Collagen synthesis during epitheliomesenchymal interactions

DEVELOPMENTAL

Collagen

BIOLOGY

22,

213-231

Synthesis

During Epitheliomesenchymal Interactions

MERTON Lt. Joseph

P. Kennedy, of Pediatrics,

(1970)

R.

BERNFIELD

Jr. Laboratories for Molecular Stanford Chiuersity School Stanford, California 94305 Accepted

November

Medicine, of Medicine,

Department

25, 1969

INTRODUCTION

Extracellular substances associated with cell surfaces and accumulating at tissue interfaces have been implicated as important agents during epitheliomesenchymal interactions (Grobstein, 1967). Ultrastructural studies of differentiating embryonic mouse pancreas epithelium cultured transfilter from salivary mesenchyme reveal collagenlike fibers at the epithelial surface (Kallman and Grobstein, 1964). When salivary, lung, and ureteric bud epithelium are treated with collagenase, a reversible alteration in epithelial contour occurs, adenomere formation is abolished, and the normal branched appearance is lost (Grobstein and Cohen, 1965; Wessells and Cohen, 1968). In addition, collagenlike fibers may be related to morphogenesis of feather germs (Stuart and Moscona, 1967; Wessells and Evans, 1968) as well as being frequently apparent ultrastructurally in epitheliomesenchymal basement membranes (Edds and Sweeny, 1961). The collagen associated with epithelium during epitheliomesenchymal interactions is postulated to be derived from both epithelial and fibroblastic cells by electron microscopic studies (Hay and Revel, 1963) and from mesenchyme by autoradiography (Kallman and Grobstein, 1965). The relationship of epithelial collagen to the morphogenetic influence of mesenchyme is unclear (Fitton Jackson, 1968). This communication describes investigations of collagen synthesis in which polypeptide hydroxyproline derived from proline is assessed during in vitro epitheliomesenchymal interactions. The results suggest that mesenchyme is a major source of epithelial collagen, that the presence of epithelial collagen is not dependent on epithelial morphogenesis, and that epithelium influences the amount of collagen in mesenchyme. 213

214

BERNFIELD

Incorporation

of

3H-3,h

- /-proline

24hrs

64 hrs v harvQ5t

Epithelium

Sl&m”f I ,

+ Total

proline OH -proline, amino acids

t Discard

Proline OH - proline

) i-CA

,

A esidue f Extract

Proline OH - proline Total protein

FIG. 1. Scheme for the handling of rudiments. At 0 hours, epithelium and mesenthyme are explanted on membrane filters either as transfilter or as isolated cultures. Mesenchyme grown alone is cultured transfilter from a plasma clot. The rudiments are incubated in standard medium and then labeled by either of two procedures. Continuous labeling: At 24 hours, cultures are transferred to medium containing

COLLAGEN

AND

EPITHELIOMESENCHYMAL METHODS

AND

INTERACTIONS

215

MATERIALS

Culture techniques. Submandibular salivary epithelium and salivary mesenchyme from 13-day-old mouse embryos were isolated, explanted and incubated as organ cultures using procedures described by Kallman and Grobstein (1965). Embryonic 11-day pancreatic epithelia of the mouse were prepared and cultured as described by Wessells (1964), except that whole rudiments were used. Mesenchyme subjacent to the thoracic mammary rudiments of 12day mouse embryos was prepared according to Kratochwil (1969). The age and desired developmental stage of the embryos were determined as described by Grobstein (1953b) and Wessells (1964). Transfilter cultures were prepared by clotting (rooster plasma) the epithelium on a membrane filter (Millipore, 22 f 3 ,J thick, 0.35 fi average pore diameter) and placing the mesenchyme transfilter without a clot. Epithelium grown alone was handled identically, except that mesenchyme was not placed on the opposite side of the filter. Mesenchyme cultured in the absence of epithelium was transfilter from a plasma clot. One set of rudiments was cultured on each filter assembly; each culture dish contained two assemblies and 1.0 ml of nutrient medium. The medium was prepared and changed daily except as otherwise indicated. Standard medium consisted of eight volumes of Eagle’s basal medium, one volume of horse serum, one volume of g-day chick embryo extract (50%), and contained 2 micromoles of glutamine, 50 units of penicillin, 50 pg of streptomycin, and 100 units of nystatin per milliliter. All incubations were at 37.5O in an atmosphere containing 5c; CO, in air and nearly saturated with water vapor. Labeling. Explants were incubated for 22 or 24 hours and then labeled with L-proline-3,4-“H by either of two procedures (see Fig. 1). Continuous labeling of transfilter cultures and of isolated epithelium and mesenchyme was performed by transferring the filter assembly to medium containing 5 &i/ml proline-“H. Cultures were 5 &i/ml proline-“H. Tissue is harvested at 64 hours. Mesenchymal labeling: At 22 hours, mesenchyme is transferred to medium containing 100 &i/ml proline-jH, and the transfilter component is incubated with fresh medium. At 24 hours, after 2 hours of mesenchymal labeling, the mesenchyme is rinsed three times and recombined with the transfilter component for reculture in standard medium. Tissue and medium are harvested at 64 hours. Details of incubation, washing, harvesting, and determination of protein-bound imino acids of tissue and acetic acid extracts of medium are described in Methods and Materials.

