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Developmental regulation of transforming growth factor β-mediated collagen synthesis in human intestinal muscle cells
GASTROENTEROLOGY 1996;110:92–101
Developmental Regulation of Transforming Growth Factor b – Mediated Collagen Synthesis in Human Intestinal Muscle Cells HILARY PERR,* PATRICIA OH,* and DANNA JOHNSON‡ *Department of Pediatrics, University of California, San Francisco, California; and ‡Department of Pathology, Medical College of Virginia, Richmond, Virginia
Background & Aims: The intestinal wall is formed by smooth muscle and the regulated deposition of specific collagen types. This study is the first to examine transforming growth factor b (TGF-b) in human fetal intestinal muscle. Studies localized TGF-b within the muscularis propria, identified the cellular source, measured TGF-b, and determined effects on collagen synthesis from 10 to 21 weeks of gestation. Methods: Localization of TGF-b within the intestinal wall and in cultured cells was determined immunohistochemically. TGF-b was measured by the CCL-64 cell growth inhibition assay. Collagen production was assayed as the uptake of 3H-proline into collagenase-digestible protein. Collagen types were identified by polyacrylamide slab gel electrophoresis and quantitated by densitometry. Experiments were performed in TGF-b (50–200 pg/mL) or anti–TGF-b (50–200 mg/mL). Results: TGF-b1 was localized in muscle cells of the muscularis propria and in culture. Muscle cells produced 340% more TGF-b at 11 weeks of gestation than at 20 weeks. At 10 weeks of gestation, TGF-b inhibited collagen production by 38%, but stimulated collagen synthesis by 70% at 21 weeks of gestation. TGF-b altered the expression of individual collagen chains in an age-specific manner. Conclusions: Smooth muscle cells secrete TGF-b during human fetal intestinal development. TGF-b stimulates or inhibits the expression of specific collagen chains depending on gestational age.
D
uring fetal development and in response to injury, the fate of the intestinal wall depends on the organization of smooth muscle cells and their product collagen. Of the 18 collagen types identified to date, types I, III, and V predominate in the muscle layers of the adult intestine.1 – 4 The matrix formed by these specific collagen types provides the scaffolding for normal muscle contraction and tissue growth and repair. Permutations in the matrix accompany inflammation, stricture formation, and neoplasia. In the fetal intestine, the matrix participates in organogenesis by modulating cell migration, attachment,5 – 11 and differentiation5,12,13 and by providing a reservoir for growth factors.14,15 We have shown pre-
viously that collagen synthesis in human fetal intestinal smooth muscle cells declines with gestational age from a maximum at 11 weeks (the earliest age studied) to adult levels by 20 weeks.16 The characteristic histological features of adult human intestine are established within the same developmental time frame. The regulation of collagen synthesis in the human intestine has been studied most extensively in adult smooth muscle cells. Exogenously administered transforming growth factor b (TGF-b), in particular, stimulates collagen production in such cells.17 Smooth muscle cells isolated from human fetal intestine secrete TGFb.18 Studies presented herein examine the role of TGFb in the development of the human intestinal muscle wall by determining effects on smooth muscle cell collagen synthesis.
Materials and Methods Reagents Dulbecco’s modified Eagle medium (DMEM), penicillin-streptomycin, phosphate-buffered saline, and fetal calf serum were obtained from GIBCO (Grand Island, NY); trypsin and crude and pure collagenase were obtained from Worthington (Freehold, NJ); tricine, hyaluronidase, bovine serum albumin, ovalbumin, leupeptin, aprotinin, pepstatin A, and Bouin’s fixative were obtained from Sigma Chemical Co. (St. Louis, MO); [3H]proline (22 Ci/mmole) was obtained from New England Nuclear (Boston, MA); ascorbate was from Fisher Scientific Co. (Fairlawn, NJ); purified TGF-b1 was from R & D Systems (Minneapolis, MN); neutralizing antibody to TGFb1 was from BioGenex Laboratories (Dublin, CA); NuSerum was from Collaborative Research (Bedford, MA); [3H]thymidine (43 Ci/mmole) was from Amersham (Arlington Heights, IL); Vectastain ABC kit was from Vector Laboratories, Inc. (Burlingame, CA); 3ⴕ,3ⴕ-diaminobenzidine HCl was from Polysciences, Inc. (Warrington, PA); and anti–TGFb1 was a generous gift from Dr. Kathleen C. Flanders from Abbreviations used in this paper: DMEM, Dulbecco’s modified Eagle medium; TGF-b, transforming growth factor b. 䉷 1996 by the American Gastroenterological Association 0016-5085/96/$3.00
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the Laboratory of Chemoprevention (National Cancer Institute, National Institutes of Health, Bethesda, MD).
Cell Isolation and Culture Intestine and skin were obtained from 10- to 21-week human abortuses as described previously.16 The protocol for this research was approved by the Institutional Review Boards of the University of California and the Medical College of Virginia according to the policies of the National Institutes of Health. Muscle cells for culture were obtained from fetal intestine as described previously.16 Dermal fibroblasts were obtained from the same fetuses to provide comparison with another collagen-producing mesenchymal cell. The cells were maintained in culture with DMEM containing 25 mmol/L tricine (pH 7.4), penicillin-streptomycin, and 10% fetal calf serum in 37⬚C incubators with circulating room air. Cells were maintained in primary culture for fewer than 3 days after isolation and then passaged once in 0.1% trypsin for use in experiments. These cultured cells have been shown to possess the phenotype and functions of corresponding cells at isolation.16
Immunochemical Staining Immunochemical staining was performed with an avidin-biotin conjugate method. Rabbit antiserum (anti–TGF-b 1–30) to a synthetic preparation of the first 30 amino acids of TGF-b1 was used.19 The antiserum was affinity purified. Segments of fetal intestine were fixed in formalin, serially cross-sectioned at 2-mm intervals, and postfixed in Bouin’s fixative. After routine tissue processing, 5-mm-thick sections were cut from paraffin blocks and mounted in gelatin-coated slides for immunohistochemistry. Primary cultures of fetal smooth muscle cells and dermal fibroblasts were plated in 8well Lab Tek chambers on glass slides (Nunc, Naperville, IL). On day 2 of culture, the chambers were washed twice with phosphate-buffered saline, air dried, fixed in cold acetone, and immunohistochemically stained as described below. The sections were deparaffinized and rehydrated. Endogenous peroxidase was blocked by incubation in 0.6% hydrogen peroxide. Permeabilization of the sections with hyaluronidase was followed by blocking of excess protein with 1% bovine serum albumin, 1% ovalbumin, and 5% normal goat serum. Primary antibody solutions (anti–TGF-b1 at 1:50 dilution) were applied to the slides and incubated overnight at 4⬚C in humidified chambers. After multiple washes in phosphate-buffered saline with 0.1% bovine serum albumin, slides were incubated with biotinylated goat anti-rabbit immunoglobulin G, washed, and then incubated with the avidin-peroxidase complex. After a final wash, slides were stained with 0.05% 3ⴕ,3ⴕdiaminobenzidine HCl and hydrogen peroxide and were counterstained with hematoxylin. Primary antibody was replaced with normal rabbit immunoglobulin G as a control.
Biosynthesis of TGF-b by Muscle Cells First passage muscle cells at 11, 15, 18, and 20 weeks of gestation and dermal fibroblasts at 15 and 20 weeks of
TGF-b REGULATION OF FETAL COLLAGEN 93
gestation were plated at 1.3 1 105 cells/cm2 in DMEM supplemented with 10% fetal calf serum. Twenty-four hours were permitted for cell attachment, and then the plates were washed three times with DMEM and 0% fetal calf serum. Cell cultures were then incubated for 24 hours in DMEM and 0% fetal calf serum with bovine serum albumin (1 mg/mL) and the following protease inhibitors: pepstatin A, aprotinin, and leupeptin (1 mg/mL). The conditioned medium was collected and stored at 070⬚C. The cell layer was washed three times with DMEM and 0% fetal calf serum; resuspended in DMEM and 0% fetal calf serum with bovine serum albumin (1 mg/mL) and protease inhibitors pepstatin A, aprotinin, and leupeptin (1 mg/mL); sonicated; and stored at 070⬚C. Experiments were repeated twice (n Å 6).
Determination of TGF-b Bioactivity Total TGF-b bioactivity of smooth muscle cell and of dermal fibroblast conditioned medium and cell lysates was determined by a modification of the CCL-64 mink lung growth inhibition assay of Danielpour et al.20 Mink lung cells are known to respond to TGF-b through inhibition of cell proliferation. CCL-64 mink lung cells were a gift from Dr. A. B. Fowler from the Medical College of Virginia. Mink lung cells were maintained in DMEM and 10% NuSerum at 37⬚C in a circulating room air incubator. Cells were passaged and plated at 6.5 1 106 cell/75 cm2 T-flask at 3-day intervals three times before use in experiments. Subconfluent cells were used in the growth inhibition assay. Cells were trypsinized, resuspended in 10% NuSerum, pelleted at 500g for 5 minutes, washed once with 10 mL of assay buffer (DMEM supplemented with 0.2% NuSerum, 10 mmol/L HEPES, pH 7.4, 25 U/mL penicillin, and 25 mg/mL streptomycin). Mink lung cells were plated at 5 1 104 cells/well (Danielpour, personal communication, March 1991). After 1 hour, TGF-b standard preparation or sample conditioned medium was added to the wells and followed by a 22-hour incubation. Mink lung cells were then pulsed for 3 hours with 0.50 mCi/well [3H]thymidine (43 Ci/ mmol). Mink lung cells were then fixed with 1 mL of methanol/acetic acid (vol/vol, 3:1). After 1 hour at room temperature, the wells were washed twice with 2 mL of 80% methanol. The label was extracted by incubation with 0.5 mL 0.05% crude pancreatic trypsin for 30 minutes, and then 0.5 mL of 1% sodium dodecyl sulfate was added. Latent activity was determined in 200-mL aliquots of the original conditioned medium or cell lysates that had been treated with 5 mL of 1.8N HCl neutralized with 7.5 mL of NaOH-HEPES and assayed as described above. Values were normalized to nanograms of DNA in the original cultures of confluent muscle cells or fibroblasts. Experiments were repeated twice (n Å 6).
Cell Proliferation Cell proliferation was determined by measuring DNA content fluorometrically as a reflection of cell number.21
Assays of Collagen Synthesis and Types Fetal human intestinal smooth muscle cells were plated onto 24-well plates to quantitate collagen synthesis by mi-
