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J. Biol. Chem., Vol. 275, Issue 43, 33929-33936, October 27, 2000

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Retinoic Acid-dependent Transforming Growth Factor-2-mediated Induction of MUC4 Mucin Expression in Human Pancreatic Tumor Cells Follows Retinoic Acid Receptor- Signaling Pathway^*

Amit Choudhury, Rakesh K. Singh^§, Nicolas Moniaux, Tarek H. El-Metwally, Jean-Pierre Aubert^¶, and Surinder K. Batra

From the  Department of Biochemistry and Molecular Biology and Eppley Institute for Research in Cancer and Allied Diseases and ^§ Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198 and ^¶ Unite 377 INSERM, Place de Verdun, 59045 Lille Cedex, France

Received for publication, June 13, 2000, and in revised form, August 9, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The MUC4 mucin is considered as the homologue of rat sialomucin complex (SMC, rat Muc4) due to its similar structural organization. Like SMC, MUC4 may also exist as two subunits: a mucin type unit known as MUC4 and a growth factor-like transmembrane subunit, MUC4. The expression of MUC4 in normal human pancreas is not detectable, but it is highly expressed in pancreatic tumor cells. In the present study, we investigated the regulation of MUC4 expression in human pancreatic tumor cells CD18/HPAF, exhibiting a high level of MUC4 transcripts and protein. When these cells were adapted to grow in the serum-free medium (CD18/HPAF-SF), the MUC4 expression was undetectable. Among several serum constituents, all-trans-retinoic acid (RA) induced the expression of MUC4 transcripts in a concentration- and time-dependent manner. The RA-mediated increase in the level of the MUC4 transcript coincided with an increased expression of transforming growth factor-2 (TGF-2) transcript. The antagonist of the retinoic acid receptor (RAR)- (Ro41-5253) abrogated the expression of MUC4 and TGF-2 induced by RA. The exogenous addition of TGF-2 also increased the MUC4 expression. The TGF--neutralizing antibody blocked the RA-induced as well as TGF-2-mediated MUC4 expression. In conclusion, induction of MUC4 expression in pancreatic carcinoma by RA is mediated through the RAR- signaling pathway, and TGF-2 may serve as an interim mediator of this regulated expression.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mucins are high molecular weight glycoproteins produced by the majority of secretory epithelial cells for the lubrication and protection of ducts and lumina within the human body (1). Twelve human mucin (MUC)¹ genes have been identified and designated as MUC1-4, MUC5AC, MUC5B, MUC6-9, and MUC11-12 (2-12). The normal distribution and pathological alterations of the apomucins are being investigated in several organs (13-19) including the pancreas (20). However, the molecular basis of the alterations that occur in mucins during the pathogenesis of different diseases is poorly understood (21).

MUC4 is a member of the membrane-bound mucin family and has been cloned from a human tracheobronchial cDNA library and from the human pancreatic tumor cell line (22-24). The NH₂ terminus of MUC4 is composed of a 27-residue signal peptide and a large domain, varying in length from 3285 to 7285 amino acid residues due to variable number of tandem repeats. The COOH terminus of MUC4 encodes 1156-residue peptide and includes two cysteine-rich domains, three epidermal growth factor-like domains, a hydrophobic transmembrane region, and two regions rich in potential N-glycosylation sites. The MUC4 mucin is considered the homologue of rat sialomucin complex (SMC, rat Muc4) due to its structural organization; however, rat Muc4 lacks the tandem repeat domain containing 16-residue repetitive units, the identifying feature of MUC4 (25). Rat Muc4 is well characterized and is composed of a highly glycosylated mucin subunit (ascites sialoglycoprotein-1) and a 120-kDa transmembrane N-glycosylated component (ascites sialoglycoprotein-2) (25). The rat Muc4 has been shown to act as a ligand for the receptor tyrosine kinase ErbB2/HER2/neu, which has been strongly implicated in breast cancer prognosis (26). Like rat Muc4, the human MUC4 can also exist as two subunits: a mucin type known as MUC4 and a growth factor like-transmembrane subunit, MUC4 (22-24). Moreover, alternate splicing generates three distinct putative types of MUC4: a family of secreted MUC4, a membrane-associated variant form, and a membrane-bound form lacking the tandem repeat domain (24, 27, 28).

