ArchsoralBid. Vol. 36, No. Printed in Great Britain. All
5, pp. 389-395, 1991 rights reserved
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SULPHATED GLYCOSAMINOGLYCAN SYNTHESIS BY DEVELOPING RAT SUBMANDIBULAR GLAND SECRETORY UNITS L. S. CUTLER,*C. P. CHRISTIANand J. K. WENDELL Department of Oral Diagnosis, School of Dental Medicine, University of Connecticut Health Center, Farmington, CT 06032, U.S.A. (Accepted 19 September 1990)
~~~-This study examined the profile of S-GAGS synthesized by presecretory and secretory units isolated from rats at 17, 18 and 21 days in u;ero and 1,6 and 35 days after birth. The units were incubated for 2 h in medium containing [35S]-sodium sulphate and then processed and analysed. Secretory units from 17-day embryonic presecretory units produced a S-GAG profile composed of approx. 73% chondroitin sulphate and 24% heparan sulphate. When cells of the embryonic units undergo cyt~ffe~ntiation to become secretory cells (I8 days in utero), there is a major change in the relative amounts of S-GAG synthesized with 54% of the S-GAG produced being heparan sulphate and 41% chondroitin sulphate. There is a progressive increase in the relative amount of heparan sulphate produced and a concomitant decline in chondroitin sulphate as the secretory compartment of the gland matures. By 35 days after birth, the secretory units produced a S-GAG protie that was greater than 85% heparan sulphate and less than 10% chondroitin sulphate. The ratio of heparan sulphate/chondroitin suiphate production was 0.36 by 17-day embryonic presecretory units and shifted to 9.1 by 35day postnatal units.
Key words: giycosaminogly~ns, development, submandibular gland, synthesis.
INTRODUCTION Proteoglycans and their associated GAG moieties may have a significant role in the morphogenesis of the submandibular glands and several other tissues (reviewed by Bernfield et al., 1984). Definitive evidence for the role of S-GAG in the branching morphogenesis of salivary glands has recently been provided by Thompson and Spooner (1982, 1983) and Spooner and co-workers (1985) who demonstrated that branching could be reversibly inhibited in vitro by blocking proteoglycan synthesis and S-GAG deposition in the basement membrane with exogenously added @-D-xyloside, an inhibitor of proteoglycan assembly. This work and that of Toole (1981, 1982) has suggested that GAGS such as hyaluronic acid and chondroitin sulphate are involved in the development of morphogenetic branching patterns. Other studies have suggested that S-GAGs, particularly heparan sulphate, are associated with cytodifferentiation (reviewed by Trelstad, 1985; Gallagher, Lynon, and Steward, 1986). Further, several studies have indicated that the GAG imposition of the extracellular matrix influences the metabolism of the embedded or surrounded cells (Gallagher et al., 1986; Trelstad, 1985; Gallager and Hampson, 1984), suggesting that GAG/proteoglycan, particularly heparan sulphate, may be responsible for stabilizing the differentiated state of many tissues. Sulphated GAGS have been shown to inhibit adenylate cyclase of the *To whom correspondence should be addressed. Abbreviations: HBSS, Hank’s balanced salt solution; (S-)GAG, (sulphated) glycosaminoglycans.
submandibular gland (Cutler and Christian, 1984) and could thus have a regulatory role in cyclic AMP-dependent processes in salivary gland development and function. The profile of GAG synthesized by adult submandibular secretory units has recently been described in work from our laboratory (Cutler, Christian and Rendell, 1987). There is only limited information on the types of GAG synthesized by the developing submandibular gland. Cohn et al. (1977) reported that the GAG profile synthesized by the early branching rudiments of the gland in the mouse was about 50% hyaluronic acid and 40% chondroitin sulphate, with the remaining 10% probably heparan sulphate (although this was not directly measured). Thompson and Spooner (1982) reported that at the time of initial morphogenetic branching, cultured rudiments of embryonic mouse submandibular gland synthesized S-GAGS that were 70% chondroitin sulphate and 30% heparan sulphate. We have found no specific information on S-GAGS in general, and heparan sulphate in particular, at important developmental stages of the submandibular gland, other than the time of initial morphogenetic branching. Neither has the relationship of S-GAG synthesis to cytodifferentiation of the acinar secretory cells been determined. Our purpose now was to characterize the types of S-GAG synthesized by the developing of rat secretory units from the submandibular gland and their precursors to determine if stage-specific S-GAG profhes were associated with important developmental periods related to secretory cytodifferentiation (reviewed by Cutler, 1989; Cutler and Chaudhry, 1974). 389
L. S. CUTLER
390 METHODS
Isolation of end buds (secretory unit precursors) and secretory units from the prenatal and postnatal glanak
The submandibular glands were removed from Sprague-Dawley rats at 17, 18 and 21 days of gestation and at 1, 6 and 35 days after birth. The sublingual gland and surrounding connective tissue were cleaned away, and the tissue immediately minced into small pieces in HBSS containing HEPES buffer @H 7.3). For embryonic and early postnatal glands, tissue from several animals was pooled to obtain enough secretory units for studies of biosynthesis. The minced tissue was transferred to a test tube with a IO-fold excess (w/v) of HBSS-HEPES containing 100 units/ml of purified collagenase (Cooper Biomedical, Freehold, NJ) and 100 units/ml of grade IV S bovine testicular hyaluronidase (Sigma Chemical Co., St Louis, MO). The tissue was incubated for 30-45 min in a 37°C water bath with moderate agitation and gassed every 10 min with 95% air-5% CO*. The resulting suspension was concentrated by gentle centrifugation (100 rev/min for 30 s), the supernate removed, the pellet resuspended in HBSSHEPES, and then sequentially washed through a series of two Teflon screens with decreasing pore sizes of 250 p and 150 p(, the acini being trapped on a 70 p screen (Cutler, Christian and Rendell, 1987). Previous studies have shown that the resulting preparation is composed of greater than 90% acini or endbuds and that more than 80% of the cells are viable by trypan-blue exclusion. Cells in this type of preparation are functionally normal with regard to the incorporation of amino acids into secretory proteins and incorporation of sulphate or glucosamine into GAG (Cutler and Rendell, 1984; Beeman and Cutler, 1985; Cutler et al., 1987). Analysis of S-GAG synthesis
The endbuds or secretory units were incubated for 2 h at 37°C with gentle agitation in HBSS-HEPES containing 50 pCi/ml of [35S]-sodium sulphate (Carrier-free) (New England Nuclear). The units were gassed with 95% air-5% CO, every 20 min during the incubation. After incubation, the secretory units were washed twice in fresh medium and then homogenized. The resulting homogenate was digested for 48 h at 50°C in predigested pronase (1.5 mg/ml, Sigma protease VI in 2.0ml of 0.2 M tris-HCl buffer, pH 8.0) (Lemkin and Farquhar, 1981). Fresh protease was added after 24 h, making the final volume 3.0 ml. The pronase digest was boiled and then dialysed for 48 h against distilled water to remove low molecular-weight breakdown products and free sulphate. The dialysate was lyophilized.
et
al.
