately the same number of terminal branches. Tissue strips were cultured in organ culture dishes, on sterilized polycarbonate membranes, at the air/medium interface. The medium consisted of serum-free, modified F-12 culture medium (Ambesi-Impiombato et al., 1980) containing dexamethasone, thyroxine, growth factors and antibiotics (Hilfer et al., 1986). Depending on the experiment, culture medium also contained 0.5 mM p-nitrophenyl-cr-D-xylopyranoside (a-xyloside), or 0.5 mM p-nitrophenyl-/3-D-xylopyranoside (P-xyloside) and either 5 pCi/ml [35S]sulfate or 10 pCi/ml [3H]glucosamine. For autoradiographic studies that did not involve xyloside treatment, the tissue was labeled in vitro for 2 h. Xylosides were added to the culture medium 1 h before addition of radioactive precursor, followed by culture for an additional 18 h in the presence of both the precursor and xyloside. Autoradiography Tissue strips were labeled in vitro as described above. Fetal lungs were labeled in vivo for 2 h by an intraperitoneal injection into the mother of 250 PCi of either [ 35S]sulfate or [ 3H]glucosamine. After labeling, the tissue was rinsed three times in unlabeled culture medium to remove unincorporated radiolabel and fixed in 2% glutaraldehyde in Tyrode’s solution for 50 min. Tissues were dehydrated in a graded series of alcohols, embedded in paraffin, and sectioned at 5 pm. Sections were deparaffinized and rehydrated through a graded alcohol series. Slides from an individual experiment were treated with nitrous acid at room temperature for 90 min to digest heparin and heparan sulfate, treated with 0.05 units of chondroitinase ABC (see below) to digest chondroitin and dermatan sulfates or kept in buffer as a control. All slides were rinsed thoroughly with distilled water and dipped in Kodak NTB-3 emulsion that was diluted 1 : 1 with distilled water prior to use. Di-

pped slides were air-dried for 20 min and stored in light-tight boxes at 4°C. Following an exposure period that was held constant for all slides, the emulsion was developed for 4 min in Rodinal (diluted 1 : 30 with distilled water) and fixed in Kodak Rapid Fix for 4 min. Slides were stained with Baker’s hematein-alum (Searls, 1967). Sections were photographed on a Leitz ortholux II microscope using epi-illumination and a polarizing cube. Proteoglycan extraction Respiratory regions were cultured in the presence of [35S]sulfate for 18 h, and washed three times with fresh culture medium to remove excess isotope. Proteoglycan was extracted by two methods (Vogel and Peterson, 1981). In the first, total proteoglycan was isolated with a mixture of Zwittergent 3-12 and guanidinium-HCl. The second method consisted of sequential extraction with trypsin, Zwittergent 3-12 and guanidinium-HCl to remove cell surface, membrane-bound and residual intracellular material, respectively. Throughout these procedures, supernatants of digested material were prepared by centrifugation at 12000 x g for 10 min at 4°C. Dialysis to remove unincorporated isotope was carried out in 4 liter volumes, at 4°C overnight against 10 mM sodium sulfate and for the following 24 h against four changes of deionized water. Dialysis solutions contained protease inhibitors as described below. Liquid scintillation counting was performed using 10 ml of cocktail in a Beckman LS-8000 counter, programmed to correct for quench and to calculate DPM. Total proteoglycan was extracted at room temperature for 30 min in buffer A (50 mM sodium acetate, pH 5.8, 0.1 M &-aminocaproic acid, 5.0 mM benzamidine-HCl, 10.0 mM disodium-EDTA, 1.0 mM phenylmethylsulfonyl fluoride) containing 4.0% Zwittergent 3-12. An equal volume of buffer A containing 8.0 M guanidinium-HCl was then added and extraction was continued overnight at 4°C on an orbital shaker oscillating at 150 RPM. Following centrifugation, supernates were collected, dialyzed to remove unincorporated isotope and lyophilized to dryness. Pelleted material was resuspended in 10% trichloroacetic acid, held

86

overnight at 4°C and recovered by centrifugation. TCA-washed pellets were counted by liquid scintillation counting following dissolution in Solusol following the manufacturer’s instructions and were found to contain less than 5% of the total nondialyzable activity. For the sequential procedure, tissues were initially extracted with 0.1% (w/v) trypsin in Hanks’ saline for 15 min at 37°C. Trypsin was then neutralized by the addition of soybean trypsin inhibitor to a final concentration of 4.5 mg/ml. Supernates were recovered following centrifugation and diluted with an equal volume of 8.0 M guanidinium-HCl containing the protease inhibitors described above. Pellets were resuspended in buffer A containing 4.0% Zwittergent 3-12 and extracted at room temperature for 30 min. Centrifugation again yielded supernates that received 8.0 M guanidinium-HCl and pellets that were dispersed in 4.0 M guanidinium-HCl. The final extracts were performed at 4“C with shaking (as above) for 18 h. Centrifugation yielded a third extract (supernate) and a pellet that was solubilized and counted (see above). Supernates were dialyzed and lyophilized to dryness. Glycosaminoglycan analysis To quantitate the incorporation of isotope per unit protein, strips of lung tissue that were fixed as above for autoradiography were placed into scintillation vials and counted. Following gross removal of most of the fluor, the remainder was evaporated at 60°C. Each vial then received 150 ~1 of 4.0 M NaOH, was recapped, and heated in a boiling water bath for 2 h to hydrolyze the tissue. Samples were neutralized with glacial acetic acid and reacted with ninhydrin reagent according to the method of Schwartz et al. (1985), using BSA as a standard. Lyophilized extracts were dissolved in 50 mM sodium acetate, pH 6.0, containing 8.0 M urea, 0.5% (v/v) Triton X-100, and 0.15 M NaCl (solution B) and applied to a 1 X 5 cm column of DEAE-Sephacel equilibrated with solution B. Samples were washed into the column with 5 ml of solution B and eluted with this solution containing a linear salt gradient of 0.15-1.5 M NaCl, at a flow rate of 2.5ml/h. One ml fractions were