94 PERR ET AL.
croassay.22 The cultures were incubated with L-[5-3H]proline (40 mCi/mL, 22 Ci/mol) for 5 hours, heated to 90⬚C to stop isotope incorporation, and frozen at 020⬚C. After thawing, the cells were sonicated and 200 mL of the contents of each well was removed for determination of DNA content.21 A 1mL solution of chick embryo carrier protein (1.6 mg/mL in 10 mmol/L cold proline) was then added to each well. Radioactive protein was separated from unincorporated L-[5-3H]proline by repeated precipitation with 5% trichloroacetic acid at 4⬚C. The remaining trichloroacetic acid was removed with ethanol/ ether (3:1). The dried residue in each well was incubated for 90 minutes with 0.5 mL of buffer containing HEPES (60 mmol, pH 7.2), CaCl2 (0.25 mmol), and N-ethylmaleimide (1.25 mmol). Protein was reprecipitated with trichloroacetic acid, and the radioactivity in the solute was measured as an incubation blank. Trichloroacetic acid was removed, and the dried residue was suspended in incubation buffer with purified bacterial collagenase.23 After a 90-minute incubation, both the soluble [3H]collagen peptides and the [3H]noncollagen protein were counted. Collagen synthesis was expressed on a per cell basis (per nanogram of DNA) and relative to total protein synthesis (relative collagen synthesis), according to a standard correction for the enriched amino acid content of collagen.24 Before studying the effects of TGF-b, baseline studies were performed in 0% and 10% fetal calf serum to characterize fetal muscle cell collagen production and cell proliferation under quiescent conditions compared with conditions known to maximize collagen synthesis in adult muscle cells.25 Twenty-one– week gestational fetal human intestinal smooth muscle cells were plated onto 24-well plates at 8 1 105 cells/well in DMEM supplemented with 10% fetal calf serum for 24 hours to permit cell attachment. The wells were washed three times with serum-free medium (0% fetal calf serum), and then the medium was changed to 10% or 0% fetal calf serum. The wells were then assayed for collagen, noncollagen protein, and DNA content at 24, 48, and 72 hours using the assay described above. Dermal fibroblasts isolated from the same fetus were studied similarly. Experiments were performed four times (n Å 4–6). For determination of collagen types, first passage intestinal smooth muscle cells were plated in 100-mm dishes. Confluent cell cultures were incubated for 6 hours in serum-free medium containing ascorbate (0.1 mmol/L), [14C]proline (0.5 mCi/mL), [3H]proline (25 mCi/mL), [3H]glycine (25 mCi/mL), and baminopropionitrile (0.1 mg/mL). Protease inhibitors (ethylenediaminetetraacetic acid [20 mmol/L], N-ethylmaleimide [8 mmol/L], and phenylmethysulfonyl fluoride [1 mmol/L]) and 0.5 mol/L acetic acid were then added. After 18 hours at 4⬚C, the combined material was dialyzed against deionized water and then against 0.4 mol/L NaCl-0.1 mol/L Tris. Chymotrypsin (0.3 mg/mL) was added for 6 hours at 10⬚C, after which N-tosyl-L-phenylalanine chloromethyl ketone (0.1 mg/mL) was added overnight to stop the reaction.26 The soluble material was dialyzed against 0.5 mol/L acetic acid (4⬚C). Collagen was precipitated in NaCl (4.5 mol/L) and then resolubilized in acetic acid. The collagen bands were separated by polyacrylamide slab gel electrophoresis27 using a urea buffer (4 mol/L),
GASTROENTEROLOGY Vol. 110, No. 1
3% stacking gel, and 6% separating gel. Types I and V collagens were identified by comparison to standards of purified collagen types I and V collagens (GIBCO BRL, Gaithersburg, MD). Type III collagen chains were identified after interruption of electrophoresis and in situ reduction with dithiothreitol28 and comparison with a purified standard (GIBCO BRL). The dried gel was then exposed to radiographic film. The proportion of collagen chains expressed was determined by densitometry of the developed film.
Effects of Exogenous and Endogenous TGF-b on Collagen Synthesis, Cell Proliferation, and Expression of Collagen Types Human intestinal smooth muscle cells at 10, 15, and 21 weeks of gestation were plated at 8 1 105 cells/well in DMEM supplemented with 10% fetal calf serum. After 24 hours, the wells were washed 31 with 0% fetal calf serum, and then the cells were exposed to 50–200 pg/mL TGF-b or 50–200 mg/mL anti–TGF-b (control). The choice of TGF-b concentrations follows preliminary studies over a range of 0.1– 1000 pg/mL TGF-b (data not shown), in which effects on collagen synthesis were noted at 50–200 pg/mL. The choice of anti–TGF-b concentrations was chosen to neutralize amounts of endogenous TGF-b that had been measured previously by bioassay (physiological concentrations of TGF-b in skin wounds approximate 5 ng/mL).29 Cells were exposed to exogenous TGF-b or neutralizing antibody for 24 hours. Collagen synthesis, noncollagen protein, and DNA content were assayed as described above. The timing and duration of exposure to TGF-b or its neutralizing antibody was based on measures of collagen synthesis in the absence of exogenous factors (0% fetal calf serum) in which collagen synthesis was maximal between 24 and 48 hours. Experiments were repeated twice (n Å 4). To determine the effect of endogenous TGF-b on collagen synthesis and cell proliferation, cells from 21-week fetal intestine were grown in culture as described above. After 24 hours, cells were exposed to serum-free medium (control) or to 50– 200 mg/mL anti–TGF-b. After a further 24 hours, collagen and DNA content were assayed. The experiments were repeated twice (n Å 4). To determine the effect of TGF-b on expression of collagen types, cells from 10- and 21-week fetal intestine were grown initially in 100-mm dishes supplemented with DMEM and 10% fetal calf serum. Once confluent, cultures were washed 31 with DMEM and 0% fetal calf serum and then exposed to either anti–TGF-b (50 mg/mL) or TGF-b (50 pg/mL) for 24 hours, including the 6-hour pulse. The duration and timing of exposure to TGF-b or its neutralizing antibody were chosen to be consistent with the duration and timing of exposure previously studied in the collagen synthesis assay. The concentrations studied were those that exerted maximal changes in the amount of collagen synthesized determined by the collagen synthesis assay. Controls remained in DMEM and 0% fetal calf serum alone. Collagen types and their relative proportions
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TGF-b REGULATION OF FETAL COLLAGEN 95
Table 1. Distribution of Intestinal TGF-b1 Immunoreactivity With Fetal Age Enterocytes
Fibroblasts
Smooth muscle
Ganglion cells
Age (wk)
Crypt
Villus
Mucosal and submucosa
Mucosa
Propria
Vascular
Muscularis propria
11 13 17 19
0–1 0–1 0–1 0–1
2 2 2 2
1 1 1 1
NP NP NP 3
3 3 3 3
2 2 2 2
NP NP 2 2
NOTE. Intensity of antibody staining was graded from 0 to 3 in enterocytes, fibroblasts, smooth muscle cells, and ganglion cells. The most intense staining occurred in the muscle cells of the muscularis propria at all ages studied. NP, structures that are not present at the particular gestational age.
were determined as described previously. Experiments were performed twice (n Å 3).
Statistical Analysis Experimental values were compared with control values by Student’s t test and considered significantly different at P values of õ0.05.
Results TGF-b Distribution and Secretion Localization of TGF-b1 throughout the developing muscle layers of human fetal intestines (11, 13, 17, and 19 weeks of gestation) was shown by immunohistochemical staining. TGF-b1 stained most strongly in the smooth muscle cells of the muscularis propria and of mesenteric vasculature at all ages studied (Table 1). The intensity and distribution of staining did not change with fetal age and was the same in the duodenum, jejunum, and ileum. Smooth muscle cells of the muscularis mucosa, a structure not present until 19 weeks of gestation, stained with equal intensity. At 17 weeks of gestation, ganglion cells appeared within the muscularis propria and showed moderate cytoplasmic reactivity for TGF-b1 (Figure 1). Weak reactivity of mucosal and submucosal stromal cells was observed at all gestational ages, with crypt enterocytes staining the least. Villus enterocytes showed moderate immunostaining for TGF-b1. The extracellular matrix showed little or no staining for TGF-b1 (Figure 1). No positive staining occurred when the primary antibody was replaced by normal rabbit immunoglobulin. Cultured intestinal smooth muscle cells isolated from the same specimens used for histochemical studies at 11, 13, 17, and 19 weeks of gestation showed immunocytochemical staining for TGF-b1 similar to muscle cells in the histological section (Figure 2). The proportion of cells staining for TGF-b1 varied with cell density. In areas of subconfluent cell growth with little adjacent cell contact, nearly 100% of cells showed mild-to-moderate, finely stippled cytoplasmic immunoreactivity for TGF-
b1. Zones of confluent cells showed minimal cytoplasmic staining in 0%–5% of cells. This pattern of staining did not change with fetal age and was similarly found in cultured fetal dermal fibroblasts (not shown). TGF-b secretion in muscle cells at 11 weeks of gestation was 81 greater than in cells from 15 to 18 weeks of gestation and more than 351 greater than cells from 20 weeks of gestation (Figure 3). Dermal fibroblasts produced more TGF-b than muscle cells at the same gestational age (Figure 4). At 15 and 20 weeks of gestation, respectively, fibroblasts synthesized 141 and 301 more TGF-b than intestinal muscle cells isolated from the same fetus. Collagen Production and Cell Proliferation Smooth muscle cells produced more collagen in serum-free medium, the largest difference in collagen production being observed at 48 hours, when collagen synthesis was 50% greater than in 10% fetal calf serum (Figure 5A). In contrast, fetal dermal fibroblasts produced the same amount of collagen in 0% and 10% fetal calf serum (Figure 5B) and less collagen per nanogram of DNA than muscle cells at all time points in culture (P õ 0.003 at 24 hours; P õ 0.001 at 48 hours; P õ 0.002 at 72 hours). No change in noncollagen protein production occurred from 24 to 72 hours in 0% fetal calf serum (88.35 cpm/ ng DNA { 6.03 at 24 hours; 73.87 { 7.20 at 48 hours; and 81.60 { 4.50 at 72 hours). However, in 10% fetal calf serum, noncollagen protein production increased initially at 24 hours (60.47 { 12.24 at 0 hours vs. 112.93 { 17.95 at 24 hours) and then steadily declined (75.13 { 4.20 at 48 hours and 33.63 { 0.90 at 72 hours). At 72 hours, noncollagen protein production was nearly 2.51 greater in 0% fetal calf serum than in 10% fetal calf serum (P õ 0.001) but otherwise was the same at earlier time points. DNA content did not change in 0% fetal calf serum (5105.06 { 136.33 ng DNA compared with control 5871.88 { 827.13). In contrast, DNA content nearly dou-
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Figure 2. Immunocytochemical detection of TGF-b1 in cultured fetal intestinal smooth muscle cells. (A ) Nearly 100% of the subconfluent cells showed cytoplasmic immunoreactivity for TGF-b1 (brown stain). (B ) In contrast, õ5% of confluent cells were immunoreactive. The bluish color represents counterstain of the nuclei with H&E. Cells from 20 weeks of gestation are shown (original magnification 401).
Figure 1. Immunohistochemical studies of TGF-b1 in human fetal intestine at 17 weeks of gestation. Cells containing TGF-b1 stain brown with anti–TGF-b1 using an avidin-biotin conjugate method as described in Materials and Methods. The bluish color represents counterstain of the background with H&E. Muscle cells of the circular and longitudinal muscle layers stained the most intensely (arrowheads). Ganglion cells, arterial smooth muscle cells, stromal cells of the mucosa and submucosa, and enterocytes of the villous tips stained moderately. Enterocytes of the crypts stained minimally (original magnification 4001).
(200 pg/mL), TGF-b inhibited noncollagen protein production 59% at 10 weeks (2.00 { 0.37 vs. 4.87 { 0.01 cpm/ng DNA; P õ 0.019) and 37% at 15 weeks (1.51 { 0.26 vs. 2.38 { 0.08 cpm/ng DNA; P õ 0.021) and stimulated noncollagen protein production 130% at
bled in 72 hours in 10% fetal calf serum (10,097.96 { 68.16). Effects of Exogenous and Endogenous TGF-b The greatest effects on collagen synthesis were mediated by 50 pg/mL TGF-b (Table 2). At 10 weeks of gestation, TGF-b inhibited collagen production by 38% compared with anti–TGF-b. In contrast, at 15 and 21 weeks of gestation, TGF-b stimulated collagen synthesis by 45% and 70%, respectively. Higher concentrations (200 pg/mL) had no effect on collagen production at 10 and 15 weeks of gestation (data not shown). Higher concentrations of TGF-b were required to produce an effect on noncollagen protein synthesis. Effects were age specific but did not show the same pattern of inhibition or stimulation observed with collagen synthesis. Noncollagen protein synthesis was inhibited by TGFb at 10 and 15 weeks of gestation and was stimulated at 21 weeks of gestation. At the highest concentration
Figure 3. Effect of fetal age on TGF-b secretion by intestinal smooth muscle cells. Twenty-four–hour collections of conditioned medium and cell lysates were assayed for total TGF-b bioactivity from primary cultures of intestinal smooth muscle cells at 11, 15, 18, and 20 weeks of gestation. TGF-b bioactivity was determined by the CCL64 mink lung epithelial cell growth inhibition assay as described in Materials and Methods. Values are expressed as mean { SEM (n Å 6). Cells at 11 weeks of gestation secreted more TGF-b than cells from 15, 18, and 20 weeks (P õ 0.001).
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TGF-b REGULATION OF FETAL COLLAGEN 97
Table 2. Effect of TGF-b on Collagen Synthesis by Fetal Human Intestinal Smooth Muscle Cells Fetal age (wk)
Anti–TGF-b
TGF-b
P value
10 15 21
3.17 { 0.45 4.26 { 0.80 11.14 { 1.72
1.60 { 0.24 7.62 { 0.75 36.36 { 3.78
õ0.042 õ0.01 õ0.0001
NOTE. Collagen synthesis (in counts per minute per nanogram of DNA) was assayed in cell cultures exposed to anti–TGF-b (50 mg/mL) or TGF-b (50 pg/mL) as described in Materials and Methods. Values are expressed as mean { SEM (n Å 4).
