Insulin, transforming growth factors, and substrates modulate growth of guinea pig pancreatic duct cells in vitro

GASTROENTEROLOGY 1995;109:944-952

Insulin, Transforming Growth Factors, and Substrates Modulate Growth of Guinea Pig Pancreatic Duct Cells In Vitro ELI BHATTACHARYYA, ASHISH PANCHAL, THOMAS J. WILKINS, JOSl~ DE ONDARZA, and SETH R. HOOTMAN Department of Physiology, Michigan State University, East Lansing, Michigan

See editorial on page 1005. Background & Aims: Little is known of the physiological mechanisms that control cellular renewal in the pancreatic excretory duct system. This study investigated the effects of potential regulatory substances on the growth of cultured guinea pig pancreatic duct epithelial monolayers. Methods: Pancreatic duct explants were cultured for 3 days on plastic and on permeable filters in the presence and absence of different substances. Growth of epithelial monolayers from these explants was measured by 5-bromo-2'-deoxyuridine incorporation and morphometric procedures. Results: Epidermal growth factor and insulin both enhanced monolayer growth and together had an additive effect. Transforming growth factor (~ enhanced and transforming growth factor 13 inhibited growth, whereas glucagon, somatostatin, pancreatic polypeptide, secretin, cerulein, bombesin, and dexamethasone had no significant effects. Monolayer growth on type I collagen-coated filters was enhanced when compared with that of monolayers grown on tissue culture plastic. Cell growth from explants on filters coated with type IV collagen and fibronectin was comparable with that on plastic, whereas growth on Matrigel- or laminin-coated filters was reduced. Conclusions: Insulin, transforming growth factors, and substrate components modulate growth of pancreatic duct epithelial cells in vitro, suggesting that they are important regulators of cell division in the excretory duct system of the intact pancreas. he exocrine pancreas in adult mammals is a highly differentiated organ that can respond to disease, surgical resection, and dietary restrictions with increased cell replication and growth.1 This response is presumably mediated by the release of cytokines within the pancreas itself and by gastrointestinal hormones, a supposition supported by studies with cultured pancreatic acinar cells. Bombesin, cholecystokinin, insulin, transforming growth factor (TGF) Or, epidermal growth factor (EGF), and fibroblast growth factor have been shown to stimu-

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late acinar cell proliferation in vitro, 2 5 whereas TGF-~ and dexamethasone have been shown to inhibit growth of cultured pancreatic acinar cells, z'6 However, the recent observation that acinar cells in culture can rapidly transdifferentiate and express ductal cell markers complicates interpretation of the results of these studies, v-9 By contrast, less is known of the control of cell growth in the excretory duct system of the exocrine pancreas, although some trophic factors have been identified. We previously developed a method for the primary culture of epithelial cells derived from the interlobular and main ducts of the guinea pig pancreas as monolayers1° and used a morphometric technique to measure cell proliferation) ~ In these studies, monolayers were grown both on tissue culture plastic and on permeable gels of type I collagen in CMRL 1066 medium containing 2.5% fetal bovine serum (FBS), 0.5 mmol/L 3-isobutyl-l-methylxanthine (IBMX), and 1 nmol/L insulin. We found EGF to be a potent mitogen, although cerulein (a stable cholecystokinin analogue), bombesin, and secretin had no appreciable effect on duct cell growth. More recently, Mangino et al. 12 have reported growth-promoting effects of secretin, bombesin, vasoactive intestinal peptide, and gastrin on cultured pancreatic duct cells of the Syrian hamster. In their studies, cells passaged 12-13 times were maintained on type I collagen-coated microporous membrane filters in Dulbecco's modified Eagle medium/ F12 medium containing 5 % Nu Serum IV (Collaborative Biomedical Products, Bedford, MA), 25 btg/mL bovine pituitary extract, 5 nmol/L triiodothyronine, 20 btg/mL EGF, and 25 [.tg/mL insulin. Whether these differences in culture conditions or differences inherent to the two species underlie the observed variation in growth-promoting effects of the various peptides tested remains unclear. Keratinocyte growth factor also has been reAbbreviations used in this paper: BrdU, 5-bromo-2'-deoxyuridine; EGF, epidermal growth factor; FBS, fetal bovine serum; IBMX, 3isobutyl-l-methylxanthine; SBTI, soybean trypsin inhibitor; TGF, transforming growth factor. © 1995 by the American Gastroenterological Association 0016-5085/95/$3.00