MUC4 is expressed at high levels in pancreatic tumors, whereas its level in a normal pancreas is undetectable (20, 24, 29). Furthermore, high levels of MUC4 expression have been detected in differentiated pancreatic tumor cell lines (24, 29). Among the various pancreatic tumor cell lines, the deduced size of the unglycosylated protein will range from 550 to 930 kDa because of the variable number of tandem repeats polymorphism (24). Other tissues reported as having undetectable levels of MUC4 expression are gall bladder biliary epithelial cells, intrahepatic bile ducts, and the liver (30). In contrast, MUC4 apomucin is expressed in numerous normal tissues such as the stomach, ovary, salivary gland, colon, lung, trachea, uterus, and prostate (31-35). MUC4 is not restricted to the specialized epithelial cells but is also expressed in ciliary cells of the respiratory tract and in intestinal absorptive cells (36). In bronchial epithelial cells, its expression is regulated at the mRNA level by the retinoic acid (37). So far, no data on MUC4 protein expression are available because of the unavailability of MUC4-specific monoclonal antibodies. Furthermore, the MUC RNA transcripts on Northern blots show a polydisperse message, with the polydispersity due to the degradation of RNA during preparation (38). The expression of MUC4 in pancreatic tumors compared with the normal pancreas suggests a link between regulation of its expression and one of the immediate cellular alterations involved in the pathogenic process.

Expression of the human mucin gene like MUC2 in airway epithelial cells has been regulated by various factors in culture, the extracellular matrix, and in certain disease states (39, 40). In human lung carcinoma, MUC5AC and MUC5B transcripts were up-regulated by exposure to irritants like acrolein and an inflammatory mediator (41). Studies in cultured human bronchial epithelial cells have demonstrated that retinoic acid was necessary for mucociliary differentiation and for the expression of mucin genes including MUC2, MUC4, MUC5AC, and MUC5B (37, 42-45).

In the present study, we investigated RA-mediated increase of MUC4 expression in pancreatic adenocarcinoma cell line CD18/HPAF. We demonstrated that the RA not only increases the MUC4 transcript and protein levels, but also the TGF-2 transcript. These events were inhibited by a retinoid that functions as RAR- antagonist. Treatment with TGF-2 increased MUC4 expression, demonstrating a linkage of MUC4 with the TGF-2-signaling pathway. Moreover, the addition of an antibody against the TGF-, which immunoneutralized the secreted TGF-2, completely abrogated the effect of RA and TGF-2 on MUC4 expression. These findings provide evidence that RA induces MUC4 through an RAR--dependent pathway, and the increase in MUC4 expression by RA requires activation of the TGF-2 pathway by autocrine or paracrine mechanisms.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- The following materials were purchased: fetal bovine serum, Dulbecco's modified Eagle's medium, Ham's F-12 nutrient mixture, Dulbecco's phosphate-buffered saline, trypsin solution, and penicillin/streptomycin solution (Life Technologies, Inc.); Genescreen nylon membranes (PerkinElmer Life Sciences); restriction enzymes and TGF-2 E_max^TM enzyme-linked immunosorbent assay system (Promega, Madison, WI); random primed labeling kit (Amersham Pharmacia Biotech); TGF-2 and TGF-1 (R&D systems, Minneapolis, MN); Vectastain universal ABC kit (Vector Laboratories, Inc., Burlingame, CA); RA, mouse IgG1,  (MOPC 21) (Sigma); mouse hybridoma (1D11.16.8) producing TGF- antibodies (ATCC, Manassas, VA).

Cell Culture-- The CD18/HPAF pancreatic tumor cells used in the study were derived from the parental heterogenous HPAF pancreatic adenocarcinoma cell line (46). CD18/HPAF cells were cultured in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. These tumor cells were slowly selected for growth in a serum-free medium (Dulbecco's modified Eagle's medium plus F-12 nutrient mixture; 1:1 ratio) by gradually decreasing the serum content in the medium at each passage. The tumor cells that are adapted to grow in a serum free medium were named CD18/HPAF-SF. These selected cells were frozen until needed. Once the cell line was thawed, the cells were grown in a Dulbecco's modified Eagle's medium plus F-12 nutrient mixture containing penicillin and streptomycin (100 µg/ml) for 15 days. CD18/HPAF-SF cells were incubated in a serum-free medium alone or with RA (1 nM to 20 µM), Ro41-5253, TGF-2, anti-TGF- antibody (IgG1), MOPC 21 antibody (IgG1) alone or in combination. The working dilution of RA was made from 100 mM stock in Me₂SO, TGF-1, and TGF-2; stocks were reconstituted according to the manufacturer's instructions.

