IPF pathogenesis is dependent upon TGFß induction of IGF-1.

Pathogenic fibrotic diseases, including idiopathic pulmonary fibrosis (IPF), have some of the worst prognoses and affect millions of people worldwide. With unclear etiology and minimally effective therapies, two-thirds of IPF patients die within 2-5 years from this progressive interstitial lung disease. Transforming Growth Factor Beta (TGFß) and insulin-like growth factor-1 (IGF-1) are known to promote fibrosis; however, myofibroblast specific upregulation of IGF-1 in the initiation and progression of TGFß-induced fibrogenesis and IPF have remained unexplored. To address this, the current study (1) documents the upregulation of IGF-1 via TGFß in myofibroblasts and fibrotic lung tissue, as well as its correlation with decreased pulmonary function in advanced IPF; (2) identifies IGF-1's C1 promoter as mediating the increase in IGF-1 transcription by TGFß in pulmonary fibroblasts; (3) determines that SMAD2 and mTOR signaling are required for TGFß-dependent Igf-1 expression in myofibroblasts; (4) demonstrates IGF-1R activation is essential to support TGFß-driven profibrotic myofibroblast functions and excessive wound healing; and (5) establishes the effectiveness of slowing the progression of murine lung fibrosis with the IGF-1R inhibitor OSI-906. These findings expand our knowledge of IGF-1's role as a novel fibrotic-switch, bringing us one step closer to understanding the complex biological mechanisms responsible for fibrotic diseases and developing effective therapies.

Hexokinase 2 couples glycolysis with the profibrotic actions of TGF-ß.

Metabolic dysregulation in fibroblasts is implicated in the profibrotic actions of transforming growth factor-ß (TGF-ß). Here, we present evidence that hexokinase 2 (HK2) is important for mediating the fibroproliferative activity of TGF-ß both in vitro and in vivo. Both Smad-dependent and Smad-independent TGF-ß signaling induced HK2 accumulation in murine and human lung fibroblasts through induction of the transcription factor c-Myc. Knockdown of HK2 or pharmacological inhibition of HK2 activity with Lonidamine decreased TGF-ß-stimulated fibrogenic processes, including profibrotic gene expression, cell migration, colony formation, and activation of the transcription factors YAP and TAZ, with no apparent effect on cellular viability. Fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibited an increased abundance of HK2. In a mouse model of bleomycin-induced lung fibrosis, Lonidamine reduced the expression of genes encoding profibrotic markers (collagenΙα1, EDA-fibronectin, α smooth muscle actin, and connective tissue growth factor) and stabilized or improved lung function as assessed by measurement of peripheral blood oxygenation. These findings provide evidence of how metabolic dysregulation through HK2 can be integrated within the context of profibrotic TGF-ß signaling.

Fatty acid synthase is required for profibrotic TGF-ß signaling.

Evidence is provided that the fibroproliferative actions of TGF-ß are dependent on a metabolic adaptation that sustains pathologic growth. Specifically, profibrotic TGF-ß signaling is shown to require fatty acid synthase (FASN), an essential anabolic enzyme responsible for the de novo synthesis of fatty acids. With the use of pharmacologic and genetic approaches, we show that TGF-ß-stimulated FASN expression is independent of Smad2/3 and is mediated via mammalian target of rapamycin complex 1. In the absence of FASN activity or protein, TGF-ß-driven fibrogenic processes are reduced with no apparent toxicity. Furthermore, as increased FASN expression was also observed to correlate with the degree of lung fibrosis in bleomycin-treated mice, inhibition of FASN was examined in a murine-treatment model of pulmonary fibrosis. Remarkably, inhibition of FASN not only decreased expression of profibrotic targets, but lung function was also stabilized/improved, as assessed by peripheral blood oxygenation.-Jung, M.-Y., Kang, J.-H., Hernandez, D. M., Yin, X., Andrianifahanana, M., Wang, Y., Gonzalez-Guerrico, A., Limper, A. H., Lupu, R., Leof, E. B. Fatty acid synthase is required for profibrotic TGF-ß signaling.

Basolateral delivery of the type I transforming growth factor beta receptor is mediated by a dominant-acting cytoplasmic motif.

