mTORC1 inhibition increases neurotensin secretion and gene expression through activation of the MEK/ERK/c-Jun pathway in the human endocrine cell line BON

The mammalian target of rapamycin (mTOR) signaling exists in two complexes: mTORC1 and mTORC2. Neurotensin (NT), an intestinal hormone secreted by enteroendocrine (N) cells in the small bowel, has important physiological effects in the gastrointestinal tract. The human endocrine cell line BON abundantly expresses the NT gene and synthesizes and secretes NT in a manner analogous to that of N cells. Here, we demonstrate that the inhibition of mTORC1 by rapamycin (mTORC1 inhibitor), torin1 (both mTORC1 and mTORC2 inhibitor) or short hairpin RNA-mediated knockdown of mTOR, regulatory associated protein of mTOR (RAPTOR), and p70 S6 kinase (p70S6K) increased basal NT release via upregulating NT gene expression in BON cells. c-Jun activity was increased by rapamycin or torin1 or p70S6K knockdown. c-Jun overexpression dramatically increased NT promoter activity, which was blocked by PD98059, an mitogen-activated protein kinase kinase (MEK) inhibitor. Furthermore, overexpression of MEK1 or extracellular signal-regulated kinase 1 (ERK1) increased c-Jun expression and NT promoter activity. More importantly, PD98059 blocked rapamycin- or torin1-enhanced NT secretion. Consistently, rapamycin and torin1 also increased NT gene expression in Hep3B cells, a human hepatoma cell line that, similar to BON, expresses high levels of NT. Phosphorylation of c-Jun and ERK1/2 was also increased by rapamycin and torin1 in Hep3B cells. Finally, we showed activation of mTOR in BON cells treated with amino acids, high glucose, or serum and, concurrently, the attenuation of ERK1/2 and c-Jun phosphorylation and NT secretion. Together, mTORC1, as a nutrient sensor, negatively regulates NT secretion via the MEK/ERK/c-Jun signaling pathway. Our results identify a physiological link between mTORC1 and MEK/ERK signaling in controlling intestinal hormone gene expression and secretion.     

Site-specific DNA methylation contributes to neurotensin/neuromedin N expression in colon cancers

The neurotensin/neuromedin N (NT/N) gene is expressed in fetal colon, repressed in newborn and adult colon, and reexpressed in approximately 25% of colon cancers. Our purpose was to determine the effect of gene methylation on NT/N silencing in colon cancers. We found that the NT/N gene was expressed in human colon cancer cell line KM12C but not in KM20 colon cancer cells. Bisulfite genomic sequencing demonstrated that all CpG dinucleotides in the region from -373 to +100 of the NT/N promoter, including a CpG site in a distal consensus AP-1 site, were methylated in KM20 but unmethylated in KM12C cells. Treatment of KM20 cells with demethylating agent 5-azacytidine induced NT/N expression, suggesting a role for DNA methylation in silencing of NT/N in colon cancers. To better elucidate the mechanisms responsible for NT/N repression by DNA methylation, we performed gel shift assays using an oligonucleotide probe corresponding to the distal AP-1 consensus sequence of the NT/N promoter. Methylation of the oligonucleotide probe inhibited protein binding to the distal AP-1 site of the NT/N promoter, suggesting a potential mechanism of NT/N gene repression in colon cancers. We show that DNA methylation plays a role in NT/N gene silencing in the human colon cancer KM20 and that NT/N expression in KM12C cells is associated with demethylation of the CpG sites. DNA methylation likely contributes to NT/N gene expression noted in human colon cancers.     