September 1995

ported to induce pancreatic ductal epithelial cell proliferation. 13 Although these studies have provided a foundation for understanding the control of cellular renewal in the excretory ducts of the pancreas, the possible effects of many other potential growth regulators on pancreatic duct cell proliferation have not been assessed. These include islet hormones, TGFs, and components of the extracellular matrix. In the present studies, we therefore examined the effects of these and several other physiological regulatory molecules on growth of epithelial cells from explants derived from the main and interlobular ducts of the guinea pig pancreas. Growth of pancreatic duct epithelial cell monolayers was determined by morphometric procedures and by labeling of the nuclei of proliferating duct cells with 5-bromo-2'-deoxyuridine (BrdU). Results of these studies indicate that insulin, TGFs, and substrate components modulate pancreatic duct cell growth.

M a t e r i a l s and M e t h o d s Materials Antibiotic and antimycotic solution, bovine serum albumin, dexamethasone, Eagle's minimum essential medium, EGF, FBS, gentamicin sulfate, glucagon, L-glutamine, insulin, IBMX, minimal Eagle's medium amino acids, somatostatin, soybean trypsin inhibitor (SBTI), and TGF-~I were purchased from Sigma Chemical Co. (St. Louis, MO). CMRL 1066 culture medium was purchased from GIBCO BRL (Grand Island, NY). Chromatographically purified collagenase (CLSPA; 962 U/mg) was from Worthington Biochemicals (Freehold, NJ). Bombesin, cerulein, pancreatic polypeptide, secretin, and TGF-O~ were purchased from Bachem California (Torrance, CA). BrdU and immunochemical reagents for its detection were purchased as part of a Cell Proliferation Kit (RPN 20) from Amersham Corp. (Arlington Heights, IL). Biocoat cell culture inserts consisting of permeable filter supports coated with rat tail collagen type I, mouse collagen type IV, human fibronectin, mouse laminin, and Matrigel (basement membrane matrix) were obtained from Collaborative Biomedical Products (Bedford, MA). All other chemicals used were of analytical grade. Male Hartley guinea pigs were obtained from the Michigan Department of Public Health (Lansing, MI).

Isolation of Pancreatic Ducts Pancreatic ducts were isolated as described previously, m Briefly, fasted male Hartley guinea pigs were killed by exsanguination following carbon dioxide gas-induced anesthesia. Pancreata were removed and dissociated by collagenase digestion and mechanical shearing. Segments of the main pancreatic duct and interlobular ducts were manually separated from acini and vascular elements and cut into short segments using iridectomy scissors. All isolation steps were performed in HEPES-buffered Ringer's solution (pH 7.4) supplemented

GROWTH REGULATION OF PANCREATIC DUCT CELLS 945

with 0.1% bovine serum albumin, 0.01% SBTI, minimal Eagle's medium essential amino acids, and 2 mmol/L t-glutamine.

Culturing of Pancreatic Duct Explants Small segments of isolated pancreatic ducts were rinsed in CMRL 1066 medium enriched with 5% FBS, 0.5 mmol/L IBMX, 0.68 mmol/L L-glutamine, 0.01% SBTI, 100 U/mL penicillin, 10 ~tg/mL streptomycin, 50 ~g/mL amphotericin B, and 50 btg/mL gentamicin sulfate and distributed randomly into the wells of 6-well culture plates in 3.0-mL aliquots of the above medium. Culture plates were placed in an incubator at 37°C under a humidified atmosphere of 5% carbon dioxide in air. When culturing explants on substrates other than tissue culture plastic, cell culture inserts were placed in the wells with 2.0 mL of medium and duct fragments were placed on the inserts with an additional 2.0 mL. Explants were cultured for 3 days to allow adherence and initiation of growth of epithelial monolayers. After this initial period, the medium was replaced with 3.0 mL of fresh CMRL 1066 supplemented with 2.5% FBS and the above concentrations of t-glutamine, IBMX, penicillin, streptomycin, amphotericin B, and gentamicin. Wells designated as controls had this medium alone, whereas other wells contained this medium supplemented individually with different potential trophic substances. Duct epithelial cells were cultured as above for an additional 3 days at 37°C in the presence or absence of these factors.