200 mg/mL). At 21 weeks of gestation, collagen synthesis was inhibited 94% by 100 mg/mL anti–TGF-b (Figure 6A). Anti–TGF-b increased DNA content in a concentration-dependent fashion and maximally by 65% at 200 mg/mL (Figure 6B). No effect on collagen synthesis was Figure 4. Comparison of TGF-b bioactivity by different mesenchymal cells from the same fetus at 15 and 20 weeks of gestation. Primary cultures of dermal fibroblasts (F ) and intestinal smooth muscle cells (M) were isolated and maintained in culture. Total TGF-b bioactivity of conditioned medium and cell lysates from 24 hours in culture were determined by the CCL-64 mink lung epithelial growth inhibition assay as described in Materials and Methods. Values are expressed as mean { SEM (n Å 6). At each age, fibroblasts secreted more TGF-b than muscle cells (P õ 0.001). Both mesenchymal cells decreased TGF-b secretion at 20 weeks compared with 15 weeks of gestation (P õ 0.001).
21 weeks (10.31 { 2.55 vs. 4.90 { 0.50 cpm/ng DNA; P õ 0.048). The effects of endogenous TGF-b on collagen synthesis and cell proliferation were studied in the presence and absence of a neutralizing antibody, anti–TGF-b (50–
Figure 5. Collagen synthesis in the absence (0% FCS) and presence (10% FCS) of fetal calf serum. Fetal human cells were isolated at 21 weeks of gestation. (A ) Intestinal smooth muscle cells. *Significance from 10% fetal calf serum: P õ 0.002 at 48 hours and P õ 0.013 at 72 hours. (B ) Dermal fibroblasts. Values are expressed as mean { SEM (n Å 4–6). Collagen production by dermal fibroblasts was the same in 0% and 10% fetal calf serum.
Figure 6. Effect of endogenous TGF-b on collagen synthesis and cell proliferation by human intestinal smooth muscle cells. Cells from 21 weeks of gestation were isolated and grown in culture as described in Materials and Methods. After 24 hours, cells were exposed to serum-free medium (control) or to 50–200 mg/mL anti–TGF-b. After another 24 hours, (A ) collagen synthesis and (B ) DNA content were assayed. *Significance from serum-free medium, P õ 0.001. Values are expressed as mean { SEM (n Å 4).
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Table 3. Effect of TGF-b on Synthesis of Collagen Types and Component Chains by Fetal Human Intestinal Smooth Muscle Cells Collagen type Chain 10 wk Control TGF-b Anti–TGF-b 20 wk Control TGF-b Anti–TGF-b
I
III
V
a1
a2
a1
a1
Total
6.265 3.966 2.853
2.102 11.12 2.848
4.836 12.05 9.167
1.017 12.32 0.595
14.22 39.456 15.463
3.519 2.081 2.79
2.234 2.592 2.76
3.498 6.112 6.16
0.58 0.0 0.0
9.831 10.785 11.71
NOTE. Cells were isolated and cultured at 10 and 20 weeks of gestation. Confluent cultures were exposed to either TGF-b (50 pg/mL) or to anti–TGF-b (50 mg/mL) for 24 hours, including the 6-hour pulse. Controls remained in serum-free medium. Collagen types and chains were identified by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and autoradiography and then quantitated by densitometry as described in Materials and Methods. Relative proportions of collagen types referred to in the text are calculated as a percent of total collagen production.
observed when the same experiment was repeated at 11 weeks of gestation (data not shown). Higher concentrations (250–1000 mg/mL) of neutralizing anti–TGF-b were then used, in consideration of the fact that cells at 11 weeks of gestation were shown to make 340% more TGF-b than cells at 20 weeks of gestation and may require more neutralizing antibody. At these increased concentrations of anti–TGF-b, cells at 11 weeks of gestation became detached from the plate. At 10 and 20 weeks of gestation, cells expressed similar proportions of types I, III, and V collagens (Table 3). At 10 weeks of gestation, muscle cells synthesized 59% type I collagen, 34% type III collagen, and 7% type V collagen. At 20 weeks of gestation, muscle cells synthesized 59% type I collagen, 36% type III collagen, and 6% type V collagen. However, the proportions of a1 and a2 chains comprising type I collagen differed with fetal age. The a1(I)/a2(I) chain ratio was approximately 3:1 at 10 weeks of gestation and 1.4:1 at 20 weeks. Addition of TGF-b or anti–TGF-b to the cultures altered the proportions and types of collagens expressed in an age-specific manner. At 10 weeks of gestation, a1(I) chains were decreased 77% by TGF-b and 59% by anti–TGF-b. At 20 weeks, a1(I) chains were decreased by 47%, but in contrast with 10 weeks of gestation, anti–TGF-b increased a1(I) chains by 50%. Effects on the expression of a2(I) chains were greatest at 10 weeks of gestation. TGF-b nearly doubled the expression of a2(I) collagen chains at 10 weeks of gestation but had no effect at 20 weeks of gestation. Anti–TGF-b did not change the amount of a2(I) chains at 10 weeks. TGF-b
exerted opposite effects on the expression of a1(III) chains depending on gestational age. TGF-b decreased a1(III) chains by 8% at 10 weeks but increased a1(III) chains by 62% at 20 weeks. Anti–TGF-b increased a1(III) chains by 74% at 10 weeks of gestation but decreased a1(III) chains by 56% at 20 weeks. The greatest effects on a1(V) chains occurred at 10 weeks of gestation, in which TGF-b increased these chains by ú240% compared with same-age controls.
Discussion This is the first study to examine the role of TGFb in regulating collagen synthesis by smooth muscle cells isolated from the human fetal intestinal wall. TGF-b inhibited or stimulated collagen synthesis in an age- and type-specific manner, suggesting that TGF-b may play a key role in modulation of intestinal development in the human fetus. TGF-b is prominent in the musculature of the developing human intestine, including circular, longitudinal, vascular, and muscularis mucosa components. This distribution profile is conserved throughout intestinal development as observed previously in animal embryos and postnatally30–32 and in contrast with fetal lung where TGF-b localizes to sites of mesenchymal-epithelial interactions.33 These observations support a role for autocrine TGF-b in the morphogenesis of the intestinal muscle wall. Synthesis of TGF-b seems to be dependent on cell density. Synthetic activity was especially notable in subconfluent areas, as observed previously in human vascular smooth muscle cells34 and bovine aortic endothelial cells.35 The current study shows that, at the same cell density, the secretion of TGF-b declined with advancing developmental age, a trend previously noted in animals. Cultures of neonatal rat aortic smooth muscle cells and lung fibroblasts secrete twofold to fourfold more TGF-b than the respective adult cells.36 Together, these studies show an inverse relationship between advancing developmental age and endogenous TGF-b secretion. Additional evidence that autocrine TGF-b plays a role in intestinal development is the observation that fetal intestinal smooth muscle cells synthesized more collagen in the absence of exogenous serum factors. This response is distinctly different from that reported for adult human intestinal smooth muscle cells (same cell type and different age) and from control fetal dermal fibroblasts (different mesenchymal cell and same developmental age). Adult intestinal smooth muscle cells require exogenous factors (10% fetal calf serum or TGF-b) to stimulate collagen synthesis.17,25 Maximal collagen synthesis accompanies cell proliferation in 10% fetal calf serum.25
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Fetal intestinal smooth muscle cells increased collagen synthesis in 0% fetal calf serum without cell proliferation, whereas dermal fibroblasts from the same fetus produced the same amount of collagen whether in 0% or 10% fetal calf serum. All three cell types proliferated only in the presence of serum. These findings emphasize that in the human intestine, fetal collagen synthesis is differently regulated than in the adult and is specific to cell type. TGF-b inhibited total collagen synthesis at 10 weeks of gestation and stimulated synthesis at 20 weeks of gestation. The magnitude and pattern of inhibition or stimulation by TGF-b was not the same for noncollagen protein synthesis, indicating that effects on collagen synthesis were selective. Selective enhancement of collagen synthesis by TGF-b has been shown in adult and fetal cells at a single developmental stage.17,36 The present study extends these findings to include inhibitory as well as stimulatory effects on collagen synthesis that are not only selective but specific to gestational age. Individual collagen types and component chains are also synthesized selectively in fetuses treated with TGFb. Studies in animal fetal organ systems have focused on the effects of exogenous TGF-b at a single gestational age. Bone cells secrete TGF-b and synthesize collagen, 90% of which is type I collagen.37 Studies in 22-day-old fetal rat bone show threefold increases in type I collagen after exposure to exogenous TGF-b.38 Fetal rat lung epithelial cells synthesize types I, III, IV, and V collagens. All types increase more than twofold after exposure to TGF-b (types I and III are greater than types IV and V).39 Collagen synthesis in primary cultures of rabbit articular chondrocytes increases in response to TGF-b without change in the proportion of specific collagen chains.40 The current investigation uses neutralizing antibodies to study the effects of endogenous TGF-b at several fetal ages in human cells. This approach shows that autocrine TGF-b exerts very different effects on matrix composition of muscle at various stages of intestinal development. The mechanisms responsible for the age-specific effects of TGF-b on collagen synthesis by human fetal intestinal smooth muscle cells are not understood. The magnitude of TGF-b secretion does not correlate with collagen synthesis at any given fetal age. One level of control may be achieved through differential expression of TGF-b receptor isoforms through which TGF-b1 may mediate specific biological functions. Three receptor subtypes have been identified41 (for review, see Roberts and Sporn42). In the epithelial Mv1Lu cells, the type I receptor influences the interaction of the cell with the extracellular matrix, whereas the type II receptor mediates the antiproliferative activity of TGF-b1.43 In the mouse embryo, type II receptor expression is greater in undifferentiated mesenchyme
TGF-b REGULATION OF FETAL COLLAGEN 99
than in more mature cells.44 Differential expression of receptor isoforms may account for uncoupling of effects on proliferation from effects on matrix but cannot explain age-specific opposing effects of TGF-b on collagen synthesis in intestinal smooth muscle cells. TGF-b enhances transcription of the proa2(I) collagen gene.45 Additional mechanisms must regulate the agespecific increases and decreases in the production of different collagen chains. The action of TGF-b may be modified by diffusible cytokines and nondiffusible factors such as matrix.42,46 Animal fetal smooth muscle cells secrete various cytokines in an age-dependent fashion.47 The distribution and types of muscle-associated matrices change during development of the intestinal wall.48,49 Consequently, the smooth muscle cell extracellular environment changes with fetal age, possibly contributing to the differential production of collagens in response to TGF-b. Collagen composition significantly influences the biomechanical and biochemical properties of the developing intestinal wall. Effective contraction and compliance of the wall arise from the specific arrangement and proportions of collagen chains.50 – 52 Tissue remodeling and inflammatory response may also be affected.53 – 56 Increased collagen deposition modifies TGF-b–induced growth suppression.57 Therefore, changes in the amount and types of collagens expressed in the presence of TGF-b must influence intestinal development and likely subsequent postnatal tissue response. In fact, neoplasia may represent a recapitulation of fetal events in postnatal life. Colon carcinoma biopsy fragments contain a peculiar form of collagen that is absent in normal adult tissues but present in fetal tissues.58 Further, fetal intestinal smooth muscle cells and neoplastic cells share a certain autonomy through endogenous production of and response to TGF-b.59,60 This study shows that TGF-b alters the synthesis of collagen chains in a type- and developmental age-specific manner. The resulting modulation in the amount and type of collagen can influence smooth muscle cells, inflammatory cells, and epithelial cells within the intestinal wall through effects on cell migration, attachment, proliferation, and differentiation. These cellular events direct normal or abnormal development, postnatal healing, and growth. Thus, understanding the developmental expression of different collagen chains induced by TGF-b in conjunction with the relatively autonomous nature of fetal intestinal smooth muscle cells may shed light on adult intestinal pathophysiology.
References 1. Vuorio E, de Crombrugghe B. The family of collagen genes. Ann Rev Biochem 1990;59:837–872.