216

BERNFIELD

incubated in this medium for an additional 40 hours before mesenthyme and epithelium were harvested. For mesenchymal labeling, mesenchyme was removed from the filter assembly and incubated in medium containing 100 &i/ml proline-“H for 2 hours. The filter assembly, containing the epithelium or plasma clot, was transferred to fresh medium and placed in the incubator. After incubation with proline-“H, the mesenchyme pieces were rinsed sequentially (5 minutes each) in LO-ml portions of warm medium, Tyrode’s solution, and medium and then replaced on the filter assembly transfilter from the epithelium or plasma clot. The rudiments were recultured for an additional 40 hours prior to harvesting. The mesenchymal free proline-“H pool and the contribution of this pool to epithelia during reculture was assessed by measuring mesenchymal and epithelial TCA-soluble proline radioactivity (Table 1). After labeling and rinsing, mesenchymal free proline-“H was about one-seventh that of proline-“H incorporated into mesenchymal protein (cf. Table 3). Ninety minutes after reculture, this TCA-SOLUBLE

PROLINE-‘H

TABLE 1 IN MESENCHYME AND EPITHELIUM, MESENCHYMAL LABELING’ TCA-soluble

Time

of harvesting

Mesenchyme (cpm)

After After

labeling reculture 90 minutes 180 minutes

and rinsing with epithelium

2 HOUR

proline-‘H Epithelium (cpm)

4960 for: 480 610

31 25

’ Transfilter cultures of salivary epithelium and mesenchyme were incubated for 22 hours, and then the mesencbyme only was labeled for 2 hours in medium containing 100 &i/ml proline-JH. The mesenchyme pieces were rinsed sequentially in. 5.0 ml of warm and gassed nutrient medium, Tyrode’s solution, and nutrient medium. Labeled mesenchymes were either frozen in Tyrode’s solution (harvested after labeling and rinsing) or recultured with epithelium for 90 or 180 minutes prior to harvesting. Treatment with TCA was as described in Methods and Materials, except that the filtrate was collected. The filtrate was lyophilized to dryness for proline-‘H assay. Aliquots of proline-“H treated in an identical manner were used for determination of recovery. Four rudiments were pooled for each determination. Radioactivity in epithelium is expressed as total counts per minute per 4 epithelia and radioactivity in mesenchyme as counts per minute per 30 fig of amino acid (the equivalent of 4 average mesenchymes) .

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AND

EPITHELIOMESENCHYMAL

INTERACTIONS

217

proline pool was further reduced lo-fold; the remaining radioactivity was quantitatively recovered in the medium, yielding a proline-“H concentration in the medium of ca. 0.001 &i/ml. Because proline is a nonessential amino acid, the specific activity of free proline-“H should continually decrease during the reculture period. Free proline-“H in epithelia and surrounding clot was low, and similar amounts were found at 90 and 180 minutes of reculture. In data not reported here, reculture for 3 hours in the presence of nonradioactive proline (1.7 pmole/ml) did not significantly diminish the epithelial and mesenchymal proline-‘H pool or the amount of mesenchymal polypeptide hydroxyproline. Harvesting of cultures. Rudiments labeled by either procedure were harvested identically. After the 40-hour reculture period, the mesenchyme pieces were rinsed in cold Tyrode’s solution and frozen in solid CO,. The plasma clot and epithelium were peeled off the filter as a single piece, rinsed in cold Tyrode’s solution, and frozen. The frozen organ rudiments were thawed and sonicated (Branson sonifier), and TCA was added to a final concentration of 10%. After 30 minutes at O”, the TCA mixture was filtered under suction through a glass fiber filter (type A, Gelman Instrument Company, Ann Arbor, Michigan). The precipitate was rapidly washed with five lo-ml aliquots of ice cold 5:; TCA. Gloves were worn to avoid touching the filters with fingers in order to prevent adventitous amino acid contamination. The filter disks were blotted and dried. An acid-extractable protein fraction of the nutrient medium was prepared by removing cells and debris by centrifugation (2000 g, 10 minutes) and mixing the clarified supernatant with ‘k volume of 50:;~ TCA. After 15 minutes at O’, the mixture was centrifuged (3000 g, 10 minutes), and the tubes were drained and wiped dry. The pellet was extracted in 3.0 ml of 0.5 N acetic acid for 60 minutes at 4”, a procedure that extracts collagen and collagenlike proteins (Lukens, 1966; Hutton and Udenfriend, 1966) as well as other proteins. The resultant suspension was centrifuged (30,000 g, 20 minutes) and the clear supernatant was dialyzed us three changes of 200 volumes of distilled water for 24 hours at 4”. Protein concentration of the retentate was estimated spectrophotometrically (Warburg and Christian, 1941), and the extract was lyophilized to dryness. Assays. The lyophilized extract of the medium and the precipitate of the tissue on the filter disks were hydrolyzed in 6 N HCl at 125O and 18 psi for 3 hours. The hydrolyzates were evaporated to dryness in vacua. and the residue and disk were extracted with 4.0 ml of dis-

