EMMPRIN regulates the canonical Wnt/beta-catenin signaling pathway, a potential role in accelerating lung tumorigenesis.

Advances in the field of tumor biology have identified that tumor cells co-opt developmental signaling pathways of embryonic stem cells and thus gain the ability to proliferate, differentiate and alter cell-cell interactions. One such pathway is the Wnt/beta-catenin signaling pathway. High levels of EMMPRIN expression have been shown to correlate with poor prognosis and metastasis in a broad range of tumors. Although a variety of functions are attributed to EMMPRIN in tumorigenesis, the specific mechanism(s) through which it can exert its effects have not been elucidated, until now. In this study, we identify EMMPRIN as a novel regulator of the canonical Wnt/beta-catenin signaling pathway in lung cancer. Increasing EMMPRIN expression levels in lung cancer epithelial cells upregulated the beta-catenin signaling pathway and silencing EMMPRIN inhibited beta-catenin signaling, cell migration, proliferation, anchorage-independent growth and tumor growth in a mouse tumor xenograft model. These results provide a compelling rationale for targeting EMMPRIN for anticancer therapies. Understanding the molecular mechanisms driving EMMPRIN-induced lung tumorigenesis will provide enormous benefits in developing new therapeutic treatments for this and other forms of cancer.

Exposure of human corneal epithelial cells to contact lenses in vitro suppresses the upregulation of human beta-defensin-2 in response to antigens of Pseudomonas aeruginosa.

Bacterial keratitis is a sight-threatening complication of contact lens wear, and Pseudomonas aeruginosa is a commonly isolated pathogen. The mechanisms by which lenses predispose the cornea to P. aeruginosa infection are unknown. Corneal epithelial cells express numerous innate defenses, some of which have bactericidal effects against P. aeruginosa. One of these is human beta-defensin-2 (hBD-2), which is upregulated in response to lipopolysaccharide or flagellin antigens. We hypothesized that prior exposure of corneal epithelia to a contact lens would interfere with upregulation of hBD-2 in response to P. aeruginosa. A novel in vitro model was used in which cultured human corneal epithelial cells were exposed to a hydrophilic contact lens for up to 3.5 days prior to challenge with a culture supernatant of P. aeruginosa antigens for 6h. Without prior lens exposure, the supernatant caused >2-fold upregulation of hBD-2 mRNA message and expression of hBD-2 peptide. Prior contact lens exposure blocked this upregulation without obvious effects on cell health. Western immunoblot and luciferase reporter studies showed that Pseudomonas-induced hBD-2 upregulation involved MyD88, c-Jun N-terminal kinase and both AP-1 and NF-kappaB transcription factors. Contact lenses did not affect surface expression of Toll-like receptor-2, -4 or -5, but did block antigen activation of AP-1, but not NF-kappaB, transcription factors. These data show that contact lenses can interfere with epithelial defense responses to bacterial antigens in vitro, and if translated in vivo, could help predispose the cornea to infection.

New HIV-drug inhibits in vitro bladder cancer migration and invasion.

OBJECTIVE: The CXCR4/CXCL12 axis appears crucial in the metastasis of bladder cancer. Our aim was to evaluate the potency of the CXCR4 antagonist, 4F-benzoyl-TE14011 (4F-bTE), as an anti-metastatic drug in this disease. In this study, we assessed the ability of 4F-bTE to inhibit tumor cell motility, invasion through extracellular matrix (ECM), matrix metalloproteinase (MMP) secretion and cytoskeletal responses to chemokine. METHODS: To assess the degree to which cells could migrate and invade ECM under various conditions, we used TCCSUP bladder cancer cells in a Boyden chamber system. To monitor actin polymerization, we stained cells on chamber slides with AlexaFluor 594 phalloidin. To measure matrix-metalloproteinase-2 and -9 (MMP) activity, we used gelatin zymography. To assess the effects of the CXCR4 antagonist 4F-bTE on each of the above parameters, we exposed bladder cancer cells either to chemokine CXCL12, alone, or to both CXCL12 and 4F-bTE. We also monitored cells for apoptotic and necrotic changes during drug treatment. RESULTS: The CXCR4 antagonist 4F-bTE markedly decreased CXCL12-induced bladder cancer cell migration and ECM invasion in Boyden chamber assays. The antagonist also blocked chemokine-induced actin polymerization as well as the induction of MMP-2 and MMP-9 in these cells. CONCLUSION: The CXCR4 antagonist 4F-bTE has the potential to inhibit expression of the metastatic phenotype and may provide therapeutic value to patients.