Delivery of biomolecules to the correct subcellular locales is critical for proper physiological function. To that end, we have previously determined that type I and II transforming growth factor beta (TGF-ß) receptors (TßRI and TßRII, respectively) localize to the basolateral domain in polarized epithelia. While TßRII targeting was shown to be regulated by sequences between amino acids 529 and 538, the analogous region(s) within TßRI is unknown. To address that question, sequential cytoplasmic TßRI truncations and point mutations identified a targeting motif between residues 158 and 163 (VxxEED) required for basolateral TßRI expression. Further studies documented that receptor internalization, down-regulation, direct recycling, or Smad signaling were unaffected by motif mutations that caused TßRI mislocalization. However, inclusion of amino acids 148-217 containing the targeting motif was able to direct basolateral expression of the apically sorted nerve growth factor receptor (NGFR, p75; extracellular and transmembrane regions) in a dominant manner. Finally, coexpression of apically targeted type I and type II TGF-ß receptors mediated Smad3 signaling from the apical membrane of polarized epithelial cells. These findings demonstrate that the absence of apical TGF-ß signaling in normal epithelia is primarily a reflection of domain-specific receptor expression and not an inability to couple with the signaling machinery.

Cell-penetrating peptides selectively targeting SMAD3 inhibit profibrotic TGF-ß signaling.

TGF-ß is considered a master switch in the pathogenesis of organ fibrosis. The primary mediators of this activity are the SMAD proteins, particularly SMAD3. In the current study, we have developed a cell-penetrating peptide (CPP) conjugate of the HIV TAT protein that is fused to an aminoterminal sequence of sorting nexin 9 (SNX9), which was previously shown to bind phosphorylated SMAD3 (pSMAD3). We determined that specifically preventing the nuclear import of pSMAD3 using the TAT-SNX9 peptide inhibited profibrotic TGF-ß activity in murine cells and human lung fibroblasts as well as in vivo with no demonstrable toxicity. TGF-ß signaling mediated by pSMAD2, bone morphogenetic protein 4 (BMP4), EGF, or PDGF was unaffected by the TAT-SNX9 peptide. Furthermore, while the TAT-SNX9 peptide prevented TGF-ß's profibrotic activity in vitro as well as in 2 murine treatment models of pulmonary fibrosis, a 3-amino acid point mutant that was unable to bind pSMAD3 proved ineffective. These findings indicate that specifically targeting pSMAD3 can ameliorate both the direct and indirect fibroproliferative actions of TGF-ß.

Profibrotic up-regulation of glucose transporter 1 by TGF-ß involves activation of MEK and mammalian target of rapamycin complex 2 pathways.

TGF-ß plays a central role in the pathogenesis of fibroproliferative disorders. Defining the exact underlying molecular basis is therefore critical for the development of viable therapeutic strategies. Here, we show that expression of the facilitative glucose transporter 1 (GLUT1) is induced by TGF-ß in fibroblast lines and primary cells and is required for the profibrotic effects of TGF-ß. In addition, enhanced GLUT1 expression is observed in fibrotic areas of lungs of both patients with idiopathic pulmonary fibrosis and mice that are subjected to a fibrosis-inducing bleomycin treatment. By using pharmacologic and genetic approaches, we demonstrate that up-regulation of GLUT1 occurs via the canonical Smad2/3 pathway and requires autocrine activation of the receptor tyrosine kinases, platelet-derived and epidermal growth factor receptors. Engagement of the common downstream effector PI3K subsequently triggers activation of the MEK and mammalian target of rapamycin complex 2, which cooperate in regulating GLUT1 expression. Of note, inhibition of GLUT1 activity and/or expression is shown to impair TGF-ß-driven fibrogenic processes, including cell proliferation and production of profibrotic mediators. These findings provide new perspectives on the interrelation of metabolism and profibrotic TGF-ß signaling and present opportunities for potential therapeutic intervention.-Andrianifahanana, M., Hernandez, D. M., Yin, X., Kang, J.-H., Jung, M.-Y., Wang, Y., Yi, E. S., Roden, A. C., Limper, A. H., Leof, E. B. Profibrotic up-regulation of glucose transporter 1 by TGF-ß involves activation of MEK and mammalian target of rapamycin complex 2 pathways.

Sorting nexin 9 differentiates ligand-activated Smad3 from Smad2 for nuclear import and transforming growth factor ß signaling.