collected and analyzed by liquid scintillation counting. Material eluting in peaks was dialyzed overnight against 4 liters of deionized water and lyophilized to dryness. The presence of heparan sulfate was determined by digestion with nitrous acid (Kosher and Searls, 1973). Each sample received equal volumes of 50% sodium nitrite and 33% acetic acid. Digestion was performed at room temperature for 30 min. Each sample then received 250 pg of carrier chondroitin sulfate, and undegraded material was precipitated with 3 vol. of absolute ethanol containing 1.33% potassium acetate. Following centrifugation, a portion of each supernate and pellet (dissolved in the Pronase buffer described below) was counted to determine the percent of the sample that was nitrous acid sensitive. Pellets were digested with Pronase to liberate GAG chains from proteoglycan; pellets were dissolved in 0.2 M Tris-HCl, pH 8.0, l/5 vol. of pronase (8.0 mg/ml stock in 0.2 M Tris-HCl, pH 8.0, predigested by the method of De la Haba and Holtzer, 1965) were added, and digestion performed at 50°C for 30 min. Buffer pH was adjusted at 50°C. Pronase digests were dialyzed against four changes of deionized water, at 4°C for 48 h and lyophilized to dryness. Pronase-digested samples were dissolved in deionized water and half of each received an equal volume of 0.1 M Tris-HCl containing 0.1 M NaCl at either pH 8.0 (for chondroitinase ABC) or pH 7.3 (for chondroitinase AC-II), respectively (Nathanson and Hay, 1980b). Digestion was carried out at 37°C for 40 min using 0.05 units of Chondroitinase ABC and 0.25 units of chondroitinase AC-II. Enzyme was then re-added at its initial concentration, and digestion continued for a total of 120 min. Digested samples and standard disaccharides were spotted onto thin-layer chromatography plates, desalted in N-butanol/absolute ethanol/ water (52 : 32 : 16) for 16 h, dried for 30 min, and developed in N-butanol/glacial acetic acid/2.0 N ammonium hydroxide (40 : 60 : 20) for 8 h at room temperature. Dissacharide digestion products were localized by spraying the plates with fluor and exposing them to X-ray film at -7O’C. Spots containing radioactivity migrating at the level of standard dissacharides were scraped into scintilla-

87

tion vials, eluted with 0.1 N HCl overnight, counted by liquid scintillation.

and

Results Autoradiographic localization of proteoglycan Lung tissue from 16- and 19-day fetuses was pulse-labeled for 2 h in culture medium containing [“Slsulfate in order to investigate by autoradiography the spatial distribution of sulfate-labeled macromolecules, presumably proteoglycan. Upon inspection of the autoradiographs, silver grains were detected over mesenchyme (Figs. 1 and 2, arrows), but to a lesser extent over the epithelium (Figs. 1 and 2, E). Silver grains were detected over both the cells and the extracellular space, and it was impossible to discern localization over one or the other component. Similarly, it was impossible to discern differential labeling at the position of the basal lamina. Thus, sulfated components of the ECM appeared to be uniformly distributed throughout these stages of lung development. The pattern of deposition during maturation of the lung in utero was determined by injecting [ 35S]sulfate into the peritoneal cavity of the mother as a control for the pattern in vitro. Labeling at 16 or 19 days of gestation, at 3 days postnatal, or in the adult, produced the same distribution of silver grains over mesenchyme and epithelium as in vitro. Labeling in utero was a great deal less intense than in vitro, perhaps being limited in the mother by rapid excretion of sulfate and, in the fetus, by poor circulation to the lung. [3H]glucosamine was used as an alternate label for glycoconjugates. It was recognized that not only proteoglycans, but also glycoproteins, would be labeled and that glucosamine would perhaps be converted into other metabolites as well, but the glucosamine would not be so readily excreted. The pattern of [3H]%lucosamine incorporation was similar to that of [ 35S]sulfate in 16- and 1Pday lungs in utero and in vitro (data not shown). Previous experiments demonstrated that /3xyloside interfered with normal maturation of the lung and decreased the incorporation of labeled sulfate into nondialysable material (Smith et al., 1990a). In an attempt to localize specific deposi-