Morphometric Procedure for Determination of Pancreatic Duct Epithelial Cell Growth To determine the effects of potential trophic factors on growth of epithelial monolayers derived from pancreatic duct explants, cultured monolayers were fixed for 6 0 - 9 0 minutes at 23°C in phosphate-buffered saline containing 2% glutaraldehyde at the end of the 3-day test period. Monolayers were then rinsed with phosphate-buffered saline and stained with 1% toluidine blue. All monolayers in each well of the 6-well culture plates were photographed at a magnification of 10× using an Olympus inverted microscope (Olympus Corp., Lake Success, NY). Areas (in square millimeters) of individual monolayers were calculated from these photomicrographs as described previously, s There was some variation in size of the original isolated duct segments introduced into the wells of culture plates. However, because the segments were distributed randomly from a common suspension in each experiment, each well received an identical range of segment sizes. For this reason, we did not routinely determine the size range of monolayers growing from these explanted segments after the initial 3-day adherence period during which no test substances were present in the culture medium.

Determination of Pancreatic Duct Epithelial Cell Growth by BrdU Incorporation At the end of the 3-day test period, the medium in all wells was replaced with 3.0 mL of Eagle's minimum essential

946

BHATTACHARYYA ET AL.

GASTROENTEROLOGY Vol. 109, No. 3

medium which, unlike CMRL 1066, lacks thymidine. This medium was supplemented with 0.5 mmol/L IBMX, 26 mmol/ L NaHCO3, 2.5% FBS, 100 U/mL penicillin, 10 p.g/mL streptomycin, 50 gg/mL amphotericin B, and a 1:500 dilution of BrdU-labeling reagent in the absence and presence of the various test substances. Cells in the monolayers were allowed to proliferate and incorporate BrdU during a further 6 hours of incubation at 37°C, after which they were fixed for 30 minutes at 23°C in 3.0 mL of acid/ethanol (90% ethanol/5% glacial acetic acid/5% distilled water). Fixed monolayers were usually stored in phosphate-buffered saline overnight at 4°C. For the cytochemical localization of sites of BrdU incorporation, an area around about 20 explants was circled with a slide marking pen (Kiyota International, Elk Grove Village, IL) before being overlayered with a 1:100 dilution of a mouse monoclonal antibody raised against BrdU diluted in Tris-buffered saline (Amersham). The monolayers were incubated with the primary antibody for 120 minutes at 23°C, rinsed with phosphatebuffered saline, and reincubated for 60 minutes at 23°C in a 1:66 dilution of a goat anti-mouse polyclonal antibody-peroxidase conjugate. After this incubation, the explants were rinsed with phosphate-buffered saline and 4 mL of 0.5 mg/mL diaminobenzidine in phosphate buffer (pH 7.4) was added to each culture well. The peroxidase reaction was allowed to proceed for 10 minutes at 23°C. Stained monolayers were rinsed with phosphate buffer (pH 7.4) and stored at 4°C until photographed. Representative areas of each of the fixed, BrdU-labeled pancreatic duct monolayers were photographed using an Olympus inverted microscope equipped with phase optics at a magnification of 50×. The negatives were printed and the number of cells in each print that had incorporated BrdU was determined, as was the number of unlabeled cells. The percentage of cells within the monolayer that had incorporated BrdU during the 6-hour incubation was calculated from these values as an expression of the rate of cellular proliferation.

Statistical Analysis Sample means were compared using Student's t test for paired samples and one-way analysis of variance for multiple samples using the INSTAT program (GraphPad, San Diego, CA). Differences were considered significant at P < 0.05.