218

BERNFIELD

tilled water. Total amino acid was determined on the extract of the disks by a micromodification of the ninhydrin procedure (Baily, 1962). Values obtained were expressed in micrograms of total amino acid, using leucine to calibrate the ninhydrin standard curve. Total amino acid per 4 salivary mesenchymes was 30.08 f 11.1 pg (N = 58) (mean f SD). Carrier proline and hydroxyproline were added to an aliquot of the extract which was assayed by a modification of a specific chemical procedure for the radioactive imino acids, proline and hydroxyproline (Juva and Prockop, 1966). Proline recovery was estimated in each set of assays from the recovery of radioactivity derived from a known amount of L-proline-3,4-“H. Recovery of hydroxyproline in each sample was determined calorimetrically on the silicic acid column effluent. The mean recovery of proline as A’-pyrroline was 54.7 f 6.5% (N = 31) and that of hydroxyproline as pyrrole was 58.3 L- 5.4% (N = 182). The contamination of the hydroxyproline fraction by proline-3H was 0.56 f 0.14% (N = 18). Results are corrected for these recoveries and for loss of isotopic “H in the conversion of proline to hydroxyproline (Juva and Prockop, 1966; Goldberg and Green, 1968). Four cultured rudiments were pooled for each determination. Attempts to harvest cultured epithelial rudiments completely free of plasma clot were unsuccessful. Trypsin treatment of the clot removed epithelial polypeptide “H-labeled proline and hydroxyproline. Urokinase activation of fibrinolysins in the clot failed to yield epithelia completely free of adhering material. Concentrated solutions of urea and guanidine HCl resulted in inconsistent dissolution of the clot and complicated further processing of the rudiment. Consequently, the entire clot including epithelium was harvested, and radioactivity of epithelia is expressed as total counts per minute proline and hydroxyproline per 4 epithelia. Mesenchyme pieces were cultured in the absence of plasma clot, and results are expressed in terms of counts per minute per 30 fig amino acid, the equivalent amount of protein hydrolyzate of 4 average mesenchymes. Materials. Various lots of L-proline-3,4-“H (5.0 Ci/Fmole) (New England Nuclear Corporation) were found to vary in their content of a volatile component, which was removed by lyophilization. This radioactive contaminant was presumably tritiated water and amounted to 0.6-0.9% of the total radioactivity. “H-proline used for tissue labeling was lyophilized to dryness at least twice before use.

COLLAGEN

AND

EPITHELIOMESENCHYMAL

INTERACTIONS

An aliquot of the dry residue migrated as a single radioactive thin-layer chromatography in two solvents.

219

spot on

RESULTS

Source of Epithelial Collagen To determine whether the collagen associated with epithelia is produced by epithelia, the capacity of epithelia and mesenchyme to synthesize collagen was investigated in continuous labeling studies. The possibility that epithelial collagen is derived from mesenthyme was evaluated by culturing epithelia transfilter from labeled mesenchyme. To study the relationship between epithelial collagen and morphogenesis, epithelia were cultured transfilter from labeled, noninducing mesenchyme. Continuous mesenchymal and epithelial labeling. Isolated and transfilter cultures of epithelia and mesenchyme were incubated for 24 hours and then grown for 40 hours in medium containing proline-“H (Table 2A). After 40 hours of continuous labeling (64 hours total incubation), all tissues contained significant quantities of macromolecular proline and all mesenchymes yielded appreciable amounts of hydroxyproline. The OH-pro/pro ratios for mesenchyme did not approach values expected for native collagen; the amount of hydroxyproline averaged approximately 9% that of proline, reflecting the incorporation of proline into noncollagenous as well as collagenous proteins. Relative to proline incorporation, polypeptide hydroxyproline in mesenchyme was 5-fold greater than in transfilter epithelia and ca. 130 times greater than in epithelia grown alone. Salivary mesenchyme in culture spread over the filter surface, and as is characteristic of this tissue, showed no features of morphogenesis. Salivary epithelia grown opposite mesenchyme underwent progressive branching (Fig. 2) and contained measurable quantities of hydroxyproline, as did the rounded, compact pancreatic epithelia. Identical cultures incubated for longer periods subsequently developed acini characteristic of these tissues. Salivary epithelia grown alone lost their characteristic shape, became vesicular and failed to undergo recognizable morphogenesis. These isolated epithelia incorporated substantial amounts of proline yet contained essentially no hydroxyproline. These data suggest that proline incorporation into polypeptide hydroxyproline is low or non-existent in