Persistent mucin glycoprotein alterations in equine recurrent airway obstruction.

Horses with the episodic asthmalike condition of recurrent airway obstruction (RAO) have bouts of inflammation and bronchoconstriction associated with indoor housing. To assess the potential differences in airway secretions between RAO-affected and control horses, methods to quantify mucus secretions were developed and applied to bronchoalveolar lavage fluid. The relative difference in the amount of mucin glycoproteins between control and RAO-affected horses was assessed with a carbohydrate side chain-specific monoclonal antibody (4E4) in an enzyme-linked immunosorbent assay and by carbohydrate-specific enzyme-linked lectin assays. Significantly increased levels of 4E4-immunoreactive glycoprotein and the mucin-associated carbohydrates fucose (alpha-1,2 linkage) and N-acetylglucosamine were detected in RAO-affected horses in acute disease. RAO-affected horses in remission maintained significantly elevated levels of alpha-1,2-fucose and N-acetylglucosamine, whereas the 4E4-immunoreactive glycoprotein levels displayed a trend toward an increase over control levels. These results indicated that persistent changes in the quantity and/or quality of mucus glycoproteins occurred in the RAO-affected horses.

ATP transduces signals from ASGM1, a glycolipid that functions as a bacterial receptor.

The flagella of the Gram-negative bacterium Pseudomonas aeruginosa serve not only for motility but also to bind bacteria to the host cell glycolipid asialoGM1 (ASGM1) through the protein flagellin. This interaction triggers defensive responses in host cells. How this response occurs is unclear because ASGM1 lacks transmembrane and cytoplasmic domains and there is little information about the downstream effectors that connect ASGM1 ligation to the initiation of host defense responses. Here, we show that ASGM1 ligation promotes ATP release from the host cell, followed by autocrine activation of a nucleotide receptor. This response links ASGM1 to cytoplasmic signaling molecules and results in activation of phospholipase C, Ca(2+) mobilization, phosphorylation of a mitogen-activated protein kinase (Erk 1/2), and activation of mucin transcription. These results indicate that bacterial interaction with host cells can trigger autocrine nucleotide signaling and suggest that agents affecting nucleotide receptors may modulate host responses to bacteria.

Regulation of mucin gene expression in human tracheobronchial epithelial cells by thyroid hormone.

We reported previously that the expression of the gene encoding MUC5AC mucin in human airway epithelial cells is controlled by retinoic acid via the retinoic acid receptor (RAR)-alpha and that 3,3',5-tri-iodothyronine (T(3)) inhibits the expression of MUC5AC. The purpose of the present study was to identify mechanisms mediating the effect of T(3). T(3) has been shown to inhibit gene expression via several mechanisms, either by enhancing or repressing the transcription of target genes or by the regulation of post-transcriptional events. Results showed that T(3) strongly inhibited MUC5AC-driven luciferase activity in normal human tracheobronchial epithelial cells that had been transiently transfected with a MUC5AC-luciferase reporter construct; however, it did not affect MUC5AC mRNA stability. These results indicate that T(3) suppresses MUC5AC expression at the transcriptional level. An analysis of deletion constructs showed that deletion of the region downstream of 3 kb resulted in markedly decreased levels of MUC5AC transcription in the absence of T(3) (i.e. under control conditions) as well as a loss of responsiveness to the inhibitory effects of T(3). This suggests that this region might contain elements important for the activation as well as the repression of MUC5AC transcription. To determine whether T(3) modulates retinoic-acid-dependent MUC5AC transcription via an alteration in the abundance of retinoid receptor proteins, we examined the type and abundance of these receptors in nuclear extracts of airway epithelial cells grown in the presence or absence of T(3). Western blots showed that T(3) markedly decreased several types of retinoid receptor while not affecting T(3) receptor proteins. Consistent with this finding were gel-shift assays revealing a decrease in RAR-retinoic acid response element complexes obtained from T(3)-treated cells. We propose that T(3) might inhibit retinoid-dependent MUC5AC expression by decreasing retinoid receptor levels and thereby decreasing the transcriptional activation of this gene for mucins.