Results BrdU Incorporation in Pancreatic Duct Monolayers Because of the relatively small amount of pancreatic duct tissue obtainable from a single guinea pig pancreas, cell proliferation in cultured duct epithelial monolayers was assessed visually by BrdU labeling rather than by biochemical analysis of [3H]thymidine incorporation. Figure 1A and B show phase-contrast and bright-field light micrographs of an explant cultured for 6 days. A confluent monolayer of epithelial cells spreads out from the ring-shaped segment of duct at center (Figure 1A). In Figure 1B, darkly stained nuclei of epithelial cells

Figure 1. Photomicrographs of a pancreatic duct explant and surrounding epithelial monolayer grown for 6 days. The monolayer was incubated in BrdU-containing medium for 6 hours at the end of the culture period and processed for immunocytochemical localization of sites of incorporation of the nucleotide into cellular DNA. (A) Phasecontrast light micrograph. (B) Bright-field light micrograph. BrdU-labeled nuclei are clearly visible in B (original magnification 38×).

that have incorporated BrdU are readily discernible. Proliferating cells are located throughout the monolayer. Although most stained nuclei showed intense deposition of reduced diaminobenzidine, staining appeared patchy and uneven in some (Figure 2). In initial experiments, the time course of BrdU incorporation was determined in monolayers cultured in the presence of 2.5% FBS, 10 nmol/L EGF, and 1 nmol/L

September 1995

insulin. The percentage of labeled cells increased with time with approximately 10% of the total population labeled after 1.5 hours of exposure to BrdU. After 6 hours, the number of cells incorporating BrdU increased to 15%-20% of the total cell population (data not shown). This latter time period was used in all subsequent experiments so that sufficient numbers of nuclei would be labeled with BrdU.

Comparison of Morphometry and BrdU Incorporation for Determination of Trophic Effects of EGF and Insulin on Pancreatic Duct Monolayers We previously showed by morphometric analysis that EGF exerts a potent trophic effect on pancreatic duct epithelial cells in vitro. 11 By contrast, although insulin has been shown to stimulate growth of cultured pancreatic acinar cells, 3 possible trophic effects of this and other islet hormones on pancreatic duct cells have not been determined. We therefore examined the effects

GROWTH REGULATION OF PANCREATIC DUCT CELLS

947

of insulin and EGF alone and in combination on BrdU incorporation by epithelial cells in cultured duct monolayers (Figure 3A). In this study, 7.8% + 0.5% of cells in control monolayers incorporated BrdU during the 6hour incubation. Addition of 1 ~lmol/L insulin and 30 nmol/L EGF to the culture medium for 3 days increased the labeling indices to 11.8% -+- 0.7% and 11.2% +

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Figure 3. Effect of insulin and EGF on growth of pancreatic duct epithelial monolayers. Growth was assessed by (A) BrdU labeling and (B) determination of monolayer areas. Pancreatic duct monolayers were cultured for 3 days at 37°C in CMRL 1066 in the absence of hormones (CON) and in the presence of 1 pmol/L insulin (INS), 30 nmol/L EGF (EGF), and 1 pmol/L insulin plus 30 nmol/L EGF (INS + EGF).The growth medium was replaced with Eagle's minimal essential medium containing the indicated substances and BrdU for the last 6 hours. Results represent means _+ SE of determinations from 12 explants in A and 1 8 - 2 5 explants in B. *Significantly different (P < 0.05) from control value. #Significantly different (P < 0.05) from values with insulin or EGF alone.