220

BERNFIELD

INCORPORATION

OF PROLINE-JH

TABLE 2 INTO MESENCHYMAL

AND EPITHELIAL

Mesenchyme Culture WV

SmeslSep

Smes/Pep /Sep

Smesl-

Smes/Sep

Expt. NO.

Proline kpm)

OH-proline (cpm)

Epithelium OH-proline proline (‘r)

A. Forty-hour continuous mesenchymal 1 24,210 2,064 8.53 2 23,280 1,689 7.26 1

24,840

1 2

-

1 2

22,830 34,350

B. Two-hour 1 2 3

2,019

3,210 2,622

Pr0lilX? (cpm)

OH-proline (cpm)

OH-proline proline (‘r)

and epithelial 8,186 8,450

labeling 163 132

1.99 1.56

8.13

10,310

144

1.40

-

13,368 12,590

6 9

0.045 0.071

14.1 7.63

mesenchymal labeling followed 16,980 2,217 13.1 18,390 1,842 10.0 32,070 4,275 13.3 2,001 2,193

PROTEIN

Smes/Pep

1 2

16,110 19,530

12.4 11.2

Mmes/Sep

1 2

11,550 12,855

1,209 960

10.5 7.47

Mmesl-

1 2

9,606 8,175

1,104 957

11.5 11.7

by 40-hour 525 360 1,128

reculture 68 47 92

13.0 13.1 8.16

985 706

95 49

9.64 6.94

530 475

66 72

12.5 15.2

-

-

A. Forty-hour continuous labeling. Transfilter cultures and cultures of epithelium and mesenchyme grown alone were incubated for 24 hours and then continuously labeled for 40 hours in medium containing 5 &i/ml proline-“H (see Fig. 1). Four rudiments were pooled for each determination. B. Two-hour mesenchymal labeling. Transfilter cultures were incubated for 22 hours and then the mesenchymal component only was labeled for 2 hours in medium containing 100 &X/ml proline-“H. The labeled mesenchymes were rinsed and then recultured with the epithelium or plasma clot in standard medium for 40 hours (see Fig. 1). Four rudiments were pooled for each determination. In all tables, radioactivity in epithelium is expressed as total counts per minute per 4 epithelia and radioactivity in mesenchyme as counts per minute per 30 fig amino acid (the equivalent of 4 average mesenchymes). Smes represents salivary mesenchyme; Sep, salivary epithelium; Pep, pancreas epithelium; Mmes, mammary mesenchyme;-represents plasma clot, and the diagonal line indicates the membrane filter.

COLLAGEN

AND

EPITHELIOMESENCHYMAL

INTERACTIONS

221

FIG. 2. Living cultures; all x 50. (A) Transfilter culture of salivary epithelium and salivary mesenchyme at 64 hours of incubation. (B) Culture of salivary epithelium incubated in the absence of transfilter mesenchyme for 64 hours. (Cl Transfilter culture of salivary epithelium and mammary mesenchyme at 64 hours of incubation. Note absence of branching and resemblance to isolated epithelium in (B). (D) Transfilter culture of pancreatic epithelium grown with salivary mesenchyme for 64 hours. (Mesenchyme was removed from the top of the filter for clarity in the picture.)