Signaling networks controlling mucin production in response to Gram-positive and Gram-negative bacteria.

Human lung cells exposed to pathogenic bacteria upregulate the production of mucin, the major macromolecular component of mucus. Generally this upregulation is beneficial for the host, however, in the lungs of cystic fibrosis patients, overproduction of mucin can lead to the plugging of pulmonary airways. Mucus plugging impedes airflow and creates an environment that is highly compartmentalized: those bacteria within the mucus layer are shielded from high doses of antibiotics whereas those outside the mucus are exposed. These conditions augment mutation rate and the development of drug resistance in bacteria that colonize the lungs of cystic fibrosis patients. While therapeutic inhibition of mucin induction would improve airflow and reduce antibiotic resistance in these patients, the challenge is to develop drugs that block excessive mucin production while leaving beneficial aspects of the response intact. To do this, we must understand the molecular mechanisms underlying mucin production. Here we review the signal transduction pathways that control mucin production in response to Gram-positive and Gram-negative bacteria.

The transcriptional responses of respiratory epithelial cells to Bordetella pertussis reveal host defensive and pathogen counter-defensive strategies.

Bordetella pertussis, the causative agent of whooping cough, has many well-studied virulence factors and a characteristic clinical presentation. Despite this information, it is not clear how B. pertussis interaction with host cells leads to disease. In this study, we examined the interaction of B. pertussis with a human bronchial epithelial cell line (BEAS-2B) and measured host transcriptional profiles by using high-density DNA microarrays. The early transcriptional response to this pathogen is dominated by altered expression of cytokines, DNA-binding proteins, and NFkappaB-regulated genes. This previously unrecognized response to B. pertussis was modified in similar but nonidentical fashions by the antiinflammatory agents dexamethasone and sodium salicylate. Cytokine protein expression was confirmed, as was neutrophil chemoattraction. We show that B. pertussis induces mucin gene transcription by BEAS-2B cells then counters this defense by using mucin as a binding substrate. A set of genes is described for which the catalytic activity of pertussis toxin is both necessary and sufficient to regulate transcription. Host genomic transcriptional profiling, in combination with functional assays to evaluate subsequent biological events, provides insight into the complex interaction of host and pathogen.

Lung mucin production is stimulated by the air pollutant residual oil fly ash.

Human and animal exposure to particulate air pollution is correlated with airway mucus hypersecretion and increased susceptibility to infection. Seeking clues to the mechanisms underlying this pathology, we examined the effect of the particulate air pollutant residual oil fly ash (ROFA) on production of the major component of mucus, mucin, and the major antibacterial protein of the respiratory tract, lysozyme. We found that following in vitro exposure to ROFA, epithelial cells showed an increase in mucin (MUC5AC) and lysozyme (LYS) steady state mRNA. This upregulation was controlled at least partly at the level of transcription as shown by reporter assays. Experiments testing the ability of the major components of ROFA to mimic these effects showed that vanadium, a metal making up 18.8% by weight, accounted for the bulk of the response. A screen of signaling inhibitors showed that MUC5AC and LYS induction by ROFA are mediated by dissimilar signaling pathways, both of which are, however, phosphotyrosine dependent. Recognizing that the ROFA constituent vanadium is a potent tyrosine phosphatase inhibitor and that mucin induction by pathogens is phophotyrosine dependent, we suggest that vanadium-containing air pollutants trigger disease-like conditions by unmasking phosphorylation-dependent pathogen resistance pathways.