tion sites of chondroitin sulfate and heparan sulfate proteoglycans, cultures of 16-day respiratory tissue either were not treated or were cultured in the presence of (Y- or /3-xyloside, labeled with [ 35S]sulfate, and digested with either chondroitinase ABC or nitrous acid, followed by autoradiography. Differential localization was not evident in autoradiographs of untreated or a-xylosidetreated tissues, digested with either chondroitinase ABC or nitrous acid. Major decreases in number of silver grains was also not evident either by visual inspection or image analysis. The same result was obtained following chondroitinase digestion after culture in P-xyloside (Fig. 3). However, sections from lungs cultured in the presence of /%xyloside that were subsequently digested with nitrous acid had almost no silver grains at all (Fig. 4, compare with Figs. 1 and 3). Similar experiments using 1Pday fetal tissue gave identical results. The percentage decrease of silver grains that resulted from culture with /3-xyloside, or by digestion with chondroitinase ABC or nitrous acid, could not be accurately determined by counting manually or by image analysis. Removal of almost all of the silver grains by the combination of culture in P-xyloside (which inhibits deposition of chondroitin sulfate proteoglycan) and digestion with nitrous acid (which eliminates heparan sulfate) indicated that the 16-day tissue contained substantial amounts of both chondroitin sulfate and heparan sulfate proteoglycans, but neither was localized to specific regions within the tissue. Based upon experiments with early salivary branching (Bernfield and Banerjee, 1982; Smith and Bernfield, 1982), it seemed possible that synthesis of proteoglycan might be uniform, but that local degradation in one region might produce a relative accumulation in another. Turnover of proteoglycan was investigated by culturing respiratory regions from fetuses of 16 and 19 days gestation, and 3 days postnatal, for 4 h in the presence of [35S]sulfate, washing three times in medium lacking radioactive precursor, and culturing in medium without label (pulse-chase). Cultures were fixed after 4, 18, and 42 h (0, 14, and 38 h chase) and autoradiographs were prepared. The tissue demonstrated a progressive decrease in the number of silver grains, but did not show a more

Figs. 1 and 2. Autoradiography Fig. 2, 19 days gestation.

of mouse lung respiratory tissue cultured 2 h in the presence of [“Slsulfate. Fig. I. 16 days gesta Ition. Photographed in: a, bright light, and b, darkfield. E, epithelium; arrows indicate mesenchyme.

89

Figs. 3 and 4. Autoradiography

of mouse lung respiratory tissue cultured as in Figs. 1 and 2, but in the presence control; Fig. 4. digested with nitrous acid.

of fi-xyloside.

Fig. 3.

90 TABLE I Turnover of [35S]]sulfate-labeled development Developmental

age

proteoglycan

during fetal lung

Chase period (h)

CPM//% protein

16-day embryonic

0 48

132.7 f 6.0 16.1 f 1.5

19-day embryonic

0 48

63.6 f 1.8 10.6 f 0.7

3-day postnatal

0 48

87.1 k 4.9 19.6 f 3.6

Strips of marginal lung tissue were pulsed with [35S]sulfate for 4 h, followed by a 48-h chase. Samples from the same culture dish were collected after the pulse and at the end of the chase, and total bound radioactivity was measured. Data are expressed as a mean+one standard deviation for three experiments that were performed at the same time, with the same culture medium and [ 35S]sulfate.

rapid metabolism of labeled material in any particular region; the silver grains were still evenly distributed (data not shown). To quantitate proteoglycan turnover, tissue strips from 16- and 1% day fetal and 3-day postnatal lungs that had been cultured for 4 h in medium containing [ 35S]sulfate were washed three times in medium without label and counted. Other tissue strips in the same dishes were similarly washed, cultured for 48 h in medium without label, and counted (Table I). The amount of radioactivity in the tissue after the chase was less than 20% of that estimated to be present at the beginning of the chase period at 16-19 days (22.5% at 3 days postnatal). Data from experiments described thus far demonstrated that: (a) the sulfate label was predominantly in the mesenchyme, both intracellularly and extracellularly, although some was also present in the epithelium; (b) there was rapid turnover of sulfate-labeled material with no change in the pattern of labeling; and (c) culture with P-xyloside or digestion with chondroitinase ABC or with nitrous acid produced little change in the distribution of the silver grains. However, a combination of culture with Pxyloside and digestion with nitrous acid removed almost all of the radioactivity.

Glycosaminoglycan analysis Extraction and analysis of proteoglycans was performed in order to obtain quantitative data. A sequential extraction protocol involving trypsin, Zwittergent 3-12, and guanidinium-HCl was used as described in Materials and Methods. Dialyzed extracts were applied to columns of DEAE-Sephacel (Fig. 5). Unbound material was found to comprise less than 10% of the DPM loaded to each column and contained less than 1% nitrous acid-sensitive material; sufficient label did not remain to permit further analysis. Bound material eluted as a single major peak, at approximately 0.35-0.4 M NaCl, irrespective of its source. Material from each peak was recovered, an aliquot was counted, and the remainder digested with nitrous acid. Undegraded material was precipitated with potassium acetate and ethanol. Radioactivity remaining in the supernatant represented nitrous acid-sensitive material (heparin and heparan sulfate) (HS; Tables II-V). Pellets remaining after nitrous acid digestion were digested with pronase to degrade core protein and divided into two aliquots. One aliquot was digested with chondroitinase ABC and the other with chondroitinase AC-II. Digestion products were separated by thin-layer chromatography, detected by fluorography, recovered and counted. Radioactive spots were detected at the origin (undigested material) (UD; Tables II-V). Above the origin, two heavily labeled spots were present that represented chondroitin 4-sulfate (4-S) and chondroitin 6-sulfate (6-S). The difference in the radioactivity between the chondroitin 4-sulfate spot obtained with chondroitinase ABC and chondoitinase AC11 was considered to represent dermatan sulfate (DS; Tables II-V). The experiment was performed twice at 16 days gestation and twice at 19 days gestation. Results are expressed as the percent of the total GAG isolated at each day of gestation (Tables II and III). The data demonstrated that the amount of undigestible material, heparan sulfate, chondroitin 6-sulfate, chondroitin 4-sulfate, and dermatan sulfate, remained roughly constant between 16 days and 19 days (Tables II and III, row labeled ‘sum’). the composition of each pool however, changed quite markedly. Whereas the three pools contained roughly equivalent proportions of