222

BERNFIELD

epithelia grown in the absence of mesenchyme and not undergoing morphogenesis. The presence of protein-bound hydroxyproline-“H in epithelial rudiments grown transfilter from mesenchyme suggests either that epithelia undergoing development are capable of collagen synthesis, or that epithelial collagen is derived from transfilter mesenchyme. Mesenchymal labeling. The relative amount of collagen in epithelia cultured transfilter from labeled mesenchyme was compared with that found after continuous labeling of epithelia (Table 2B). The rudiments were cultured for 22 hours prior to mesenchymal labeling. Mesenchyme labeled for 2 hours and then recultured with epithelia for 40 hours contained about 12% hydroxyproline relative to proline, a value approximating that observed when mesenchyme were continuously labeled for 40 hours (cf. Table 2A). In contrast, epithelia grown transfilter from labeled mesenchyme showed about a 6-fold increase in OH-pro/pro ratio compared with epithelia continuously labeled as transfilter cultures. Although these data suggest that mesenchyme is a source of epithelial collagen, the radioactivity in the epithelia possibly may be derived from the mesenchymal free proiine-“H pool, from degradation of mesenchymal polypeptide proline-“H, or from the low level of isotope in the medium (cf. Methods and Materials). However, the proline thus presented to the epithelia would be incorporated similarly to that in the continuous labeling studies and would not account for the marked increase in the epithelial OH-pro/pro ratio noted when mesenchyme only is labeled. The amount of free hydroxyproline is irrelevant because polypeptide hydroxyproline is not derived directly from the free amino acid pool, but from proline present in collagenous proteins (Udenfriend, 1966). To determine whether the presence of epithelial collagen is dependent upon epithelial morphogenesis, salivary epithelia were cultured transfilter from collagen-producing mesenchymes that do not support salivary epithelial development. Salivary epithelia cultivated with mammary mesenchyme remain viable, but do not branch, or form adenomeres or acini (Fig. 2) (Kratochwil, 1969). Salivary epithelia grown transfilter from labeled mammary mesenchyme demonstrated nearly identical OH-pro/pro ratios and amounts of collagen as developing epithelia grown transfilter from labeled salivary mesenchyme (Table 2B). The OH-pro/pro ratio of mammary mesenthyme cultured with epithelium was similar to that of mammary mesenchyme grown alone.

COLLAGEN

Effect

AND

EPITHELIOMESENCHYMAL

223

INTERACTIONS

of Epithelium on Mesenchymal Collagen

Epithelium induces a morphological effect on mesenchyme during kidney (Grobstein, 1953a) and tooth (Koch, 1967) development. Cultured transfilter from salivary (Grobstein, 1967) or pancreatic epithelium (Golosow and Grobstein, 1962)) salivary mesenchyme exerts an inductive effect on the epithelium but does not itself undergo a microscopically recognizable alteration in morphology. To determine whether a “reciprocal” interaction of epithelium on salivary mesenchyme could be detected, collagen synthesis was studied in salivary mesenchyme cultured in the presence and the absence of epithelium (Table 3). Mesenchyme of both culture types incorporated similar amounts of proline-“H into polypeptide proline when harvested after the P-hour label period. However, the percentage of hydroxyproline in mesenchyme cultured with epithelia prior to labeling was slightly, but significantly (P < 0.005) increased relative to mesenchyme cultured alone. The difference in polypeptide hydroxyproline between mesenchyme cultured with and without epithelium became more exaggerated (2-fold difference in the OH-

EFFECT

TABLE 3 OF EPITHELIUM ON INCORPORATION OF PROLINE-‘H INTO MESENCHYMAL PROTEIN ~-HOUR MESENCHYMAL LABELINGS Harvest

Culture

me

“L

Expts.

Proline (cpm)

OHproline (cpm)

SmeslSep

5

31,600 zt 3200

1960 * 330

Smesl-

4

31,900 * 3700

1410 * 210

-

Harvest after 40.hour reculture period

after label period

No. nf

OH-praline proline (‘<)

Praline (cpd

OHpdine Icpm)

OH-proline praline I’,)

6.17 zt 0.47b

19,100 +z 2500

2200 It 400

11.6 zt 1.9

4.41

12,400 * 5700

650 f 320

+ 0.23’

5.12 i 0.35’

” Transfilter cultures of mesenchyme with epithelium or plasma clot were incubated for 22 hours, and then the mesenchymal component only was labeled for 2 hours in medium containing 100 &i/ml proline-“H. Labeled mesenchymes were rinsed and then either harvested or recultured with the transfilter component in standard medium for 40 hours (see Fig. 1). Four mesenchymes were pooled for each determination. Radioactivity is expressed as counts per minute per 30 fig of amino acid (the equivalent of 4 average mesenchymes). Values are means + standard deviation. Notations are as in the legend to Table 2. ’ Significance of the difference; P < 0.005. ’ Significance of the difference; P < 0.001.