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2. Van der Rest M, Garrone R. Collagen family of proteins. FASEB J 1991;5:2814–2823. 3. Li K, Tamai K, Tan EML, Uitto J. Cloning of type XVII collagen. Complementary and genomic DNA sequences of mouse 180 kilodalton bullous pemphigoid antigen (BPAG2) predict an interrupted collagenous domain, a transmembrane segment, and unusual features in the 5ⴕ-end of the gene and the 3ⴕ untranslated region of the mRNA. J Biol Chem 1993;268:8825–8834. 4. Graham MF, Diegelmann RF, Elson CO, Lindblad WJ, Gotschalk N, Gay S, Gay R. Collagen content and types in the intestinal strictures of Crohn’s disease. Gastroenterology 1988;94:257– 265. 5. Bernfield M, Banerjee SD, Koda JA, Rapraeger AC. Remodeling of the basement membrane as a mechanism of morphogenetic tissue interaction. In: Trelstad RL, ed. The role of the extracellular matrix in development. New York: Liss, 1984:545–572. 6. Furcht LT. Critical factors controlling angiogenesis: cell products, cell matrix, and growth factors. Lab Invest 1986;55:505–509. 7. Lacovara J, Cramer EB, Quigley JP. Fibronectin enhancement of directed migration of B16 melanoma cells. Cancer Res 1984;44:1657–1663. 8. McCarthy JB, Hagen ST, Furcht LT. Human fibronectin contains distinct adhesion- and motility-promoting domains for metastatic melanoma cells. J Cell Biol 1986;102:179–188. 9. Montesano R, Orci L, Vassalli P. In vitro rapid organization of endothelial cells into capillary-like networks is promoted by collagen matrices. J Cell Biol 1983;97:1648–1652. 10. Nabeshima K, Kataoka H, Koono M. Enhanced migration of tumor cells in response to collagen degradation products and tumor cell collagenolytic activity. Invasion Metastasis 1986;6:270–286. 11. Thesleff I, Barrach HJ, Fiodart JM, Vaheri A, Pratt RM, Martin GR. Changes in the distribution of type IV collagen, laminin, proteoglycan and fibronectin during mouse tooth development. Dev Biol 1981;81:182–192. 12. Form DM, Pratt BM, Madri JA. Endothelial cell proliferation during angiogenesis in vitro modulation by basement membrane components. Lab Invest 1986;55:521–530. 13. Hosick HL, Inaguma Y, Kusakabe M, Sakakura T. Morphogenesis of mouse mammary epithelium in vivo in response to biomatrix prepared from a stimulatory fetal mesenchyme. Dev Growth Differ 1988;30:229–240. 14. Ekblom P. Basement membrane proteins and growth factors in kidney differentiation. In: Trelstad RL, ed. The role of the extracellular matrix in development. New York: Liss, 1984:173–206. 15. Gospadarowicz D, Ferrara N, Schweigerer L, Neafield G. Structural characterization and biological functions of fibroblast growth factor. Endocr Rev 1981;8:95–108. 16. Perr HA, Grider JR, Mills AS, Kornstein M, Turner DA, Diegelmann RF, Graham MF. Collagen production by human smooth muscle cells isolated during intestinal organogenesis. Anat Embryol 1992;185:517–527. 17. Graham MF, Bryson GR, Diegelmann RF. Transforming growth factor–b1 selectively augments collagen synthesis by human intestinal smooth muscle cells. Gastroenterology 1990;99:447– 453. 18. Perr HA, Johnson D. Human fetal intestinal smooth muscle cells secrete TGF-b and synthesize collagen without cell proliferation (abstr). J Cell Biochem 1993;17E (Suppl):181. 19. Flanders KC, Thompson NL, Lissel DS, Van Obberghen-Schilling E, Baker CC, Kass ME, Ellingsworth LR, Roberts AB, Sporn MB. Transforming growth factor–b1: histochemical localization with antibodies to different epitopes. J Cell Biol 1989;108:653–660. 20. Danielpour D, Dart LL, Flanders KC, Roberts AB, Sporn MB. Immunodetection and quantitation of the two forms of transforming growth factor–beta (TGF-b1 and TGF-b2) secreted by cells in culture. J Cell Physiol 1989;138:79–86.
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21. Labarca C, Paigen K. A simple, rapid and sensitive DNA assay procedure. Anal Biochem 1980;102:344–352. 22. Diegelmann RF, Bryson GR, Flood LC, Graham MF. Microassay to quantitate collagen synthesis by cells in culture. Anal Biochem 1990;186:296–300. 23. Peterkofsky B, Diegelmann RF. The use of a mixture of proteinase-free collagenase for the specific assay of radioactive collagen in the presence of other proteins. Biochemistry 1971;10: 988–994. 24. Diegelmann RF, Peterkofsky B. Collagen biosynthesis during connective tissue development in chick embryo. Dev Biol 1972; 28:443–453. 25. Graham MF, Drucker DEM, Diegelmann RF, Elson CO. Collagen synthesis by human intestinal smooth muscle cells in culture. Gastroenterology 1987;92:400–405. 26. Clark CC, Richards CF. Isolation and partial characterization of precursors to minor cartilage collagens. Coll Relat Res 1985;5:205–233. 27. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–685. 28. Sykes B, Puddle B, Francis M, Smith R. The estimation of two collagens from human dermis by interrupted gel electrophoresis. Biochem Biophys Res Commun 1976;72:1472–1480. 29. Cromack DT, Sporn MB, Roberts AF, Merino MF, Dart LL, Norton JA. Transforming growth factor beta levels in rat wound chambers. J Surg Res 1987;42:622–628. 30. Pelton RW, Saxena B, Jones M, Moses HL, Gold LI. Immunohistochemical localization of TGF-b1, TGF-b2, and TGF-b3 in the mouse embryo: expression patterns suggest multiple roles during embryonic development. J Cell Biol 1991;115:1091–1105. 31. Barnard JA, Beauchamp RD, Coffey RJ Jr., Moses HL. Regulation of intestinal epithelial cell growth transforming growth factor type beta. Proc Natl Acad Sci USA 1989;86:1578–1582. 32. Barnard JA, Warwick GJ, Gold LI. Localization of transforming growth factor beta isoforms in the normal murine small intestine and colon. Gastroenterology 1993;105:67–73. 33. Heine UI, Munoz EF, Flanders KC, Roberts AB, Sporn MB. Colocalization of TGF-beta and collagen I and III, fibronectin and glycosaminoglycans during lung branching morphogenesis. Development 1990;109:29–36. 34. Nikol S, Isner JM, Pickering G, Kearney M, Leclerc G, Weir L. Expression of transforming growth factor–b1 is increased in human vascular restenosis lesions. J Clin Invest 1992;90:1582– 1592. 35. Merrilees MJ, Sodek J. Synthesis of TGF-b1 by vascular endothelial cells is correlated with cell spreading. J Vasc Res 1992;29: 376–384. 36. McGowan SE. Influences of endogenous and exogenous TGF-b on elastin in rat lung fibroblasts and aortic smooth muscle cells. Am J Physiol 1992;263:L257–L263. 37. Hunter GK. In vitro studies on matrix-mediated mineralization. In: Hall BK, ed. Bone. Volume 4. Boca Raton, FL: CRC, 1991: 225–247. 38. Centrella M, Casinghino S, Ignotz R, McCarthy TL. Multiple regulatory effects by transforming growth factor–b on type I collagen levels in osteoblast-enriched cultures from fetal rat bone. Endocrinology 1992;131:2863–2872. 39. DiMari SJ, Howe AM, Haralson MA. Effects of transforming growth factor–b on collagen synthesis by fetal rat lung epithelial cells. Am J Respir Cell Mol Biol 1992;4:455–462. 40. Galera P, Vivien D, Pronost S, Bonaventure J, Redini F, Loyau G, Pujol J-P. Transforming growth factor–b1 (TGF-b1) up-regulation of collagen type II in primary cultures of rabbit articular chondrocytes (RAC) involves increased mRNA levels without affecting mRNA stability and procollagen processing. J Cell Physiol 1992;153:596–606. 41. Cheifetz S, Weatherbee JA, Tsang ML-S, Anderson JK, Mole JE,
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42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
Lucas R, Massague J. The transforming growth factor–b system, a complex pattern of cross-reactive ligands and receptors. Cell 1987;48:409–415. Roberts AB, Sporn MB. The transforming growth factor–bs. In: Sporn MB, Roberts AB, eds. Handbook of experimental pharmacology. Peptide growth factors and their receptors. Heidelberg, Germany: Springer–Verlag, 1990:419–472. Chen R-H, Reinhard E, Derynck R. Inactivation of the type II receptor reveals two receptor pathways for the diverse TGF-b activities. Science 1993;260:1335–1338. Lawler S, Candia AF, Ebner R, Shum L, Lopez AR, Moses H, Wright CVE, Derynck R. The murine type II TGF-b receptor has a coincident embryonic expression and binding preference for TGFb1. Development 1994;120:165–175. Rossi P, Karsenty G, Roberts AB, Roche NS, Sporn MB, de Crombrugghe B. A nuclear factor 1 binding site mediates the transcriptional activation of type I collagen by transforming growth factor– b. Cell 1988;52:405–414. Battegay EJ, Raines EW, Seifert RA, Bowen-Pope DF, Ross RR. TGF-b induces bimodal proliferation of connective tissue cells via complex control of an autocrine PDGF loop. Cell 1990;63:515– 524. Seifert RA, Schwartz SM, Bowen-Pope DF. Developmentally regulated production of platelet-derived growth factor–like molecules. Nature 1984;311:669–671. Simon-Assmann P, Kedinger M, Haffen K. Immunocytochemical localization of extracellular-matrix proteins in relation to rat intestinal morphogenesis. Differentiation 1986;32:59–66. Simon-Assmann P, Duclos B, Orian-Roussear V, Arnold C, Mathelin C, Engvall E, Kedinger M. Differential expression of laminin isoforms and alpha 6–beta 4 integrin subunits in the developing human and mouse intestine. Dev Dyn 1994;201:71–85. Fleischmajer R, Timpl R, Tuderman L, Raisher L, Wiestner M, Perlish JS, Graves PN. Ultrastructural identification of extension aminopeptides of type I and III collagens in human skin. Proc Natl Acad Sci USA 1981;78:7360–7364. Broek DL, Madri J, Eikenberry EF, Brodsky B. Characterization of
TGF-b REGULATION OF FETAL COLLAGEN 101
52.
53.
54.
55.
56.
57.
58.
59. 60.
the tissue form of type V collagen from chick bone. J Biol Chem 1985;260:555–562. Martinez-Hernandez A, Gay S, Miller EJ. Ultrastructural localization of type V collagen in rat kidney. J Cell Biol 1982;92:343– 349. Jaffe R, Deykin D. Evidence for a structural requirement for the aggregation of platelets by collagen. J Clin Invest 1974;53:875– 883. Balleisen L, Marx R, Kuhn K. Platelet-collagen interaction. The influence of native and modified collagen (type I) on the aggregation of human platelets. Haemostasis 1976;5:155–164. Stenn KS, Madri JA, Roll FJ. Migrating epidermis produces AB2 collagen and requires continual collagen synthesis for movement. Nature 1979;277:229–232. Grotendorst GR, Seppa HE, Keinman HK, Martin GR. Attachment of smooth muscle cells to collagen and their migration toward platelet-derived growth factor. Proc Natl Acad Sci USA 1981;78:3669–3672. Nugent MA, Newman JJ. Inhibition of normal rat kidney cell growth by transforming growth factor–b is mediated by collagen. J Biol Chem 1989;264:18060–18067. Pucci-Minafra I, Luparello C, Andriolo M, Basirico L, Aquino A, Minafra S. A new form of tumor and fetal collagen that binds laminin. Biochemistry 1993;32:7421–7427. Roberts AB, Sporn MB. Transforming growth factors. Cancer Surv 1985;4:683–705. Sporn MB, Roberts AB. Peptide growth factors and inflammation, tissue repair, and cancer. J Clin Invest 1986;78:329–332.
Received October 25, 1994. Accepted September 1, 1995. Address requests for reprints to: Hilary Perr, M.D., Department of Pediatrics, Box 0136, University of California, San Francisco, California 94143. Fax: (415) 476-1343. Supported by Clinical Investigator Award DK01849 from the National Institutes of Health and by the Tooley Foundation. The authors thank Drs. D. Mongomery Bissell, M. Michael Thaler, and F. J. Roll for editorial assistance and expert advice and Dr. Theodore J. Gradman for statistical consultation.