Transforming growth factor ß (TGFß) is a pleiotropic protein secreted from essentially all cell types and primary tissues. While TGFß's actions reflect the activity of a number of signaling networks, the primary mediator of TGFß responses are the Smad proteins. Following receptor activation, these cytoplasmic proteins form hetero-oligomeric complexes that translocate to the nucleus and affect gene transcription. Here, through biological, biochemical, and immunofluorescence approaches, sorting nexin 9 (SNX9) is identified as being required for Smad3-dependent responses. SNX9 interacts with phosphorylated (p) Smad3 independent of Smad2 or Smad4 and promotes more rapid nuclear delivery than that observed independent of ligand. Although SNX9 does not bind nucleoporins Nup153 or Nup214 or some ß importins (Imp7 or Impß), it mediates the association of pSmad3 with Imp8 and the nuclear membrane. This facilitates nuclear translocation of pSmad3 but not SNX9.

Profibrotic TGFß responses require the cooperative action of PDGF and ErbB receptor tyrosine kinases.

Transforming growth factor ß (TGFß) has significant profibrotic activity both in vitro and in vivo. This reflects its capacity to stimulate fibrogenic mediators and induce the expression of other profibrotic cytokines such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF/ErbB) ligands. Here we address both the mechanisms by which TGFß induced ErbB ligands and the physiological significance of inhibiting multiple TGFß-regulated processes. The data document that ErbB ligand induction requires PDGF receptor (PDGFR) mediation and engages a positive autocrine/paracrine feedback loop via ErbB receptors. Whereas PDGFRs are essential for TGFß-stimulated ErbB ligand up-regulation, TGFß-specific signals are also required for ErbB receptor activation. Subsequent profibrotic responses are shown to involve the cooperative action of PDGF and ErbB signaling. Moreover, using a murine treatment model of bleomycin-induced pulmonary fibrosis we found that inhibition of TGFß/PDGF and ErbB pathways with imatinib plus lapatinib, respectively, not only prevented myofibroblast gene expression to a greater extent than either drug alone, but also essentially stabilized gas exchange (oxygen saturation) as an overall measure of lung function. These observations provide important mechanistic insights into profibrotic TGFß signaling and indicate that targeting multiple cytokines represents a possible strategy to ameliorate organ fibrosis dependent on TGFß.

ERBB receptor activation is required for profibrotic responses to transforming growth factor beta.

Engagement of the transforming growth factor-ß (TGF-ß) receptor complex activates multiple signaling pathways that play crucial roles in both health and disease. TGF-ß is a key regulator of fibrogenesis and cancer-associated desmoplasia; however, its exact mode of action in these pathologic processes has remained poorly defined. Here, we report a novel mechanism whereby signaling via members of the ERBB or epidermal growth factor family of receptors serves as a central requirement for the biological responses of fibroblasts to TGF-ß. We show that TGF-ß triggers upregulation of ERBB ligands and activation of cognate receptors via the canonical SMAD pathway in fibroblasts. Interestingly, activation of ERBB is commonly observed in a subset of fibroblast but not epithelial cells from different species, indicating cell type specificity. Moreover, using genetic and pharmacologic approaches, we show that ERBB activation by TGF-ß is essential for the induction of fibroblast cell morphologic transformation and anchorage-independent growth. Together, these results uncover important aspects of TGF-ß signaling that highlight the role of ERBB ligands/receptors as critical mediators in fibroblast responses to this pleiotropic cytokine.

Distinct roles for mammalian target of rapamycin complexes in the fibroblast response to transforming growth factor-beta.

Transforming growth factor-beta (TGF-beta) promotes a multitude of diverse biological processes, including growth arrest of epithelial cells and proliferation of fibroblasts. Although the TGF-beta signaling pathways that promote inhibition of epithelial cell growth are well characterized, less is known about the mechanisms mediating the positive response to this growth factor. Given that TGF-beta has been shown to promote fibrotic diseases and desmoplasia, identifying the fibroblast-specific TGF-beta signaling pathways is critical. Here, we investigate the role of mammalian target of rapamycin (mTOR), a known effector of phosphatidylinositol 3-kinase (PI3K) and promoter of cell growth, in the fibroblast response to TGF-beta. We show that TGF-beta activates mTOR complex 1 (mTORC1) in fibroblasts but not epithelial cells via a PI3K-Akt-TSC2-dependent pathway. Rapamycin, the pharmacologic inhibitor of mTOR, prevents TGF-beta-mediated anchorage-independent growth without affecting TGF-beta transcriptional responses or extracellular matrix protein induction. In addition to mTORC1, we also examined the role of mTORC2 in TGF-beta action. mTORC2 promotes TGF-beta-induced morphologic transformation and is required for TGF-beta-induced Akt S473 phosphorylation but not mTORC1 activation. Interestingly, both mTOR complexes are necessary for TGF-beta-mediated growth in soft agar. These results define distinct and overlapping roles for mTORC1 and mTORC2 in the fibroblast response to TGF-beta and suggest that inhibitors of mTOR signaling may be useful in treating fibrotic processes, such as desmoplasia.