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36,000

‘24,000

E 0”

LG d c

12,000

6,000

I

I

I

15

30

45

Hours

600

of reculturcz

L

0

I

15

I

30

I

45

al-%x labeling

FIG. 3. Proline and hydroxyproline of mesenchyme as a function of transfilter component and time. Transfilter cultures were incubated for 22 hours and then the mesenchymal component only was labeled for 2 hours in medium containing 100 &i/ml proline-“H. The labeled mesenchymes were rinsed and then recultured with the transfilter component in standard medium. The times indicated on the abscissa are hours following reculture. At the intervals indicated, mesenchymes were harvested for determination of radioactive, protein-bound imino acids. Four mesenchymes were pooled for each determination. Radioactivity is expressed as counts per minute per 30 rg of amino acid (the equivalent of 4 average mesenchymes). Note that there is a IO-fold difference in the ordinates for proline and hydroxyproline. Notations are as in the legend to Table 2.

pro/pro ratio) when the mesenchyme were assayed after the 40hour reculture period. This difference between mesenchyme cultured with and without epithelia was not seen after continuous labeling (Table 2A) or with mammary mesenchyme (Table 2B). Time course of mesenchymal collagen synthesis. In the experiments of Table 3, total mesenchymal radioactivity is lower after reculture than after the labeling period. The loss of radioactivity observed after 40 hours of reculture is predominantly due to a decrease in the amount of polypeptide proline. Polypeptide hydroxyproline decreases in mesenchyme cultured alone, but undergoes little apparent change in mesenchyme cultured transfilter from

COLLAGEN

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225

INTERACTIONS

epithelium. The alterations in proline and hydroxyproline during reculture were investigated by harvesting mesenchyme cultured transfilter from plasma clot or epithelium at intervals after labeling. The results of a typical experiment are presented in Fig. 3. As expected, mesenchyme of both culture types lost proline radioactivity during the reculture period. Polypeptide hydroxyproline of mesenthyme grown transfilter from epithelia increased at 10, 20, and 30 hours to levels greater than two standard deviations above the mean of those observed immediately after labeling (cf. Table 3) and then decreased, reaching a value at 45 hours that agrees well with Table 3. A similar increase in polypeptide hydroxyproline was not observed in mesenchyme grown alone; values at 15 and 30 hours were within two standard deviations of the amount observed after labeling and decreased at 45 hours to a value similar to those observed previously at 40 hours (Table 3). Polypeptide proline and hydroxyproline in the culture medium. Differential secretion of polypeptide proline or hydroxyproline into the culture medium during incubation possibly could account for the difference in percentage of hydroxyproline between mesenchyme TABLE Acm

Culture

EXTRACTABLE

type

PROTEIN ~-HOUR

Expt.

No.

OF MEDIUM MESENCHYMAL

Total

protein (mg)

4 AFTER

40 HOURS’

RECULTURE,

LABELING”

Proline (cpmhg protein)

OH-proline (cpmhg protein)

SmeslSep

1 2

9.10 6.00

376 493

23.1 23.7

SmeslPep

1 2

7.40 8.10

492 397

31.5 23.3

Smes/-

1 2

10.6 7.90

296 266

24.5 26.2

” Transfilter cultures were incubated for 22 hours and then the mesenchymal component only was labeled for 2 hours in medium containing 100 &i/ml proline-“H. Labeled mesenchymes were rinsed and then recultured with the transfilter component in standard medium for 40 hours (see Fig. 1). After 40 hours reculture, a dialyzed acetic acid extract was prepared from a cell-free TCA-precipitate of the nutrient medium. The total protein in the extract indicates the extraction efficiency and recovery of proteins from the medium. Nutrient medium from 2 dishes (from 4 transfilter cultures) were pooled for each determination. Notations are as in the legend to Table 2.

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BERNFIELD

grown in the presence and the absence of epithelium. An extract of the nutrient medium from cultures of labeled mesenchyme grown with and without epithelia was examined for polypeptide imino acids after 40 hours reculture. As the bulk of proteins in the medium are horse serum and chick embryo proteins, the amount of protein extracbed serves as an index of extraction efficiency and recovery (Table 4). The polypeptide hydroxyproline content of the extract is similar for each culture type, and there are slight differences between culture types in the amount of proline. DISCUSSION

The present studies attempt to define the source of collagen during in vitro epitheliomesenchymal interactions, clarify its role in these interactions and describe a possible means by which collagen synthesis may be regulated. In this study, collagen synthesis has been appraised by the presence of polypeptide hydroxyproline derived from radioactive proline. This method for assessment of collagen does not distinguish between true collagen, collagen precursors, and hydroxyproline-poor collagen. Experiments were designed to determine the source of the collagen associated with epithelia. Collagen synthesis was evaluated in transfilter cultures during continuous labeling of both components and during culture of epithelia transfilter from labeled mesenchyme. The cultures were incubated 22-24 hours prior to either type of labeling, and then recultured for 40 hours to assess any possible transfer of polypeptide material during morphogenesis in vitro. Epithelia continuously labeled and cultured in the absence of mesenchyme did not undergo morphogenesis and contained trace amounts of radioactive hydroxyproline. Mesenchyme grown without epithelia incorporated considerable amounts of proline into polypeptide hydroxyproline. Transfilter salivary cultures demonstrated epithelial branching and adenomere formation. Continuous labeling of such cultures showed that mesenchyme contain considerably more hydroxyproline (relative to proline) than epithelia. No conclusion regarding the source of epithelial collagen can be made from these data, because epithelia cultured alone may be markedly different from epithelia grown transfilter from mesenchyme in their ability to synthesize collagen. If developing epithelia produce collagen, then epithelia grown transfilter from labeled mesenchyme might synthesize polypeptide hydroxyproline from radioactive proline derived from the transfer