The distribution of [3sS]-sulphate in different GAG species and glycopeptides was determined by a modification of the procedures of Hart (1976, 1978) as described by Lemkin and Farquhar (1981) and shown to be effective in the study of GAG synthesis by these glands (Cutler et al., 1987). Initial separation of total GAG from glycopeptides and unincorporated label was accomplished by gel filtration chromatography on a Sephadex G-50 column (1 x 120 cm). The lyophilized samples were suspended in and eluted with 0.1 M ammonium acetate in 20% (v/v) ethanol; 1.75 ml fractions were collected and 0.4 ml samples of each fraction were counted to determine their radioactivity. After chromatography of the initial sample to separate GAG from glycopeptides and free sulphate, all of the GAGcontaining fractions were pooled (total GAG fraction), lyophilized and subjected to a series of enzymatic and chemical degradative procedures, each followed by chromatography over Sephadex G-50 (1 x 12Ocm). Samples from cells incubated with sulphate were first chondroitinase ABC digested. The total GAG fraction was digested with 0.17 units/ml of Proteus vulgaris chondroitinase ABC (Seikagaku Kogyo Co. Ltd, Tokyo, Japan) in 3.0 ml of 0.01 M tri-HCI buffer, pH 8.0 for 24 h at room temperature. This enzyme degrades chondroitin 4,-6 and dermatan sulphates. The treated material was chromatographed on a Sephadex G-50 column (1 x 120 cm) and fractions counted to determine radioactivity (as described above) and to separate resistant GAG from degradation products. The resistant GAG fractions were pooled, lyophilized, and then subjected to nitrous acid oxidation for 3 h at room temperature to degrade the N-sulphated GAG, heparan sulphate. The degradation products were separated from any residual, resistant GAG by chromatography of Sephadex G-50 as before. Fractions were counted to determine radioactivity as described. This sequential procedure provided a profile of the S-GAGS synthesized by secretory unit precursors as well as by embryonic and maturing secretory units. Two or three experiments were run for each developmental stage. The data are reported as the mean for the experiments run.
RESULTS
Isolation of endbuds and secretory units
The procedure resulted in a preparation which was composed of 90-95% secretory units (acini or end buds) as determined by differential counting of random samples from 6 different preparations. The isolated units had normal appearances (Plate Fig. 1)
Plate 1 Fig. 1. Micrographs showing a typical secretory-unit preparation. These micrographs were taken of a preparation made from 21-day prenatal submandibular tissue. (A) A phase-contrast micrograph of the isolated secretory units. x 200. (B) A light micrograph of secretory units. Most of the cells appear normal. However, some cells at the periphery of one of the units appear swollen and are probably non-viable (arrows). Such cells would not exclude trypan blue. x 300. (C) An electron micrograph showing secretory cells from a typical isolated unit. The ultrastructure of the cells is normal. x 3000
Glycosamin~glycans
in developing salivary glands
Plate 1
391
L. S. CUTLER et al.
392
and more than 80% of the cells within the units were viable by trypan blue exclusion.
Table 1. Incorporation of [35S]-sulphate into GAG and glycopeptides by developing secretory units of submandibular glands (SMG)
Projile of GAG and glycopeptides
Typical GAG and glycopeptide profiles from 17day prenatal endbuds, 18- and 21-day prenatal, and l-6- and 35-day postnatal secretory units are shown in Text Fig. 2. The GAGS were eluted in fractions 19-26, glycopeptides were eluted in fractions 3346, and free sulphate after fraction 58. The percentage of the radioactivity present in the GAG and glycopeptide fractions is shown in Table 1. Secretory units from prenatal and neonatal secretory units incorporated [35S]-sulphate into both GAG and glycopeptides. Prenatal (18- and 21-day) and 35-day postnatal secretory units incorporated 82.4 and 71.2% of the [35S]-sulphate into GAG and only 17.6, 23.3 and 28.8% into glycopeptides respectively, while 17-day prenatal and l- and 6-day postnatal units incorporated 22, 40.4 and 36.1% of the [35S]sulphate into GAG and 78, 59.6, and 63.9% glycopeptides. Distribution of [“S]-sulphate in speciJic GAG fractions Chondroitin sulphate. The total GAG fractions
eluted from the initial Sephadex G-50 column (Text Fig. 2) were treated with chondroitinase ABC, and the resistant GAG and digestion products separated by chromatography on a Sephadex G-50 column. Approximately 73% of the sulphated GAG from the 17-day prenatal presecretory units was sensitive to
600 500
400 300 200 100
600 500 400 300 200 loo
[35S]-Sulphate
(%)
Glycopeptides
GAG
Prenatal SMG secretory units 78.0 17.6 23.3
22.0 82.4 76.7
17 days (2) 18 days (2) 21 days (3)
Neonatal SMG secretory units 1 day (3) 6 days (3) 35 days (3)
40.4
59.6
36.1 71.2
63.9 28.8
Total GAG and glycopeptides were collected from developing secretory units in vitro. GAG and glycopeptides were separated by gel chromatography. The percentage radioactivity in the GAG and glycopeptide fractions was calculated by dividing the c.p.m. in the GAG or glycopeptide peaks by the total c.p.m. of the GAG + glycopeptide peaks x 100. Data are reported as the mean of the experiments run. The number of experiments for each time point is shown in parentheses.
