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Am J Physiol Cell Physiol 291: C546-C554, 2006. First published May 10, 2006; doi:10.1152/ajpcell.00011.2006
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GROWTH, DIFFERENTIATION, AND APOPTOSIS

Biphasic regulation by bile acids of dermal fibroblast proliferation through regulation of cAMP production and COX-2 expression level

Jian Ping Meng,1 Susan Ceryak,2 Zaheer Aratsu,1 Loren Jones,1 Lauren Epstein,1 and Bernard Bouscarel1,3

1Gastroenterology Research Laboratory, Department of Biochemistry and Molecular Biology, 2Department of Pharmacology and Physiology, and 3Department of Medicine, The George Washington University Medical Center, Washington, District of Columbia

Submitted 11 January 2006 ; accepted in final form 25 March 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have previously reported that the bile acids chenodeoxycholate (CDCA) and ursodeoxycholate (UDCA) decreased PGE1-induced cAMP production in a time- and dose-dependent manner not only in hepatocytes but also in nonhepatic cells, including dermal fibroblasts. In the present study, we investigated the physiological relevance of this cAMP modulatory action of bile acids. PGE1 induced cAMP production in a time- and dose-dependent manner. Moreover, PGE1 (1 µM), forskolin (1–10 µM), and the membrane-permeable cAMP analog CPT-cAMP (0.1–10 µM) decreased dermal fibroblast proliferation in a dose-dependent manner with a maximum inhibition of ~80%. CDCA alone had no significant effect on cell proliferation at a concentration up to 25 µM. However, CDCA significantly reduced PGE1-induced cAMP production by 80–90% with an EC50 of ~20 µM. Furthermore, at concentrations ≤25 µM, CDCA significantly attenuated the PGE-1-induced decreased cell proliferation. However, at concentrations of 50 µM and above, while still able to almost completely inhibit PGE-1-induced cAMP production, CDCA, at least in part through an increased cyclooxygenase-2 (COX-2) expression level and PGE2 synthesis, produced a direct and significant decrease in cell proliferation. Indeed, the CDCA effect was partially blocked by ~50–70% by both indomethacin and dexamethasone. In addition, overexpression of COX-2 cDNA wild type resulted in an increased efficacy of CDCA to block cell proliferation. The effects of CDCA on both cAMP production and cell proliferation were similar to those of UDCA and under the same conditions cholate had no effect. Results of the present study underline pathophysiological consequences of cholestatic hepatobiliary disorders, in which cells outside of the enterohepatic circulation can be exposed to elevated bile acid concentrations. Under these conditions, low bile acid concentrations can attenuate the negative hormonal control on cell proliferation, resulting in the stimulation of cell growth, while at high concentrations these bile acids provide for a profound and prolonged inhibition of cell proliferation.

chenodeoxycholic acid; cyclic adenosine monophosphate

cAMP, which can be induced by the prostaglandins (PG) PGE1 and PGE2, is known to have both proliferative and antiproliferative effects, according to cell type. For example, cAMP stimulates the proliferation of cells, such as hepatocytes and 3T3 cells, while inhibiting the proliferation of normal human skin fibroblasts, gastric cells, and hepatic stellate cells (20, 30, 38, 55). The PGE2 is also a potent inhibitor of fibroblast migration, proliferation and collagen synthesis (23, 24, 54). PGs are derived from arachidonic acid through the cyclooxygenase (COX) pathway. COX catalyzes the rate-limiting oxidative cyclization of arachidonic acid to prostaglandin endoperoxide, which is the immediate precursor of PGs, thromboxane, and prostacyclin (40). Two isoforms of COX have been recognized. COX-1 (72 kDa) is expressed constitutively, whereas COX-2 (72/74 kDa) is induced by growth factors, oncogenes, tumor promoters, and proinflammatory cytokines (see Ref. 4 for review). Furthermore, while COX-1 is expressed in most tissues, COX-2 is usually quiescent or absent in most tissues but can be rapidly expressed in large amounts by physiological, pathological, and mitogenic stimuli (32, 50). The regulation of COX-2 activity occurs principally through control of gene expression and is cell type specific. For example, TGF-beta augments COX-2 induction by IL-1 in fibroblasts while it inhibits COX-2 induction in macrophages (49). Therefore, cAMP and COX-2 are key factors in the regulation of cell proliferation by PG.