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227

of free proline across the filter or from label in the medium. The amount of hydroxyproline relative to proline incorporated into these epithelia would be similar to that seen in the continuous labeling experiments. If collagen were synthesized in the mesenthyme and transported to the epithelium, the OH-pro/pro ratio of the epithelium might approach that of native collagen, or at least be similar to that of the mesenchyme. The percentage of OH-pro/ pro of epithelia grown transfilter from labeled mesenchyme was found to be equivalent to that of the mesenchyme and severalfold greater than that of continuously labeled epithelia. Continuously labeled developing epithelia showed proline levels lo- to 20-fold greater than epithelia labeled from mesenchyme, although hydroxyproline levels were only 2- to 3-fold greater. In contrast, the mesenchymal OH-pro/pro ratio was similar in the two types of labeling. These data suggest that collagenous proteins (and possibly noncollagenous proteins) traverse the filter membrane. Other evidence suggesting that collagenous proteins are transported transfilter from mesenchyme to epithelium is provided by the experiments in which labeled mammary mesenchyme were cultured transfilter from salivary epithelium. Salivary epithelia cultured in this manner did not undergo morphogenesis, yet contained a similar proportion of hydroxyproline relative to proline as growing and branching epithelia cultured with salivary mesenchyme. Although these data suggest that collagen synthesized in the mesenchyme is transported across the filter to the epithelium, they do not exclude the possibility that the epithelium contributes proteins to its surface-associated material, nor do they rule out the synthesis of collagen by epithelia. Because of the small pool of free proline-“H in mesenchyme and epithelium soon after reculture, and the low concentration of free proline in the medium, it is unlikely that epithelial hydroxyproline is derived from these sources. Turnover of mesenchymal proteins may contribute to the radioactivity of epithelial proteins; however, free hydroxyproline cannot be utilized in de nouo collagen synthesis. It is doubtful that the epithelia synthesize appreciable amounts of nonhydroxylated protocollagen, because Goldberg and Green (1968) have shown that the exceedingly small amounts of protocollagen synthesized in nonfibroblastic cell lines are fully hydroxylated. Labeled mesenchymal collagen may form soluble subunits that are reaggregated (without de nouo synthesis) on the epithelial surface (Klein and Weiss, 1966). The present results are in agreement with the studies of Kallman

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and Grobstein (1965). On the basis of autoradiographic localization of radioactivity after mesenchymal labeling with proline-“H, these workers suggested that collagenous proteins synthesized in the mesenchyme were transported across the filter membrane and were deposited at the surface of the epithelium. Radioactivity derived from proline-“H was not localized at the epithelial surface when epithelium was labeled and cultured transfilter from mesenchyme. Collagen or collagenlike proteins are associated with the surface of developing epithelia (Grobstein, 1967), yet the function of these proteins in morphogenesis is unknown. Wessells and Cohen (1968) report that in vitro morphogenesis of ureteric bud, lung, and salivary, but not pancreatic epithelium are altered by collagenase treatment. They conclude that collagenase-sensitive materials may be essential to normal morphogenesis in instances where epithelial development is highly dependent upon a specific mesenchyme and where epithelial branching is predominant. Salivary epithelia specifically require homologous mesenchyme for development, and in organ culture undergo progressive branching prior to acinus formation. Pancreatic epithelia development is supported by mesenchyme from many sources (Golosow and Grobstein, 1962), and these epithelia form acini without branching or decided alteration in surface contour. Nevertheless, in the studies reported here, similar amounts of hydroxyproline were found associated with pancreatic and salivary epithelia cultured transfilter from salivary mesenchyme and there was little difference in OH-pro/pro ratios between the two epithelial types. Though the two types of epithelia may have distinct requirements for the site or type of collagen, the difference in sensitivity of pancreatic and salivary epithelium to collagenase is probably not due to the amount of collagenlike protein. Similar values for hydroxyproline and OH-pro/pro ratio were observed in the rounded, unbranched salivary epithelia grown transfilter from labeled mammary mesenchyme. Therefore, the quantity of epithelial collagen is also apparently independent of epithelial branching, albeit the localization of collagen may be critical. Since epithelial development is apparently not required for transfilter passageof collagen and inasmuch as inducing as well as noninducing mesenchymes transfer collagen across the filter membrane to the epithelium, the morphogenetic effect of mesenchyme is probably not exclusively mediated via collagen. Morphogenesis of a variety of organs involves interactions between