Control of mucin transcription by diverse injury-induced signaling pathways.

Mucin production is an evolutionarily ancient defense mechanism that is retained in mammals and operates at all mucosal surfaces to protect the host against pathogens and irritants. As in lower organisms, the mammalian mucosa (epithelium) produces mucin in response to diverse insults. Our studies aim to understand the intracellular signaling and gene regulation mechanisms mediating mucin production in response to clinically important insults. To date, we find that the signaling pathway triggered by each type of insult is distinct. Relatively common, however, is the involvement of the protein tyrosine kinase c-Src, the MAP kinase kinase MEK 1/2, and the transcription factor NF-kappaB. Basbaum C, Lemjabbar H, Longphre M, Li D, Gensch E, McNamara N. Control of mucin transcription by diverse injury-induced signaling pathways.

Allergen-induced IL-9 directly stimulates mucin transcription in respiratory epithelial cells.

A hallmark of asthma is mucin overproduction, a condition that contributes to airway obstruction. The events responsible for mucin overproduction are not known but are thought to be associated with mediators of chronic inflammation. Others have shown that T-helper 2 (Th2) lymphocytes are required for mucous cell metaplasia, which then leads to mucin overproduction in animal models of allergy. We hypothesized that Th2 cell mediators are present in asthmatic airway fluid and directly stimulate mucin synthesis in airway epithelial cells. Results in cultured airway epithelial cells showed that samples of asthmatic fluid stimulated mucin (MUC5AC) synthesis severalfold more potently than non-asthmatic fluid. Consistent with this, lavage fluid from the airways of allergen-challenged dogs stimulated mucin synthesis severalfold more potently than that from non-allergen-challenged dogs. Fractionation of dog samples revealed 2 active fractions at <10 kDa and 30-100 kDa. Th2 cytokines in these molecular weight ranges are IL-9 (36 kDa), IL-5 (56 kDa), and IL-13 (10 kDa). Antibody blockade of ligand-receptor interaction for IL-9 (but not IL-5 or IL-13) inhibited mucin stimulation by dog airway fluid. Furthermore, recombinant IL-9, but not IL-5 or IL-13, stimulated mucin synthesis. These results indicate that IL-9 may account for as much as 50-60% of the mucin-stimulating activity of lung fluids in allergic airway disease.

Myeloid ELF-1-like factor up-regulates lysozyme transcription in epithelial cells.

Lysozyme is an important component of innate immunity against common pathogens at mucosal surfaces. We previously cloned and characterized the bovine lysozyme 5A (lys5A) promoter with the purpose of determining cis- and trans-acting elements controlling airway epithelial cell-specific expression. We found that such expression is controlled by protein binding to an ETS consensus sequence located approximately at -46 to -40 bp from the transcription start site. The identity of the ETS-related protein responsible for gene transactivation was unknown. In this study, we screened six ETS-related proteins by transient transfection into epithelial cells and fibroblasts. Results showed that among these factors, the myeloid Elf-1-like factor (MEF) was the most potent. Gel shift analysis of epithelial cell nuclear extracts using a lys5A probe including the ETS-binding site (-50/-31) yielded a single band with retarded mobility. This band was supershifted by an antibody directed against MEF. Supporting the possibility that MEF is responsible for functional transactivation of lysozyme in epithelial cells, we found that antisense MEF mRNA decreased lys5A promoter activity and that MEF overexpression in stably transfected cells increased lysozyme mRNA and protein expression. We conclude that MEF is required for epithelial cell transactivation of lysozyme.

Long-lasting effects of chronic ozone exposure on rat nasal epithelium.