Cigarette smoke-induced differential expression of the genes involved in exocrine function of the rat pancreas.

OBJECTIVE: Little is known about the molecular and biological aspects of the epidemiological association between smoking and pancreatic pathology, such as chronic pancreatitis and pancreatic cancer. Recently, we reported that tobacco smoke exposure induced morphological alterations in the rat pancreas. Here, we have investigated the alterations in the expression of genes associated with exocrine pancreatic function and cellular differentiation upon exposure to cigarette smoke. METHODS: Female rats were exposed to environmental smoke inhalation for 2 d/wk (70 min/d) for 12 weeks. The expression profiles of trypsinogen, pancreas-specific trypsin inhibitor, cholecystokinin A receptor, cystic fibrosis transmembrane conductance regulator (CFTR), carbonic anhydrase, and Muc1 and Muc4 mucins transcripts were analyzed by RNA slot blot analysis. Muc4 expression was also examined by immunohistochemistry. RESULTS: Our data revealed that the ratio of trypsinogen to that of the protective pancreas-specific trypsin inhibitor was elevated upon cigarette smoke exposure. The expression of carbonic anhydrase and CFTR remained unaltered when inflammatory signs were not detected in histological examinations. On the other hand, when pancreatic inflammation was present, the levels of CFTR and carbonic anhydrase were increased, indicating ductal and/or centroacinar cell involvement. No changes in the expression of Muc1 and Muc4 mucins were observed. CONCLUSIONS: Our data show that cigarette smoke exposure leads to an increased vulnerability to pancreatic self-digestion. Moreover, the concomitant involvement of pancreatic ducts occurs only when focal pancreatic inflammation is present.

MUC4-expressing pancreatic adenocarcinomas show elevated levels of both T1 and T2 cytokines: potential pathobiologic implications.

OBJECTIVE: The human MUC4 mucin plays an important role in the pathogenesis of pancreatic cancer. Recently, we have demonstrated that MUC4 expression in pancreatic tumor cells is regulated by interferon-gamma (IFNgamma) and by retinoic acid via transforming growth factor beta 2 (TGFbeta-2). In the present study, we established the pathobiological association of various cytokines and MUC4 in pancreatic tumor tissues and tumor cell lines. METHODS: Using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and/or immunohistochemical analyses, we examined the expression of MUC4, IFNgamma, TGFbetas, and several immunologically relevant cytokines in a panel of 11 pancreatic adenocarcinomas (PA), three normal pancreatic (NP) tissue specimens, and 11 pancreatic tumor cell lines. RESULTS: Our data revealed that both MUC4 and IFNgamma were expressed at moderate to high levels in the majority of PA, while being undetectable in NP. Moreover, transcript for interleukin 2 (IL-2), a known marker of activated T helper 1 (TH1) lymphocytes, exhibited an expression profile similar to IFNgamma, suggesting a role of these immune effector cells as a potential source of IFNgamma in PA. Of note, IFNgamma protein was detected in the inflamed tissues neighboring tumor areas. Furthermore, TGFbetas were expressed by most cell lines and frequently upregulated in PA compared with NP. Interestingly, both IL-12 and IL-10, two key cytokines of the TH1 and TH2 pathways, respectively, were expressed at higher levels in PA relative to NP. CONCLUSIONS: These observations support the potential implication of IFNgamma and TGFbetas in MUC4 regulation in vivo and suggest a complex interaction of TH1 and TH2 signaling in the pancreatic tumor microenvironment. These findings may provide useful insights into the pathobiology of pancreatic cancer.

Regulation of mucin expression: mechanistic aspects and implications for cancer and inflammatory diseases.