91

DPM at 16 days (Table II, column labeled total), trypsin-sensitive DPM increased at 19 days while material extracted with guanidine decreased (Table III).

40

I

.

irypsin 30

-

20

-

10

-

‘I

.

.*“\_

I -\--oL=-• 300

I,.iI 5.

‘\.. %.....

,

, 0.8 Trypsin-Zwitt

/

,

I

BOUND

,’

0

-0.6

t

O

1: 0.2

1

0.0

80

(

,

Fraction

Fig. 5. Fractionation of [35S]sulfate-labeled material on DEAE-Sephacel. Proteoglycan, extracted sequentially with trypsin, Zwittergent 3-12, and 4.0 M guanidinium-HCl was initially fractionated by ion-exchange chromatography. Both 16- and 19-day extracts chromatographed similarly; the figure shows a representative profile from a 19-day extract. Each extract resolved as two pools. The first pool eluted as unbound material while the second &ted as bound material at an ionic strength of approximately 0.4 M NaCI. Insufficient dpm were recovered in the unbound pools to permit further analysis. Bound material was recovered and analyzed for its glycosaminoglycan composition.

Chondroitinase-sensitive proteoglycan (6-S + 4S + DS) that could be extracted with trypsin (extracellular) was found to increase from 18 to 28% of the total chondroitin sulfate between 16 and 19 days, while the guanidine-extractable pool decreased from 16 to 3%. Nitrous acid-sensitive proteoglycan differed in that the Zwittergent 3-12 and guanidine-HCl-extractable pools were found to have altered compositions. The Zwittergent-extractable pool increased by 14% at 19 days, whereas the guanidine-extractable pool decreased by an identical amount. The effect of P-xyloside on the pattern of GAG synthesis by respiratory regions from mice of 16 and 19 days gestation was also determined. For each experiment, respiratory strips were labeled in vitro in either control medium or medium containing a-xyloside or /3-xyloside. After 18-h culture, total proteoglycan was extracted from the tissue layer and medium and analyzed for its glycosaminoglycan content. The experiment was done three times at 16 days gestation and once at 19 days. The results obtained in each experiment were calculated as a percentage of the total GAG in the medium plus tissue of control cultures not containing xyloside. The percentages from the different experiments at 16 days were averaged (Table IV). The results indicated that /3-xyloside treatment of 16-day cultures caused a 50% inhibition of incorporation into the tissue and a 7-fold stimulation of incorporation into the medium (Table IV, column labeled ‘total’). At 19 days of gestation, P-xyloside elicited a slight stimulation of incorporation into the tissue and a 12-fold stimulation of incorporation into the medium (Table V, column labeled ‘total’). In the salivary gland, P-xyloside caused no inhibition of sulfate uptake into the tissue layer, but roughly half of the sulfated material in the tissue layer was determined to be GAG-xyloside (Thompson and Spooner, 1982). A similar effect may have occured in the lung at 19 days of gestation. a-Xyloside caused very little change in the incorporation into either the tissue or the medium at either 16 or 19 days of gestation. At 16 and 19 days of gestation, /3-xyloside caused essentially no change in the incorporation into nitrous acid-sensitive material (HS). Almost

92 TABLE

II

GIycosaminogIycan

composition

of 16-day

respiratory

tissue

Extract

HS

UD

6-S

4-S

DS

Total

Trypsin Zwittergent Guanidine Sum

17+2.9 8f0.7 16*0.7 41+ 4.2

4+3.5 10*0.7 3kO.2 17*4.4

s*o.1 4*0.7 11+0.7 23k1.4

8kO.7 3*0.7 2*1.5 13 f 2.9

2*0.5 1+0.8 3+1.4 6+2.8

39 + 4.2 26k4.2 35 f 0.7 100

Data are expressed as the mean percent of two experiments f one standard deviation. 6-S, chondroitin 6-sulfate; 4-S chondroitin Csulfate; DS, dermatan sulfate.

all of the change was due to incorporation into undigested material and into the chondroitin sulfates. At 16 days gestation, P-xyloside caused a 70% decrease in chondroitinase ABC-sensitive material and a roughly 50% decrease in indigestible material in the tissue; there was an S-fold increase in chondroitinase ABC-sensitive material and a more than 4-fold increase in indigestible

TABLE

material;

HS, heparan

sulfate;

material in the medium. The effect may have been greater than this, because residual GAG-xyloside may have remained in the tissue layer. The results from biochemical analysis support and extend the results from autoradiography. The biochemical analysis indicates that: (a) chondroitinase ABCsensitive proteoglycan is predominantly extracellular, particularly at 19 days gestation; (b) chondroi-

III

Glycosaminoglycan

composition

of 19-day

respiratory

tissue

Extract

HS

UD

6-S

4-S

Trypsin Zwittergent Guanidinium Sum

16+1.4 22 f 2.1 250.7 4Ok4.2

7k4.2 llf2.8 1*0.7 19k7.8

8 f 1.4 5f1.4 2kO.7 15*3.5

12 4 0.5 16

Data are expressed

TABLE

UD, undigested

as the mean percent

of two experiments

+ one standard

DS *1.4 *2.1 + 0.1 k3.7

deviation.