Bile acids are known to be co-carcinogenic agents. They are synthesized in the liver and secreted into bile conjugated mainly to either glycine (G) or taurine (T). Under physiological conditions, while chiefly confined to the enterohepatic circulation, bile acid concentration in the systemic circulation can increase postprandially two- to threefold from a fasting level of ~1–3 µM (2, 44). In the serum, bile acids are mainly in the amidated form. The level of unconjugated bile acids exhibits a diurnal variation, attaining a maximum concentration of 30–40% of the total serum bile acids after breakfast (44). In several pathological conditions, tissues outside of the enterohepatic circulation can come in contact with high concentrations of both unconjugated and conjugated bile acids. For example, in cholestatic hepatobiliary disorders, bile acids accumulate in the systemic circulation, resulting in a 20- to 100-fold increase in serum bile acid concentration (29). Under these conditions, serum levels of unconjugated bile acids can increase dramatically, particularly if portal cirrhosis is present (29). Finally, in patients with stagnant loop syndrome, serum unconjugated bile acid levels increase due to bacterial overgrowth in the small intestine (27).

Sodium-dependent bile acid transporters have been reported to be functionally present mainly in the liver, ileum, and kidney (1, 46). However, bile acids can enter cells by a sodium-independent transporter, as well as by passive diffusion, which is a function of their respective hydrophobicity (11, 53). Thus these observations underline the relevance that increases of either or both conjugated and unconjugated bile acids in the systemic circulation could have in their accumulation in extrahepatic tissues (12, 19, 34). This is in light of considerable evidence both in human and in animal models that cholestasis is associated with increased deposition of bile acids in extrahepatic tissues, with the skin being one of the predominant ones (5, 12, 16, 43).

Both the physiological and the pathophysiological effects of bile acids have been well documented for the liver and intestine, while they are less understood for tissues outside of the enterohepatic circulation, despite their exposure to bile acids, which is particularly significant in hepatobiliary diseases. Therefore, the present study was designed to investigate the chronic effect of bile acids on stimulated cAMP synthesis in human dermal fibroblasts. Moreover, the functional consequence of bile acid-induced inhibition of stimulated cAMP on fibroblast proliferation was assessed. We show that physiological concentrations of bile acids inhibit stimulated cAMP formation after chronic exposure and that the cAMP-induced inhibition of cell proliferation is partially abrogated in the presence of bile acids. However, at concentrations >50 µM, certain bile acids like chenodeoxycholic acid (CDCA) directly inhibit cell proliferation through an increased COX-2 expression and PGE2 synthesis.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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Materials. Ursodeoxycholic acid (UDCA) was supplied by Tanabe (Tokyo, Japan), and CDCA was supplied by Dr. Falk (Pharma, Freiburg, Germany). Deoxycholic acid (DCA) and cholic acid (CA) were purchased from Steraloids (Wilton, NH). All bile acids used were 98–99% pure, as judged by gas-liquid chromatography. Prostaglandin (PG) E1, forskolin (FK), and 8-(4-chlorophenylthio)-cAMP sodium salt (cpt-cAMP) were purchased from Calbiochem (San Diego, CA). PGE1, PGE2, 1OH-PGE1 were from Cayman (Ann Arbor, MI). RPMI and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma (St. Louis, MO). [125I]cAMP was purchased from Linco Research Products (St. Charles, MO), and [methyl-3H]thymidine (specific activity, 80 Ci/mmol) was from Amersham (Piscataway, NJ). Dulbecco’s modified Eagle’s minimum essential medium (DMEM; CellGro) was purchased from Fisher Scientific (Pittsburgh, PA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT). Other chemicals were from either Sigma or Fisher Scientific and were of the highest purity available.

Culture of human skin fibroblasts. Human skin fibroblasts obtained from forearm skin biopsy were purchased from Coriell Institute for Medical Research (Camden, NJ). The cells were cultured, as previously described (8), in DMEM with 1% L-glutamine, 2% essential and nonessential amino acids, 1% penicillin and streptomycin, and 10% FBS. The cells were plated at a density of 3–5 x 104 cells·ml·well–1 in 12-well plates. Four to eight hours before an experiment, the cells were cultured in DMEM, containing either 0.1% FBS for cAMP determination or 1% FBS to measure cell proliferation.

cAMP determination. Cells were either incubated alone or with the sodium salt of the respective bile acid for the designated period of time. cAMP synthesis was stimulated either simultaneously, or following bile acid preincubation, with PGE1 in the presence or absence of 50 µM IBMX, for the indicated period of time. Following incubation with the designated agents, cAMP was measured in cellular HClO4 extracts, as well as in the medium, by radioimmunoassay as previously described (8), using the method of Gettys et al. (15). The results were expressed either per milligram of total cell protein as determined by the BCA assay (Pierce, Rockford, IL) or as percentage of the maximum obtained by incubating the cells with PGE1 + IBMX alone.