ABC treatment. This percentage of chondroitinase ABC-sensitive material in these early rudiments is consistent with the figure reported for the mouse submandibular gland by Thompson and Spooner (1982). The percentage of S-GAG which was sensitive to this treatment decreased significantly as secretory activity became apparent in the 18-day prenatal units. The chondroitinase ABC-sensitive material synthesized by ll-day prenatal units dropped to 41% of the S-GAG. The portion of the S-GAG produced that was sensitive to chondroitinase ABC treatment progressively decreased as acinar maturation proceeded. Chondroitin sulphate represented approximately 25-29% of the GAG produced by 21-day prenatal and l-day postnatal units, 18% of that made by 6-day units and only about 9% of that produced by 35-day postnatal secretory units (Table 2 and Text Fig. 3). Heparan sulphate. The S-GAG fractions that were resistant to chondroitinase ABC digestion were broken down by nitrous acid oxidation and rechromatographed on Sephadex G-50. Most of the chondroitinase-resistant GAG was broken down by this means. Only 24% of the S-GAG synthesized by 17-day prenatal endbuds was sensitive to nitrous acid chondroitinase
Table 2. Incorporation of [r5S]-sulphate into specific GAG by developing secretory units of submandibular glands [35S]-Sulphate (%) Prenatal Postnatal FRACTION NUMBER Fig. 2. Separation of GAG and glycopeptides by filtration on a Sephadex G-50 gel filtration column (1 x 120 cm) after pronase digestion (see Methods). The S-GAG eluted in the void volume in fractions 19-24. The glycopeptides were retarded and eluted in the included volume in fractions 3546. (A) Profile from 17-day embryonic presecretory units. (B) Profile from earliest differentiated secretory units at 18 days in utero (C) Profile from 21-day embryonic secretory units. (D) Profile from l-day postnatal units. (E) Profile from 6-day postnatal secretory units. (F) Profile from 35day postnatal secretory units.
GAG
Type
Chondroitin sulphates and dermatan sulphate Heparan sulphate Enzyme/nitrous acid resistant material
17
18
21
1
6
35
73 26
41.6 53.3
28.9 63.9
25.7 66.3
18.1 78.9
9.4 85.3
1
4.1
7.2
8
3
5.3
Newly synthesized [r’s]-sulphate labelled GAG from sccretory units in vitro. The figures are reported as the percentage of the total GAG synthesized. Data are reported as the mean of the experiments for each developmental time point.
~lyco~mino~~ans
o-i,, 14i8!1 I.1
PRENArAL
6
I
6
,
I
3
in developing salivary glands
1
14 21 28 35 POSTNATAL
AGE
Fig. 3. Graphic presentation of the change in the relative amounts of chon~oitin suiphate (dashed line) and heparan sulphate (solid line) produced in vitro by presecretory and secretory units of different developmental ages. The data are the mean of the experiments and are expressed as the percentage of the total S-GAG produced.
degradation. The proportion of heparan sulphate synthesized increased substantially at 18 days in utero and represented 54% of the S-GAG produced by these secretory units. As the units matured, they produced a S-GAG profile characterized by an increased content of heparan sulphate: at 21 days in utero and 1 day after birth, this represented about 66% of the S-GAG; at 6 days after birth, it was 79% of the S-GAG, and; by 35 days of age, it comprised 85% of the S-GAG (Table 2 and Text Fig. 3). Emzyme- and nitrous acid-resistant material A small amount of sulphate-containing material (l-8%) in all of the GAG samples from neonatal and prenatal secretory units was resistant to chondroitinase ABC and nitr0u.s acid digestion. The nature of this material was not determined. IXSCUSSION
We demonstrate a significant reduction in the proportion of chondroitin sulphate synthesized by the isolated endbuds of rat submandibular glands between day 17 and day 18 in utero. There is a concomitant rise in the proportion of heparan sulphate. This change in the profile of S-GAG produced by the developing endbuds is coordinated with the beginning of the cytodifferentiation of the endbuds into secretory cells. At this time, these cells develop the organelles required for the production and packaging of exocrine proteins (reviewed by Cutler, 1989; Cutler and Chaudhry, 1974). On the 18th day of gestation, the first recognizable secretory granules are found in the apical cytoplasm of the differentiating secretory cells and these cells develop the characteristic apical-basal polarity of organelles associated with exocrine secretory activity. This observation suggests that as the c~ellsundergo cytodifferentiation they alter the pattern of S-GAG that they produce. This shift in the pattern of S-GAG production is important because the synthesis of chondroitin sulphate and its deposition in the basement membrane surrounding the early rudiment of the gland is re-
393
quired for development of the morphogenetic branching pattern (Banerjee, Cohn and Bernfield, 1977; Bemfield and Banerjee, 1972; Bemfield, Banerjee and Cohn, 1972; Cohn, Banerjee and Bemfield, 1977; Thompson and Spooner, 1982, 1983). It is interesting that even though there is a dramatic drop in the relative amount of chondroitin sulphate produced, the arborized pattern of the glandular rudiment is maintained once it has been well established. Bemfield and co-workers (Rapraeger and Bemfield, 1983; Bemfield er al., 1984; Koda et al., 1985) have suggested that an increase in heparan sulphate production and its deposition on the cell surface or in the basement membrane might serve to stabilize the gland’s branched architecture. We now provide evidence that the proportion of heparan sulphate synthesized is increased at the time and in the fashion required to satisfy this hypothesis. Increases in the proportion of heparan sulphate synthesized in developing tissues have been associated with the onset and stabilization of cellular cytodifferentiation in several systems (Trelstad, 1985; Gallagher and Hampson, 1984; Morriss-Kay and Crutch, 1982). Defects in its production or deposition have been associated with various pathological conditions that are characterized by alterations in differentiated functions of the glands, for example, cystic fibrosis (Ben-Yoseph and Nadler, 1982). A change from a heparan sulphate-~ch GAG profile to one high in chondroitin sulphate is characteristic of neoplastic transformation of several tissues including salivary glands (Takeuchi et al., 1975, 1978, 1981) and a shift from a heparan-rich to a chondroitin-~ch extracellular matrix appears to be associated with a loss of cellular differentiation and destabilization of both tissue structure and function (reviewed by Trelstad, 1985; Gallagher and Hampson, 1984). It is important to remember that our study conditions did not allow differentiation between heparan sulphate that was at the cell surface or in the basement membrane because we only measured the total heparan sulphate. Unique ~pulations of heparan sulphate in specific locations may modulate different aspects of morphological and/or cytological differentiation. Thus, the rapid rise in heparan sulphate found in the submandibular rudiment at 18 days in utero may be directly related to the secretory cell differentiation which occurs at this point in the gland’s development. The relatively rapid rise in the proportion of heparan sulphate and the decrease in the chondroitin sulphate of the GAG profile produced by the developing secretory units begins to form a plateau at 6 days after birth. This plateau may reflect the fact that, at this time, the acini become function~ly differentiated and, with the exception of some minor modifications in the nature of the secretory product, cytodifferentiation is complete (Cutler and Chaudhry, 1974; Cutler, 1989). There is a slow but progressive increase in the heparan sulphate content of the GAG profile between 6 and 35 days, which continues until full glandular maturation is achieved at about 90 days of age (Cutler et al., 1987). Fully matured secretory units from the adult gland produce a S-GAG profile that is 90% heparan and only 4% chondroitin sulphate (Cutler et al., 1987). The ratio