Ozone, the principal oxidant pollutant in photochemical smog, causes airway epithelial injury in the upper and lower respiratory tract of laboratory animals. We have recently reported that long-term inhalation exposure to ozone causes mucous-cell metaplasia (MCM) in the surface epithelium lining the nasal airways of F344 rats. The principal objective of the present study was to determine the persistence of ozone-induced MCM in the nasal epithelium after the end of a chronic exposure. Male F344/N rats were exposed to 0, 0.25, or 0.5 ppm ozone, for 8 h/d, 7 d/wk for 13 wk. Animals were killed 8 h, 4 wk, or 13 wk after the end of the chronic exposure. Ozone-related alterations in the nasal epithelium were qualitatively and quantitatively characterized through histochemistry, image analysis, and morphometric techniques. Some rats were exposed for an additional 8 h to 0.5 ppm ozone at 13 wk after the end of the chronic exposure to determine whether previous ozone exposure results in persistent changes in the sensitivity of nasal epithelium to acute injury. At the end of the chronic exposure, hyperplasia was present in the nasal epithelium of rats exposed to 0.25 and 0.5 ppm ozone. By 13 wk postexposure, this proliferative alteration was still evident only in the rats exposed to 0.5 ppm ozone. Ozone-induced MCM with associated intraepithelial mucosubstances was evident only in the nasal tissues of rats exposed to 0.5 ppm ozone. Though attenuated, these alterations in the nasal mucous apparatus were still detectable at 13 wk after the end of the exposure. At this same time after the chronic exposure, an acute (8 h) exposure to 0.5 ppm ozone induced an additional increase of mucosubstances in the nasal epithelium of rats previously exposed to 0.5 ppm ozone, but not in rats chronically exposed to 0 or 2.5 ppm ozone. The persistent nature of the ozone-induced MCM in rats documented in this report suggests that ozone exposure may have the potential to induce similar long-lasting alterations in the airways of humans.

[Novel transcription factor MEF is associated with the function of lung epithelial cells].

Lysozyme is an important component of innate immunity against common pathogens at mucosal surfaces. We previously cloned and characterized the bovine lysozyme 5A (lys5A) promoter with the purpose of determining cis- and trans-acting elements controlling airway epithelial cell-specific expression. We found that such expression is controlled by protein binding to an ETS consensus sequence located approximately at -46 to -40 bp from the transcription start site. The identity of the ETS-related protein responsible for gene transactivation was unknown. In this study, we screened six ETS-related proteins by transient transfection into epithelial cells and fibroblasts. Results showed that among these factors, the myeloid Elf-I-like factor (MEF) was the most potent. Gel shift analysis of epithelial cell nuclear extracts using a lys5A probe including the ETS-binding site (-50/-31) yielded a single band with retarded mobility. This band was super-shifted by an antibody directed against MEF. Supporting the possibility that MEF is responsible for functional transactivation of lysozyme in epithelial cells, we found that antisense MEF mRNA decreased lys5A promoter activity and that MEF overexpression in stably transfected cells increased lysozyme mRNA and protein expression. We conclude that MEF is required for epithelial cell transactivation of lysozyme.

Altered mucin expression is a field change that accompanies mucinous (colloid) breast carcinoma histogenesis.

