Differential gene expression in human conducting airway surface epithelia and submucosal glands.
ABSTRACT Human conducting airways contain two anatomically distinct epithelial cell compartments: surface epithelium and submucosal glands (SMG). Surface epithelial cells interface directly with the environment and function in pathogen detection, fluid and electrolyte transport, and mucus elevation. SMG secrete antimicrobial molecules and most of the airway surface fluid. Despite the unique functional roles of surface epithelia and SMG, little is known about the differences in gene expression and cellular metabolism that orchestrate the specialized functions of these epithelial compartments. To approach this problem, we performed large-scale transcript profiling using epithelial cell samples obtained by laser capture microdissection (LCM) of human bronchus specimens. We found that SMG expressed high levels of many transcripts encoding known or putative innate immune factors, including lactoferrin, zinc alpha-2 glycoprotein, and proline-rich protein 4. By contrast, surface epithelial cells expressed high levels of genes involved in basic nutrient catabolism, xenobiotic clearance, and ciliated structure assembly. Selected confirmation of differentially expressed genes in surface and SMG epithelia demonstrated the predictive power of this approach in identifying genes with localized tissue expression. To characterize metabolic differences between surface epithelial cells and SMG, immunostaining for a mitochondrial marker (isocitrate dehydrogenase) was performed. Because greater staining was observed in the surface compartment, we predict that these cells use significantly more energy than SMG cells. This study illustrates the power of LCM in defining the roles of specific anatomic features in airway biology and may be useful in examining how disease states alter transcriptional programs in the conducting airways.
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ABSTRACT: Zinc-alpha 2-glycoprotein (Zn alpha 2gp) is widely distributed in body fluids and in various epithelia; its gene has been completely sequenced, but its function has long remained elusive. We have found that Zn alpha 2gp has RNase activity, comparable to onconase but two orders of magnitude less than RNase A. The RNase activity of Zn alpha 2gp is characterized by maxima in pH at 7.5, in ionic strength at 50 mM NaCl, and in temperature at 60 degreesC. It is strongly inhibited by ZnCl2, but unaffected by MgCl2. It is partially inactivated (down to 20%) by the placental RNase inhibitor. On synthetic polyribonucleotide substrates, the RNase activity of Zn alpha 2gp is specific for pyrimidine residues [poly(C) and poly(U) equally] and cleaves only single-stranded RNA. For onconase, it has been demonstrated that the RNase activity depends on pyroglutamic acid (pyr 1) as the N-terminus; Zn alpha 2gp also has pyr 1, while RNase A does not. Alignment of the amino acid sequences of Zn alpha 2gp and onconase or RNase A reveals only modest matches. Despite the more substantial overall structural homology of Zn alpha 2gp to class I major histocompatibility complex proteins, Zn alpha 2gp has not been proven to be associated with the immune response and, conversely, we could not detect RNase activity in six class I HLA heavy chains.Archives of Biochemistry and Biophysics 08/1998; 355(2):160-4. · 2.93 Impact Factor
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ABSTRACT: The expression of individual xenobiotic-metabolizing cytochrome P450 (CYP) genes in human placenta was studied at the mRNA level by reverse transcriptase-polymerase chain reaction (RT-PCR). mRNAs of CYP1A1, CYP2E1, CYP2F1, CYP3A3/4, CYP3A5, and CYP4B1 were detected by RT-PCR, and CYP1A2, CYP2A6/7, CYP2B6/7, CYP2C8-19, CYP2D6, and CYP3A7 were not detected. Several enzyme activity assays and immunoblots were used to further characterize expression of forms producing detectable mRNA transcripts. The catalytic activities of 7-ethoxycoumarin O-deethylase (ECOD), 7-ethoxyresorufin O-deethylase (EROD) and aryl hydrocarbon hydroxylase (AHH) were substantially increased in response to maternal cigarette smoking, and paralleled the amount of CYP1A1 mRNA and protein. Aromatase activities were slightly lower in placentas exposed to cigarette smoke compared with nonexposed placentas. These data show that several xenobiotic-metabolizing CYP genes are expressed in human placenta at a low level. The significance of such low-level expression is unknown, but it may have local physiological or toxic consequences.Biochemical Pharmacology 03/1996; · 4.70 Impact Factor
Article: NAD(P)H:quinone oxidoreductase1 (DT diaphorase) specifically prevents the formation of benzo[a]pyrene quinone-DNA adducts generated by cytochrome P4501A1 and P450 reductase.[show abstract] [hide abstract]
ABSTRACT: Monkey kidney COS1 cells transiently transfected with plasmids pMT2-cytochrome P450 1A1 (CYP1A1), pMT2-cytochrome P450 reductase (P450 reductase), and pMT2-NAD(P)H:quinone oxidoreductase1 (NQO1 or DT diaphorase), individually or in combination, expressed significantly elevated levels of the respective enzyme(s). The transfected cells were homogenized to break cell membranes without affecting the nuclei and incubated with benzo[a]pyrene (BP) to determine the role of cDNA-encoded enzymes in metabolic activation and/or detoxification of BP. These studies were performed by measuring the capacity of the transfected cells to form DNA adducts as determined by 32P postlabeling and protein adduct detection. Cotransfection of the COS1 cells with cDNAs encoding CYP1A1 and P450 reductase resulted in eight distinct BP-DNA adducts. Inclusion of cDNA encoding NQO1 along with CYP1A1 and P450 reductase in transfection reduced the number of DNA adducts to six. The two lost DNA adducts were specifically eliminated due to the presence of cDNA-derived NQO1 activity. Subsequent experiments with BP-1,6-quinone, BP-3,6-quinone, and BP-6,12-quinone identified these two adducts as those of BP quinones. In an in vitro system, BP-3,6-quinone produced two adducts with deoxyguanosine (dG) but not with dA, dC, and dT. Furthermore, the positions of BP-3,6-quinone-dG adducts on TLC plate correspond to those that are prevented by cDNA-derived NQO1, thus identifying these adducts as BP quinones of dG. In addition, NQO1 reduced the amount of protein-BP adducts generated by CYP1A1 and P450 reductase into transfected COS1 cells. These results show that semiquinones can directly bind to DNA and demonstrate that NQO1 activity can specifically reduce the binding of quinone metabolites of BP generated by CYP1A1 and P450 reductase to DNA and protein.Proceedings of the National Academy of Sciences 09/1994; 91(18):8413-7. · 9.68 Impact Factor