Cell proliferation assessment. Cells were seeded at 3–5 x 104 cells/well in 12-well plates. After 24 h, the culture medium was replaced with medium containing 1% FBS 4 h before preincubation with the respective bile acid or tested agents. Cells were incubated for an additional 24 h after PGE1 addition, and the uptake of [methyl-3H]thymidine was determined as an index of cell proliferation. Briefly, 1 µCi [methyl-3H]thymidine was added to each well, and the cells were incubated for an additional 4 h. Cells were then washed, harvested with trypsin, and pipetted onto glass fiber filters (Whatman, GF/B). Filters were washed four times with Tris-buffered saline (20 mM Tris·HCl and 150 mM NaCl, pH 7.4), and cellular [3H]thymidine incorporation was measured by scintillation counting (model SL6000; Beckman, Palo Alto, CA).

RNA extraction and reverse transcription. Total RNA was extracted from fibroblasts using RNA Bee (Tel-Test, Friendswood, TX). Oligo dT and Superscript III were used for transcription containing 1–2 µg of RNA. Reverse transcription was conducted at 50°C for 60 min after RNAse H treatment for 30 min. DNA (5–10 ng) was used for PCR reaction using Taq DNA polymerase (GIBCO-BRL). PCR reactions were conducted at 94°C for 3 min for denaturation, followed by 94°C for 45 s, 55°C for 30 s, 72°C for 40–60 s, for either 24–27 cycles for GAPDH or 27–32 cycles for human COX2, followed by a final extension at 72°C for 1 min. The reaction products were analyzed by electrophoresis on 1.5% agarose gels. The gels were analyzed by densitometric scanning with the use of photo imaging (Molecular Dynamics, Sunnyvale, CA). The densities of the COX-2 bands were normalized with the respective GAPDH bands.

Immunoblot analysis and PGE2 synthesis. Total cellular homogenates were prepared from cultured fibroblasts in modified RIPA buffer (0.5 M Tris·HCl, pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid, 10% Nonidet P-40, and 10 mM EDTA) containing one tablet of protease inhibitor (Roche, Indianapolis, IN). In addition, 1 mM of Na3VO4 and 1 mM of NaF were used in all the buffer preparations when phosphorylation studies were performed. Protein samples (20–30 µg) of the respective cellular fractions were separated by SDS-PAGE, using a mini-gel apparatus (Invitrogen, Carlsbad, CA) and transferred to Hybond-P Amersham Biosciences (Arlington Heights, IL) membranes using the Invitrogen transfer apparatus according to the manufacturer’s directions. The protein-containing membranes were blocked in 5% casein and 0.1% Tween (pH 7.4) and further incubated overnight at 4°C with either goat anti-human COX-2 (1:500), rabbit anti-human PKC{alpha} (1:1,000), anti-phospho P38 (1:500), anti-P38 (1:1,000), or anti-beta-actin (1:5,000) antibodies. The protein content was visualized using horseradish peroxidase-conjugated corresponding secondary antibodies, followed by enhanced chemiluminescence (Amersham), and analyzed densitometrically. The immunoreactive signals were normalized against that of beta-actin. The production of PGE2 was determined spectrometrically (Spectra-MAX, Molecular Dynamics) using an ELISA assay kit according to the manufacturer’s protocol (Cayman).

Statistical analysis. Except as otherwise indicated, the results were expressed as means ± SE. The statistical significance was determined by either Student’s t-test or ANOVA when more than two groups were compared.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Comparative effect of various agents on cAMP production. Several hormones and agents were investigated for their ability to significantly stimulate cAMP synthesis in human dermal fibroblasts. Histamine, PGE1, 1OH-PGE1, and PGE2 were found to stimulate cAMP synthesis in these cells as previously reported (10). Among the different agents tested, PGE1 was the most potent to stimulate cAMP production. Furthermore, the PGE1-induced cAMP production was time dependent with little receptor desensitization up to 24 h. The basal level of cAMP was 28–45 pmol/mg protein and was increased to 2,200 pmol/mg protein after 2 h of incubation with 1 µM PGE1 and 50 µM IBMX. cAMP production was induced by 1OH-PGE1 a EP2/EP4 agonist and little effect was observed with either sulprostone or misoprostol an EP1/EP3 and EP2/EP3 agonist, respectively (data not shown).

Comparative effect of bile acids on PGE1-induced cAMP production. Two-hour incubation of human skin fibroblasts with the dihydroxy bile acids DCA, CDCA, and UDCA had no effect on the basal cellular level of cAMP synthesis (data not shown). However, DCA, CDCA, and UDCA dramatically inhibited PGE1-induced cAMP production after 2-h incubation (Fig. 1A). The approximate 80-fold increase in cAMP induced by 1 µM PGE1 plus 50 µM IBMX exposure was inhibited by 50% at a concentration (EC50) of 4.6 µM by DCA and of 20 to 25 µM by CDCA and UDCA, respectively (Table 1). Under these conditions, 50–100 µM of either DCA or CDCA almost completely (96%-98%) inhibited the maximum production of cAMP. Furthermore, as previously reported (8, 9), bile acids inhibited PGE1-induced cAMP production to a similar extent in the presence and absence of IBMX following incubation with the fibroblasts for 2 h (data not shown). Thus the effect of the bile acid is not at the level of the phophodiesterase and cAMP breakdown. Therefore, IBMX is not required for the bile acid to induce this inhibitory effect.