948 BHATTACHARYYAET AL.

1.2%, representing increases over the control value of 52% and 45%, respectively. When EGF and insulin were both added to the culture medium, the labeling index increased to 16.0% + 1.0%, representing a doubling of the control value. The increase in percentage of cells labeled with BrdU in the presence of both EGF and insulin (106%) was not significantly different from the additive sum of the increases caused by the two trophic factors alone (97%). The areas of monolayers in this experiment also were determined to assess the correspondence of BrdU labeling and morphometric analysis for measuring duct cell growth. Figure 3B shows the increase in monolayer area with 3-day exposure to EGF, insulin, and a combination of EGF and insulin. In the presence of 30 nmol/L EGF, the average monolayer area increased from 0.87 -+- 0.06 to 1.40 + 0.10 m m 2 , an increase of 61%. W i t h 1 btmol/ L insulin, this value increased to 1.61 + 0.12 mm 2, an increase of 84%. Average monolayer area in explants incubated with both insulin and EGF was 2.25 + 0.15 m m 2. Again, the increase evoked by the combination of the two trophic factors (157%) was not significantly different than the additive sum of the increases elicited by EGF and insulin alone (145%). These studies reconfirmed the trophic effect of EGF found in our earlier studies and showed that insulin also stimulates growth of pancreatic duct epithelial cells. The observation that maximally effective concentrations of both peptides produce additive stimulation of growth suggests that separate intracellular pathways are activated. The studies also showed that morphometric analysis and BrdU labeling each provide a valid method for quantitating cell proliferation in pancreatic duct monolayers.

Effects of Potential Growth Regulatory Substances on BrdU Labeling of Pancreatic Duct Cells BrdU labeling of monolayer cultures grown for 3 days in the absence and presence of a number of potential growth regulatory substances in addition to EGF and insulin also was performed (Table 1). Significant variability was found from experiment to experiment in the percentage of labeled cells in control monolayers. For this reason, paired control wells were included when assessing the effects of each tested substance. Addition of bombesin, dexamethasone, glucagon, pancreatic polypeptide, secretin, and somatostatin to the culture medium had no significant effect on the percentage of BrdUlabeled cells compared with controls. Insulin, EGF, and TGF-Oq however, each showed potent mitogenic activity as shown by increases in BrdU labeling indices of 57%,

GASTROENTEROLOGYVol. 109, No. 3

36%, and 44%, respectively. In contrast, TGF-~ inhibited growth of pancreatic duct cells as shown by a decrease in the BrdU labeling index of 51% from the control value. A marginally significant decrease in BrdU labeling also was observed when cerulein was added to the culture medium.

Dose Effect of Insulin and Other Islet Hormones The dose dependence of the trophic effect of insulin on cultured pancreatic duct cells measured morphometrically is shown in Figure 4. Very little increase in monolayer area was found below a concentration of 1 nmol/L insulin, but a sharp increase in growth was observed from 1 to 100 nmol/L. The maximal effect of insulin was found at a concentration of 1 t.tmol/L (Table 2) with a 50% effective concentration of 8 nmol/L. Although BrdU labeling studies (Table 1) indicated that the three other islet hormones do not exert trophic effects on cultured pancreatic duct epithelial cells, we reexamined their possible effects on monolayer area at concentrations ranging from 0.1 nmol/L to 1 btmol/L using morphometric analysis. Glucagon, pancreatic polypeptide, and somatostatin had no significant effects on the areas of pancreatic duct monolayers up to a concentration of 1 ~tmol/L (Table 2), confirming the results of the BrdU labeling studies.

Effects of TGF-(x, TGF-13, and Dexamethasone on Pancreatic Duct Monolayer Growth Previous studies have shown inhibition of pancreatic acinar cell growth by glucocorticoids and TGF-~. 3'6 W e therefore examined the effects of these substances and TGF-O~ on growth of monolayers of cultured guinea pig pancreatic duct epithelial ceils. Three-day exposure of cultured duct explants to TGF-O~ caused a dose-dependent increase in monolayer area with a 50% effective concentration of 0.8 nmol/L (Figure 5). The dose response curve was biphasic with maximal growth stimulation found at 10 nmol/L (Table 2), as was observed for EGF in an earlier study. 11 By contrast, addition of TGF-~ to the culture medium caused a dose-dependent decrease in monolayer area with a 50% inhibitory concentration of 0.5 pmol/L (Figure 6 and Table 2). Although dexamethasone is a potent inhibitor of pancreatic acinar cell growth, 3 it did not seem to modulate growth of cultured guinea pig pancreatic duct epithelial ceils in vitro at concentrations up to 1 btmol/L (Table 2).