Mucins are large multifunctional glycoproteins whose primary functions are to protect and lubricate the surfaces of epithelial tissues lining ducts and lumens within the human body. Several lines of evidence also support the involvement of mucins in more complex biological processes such as epithelial cell renewal and differentiation, cell signaling, and cell adhesion. Recent studies have uncovered the role of select mucins in the pathogenesis of cancer, underscoring the importance of a detailed knowledge about mucin biology. Under normal physiological conditions, the production of mucins is optimally maintained by a host of elaborate and coordinated regulatory mechanisms, thereby affording a well-defined pattern of tissue-, time-, and developmental state-specific distribution. However, mucin homeostasis may be disrupted by the action of environmental and/or intrinsic factors that affect cellular integrity. This results in an altered cell behavior that often culminates into a variety of pathological conditions. Deregulated mucin production has indeed been associated with numerous types of cancers and inflammatory disorders. It is, therefore, crucial to comprehend the underlying basis of molecular mechanisms controlling mucin production in order to design and implement adequate therapeutic strategies for combating these diseases. Herein, we discuss some physiologically relevant regulatory aspects of mucin production, with a particular emphasis on aberrations that pertain to pathological situations. Our views of the achievements, the conceptual and technical limitations, as well as the future challenges associated with studies of mucin regulation are exposed.

Chronic pancreatic inflammation induced by environmental tobacco smoke inhalation in rats.

OBJECTIVE: Despite a strong epidemiological association between cigarette smoking and pancreatic diseases, such as pancreatic cancer and chronic pancreatitis, the effects of long-term cigarette smoke inhalation on the pancreas have not been clearly determined. In the present study, we investigated the effect of cigarette smoke inhalation on the pancreas. METHODS: Thirty-six female Sprague Dawley rats were exposed to two different doses of environmental tobacco smoke averaging 100 mg or 160 mg/m3 total suspended particulate matter (TSP) per m3 for 70 min twice a day for 12 wk. The animals were sacrificed and examined for the effects of tobacco smoke exposure on pancreatic morphology and function. RESULTS: In 58% (7/12) of the animals, exposure to 160 mg/m3 TSP cigarette smoke induced a chronic pancreatic inflammatory process with fibrosis and scarring of pancreatic acinar structures. Animals with fibrotic alterations showed an induction of pancreatic pro-collagen 1 gene expression, and the infiltration of immune cells was accompanied by the expression of the inflammatory mediators MIP-1alpha, IL-1beta, and TGF-beta in 33% (4/12) of the animals. Acinar cell stress was manifested by a significant up-regulation of pancreatitis-associated protein expression (PAP) in smoke-exposed animals (smoke-exposed 6,932 +/- 1,236 vs control 3,608 +/- 305, p < 0.05). Possibly contributing to the morphological damage to the exocrine pancreas, the inhalation of cigarette smoke induced trypsinogen and chymotrypsinogen gene expression and, furthermore, reduced pancreatic enzyme content. CONCLUSIONS: This study provides experimental evidence of morphological pancreatic damage induced by the inhalation of cigarette smoke, which is likely to be mediated by alterations of acinar cell function.

Synergistic induction of the MUC4 mucin gene by interferon-gamma and retinoic acid in human pancreatic tumour cells involves a reprogramming of signalling pathways.

The transmembrane mucin, MUC4, is aberrantly expressed with a high incidence in human pancreatic adenocarcinomas and plays an important role in the pathogenesis of the disease. Our recent studies have shown that interferon-gamma (IFNgamma) and retinoic acid (RA) are important regulators of MUC4 in pancreatic tumour cells. Induction of MUC4 by IFNgamma occurs via a novel pathway involving upregulation of the signal transducer and activator of transcription 1 (STAT-1), whereas its stimulation by RA requires mediation by the transforming growth factor beta-2 (TGFbeta-2). In this study, we have investigated the molecular mechanisms underlying the interaction of IFNgamma and RA in MUC4 regulation in pancreatic tumour cells. We demonstrate that these reagents exert a synergistic induction of MUC4. Interestingly, while the upregulation of STAT-1 by IFNgamma is partially inhibited by RA, IFNgamma is shown to repress RA-driven TGFbeta-2 induction, pointing to the involvement of alternative mechanism(s) in IFNgamma-RA synergism. Moreover, a dose-dependent and cooperative induction of MUC4 promoter activity suggests a regulation at the transcriptional level, most likely by STAT-1 and RAR/RXR (RA receptor/retinoic X receptor) or other IFNgamma/RA-induced secondary intermediate effectors. Our findings provide potential mechanisms that may account for the aberrant expression of MUC4 in pancreatic tumour cells and expose a novel molecular mechanism of gene induction, whereby a reprogramming of signalling pathway through alternative route(s) operates during a synergistic interaction of biological modifiers.

ErbB2 growth factor receptor, a marker for neuroendocrine cells?