Menin and JunD regulate gastrin gene expression through proximal DNA elements

Mutations in the MEN1 gene correlate with multiple endocrine neoplasia I (MEN1). Gastrinomas are the most malignant of the neuroendocrine tumors associated with MEN1. Because menin and JunD proteins interact, we examined whether JunD binds to and regulates the gastrin gene promoter. Both menin and JunD are ubiquitous nuclear proteins that we showed colocalize in the gastrin-expressing G cells of the mouse antrum. Transfection with a JunD expression vector alone induced endogenous gastrin mRNA in AGS human gastric cells, and the induction was blocked by menin overexpression. We mapped repression by menin to both a nonconsensus AP-1 site and proximal GC-rich elements within the human gastrin promoter. Chromatin immunoprecipitation assays, EMSAs, and DNA affinity precipitation assays documented that JunD and Sp1 proteins bind these two elements and are both targets for menin regulation. Consistent with menin forming a complex with histone deacetylases, we found that repression of gastrin gene expression by menin was reversed by trichostatin A. In conclusion, proximal DNA elements within the human gastrin gene promoter mediate interactions between JunD, which induces gastrin gene expression and menin, which suppresses JunD-mediated activation.     

Human T-cell leukemia virus type 1 Tax transactivates the matrix metalloproteinase 7 gene via JunD/AP-1 signaling

Adult T-cell leukemia (ATL) is a T-cell malignancy associated with human T-cell leukemia virus type 1 (HTLV-1) and characterized by visceral invasion. Degradation of the extracellular matrix by matrix metalloproteinases (MMPs) is a crucial process in invasion of tumors and metastasis. MMP-7 (or matrilysin), is a “minimal domain MMP” with proteolytic activity against components of the extracellular matrix. To determine the involvement of MMP-7 in visceral spread in ATL, this study investigated MMP-7 expression in ATL. MMP-7 expression was identified in HTLV-1-infected T-cell lines, peripheral blood ATL cells and ATL cells in lymph nodes, but not in uninfected T-cell lines or normal peripheral blood mononuclear cells. MMP-7 expression was induced following infection of a human T-cell line with HTLV-1, and specifically by the viral protein Tax. Functionally, MMP-7 promoted cell migration of HTLV-1-infected T cells. The MMP-7 promoter activity was increased by Tax and reduced by deletion of the activator protein-1 (AP-1) binding site. Electrophoretic mobility shift assay showed high levels of AP-1 binding proteins, including JunD, in HTLV-1-infected T-cell lines and ATL cells, and Tax elicited JunD binding to the MMP-7 AP-1 element. Tax-induced MMP-7 activation was inhibited by dominant negative JunD and augmented by JunD/JunD homodimers. Short interfering RNA against JunD inhibited MMP-7 mRNA expression in HTLV-1-infected T-cell lines. These results suggest that the induction of MMP-7 by Tax is regulated by JunD and that MMP-7 could facilitate visceral invasion in ATL.     

Phorbol ester-mediated neurotensin secretion is dependent on the PKC-alpha and -delta isoforms

Neurotensin (NT) plays an important role in gastrointestinal secretion, motility, and growth. The mechanisms regulating NT secretion are not entirely known. Our purpose was to define the role of the PKC signaling pathway in secretion of NT from BON cells, a human pancreatic carcinoid cell line that produces and secretes NT peptide. We demonstrated expression of all 11 PKC isoforms at varying levels in untreated BON cells. Expression of PKC-alpha, -beta2, -delta, and -mu isoforms was most pronounced. Immunofluorescent staining showed PKC-alpha and -mu expression throughout the cytoplasm and in the membrane. Also, significant fluorescence of PKC-delta was noted in the nucleus and cytoplasm. Treatment with PMA induced translocation of PKC-alpha, -delta, and -mu from cytosol to membrane. Activation of PKC-alpha, -delta, and -mu was further confirmed by kinase assays. Addition of PKC-alpha inhibitor G?-6976 at a nanomolar concentration, other PKC inhibitors G?-6983 and GF-109203X, or PKC-delta-specific inhibitor rottlerin significantly inhibited PMA-mediated NT release. Overexpression of either PKC-alpha or -delta increased PMA-mediated NT secretion compared with control cells. We demonstrated that PMA-mediated NT secretion in BON cells is associated with translocation and activation of PKC-alpha, -delta, and -mu. Furthermore, inhibition of PKC-alpha and -delta blocked PMA-stimulated NT secretion, suggesting a critical role for these isoforms in NT release.