Figure 1
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  		Fig. 1. Dose-dependent effect of bile acids on PGE1-induced cAMP formation. Cultured human dermal fibroblasts were incubated at 37°C for either 2 h (A) or 20 h (B) with 1 µM PGE1 and 50 µM 3-isobutyl-1-methylxanthine (IBMX), as well as in the presence of increasing concentrations of the indicated bile acids. At the end of this period, the total cellular cAMP level was determined by radioimmunoassay. Results are expressed as a percentage of the maximum level of cAMP (~2,200 pmol/mg protein at 2 h, and 7,500 pmol/mg protein at 20 h), corrected for the basal level (28–45 pmol/mg protein), and determined in the presence of PGE1 and IBMX and in the absence of bile acids. Results are means ± SE of 3–4 determinations. CA, cholic acid; DCA, deoxycholic acid; CDCA, chenodeoxycholic acid; UDCA, ursodeoxycholic acid.

 

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  		Table 1. Bile acid concentrations eliciting 50% inhibition of the maximum PGE1-induced cAMP synthesis

 
After 20 h of exposure of the fibroblasts to DCA, CDCA, or UDCA, and in the presence of 1 µM PGE1 and 50 µM IBMX, the cellular cAMP formation was inhibited to the same extent as that measured after 2 h (Fig. 1B). However, as previously observed in acute studies (8, 9), CA remained without effect on PGE-1-induced cAMP formation. Furthermore, the basal level of cAMP at ~45 pmol/mg protein, was not affected by prolonged exposure of the cells to any of the bile acids studied (data not shown). The maximum cAMP formation, in the presence of PGE1 and IBMX, at 7,500 pmol/mg protein, was inhibited by 50% at similar respective EC50 concentration that was observed after 2 h of exposure (Table 1). The respective level of inhibition at a bile acid concentration of 100 µM was also similar as that observed at 2 h, suggesting that the bile acid inhibitory effect was maximum by 2 h and persisted for at least 20 h. Under the same 20-h conditions, the increased cAMP production induced by 1 µM 1OH-PGE1 was decreased by CDCA in a dose-dependent manner with an almost complete inhibition with CDCA concentrations >25 µM (Fig. 2).


Figure 2
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  		Fig. 2. Dose-dependent effect of CDCA on 1-OH-PGE1-induced cAMP formation. Fibroblasts were incubated for 20 h with 1 µM 1OH-PGE1 and 50 µM IBMX, as well as in the presence of increasing concentrations (1–100 µM) of CDCA. See Fig. 1 for details. Result is a representative experiment performed in quadruplicate. *P < 0.01, significantly different from control determined in the absence of bile acid.

 
To determine whether the bile acid effect to inhibit cAMP production was dependent on the initial presence of PGE1, the fibroblasts were incubated first with CDCA for 20–24 h and then with PGE 1 + IBMX for 8 min before the cellular cAMP production was assessed. Under these conditions, CDCA decreased PGE1-induced cAMP production by 70% compared with >90% when both CDCA and PGE1 are chronically incubated together (data not shown).

Effect of cAMP synthesis-stimulating agents on cell proliferation. The various agents tested, which stimulate cAMP production, including IBMX, PGE1, 1OH-PGE1, FK, and the nonhydrolyzable, cell-permeable cAMP analog cpt-cAMP, were able to significantly inhibit human dermal fibroblast proliferation, as measured by thymidine incorporation. As shown in Fig. 3A, 20–24 h incubation with 50 µM IBMX, a phosphodieasterase inhibitor that prevents cAMP breakdown, inhibited cell growth by 40%. Furthermore, this inhibitory effect was mimicked by 1 µM of both PGE1 and 1-OH-PGE after 20–24 h incubation, and the PGE1 effect was potentiated by IBMX. Although not shown, little inhibition of cell proliferation was observed when sulprostone, an EP1/EP3 agonist, and misoprostol, an EP2/EP3 agonist, were tested. Likewise, FK, an activator of adenylate cyclase, decreased cell growth to 40% of the control, whereas cpt-cAMP evoked a dose-dependent decrease in [3H]thymidine DNA incorporation, with a maximum inhibition of ~80%. These results confirm the inhibitory effect of cAMP on cell proliferation in human dermal fibroblasts. However, under the same conditions, 1 µM of either 15{Delta}PGJ2 or PJD2 did not significantly affect fibroblast proliferation (data not shown).