BACKGROUND/AIMS: The overexpression of ErbB2 in pancreatic cancer has been reported with a varying incidence ranging between 1 and 80%. Our routine examination, however, revealed a consistently strong immunoreactivity of three anti-ErbB2 growth factor receptor antibodies in pancreatic islets and intrapancreatic ganglia. To validate our findings and to understand the reasons for the reported differences in the frequency of ErbB2 overexpression in pancreatic cancer, the following studies were performed. MATERIALS AND METHODS: Tissue samples from 12 normal pancreata, 7 surgical chronic pancreatitis cases, 21 primary pancreatic adenocarcinomas, 9 metastatic pancreatic adenocarcinomas, and 4 islet cell tumors were subjected to immunohistochemical examination using antibodies from three manufacturers. Cultured human islet cells and pancreatic cancer cell lines, as well as samples from the gastrointestinal tract, the CNS, and the adrenal gland were included in the study. For comparison, mammary cancer tissue and mammary cancer cells, as well as selected tissues from Syrian golden hamsters, were used. To verify the results, Western blot and Northern slot-blot analyses were performed. RESULTS: Pancreatic cancer cells, in vitro and in vivo, showed a remarkable heterogeneity in the immunostaining of ErbB2, ranging from very faintly to strongly stained. On the other hand, in both humans and hamsters, a consistently strong immunostaining was found in the Langerhans' islets, in the ganglia of intrapancreatic and extrapancreatic nerves, as well as in the CNS, spinal cord and adrenal gland. CONCLUSIONS: ErbB2 appears to play an important role in neuroendocrine tissues and is probably involved in the development and functional regulation of these cells. The concomitant expression of these factors and islet cell hormones very likely results in the activation of multiple growth-promoting pathways in pancreatic cancer and its aggressive behavior.

Generation and characterization of anti-MUC4 monoclonal antibodies reactive with normal and cancer cells in humans.

We have previously cloned the full-length cDNA (approximately 28 Kb) and established the complete genomic organization (25 exons/introns over 100 kb) of the human MUC4 mucin. This large molecule is predicted to protrude over 2 microm above the cell surface, in which MUC4alpha is an extracellular mucin-type glycoprotein subunit and MUC4beta is the transmembrane subunit. Over two thirds of the encoded protein sequence consists of 16-amino-acid tandem repeats (TR), which are flanked by unique sequences. In this study we generated and characterized monoclonal antibodies (MAbs) directed against the TR region of MUC4. Mice were immunized with a KLH-conjugated MUC4 TR peptide, STGDTTPLPVTDTSSV. Several clones were purified by three rounds of limited dilutions and stable clones presenting a sustained antibody production were selected for subsequent characterization. Antibodies were tested for their reactivity and specificity to recognize the MUC4 peptide and further screened by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. One of the MAbs (8G7) was strongly reactive against the MUC4 peptide and with native MUC4 from human tissues or pancreatic cancer cells in Western blotting, immunohistochemistry, and confocal analysis. Anti-MUC4 MAb may represent a powerful tool for the study of MUC4 function under normal and pathological conditions and for diagnosis of solid tumors including those in the breast, pancreas, lungs, and ovaries.

Purification and characterization of a human pancreatic adenocarcinoma mucin.

Pancreatic mucins consist of core proteins that are decorated with carbohydrate structures. Previous studies have identified at least two physically distinct populations of mucins produced by a pancreatic adenocarcinoma cell line (HPAF); one is the MUC1 core protein, which includes an oligosaccharide structure identified by a monoclonal antibody (MAb) recognizing the DU-PAN-2 epitope. In this study, we purified and characterized a second mucin fraction, which also shows reactivity with the DU-PAN-2 antibody, but which has an amino acid composition that is not consistent with the MUC1 core protein. This new mucin was purified by ammonium sulfate precipitation, molecular sieve chromatography, and density gradient centrifugation. It eluted in the void volume of a Sepharose 4B column together with an associated low molecular weight protein, which could be further resolved. The mucin is highly polyanionic due to numerous sulfated and sialylated saccharide chains. Carbohydrate analyses of the purified mucin showed the presence of galactose, glucosamine, galactosamine, and sialic acid, but no mannose, glucose, or uronic acid. The purified and deglycosylated mucin shows no reactivity with anti-MUC1 apomucin antibody, but reacts with antiserum against deglycosylated tracheal mucins and antiserum against the MUC4 tandem repeat peptide. Analysis of mucin expression in HPAF cells revealed high levels of MUC1 and MUC4 mRNA, and moderate levels of MUC5AC and MUC5B mRNA. The amino acid composition of the purified mucin shows a high degree of similarity to the MUC4 core protein.