Isolation of RNA-- Total cellular RNA from the tumor cells was isolated by guanidine isothiocyanate cesium chloride cushion ultracentrifugation. Cells were washed twice with ice-cold phosphate-buffered saline (pH 7.4) and lysed with a solution containing 4 M guanidine isothiocyanate, 0.05 M sodium acetate, and 250 mM 2-mercaptoethanol. Total RNA was recovered via sedimentation through a 5.7 M CsCl in 0.025 M sodium acetate cushion in a Beckman SW40Ti rotor centrifuged at 32,000 rpm for 18 h. RNA pellets were resuspended in 0.3 M sodium acetate and precipitated with ethanol.

Northern Blotting-- Total RNA (20 µg) was fractionated by electrophoresis on 1.0% agarose gels containing 0.66 M formaldehyde and transferred to nitrocellulose via capillary blotting. The cDNA probes were labeled with [³²P]dCTP using a random primed labeling kit and were separated from the free label by sephadex G-50 column chromatography. Prehybridization and hybridization of blots were carried out in a solution of 5× SSPE, 50% formamide, 5× Denhardt's reagent, 200 µg/ml sheared salmon sperm DNA, and a minimum of 10⁶ cpm/ml of probe at 42 °C for 18 h. Blots were washed twice with 2× SSC containing 0.1% SDS at room temperature for 15 min, followed by four washes with 0.2× SSC, 0.1% SDS at 60 °C.

The cDNA probes used in this analysis were a 500-bp human MUC4 and 780-bp human glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The MUC4 cDNA clone was isolated from the HPAF cDNA library that contains 10 complete tandem repeat units (AF177925). It shares a 98% similarity with the published MUC4 tandem repeat sequence (5). The GAPDH probe has 31 bp of 5'-untranslated region and the region coding the first 250 amino acid residues of the protein.

Expression of the MUC4 mRNA was quantified by densitometry of autoradiograms using the ImageQuant software program (Molecular Dynamics, Inc., Sunnyvale, CA). The MUC4 signal on the Northern blot appears as a smear. Each sample measurement was calculated as the ratio of the average areas between MUC4-specific smear and 1.3 kilobase pairs GAPDH band in the linear range of the autoradiogram as reported earlier (29).

Immunohistochemical Analysis-- The subconfluent monolayer of cells cultured in eight-chambered slides (Nunc, Inc., Naperville, IL) was used for the immunohistochemical analysis. After the monolayer was washed, cells were fixed with ice-cold acetone and methanol mixture (1:1) for 10 min. Immunostaining was performed on the fixed cells with the Vectastain universal ABC kit. The cell monolayer was blocked with normal blocking serum for 1 h. Following blocking, the samples were incubated at 4 °C overnight with either anti-MUC4 rabbit antiserum raised against 16-amino acid tandem repeat (Ser-Thr-Gly-Asp-Thr-Thr-Pro-Leu-Pro-Val-Thr-Asp-Thr-Ser-Ser-Val) peptide or preimmune rabbit serum serving as a negative control. To block the positive staining, the primary test antisera (1:100 dilution) were incubated with different concentrations of tandem repeat peptide ranging from 100 through 0.001 mg/ml and were incubated for 30 min at room temperature. Samples were incubated with the secondary antibody for 1 h at room temperature and washed with Dulbecco's phosphate-buffered saline (Life Technologies). The cells were incubated with ABC reagent for 30 min followed by treatment with peroxidase substrate solution until the desired stain intensity was developed.