Figure 3
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  		Fig. 3. Effect of cAMP-modulating agents and bile acids on cell proliferation. Fibroblasts were incubated in the absence and presence of IBMX, 10H-PGE1, forskolin (FK), a membrane-permeable cAMP analog (cpt-cAMP) and PGE1 (A) or increasing concentrations of CDCA, UDCA, and CA (B). These agents were present for 20 h. One µCi [methyl-3H]thymidine was then added to each well and maintained at 37°C for an additional 4 h. Finally, the cells were harvested, washed, and filtered, and the uptake of [3H]thymidine was determined as an index of cell proliferation and expressed as percentage of control, i.e., in the absence of added agents. Results are means ± SE of 3–6 experiments performed in triplicate or quadruplicate. CTL, control; cpt-cAMP, 8-(4-chlorophenylthio)-cAMP. The concentrations of the various agents tested are in µM. *P < 0.05, significantly different from control; **P < 0.01, significantly different from control.

 
Effect of bile acid on the modulation of cell proliferation by PGE-1. As reported in Fig. 3B, only concentrations of CDCA >25 µM were able to directly and significantly inhibit fibroblasts proliferation by 35–45%. Under these conditions, 50 and 100 µM UDCA decreased cell proliferation by 50 and 65%, respectively, whereas 25 µM CA had no significant inhibitory effect (Fig. 3B). However, in keeping with its observed effect on PGE1-induced cAMP formation, 10 µM CDCA preincubation was associated with a significant attenuation of PGE1-induced growth arrest (Fig. 4A). Moreover, UDCA, which has a similar effect on PGE1-induced cAMP formation, was also able to significantly attenuate the PGE1 effect, while CA was again without effect (Fig. 4A). Finally, CDCA also induced a dose-dependent reversal of the inhibition of cell proliferation induced by 0.5 µM of the EP2/EP4-specific agonist 1-OH-PGE (Fig. 4B). Collectively, these results suggest that bile acids, at physiologically relevant concentrations that reduce PGE1-induced cAMP production, are also able to reverse the cAMP-mediated PGE1-induced inhibition of cell proliferation.


Figure 4
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  		Fig. 4. Combined effect of PGE1 and bile acids on cell proliferation. Fibroblasts were incubated in the absence and presence of IBMX and either PGE1 (A) or 1OH-PGE1 (B), as well as various concentrations of CDCA, UDCA, or CA for 20 h. One µCi [methyl-3H]thymidine was then added to each well and maintained at 37°C for an additional 4 h. The cells were processed as described in the legend of Fig. 3. In addition, see legend of Fig. 1 for abbreviations. Results are means ± SE of 3–4 determinations. *P < 0.05, significantly different from respective control determined in the absence of bile acid; **P < 0.05, significantly different from control; #P < 0.05, significantly different from 1OH-PGE1 without CDCA.

 
Role of COX-2 on regulation of cell proliferation by cAMP and bile acid. To study the role of COX in the regulation of cell proliferation by cAMP and bile acids, the fibroblasts were incubated with 1 µM indomethacin, a nonspecific COX inhibitor. Indomethacin had no effect alone at this concentration but significantly reduced the inhibitory effect of PGE1, FK, and 50 µM CDCA by 25–65% (Fig. 5A). Moreover, incubation of fibroblasts with 2 µM NS-398, a specific COX-2 inhibitor reduced the effect of PGE1 and FK by 40–60% and that of CDCA by ~42% (Fig. 5B). Furthermore, preincubation of the cells with 1 µM dexamethasone, which prevents COX-2 protein synthesis, results in an almost complete abrogation of the inhibitory effect of PGE1 and PGE2 on cell proliferation, while it significantly reduces that of 50–100 µM CDCA by >45% (Fig. 5C). Together, these results suggest that at least COX-2 is involved in the inhibition of cell proliferation by PGE and higher concentrations of certain bile acids.


Figure 5
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  		Fig. 5. Effect of COX-2 inhibitors on cell proliferation in the presence of cAMP modulating agents and CDCA. Fibroblasts were incubated in the absence and presence of 1 µM indomethacin (A), 2 µM NS-398 (B), or 1 µM dexamethasone (DEX; C) 2–4 h before the addition of either the cAMP modulating agents (PGE1, PGE2, or FK) and CDCA for 20 h. One µCi [methyl-3H]thymidine was then added to each well and maintained at 37°C for an additional 4 h. The cells were processed as described in Fig. 3. Results are means ± SE of 4 determinations. *P < 0.01, significantly different from control; **P < 0.05, significantly different from respective condition without COX-2 inhibitor.