L. S. CurLEn et al.
394
of heparan sulphate/chondroitin sulphate production by 17-day embryonic presecretory units was 0.36. This ratio shifted to 1.3 for ll-day embryonic secretory units, 9.1 for 35-day postnatal units, and 22.5 for fully mature secretory units (Cutler et al., 1987). The proportion of glycopeptides produced by presecretory units (17-day prenatal) and early neonatal (during the first postnatal week) secretory units was greatly increased when compared to other prenatal and older postnatal and adult (Cutler et al., 1987) units. Glycopeptides and GAG have been implicated in promoting neurite outgrowth and in the developmental association of specific nerve terminals with cell surface neuroreceptors (Anglister and McMahan, 1984; Gallagher et al., 1986). Unusual epithelialnerve contacts have been reported in the l&17day embryonic submandibular gland (Cutler and Chaudhry, 1973). Further, during the first postnatal week, catecholamine-containing nerve processes penetrate the developing parenchyma of the gland and establish functional connections with the acinar secretory cells (Bottaro and Cutler, 1984). It would be appealing to relate the high proportion of glycopeptides produced at these developmental times to the processes of nerve ingrowth and the establishment of functional neural connections in the neonatal submandibular gland. However, such relationships can only be inferred from the current information. Acknowledgement-This
work was supported by NIH grant
DE 05632.
innervation of the rat submandibular gland. Archs oral Biol. 29, 237-242.
Cohn R. H., Banerjee S. D. and Bemtield M. R. (1977) Basal lamina of embryonic salivary epithelial: Nature of glycosaminoglycan and organization of extracellular materials. J. cell Biol. 73, 464478. Cutler L. S. (1980) The dependent and independent relationships between cytodifferentiation and morphogenesis in developing salivary gland secretory cells. Anaf. Rec. 196, 341-347. Cutler L. S. (1989) Functional differentiation of salivarv glands. In: Handbook of Physiology: Salivary, Pancreatic, Gasrric. and Hewtobiliarv Secretion. Vol. 3 (Edited bv t Forte) pp. 413220. Am.-Physiol. Sot. Press; New Y&k: Cutler L. S. and Chaudhry A. P. (1973) Intercellular contacts at the epithelial-mesenc!tymal interface during prenatal development of the rat submandibular gland. Dev. Biol. 33, 229-240.
Cutler L. S. and Chaudhry A. P. (1974) Cytodifferentiation of the acinar cells of the rat submandibular gland. Dev. Biol. 41, 3141.
Cutler L. S. and Christian C. P. (1984) Inhibition of rat salivary gland adenylate cyclase by glycosaminoglycans and high molecular weight polyanions. Archs oral Biol. 29, 629633.
Cutler L. S. and Rendell J. K. (1984) Glycosaminoglycan synthesis by developing and adult submandibular gland secretory units. J. dent. Res. 63, 302. Cutler L. S., Christian C. P. and Rendell J. K. (1987) Glycosaminoglycan synthesis by adult rat submandibular gland secretory units. Archs oral Biol. 32, 413-419. Gallagher J. T. and Hampson I. N. (1984) Proteoglycans in cellular differentiation and neoplasia. Biochem. Sot. Transact. 12, 541-543.
Gallagher J. T., Lyon M. and Steward W. P. (1986) Structure and function of heparan sulfate proteoglycans. Biochem. J. 236, 313-325.
REFERENCES
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pp. 387400. Karger, Base]. Bemfield M. R. and Banerjee S. D. (1972) Acid mucopolysaccharide (glycosaminoglycan) at the epithelialmesenchymal interface of mouse embryo salivary glands. J. cell Biol. 52, 664-673.
Bernfield M. R., Banerjee S. D. and Cohn R. H. (1972) Dependence of salivary epithelial morphology and branching morphogenesis upon acid mucopolysaccharide-protein (proteoglycan) at the epithelial surface. J. cell Biol. 52, 674689.
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Hart G. W. (1976) Biosynthesis of glycosaminoglycans during cornea1 development. J. biol. Chem. 251, 65134521. Hart G. W. (1978) Biosynthesis of glycosaminoglycans by separated tissues of the embryonic chick cornea. Dev. Biol. 52, 78-79.
Koda J. E., Rapraeger A. and Bernfield M. (1985) Heparan sulfate proteoglycans from mouse mammary epithelial cells. J. biol. Chem. 260, 8157-8162. Lemkin M. C. and Farquhar M. G. (1981) Sulfated and nonsulfated glycosaminoglycans and glycopeptides are synthesized by kidney in uivo and incorporated into glomerular basement membranes. Proc. natn. Acad. Sci. U.S.A. 78, 17261730.
Morriss-Kay G. M. and Crutch B. (1982) Culture of rat embryos with fi-o-xyloside: evidence of a role for proteoglycans in neurulation. Anat. J. 134: 491-506. Rapraeger A. C. and Bernfield M. (1983) Heparan sulfate proteoglycans from mouse mammary epithelial cells. J. biol. Chem. 258: 3632-3636.