Reverse Transcription-PCR (RT-PCR)-- Total RNA (0.5 µg) from the CD18/HPAF cells and the control normal tissues was reverse transcribed using the first strand cDNA synthesis kit (PerkinElmer Life Sciences) and oligo(dT) primers according to the manufacturer's instructions. Each target gene was co-amplified with the same GAPDH primers. Amplifications were performed in a programmable thermal controller (PTC-100, MJ Research, Inc., Watertown, MA). PCR amplification reactions were conducted in 50-µl reaction volumes containing 5 µl of 10× PerkinElmer buffer, 5 µl of 10 mM deoxynucleoside triphosphates, 2 µl of first strand pancreatic cell line cDNA, 5 µl of 25 mM MgCl₂, 10 pmol of each primer, and 2 units of Taq DNA polymerase (Ampli Taq Gold; PerkinElmer Life Sciences). The mixture was denatured at 96 °C for 10 min, followed by 30 cycles at 96 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min. The final elongation step was extended for an additional 15 min. The sequence of the PCR product was confirmed by the dideoxy-mediated chain termination method. The positive controls for the various mucin genes were as follows: for MUC1 and MUC4, HPAF/CD18; for MUC6, the normal pancreas; for MUC2 and MUC3, the small intestine; for MUC5AC and -B, the trachea; and for MUC7, salivary gland (3, 9, 29, 47-49). The sequence of the PCR product was confirmed by the dideoxy-mediated chain termination method.

Oligonucleotide primers for MUC1, -2, -3, -4, -5AC, -5B, -6, and -7, RAR-, RAR-, RAR-, TGF-1, and TGF-2 are designed from the published sequences in GenBank^TM as shown in Table I. PCR products were run on 1% agarose gels, stained with ethidium bromide, and scanned on a Nucleo Vision^TM 760 Imaging Workstation. Amplified products were quantified for each sample using the gel expert^TM 3.5 software suite (Nucleotech Corporation, San Mateo, CA). The densitometric values were calculated for gene-specific product and GAPDH for each reaction. The value for the mucin gene-specific product is expressed per unit of GAPDH to account for any differences in starting amounts of RNA. The lowest densitometric value (weakest band) for GAPDH is taken as a unit. For convenience, the corrected densitometric scores for different products were categorized in three different ranges: high (+++), moderate (++), and weak (+). Each value was determined as the mean of four densitometry readings.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Serum-dependent Regulation of MUC4 Expression in CD18/HPAF-SF Tumor Cells-- The CD18/HPAF cells showed a high level of MUC4 expression (wild type level) by Northern blotting, where the MUC4 tandem repeat cDNA probe hybridized to a >10-kilobase transcript (Fig. 1A). When the cells were adapted to grow over a period of time in the serum-free medium, no MUC4 mRNA expression was detected (these tumor cells are named as CD18/HPAF-SF). The MUC4 expression in CD18/HPAF-SF cells was resumed within 24 h of culture with fetal bovine serum. Furthermore, a similar effect on MUC4 expression was observed with blood plasma. The level of GAPDH transcript was measured for an internal control and was comparable in test and controls (Fig. 1).

View larger version (65K): [in this window] [in a new window]   Fig. 1.   Northern and immunohistochemical analysis of serum-dependent regulation of MUC4 expression. A, Northern blot analysis of 20 µg of total RNA from the CD18/HPAF cells and CD18/HPAF-SF cells alone or treated for 24 h with either serum or plasma. Blots were probed with human MUC4 tandem repeat (a) and GAPDH (b) (used as a loading control) cDNA probes. Densitometric values ± S.E. for the MUC4, normalized to GAPDH band intensity in three different experiments, were determined by using the Molecular Dynamics ImageQuant software program. B, immunohistochemical analysis of MUC4 using an anti-MUC4 polyclonal antiserum. Cells showed immunoreactivity to anti-MUC4 antiserum (1:100 dilution) (a and d), whereas CD18/HPAF-SF cells remained unstained (c). Specific staining was blocked in CD18/HPAF cells by preincubation of the MUC4-antiserum with the tandem repeat peptide (b). Original magnification, × 200 (a-d). FBS, fetal bovine serum.

Due to the large size of the MUC4 protein (1680-2800 kDa, unglycosylated), and difficulty in resolving the protein by SDS-PAGE, its expression was evaluated by immunohistochemistry under similar experimental conditions using anti-MUC4 pAb, raised against MUC4 tandem repeat peptide. The expression of MUC4 protein correlated with the transcripts. The positive staining in the cells grown in serum-containing medium was specifically blocked by preincubation of the MUC4 antiserum with the tandem repeat peptide (Fig. 1B).