Developmental Regulation of Transforming Growth Factor b – Mediated Collagen Synthesis in Human Intestinal Muscle Cells HILARY PERR,* PATRICIA OH,* and DANNA JOHNSON‡ *Department of Pediatrics, University of California, San Francisco, California; and ‡Department of Pathology, Medical College of Virginia, Richmond, Virginia
Background & Aims: The intestinal wall is formed by smooth muscle and the regulated deposition of specific collagen types. This study is the first to examine transforming growth factor b (TGF-b) in human fetal intestinal muscle. Studies localized TGF-b within the muscularis propria, identified the cellular source, measured TGF-b, and determined effects on collagen synthesis from 10 to 21 weeks of gestation. Methods: Localization of TGF-b within the intestinal wall and in cultured cells was determined immunohistochemically. TGF-b was measured by the CCL-64 cell growth inhibition assay. Collagen production was assayed as the uptake of 3H-proline into collagenase-digestible protein. Collagen types were identified by polyacrylamide slab gel electrophoresis and quantitated by densitometry. Experiments were performed in TGF-b (50–200 pg/mL) or anti–TGF-b (50–200 mg/mL). Results: TGF-b1 was localized in muscle cells of the muscularis propria and in culture. Muscle cells produced 340% more TGF-b at 11 weeks of gestation than at 20 weeks. At 10 weeks of gestation, TGF-b inhibited collagen production by 38%, but stimulated collagen synthesis by 70% at 21 weeks of gestation. TGF-b altered the expression of individual collagen chains in an age-specific manner. Conclusions: Smooth muscle cells secrete TGF-b during human fetal intestinal development. TGF-b stimulates or inhibits the expression of specific collagen chains depending on gestational age.
D
uring fetal development and in response to injury, the fate of the intestinal wall depends on the organization of smooth muscle cells and their product collagen. Of the 18 collagen types identified to date, types I, III, and V predominate in the muscle layers of the adult intestine.1 – 4 The matrix formed by these specific collagen types provides the scaffolding for normal muscle contraction and tissue growth and repair. Permutations in the matrix accompany inflammation, stricture formation, and neoplasia. In the fetal intestine, the matrix participates in organogenesis by modulating cell migration, attachment,5 – 11 and differentiation5,12,13 and by providing a reservoir for growth factors.14,15 We have shown pre-
viously that collagen synthesis in human fetal intestinal smooth muscle cells declines with gestational age from a maximum at 11 weeks (the earliest age studied) to adult levels by 20 weeks.16 The characteristic histological features of adult human intestine are established within the same developmental time frame. The regulation of collagen synthesis in the human intestine has been studied most extensively in adult smooth muscle cells. Exogenously administered transforming growth factor b (TGF-b), in particular, stimulates collagen production in such cells.17 Smooth muscle cells isolated from human fetal intestine secrete TGFb.18 Studies presented herein examine the role of TGFb in the development of the human intestinal muscle wall by determining effects on smooth muscle cell collagen synthesis.
Materials and Methods Reagents Dulbecco’s modified Eagle medium (DMEM), penicillin-streptomycin, phosphate-buffered saline, and fetal calf serum were obtained from GIBCO (Grand Island, NY); trypsin and crude and pure collagenase were obtained from Worthington (Freehold, NJ); tricine, hyaluronidase, bovine serum albumin, ovalbumin, leupeptin, aprotinin, pepstatin A, and Bouin’s fixative were obtained from Sigma Chemical Co. (St. Louis, MO); [3H]proline (22 Ci/mmole) was obtained from New England Nuclear (Boston, MA); ascorbate was from Fisher Scientific Co. (Fairlawn, NJ); purified TGF-b1 was from R & D Systems (Minneapolis, MN); neutralizing antibody to TGFb1 was from BioGenex Laboratories (Dublin, CA); NuSerum was from Collaborative Research (Bedford, MA); [3H]thymidine (43 Ci/mmole) was from Amersham (Arlington Heights, IL); Vectastain ABC kit was from Vector Laboratories, Inc. (Burlingame, CA); 3ⴕ,3ⴕ-diaminobenzidine HCl was from Polysciences, Inc. (Warrington, PA); and anti–TGFb1 was a generous gift from Dr. Kathleen C. Flanders from Abbreviations used in this paper: DMEM, Dulbecco’s modified Eagle medium; TGF-b, transforming growth factor b. 䉷 1996 by the American Gastroenterological Association 0016-5085/96/$3.00
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the Laboratory of Chemoprevention (National Cancer Institute, National Institutes of Health, Bethesda, MD).
Cell Isolation and Culture Intestine and skin were obtained from 10- to 21-week human abortuses as described previously.16 The protocol for this research was approved by the Institutional Review Boards of the University of California and the Medical College of Virginia according to the policies of the National Institutes of Health. Muscle cells for culture were obtained from fetal intestine as described previously.16 Dermal fibroblasts were obtained from the same fetuses to provide comparison with another collagen-producing mesenchymal cell. The cells were maintained in culture with DMEM containing 25 mmol/L tricine (pH 7.4), penicillin-streptomycin, and 10% fetal calf serum in 37⬚C incubators with circulating room air. Cells were maintained in primary culture for fewer than 3 days after isolation and then passaged once in 0.1% trypsin for use in experiments. These cultured cells have been shown to possess the phenotype and functions of corresponding cells at isolation.16
Immunochemical Staining Immunochemical staining was performed with an avidin-biotin conjugate method. Rabbit antiserum (anti–TGF-b 1–30) to a synthetic preparation of the first 30 amino acids of TGF-b1 was used.19 The antiserum was affinity purified. Segments of fetal intestine were fixed in formalin, serially cross-sectioned at 2-mm intervals, and postfixed in Bouin’s fixative. After routine tissue processing, 5-mm-thick sections were cut from paraffin blocks and mounted in gelatin-coated slides for immunohistochemistry. Primary cultures of fetal smooth muscle cells and dermal fibroblasts were plated in 8well Lab Tek chambers on glass slides (Nunc, Naperville, IL). On day 2 of culture, the chambers were washed twice with phosphate-buffered saline, air dried, fixed in cold acetone, and immunohistochemically stained as described below. The sections were deparaffinized and rehydrated. Endogenous peroxidase was blocked by incubation in 0.6% hydrogen peroxide. Permeabilization of the sections with hyaluronidase was followed by blocking of excess protein with 1% bovine serum albumin, 1% ovalbumin, and 5% normal goat serum. Primary antibody solutions (anti–TGF-b1 at 1:50 dilution) were applied to the slides and incubated overnight at 4⬚C in humidified chambers. After multiple washes in phosphate-buffered saline with 0.1% bovine serum albumin, slides were incubated with biotinylated goat anti-rabbit immunoglobulin G, washed, and then incubated with the avidin-peroxidase complex. After a final wash, slides were stained with 0.05% 3ⴕ,3ⴕdiaminobenzidine HCl and hydrogen peroxide and were counterstained with hematoxylin. Primary antibody was replaced with normal rabbit immunoglobulin G as a control.
Biosynthesis of TGF-b by Muscle Cells First passage muscle cells at 11, 15, 18, and 20 weeks of gestation and dermal fibroblasts at 15 and 20 weeks of
TGF-b REGULATION OF FETAL COLLAGEN 93
gestation were plated at 1.3 1 105 cells/cm2 in DMEM supplemented with 10% fetal calf serum. Twenty-four hours were permitted for cell attachment, and then the plates were washed three times with DMEM and 0% fetal calf serum. Cell cultures were then incubated for 24 hours in DMEM and 0% fetal calf serum with bovine serum albumin (1 mg/mL) and the following protease inhibitors: pepstatin A, aprotinin, and leupeptin (1 mg/mL). The conditioned medium was collected and stored at 070⬚C. The cell layer was washed three times with DMEM and 0% fetal calf serum; resuspended in DMEM and 0% fetal calf serum with bovine serum albumin (1 mg/mL) and protease inhibitors pepstatin A, aprotinin, and leupeptin (1 mg/mL); sonicated; and stored at 070⬚C. Experiments were repeated twice (n Å 6).
Determination of TGF-b Bioactivity Total TGF-b bioactivity of smooth muscle cell and of dermal fibroblast conditioned medium and cell lysates was determined by a modification of the CCL-64 mink lung growth inhibition assay of Danielpour et al.20 Mink lung cells are known to respond to TGF-b through inhibition of cell proliferation. CCL-64 mink lung cells were a gift from Dr. A. B. Fowler from the Medical College of Virginia. Mink lung cells were maintained in DMEM and 10% NuSerum at 37⬚C in a circulating room air incubator. Cells were passaged and plated at 6.5 1 106 cell/75 cm2 T-flask at 3-day intervals three times before use in experiments. Subconfluent cells were used in the growth inhibition assay. Cells were trypsinized, resuspended in 10% NuSerum, pelleted at 500g for 5 minutes, washed once with 10 mL of assay buffer (DMEM supplemented with 0.2% NuSerum, 10 mmol/L HEPES, pH 7.4, 25 U/mL penicillin, and 25 mg/mL streptomycin). Mink lung cells were plated at 5 1 104 cells/well (Danielpour, personal communication, March 1991). After 1 hour, TGF-b standard preparation or sample conditioned medium was added to the wells and followed by a 22-hour incubation. Mink lung cells were then pulsed for 3 hours with 0.50 mCi/well [3H]thymidine (43 Ci/ mmol). Mink lung cells were then fixed with 1 mL of methanol/acetic acid (vol/vol, 3:1). After 1 hour at room temperature, the wells were washed twice with 2 mL of 80% methanol. The label was extracted by incubation with 0.5 mL 0.05% crude pancreatic trypsin for 30 minutes, and then 0.5 mL of 1% sodium dodecyl sulfate was added. Latent activity was determined in 200-mL aliquots of the original conditioned medium or cell lysates that had been treated with 5 mL of 1.8N HCl neutralized with 7.5 mL of NaOH-HEPES and assayed as described above. Values were normalized to nanograms of DNA in the original cultures of confluent muscle cells or fibroblasts. Experiments were repeated twice (n Å 6).
Cell Proliferation Cell proliferation was determined by measuring DNA content fluorometrically as a reflection of cell number.21
Assays of Collagen Synthesis and Types Fetal human intestinal smooth muscle cells were plated onto 24-well plates to quantitate collagen synthesis by mi-
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croassay.22 The cultures were incubated with L-[5-3H]proline (40 mCi/mL, 22 Ci/mol) for 5 hours, heated to 90⬚C to stop isotope incorporation, and frozen at 020⬚C. After thawing, the cells were sonicated and 200 mL of the contents of each well was removed for determination of DNA content.21 A 1mL solution of chick embryo carrier protein (1.6 mg/mL in 10 mmol/L cold proline) was then added to each well. Radioactive protein was separated from unincorporated L-[5-3H]proline by repeated precipitation with 5% trichloroacetic acid at 4⬚C. The remaining trichloroacetic acid was removed with ethanol/ ether (3:1). The dried residue in each well was incubated for 90 minutes with 0.5 mL of buffer containing HEPES (60 mmol, pH 7.2), CaCl2 (0.25 mmol), and N-ethylmaleimide (1.25 mmol). Protein was reprecipitated with trichloroacetic acid, and the radioactivity in the solute was measured as an incubation blank. Trichloroacetic acid was removed, and the dried residue was suspended in incubation buffer with purified bacterial collagenase.23 After a 90-minute incubation, both the soluble [3H]collagen peptides and the [3H]noncollagen protein were counted. Collagen synthesis was expressed on a per cell basis (per nanogram of DNA) and relative to total protein synthesis (relative collagen synthesis), according to a standard correction for the enriched amino acid content of collagen.24 Before studying the effects of TGF-b, baseline studies were performed in 0% and 10% fetal calf serum to characterize fetal muscle cell collagen production and cell proliferation under quiescent conditions compared with conditions known to maximize collagen synthesis in adult muscle cells.25 Twenty-one– week gestational fetal human intestinal smooth muscle cells were plated onto 24-well plates at 8 1 105 cells/well in DMEM supplemented with 10% fetal calf serum for 24 hours to permit cell attachment. The wells were washed three times with serum-free medium (0% fetal calf serum), and then the medium was changed to 10% or 0% fetal calf serum. The wells were then assayed for collagen, noncollagen protein, and DNA content at 24, 48, and 72 hours using the assay described above. Dermal fibroblasts isolated from the same fetus were studied similarly. Experiments were performed four times (n Å 4–6). For determination of collagen types, first passage intestinal smooth muscle cells were plated in 100-mm dishes. Confluent cell cultures were incubated for 6 hours in serum-free medium containing ascorbate (0.1 mmol/L), [14C]proline (0.5 mCi/mL), [3H]proline (25 mCi/mL), [3H]glycine (25 mCi/mL), and baminopropionitrile (0.1 mg/mL). Protease inhibitors (ethylenediaminetetraacetic acid [20 mmol/L], N-ethylmaleimide [8 mmol/L], and phenylmethysulfonyl fluoride [1 mmol/L]) and 0.5 mol/L acetic acid were then added. After 18 hours at 4⬚C, the combined material was dialyzed against deionized water and then against 0.4 mol/L NaCl-0.1 mol/L Tris. Chymotrypsin (0.3 mg/mL) was added for 6 hours at 10⬚C, after which N-tosyl-L-phenylalanine chloromethyl ketone (0.1 mg/mL) was added overnight to stop the reaction.26 The soluble material was dialyzed against 0.5 mol/L acetic acid (4⬚C). Collagen was precipitated in NaCl (4.5 mol/L) and then resolubilized in acetic acid. The collagen bands were separated by polyacrylamide slab gel electrophoresis27 using a urea buffer (4 mol/L),
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3% stacking gel, and 6% separating gel. Types I and V collagens were identified by comparison to standards of purified collagen types I and V collagens (GIBCO BRL, Gaithersburg, MD). Type III collagen chains were identified after interruption of electrophoresis and in situ reduction with dithiothreitol28 and comparison with a purified standard (GIBCO BRL). The dried gel was then exposed to radiographic film. The proportion of collagen chains expressed was determined by densitometry of the developed film.