 
Effect of bile acids on COX-2 mRNA expression and PG2 synthesis. A recent report (25) demonstrating that the PGE-2 induced inhibition of fibroblast proliferation was abrogated in COX-2 but not COX-1 knockout mice supports a fundamental role of COX-2 and PG synthesis in the regulation of cell proliferation. Therefore, we undertook experiments to study the COX-1 and COX-2 mRNA expression level by RT-PCR in human dermal fibrobalts after exposure to different agents. The basal COX-1 mRNA expression was not affected by any of the agents tested (data not shown). The basal COX-2 mRNA level was barely detectable (Fig. 6A). Furthermore, while 1 µM PGE1 and 10 µM CDCA induced a slight but statistically insignificant increase in COX-2 mRNA expression, 100 µM of either CDCA, UDCA, or DCA stimulated COX-2 mRNA expression by 8-, 6-, and 4-fold, respectively (Fig. 6, A and B); 200 nM PMA was used as control. Furthermore, although not shown, the increase in COX-2 mRNA expression by the bile acids was time and dose dependent, i.e., a maximum effect observed at 12 h and with 200–400 µM CDCA. CDCA and PMA stimulated COX-2 protein expression by ~4- and ~9-fold, respectively, 8 and 12 h after their addition to the cell culture (Fig. 6B). In addition, this COX-2 transcriptional stimulation was associated with an increased PGE2 synthesis. Indeed, whereas 10 µM CDCA had no effect on PGE2 synthesis, 10 µM FK and 100 µM CDCA significantly increased prostaglandin synthesis by 20–30% and 80%, respectively (Fig. 7). After 24 h of incubation, 300 µM DCA increased PGE2 synthesis by over fivefold.


Figure 6
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  		Fig. 6. COX-2 mRNA and protein expression level. A: fibroblasts were incubated with 100 µM of the indicated bile acids, 100 nM PMA, or 1 µM PGE1 for 12–14 h. Total cellular RNA was extracted using the Total RNA Mini kit (Qiagen, Valencia, CA). The oligonucleotide primers used were the following (forward CAAGTCCCTGAGCATCTACG and reverse CATAGGGCTTCAGCATAAAGC). The PCR mixture contained the RNA, 10 x Hotstar Taq DNA polymerase reaction buffer (Qiagen), 15 mM MgCl2, 1 µl of the oligonucleotide primer mixture (containing each target primer at 10 µM and the internal control primers at 1 µM), and 10 mM deoxynucleoside triphosphates. The following amplification conditions were used: denaturation and enzyme activation at 94°C for 3 min, followed by 27 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 60 s. The reaction products were analyzed by electrophoresis on 1.5% agarose gels. P < 0.05, significantly different from control, for CDCA, UDCA and PMA; P < 0.01, significantly different from control for DCA. B: Cox-2 protein expression following incubation of fibroblasts with various agents for 24 h was determined by immunoblotting using a specific anti-Cox-2 primary antibody and a horseradish peroxidase-labeled secondary antibody. The representative gels and membranes were analyzed by densitometric scanning (ImageQuant software; Molecular Dynamics). The values expressed between parentheses are fold increase over control. The arbitrary value for control was set at 1. The results are representative of at least three different experiments. Results from the mean of the three experiments were analyzed by one-way ANOVA and Bonferroni’s posttest. P < 0.001, significantly different from CTL for CDCA and PMA.

 

Figure 7
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  		Fig. 7. Effect of bile acids and FK on PGE2 synthesis. Fibroblasts were stimulated with the indicated bile acid and 10 µM FK for 6 h. PGE2 synthesis was measured in the cultured medium using an immunoassay kit from Cayman. Results are means ± SE from 3 different experiments. *P < 0.01, significantly different from control; #P < 0.001, significantly different from control.

 
To further confirm the role of COX-2 on the CDCA-induced inhibition of cell proliferation, the fibroblasts were transfected with wild-type COX-2 cDNA using the procedure described by Amaxa (Gaithersburg, MD). Under these conditions, the transfection efficiency measured by fluorescent microscopy using GFP cDNA and expressed as a ratio of the fluorescent cells to the total cell expression was always >50%. Thirty-five to forty-eight hours after COX-2 cDNA transfection, a concentration of 20 µM CDCA, which was without significant effect on cell proliferation, decreased fibroblast proliferation by 80%, while the 68% inhibition observed with 100 µM CDCA was almost complete at 99% after COX-2 overexpression (Fig. 8).