RA-induced MUC4 Expression in CD18/HPAF-SF Cells-- MUC4 mRNA expression was evaluated after treatment of CD18/HPAF-SF cells with estradiol-17, progesterone, dexamethasone, hydrocortisone, insulin, epidermal growth factor, tumor necrosis factor-, TGF-, and retinoids. We observed that retinoic acid (RA, 9-cis-retinoic acid, and 13-cis-retinoic acid) up-regulated the expression of MUC4 in CD18/HPAF-SF tumor cells. Treatment with RA resulted in a time-dependent increase in the level of MUC4 mRNA from day 1 to day 4 (Fig. 2A). The RA-induced increase of MUC4 expression was dose-dependent (from 1 nM to 20 µM concentration); however, the concentration of 10 µM and above was found toxic for the cells (Fig. 2B). We have therefore used RA at 1 µM or lower concentrations in our subsequent experiments.

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Northern analysis of the MUC4 gene on total cellular RNA prepared from CD18/HPAF-SF cells. A, expression was examined in cells treated with RA for different time periods or with medium alone (0 h). B, expression was examined in cells treated for 48 h with medium alone (0) or with the indicated concentration of RA. Blots were probed with human MUC4 tandem repeat (a) and GAPDH (b) (used as a loading control) cDNA probes. Densitometric values ± S.E. for the MUC4, normalized to GAPDH band intensity in three different experiments, were determined by using the Molecular Dynamics ImageQuant software program.

It is known that the action of RA is mediated through the nuclear retinoic acid receptors (RARs). We investigated the level of retinoic acid receptor (RAR-, -, -) mRNA in CD18/HPAF-SF cells treated with RA (1 µM) and untreated (medium alone) by RT-PCR. Appropriate amplification products were obtained: 372, 383, and 266 bp, for RAR-, -, and -, respectively (Fig. 3). The results indicate that the levels of all the RAR isoforms were similar in RA-treated and untreated cultures.

View larger version (48K): [in this window] [in a new window]   Fig. 3.   RT-PCR analysis of RAR-, -, and - mRNA expression in RA-treated CD18/HPAF-SF cells. Subconfluent CD18/HPAF-SF cultures were exposed for 48 h to 1 µM concentration of RA, the total RNA was isolated, and levels of RAR-, -, and - were analyzed by RT-PCR. Amplified products were quantified for each sample using the gel expertTM 3.5 software suite. The densitometric values were calculated for gene specific product and GAPDH for each reaction. The value for RAR isoform-specific product is expressed per unit of GAPDH to account for any differences in starting amounts of RNA. kb, kilobase pair.

RA-induced MUC4 Expression Mediated through RAR--- The contribution of RAR--dependent signaling pathway to the increased MUC4 mRNA levels induced by RA was analyzed. The CD18/HPAF-SF cells were treated for 48 h with 50 nM RA and different doses of Ro41-5253 (a synthetic retinoid) that functions as an RAR- antagonist (50). When the Ro41-5253 (1 µM) was used in molar excess to RA (50 nM), it abrogated the RA-induced MUC4 expression (Fig. 4). However, the Ro41-5253 (in molar excess) in combination with 1 µM RA was found toxic to the cells. Consistent with our mRNA data, similar results were obtained at protein level, evaluated by immunohistochemistry using the anti-MUC4 pAb. An intense MUC4 protein staining was observed after treatment with 50 nM RA (data not shown). The intensity of staining was similar to that found with the CD18/HPAF cells (Fig. 1B, a). However, no MUC4 staining was observed in the cells co-cultured with 50 nM RA and 1 µM Ro41-5253 (data not shown).

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Northern analysis of the MUC4 expression in CD18/HPAF-SF cells. A, total cellular RNA prepared from the tumor cells treated for 48 h with 50 nM RA alone or in the presence of different doses of the RAR- antagonist Ro41-5253. B, densitometric values ± S.E. for the MUC4, normalized to GAPDH band intensity in three different experiments, was determined by using the Molecular Dynamics ImageQuant software program.