Total

8 1 0.6 10 Abbreviations

f2.8 *0.7 f 0.2 *I.7

51 f8.5 43*3.5 6*1.4 100

as in the legend

to Table II.

IV

Glycosaminoglycan composition of control normalized to that of the control

and xyloside

treated

cultures

of 16-day

respiratory

tissue expressed

Total

Fraction

HS

UD

6-S

4-s

Conrrol Tissue Medium

12k1.4 2+0.7

18k1.4 5 f 2.1

18_+ 2.1 lo+ 0.7

15+ 6+

0.7 1.5

9il.4 4*1.4

72 27

a -Xyloside Tissue Medium

11*3.0 2*1.2

2Ok5.8 5 + 1.0

14* 7+

13* 55

3.1 0.6

9f2.3 2kO.6

67 21

/I -Xyloside Tissue Medium

11 f 3.0 5*1.5

9k2.1 23 + 5.6

6k 1.4 63k16

2*1.0 34 f 8.5

34 196

Data expressed

as the mean f one standard

deviation

3.5 1.5

of three experiments.

DS

as a percentage

6k 1.1 71*11 Abbreviations

as in the legend

to Table II.

and

93

TABLE V Glycosaminoglycan composition of control and xyloside treated cultures of 19-day respiratory tissue expressed as a percentage and normalized to that of the control 6-S

4-S

12 20

18 9

10 3

16 15

16 9

8 58

Fraction

HS

UD

Corm01 Tissue Medium

12 1

a -Xyloside Tissue Medium t9 -Xyloside Tissue Medium

DS

Total

17 6

3 2

62 38

13 9

11 7

4 2

54 36

24 157

15 162

7 77

70 463

Data are derived from a single experiment. Abbreviations as

in

the legend to Table II.

tinase ABC-sensitive material and nitrous acidsensitive material were each about 40% of the total GAG, explaining why the effect of digestion was not easily perceived in the autoradiographs; and (c) at 16 days gestation, p-xyloside caused at least a 70% inhibition of synthesis of chondroitinase ABC-sensitive proteoglycan, but no detectable inhibition of synthesis of nitrous acid-sensitive material.

Discussion Maturation of the respiratory epithelium from fetal mouse lung requires that it be combined with respiratory mesenchyme (Hilfer et al., 1985). Mesenchyme may be involved in several ways, but there is reason to believe that its influence is mediated in part through an effect on proteoglycan synthesis. Addition of P-xyloside to the culture medium severely retards differentiation of the respiratory region of 16-day lungs (Smith et al., 1990a). However, P-xyloside causes no toxicity recognizable in transmission electron micrographs, does not change the rate of DNA accumulation (Smith et al., 1990a), and does not affect the rate of protein synthesis (unpublished observations). In the present experiments, we have found that /3-xyloside inhibits the deposition of chondroitin

sulfate proteoglycan into the tissue, and to a lesser extent material that remains undigestible by both nitrous acid and chondroitinase ABC. However, autoradiographs of sections digested with nitrous acid (to reveal the distribution of chondroitin sulfates) do not indicate that these proteoglycans are differentially localized to a particular region of the tissue. Silver grains are observed over cells and extracellular spaces of the mesenchyme, and to a lesser extent over epithelium. Autoradiographs prepared after a pulse-chase with [35S]sulfate did not supply any evidence for differential metabolism of chondroitin sulfates, either at the epithelial/mesenchymal interface or elsewhere in the tissue. Much of the chondroitin sulfates must be in the extracellular space because much of it can be extracted by digestion with trypsin, and the amount of chondroitin sulfates that can be extracted with trypsin increases between 16 and 19 days. The structure of lung connective tissue undergoes a striking change between 16 days of gestation (pseudoglandular stage) and birth at 20 days (saccular stage). At the pseudoglandular stage, mesenchyme forms dense, cellular partitions between terminal epithelial branches (Ten HaveOpbroek, 1979; Hilfer, 1983). The extracellular space is minimal and contains little fibrillar or granular material. The basal lamina forms a thin layer proximally and is discontinuous at the distal tips, especially where branching occurs (Bluemink et al., 1976; Grant et al., 1983). During progression to the saccular stage, the mesenchyme is converted to thin septal partitions containing few fibroblasts and a thin layer of fibrillar extracellular matrix adjacent to a thin, continuous basal lamina. This change in organization must reflect differentiation of the mesenchyme in addition to differentiation of the epithelium. However, mesenchymal differentiation is difficult to appraise. It may be that the synthesis of a specific extracellular proteoglycan is an essential component of mesenchymal differentiation, and that synthesis of this proteoglycan is required for formation of thin septal partitions. Deposition of such a chondroitin sulfate proteoglycan would be difficult to detect in the autoradiographs because it would not be the only chondroitin sulfate pro-