Figure 8
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  		Fig. 8. Dose-dependent effect of CDCA on cell proliferation following COX-2 overexpression. Fibroblasts were transfected with either the empty PGL3 vector or the WT COX-2 cDNA containing PGL3 vector following the procedure described by Amaxa (Gaithersburg, MD). Sixteen to twenty hours later, the cells were incubated in the absence and presence of increasing concentrations (20–200 µM) CDCA for 18–20 h and the uptake of [3H]thymidine was determined as described in Fig. 3. Results are the means ± SE of a representative experiment. Results were analyzed by one-way ANOVA and Bonferroni’s posttest. *P < 0.001, significantly different from respective control.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results from the present study clearly demonstrate that chronic exposure of human dermal fibroblasts to dihydroxy bile acids causes an almost complete inhibition (>90%) of the PGE1-induced cAMP production. This inhibitory effect was maximal after 2 h for CDCA, UDCA, or DCA and plateaued for at least 20 h, suggesting that the bile acid inhibitory effect was not significantly desensitized even after chronic exposure. The loss of responsiveness of the G protein-coupled receptor signal transduction is an integral part of the adaptive receptor regulatory mechanism to prevent either overstimulation or signal termination, as previously reported for the beta-adrenergic receptor (see Ref. 22 for review). Furthermore, the fact that the bile acids are able to attenuate PGE1-induced cAMP production even when added before PGE1 stimulation supports a mechanism independent of both increased cAMP production and activated cAMP-dependent protein kinase.

In the present study, the respective EC50 for UDCA and CDCA was similar at both 2 and 20 h, at ~20 µM, whereas that for DCA was just ~5 µM. These EC50 concentrations are clearly attainable in the systemic circulation during cholestasis, and possibly, even postprandially (2, 29, 44), and further underline the relevance of this bile acid effect. Even after 20 h of exposure, CA was without effect on stimulated cAMP formation, which is consistent with our previous reports (8, 9), suggesting that the potency of the bile acid to inhibit stimulated cAMP formation varied with its hydroxylation state. On the other hand, the present results also differ somewhat from our previous report (9). While in isolated hepatocytes the order of potency among the bile acids tested to inhibit stimulated cAMP formation was UDCA>CDCA≥DCA, in dermal fibroblasts, the order of potency is the following: DCA>CDCA≥UDCA. Different hypotheses can be proposed to explain this discrepancy in bile acid potency between these two cellular models. First, although the PGE1 and glucagon receptors are coupled to cAMP synthesis in fibroblasts and hepatocytes, respectively, either or both the mechanism of desensitization or/and the cellular machinery available to desensitize the respective receptor signaling cascade may be different. In addition, the difference in relative potency of the dihydroxy bile acids in fibroblasts and hepatocytes may reflect the respective levels of intracellular bile acid-binding proteins in these two cell types (47).

There is considerable evidence in human and in animal models, that cholestasis is associated with increased deposition of bile acids in the skin (5, 12, 16, 19, 34, 43). In two hamster models of hepatic failure, namely bile duct ligation and functional hepatectomy, we have shown that bile acids were targeted to several tissues outside of the enterohepatic circulation, most notably, the skin (12). Thus, one could imagine that in the event of hepatobiliary disorders, which result in cholestasis, similar effects may be observed in vivo, as those observed in situ, in the present study. Furthermore, under conditions of impaired liver function and decreased bile secretion, such as those found in patients with portal cirrhosis (29) and in infants with extrahepatic biliary atresia and neonatal hepatitis (21), serum concentrations of unconjugated CDCA could reach a level ≥20 µM, which is shown in the present study to completely inhibit the PGE1/G protein-coupled receptor signaling response.

Elevated serum bile acid levels under cholestatic conditions have been associated with hepatotoxicity (17, 18), hepatic fibrosis (33), pruritus (39), cardiomyopathy (28), and vasodilation (7). Ligation of the common bile duct in rodents is associated with increased fibroblast proliferation and fibrosis (52). This condition is also associated with a delayed or impaired wound healing of the skin combined with a weaker scar (3, 13). Bile and bile acids in particular have been suggested to be central to the pathologies associated to disorders of hepatic, coagulation, renal, skin, or immune function coincident with deep jaundice, which can be reduced via biliary drainage (36).

Results from the present study are suggesting that bile acids affect fibroblast proliferation at least in part through modulation of the PGE1-induced cAMP production (see Fig. 9). Previously, Okuyama et al. (37) have reported that prostaglandins inhibit cell proliferation through the EP2/EP4 receptor population, which involves cAMP. Furthermore, our data support this inhibitory effect by PGE1, PGE2, and 1-OH-PGE, a EP2/EP4 specific agonist. Although not shown, we observed virtually no inhibition of cell proliferation with sulprostone, an EP1/EP3 agonist, or misoprostol, an EP2/EP3 agonist, thus suggesting a predominant EP4 receptor-dependent growth inhibitory mechanism in these cells. Moreover, the ability of the bile acid to abrogate the PG effect was observed with both PGE1 and 1-OH-PGE1. Finally, the bile acid effect was observed with CDCA and UDCA, but not CA, in keeping with their respective effect on inhibition of cAMP production.