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epithelial primordia and their closely associated mesenchyme (Grobstein, 1967). One aspect of the interaction is the apparent ability of epithelium to increase collagen in mesenchyme. Salivary mesenchyme cultured transfilter from salivary epithelia incorporated substantially more proline into hydroxyproline than mesenchyme cultured alone. Since pancreas epithelia influenced salivary mesenthyme to a similar extent (cf. Table 2B), the increase in mesenchymal collagen is not due to a specific interaction between salivary epithelium and its own mesenchyme. The morphological correlate of the increase in mesenchymal collagen may be the conspicuous concentric orientation of mesenchymal cells and intercellular fibers around epithelial structures observed by Borghese (1950) and earlier workers in developing explants of whole salivary glands and in uiuo. The OH-pro/pro ratio of mammary mesenchyme was unaffected by the presence or absence of epithelia. The lack of an epithelial effect on mammary mesenchyme may be due to a qualitative difference between salivary and mammary mesenchyme or due to the lack of epithelial development. The effect of epithelia on mesenchyme could be due to an increase in protocollagen synthesis or hydroxylation or to a decrease in collagen degradation or secretion. A significant difference in OH-pro/pro ratio between mesenchyme grown in the presence and absence of epithelium was evident immediately after the 2-hour label period, suggesting that the effect of epithelium on mesenchyme began during the 22-hour culture period prior to labeling. During reculture the difference between the mesenchyme types became more distinct. Polypeptide hydroxyproline increased in mesenchyme grown tram+ filter from epithelia whereas polypeptide proline decreased similarly in both mesenchymal types, suggesting that the presence of epithelium influences hydroxyproline-containing proteins. Mesenthymes grown in the presence or absence of epithelium probably secrete similar amounts of collagen into the medium. At 46 hours of rerulture, the amount of polypeptide hydroxyproline in an acid extract of the medium was identical in both culture types. SUMMARY

The synthesis of collagenous proteins was examined in transfilter cultures of mouse embryonic mesenchyme and epithelium. Tissues were analyzed for polypeptide proline and hydroxyproline derived from proline-“H. Mesenchyme grown in the presence or absence of

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epithelium synthesized appreciable amounts of collagen, although radioactive collagen was not detected in epithelia grown in the absence of mesenchyme. The formation of epithelial collagen was investigated in transfilter cultures by continuous labeling of the rudiments and after mesenchymal labeling. After continuous labeling, pancreatic and salivary epithelia grown in association with salivary mesenchyme contained radioactive collagen. Compared to those exposed to continuous label, epithelia grown transfilter from labeled mesenchyme showed a 6-fold increase in the relative amount of radioactive collagen. Salivary epithelia cultured transfilter from labeled mammary mesenchyme did not undergo morphogenesis, yet contained a similar amount of radioactive collagen as pancreatic and salivary epithelia grown transfilter from labeled salivary mesenchyme. The label in the epithelia could not be accounted for by the transfer of free proline-“H from the mesenchyme. The results suggest that collagen synthesized in the mesenchyme is transported across the filter to the epithelium, but that the transfilter passage of collagenous proteins is not dependent upon epithelial morphogenesis. Developing epithelia were shown to regulate the amount of mesenchymal collagen. The relative amount of radioactive collagen in salivary mesenchyme cultured transfilter from pancreatic 0: salivary epithelium was significantly greater than in mesenchyme grown in the absence of epithelium. Equivalent quantities of polypeptide hydroxyproline were found in extracts of the nutrient medium from each culture type. No difference in the amount of radioactive collagen was detected between mammary mesenchyme cultured in the presence or absence of salivary epithelium. This work was supported, in part, by grants from The National Foundation and from the National Institutes of Health (HD 02147). A portion of these studies were performed while the author was a Research Investigator of the National Institute of Child Health and Human Development at the University of California, San Diego. The author wishes to thank Drs. Clifford Grobstein, Paul Fell, and Klaus Kratochwil for helpful discussions. The technical assistance of Mr. James Williams is gratefully acknowledged. REFERENCES BAILY, J. L. (1962). “Techniques in Protein Chemistry,” p. 73. Elsevier, New York. BORGHESE, E. (1950). The development in vitro of the submandibular and sublingual

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EDDS,

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differentiation Structure”

of (D.

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