Cells dividing at the time of carcinogen exposure are at particular risk for neoplasia. Tobacco smoke contains numerous carcinogens, and we find that smoke, in the absence of exogenous growth factors, is capable of stimulating cell proliferation. The smoke-triggered mechanism includes the generation of oxygen radicals, which in turn stimulate tumor necrosis factor ␣-converting enzyme (a disintegrin and metalloproteinase (ADAM) 17) to cleave transmembrane amphiregulin, a ligand for the epidermal growth factor receptor (EGFR). The binding of amphiregulin to EGFR then stimulates proliferation of lung epithelial cells. These results shed light on the pathogenesis of lung cancer, suggest novel drug targets for the reduction of cancer risk in smokers, and provide insight into how EGFR integrates responses to diverse noxious stimuli.

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 endspolymerase 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 NFB and Sp 1. Within 4 kb of the transcription start site are elements mediating transcriptional up-regulation in response to bacterial exoproducts.Mucin is a glycoprotein secreted from epithelial cells at many body surfaces. In the airways, mucin interacts with cilia to trap and clear pathogens and irritants. This mucociliary mechanism is impaired when mucin is produced excessively as in cystic fibrosis, chronic bronchitis, and asthma. Mucociliary impairment leads to airway mucus plugging, which promotes chronic infection, airflow obstruction, and sometimes death.Nine mucin genes are known to be expressed in man: MUC5AC, MUC5B,. The mRNAs encoding two of them, MUC2 and MUC5AC, have been shown to be up-regulated in cystic fibrosis airways (13,14) 1 and likely contribute to the airway mucus plugging characteristic of this disease. Insofar as DNA-RNA transcription is controlled by mechanisms amenable to pharmaceutical intervention, an understanding of mucin transcription may suggest ways of inhibiting mucin overproduction.Both MUC2 and MUC5AC map to chromosome 11p15.5 and may have arisen from a common ancestral gene. The structure of MUC2 is known. Its central region, comprising Ͼ50% of the polypeptide, contains two tandem repeat sequences rich in threonine, serine, and proline (4, 17); this is flanked up-and downstream by cysteine-rich regions (17, 18). The threonine and serine residues represent O-glycosylation sites, whereas the cysteine residues are thought to mediate intermolecular interactions underlying mu...

In smokers' lungs, excessive mucus clogs small airways, impairing respiration and promoting recurrent infection. A breakthrough in understanding this pathology was the realization that smoke could directly stimulate mucin synthesis in lung epithelial cells and that this phenomenon was dependent on the cell surface receptor for epidermal growth factor, EGFR. Distal steps in the smoke-triggered pathway have not yet been determined. We report here that the predominant airway mucin (MUC5AC) undergoes transcriptional up-regulation in response to tobacco smoke; this is mediated by an AP-1-containing response element, which binds JunD and Fra-2. These transcription factors require phosphorylation by upstream kinases JNK and ERK, respectively. Whereas ERK activation results from the upstream activation of EGFR, JNK activation is chiefly EGFR-independent. Our experiments demonstrated that smoke activates JNK via a Src-dependent, EGFRindependent signaling cascade initiated by smoke-induced reactive oxygen species. Taken together with our earlier results, these data indicate that the induction of mucin by smoke is the combined effect of mutually independent, reactive oxygen species activation of both EGFR and JNK.The primary cause of morbidity in chronic bronchitis is mucin overproduction, a phenomenon for which the molecular pathogenesis is unknown. Inflammatory cells are abundant in smokers' airways (1-3) and are capable of stimulating mucin production (4 -7), suggesting that at least some of the excessive mucin in smokers' lungs is secondary to inflammation.In addition, however, smoke itself can induce mucin synthesis in lung cells (8,9). The question of how this occurs is complex in that smoke, a composite of irritant molecules including acetaldehyde, hydroquinone, formaldehyde, benzo-[a]pyrene, cresol, nicotine, catechol, acrolein, coumarin, anthracene, nitrogen oxides, and heavy metals (10, 11) may act on lung epithelial cells in diverse ways. For example, the induction of cytochrome P450 by tobacco smoke (12) is mediated by binding of the aryl hydrocarbon nuclear receptor to a dioxin response element in the 5Ј-flank of the gene, but the induction of the ␥-glutamylcysteine synthetase heavy subunit (␥-GCS-HS) gene is mediated by the binding of a c-Jun/c-Jun homodimer to an AP-1-like response element (13).Previous reports have implicated the receptor for epidermal growth factor (EGFR) 1 in the induction of mucin gene MUC5AC by smoke (9). Consistent with a role for EGFR in mucin induction, an EGF response element has been identified 200 bp upstream of the MUC5AC gene (14). The response of this element to EGFR ligands EGF and transforming growth factor-␣ is mediated by Sp1. One might predict from these data that the induction of MUC5AC by smoke would depend on interaction between the EGF response element at Ϫ200 bp and Sp1.In contrast, in the present study we show that MUC5AC is controlled principally by a smoke response element ϳ3 kb upstream of the EGF response element. This element is AP-1-dependent and is bound by Ju...

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.

Headache disorders are highly prevalent and debilitating, with limited treatment options. Previous studies indicate that many proinflammatory immune cells contribute to headache pathophysiology. Given the well-recognized role of regulatory T (Treg) cells in maintaining immune homeostasis, we hypothesized that enhancing Treg function may be effective to treat multiple headache disorders. In a mouse model of chronic migraine, we observed that repeated nitroglycerin (NTG, a reliable trigger of migraine in patients) administration doubled the number of CD3+ T cells in the trigeminal ganglia without altering the number of Treg cells, suggesting a deficiency in Treg-mediated immune homeostasis. We treated mice with low-dose interleukin-2 (ld-IL2) to preferentially expand and activate endogenous Treg cells. This not only prevented the development of NTG-induced persistent sensitization but also completely reversed the established facial skin hypersensitivity resulting from repeated NTG administration. The effect of ld-IL2 was independent of mouse sex and/or strain. Importantly, ld-IL2 treatment did not alter basal nociceptive responses, and repeated usage did not induce tolerance. The therapeutic effect of ld-IL2 was abolished by Treg depletion and was recapitulated by Treg adoptive transfer. Furthermore, treating mice with ld-IL2 1 to 7 days after mild traumatic brain injury effectively prevented as well as reversed the development of behaviors related to acute and chronic post-traumatic headache. In a model of medication overuse headache, Ld-IL2 completely reversed the cutaneous hypersensitivity induced by repeated administration of sumatriptan. Collectively, this study identifies ld-IL2 as a promising prophylactic for multiple headache disorders with a mechanism distinct from the existing treatment options.

Allele losses and mutations have been examined in 38 cases of primary hepatocellular carcinomas (HCC) from different geographic areas of China by Southern, single-strand conformational polymorphism (SSCP) and direct DNA sequencing analyses. Two of 12 samples from Qi-Dong and six of 18 HCCs from Shanghai showed loss of heterozygosity (LOH) at the loci on chromosome 17p13.3. All of the nine mutations in the p53 gene detected in HCC from Qi-Dong were clustered at the third base of codon 249, i.e. G:C to T:A, leading to an arginine to serine change. In contrast, 18 HCC samples from Shanghai contained three mutations at codons 249, 255 and 279. These results suggested a relationship between the spectrum of p53 aberration and environmental risk factors in these two geographic areas. Since no correlation between the state of HBV DNA and p53 aberration was observed, other factors such as dietary exposure to aflatoxin B1 (AFB1) might be responsible for the mutational hotspot at codon 249 in HCCs from Qi-Dong area.

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