94

teoglycan in the lung, and because the septa become too thin for the silver grains to be readily localized to the extracellular matrix in light micrographs. We conclude from the present results that the influence of b-xyloside on normal maturation of the lung must be through a chondroitin sulfate proteoglycan located in the extracellular matrix of the walls between saccules. Chondroitin sulfate proteoglycans have been isolated from adult human lung fibroblasts (David et al., 1989). Some of these proteoglycans are lipophilic and are assumed to be membrane-associated, while others are not. In sections of the adult lung, filaments detectable with cuprolinic blue that are sensitive to chondroitinase ABC, but not AC, are located adjacent to collagen fibrils in the extracellular matrix (Van Kuppevelt et al., 1984, 1988); similar filaments at the boundary between large extracellular matrix structures are sensitive to both enzymes (Rutten et al., 1987). Adult human lung fibroblasts synthesize a small, hydrophobic heparan sulfate proteoglycan associated with cell membranes and a large hydrophilic heparan sulfate that is present in the extracellular matrix (Lories et al., 1986; Heremans et al., 1988). Little work has been done on proteoglycans of the embryonic and fetal lung. The basal lamina stains with ruthenium red (Grant et al., 1983; Gallagher, 1986) and contains heparan sulfate. In the basal lamina, there is no significant loss of ruthenium red staining after digestion with either Streptomyces hyaluronidase or chondroitinase ABC (Grant et al., 1983), and radioactive sulfate incorporated at the epithelial/mesenchymal interface is sensitive to nitrous acid but not to chondroitinase digestion (Spooner et al., 1988). Chondroitinasesensitive radioactivity is found, however, in the extended matrix during initial outgrowth and primary branching. Incorporation of sulfate into material within the mesenchyme is inhibited by P-xyloside but not by cr-xyloside at nontoxic concentrations, whereas the same concentrations do not affect incorporation into the basal lamina and have no effect on branching. Specific inhibition of terminal branching by P-xyloside (Smith et al., 1990a; this study) and lack of inhibition of primary branching (Spooner et al., 1988) support the suggestion that the basal lamina is involved in

initial outgrowth and primary branching while changes in the extended matrix are involved in alveolar sac formation during terminal maturation at fetal stages. It is well established that morphogenesis and cytodifferentiation of many epithelial organs is strongly dependent on the mesenchymal component of the organ primordium (reviewed recently in Sanders, 1988). It is generally assumed that one way the mesenchyme exerts its influence is by controlling the composition of the extracellular matrix, either through its own synthetic activity or by degrading specific extracellular components produced by the epithelium. In the embryonic lung and salivary primordia, perturbation of collagen synthesis results in inhibition of branching (Alescio, 1973; Spooner and Faubion, 1980). In the salivary, formation of an initial cleft coincides with the deposition of fine collagen fibrils within the groove (Nakanishi et al., 1986). Perturbation of chondroitin sulfate proteoglycan synthesis by &xyloside inhibits branching morphogenesis of the salivary gland (Thompson and Spooner, 1982, 1983; Spooner and Thompson-Pletscher; 1986) and inhibits tubulogenesis in the kidney (Platt et al., 1987). In the salivary, incorporation of radioactive sulfate and glucosamine into the basement membrane occurs at the same rate at bud tips and clefts, but the material in the clefts turns over more slowly than at the bud tips. Turnover is dependent upon the mesenchyme (Banerjee et al., 1977; Bernfield and Banerjee, 1978; 1982); all of the action of the mesenchyme seems to be involved in metabolism of the extracellular matrix at the epithelial/mesenchymal interface. The respiratory epithelium does not progress beyond the pseudoglandular stage when it is combined with mesenchyme from the trachea and cultured for 5 days (Hilfer et al., 1985) if it is cultured in the absence of corticosteroid (Smith et al., 1990b) or if the respiratory regions are cultured in the presence of fi-xyloside (Smith et al., 1990a). It is well established that the respiratory mesenchyme is stimulated by corticosteroids to produce fibroblast pneumocyte factor (Smith, 1979; Smith and Sabry, 1983). It would appear probable that the respiratory mesenchyme can respond to corticosteroids in other ways as well,

95

specifically by producing a characteristic chondroitin sulfate proteoglycan. We suggest that inhibited synthesis of this chondroitin sulfate proteoglycan prevents thinning of the connective tissue to form septa (saccule formation). If saccule formation does not occur, the respiratory epithelial cells cannot differentiate as type I and type II cells. Respiratory mesenchyme appears mandatory for normal morphogenesis and differentiation of respiratory epithelium, due to synthesis of a chondroitin sulfate proteoglycan required for normal septum formation.

Acknowledgements Supported by NIH grants HL283303 to S.R.H. and R.L.S. and AR37940 to M.A.N., a Biomedical Research Support Grant RR07115 to Temple University, a Temple University Grant-in-Aid, and by the Foundation of the University of Medicine and Dentistry of New Jersey.