Regulation of Expression of MUC Genes in RA-treated CD18/HPAF-SF Cells-- Expression of eight mucin genes was investigated by RT-PCR using total RNA isolated from the RA-treated (48 h) cultures. The level of MUC4 in RA-treated (1 µM) cultures was comparable with the wild type levels (Table II). MUC2 showed an expression pattern similar to MUC4 but at lower level. MUC7 was not expressed in CD18/HPAF cells but was induced by RA in CD18/HPAF-SF cells. RA had no influence on the expression of MUC1 and MUC5B, since their expression remained unchanged in treated and untreated cells. No detectable expression of MUC3, MUC5AC, and MUC6 was observed in RA-treated and untreated cultures, but the positive controls (described under "Experimental Procedures") showed appropriate amplification.

View this table: [in this window] [in a new window]   Table II RT-PCR analysis of mucin gene expression +++, high; ++, moderate; +, low; , undetectable.

RA-induced Increase Of TGF-2 in CD18/HPAF-SF Cells-- The expression of TGF2 and TGF1 mRNA was measured by RT-PCR using total RNA. The amplified products for TGF2 and TGF1 were appropriately visualized at 600 and 186 bp, respectively. A high level of TGF2 expression was observed in RA-treated cells, but no detectable expression was found in untreated cells (Fig. 5A). No TGF2 expression was seen in the cells cultured in the presence of RA plus Ro41-5253 (in molar excess); however, there was no effect on the TGF1 expression. TGF1 was expressed in both RA (50 nM)-treated and untreated (medium alone) samples. Furthermore, treatment with TGF2 (20 pg/ml) up-regulated the expression of TGF2 transcripts, whereas under similar conditions TGF1 levels remained unaltered.

View larger version (56K): [in this window] [in a new window]   Fig. 5.   Analysis of TGF-2 expression in RA-treated CD18/HPAF-SF cells. The CD18/HPAF culture (50% confluent) was exposed for 48 h to the indicated culture conditions. Total RNA was isolated, and TGF-2 or TGF-1 was co-amplified with GAPDH mRNA in each reaction by RT-PCR.

TGF-2 Increases the MUC4 Expression in CD18/HPAF-SF Cells-- The effect of the exogenous addition of TGF-2 on MUC4 mRNA and protein levels was examined. A dose-dependent study was carried out using 10-100 pg/ml concentrations of TGF-2. The MUC4 expression increased from 0 to 20 pg/ml concentration (Fig. 6). TGF-2 not only increased MUC4 mRNA but also increased the MUC4 protein expression at a concentration of 20 pg/ml after treatment for 48 h as analyzed by immunohistochemistry using anti-MUC4 pAb (data not shown).

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Northern analysis of MUC4 expression in TGF-2-treated CD18/HPAF-SF cells. A, total cellular RNA was prepared from the cells treated for 48 h with medium alone or with the indicated concentration of TGF-2. B, densitometric values ± S.E. for the MUC4, normalized to GAPDH band intensity in three different experiments, was determined by using the Molecular Dynamics ImageQuant software program.

TGF--neutralizing Antibody Abrogates RA- and TGF-2-induced MUC4 Expression in CD18/HPAF-SF Cells-- The specificity of the TGF-2-mediated increase in MUC4 expression in these tumors and the role of TGF-2 in a retinoid-mediated increase of MUC4 expression were assessed by use of a TGF--neutralizing antibody. The tumor cell culture (80% confluent) was treated with RA (50 nM), TGF2 (20 pg/ml) alone or in the presence of 100 µg/ml anti-TGF- monoclonal antibody (IgG1 isotype, binds to TGF-1 and TGF-2). Controls were mouse IgG1 isotype antibody (mouse MOPC 21, CMAb). The addition of anti-TGF- monoclonal antibody along with the RA treatment completely abrogated the RA (50 nM)-induced MUC4 expression in the CD18/HPAF-SF cells, but controls with CMAb showed a high level of MUC4 expression (Fig. 7). Furthermore, the incubation of the antibody with the TGF-2 (20 pg/ml)-treated culture also resulted in loss of TGF-2-induced MUC4 expression.