Spooner B. S., Bassett K. and Stokes B. (1985) Sulfated glycosaminoglycan deposition and processing at the basal epithelial surface in branching and /7-o-xyloside-inhibited embryonic salivary glands. Dev. Biol. 109, 177-183. Takeuchi J., Sobue M., Yoshida M., Esaki T. and Katoh Y. (1975) Pleomorphic adenoma of the salivary gland with special reference to histochemical and electron microscopic studies and biochemical analysis of glycosaminoglycans in viva and in vitro. Cancer Xi, 1771-1784. Takeuchi J., Sobue M., Yoshida M. and Sato E. (1978) Glycosaminoglycans-synthetic activity of pleomorphic adenoma, adenoid cystic carcinoma and non-neoplastic tubulo-acinar cells of the salivary gland. Cancer 42, 202-208. Takeuchi J., Sobue M., Sato E., Yoshida M., Uchibori N. and Miura K. (198 1) A high level of glycosaminoglycan-
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in developing salivary glands
synthesis of squamous cell carcinoma of the parotid gland. Cancer 41, 2030-2035. Thompson H. A. and Spooner B. S. (1982) Inhibition of branching morphogenesis and alteration of glycosaminoglycan biosynthesis in salivary glands treated with B-Dxyloside. Dev. Biol. 89, 417-424. Thompson H. A. and Spooner B. S. (1983) Proteoglycan and glycosaminoglycan synthesis in embryonic mouse salivary glands: Effects of B-D-xyloside, an inhibi-
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5, pp. 389-395, 1991 rights reserved
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~3-99~9~91 $3.00 + 0.00 1991 PergamonPressplc
SULPHATED GLYCOSAMINOGLYCAN SYNTHESIS BY DEVELOPING RAT SUBMANDIBULAR GLAND SECRETORY UNITS L. S. CUTLER,*C. P. CHRISTIANand J. K. WENDELL Department of Oral Diagnosis, School of Dental Medicine, University of Connecticut Health Center, Farmington, CT 06032, U.S.A. (Accepted 19 September 1990)
~~~-This study examined the profile of S-GAGS synthesized by presecretory and secretory units isolated from rats at 17, 18 and 21 days in u;ero and 1,6 and 35 days after birth. The units were incubated for 2 h in medium containing [35S]-sodium sulphate and then processed and analysed. Secretory units from 17-day embryonic presecretory units produced a S-GAG profile composed of approx. 73% chondroitin sulphate and 24% heparan sulphate. When cells of the embryonic units undergo cyt~ffe~ntiation to become secretory cells (I8 days in utero), there is a major change in the relative amounts of S-GAG synthesized with 54% of the S-GAG produced being heparan sulphate and 41% chondroitin sulphate. There is a progressive increase in the relative amount of heparan sulphate produced and a concomitant decline in chondroitin sulphate as the secretory compartment of the gland matures. By 35 days after birth, the secretory units produced a S-GAG protie that was greater than 85% heparan sulphate and less than 10% chondroitin sulphate. The ratio of heparan sulphate/chondroitin suiphate production was 0.36 by 17-day embryonic presecretory units and shifted to 9.1 by 35day postnatal units.
Key words: giycosaminogly~ns, development, submandibular gland, synthesis.
INTRODUCTION Proteoglycans and their associated GAG moieties may have a significant role in the morphogenesis of the submandibular glands and several other tissues (reviewed by Bernfield et al., 1984). Definitive evidence for the role of S-GAG in the branching morphogenesis of salivary glands has recently been provided by Thompson and Spooner (1982, 1983) and Spooner and co-workers (1985) who demonstrated that branching could be reversibly inhibited in vitro by blocking proteoglycan synthesis and S-GAG deposition in the basement membrane with exogenously added @-D-xyloside, an inhibitor of proteoglycan assembly. This work and that of Toole (1981, 1982) has suggested that GAGS such as hyaluronic acid and chondroitin sulphate are involved in the development of morphogenetic branching patterns. Other studies have suggested that S-GAGs, particularly heparan sulphate, are associated with cytodifferentiation (reviewed by Trelstad, 1985; Gallagher, Lynon, and Steward, 1986). Further, several studies have indicated that the GAG imposition of the extracellular matrix influences the metabolism of the embedded or surrounded cells (Gallagher et al., 1986; Trelstad, 1985; Gallager and Hampson, 1984), suggesting that GAG/proteoglycan, particularly heparan sulphate, may be responsible for stabilizing the differentiated state of many tissues. Sulphated GAGS have been shown to inhibit adenylate cyclase of the *To whom correspondence should be addressed. Abbreviations: HBSS, Hank’s balanced salt solution; (S-)GAG, (sulphated) glycosaminoglycans.