Bibliography Alescio, T.: Effect of a proline analogue, azetidine-2-carboxylic acid, on the morphogenesis of mouse embryonic lung. J. Embryol. Exp. Morphol. 29, 439-51 (1973). Ambesi-Impiombato, F.S., L.A.M. Parks and H.G. Coon: Culture of hormone-dependent functional epithelial cells from rat thyroids. Proc. Natl. Acad. Sci. USA 77, 3455-3459 (1980). Banejee, SD., R.H. Cohn and M.R. Bemfield: Basal lamina of embryonic salivary epithelia. Production by the epithelium and role in maintaining lobular morphology. J. Cell Biol. 73, 445-463 (1977). Bernfield, M.R. and N.K. Wessells: Intra- and extracellular control of epithelial morphogenesis. Dev. Biol. (Suppl.) 4, 195-249 (1970). Bernfield, M.R., R.H. Cohn and S.D. Banejee: Glycosaminoglycans and epithehal organ formation. Am. Zool. 13, 1067-1083 (1973). Bernfield, M.R. and S.D. Banejee: The basal lamina in epithelial-mesenchymal morphogenetic interactions. In: Proceeding, First International Symposium on the Biology and Chemistry of Basement Membranes, Philadelphia, ed. N.A. Kefaliedes (Academic Press, New York) pp 137-148 (1978). Bernfield, M.R. and S.D. Bane+: The turnover of basal lamina glycosaminoglycan correlates with epithelial morphogenesis. Dev. Biol. 90, 2291-2305 (1982).

Bluemink, J.G., P. Van Maurik and K.A. Lawson: Intimate cell contacts at the epithelial/mesenchymal interface in embryonic mouse lung. J. Ultrastruct. Res. 55, 257-270 (1976). David, G., V. Lories, A. Heremans, B. Van Der Schueren, J.J. Cassiman, and H. Van Den Berghe: Membrane-associated chondroitin sulfate proteoglycan of human lung fibroblasts. J. Cell Biol. 108, 1165-1175 (1989). De la Haba, G. and Holtzer, H.: Chondroitin sulfate: inhibition of synthesis by puromycin. Science 149, 1263-1265 (1965). Fukuda, Y., Y. Masluda, J.-I. Kishi, Y. Hashimoto, T. Hayakawa, H. Nogawa and Y. Nakanishi: The role of interstitial collagens in cleft formation of mouse embryonic submandibular gland during initial branching. Development 103, 259-267 (1988). Gallagher, B.C.: Basal laminar thining in branching morphogenesis of the chick lung as demonstrated by lectin probes. J. Embryol. Exp. Morphol. 94, 173-188 (1986). Grant, M.M., N.R. Cutts and J.S. Brody: Alterations in lung basement membrane during fetal growth and type II cell development. Dev. Biol. 97, 173-183 (1983). Grobstein, C.: Interactive processes in cytodifferentiation. J. Cell Comp. Physiol. (Suppl. 1) 60, 35-48 (1962). Heremans, A., J.-J. Cassiman, H. Van Den Berghe and G. David: Heparan sulfate proteoglycan from the extracellular matrix of human lung fibroblasts. J. Biol. Chem. 263, 4731-4739 (1988). Hilfer, S.R.: Development of terminal buds in the fetal mouse lung. Scanning Electron Microscopy III (SEM, Inc. Publishers, Chicago, IL) pp. 1387-1401 (1983). Hilfer, S.R., R.M. Rayner and J.W. Brown: Mesenchymal control of branching pattern in the fetal mouse lung. Tissue Cell 17, 525-538 (1985). Hilfer, S.R., S.L. Schneck and J.W. Brown: The effect of culture conditions on cytodifferentiation of fetal mouse lung respiratory passageways. Exp. Lung Res. 10, 115-136 (1986). Kosher, R.A. and Searls, R.L.: Sulfated mucopolysaccharides during the development of Rum pipiens. Dev. Biol. 32, 50-68 (1973). Lories, V., G. David, J.-J. Cassiman and H. Van Den Berghe: Heparan sulfate proteoglycans of human lung fibroblasts. Occurrence of distinct membrane, matrix, and secreted forms. Eur. J. Biochem. 158, 351-360 (1986). Nakanishi, Y., F. Sugiura, J. Kishi and T. Hayakawa: Collagenase inhibitor stimulates cleft formation during early morphogenesis of mouse salivary gland. Dev. Biol. 113, 201-206 (1986a). Nakanishi, Y., F. Sugiura, J. Kishi and T. Hayakawa: Local effects of implanted Elvax chips containing collagenase inhibitor and bacterial collagenase on branching morphogenesis of mouse embryonic submandibular glands in vitro. Zool. Sci. 3, 479-486 (1986b). Nathanson, M.A. and Hay, E.D.: Analysis of cartilage differentiation from skeletal muscle grown on bone matrix. II. Chondroitin sulfate synthesis and reaction to exogenous glycosaminoglycans. Dev. Biol. 78, 332-350 (1980).

