title: Isolation and characterization of the cDNA for mouse neutrophil collagenase: demonstration of shared negative regulatory pathways for neutrophil secondary granule protein gene expression.
abstract: A characteristic of normal neutrophil maturation is the induction of secondary granule protein (SGP) mRNA expression. Several leukemic human cell lines mimic normal morphologic neutrophil differentiation but fail to express SGPs, such as lactoferrin (LF) and neutrophil gelatinase (NG). In contrast, two murine cell lines (32D C13 and MPRO) are able to differentiate into neutrophils and induce expression of LF and NG. Therefore, to study the normal regulation and function of these genes, the corresponding murine homologs must be isolated. Using cDNA representational difference analysis (RDA) to compare a committed myeloid progenitor cell line (EPRO) with the multipotent stem cell line from which it was derived (EML), we isolated a fragment bearing homology to human neutrophil collagenase (hNC). Here, we describe the cloning and characterization of a full-length ( approximately 2 kb) clone that exhibits nearly 65% nucleotide and 73% amino acid identity to hNC. Ribonuclease protection analysis (RPA) of the tissues and cell lines shows that mouse NC (mNC) is expressed only in cell lines exhibiting neutrophilic characteristics, further confirming its identity as the mouse homolog of hNC. Furthermore, we have demonstrated a shared negative regulatory pathway for this and other SGP genes. We have previously shown that CCAAT displacement protein (CDP/cut) binds to a specific region of the LF promoter, and overexpression of CDP blocks G-CSF-induced upregulation of LF gene expression in 32D C13 cells. We show here that in these cells, upregulation of both NC and NG is also blocked. CDP is thus the first identified transcription factor that is a candidate for mediating the shared regulation of neutrophil SGP protein genes.
authors.affiliation: Department of Biology and Section of Hematology, Yale University School of Medicine, New Haven, CT 06510, USA.
authors.forename: N D
authors.initials: ND
authors.lastname: Lawson
authors.forename: A
authors.initials: A
authors.lastname: Khanna-Gupta
authors.forename: N
authors.initials: N
authors.lastname: Berliner
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017364:Collagenases / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D018093:Gelatinases / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018398:Homeodomain Proteins; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000201:enzymology*; D009687:Nuclear Proteins / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors
chemical_list: C080632:CUX1 protein, human; C000624101:Cux1 protein, mouse; D018076:DNA, Complementary; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D017364:Collagenases; D018093:Gelatinases