Effects of Exogenous and Endogenous TGF-b on Collagen Synthesis, Cell Proliferation, and Expression of Collagen Types Human intestinal smooth muscle cells at 10, 15, and 21 weeks of gestation were plated at 8 1 105 cells/well in DMEM supplemented with 10% fetal calf serum. After 24 hours, the wells were washed 31 with 0% fetal calf serum, and then the cells were exposed to 50–200 pg/mL TGF-b or 50–200 mg/mL anti–TGF-b (control). The choice of TGF-b concentrations follows preliminary studies over a range of 0.1– 1000 pg/mL TGF-b (data not shown), in which effects on collagen synthesis were noted at 50–200 pg/mL. The choice of anti–TGF-b concentrations was chosen to neutralize amounts of endogenous TGF-b that had been measured previously by bioassay (physiological concentrations of TGF-b in skin wounds approximate 5 ng/mL).29 Cells were exposed to exogenous TGF-b or neutralizing antibody for 24 hours. Collagen synthesis, noncollagen protein, and DNA content were assayed as described above. The timing and duration of exposure to TGF-b or its neutralizing antibody was based on measures of collagen synthesis in the absence of exogenous factors (0% fetal calf serum) in which collagen synthesis was maximal between 24 and 48 hours. Experiments were repeated twice (n Å 4). To determine the effect of endogenous TGF-b on collagen synthesis and cell proliferation, cells from 21-week fetal intestine were grown in culture as described above. After 24 hours, cells were exposed to serum-free medium (control) or to 50– 200 mg/mL anti–TGF-b. After a further 24 hours, collagen and DNA content were assayed. The experiments were repeated twice (n Å 4). To determine the effect of TGF-b on expression of collagen types, cells from 10- and 21-week fetal intestine were grown initially in 100-mm dishes supplemented with DMEM and 10% fetal calf serum. Once confluent, cultures were washed 31 with DMEM and 0% fetal calf serum and then exposed to either anti–TGF-b (50 mg/mL) or TGF-b (50 pg/mL) for 24 hours, including the 6-hour pulse. The duration and timing of exposure to TGF-b or its neutralizing antibody were chosen to be consistent with the duration and timing of exposure previously studied in the collagen synthesis assay. The concentrations studied were those that exerted maximal changes in the amount of collagen synthesized determined by the collagen synthesis assay. Controls remained in DMEM and 0% fetal calf serum alone. Collagen types and their relative proportions
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TGF-b REGULATION OF FETAL COLLAGEN 95
Table 1. Distribution of Intestinal TGF-b1 Immunoreactivity With Fetal Age Enterocytes
Fibroblasts
Smooth muscle
Ganglion cells
Age (wk)
Crypt
Villus
Mucosal and submucosa
Mucosa
Propria
Vascular
Muscularis propria
11 13 17 19
0–1 0–1 0–1 0–1
2 2 2 2
1 1 1 1
NP NP NP 3
3 3 3 3
2 2 2 2
NP NP 2 2
NOTE. Intensity of antibody staining was graded from 0 to 3 in enterocytes, fibroblasts, smooth muscle cells, and ganglion cells. The most intense staining occurred in the muscle cells of the muscularis propria at all ages studied. NP, structures that are not present at the particular gestational age.
were determined as described previously. Experiments were performed twice (n Å 3).
Statistical Analysis Experimental values were compared with control values by Student’s t test and considered significantly different at P values of õ0.05.
Results TGF-b Distribution and Secretion Localization of TGF-b1 throughout the developing muscle layers of human fetal intestines (11, 13, 17, and 19 weeks of gestation) was shown by immunohistochemical staining. TGF-b1 stained most strongly in the smooth muscle cells of the muscularis propria and of mesenteric vasculature at all ages studied (Table 1). The intensity and distribution of staining did not change with fetal age and was the same in the duodenum, jejunum, and ileum. Smooth muscle cells of the muscularis mucosa, a structure not present until 19 weeks of gestation, stained with equal intensity. At 17 weeks of gestation, ganglion cells appeared within the muscularis propria and showed moderate cytoplasmic reactivity for TGF-b1 (Figure 1). Weak reactivity of mucosal and submucosal stromal cells was observed at all gestational ages, with crypt enterocytes staining the least. Villus enterocytes showed moderate immunostaining for TGF-b1. The extracellular matrix showed little or no staining for TGF-b1 (Figure 1). No positive staining occurred when the primary antibody was replaced by normal rabbit immunoglobulin. Cultured intestinal smooth muscle cells isolated from the same specimens used for histochemical studies at 11, 13, 17, and 19 weeks of gestation showed immunocytochemical staining for TGF-b1 similar to muscle cells in the histological section (Figure 2). The proportion of cells staining for TGF-b1 varied with cell density. In areas of subconfluent cell growth with little adjacent cell contact, nearly 100% of cells showed mild-to-moderate, finely stippled cytoplasmic immunoreactivity for TGF-
b1. Zones of confluent cells showed minimal cytoplasmic staining in 0%–5% of cells. This pattern of staining did not change with fetal age and was similarly found in cultured fetal dermal fibroblasts (not shown). TGF-b secretion in muscle cells at 11 weeks of gestation was 81 greater than in cells from 15 to 18 weeks of gestation and more than 351 greater than cells from 20 weeks of gestation (Figure 3). Dermal fibroblasts produced more TGF-b than muscle cells at the same gestational age (Figure 4). At 15 and 20 weeks of gestation, respectively, fibroblasts synthesized 141 and 301 more TGF-b than intestinal muscle cells isolated from the same fetus. Collagen Production and Cell Proliferation Smooth muscle cells produced more collagen in serum-free medium, the largest difference in collagen production being observed at 48 hours, when collagen synthesis was 50% greater than in 10% fetal calf serum (Figure 5A). In contrast, fetal dermal fibroblasts produced the same amount of collagen in 0% and 10% fetal calf serum (Figure 5B) and less collagen per nanogram of DNA than muscle cells at all time points in culture (P õ 0.003 at 24 hours; P õ 0.001 at 48 hours; P õ 0.002 at 72 hours). No change in noncollagen protein production occurred from 24 to 72 hours in 0% fetal calf serum (88.35 cpm/ ng DNA { 6.03 at 24 hours; 73.87 { 7.20 at 48 hours; and 81.60 { 4.50 at 72 hours). However, in 10% fetal calf serum, noncollagen protein production increased initially at 24 hours (60.47 { 12.24 at 0 hours vs. 112.93 { 17.95 at 24 hours) and then steadily declined (75.13 { 4.20 at 48 hours and 33.63 { 0.90 at 72 hours). At 72 hours, noncollagen protein production was nearly 2.51 greater in 0% fetal calf serum than in 10% fetal calf serum (P õ 0.001) but otherwise was the same at earlier time points. DNA content did not change in 0% fetal calf serum (5105.06 { 136.33 ng DNA compared with control 5871.88 { 827.13). In contrast, DNA content nearly dou-
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Figure 2. Immunocytochemical detection of TGF-b1 in cultured fetal intestinal smooth muscle cells. (A ) Nearly 100% of the subconfluent cells showed cytoplasmic immunoreactivity for TGF-b1 (brown stain). (B ) In contrast, õ5% of confluent cells were immunoreactive. The bluish color represents counterstain of the nuclei with H&E. Cells from 20 weeks of gestation are shown (original magnification 401).
Figure 1. Immunohistochemical studies of TGF-b1 in human fetal intestine at 17 weeks of gestation. Cells containing TGF-b1 stain brown with anti–TGF-b1 using an avidin-biotin conjugate method as described in Materials and Methods. The bluish color represents counterstain of the background with H&E. Muscle cells of the circular and longitudinal muscle layers stained the most intensely (arrowheads). Ganglion cells, arterial smooth muscle cells, stromal cells of the mucosa and submucosa, and enterocytes of the villous tips stained moderately. Enterocytes of the crypts stained minimally (original magnification 4001).
(200 pg/mL), TGF-b inhibited noncollagen protein production 59% at 10 weeks (2.00 { 0.37 vs. 4.87 { 0.01 cpm/ng DNA; P õ 0.019) and 37% at 15 weeks (1.51 { 0.26 vs. 2.38 { 0.08 cpm/ng DNA; P õ 0.021) and stimulated noncollagen protein production 130% at
bled in 72 hours in 10% fetal calf serum (10,097.96 { 68.16). Effects of Exogenous and Endogenous TGF-b The greatest effects on collagen synthesis were mediated by 50 pg/mL TGF-b (Table 2). At 10 weeks of gestation, TGF-b inhibited collagen production by 38% compared with anti–TGF-b. In contrast, at 15 and 21 weeks of gestation, TGF-b stimulated collagen synthesis by 45% and 70%, respectively. Higher concentrations (200 pg/mL) had no effect on collagen production at 10 and 15 weeks of gestation (data not shown). Higher concentrations of TGF-b were required to produce an effect on noncollagen protein synthesis. Effects were age specific but did not show the same pattern of inhibition or stimulation observed with collagen synthesis. Noncollagen protein synthesis was inhibited by TGFb at 10 and 15 weeks of gestation and was stimulated at 21 weeks of gestation. At the highest concentration
Figure 3. Effect of fetal age on TGF-b secretion by intestinal smooth muscle cells. Twenty-four–hour collections of conditioned medium and cell lysates were assayed for total TGF-b bioactivity from primary cultures of intestinal smooth muscle cells at 11, 15, 18, and 20 weeks of gestation. TGF-b bioactivity was determined by the CCL64 mink lung epithelial cell growth inhibition assay as described in Materials and Methods. Values are expressed as mean { SEM (n Å 6). Cells at 11 weeks of gestation secreted more TGF-b than cells from 15, 18, and 20 weeks (P õ 0.001).
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TGF-b REGULATION OF FETAL COLLAGEN 97
Table 2. Effect of TGF-b on Collagen Synthesis by Fetal Human Intestinal Smooth Muscle Cells Fetal age (wk)
Anti–TGF-b
TGF-b
P value
10 15 21
3.17 { 0.45 4.26 { 0.80 11.14 { 1.72
1.60 { 0.24 7.62 { 0.75 36.36 { 3.78
õ0.042 õ0.01 õ0.0001
NOTE. Collagen synthesis (in counts per minute per nanogram of DNA) was assayed in cell cultures exposed to anti–TGF-b (50 mg/mL) or TGF-b (50 pg/mL) as described in Materials and Methods. Values are expressed as mean { SEM (n Å 4).