Mucinous carcinomas of the breast, so-called colloid carcinomas, exhibit better prognoses than their nonmucinous breast counterparts. This biological difference exhibited by mucinous breast carcinomas prompted us to examine the relationship of mucin expression to colloid carcinoma histogenesis. We studied 50 colloid carcinomas, 50 noncolloid cancers, and 50 normal breasts by hematoxylin-eosin (H&E) and Alcian blue staining, mucin immunohistochemistry, in situ hybridization with a battery of MUC riboprobes, and ancillary digital image analysis. We observed luminal mucin in normal ducts in 80% of colloid carcinomas compared with 10% of noncolloid carcinomas and 6% of normal breasts (P < .01). In the cases of colloid carcinoma that showed mucin-filled ducts, luminal mucin was observed in 40% of the normal ducts and acini, 40% to 75% of the ducts involved by hyperplasia, atypical ductal hyperplasia (ADH) and ductal carcinoma in situ (DCIS), respectively, and in 50% of the co-incidental areas of cysts (mucoceles), adenosis, fibroadenoma, and intraductal papilloma (P < .01). Immunohistochemistry showed that colloid carcinomas showed strong MUC2 cytoplasmic immunoreactivity and decreased MUC1 immunoreactivity compared with noncolloid carcinomas. In situ hybridization studies indicated fivefold increased MUC2 signals and twofold increased MUC5 signals within adjacent and remote normal epithelium in only the colloid carcinoma cases (P < .01; P < .05). In these cases of colloid carcinoma, these increased MUC2 and MUC5 signals were also observed in areas of hyperplasia, ADH, DCIS, and invasive carcinoma. In contrast, the noncolloid carcinomas showed fivefold increased MUC1 signals but no increases in MUC2 or MUC5. In mixed colloid/noncolloid carcinomas, the colloid areas had identical mucin expression patterns as the pure colloid carcinomas, but there was a loss of MUC2 and MUC5 expression and a gain of MUC1 expression in the noncolloid areas that was therefore identical to the pattern observed in pure noncolloid carcinoma. In this study, we conclude that the altered expression of mucin so characteristic of colloid carcinoma is also a field change present in adjacent and remote normal breast epithelium.

Mucin gene (MUC 2 and MUC 5AC) upregulation by Gram-positive and Gram-negative bacteria.

Bacterial infection of the lung is associated with mucin overproduction. In partial explanation of this phenomenon, we recently reported that supernatant from the Gram-negative organism Pseudomonas (P.) aeruginosa contained an activity that upregulated transcription of the MUC 2 mucin gene [J.-D. Li, A. Dohrman, M. Gallup, S. Miyata, J. Gum, Y. Kim, J. Nadel, A. Prince, C. Basbaum, Transcriptional activation of mucin by P. aeruginosa lipopolysaccharide in the pathogenesis of cystic fibrosis lung disease, Proc. Natl. Acad. Sci. U.S.A., 94 (1997) 967-972]. The purpose of the present study was to determine whether mucin genes other than MUC 2 are so regulated and whether Gram-positive organisms also contain mucin stimulatory activity. Results from in situ hybridization and RNase protection assays showed that P. aeruginosa upregulates MUC 5AC as well as MUC 2 in both bronchial explants and cultured airway epithelial cells. The upregulation of both genes by P. aeruginosa can be mimicked by lipopolysaccharide (LPS) and can be blocked by the tyrosine kinase inhibitor genistein. In addition, both genes are upregulated by a variety of Gram-positive as well as Gram-negative organisms showing the same rank order of potency. These data indicate the existence of a general mechanism by which epithelial cells respond to the presence of bacteria by increasing mucin synthesis.

Activation of NF-kappaB via a Src-dependent Ras-MAPK-pp90rsk pathway is required for Pseudomonas aeruginosa-induced mucin overproduction in epithelial cells.

Cystic fibrosis (CF) is an autosomal recessive disorder, the most common lethal genetic disease in Caucasians. Respiratory disease is the major cause of morbidity and mortality. Indeed, 95% of CF patients die of respiratory failure. Pseudomonas aeruginosa, an opportunistic pathogen, chronically infects the lungs of over 85% of CF patients. It is ineradicable by antibiotics and responsible for airway mucus overproduction that contributes to airway obstruction and death. The molecular mechanisms underlying this pathology are unknown. Here we show that P. aeruginosa activates a c-Src-Ras-MEK1/2-MAPK-pp90rsk signaling pathway that leads to activation of nuclear factor NF-kappaB (p65/p50). Activated NF-kappaB binds to a kappaB site in the 5'-flanking region of the MUC2 gene and activates MUC2 mucin transcription. These studies bring new insight into bacterial-epithelial interactions and more specifically into the molecular pathogenesis of cystic fibrosis. Understanding these signaling and gene regulatory mechanisms opens up new therapeutic targets for cystic fibrosis.

Cloning of the amino-terminal and 5'-flanking region of the human MUC5AC mucin gene and transcriptional up-regulation by bacterial exoproducts.