submandibular gland (Cutler and Christian, 1984) and could thus have a regulatory role in cyclic AMP-dependent processes in salivary gland development and function. The profile of GAG synthesized by adult submandibular secretory units has recently been described in work from our laboratory (Cutler, Christian and Rendell, 1987). There is only limited information on the types of GAG synthesized by the developing submandibular gland. Cohn et al. (1977) reported that the GAG profile synthesized by the early branching rudiments of the gland in the mouse was about 50% hyaluronic acid and 40% chondroitin sulphate, with the remaining 10% probably heparan sulphate (although this was not directly measured). Thompson and Spooner (1982) reported that at the time of initial morphogenetic branching, cultured rudiments of embryonic mouse submandibular gland synthesized S-GAGS that were 70% chondroitin sulphate and 30% heparan sulphate. We have found no specific information on S-GAGS in general, and heparan sulphate in particular, at important developmental stages of the submandibular gland, other than the time of initial morphogenetic branching. Neither has the relationship of S-GAG synthesis to cytodifferentiation of the acinar secretory cells been determined. Our purpose now was to characterize the types of S-GAG synthesized by the developing of rat secretory units from the submandibular gland and their precursors to determine if stage-specific S-GAG profhes were associated with important developmental periods related to secretory cytodifferentiation (reviewed by Cutler, 1989; Cutler and Chaudhry, 1974). 389
L. S. CUTLER
390 METHODS
Isolation of end buds (secretory unit precursors) and secretory units from the prenatal and postnatal glanak
The submandibular glands were removed from Sprague-Dawley rats at 17, 18 and 21 days of gestation and at 1, 6 and 35 days after birth. The sublingual gland and surrounding connective tissue were cleaned away, and the tissue immediately minced into small pieces in HBSS containing HEPES buffer @H 7.3). For embryonic and early postnatal glands, tissue from several animals was pooled to obtain enough secretory units for studies of biosynthesis. The minced tissue was transferred to a test tube with a IO-fold excess (w/v) of HBSS-HEPES containing 100 units/ml of purified collagenase (Cooper Biomedical, Freehold, NJ) and 100 units/ml of grade IV S bovine testicular hyaluronidase (Sigma Chemical Co., St Louis, MO). The tissue was incubated for 30-45 min in a 37°C water bath with moderate agitation and gassed every 10 min with 95% air-5% CO*. The resulting suspension was concentrated by gentle centrifugation (100 rev/min for 30 s), the supernate removed, the pellet resuspended in HBSSHEPES, and then sequentially washed through a series of two Teflon screens with decreasing pore sizes of 250 p and 150 p(, the acini being trapped on a 70 p screen (Cutler, Christian and Rendell, 1987). Previous studies have shown that the resulting preparation is composed of greater than 90% acini or endbuds and that more than 80% of the cells are viable by trypan-blue exclusion. Cells in this type of preparation are functionally normal with regard to the incorporation of amino acids into secretory proteins and incorporation of sulphate or glucosamine into GAG (Cutler and Rendell, 1984; Beeman and Cutler, 1985; Cutler et al., 1987). Analysis of S-GAG synthesis
The endbuds or secretory units were incubated for 2 h at 37°C with gentle agitation in HBSS-HEPES containing 50 pCi/ml of [35S]-sodium sulphate (Carrier-free) (New England Nuclear). The units were gassed with 95% air-5% CO, every 20 min during the incubation. After incubation, the secretory units were washed twice in fresh medium and then homogenized. The resulting homogenate was digested for 48 h at 50°C in predigested pronase (1.5 mg/ml, Sigma protease VI in 2.0ml of 0.2 M tris-HCl buffer, pH 8.0) (Lemkin and Farquhar, 1981). Fresh protease was added after 24 h, making the final volume 3.0 ml. The pronase digest was boiled and then dialysed for 48 h against distilled water to remove low molecular-weight breakdown products and free sulphate. The dialysate was lyophilized.
et
al.
The distribution of [3sS]-sulphate in different GAG species and glycopeptides was determined by a modification of the procedures of Hart (1976, 1978) as described by Lemkin and Farquhar (1981) and shown to be effective in the study of GAG synthesis by these glands (Cutler et al., 1987). Initial separation of total GAG from glycopeptides and unincorporated label was accomplished by gel filtration chromatography on a Sephadex G-50 column (1 x 120 cm). The lyophilized samples were suspended in and eluted with 0.1 M ammonium acetate in 20% (v/v) ethanol; 1.75 ml fractions were collected and 0.4 ml samples of each fraction were counted to determine their radioactivity. After chromatography of the initial sample to separate GAG from glycopeptides and free sulphate, all of the GAGcontaining fractions were pooled (total GAG fraction), lyophilized and subjected to a series of enzymatic and chemical degradative procedures, each followed by chromatography over Sephadex G-50 (1 x 12Ocm). Samples from cells incubated with sulphate were first chondroitinase ABC digested. The total GAG fraction was digested with 0.17 units/ml of Proteus vulgaris chondroitinase ABC (Seikagaku Kogyo Co. Ltd, Tokyo, Japan) in 3.0 ml of 0.01 M tri-HCI buffer, pH 8.0 for 24 h at room temperature. This enzyme degrades chondroitin 4,-6 and dermatan sulphates. The treated material was chromatographed on a Sephadex G-50 column (1 x 120 cm) and fractions counted to determine radioactivity (as described above) and to separate resistant GAG from degradation products. The resistant GAG fractions were pooled, lyophilized, and then subjected to nitrous acid oxidation for 3 h at room temperature to degrade the N-sulphated GAG, heparan sulphate. The degradation products were separated from any residual, resistant GAG by chromatography of Sephadex G-50 as before. Fractions were counted to determine radioactivity as described. This sequential procedure provided a profile of the S-GAGS synthesized by secretory unit precursors as well as by embryonic and maturing secretory units. Two or three experiments were run for each developmental stage. The data are reported as the mean for the experiments run.
RESULTS
Isolation of endbuds and secretory units
The procedure resulted in a preparation which was composed of 90-95% secretory units (acini or end buds) as determined by differential counting of random samples from 6 different preparations. The isolated units had normal appearances (Plate Fig. 1)
Plate 1 Fig. 1. Micrographs showing a typical secretory-unit preparation. These micrographs were taken of a preparation made from 21-day prenatal submandibular tissue. (A) A phase-contrast micrograph of the isolated secretory units. x 200. (B) A light micrograph of secretory units. Most of the cells appear normal. However, some cells at the periphery of one of the units appear swollen and are probably non-viable (arrows). Such cells would not exclude trypan blue. x 300. (C) An electron micrograph showing secretory cells from a typical isolated unit. The ultrastructure of the cells is normal. x 3000
Glycosamin~glycans
in developing salivary glands
Plate 1
391
L. S. CUTLER et al.
392
and more than 80% of the cells within the units were viable by trypan blue exclusion.