Effects of Different Culture Substrates on Growth of Pancreatic Duct Monolayers To test the effects of different substrates on guinea pig pancreatic duct epithelial cell growth, explants were

September 1995

GROWTH REGULATION OF PANCREATIC DUCT CELLS 949

Table 1. Effects of Potential Growth Regulatory S u b s t a n c e s on BrdU Labeling of Cultured Guinea Pig Pancreatic Duct Epithelial Monolayers Percent of cells labeled Test substance

Concentration

Cerulein Bombesin Secretin Glucagon Insulin Somatostatin Pancreatic polypeptide EGF TGF-c( TGF-13 Dexamethasone

1 pmol/L 10 nmol/L 1 pmol/L 1 pmol/L 1 pmol/L 1 pmol/L 1 pmol/L 30 nmol/L 10 nmol/L 0.1 nmol/L 0.1 pmol/L

Control 14.6 13.5 16.2 12.2 8.4 16.8 6.5 8.4 7.9 9.5 10.8

+ ± ± + + ± + ± + ± ±

0.3 0.5 0.5 0.9 0.4 0.2 0.2 0.4 0.3 0.6 0.5

Experimental (22) (21) (21) (24) (27) (20) (12) (27) (37) (17) (24)

13.6 12.2 15.8 11.1 13.2 17.6 5.9 11.4 11.3 4.6 10.3

+ 0.4 _+ 0.5 _+ 0.3 _+ 0.8 +_ 0.5 ± 0.4 +_ 0.4 + 0.7 +_ 0.4 ± 0.3 ± 0.6

(16) (20) (20) (31) (31) (21) (20) (22) (40) (21) (23)

P value 0.03 a 0.09 0.56 0.37 0.0001 a 0.09 0.20 0.0002 a 0.00013 0.0001 `7 0.56

NOTE. Pancreatic duct epithelial monolayers were cultured for 3 days in the presence and absence of the indicated test substances. BrdU was added to the culture medium during the last 6 hours of the test period. Results represent means ± SE of the percentage of cells labeled in the number of individual monolayers indicated in parentheses and are from 2 - 3 separate experiments. aExperimental values that are significantly different (P < 0.05) than their respective controls.

seeded onto permeable filters coated with type I collagen, type IV collagen, fibronectin, laminin, and Matrigel and onto tissue culture plastic and were cultured in C M R L 1066 medium for 6 days at 37°C (Figure 7). Explants readily attached to and grew on type I collagen-, type IV collagen-, and fibronectin-coated filters. By compari-

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son, the efficiency of attachment of duct explants to laminin- or Matrigel-coated filters was much lower, and the resulting monolayers formed were substantially smaller than those that grew from explants seeded onto the other substrates. Average areas of monolayers grown on type IV collagen- and fibronectin-coated filters were not different from those of monolayers grown on tissue culture plastic, although those grown on a substrate of type I collagen were significantly larger.

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Insulin (log Molar) Figure 4. Dose dependence of the effect of insulin on the areas of cultured pancreatic duct epithelial monolayers. Monolayers were incubated for 3 days in CMRL 1066 with or without the indicated concentrations of insulin. Monolayer areas were determined at the end of this period by morphometric procedures. Results represent the means _+ SE of determinations of areas of 5 3 - 8 0 monolayers for each concentration. *Significantly different (P < 0.05) from the control value.