96 Parry, G., D. Farson, B. Cullen and M.J. Bissell: pNitrophenyl+-o-xyloside modulates proteoglycan synthesis and secretory differentiation in mouse mammary epithelial cell cultures. In Vitro 24, 1217-1222 (1988). Platt, J.L., D.M. Brown, K. Granlund, T.R. Oegema and D.J. Klein: Proteoglycan metabolism associated with mouse metanephric development: morphologic and biochemical effect of /I-xyloside. Dev. Biol. 123, 293-306 (1987). Rutten, T.L.M., T.H.M.S.M. van Kuppevelt, H.M.J. Janssen and C.M.A. Kuyper: Ultrastructural localization of a chondroitinase-sensitive, cuprolinic blue-positive filament in developing mouse lung. Eur. J. Cell Biol. 45, 256-261 (1987). Rutter, W.J., N.K. Wessells and C. Grobstein: Control of specific synthesis in the developing pancreas. National Cancer Institute Monograph No. 13, 51-65 (1964). Sanders. E.J.: The roles of epithelial-mesenchymal cell interactions in developmental systems. B&hem. Cell Biol. 66, 530-540 (1988). Schwartz, D.E., Choi, Y., Sandell, L. and Hanson, W.R.: Quantitative analysis of collagen, protein and DNA in fixed, paraffin-embedded and sectioned tissue. Histochem. J. 17, 655-663 (1985). Schwartz, N.B.: Regulation of chondroitin sulfate synthesis. J. Biol. Chem. 252, 6316-6321 (1977). Searls, R.L.: The role of cell migration in the development of the embryonic chick limb bud. J. Exp. Zool. 16, 39-50 (1967). Smith, B.T. (1979) Lung maturation in the fetal rat: acceleration by injection of fibroblast-pneumocyte factor. Science 204, 1094-1096 (1967). Smith, B.T. and K. Sabry: Glucocorticoid-thyroid synergism in lung maturation: a mechanism involving epithelialmesenchymal interaction. Proc. Natl. Acad. Sci. USA 80, 1951-1983 (1983). Smith, CL, S.R. Hilfer, R.L. Searls, M.A. Nathanson and MI. Allodoli: Effects of P-D-xyloside on differentiation of the respiratory epithelium in the fetal mouse lung. Dev. Biol. 138,42-52 (1990a). Smith, CL, R.L. Sea& and S.R. Hilfer: The effects of hormones on functional differentiation of mouse respiratory epithelium. Exp. Lung Res., 16, 191-209 (1990b). Smith, R.L. and M.R. Bemfield: Salivary morphogenesis: degradation of epithelial GAG by embryonic mesenchyme. J. Cell Biol. 75, 106a (1982). Spooncer, E., J.T. Gallagher, F. Krizsa and T.M. Dexter: Regulation of haemopoiesis in long-term bone marrow cultures. IV. Glycosaminoglycan synthesis and the stimula-

tion of haemopoiesis by B-D-xylosides. J. Cell Biol. 96, 510-514 (1983). Spooner, B.S. and J. Faubion: Collagen involvement in branching morphogenesis of embryonic lung and salivary gland. Dev. Biol. 77, 84-102 (1980). Spooner, B.S. and H.A. Thompson-Pletscher: Matrix accumulation and the development of form: proteoglycans and branching morphogenesis. In ‘Regulation of Matrix Accumulation’, ed. R.P. Mecham, Biology of Extracellular Matrix, Vol. 1, (Academic Press, Orlando, FL) pp. 399-444 (1986). Spooner, B.S., B. Stokes and K. Bassett.: Glycosaminoglycan deposition and processing at the basal epithelial surface of morphogenetically active and /3-xyloside-inhibited embryonic salivary glands. Dev. Biol. 109, 177-183 (1985). Spooner, B., K. Bassett and B. Spooner, Jr.: Sulfated GAG and lung development. J. Cell Biol. 107, 59a (1988). Ten Have-Opbroek, A.A.W.: The development of the lung in mammals: an analysis of concepts and findings. Am. J. Anat. 162, 201-219 (1979). Thompson, H.A. and B.S. Spooner: Inhibition of branching morphogenesis and alteration of glycosaminoglycan biosynthesis in salivary glands treated with /I-D-xyloside. Dev. Biol. 89, 417-424 (1982). Thompson, H.A. and B.S. Spooner: Proteoglycan and glycosaminoglycan synthesis in embryonic mouse salivary glands: Effects of B-D-xyloside, an inhibitor of branching morphogenesis. J. Cell Biol. 96, 1443-1450 (1983). Van Kuppevelt, T.H.M.S.M., J.G.W. Domen, G.P.M. Cremers and C.M.A. Kuyper: Staining of proteoglycans in mouse lung alveoli. I. Ultrastructural localization of anionic sites. Histochem. J. 16, 657-669 (1984). Van Kuppevelt, T.H.M.S.M., H.M.J. Janssen, H.M. van Beuningen, K.-S. Cheung, M.M.A. Schijen, C.M.A. Kuyper and J.H. Veerkamp: Isolation and characterization of a collagen fibril-associated dermatan sulfate proteoglycan from bovine lung. B&him. Biophys. Acta 926, 296-309 (1988). Vogel, K.G. and Peterson, D.W.: Extracellular, surface, and intracellular proteoglycans produced by human embryo lung fibroblasts in cell culture (IMR-90). J. Biol. Chem. 256, 13235-13242 (1981). Wessells, N.K.: Problems in the analysis of determination, mitosis, and differentiation. In ‘Epithelial-Mesenchymal Interactions’, ed. R. Fleischmajer (Williams and Wilkins) pp. 132-151 (1968). Wessells, N.K.: Mammalian lung development: interactions in formation and morphogenesis of tracheal buds. J. Exp. Zool. 175, 455-466 (1970).