Table 1. Incorporation of [35S]-sulphate into GAG and glycopeptides by developing secretory units of submandibular glands (SMG)
Projile of GAG and glycopeptides
Typical GAG and glycopeptide profiles from 17day prenatal endbuds, 18- and 21-day prenatal, and l-6- and 35-day postnatal secretory units are shown in Text Fig. 2. The GAGS were eluted in fractions 19-26, glycopeptides were eluted in fractions 3346, and free sulphate after fraction 58. The percentage of the radioactivity present in the GAG and glycopeptide fractions is shown in Table 1. Secretory units from prenatal and neonatal secretory units incorporated [35S]-sulphate into both GAG and glycopeptides. Prenatal (18- and 21-day) and 35-day postnatal secretory units incorporated 82.4 and 71.2% of the [35S]-sulphate into GAG and only 17.6, 23.3 and 28.8% into glycopeptides respectively, while 17-day prenatal and l- and 6-day postnatal units incorporated 22, 40.4 and 36.1% of the [35S]sulphate into GAG and 78, 59.6, and 63.9% glycopeptides. Distribution of [“S]-sulphate in speciJic GAG fractions Chondroitin sulphate. The total GAG fractions
eluted from the initial Sephadex G-50 column (Text Fig. 2) were treated with chondroitinase ABC, and the resistant GAG and digestion products separated by chromatography on a Sephadex G-50 column. Approximately 73% of the sulphated GAG from the 17-day prenatal presecretory units was sensitive to
600 500
400 300 200 100
600 500 400 300 200 loo
[35S]-Sulphate
(%)
Glycopeptides
GAG
Prenatal SMG secretory units 78.0 17.6 23.3
22.0 82.4 76.7
17 days (2) 18 days (2) 21 days (3)
Neonatal SMG secretory units 1 day (3) 6 days (3) 35 days (3)
40.4
59.6
36.1 71.2
63.9 28.8
Total GAG and glycopeptides were collected from developing secretory units in vitro. GAG and glycopeptides were separated by gel chromatography. The percentage radioactivity in the GAG and glycopeptide fractions was calculated by dividing the c.p.m. in the GAG or glycopeptide peaks by the total c.p.m. of the GAG + glycopeptide peaks x 100. Data are reported as the mean of the experiments run. The number of experiments for each time point is shown in parentheses.
ABC treatment. This percentage of chondroitinase ABC-sensitive material in these early rudiments is consistent with the figure reported for the mouse submandibular gland by Thompson and Spooner (1982). The percentage of S-GAG which was sensitive to this treatment decreased significantly as secretory activity became apparent in the 18-day prenatal units. The chondroitinase ABC-sensitive material synthesized by ll-day prenatal units dropped to 41% of the S-GAG. The portion of the S-GAG produced that was sensitive to chondroitinase ABC treatment progressively decreased as acinar maturation proceeded. Chondroitin sulphate represented approximately 25-29% of the GAG produced by 21-day prenatal and l-day postnatal units, 18% of that made by 6-day units and only about 9% of that produced by 35-day postnatal secretory units (Table 2 and Text Fig. 3). Heparan sulphate. The S-GAG fractions that were resistant to chondroitinase ABC digestion were broken down by nitrous acid oxidation and rechromatographed on Sephadex G-50. Most of the chondroitinase-resistant GAG was broken down by this means. Only 24% of the S-GAG synthesized by 17-day prenatal endbuds was sensitive to nitrous acid chondroitinase
Table 2. Incorporation of [r5S]-sulphate into specific GAG by developing secretory units of submandibular glands [35S]-Sulphate (%) Prenatal Postnatal FRACTION NUMBER Fig. 2. Separation of GAG and glycopeptides by filtration on a Sephadex G-50 gel filtration column (1 x 120 cm) after pronase digestion (see Methods). The S-GAG eluted in the void volume in fractions 19-24. The glycopeptides were retarded and eluted in the included volume in fractions 3546. (A) Profile from 17-day embryonic presecretory units. (B) Profile from earliest differentiated secretory units at 18 days in utero (C) Profile from 21-day embryonic secretory units. (D) Profile from l-day postnatal units. (E) Profile from 6-day postnatal secretory units. (F) Profile from 35day postnatal secretory units.
GAG
Type
Chondroitin sulphates and dermatan sulphate Heparan sulphate Enzyme/nitrous acid resistant material
17
18
21
1
6
35
73 26
41.6 53.3
28.9 63.9
25.7 66.3
18.1 78.9
9.4 85.3
1
4.1
7.2
8
3
5.3
Newly synthesized [r’s]-sulphate labelled GAG from sccretory units in vitro. The figures are reported as the percentage of the total GAG synthesized. Data are reported as the mean of the experiments for each developmental time point.
~lyco~mino~~ans
o-i,, 14i8!1 I.1
PRENArAL
6
I
6
,
I
3
in developing salivary glands
1
14 21 28 35 POSTNATAL
AGE
Fig. 3. Graphic presentation of the change in the relative amounts of chon~oitin suiphate (dashed line) and heparan sulphate (solid line) produced in vitro by presecretory and secretory units of different developmental ages. The data are the mean of the experiments and are expressed as the percentage of the total S-GAG produced.