Although the excretory duct system of the exocrine pancreas constitutes only a small percentage of the total pancreatic mass, it plays a central role in the elaboration of pancreatic juice and constitutes an important site of dysfunction in several pancreatic diseases, including pancreatitis, pancreatic cancer, and cystic fibrosis. ~* For these reasons, a substantial amount of effort has been expended in an attempt to elucidate the cellular and subcellular mechanisms that subserve regulated secretion of electrolytes by the pancreatic duct epithelium. I~ By contrast, however, comparatively little attention has been paid to the physiological mechanisms that control cellular proliferation in the duct system of the pancreas. Because of the importance of the excretory duct system to the physiological function of the pancreas and to the initiation and progression of the various pathologies to which the pancreas is subject, elucidation of the physiological regulators of cell turnover in this tissue is of prime importance. The recent development of preparations of isolated pancreatic ducts from which primary cultures of epithelial cells can be established has made in vitro screening of potential growth-regulating substances pos-

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GASTROENTEROLOGY Vol. 1 0 9 , No. 3

T a b l e 2. Effects of Potential Growth Regulatory S u b s t a n c e s on Areas of Cultured Guinea Pig Pancreatic Duct Epithelial

Monolayers Monolayer area Test substance

Concentration

Insulin Glucagon Somatostatin Pancreatic polypeptide TGF-(~ TGF-~ Dexamethasone

1 gmol/L 1 pmol/L 1 pmol/L 1 I~mol/L 10 nmol/L 1 nmol/L 1 pmol/L

(mm2)

Control 0.68 0.80 1.06 1.23 0.96 0.95 0.99

Experimental

_+ 0.03 (69) + 0.05 (78) _+ 0.08 (64) _+ 0.7 (81) _+ 0.05 (88) _+ 0.05 (57) + 0.06 (66)

1.13 0.92 0.93 1.34 1.41 0.55 0.89

_ 0.06 _+ 0.06 _+ 0.06 +_ 0.06 +_ 0.09 _ 0.03 _+ 0.05

P value

(66) (85) (99) (71) (89) (64) (91)

0.0001 a 0.13 0.19 0.24 0.0001 a 0.0001 a 0.20

NOTE. Pancreatic duct epithelial monolayers were cultured for 3 days in the presence and absence of the indicated test substances. Results represent means _+ SE of the areas of the number of monolayers indicated in parentheses. aExperimental values that are significantly different (P < 0.05) than their respective controls.

sible. ~6 As noted above, using primary cultures of epithelial cells derived from guinea pig main and interlobular ducts, we earlier established EGF as a potent trophic factor) ~ In the current studies, we extended these investigations using morphometric analysis and the newly developed technique of BrdU labeling to determine the effects of a number of potential regulators on pancreatic duct epithelial cell growth in vitro. Bombesin and cerulein have been shown to exert trophic effects on mouse pancreatic acinar cells in culture, 2'4 and infusion of cerulein in intact rats has been reported to increase the mitotic labeling indices of both

pancreatic acinar and intralobular duct cells, lv'~8 In our earlier studies, 11 however, we found no effect of these two peptides on growth of cultured guinea pig main and interlobular pancreatic duct epithelial cells. In the current studies, we confirmed this observation using BrdU labeling. As shown in Table 1, neither cerulein nor bombesin increased the BrdU labeling indices of duct epithelial cells. Although secretin is the primary postprandial endocrine regulator of pancreatic duct bicarbonate secretion .5 and has been shown to exert a trophic effect on cultured hamster duct epithelial cells, 12 we likewise observed no effect of this hormone

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September 1995

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0.0 TCP COL-ICOL-IV FIB LAM MATR Rgure 7. Effects of different culture substrates on growth of cultured pancreatic duct epithelial monolayers. Duct explants were seeded onto tissue culture plastic (TCP) and onto porous filters coated with type I collagen (COL-I), type IV collagen (COL-IV), fibronectin (FIB), laminin (I_AM),and Matrigel (MATR) and cultured for 6 days at 37°C in CMRL 1066 medium. At this time, the resulting monolayers were fixed and their areas determined morphometrically. Results represent means _+ SE of determinations of areas for the following numbers of monolayers: 77 on tissue culture plastic, 43 on type I collagen, 64 on type IV collagen, 74 on fibronectin, 25 on laminin, and 14 on Matrigel. *Significantly different (P < 0.05) from tissue culture plastic value.