200 mg/mL). At 21 weeks of gestation, collagen synthesis was inhibited 94% by 100 mg/mL anti–TGF-b (Figure 6A). Anti–TGF-b increased DNA content in a concentration-dependent fashion and maximally by 65% at 200 mg/mL (Figure 6B). No effect on collagen synthesis was Figure 4. Comparison of TGF-b bioactivity by different mesenchymal cells from the same fetus at 15 and 20 weeks of gestation. Primary cultures of dermal fibroblasts (F ) and intestinal smooth muscle cells (M) were isolated and maintained in culture. Total TGF-b bioactivity of conditioned medium and cell lysates from 24 hours in culture were determined by the CCL-64 mink lung epithelial growth inhibition assay as described in Materials and Methods. Values are expressed as mean { SEM (n Å 6). At each age, fibroblasts secreted more TGF-b than muscle cells (P õ 0.001). Both mesenchymal cells decreased TGF-b secretion at 20 weeks compared with 15 weeks of gestation (P õ 0.001).
21 weeks (10.31 { 2.55 vs. 4.90 { 0.50 cpm/ng DNA; P õ 0.048). The effects of endogenous TGF-b on collagen synthesis and cell proliferation were studied in the presence and absence of a neutralizing antibody, anti–TGF-b (50–
Figure 5. Collagen synthesis in the absence (0% FCS) and presence (10% FCS) of fetal calf serum. Fetal human cells were isolated at 21 weeks of gestation. (A ) Intestinal smooth muscle cells. *Significance from 10% fetal calf serum: P õ 0.002 at 48 hours and P õ 0.013 at 72 hours. (B ) Dermal fibroblasts. Values are expressed as mean { SEM (n Å 4–6). Collagen production by dermal fibroblasts was the same in 0% and 10% fetal calf serum.
Figure 6. Effect of endogenous TGF-b on collagen synthesis and cell proliferation by human intestinal smooth muscle cells. Cells from 21 weeks of gestation were isolated and grown in culture as described in Materials and Methods. After 24 hours, cells were exposed to serum-free medium (control) or to 50–200 mg/mL anti–TGF-b. After another 24 hours, (A ) collagen synthesis and (B ) DNA content were assayed. *Significance from serum-free medium, P õ 0.001. Values are expressed as mean { SEM (n Å 4).
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Table 3. Effect of TGF-b on Synthesis of Collagen Types and Component Chains by Fetal Human Intestinal Smooth Muscle Cells Collagen type Chain 10 wk Control TGF-b Anti–TGF-b 20 wk Control TGF-b Anti–TGF-b
I
III
V
a1
a2
a1
a1
Total
6.265 3.966 2.853
2.102 11.12 2.848
4.836 12.05 9.167
1.017 12.32 0.595
14.22 39.456 15.463
3.519 2.081 2.79
2.234 2.592 2.76
3.498 6.112 6.16
0.58 0.0 0.0
9.831 10.785 11.71
NOTE. Cells were isolated and cultured at 10 and 20 weeks of gestation. Confluent cultures were exposed to either TGF-b (50 pg/mL) or to anti–TGF-b (50 mg/mL) for 24 hours, including the 6-hour pulse. Controls remained in serum-free medium. Collagen types and chains were identified by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and autoradiography and then quantitated by densitometry as described in Materials and Methods. Relative proportions of collagen types referred to in the text are calculated as a percent of total collagen production.
observed when the same experiment was repeated at 11 weeks of gestation (data not shown). Higher concentrations (250–1000 mg/mL) of neutralizing anti–TGF-b were then used, in consideration of the fact that cells at 11 weeks of gestation were shown to make 340% more TGF-b than cells at 20 weeks of gestation and may require more neutralizing antibody. At these increased concentrations of anti–TGF-b, cells at 11 weeks of gestation became detached from the plate. At 10 and 20 weeks of gestation, cells expressed similar proportions of types I, III, and V collagens (Table 3). At 10 weeks of gestation, muscle cells synthesized 59% type I collagen, 34% type III collagen, and 7% type V collagen. At 20 weeks of gestation, muscle cells synthesized 59% type I collagen, 36% type III collagen, and 6% type V collagen. However, the proportions of a1 and a2 chains comprising type I collagen differed with fetal age. The a1(I)/a2(I) chain ratio was approximately 3:1 at 10 weeks of gestation and 1.4:1 at 20 weeks. Addition of TGF-b or anti–TGF-b to the cultures altered the proportions and types of collagens expressed in an age-specific manner. At 10 weeks of gestation, a1(I) chains were decreased 77% by TGF-b and 59% by anti–TGF-b. At 20 weeks, a1(I) chains were decreased by 47%, but in contrast with 10 weeks of gestation, anti–TGF-b increased a1(I) chains by 50%. Effects on the expression of a2(I) chains were greatest at 10 weeks of gestation. TGF-b nearly doubled the expression of a2(I) collagen chains at 10 weeks of gestation but had no effect at 20 weeks of gestation. Anti–TGF-b did not change the amount of a2(I) chains at 10 weeks. TGF-b
exerted opposite effects on the expression of a1(III) chains depending on gestational age. TGF-b decreased a1(III) chains by 8% at 10 weeks but increased a1(III) chains by 62% at 20 weeks. Anti–TGF-b increased a1(III) chains by 74% at 10 weeks of gestation but decreased a1(III) chains by 56% at 20 weeks. The greatest effects on a1(V) chains occurred at 10 weeks of gestation, in which TGF-b increased these chains by ú240% compared with same-age controls.
Discussion This is the first study to examine the role of TGFb in regulating collagen synthesis by smooth muscle cells isolated from the human fetal intestinal wall. TGF-b inhibited or stimulated collagen synthesis in an age- and type-specific manner, suggesting that TGF-b may play a key role in modulation of intestinal development in the human fetus. TGF-b is prominent in the musculature of the developing human intestine, including circular, longitudinal, vascular, and muscularis mucosa components. This distribution profile is conserved throughout intestinal development as observed previously in animal embryos and postnatally30–32 and in contrast with fetal lung where TGF-b localizes to sites of mesenchymal-epithelial interactions.33 These observations support a role for autocrine TGF-b in the morphogenesis of the intestinal muscle wall. Synthesis of TGF-b seems to be dependent on cell density. Synthetic activity was especially notable in subconfluent areas, as observed previously in human vascular smooth muscle cells34 and bovine aortic endothelial cells.35 The current study shows that, at the same cell density, the secretion of TGF-b declined with advancing developmental age, a trend previously noted in animals. Cultures of neonatal rat aortic smooth muscle cells and lung fibroblasts secrete twofold to fourfold more TGF-b than the respective adult cells.36 Together, these studies show an inverse relationship between advancing developmental age and endogenous TGF-b secretion. Additional evidence that autocrine TGF-b plays a role in intestinal development is the observation that fetal intestinal smooth muscle cells synthesized more collagen in the absence of exogenous serum factors. This response is distinctly different from that reported for adult human intestinal smooth muscle cells (same cell type and different age) and from control fetal dermal fibroblasts (different mesenchymal cell and same developmental age). Adult intestinal smooth muscle cells require exogenous factors (10% fetal calf serum or TGF-b) to stimulate collagen synthesis.17,25 Maximal collagen synthesis accompanies cell proliferation in 10% fetal calf serum.25
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Fetal intestinal smooth muscle cells increased collagen synthesis in 0% fetal calf serum without cell proliferation, whereas dermal fibroblasts from the same fetus produced the same amount of collagen whether in 0% or 10% fetal calf serum. All three cell types proliferated only in the presence of serum. These findings emphasize that in the human intestine, fetal collagen synthesis is differently regulated than in the adult and is specific to cell type. TGF-b inhibited total collagen synthesis at 10 weeks of gestation and stimulated synthesis at 20 weeks of gestation. The magnitude and pattern of inhibition or stimulation by TGF-b was not the same for noncollagen protein synthesis, indicating that effects on collagen synthesis were selective. Selective enhancement of collagen synthesis by TGF-b has been shown in adult and fetal cells at a single developmental stage.17,36 The present study extends these findings to include inhibitory as well as stimulatory effects on collagen synthesis that are not only selective but specific to gestational age. Individual collagen types and component chains are also synthesized selectively in fetuses treated with TGFb. Studies in animal fetal organ systems have focused on the effects of exogenous TGF-b at a single gestational age. Bone cells secrete TGF-b and synthesize collagen, 90% of which is type I collagen.37 Studies in 22-day-old fetal rat bone show threefold increases in type I collagen after exposure to exogenous TGF-b.38 Fetal rat lung epithelial cells synthesize types I, III, IV, and V collagens. All types increase more than twofold after exposure to TGF-b (types I and III are greater than types IV and V).39 Collagen synthesis in primary cultures of rabbit articular chondrocytes increases in response to TGF-b without change in the proportion of specific collagen chains.40 The current investigation uses neutralizing antibodies to study the effects of endogenous TGF-b at several fetal ages in human cells. This approach shows that autocrine TGF-b exerts very different effects on matrix composition of muscle at various stages of intestinal development. The mechanisms responsible for the age-specific effects of TGF-b on collagen synthesis by human fetal intestinal smooth muscle cells are not understood. The magnitude of TGF-b secretion does not correlate with collagen synthesis at any given fetal age. One level of control may be achieved through differential expression of TGF-b receptor isoforms through which TGF-b1 may mediate specific biological functions. Three receptor subtypes have been identified41 (for review, see Roberts and Sporn42). In the epithelial Mv1Lu cells, the type I receptor influences the interaction of the cell with the extracellular matrix, whereas the type II receptor mediates the antiproliferative activity of TGF-b1.43 In the mouse embryo, type II receptor expression is greater in undifferentiated mesenchyme
TGF-b REGULATION OF FETAL COLLAGEN 99
than in more mature cells.44 Differential expression of receptor isoforms may account for uncoupling of effects on proliferation from effects on matrix but cannot explain age-specific opposing effects of TGF-b on collagen synthesis in intestinal smooth muscle cells. TGF-b enhances transcription of the proa2(I) collagen gene.45 Additional mechanisms must regulate the agespecific increases and decreases in the production of different collagen chains. The action of TGF-b may be modified by diffusible cytokines and nondiffusible factors such as matrix.42,46 Animal fetal smooth muscle cells secrete various cytokines in an age-dependent fashion.47 The distribution and types of muscle-associated matrices change during development of the intestinal wall.48,49 Consequently, the smooth muscle cell extracellular environment changes with fetal age, possibly contributing to the differential production of collagens in response to TGF-b. Collagen composition significantly influences the biomechanical and biochemical properties of the developing intestinal wall. Effective contraction and compliance of the wall arise from the specific arrangement and proportions of collagen chains.50 – 52 Tissue remodeling and inflammatory response may also be affected.53 – 56 Increased collagen deposition modifies TGF-b–induced growth suppression.57 Therefore, changes in the amount and types of collagens expressed in the presence of TGF-b must influence intestinal development and likely subsequent postnatal tissue response. In fact, neoplasia may represent a recapitulation of fetal events in postnatal life. Colon carcinoma biopsy fragments contain a peculiar form of collagen that is absent in normal adult tissues but present in fetal tissues.58 Further, fetal intestinal smooth muscle cells and neoplastic cells share a certain autonomy through endogenous production of and response to TGF-b.59,60 This study shows that TGF-b alters the synthesis of collagen chains in a type- and developmental age-specific manner. The resulting modulation in the amount and type of collagen can influence smooth muscle cells, inflammatory cells, and epithelial cells within the intestinal wall through effects on cell migration, attachment, proliferation, and differentiation. These cellular events direct normal or abnormal development, postnatal healing, and growth. Thus, understanding the developmental expression of different collagen chains induced by TGF-b in conjunction with the relatively autonomous nature of fetal intestinal smooth muscle cells may shed light on adult intestinal pathophysiology.
References 1. Vuorio E, de Crombrugghe B. The family of collagen genes. Ann Rev Biochem 1990;59:837–872.
