DEVELOPMENTAL

BIOLOGY

145,374-378

(1991)

BRIEF NOTE Transforming Growth Factor-P Modulation of Glycosaminoglycan Production by Mesenchymal Cells of the Developing Murine Secondary Palate Daniel Baugh, Institute, Jefferson Medical College, Thomas Jefferson

University,

Philadelphia,

Pennsylvania 19107

Accepted February 25, 1991 Development of the mammalian secondary palate requires proper production of the extracellular matrix, particularly glycosaminoglycans (GAGS) and collagen. Endogenous factors that regulate the metabolism of these molecules are largely undefined. A candidate for a locally derived molecule would be transforming growth factor pi (TGFP,) by virtue of its potency as a modulator of extracellular matrix metabolism by several cell lines. We have thus attempted to assign a regulatory role for TGF& in modulation of GAG production and degradation by mesenchymal cells of the murine embryonic palate (MEPM). Treatment with TGFB, or TGF/3,, but not IGF-II, resulted in a stimulation of total GAG synthesis. Furthermore, cells treated with both TGF& and TGFo showed a synergistic increase in GAG synthesis if pretreated with TGF& but not TGFa. Simultaneous stimulation with TGF& and TGF& did not elicit a synergistic response. These studies demonstrate the ability of TGFB, synthesized by embryonic palatal cells, to specifically stimulate GAG synthesis by MEPM cells. Other growth factors present in the developing craniofacial region may also modulate TGFB-induced GAG synthesis, a biosynthetic process critical to normal development of the embryonic pal:c) 1991 Academic Press. Inc ate.

INTRODUCTION

An increasing body of evidence indicates that growth factors are regulators of proliferation and differentiation in several cell lines and systems (Rizzino, 1988; Derynck, 1988; Rifkin and Moscatelli, 1989). Specifically, the transforming growth factor superfamily has been shown to potentiate and inhibit cell growth (Muller et al., 1987), regulate tissue differentiation (Masui et al., 1986; Kulyk et ah, 1989; Akhurst et ab, 1990), and alter extracellular matrix synthesis and degradation (Ignotz and Massague, 1986; Bassols and Massague, 1988; KeskiOja et al., 1988; Bachem et al., 1989). Interactions between, and modulations of responses to, polypeptide growth factors, especially transforming growth factor & (TGFP,), have been demonstrated in several culture systems (Bascom et ab, 1989; Ranganathan and Getz, 1990). In addition, the distribution of TGF& and other growth factors and their mRNA has been demonstrated during virtually every phase of murine development (Lund et ah, 1986; Heine et al., 198’7;Lehnert and Akhurst, 1988; Rappolee et al., 1988). Migration of neural crest cells into the first branchial arch and subsequent medial growth of the maxillary i To whom 001%1606/91 Copyright All rights

correspondence

should

$3.00

B 1991 by Academic Press, Inc. of reproduction in any form reserved.

he addressed 374

processesinto the primitive oral cavity result in the formation of the palatal processes. Between Days 12 and 14 (D12-D14) of murine gestation the palatal processes lengthen, increase their production of extracellular matrix molecules, and move into a position between the tongue and nasal septum. On Day 14 of gestation these processes fuse with each other and the nasal septum. Medial edge epithelial cells of the palate are subsequently removed, leaving a homogeneously mesenchyma1 palate. Specific extracellular matrix components, including glycosaminoglycans (GAGS), primarily hyaluranic acid (HA), and chondroitin sulfate, collagen, tenascin, and fibronectin are distributed throughout the mesenchymal core of the palate. Temporal and spatial distributions for these matrix components, as well as the necessity for proper matrix metabolism during palatal development, have been described (Hassell and Orkin, 1976; Pratt et al., 1973; Brinkley and Morris-Wiman, 1984; Sharpe and Ferguson, 1988). Much of organogenesis is thought to involve the actions of locally derived factors which regulate cell proliferation and differentiation. Candidates for these locally derived agents are the polypeptide growth factors by virtue of their ubiquitous presence in nearly all tissues and the presence of cell surface receptors for these agents on most cell types (Hollenberg, 1989; Mummery