on BrdU labeling of guinea pig duct cells in the current studies. We also examined the effects of all four islet hormones on growth of cultured duct epithelial cells in the current studies because, although the importance of islet hormones to growth and maintenance of differentiated function of pancreatic acinar cells has been well established, 19 little is known of the functional relationship between pancreatic islets and cells of the excretory ducts. Islets arise during embryonic development from endocrine cells located in the pancreatic duct system, and islet cells are found within the duct epithelium in adult mammals, 2°-22 suggesting the existence of a physiologically relevant islet-duct axis. Insulin previously has been shown to exert trophic effects on pancreatic acinar cells 3 and on acinarderived AR42J tumor cells, 23 whereas somatostatin analogues have been reported to inhibit growth of both acinar and ductal carcinomas. 24'25 However, the effects of these two hormones and of glucagon and pancreatic polypeptide on growth of normal pancreatic duct cells have not been investigated. Results shown in Tables 1 and 2 and Figures 3 and 4 indicate that insulin is the only islet hormone with appreciable growth-promoting activity. Glucagon, somatostatin, and pancreatic polypeptide caused no significant changes in BrdU labeling indices or in pancreatic duct epithelial monolayer areas at concentrations up to 1 ~lmol/L. These observations for the first time establish insulin as an important regulator of growth in the pancreatic excretory duct system and

951

help explain the basis for exocrine pancreatic insufficiency in many diabetic patients. 26'27 In the current studies, we also examined the effects of TGF-O~ and TGF-~ on guinea pig pancreatic duct cell growth. TGF-O~, which binds to and activates the EGF receptor, is expressed by normal pancreatic duct epithelial cells 28 and has been reported to be overexpressed in ductal adenocarcinomas and during chronic pancreatitis, 29'3° suggesting that an autocrine mechanism involving this growth factor may contribute to the severity of these diseases. By contrast, TGF-~ is expressed primarily in acinar and stromal cells of the intact pancreas 3~ and has been shown to strongly inhibit growth of cultured mouse acinar cells 6 and rat duct cells. 3~'33 Results of the current studies show that TGF-0~ is a trophic factor as well for guinea pig pancreatic duct epithelial cells with a potency equal to that of EGF and that TGF-~ is a potent inhibitor of guinea pig pancreatic duct cell growth. These observations strengthen the view that TGFs play important roles as autocrine and paracrine regulators of cell growth in the excretory duct system of the normal pancreas. Growth of the duct epithelium also may be regulated by components of the extracellular matrix in addition to soluble growth factors. Laminin and type IV collagen are major components of epithelial basement membranes, whereas type I collagen and fibronectin are molecular components of fibrous connective tissue) 4 All four molecules are present in the exocrine pancreas. 35 Mollenhauer et al. earlier reported that proliferation of PaTu II pancreatic carcinoma cells was enhanced when these cells were cultured on surfaces coated with fibronectin or type I collagen and inhibited when cultured on a laminincoated surface) 6 Results of the current studies (Figure 7) indicate that type I collagen exerts a strong trophic effect on normal pancreatic duct epithelial cells, whereas laminin inhibits growth. Proliferation of guinea pig pancreatic duct epithelial cells also was significantly inhibited when duct explants were established on Matrigel, an artificial basement membrane containing laminin and type IV collagen. These results indicate that the basement membrane, which underlies and supports the epithelium of the pancreatic duct system, exerts a net suppressive effect on cellular proliferation, whereas cell growth is encouraged by contact of duct cells with components of the surrounding connective tissue. Type I collagen is not normally encountered by duct epithelial cells unless the integrity of the basement membrane is compromised, which may happen during invasion of the surrounding connective tissue stroma by ductal-derived rumors. The strong growth-promoting effects of type I collagen on duct epithelial cells may therefore contribute

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to the observed invasiveness and characteristically rapid metastasis of ductal adenocarcinomas.

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Received January 16, 1995. Accepted April 11, 1995. Address requests for reprints to: Seth R. Hootman, Ph.D., Department of Physiology, Michigan State University, 108 Giltner Hall, East Lansing, Michigan 48824. Fax: (517) 355-5125. Supported by National Institute of Diabetes and Digestive and Kidney Diseases grant RO1-DK46607.