To obtain gene regulatory sequence for the mucin gene MUC5AC, we have isolated the MUC5AC amino terminus cDNA and 5'-flanking region. This was possible through the use of rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) in which the 5' sequence of the human gastric mucin cDNA HGM-1 (1) was used to design the first MUC5AC-specific primer. Primers for subsequent rounds of RACE were designed from the 5'-ends of amplified RACE products. After five rounds of RACE-PCR, we could no longer generate upstream extensions of the cDNA and hypothesized that we had reached the 5'-end. Primer extension and RNase protection analysis confirmed this. Combined nucleotide sequence for the RACE-PCR products was 3.3 kb with an open reading frame encoding 1100 amino acids. A putative translation start site was found at nucleotide +48. This was followed by a 45 nucleotide putative signal sequence. This amino-terminal sequence contains no tandem repeats but is >60% similar to the amino-terminal nucleotide sequence of MUC2. The positions of cysteine residues in this MUC2-similar region are almost 100% conserved between the two genes. Northern analysis showed expression of cognate RNA in the stomach and airway but not muscle and esophagus. This pattern was the same as that obtained using previously reported 3'-MUC5AC sequences. We have cloned approximately 4 kb of genomic DNA upstream of the transcription start site and have sequenced 1366 nucleotides containing a TATA box, a CACCC box, and putative binding sites for NFkappaB and Sp 1. Within 4 kb of the transcription start site are elements mediating transcriptional up-regulation in response to bacterial exoproducts.

Tumor-derived EMMPRIN (extracellular matrix metalloproteinase inducer) stimulates collagenase transcription through MAPK p38.

EMMPRIN (extracellular matrix metalloproteinase inducer) stimulates fibroblast metalloproteinases (MMP) 1, 2 and 3 (Kataoka et al. (1993) Cancer Res. 53, 3154-3158). Here we focus on MMP-1, showing that in lung tumors, MMP-1's cognate mRNA is strongly expressed in stromal fibroblasts adjacent to EMMPRIN-expressing tumor cells. In vitro, EMMPRIN upregulates MMP-1 mRNA expression in a concentration-dependent manner, with a peak accumulation at 24 h. The response is genistein-sensitive, suggesting it is dependent on tyrosine kinase activity. Analysis of tyrosine phosphorylation-dependent MAP kinases ERK 1/2, SAPK/JNK, and p38 showed that the activity of p38 but not that of the other 2 kinases was elevated in response to EMMPRIN. That p38 activity was required for EMMPRIN stimulation of MMP-1 was evident from results showing that the p38 inhibitor SB203580 blocked this response. This is the first available information regarding the mechanism by which tumor-associated molecules upregulate MMP synthesis in stromal fibroblasts.

Sp1 protein contributes to airway-specific rat MUC 2 mucin gene transcription.

We have shown increases in the abundance of airway mucin mRNA during the pathogenesis of chronic obstructive pulmonary disease in rat models (Jany et al., 1991) and now seek to determine the underlying mechanisms. As transcriptional modulation may be involved, we provide here a functional analysis of the 5' flanking region of a rat mucin gene (MUC 2). Using deletion mutants to bp -859, we constructed expression cassettes in CAT vectors and transfected them into two MUC 2-expressing cell lines, SPOC 1, a rat airway epithelial cell line and IEC-6, a rat intestinal epithelial cell line, and into one MUC 2 non-expressing cell line, FR, a rat skin fibroblast cell line. Results indicated that nucleotides -59 to -40 mediated high level expression in SPOC 1, but not in the other cells. Used as a probe in gel shift assays, fragment -59/-40 formed complexes of differing mobilities when incubated with nuclear protein extracts from the three cell types. Mutation of the putative Sp1 binding site in the probe sequence interfered with protein binding in all three cell types, but anti-Sp1 antibody supershifted a band formed only by airway cell extracts. A model of airway cell-specific MUC 2 transcription is proposed.