BRIEF NOTE

et ul., 1990). Recent studies have demonstrated the presence of several growth factors in the tissues of developing craniofacial structures (Heine et al., 1987; Beck et al., 1987; Stylianopoulou et al., 1988; Lehnert and Akhurst, 1988; Pelton et al., 1989,199O; Fitzpatrick et al, 1990; Gehris et al., 1991). Embryonic palatal tissue grown in organ culture exhibits epithelial differentiation identical to that seen in vitro (Smiley and Koch, 1972; Shiota et al., 1990). Thus, in this system, locally derived soluble factors may be responsible for differentiation of the medial edge palatal epithelium. The localization of TGFO protein (Gehris et al., 1991) and its mRNA (Fitzpatrick et al., 1990; Pelton et al., 1990) in these cells during their differentiation supports such a notion. The objective of these studies was to determine whether TGFP,, a locally derived factor present in murine embryonic palatal tissue (Sharpe and Ferguson, 1988; Gehris et al., 1991), can regulate GAG synthesis by embryonic palate mesenchymal cells. By measuring synthesis as well as degradation of total GAGS by confluent primary cultures of murine embryonic palate mesenchyma1 (MEPM) cells, we have attempted to assign a specificity of response to this growth factor. MATERIALS

AND

METHODS

Cell Culture Mature male and female ICR (Ace Laboratories) mice, housed in a climate-controlled, 12 hr light cycled room, were mated overnight. Females exhibiting a vaginal plug in the morning were considered as being in DO of gestation. On D13 of gestation, females were sacrificed by cervical dislocation, and embryos dissected free of the extraembryonic membranes and placed in sterile calcium/magnesium-free phosphate-buffered saline. Palatal shelves, along with some underlying maxillary mesenchyme, were dissected, minced, and trypsinized to obtain a cell suspension. Primary cultures of MEPM cells were then seeded at a plating density of 2 X lo5 cells per 35-mm Falcon tissue culture dish (Fisher Scientific). Cultures were maintained in Opti-MEM (GIBCO) containing 5% fetal bovine serum, 500 units/ml penicillin, 500 cc.g/ml streptomycin, and 12.5 Fg/ml Fungizone (GIBCO) in an atmosphere of 5% CO,/95% air at 37°C until confluent. Treatment Regimen In all cases the treatment medium was a serum-free Dulbecco’s modified Eagle’s medium and Ham’s F-12 nutrient broth (1:l; DME/F-12; Sigma Chemical Co.), pH 7.4, containing antibiotics and antimycotics. Prior to treatment with growth factors, primary cultures were rinsed well with DME/F-12 to remove any traces of serum and allowed to equilibrate overnight. For all total GAG measurements, cells were exposed to growth fac-

375

tor for 24 hr. Transforming growth factors & and &. (TGFP,, TGF&) and platelet-derived growth factor (PDGF) were purchased from R&D Systems (Minneapolis, MN). Transforming growth factor cy (TGFcu) was purchased from Peninsula Labs (Belmont, CA) and insulin-like growth factor II (IGF-II) was purchased from Bachem Inc. (Torrance, CA). To analyze possible synergistic interactions between TGFP, and other developmentally relevant growth factors, cells were pretreated for 2 hr with either a dose of TGF& which significantly stimulated GAG production (10 rig/ml) or another growth factor at a dose (1 rig/ml) which did not itself stimulate GAG synthesis. Cells were then rinsed once with DME/F-12 and incubated for an additional 22 hr in the presence of both growth factors. Analysis of Glycosaminoglycans Confluent cells were treated with growth factors for 24 hr, the last 18 of which included the presence of 4 &i/ml D-[6-3H(l\r)]glucosamine hydrochloride (20-30 Ci/mmole; DuPont NEN). The media fraction was collected, GAGS were analyzed by cetylperidinium chloride (CPC) precipitation, and DNA in the cell layer was quantified by the method of Kissane and Robins (1958). GAG synthesis is presented as pronase-digested, nondialyzable, CPC-precipitable, [3Hlglucosamineincorporated DPM/pg DNA. The media fraction was heat-inactivated, protease-digested, and excess protein precipitated in the presence of 10% trichloroacetic acid. The supernatant was then extensively dialyzed against running water to remove any unincorporated glucosamine followed by dialysis in PBS (1:2). Carrier hyaluranic acid (50 pg/ml) and chondroitin sulfate (50 pug/ ml) were added to the dialysis retentates and GAGS precipitated by incubation with 0.2% CPC at 37°C for 1 hr. CPC-precipitable material (GAGS) was collected by centrifugation, dissolved in methanol, and counted in a scintillation spectrometer. For qualitative analysis, dialysis retentates were chromatographed at room temperature on a diethylaminoethyl (DEAE)-Sephacel column (15 X 60 mm) equilibrated with 0.05 M Tris, pH 7.2, and eluted with a linear gradient of 0.0 to 1.0 NaCl in 0.05 M Tris, pH 7.2. Individual fractions were counted for radioactivity, and identity of specific GAGS was determined by the salt concentration at which they eluted from DEAE (Pratt et al., 1973). In order to determine the rate of overall GAG degradation, confluent MEPM cells were pulsed for 3 hr with 10 pCi/ml D-[6-3H(N)]glucosamine hydrochloride. Dishes were rinsed once and chased with either DME/ F-12 or media containing 10 rig/ml TGF& both also containing an excess (10 mM) of unlabeled glucosamine hy-