Figure 9
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  		Fig. 9. Effect of PGE1/PGE2 and CDCA on cell proliferation. Fibroblasts stimulated by PGE1 or PGE2 will, through activation of the EP2/EP4 receptor, and mediated through the trimeric stimulatory G protein (Gs), activate the adenylyl cyclase (AC) and cAMP synthesis. In turn, cAMP will activate the cAMP-dependent kinase (PKA) and COX-2 increased protein expression. COX-2 activation will result in an increased PGE2 synthesis from arachidonic acid. The bile acid, CDCA, has a dual effect decreasing the production of cAMP and stimulating COX-2 protein expression and activity. {alpha}s, beta, and {gamma}; stimulatory trimeric G protein. Solid arrows, stimulation; broken arrows, inhibition.

 
The implications for bile acid-induced modulation of PGE-1-induced cAMP formation and the consequent effects on PGE-1 induced cell proliferation are significant. We hypothesize that an increased deposition of bile acids not only in the liver but also in the skin could lead to tissue injury and the alteration of the balance between cell proliferation and cell death induced by various hormones, cytokines, and growth factors. In addition, PGE1 has been suggested to play an important role in tissue regeneration, due to its ability to regulate hepatocyte growth factor expression in human dermal fibroblasts in a cAMP-dependent fashion (31). Finally, PGE1 has been shown to inhibit collagenase gene expression in human skin fibroblasts (14). It is tempting to speculate that the bile acid-induced modulation of stimulated cAMP formation and cell proliferation could similarly occur in mesenchyme-derived cells of the liver under certain liver disease conditions.

Most studies have found COX-2 to be primarily, if not exclusively, located in the stromal compartment in human and rodent tissues (45). In the skin, COX-2 is mostly expressed in keratinocytes (26). Topical PGE2 application to the skin results in an increased expression of COX-2 (41). Furthermore, co-culture of dermal fibroblasts with keratinocytes resulted in an increased COX-2 activity in fibroblasts (42), supporting cross-talk between the dermis and epidermis. The results of the present study suggest that COX-2 expression can also be induced in fibroblasts. Indeed, the direct incubation of fibroblasts with concentrations >20 µM CDCA results in a significant increase in COX-2 mRNA and protein level. This supports reports showing that bile acid-induced increased COX-2 expression and PGE2 synthesis in various cell lines, including human pancreatic cancer cell lines (51), human pharyngeal cells (48), and colonic cell lines (57, 58). In the skin, the stimulation of PGE2 by bile acids could have not only an autocrine but also a paracrine effect. This increased COX-2 expression and PGE2 synthesis could in turn influence the cellular cAMP production as previously demonstrated in fibroblasts (8) and the phenotype not only of the fibroblasts but of the keratinocytes, as well by affecting proliferation, apoptosis, intercellular adhesion, and extracellular matrix production, which are key steps in the pathology of fibrosis.

Furthermore, in support of the findings from the present study, it is worthwhile to mention that several other reports associate COX-2 overexpression to suppression of tumor development. Indeed, Wilson and Potten (56) have reported that topically applied PGE2 inhibited tumor promotion in Min/+ mice. Furthermore, Lama et al. (25) have shown that fibroblasts from COX-1 knockout but not from COX-2 knockout mice were able to synthesize PGE2 and to inhibit cell proliferation. Finally, Bol et al. (6) have reported that COX-2 overexpression conferred resistance to skin tumor development to the K14.COX-2 trangenic mice. Therefore, these studies would support a biphasic effect of bile acid, i.e., a high concentration will decrease cell proliferation and potentially tumor formation, whereas low doses that can be reached under mild cholestatic conditions in the skin could perturb the local hormonal balance leading to increased cell proliferation. Furthermore, pathological conditions would be consequential to the loss by the cells of a normal response of COX-2 gene expression to extracellular agents, including prostanoids.

The biphasic effect of CDCA on cell proliferation is of interest in light of recent findings by Nishioka et al. (35). These authors suggest that CDCA stimulated proliferation of squamous cell carcinomas at low concentrations, while having a profound effect on the inhibition of cell proliferation at concentrations >30 µM. Those results are in agreement with the present findings as far as the regulation of cell proliferation is concerned. However, these authors have suggested that the bile acid effect on cell proliferation was independent of the bile acid-induced increase COX-2 expression. Those results are in contrast to those of the present study in normal diploid dermal fibroblasts, which demonstrates that the bile acid effect on cell proliferation was at least partially inhibited by both indomethacin and NS-398 while enhanced after COX-2 overexpression, supporting at least a partial COX-2 mediated inhibition of cell proliferation.

In summary, these findings underline the importance of potential pathophysiological mechanisms by which significant increase in particular systemic bile acids may not only result in cytotoxicity but also lead to changes in the response of extrahepatic tissues to hormones and transmittors, which are regulated by second messengers.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-46954 and DK-56108.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. Bouscarel, Gastroenterology Research Laboratory, The George Washington Univ. Medical Center, 2300 I St. NW, 523 Ross Hall, Washington, DC 20037 (e-mail: bbouscarel{at}mfa.gwu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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