degradation. The proportion of heparan sulphate synthesized increased substantially at 18 days in utero and represented 54% of the S-GAG produced by these secretory units. As the units matured, they produced a S-GAG profile characterized by an increased content of heparan sulphate: at 21 days in utero and 1 day after birth, this represented about 66% of the S-GAG; at 6 days after birth, it was 79% of the S-GAG, and; by 35 days of age, it comprised 85% of the S-GAG (Table 2 and Text Fig. 3). Emzyme- and nitrous acid-resistant material A small amount of sulphate-containing material (l-8%) in all of the GAG samples from neonatal and prenatal secretory units was resistant to chondroitinase ABC and nitr0u.s acid digestion. The nature of this material was not determined. IXSCUSSION
We demonstrate a significant reduction in the proportion of chondroitin sulphate synthesized by the isolated endbuds of rat submandibular glands between day 17 and day 18 in utero. There is a concomitant rise in the proportion of heparan sulphate. This change in the profile of S-GAG produced by the developing endbuds is coordinated with the beginning of the cytodifferentiation of the endbuds into secretory cells. At this time, these cells develop the organelles required for the production and packaging of exocrine proteins (reviewed by Cutler, 1989; Cutler and Chaudhry, 1974). On the 18th day of gestation, the first recognizable secretory granules are found in the apical cytoplasm of the differentiating secretory cells and these cells develop the characteristic apical-basal polarity of organelles associated with exocrine secretory activity. This observation suggests that as the c~ellsundergo cytodifferentiation they alter the pattern of S-GAG that they produce. This shift in the pattern of S-GAG production is important because the synthesis of chondroitin sulphate and its deposition in the basement membrane surrounding the early rudiment of the gland is re-
393
quired for development of the morphogenetic branching pattern (Banerjee, Cohn and Bernfield, 1977; Bemfield and Banerjee, 1972; Bemfield, Banerjee and Cohn, 1972; Cohn, Banerjee and Bemfield, 1977; Thompson and Spooner, 1982, 1983). It is interesting that even though there is a dramatic drop in the relative amount of chondroitin sulphate produced, the arborized pattern of the glandular rudiment is maintained once it has been well established. Bemfield and co-workers (Rapraeger and Bemfield, 1983; Bemfield er al., 1984; Koda et al., 1985) have suggested that an increase in heparan sulphate production and its deposition on the cell surface or in the basement membrane might serve to stabilize the gland’s branched architecture. We now provide evidence that the proportion of heparan sulphate synthesized is increased at the time and in the fashion required to satisfy this hypothesis. Increases in the proportion of heparan sulphate synthesized in developing tissues have been associated with the onset and stabilization of cellular cytodifferentiation in several systems (Trelstad, 1985; Gallagher and Hampson, 1984; Morriss-Kay and Crutch, 1982). Defects in its production or deposition have been associated with various pathological conditions that are characterized by alterations in differentiated functions of the glands, for example, cystic fibrosis (Ben-Yoseph and Nadler, 1982). A change from a heparan sulphate-~ch GAG profile to one high in chondroitin sulphate is characteristic of neoplastic transformation of several tissues including salivary glands (Takeuchi et al., 1975, 1978, 1981) and a shift from a heparan-rich to a chondroitin-~ch extracellular matrix appears to be associated with a loss of cellular differentiation and destabilization of both tissue structure and function (reviewed by Trelstad, 1985; Gallagher and Hampson, 1984). It is important to remember that our study conditions did not allow differentiation between heparan sulphate that was at the cell surface or in the basement membrane because we only measured the total heparan sulphate. Unique ~pulations of heparan sulphate in specific locations may modulate different aspects of morphological and/or cytological differentiation. Thus, the rapid rise in heparan sulphate found in the submandibular rudiment at 18 days in utero may be directly related to the secretory cell differentiation which occurs at this point in the gland’s development. The relatively rapid rise in the proportion of heparan sulphate and the decrease in the chondroitin sulphate of the GAG profile produced by the developing secretory units begins to form a plateau at 6 days after birth. This plateau may reflect the fact that, at this time, the acini become function~ly differentiated and, with the exception of some minor modifications in the nature of the secretory product, cytodifferentiation is complete (Cutler and Chaudhry, 1974; Cutler, 1989). There is a slow but progressive increase in the heparan sulphate content of the GAG profile between 6 and 35 days, which continues until full glandular maturation is achieved at about 90 days of age (Cutler et al., 1987). Fully matured secretory units from the adult gland produce a S-GAG profile that is 90% heparan and only 4% chondroitin sulphate (Cutler et al., 1987). The ratio
L. S. CurLEn et al.
394
of heparan sulphate/chondroitin sulphate production by 17-day embryonic presecretory units was 0.36. This ratio shifted to 1.3 for ll-day embryonic secretory units, 9.1 for 35-day postnatal units, and 22.5 for fully mature secretory units (Cutler et al., 1987). The proportion of glycopeptides produced by presecretory units (17-day prenatal) and early neonatal (during the first postnatal week) secretory units was greatly increased when compared to other prenatal and older postnatal and adult (Cutler et al., 1987) units. Glycopeptides and GAG have been implicated in promoting neurite outgrowth and in the developmental association of specific nerve terminals with cell surface neuroreceptors (Anglister and McMahan, 1984; Gallagher et al., 1986). Unusual epithelialnerve contacts have been reported in the l&17day embryonic submandibular gland (Cutler and Chaudhry, 1973). Further, during the first postnatal week, catecholamine-containing nerve processes penetrate the developing parenchyma of the gland and establish functional connections with the acinar secretory cells (Bottaro and Cutler, 1984). It would be appealing to relate the high proportion of glycopeptides produced at these developmental times to the processes of nerve ingrowth and the establishment of functional neural connections in the neonatal submandibular gland. However, such relationships can only be inferred from the current information. Acknowledgement-This
work was supported by NIH grant
DE 05632.
innervation of the rat submandibular gland. Archs oral Biol. 29, 237-242.
Cohn R. H., Banerjee S. D. and Bemtield M. R. (1977) Basal lamina of embryonic salivary epithelial: Nature of glycosaminoglycan and organization of extracellular materials. J. cell Biol. 73, 464478. Cutler L. S. (1980) The dependent and independent relationships between cytodifferentiation and morphogenesis in developing salivary gland secretory cells. Anaf. Rec. 196, 341-347. Cutler L. S. (1989) Functional differentiation of salivarv glands. In: Handbook of Physiology: Salivary, Pancreatic, Gasrric. and Hewtobiliarv Secretion. Vol. 3 (Edited bv t Forte) pp. 413220. Am.-Physiol. Sot. Press; New Y&k: Cutler L. S. and Chaudhry A. P. (1973) Intercellular contacts at the epithelial-mesenc!tymal interface during prenatal development of the rat submandibular gland. Dev. Biol. 33, 229-240.
Cutler L. S. and Chaudhry A. P. (1974) Cytodifferentiation of the acinar cells of the rat submandibular gland. Dev. Biol. 41, 3141.
Cutler L. S. and Christian C. P. (1984) Inhibition of rat salivary gland adenylate cyclase by glycosaminoglycans and high molecular weight polyanions. Archs oral Biol. 29, 629633.
Cutler L. S. and Rendell J. K. (1984) Glycosaminoglycan synthesis by developing and adult submandibular gland secretory units. J. dent. Res. 63, 302. Cutler L. S., Christian C. P. and Rendell J. K. (1987) Glycosaminoglycan synthesis by adult rat submandibular gland secretory units. Archs oral Biol. 32, 413-419. Gallagher J. T. and Hampson I. N. (1984) Proteoglycans in cellular differentiation and neoplasia. Biochem. Sot. Transact. 12, 541-543.
Gallagher J. T., Lyon M. and Steward W. P. (1986) Structure and function of heparan sulfate proteoglycans. Biochem. J. 236, 313-325.
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