376

DEVELOPMENTALBIOLOGY TABLE 1 DETERMINATIONOF SERUM-FREECULTURE CONDITIONS

Treatment

Direct

Cell viability

cell count

DPM/~g

DNA

570MEM 23.5 hi48.5 hr

2.0 x 106 + 3.3 x 106

98%

k 4%

1271 + 217 1596 k 22

2.6 x 10’ -+ 3.3 x lo5 1.9 x 106 t 3.1 x 106

97% 95%

i: 2.9% t 4.4%

1122 f t59 2

30 12

2.8 x lo6 2 5.6 x lo5 2.5 x lo6 k 6.8 x lo5

99% 98%

i 0.8% k 1.6%

t58 i t50 ?I

7 2

DME/F-12 23.5 hr 48.5 hr

DME/F-12+ supplements 23.5 hr 48.5 hr

Note. GD13 (Plug Day = 0) palatal shelves were dissected, minced, trypsinized, and seeded into monolayer cultures at a plating density of 2 X lo5 cells/ml as described under Materials and Methods. Cultures were maintained in Opti-MEM containing 5% fetal bovine serum until confluent. Serum-free treatment was a 1:l mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 nutrient mixture (DME/F12). DME/F-l2+ supplements included 5 pg/ml insulin, 50 pg/ml transferrin, 100 ~Mputrescine, and 30 nMsodium selenite. Direct cell counts were obtained from cell suspensions using a hemocytometer. Cell viability was determined by Erythrosin B dye exclusion. Total GAG was measured by CPC-precipitation as described under Materials and Methods. GAG values are represented as DPM of CPC precipitable material per microgram DNA. t These values are statistically different (P-value < 0.01) from the 5% MEM control group. All statistical parameters were calculated by the Student’s t test.

drochloride. GAGS were quantified at intervals up to 72 hr and the slopes of the generated lines were taken to indicate the rate of GAG degradation. RESULTS

AND

V0~~~~145,1991

al, 1989) synthesis. Treatment of confluent MEPM cells with TGFP, or TGF& (l-100 rig/ml) resulted in a dosedependent increase in total GAG production. Glycosaminoglycan metabolism, however, represents both synthesis and degradation. In order to determine the rate of GAG degradation in response to TGFP treatments, cells were treated with 10 rig/ml TGFP, and pulse-chased with [3H]glucosamine. The rate of loss of [3H]glucosamine-labeled GAGS in TGFP-treated MEPM cells was similar to that of untreated controls. These data indicate that TGFP treatment did not result in a diminution in the rate of GAG degradation and that the effect of TGFP, on total GAG production is to specifically increase synthesis. MEPM cells did not respond to IGF-II (l-100 rig/ml) with any significant increase in GAG synthesis, indicating that the response to the TGFPs is specific and the degree of response is directly proportional to the amount of growth factor present. Qualitative analysis of TGFP-stimulated synthesis of palatal GAGS by DEAE anion exchange chromatography indicated an increase of approximately 150% in total GAG synthesis with a reduction in the ratio of hyaluranic acid/sulfated GAGS (Table 2). Synthesis of sulfated GAGS was increased in response to TGFP treatment by 37% (Table 2), a response also seen in other cell types (Bassols and Massague, 1988; Bachem et al., 1989). These data contrast somewhat with a previous report by Sharpe and Ferguson (1988), who demonstrated TGFP-induction of both HA and chondroitin sulphate synthesis in MEPM cells. These studies, however,

