Genomic cloning and promoter analysis of macrophage inflammatory protein (MIP)-2, MIP-1 alpha, and MIP-1 beta, members of the chemokine superfamily of proinflammatory cytokines.
Macrophage inflammatory protein (MIP)-2, MIP-1 alpha, and MIP-1 beta all belong to the newly recognized "chemokine" superfamily of structurally related, activation-inducible cytokines with inflammatory and growth regulatory activities. We report the isolation and sequencing of genomic clones for murine MIP-2 and murine MIP-1 beta, and analyze their regulatory sequences in comparison with each other and with several other members of the chemokine family. The murine (mu)MIP-2 genomic clone displays the canonical four exon/three intron structure typical of other genes in the chemokine alpha subfamily (e.g., IL-8). Potential cis regulatory elements in the proximal promoter region were highly conserved between muMIP-2 and its three most closely related human homologs: human (hu)GRO-alpha, huGRO-beta, and huGRO-gamma. A mouse macrophage cell line, RAW 264.7, was transfected with a growth hormone reporter construct driven by a proximal fragment of the muMIP-2 5' promoter, and nested deletion mutant analysis localized the LPS responsive element to a region that contains a conserved NF kappa B consensus motif and lies 51 to 70 bp 5' from the transcription start site. In contrast to that of MIP-2, the muMIP-1 beta genomic clone exhibited the three exon/two intron structure characteristic of the chemokine beta family members (e.g., MCP-1). A comparison of the promoters for muMIP-1 beta and muMIP-1 alpha reveals a conserved CK-1 element, but transient expression studies in RAW 264.7 macrophages with proximal fragments of either the muMIP-1 beta or the muMIP-1 alpha 5' promoter fused to a human growth hormone reporter gene link LPS-inducibility in both to promoter segments near to, but not identical with, the consensus CK-1 sequence. Proximal 5' promoter fragments cloned from both the MIP-1 alpha and MIP-1 beta genes unexpectedly conferred constitutive expression on the fused reporter gene sequences in macrophage-like cells, but initial 5' deletion analysis did not link this responsiveness to known sequence motifs. The muMIP-1 beta promoter, but not the muMIP-1 alpha promoter, was constitutively active in B16 mouse melanoma cells, and both promoters were active in the myelomonocytic cell line WEHI 3B(A)d-, the muMIP-1 alpha promoter being three times stronger.
Picower Institute for Medical Research, Manhasset, NY 11030.
U
U
Widmer
K R
KR
Manogue
A
A
Cerami
B
B
Sherry
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D054407:Chemokine CCL4; D054426:Chemokine CXCL2; D003001:Cloning, Molecular; D016207:Cytokines / Q000235:genetics* / Q000302:isolation & purification; D006133:Growth Substances / Q000235:genetics; D006801:Humans; D007249:Inflammation / Q000276:immunology; D019402:Macrophage Inflammatory Proteins; D051379:Mice; D008969:Molecular Sequence Data; D015846:Monokines / Q000235:genetics* / Q000302:isolation & purification; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000276:immunology*; D012689:Sequence Homology, Nucleic Acid
D054407:Chemokine CCL4; D054426:Chemokine CXCL2; D016207:Cytokines; D006133:Growth Substances; D019402:Macrophage Inflammatory Proteins; D015846:Monokines