View larger version (32K): [in this window] [in a new window]   Fig. 7.   RT-PCR analysis to study the effect of TGF--neutralizing antibody (100 µg/ml) on the RA (50 nM)- and TGF-2 (20 pg/ml)-induced MUC4 mRNA expression in CD18/HPAF-SF cells. A, 50% confluent cultures were exposed for 48 h to the indicated culture conditions. Total RNA was isolated, and MUC4 and GAPDH mRNA are co-amplified in each reaction by RT-PCR. B, amplified products were quantified for each sample using the gel expertTM 3.5 software suite. The densitometric values were calculated for gene-specific product and GAPDH for each reaction. The value for MUC4 gene-specific product is expressed per unit of GAPDH to account for any differences in starting amounts of RNA. kb, kilobase pair.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We report, for the first time, the regulation of MUC4 expression in human pancreatic tumor cells. The MUC4 gene is highly expressed in pancreatic tumors and cell lines but has no detectable expression in a normal pancreas (20, 29). A clonal pancreatic tumor cell line (CD18/HPAF) that produces high amounts of MUC4 mRNA (29, 46) was used. The expression of MUC4 was serum-dependent, and the blood plasma mimics the serum effect, ruling out the involvement of clotting factors in its regulation. The SMC (rat Muc4) transcripts have similarly been shown up-regulated in primary rat mammary epithelial cells (MECs) by serum (51).

Several serum factors were analyzed for their ability to increase the MUC4 expression in CD18/HPAF-SF tumor cells, including steroids, growth factors, cytokines, and retinoic acid. We observed that the naturally occurring retinoid RA increases the expression of MUC4 mRNA and protein in a concentration- and time-dependent fashion. In airway epithelial cells, it has been demonstrated that the components of culture medium, like hormones, and growth factors regulate mucin production (44, 52). In bronchial epithelial cells, a serum component, the RA, has been reported essential for the mucociliary differentiation, maintenance of mucociliary phenotype, and the expression of a variety of mucin (37). The ultrastructural analysis of the RA-treated CD18/HPAF-SF cells exhibited differentiated morphology with large luminal spaces,² similar to CD18/HPAF tumor cells that showed high levels of MUC4 expression (24, 46). In cultured bronchial epithelial cells, an RA-dependent increase of MUC2, MUC4, MUC5AC, and MUC5B transcript has been reported (37, 45). In pancreatic adenocarcinoma cells (CD18/HPAF-SF), the RA also increased the expression of MUC2 and MUC7, but at low levels.

Retinoic acids are ligands for the nuclear retinoic acid receptors (RAR-, -, -) and retinoid X receptors (RXR-, -, -). Both RAR and RXR act as ligand-activated transcription factors, controlling gene transcription initiated from promoters of retinoid-regulated genes by interacting with cis-acting DNA elements, also called retinoic acid-responsive elements. The RA is a naturally acting ligand for the RAR in vivo (53). In CD18/HPAF-SF cells, the expression of RAR-, -, and - mRNA in RA-treated as well as in untreated cells was found similar. An antagonist to RAR- isotype (Ro41-5253, 10⁶ M) blocked the RA (50 nM)-induced expression of MUC4 mRNA and protein. The Ro41-5253 at a lower concentration (10⁸ and 10⁹ M) was not effective in abrogating the RA (50 nM)-induced MUC4 expression, suggesting that a molar excess of the antagonist was required to efficiently compete and block the action of RA on RAR-. The RAR- is also reported to be the main RAR form involved in the RA-dependent regulation of MUC2 and MUC5AC transcripts in human tracheobronchial epithelial cells (54).

We also observed an RA-induced expression of TGF-2 transcripts and activated TGF-2² in these tumor cells, but no effect was found on the TGF-1 transcripts. Co-incubating the tumor cells with RA and Ro41-5253 (in molar excess) abrogated the RA-induced expression of not only MUC4 but also TGF-2 transcripts. TGF-2 appeared to act as a mediator of RA action and was confirmed by the abrogation of RA-induced MUC4 expression by TGF--neutralizing antibody. Similarly, in epidermal cells, one of the major effects of RA was the specific induction of TGF-2 that was further supported by the evidence that TGF-2 can act as local mediator of RA action (55). However, these effects of RA on the induction of TGF-2 expression are not believed to be direct effects on the TGF-2 promoter (56). A positive correlation between the MUC4 and TGF-2 transcript level was observed; treatments that resulted in an increased MUC4 expression were also found to up-regulate the TGF-2 transcript levels.