TABLE

DISCUSSION

EFFECTOFTGF&

In order to precisely define the effects of TGF& and other growth factors on production of GAGS by MEPM cells, we first defined a treatment media devoid of serum that would sustain cell viability. Determination of cell growth and cell viability by dye exclusion indicated that confluent primary cultures of MEPM cells were equivalent in viability whether treated with MEM containing 5% serum, DME/F-12 alone, or DME/F-12 supplemented as described (Table 1). In the presence of serum, MEPM cells were able to synthesize lo- to 20-fold more GAGS than under serum-free conditions (Table 1). This emphasized the importance of a serum-free environment in which to assessthe ability of growth factors to modulate MEPM GAG synthesis since serum itself contained factors capable of stimulating GAG synthesis. Based on these results we chose to use DME/F-12 alone for subsequent studies. TGFP, alters extracellular matrix production in several cell lines, specifically by increasing fibronectin, collagen (Ignotz and Massague, 1986; Thompson et al., 1988), and GAG (Bassols and Massague, 1988; Bachem et

2

ON SPECIFIC GAGS SYNTHESIZEDBYMEPM

*% of Total DPM/pg DNA incorporated into GAG % as hyaluronic acid % as total sulfated GAGS **% Increase of Total GAG Sulfated GAG

CELLS

DME/F-12 treated

TGW’, treated

12.0% 67.3% 32.7%

30.0% 55.2% 44.8%

per 50%/37%

Note. Confluent monolayers of MEPM cells were treated in serumfree DME/F-12 containing 100 rig/ml TGFP, for 24 hr the last 18 hr of which they were labeled with 4 &i/ml D-[6-aH(N)jglucosamine hydrochloride. The medium was then collected, heat inactivated, and dialyzed extensively against 50 mM Tris-HCl, pH 7.2, until all unincorporated counts were removed. Eluted fractions were lyophilized and resuspended in a final volume such that 100 ~1 contained at least 10,000 CPM. Specific GAG profiles were determined by the salt concentration (0 to 1 M NaCl) at which they eluted from a DEAE anion exchange column. * Percentage DPM/pg DNA was determined from the total DPM/ pg DNA loaded and the total DPMs/pg DNA eluted in each peak. **Percentage increase over control was determined by: control value/measured value = fold; (l/fold ~ 1) X 100 = % increase.

BRIEFNOTE

GROWTH

FACTOR

Group TGF& + TGFtv TGFty/TGF/j TGF& /TGFtu TGF/j, + IGF-II IGF-II/TGF& TGF& /IGF-II TGF& + TGF& TGF/&/TGF,Y, TGF&/TGF& TGF& + PDGF PDGF/TGF& TGF&/PDGF

TABLE 3 INTERACTIONSIN GLYCOSAMINOGLYCAN BY MEPM CELLS DPMlpg 1458 1643 2682 1336 1746 2436 1311 1734 1519 1363 1326 1941