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2. Van der Rest M, Garrone R. Collagen family of proteins. FASEB J 1991;5:2814–2823. 3. Li K, Tamai K, Tan EML, Uitto J. Cloning of type XVII collagen. Complementary and genomic DNA sequences of mouse 180 kilodalton bullous pemphigoid antigen (BPAG2) predict an interrupted collagenous domain, a transmembrane segment, and unusual features in the 5ⴕ-end of the gene and the 3ⴕ untranslated region of the mRNA. J Biol Chem 1993;268:8825–8834. 4. Graham MF, Diegelmann RF, Elson CO, Lindblad WJ, Gotschalk N, Gay S, Gay R. Collagen content and types in the intestinal strictures of Crohn’s disease. Gastroenterology 1988;94:257– 265. 5. Bernfield M, Banerjee SD, Koda JA, Rapraeger AC. Remodeling of the basement membrane as a mechanism of morphogenetic tissue interaction. In: Trelstad RL, ed. The role of the extracellular matrix in development. New York: Liss, 1984:545–572. 6. Furcht LT. Critical factors controlling angiogenesis: cell products, cell matrix, and growth factors. Lab Invest 1986;55:505–509. 7. Lacovara J, Cramer EB, Quigley JP. Fibronectin enhancement of directed migration of B16 melanoma cells. Cancer Res 1984;44:1657–1663. 8. McCarthy JB, Hagen ST, Furcht LT. Human fibronectin contains distinct adhesion- and motility-promoting domains for metastatic melanoma cells. J Cell Biol 1986;102:179–188. 9. Montesano R, Orci L, Vassalli P. In vitro rapid organization of endothelial cells into capillary-like networks is promoted by collagen matrices. J Cell Biol 1983;97:1648–1652. 10. Nabeshima K, Kataoka H, Koono M. Enhanced migration of tumor cells in response to collagen degradation products and tumor cell collagenolytic activity. Invasion Metastasis 1986;6:270–286. 11. Thesleff I, Barrach HJ, Fiodart JM, Vaheri A, Pratt RM, Martin GR. Changes in the distribution of type IV collagen, laminin, proteoglycan and fibronectin during mouse tooth development. Dev Biol 1981;81:182–192. 12. Form DM, Pratt BM, Madri JA. Endothelial cell proliferation during angiogenesis in vitro modulation by basement membrane components. Lab Invest 1986;55:521–530. 13. Hosick HL, Inaguma Y, Kusakabe M, Sakakura T. Morphogenesis of mouse mammary epithelium in vivo in response to biomatrix prepared from a stimulatory fetal mesenchyme. Dev Growth Differ 1988;30:229–240. 14. Ekblom P. Basement membrane proteins and growth factors in kidney differentiation. In: Trelstad RL, ed. The role of the extracellular matrix in development. New York: Liss, 1984:173–206. 15. Gospadarowicz D, Ferrara N, Schweigerer L, Neafield G. Structural characterization and biological functions of fibroblast growth factor. Endocr Rev 1981;8:95–108. 16. Perr HA, Grider JR, Mills AS, Kornstein M, Turner DA, Diegelmann RF, Graham MF. Collagen production by human smooth muscle cells isolated during intestinal organogenesis. Anat Embryol 1992;185:517–527. 17. Graham MF, Bryson GR, Diegelmann RF. Transforming growth factor–b1 selectively augments collagen synthesis by human intestinal smooth muscle cells. Gastroenterology 1990;99:447– 453. 18. Perr HA, Johnson D. Human fetal intestinal smooth muscle cells secrete TGF-b and synthesize collagen without cell proliferation (abstr). J Cell Biochem 1993;17E (Suppl):181. 19. Flanders KC, Thompson NL, Lissel DS, Van Obberghen-Schilling E, Baker CC, Kass ME, Ellingsworth LR, Roberts AB, Sporn MB. Transforming growth factor–b1: histochemical localization with antibodies to different epitopes. J Cell Biol 1989;108:653–660. 20. Danielpour D, Dart LL, Flanders KC, Roberts AB, Sporn MB. Immunodetection and quantitation of the two forms of transforming growth factor–beta (TGF-b1 and TGF-b2) secreted by cells in culture. J Cell Physiol 1989;138:79–86.
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21. Labarca C, Paigen K. A simple, rapid and sensitive DNA assay procedure. Anal Biochem 1980;102:344–352. 22. Diegelmann RF, Bryson GR, Flood LC, Graham MF. Microassay to quantitate collagen synthesis by cells in culture. Anal Biochem 1990;186:296–300. 23. Peterkofsky B, Diegelmann RF. The use of a mixture of proteinase-free collagenase for the specific assay of radioactive collagen in the presence of other proteins. Biochemistry 1971;10: 988–994. 24. Diegelmann RF, Peterkofsky B. Collagen biosynthesis during connective tissue development in chick embryo. Dev Biol 1972; 28:443–453. 25. Graham MF, Drucker DEM, Diegelmann RF, Elson CO. Collagen synthesis by human intestinal smooth muscle cells in culture. Gastroenterology 1987;92:400–405. 26. Clark CC, Richards CF. Isolation and partial characterization of precursors to minor cartilage collagens. Coll Relat Res 1985;5:205–233. 27. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–685. 28. Sykes B, Puddle B, Francis M, Smith R. The estimation of two collagens from human dermis by interrupted gel electrophoresis. Biochem Biophys Res Commun 1976;72:1472–1480. 29. Cromack DT, Sporn MB, Roberts AF, Merino MF, Dart LL, Norton JA. Transforming growth factor beta levels in rat wound chambers. J Surg Res 1987;42:622–628. 30. Pelton RW, Saxena B, Jones M, Moses HL, Gold LI. Immunohistochemical localization of TGF-b1, TGF-b2, and TGF-b3 in the mouse embryo: expression patterns suggest multiple roles during embryonic development. J Cell Biol 1991;115:1091–1105. 31. Barnard JA, Beauchamp RD, Coffey RJ Jr., Moses HL. Regulation of intestinal epithelial cell growth transforming growth factor type beta. Proc Natl Acad Sci USA 1989;86:1578–1582. 32. Barnard JA, Warwick GJ, Gold LI. Localization of transforming growth factor beta isoforms in the normal murine small intestine and colon. Gastroenterology 1993;105:67–73. 33. Heine UI, Munoz EF, Flanders KC, Roberts AB, Sporn MB. Colocalization of TGF-beta and collagen I and III, fibronectin and glycosaminoglycans during lung branching morphogenesis. Development 1990;109:29–36. 34. Nikol S, Isner JM, Pickering G, Kearney M, Leclerc G, Weir L. Expression of transforming growth factor–b1 is increased in human vascular restenosis lesions. J Clin Invest 1992;90:1582– 1592. 35. Merrilees MJ, Sodek J. Synthesis of TGF-b1 by vascular endothelial cells is correlated with cell spreading. J Vasc Res 1992;29: 376–384. 36. McGowan SE. Influences of endogenous and exogenous TGF-b on elastin in rat lung fibroblasts and aortic smooth muscle cells. Am J Physiol 1992;263:L257–L263. 37. Hunter GK. In vitro studies on matrix-mediated mineralization. In: Hall BK, ed. Bone. Volume 4. Boca Raton, FL: CRC, 1991: 225–247. 38. Centrella M, Casinghino S, Ignotz R, McCarthy TL. Multiple regulatory effects by transforming growth factor–b on type I collagen levels in osteoblast-enriched cultures from fetal rat bone. Endocrinology 1992;131:2863–2872. 39. DiMari SJ, Howe AM, Haralson MA. Effects of transforming growth factor–b on collagen synthesis by fetal rat lung epithelial cells. Am J Respir Cell Mol Biol 1992;4:455–462. 40. Galera P, Vivien D, Pronost S, Bonaventure J, Redini F, Loyau G, Pujol J-P. Transforming growth factor–b1 (TGF-b1) up-regulation of collagen type II in primary cultures of rabbit articular chondrocytes (RAC) involves increased mRNA levels without affecting mRNA stability and procollagen processing. J Cell Physiol 1992;153:596–606. 41. Cheifetz S, Weatherbee JA, Tsang ML-S, Anderson JK, Mole JE,
January 1996
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
Lucas R, Massague J. The transforming growth factor–b system, a complex pattern of cross-reactive ligands and receptors. Cell 1987;48:409–415. Roberts AB, Sporn MB. The transforming growth factor–bs. In: Sporn MB, Roberts AB, eds. Handbook of experimental pharmacology. Peptide growth factors and their receptors. Heidelberg, Germany: Springer–Verlag, 1990:419–472. Chen R-H, Reinhard E, Derynck R. Inactivation of the type II receptor reveals two receptor pathways for the diverse TGF-b activities. Science 1993;260:1335–1338. Lawler S, Candia AF, Ebner R, Shum L, Lopez AR, Moses H, Wright CVE, Derynck R. The murine type II TGF-b receptor has a coincident embryonic expression and binding preference for TGFb1. Development 1994;120:165–175. Rossi P, Karsenty G, Roberts AB, Roche NS, Sporn MB, de Crombrugghe B. A nuclear factor 1 binding site mediates the transcriptional activation of type I collagen by transforming growth factor– b. Cell 1988;52:405–414. Battegay EJ, Raines EW, Seifert RA, Bowen-Pope DF, Ross RR. TGF-b induces bimodal proliferation of connective tissue cells via complex control of an autocrine PDGF loop. Cell 1990;63:515– 524. Seifert RA, Schwartz SM, Bowen-Pope DF. Developmentally regulated production of platelet-derived growth factor–like molecules. Nature 1984;311:669–671. Simon-Assmann P, Kedinger M, Haffen K. Immunocytochemical localization of extracellular-matrix proteins in relation to rat intestinal morphogenesis. Differentiation 1986;32:59–66. Simon-Assmann P, Duclos B, Orian-Roussear V, Arnold C, Mathelin C, Engvall E, Kedinger M. Differential expression of laminin isoforms and alpha 6–beta 4 integrin subunits in the developing human and mouse intestine. Dev Dyn 1994;201:71–85. Fleischmajer R, Timpl R, Tuderman L, Raisher L, Wiestner M, Perlish JS, Graves PN. Ultrastructural identification of extension aminopeptides of type I and III collagens in human skin. Proc Natl Acad Sci USA 1981;78:7360–7364. Broek DL, Madri J, Eikenberry EF, Brodsky B. Characterization of
TGF-b REGULATION OF FETAL COLLAGEN 101
52.
53.
54.
55.
56.
57.
58.
59. 60.
the tissue form of type V collagen from chick bone. J Biol Chem 1985;260:555–562. Martinez-Hernandez A, Gay S, Miller EJ. Ultrastructural localization of type V collagen in rat kidney. J Cell Biol 1982;92:343– 349. Jaffe R, Deykin D. Evidence for a structural requirement for the aggregation of platelets by collagen. J Clin Invest 1974;53:875– 883. Balleisen L, Marx R, Kuhn K. Platelet-collagen interaction. The influence of native and modified collagen (type I) on the aggregation of human platelets. Haemostasis 1976;5:155–164. Stenn KS, Madri JA, Roll FJ. Migrating epidermis produces AB2 collagen and requires continual collagen synthesis for movement. Nature 1979;277:229–232. Grotendorst GR, Seppa HE, Keinman HK, Martin GR. Attachment of smooth muscle cells to collagen and their migration toward platelet-derived growth factor. Proc Natl Acad Sci USA 1981;78:3669–3672. Nugent MA, Newman JJ. Inhibition of normal rat kidney cell growth by transforming growth factor–b is mediated by collagen. J Biol Chem 1989;264:18060–18067. Pucci-Minafra I, Luparello C, Andriolo M, Basirico L, Aquino A, Minafra S. A new form of tumor and fetal collagen that binds laminin. Biochemistry 1993;32:7421–7427. Roberts AB, Sporn MB. Transforming growth factors. Cancer Surv 1985;4:683–705. Sporn MB, Roberts AB. Peptide growth factors and inflammation, tissue repair, and cancer. J Clin Invest 1986;78:329–332.
Received October 25, 1994. Accepted September 1, 1995. Address requests for reprints to: Hilary Perr, M.D., Department of Pediatrics, Box 0136, University of California, San Francisco, California 94143. Fax: (415) 476-1343. Supported by Clinical Investigator Award DK01849 from the National Institutes of Health and by the Tooley Foundation. The authors thank Drs. D. Mongomery Bissell, M. Michael Thaler, and F. J. Roll for editorial assistance and expert advice and Dr. Theodore J. Gradman for statistical consultation.