In pancreatic tumor cell lines, expression of MUC4 has been reported undetectable in poorly differentiated cell lines and with an elevated level of expression in moderately to well differentiated cell lines (24, 29). A differentiation-dependent induction of TGF-2 was detected in F9 and PC-13 MECs (57). The TGF- can exert either positive or negative effects on neoplastic cells. The in vitro growth of many tumor cells was inhibited by picomolar concentrations of exogenous TGF- (58-62). Furthermore, in MECs the SMC expression is inhibited by TGF- by a posttranslational mechanism; however, these regulatory processes are altered in the ascites tumor cells (51, 63). In contrast to its inhibitory effects, TGF- can also promote the growth and/or invasiveness of several different tumors (64, 65). In pancreatic cancer, enhanced expression of TGF- isoforms, especially TGF-2, was found associated with disease progression (65). However, the mechanisms regulating the levels of TGF- are poorly understood. We found that, exogenously supplied, the TGF-2 can by itself up-regulate the MUC4 expression in the tumor cells. However, its other isoform, the TGF-1, showed no effect on MUC4 expression in these tumor cells (data not shown). Similarly, TGF-1 had no effect on the expression of rat SMC (rat Muc4) transcript and protein levels in mammary adenocarcinoma cells (51). In contrast, in normal mammary gland cells, TGF-1 showed no effect on SMC transcript, but the protein expression was down-regulated.

In most if not all cases, TGF- is secreted as an inactive (latent) high molecular weight complex, composed of TGF-, the amino-terminal part of the TGF- precursor, and the latent TGF--binding proteins (66). In the absence of latent TGF--binding proteins, TGF- is secreted very slowly, and considerable amounts of TGF- remain inside the cell (67). We observed that the treatment of CD18/HPAF-SF cells with RA increased not only the MUC4 mRNA levels but also TGF-2 transcripts in the cells and the activated TGF-2 in the culture supernatant.² The expression of TGF-1 mRNA was found similar in RA-treated and -untreated cells. Furthermore, the RA-induced expression of TGF-2 transcripts in CD18/HPAF cell was abrogated in the presence of a molar excess of the RAR- antagonist (Ro41-5253); however, there was no effect on the TGF-1 expression. Hence, the results suggest that the TGF-2 is a local mediator of the RA action on MUC4 expression.

In summary, we report that expression of the MUC4 mucin in the CD18/HPAF cell is regulated by RA and TGF-2, which acts as a local mediator of RA action. This action of RA is mediated through the RAR--dependent signaling pathway. Additional investigations on the promoter of this gene and the analysis of different regulatory elements will provide better understanding of the regulation of this gene under normal and pathological conditions.

	ACKNOWLEDGEMENTS

We thank Erik Moore (University of Nebraska Medical Center) and Evelyne Destailleur (INSERM) for technical support. The generous gift of anti-MUC4 rabbit polyclonal antiserum form Dr. Sandra J Gendler (Mayo Clinic Scottsdale, Scottsdale, AZ) is acknowledged. We also thank the Molecular Biology Core Facility, UNMC, for oligonucleotide synthesis and DNA sequencing and Kristi L. W. Berger (Eppley Institute) for editorial assistance.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants CA 78590 (to S. K. B) and CA72781 (to R. K. S).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.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, 984525 Nebraska Medical Center, Omaha, NE 68198-4525. Tel.: 402-559-5455; Fax: 402-559-6650; E-mail: sbatra@unmc.edu.

Published, JBC Papers in Press, August 10, 2000, DOI 10.1074/jbc.M005115200

² A. Choudhury, and S. K. Batra, unpublished results.

	ABBREVIATIONS

The abbreviations used are: MUC, mucin; RA, all-trans-retinoic acid; SMC, sialomucin complex; TGF, transforming growth factor; SF, serum free; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; RAR, retinoic acid receptor; bp, base pair(s); GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MEC, mammary epithelial cell.

	REFERENCES

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