DNA

SD f 32 f 61 t23’7 f 92 +4m *737 &104 + 24 * 18 f 59 &316 +331

SYNTHESIS

P value

0.018 *0.001 0.220 0.067 0.027 0.033

no P value 0.041

!v’otc~. Confluent primary cultures of MEPM cells were rinsed with DME/F-12, allowed to equilibrate in serum-free media overnight, and, then treated with a dose of TGF-@, known to stimulate synthesis of GAGS (10 rig/ml) or a dose of another growth factor (1 rig/ml) which did not stimulate GAG synthesis. Test groups were pretreated for 2 hr with, for example, TGF-P, (TGF&/TGFn), media removed, dishes rinsed, and cells treated for the remaining 22 hr with both growth factors. TGF& + TGFn represents a theoretical calculated additive value for treatment with two different growth factors. All values represent DPM CPC-precipitable material per microgram DNA, with control (DME/F-12) values subtracted as background. * Signifies a statistically significant P value calculated by the HSDSTATS Student’s t test between the theoretically calculated additive value (i.e., TGF& + TGFtr) and the test value (i.e., pretreatment with TGF& followed hg 22 hr in the presence of TGF& and TGFn (TGF&/ TGFo) ).

were conducted using subconfluent cells in the presence of serum which may account for the apparent discrepancy. Indeed, these authors report that under serumfree conditions, similar to those used in the present study, only a low molecular weight class of Streptomyces hyaluronidase-sensitive material was preferentially stimulated by TGFP. To investigate whether TGF& can interact with other growth factors in modulating GAG production by MEPM cells, confluent primary cultures were exposed to either a stimulatory dose of TGFP, (10 rig/ml), a noneffective dose of another growth factor (1 rig/ml), or pretreatment with one growth factor (2 hr) followed by combined treatment for an additional (22 hr). The combined treatment groups were compared to a theoretical additive value based on the data derived from treatment with a single growth factor. In the case of TGFcu, pretreatment with TGFP, resulted in a synergistic increase in total GAG production (Table 3). An explanation for this phenomenon may be provided by the observation that TGFn binds to the EGF receptor in the developing mouse embryo (Next et al., 1980) and that TGFP, recruits EGF receptors (Thompson et ah, 1988). Moreover, Fernandez-Pol et ah, (1989) have shown that TGF& and

377

TGFa treatment can increase TGFa induction of EGF receptors. Therefore, the synergistic increase in total GAG synthesis by MEPM cells seen with combined TGF& and TGFa treatment could be a function of increased numbers of EGF cell surface receptors. Indeed, Ranganathan and Getz (1990) have reported the ability of TGF& pretreatment to synergistically increase the transcription of an EGF-dependent panel of genes. Binding to an increased number of cell surface EGF receptors could be responsible for the increase in total GAGS produced by the MEPM cells in the presence of TGFol and TGFB,. A modulation of response was also seen when cells were treated with PDGF and IGF-II, although the degree of synergy was not as great (Table 3). In the case of pretreatment with TGFP, or TGFP,, neither response was significantly greater than the additive value (TGFP, + TGFP,). Pretreatment for 2 hr with TGFP,, followed by a single growth factor exposure, did not show any alteration in the synthesis of total GAGS when compared to the appropriate control (data not shown). Thus, for MEPM cells, the response to TGFB, is not a transient one. A possible explanation for the nonsynergistic response seen with TGF& and TGF& treatment could be the presence of regulatory mechanisms to limit the effects of TGF/3 on cellular connective tissue synthesis. For example, the demonstration of TGFP-induced prostaglandin E, synthesis acting as a regulatory check on extracellular matrix synthesis (Diaz et al., 1989) is particularly relevant in that embryonic palatal tissue is capable of synthesizing significant quantities of PGE, (Alam et al., 1982). Thus, the response of the MEPM cells to the TGF/3s may reflect, in part, negative regulatory control of cellular responsiveness. Whether the direct mechanism of stimulation of GAGS by mesenchymal cells of the developing murine secondary palate includes the second messenger system of a specific receptor type remains to be seen. Receptor types I, II, and III are all present in MEPM cells (Linask et a,Z., 1991). The important notion from these experiments remains that TGFP,, a locally derived factor, is capable of regulating a matrix response in vitro characteristic of the in ,vivo situation. Furthermore, TGFP, can modulate the response of MEPM cells to produce GAG and to react to other locally derived factors which are present (Gehris et al., 1991). From the data presented here, interactions among several growth factors, including the TGFPs, appear to be a significant factor in regulating the synthesis of this extracellular matrix constituent, known to be critical for proper development of the palate. This work was supported in part by NIH Grant DE09540 and DE08199. M.D. was supported in part by NIH Training Grant HD07326.

378

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