Related Experiment Videos
Molecular cloning of the MCP-3 chemokine gene and regulation of its expression
A Minty1, P Chalon, J C Guillemot
1Sanofi Elf Bio Recherches, Labège, France.
Abstract:
We have isolated a cDNA (NC28) transcribed from a mRNA which is transiently induced in U937 promonocytic cells by PMA and super-induced by cycloheximide. NC28 cDNA encodes a new member of the chemokine family, MCP-3, recently purified from MG-63 osteosarcoma cells by Van Damme et al. [1]. The MCP-3 protein sequence shows 74% identity with human monocyte chemoattractant protein 1 (MCP-1) and, like MCP-1, recombinant MCP-3 protein shows chemotactic activity for monocytes but not for neutrophils. However the secreted MCP-3 protein differs from MCP-1 in being N-glycosylated. The 3' noncoding regions of MCP-3 and MCP-1 mRNAs are more diverged (44%), allowing specific cDNA probes to be made, and indicating that the two genes are evolutionarily distant. Sequence comparisons of the 3' noncoding regions suggest that MCP-3 may be the human homologue of the mouse MARC gene [2], and that MCP-1 and MCP-3 genes arose by a gene duplication event before the mammalian radiation. Both MCP-1 and MCP-3 mRNAs are expressed by PBMC, principally by monocytes, with MCP-1 mRNA being expressed at levels 2-4 times that of MCP-3 mRNA. However, while MCP-1 mRNA is also expressed at high levels in fibroblast or astrocytoma cell lines after IL-1 and TNF stimulation, MCP-3 mRNA is expressed only at very low levels in these cells. The cellular origin of MCP-3 is thus more restricted than that of MCP-1. In our experiments on PBMC, LPS is not a consistent inducer of MCP-1 and MCP-3 mRNAs. In some experiments, it actually decreases levels of these two mRNAs, while concomitantly increasing IL-6 and TNF-alpha mRNA levels. Levels of MCP-1 and MCP-3 mRNAs in PBMC are both increased by IFN-gamma, although IL-6 mRNA is not induced. They are also increased by PHA-P and are decreased, in most cases, by IL-13 [3]. MCP-1 and MCP-3 mRNAs are thus co-ordinately regulated in monocytes in response to a number of inducing or inhibitory agents, in a manner differing in several respects from that of other monokines such as IL-6.
Insights
We identified a new chemokine, MCP-3, which attracts monocytes but not neutrophils. MCP-3 and MCP-1 chemokines are co-regulated in monocytes, with MCP-3 having a more restricted cellular origin.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- Chemokines are crucial signaling proteins in the immune system.
- Monocyte chemoattractant protein 1 (MCP-1) plays a key role in monocyte recruitment.
- Understanding novel chemokine functions and regulation is vital for immune response studies.
Purpose of the Study:
- To isolate and characterize a novel chemokine, designated NC28, identified in U937 cells.
- To investigate the functional and regulatory relationship between the novel chemokine MCP-3 and MCP-1.
- To explore the evolutionary origins and cellular expression patterns of MCP-3.
Main Methods:
- Isolation of NC28 cDNA from PMA-induced U937 cells.
- Recombinant protein expression and functional assays (chemotaxis).
- mRNA expression analysis in various cell types and in response to different stimuli (LPS, IFN-gamma, IL-13).
- Sequence comparison of 3' noncoding regions to infer evolutionary relationships.
Main Results:
- NC28 cDNA encodes MCP-3, a novel chemokine with 74% sequence identity to MCP-1.
- Recombinant MCP-3 exhibits chemotactic activity for monocytes but not neutrophils and is N-glycosylated.
- MCP-3 and MCP-1 mRNAs are co-ordinately regulated in peripheral blood mononuclear cells (PBMCs), primarily monocytes.
- MCP-3 mRNA expression is more restricted to monocytes compared to MCP-1, which is also found in fibroblasts and astrocytoma cells.
- Sequence analysis suggests MCP-3 is the human homologue of mouse MARC and that MCP-1 and MCP-3 arose from gene duplication.
Conclusions:
- MCP-3 is a novel, N-glycosylated chemokine with monocyte-specific chemoattractant activity.
- MCP-3 and MCP-1 display coordinated regulation in monocytes but differ in cellular expression patterns and evolutionary origins.
- The distinct characteristics of MCP-3 suggest specialized roles in immune responses compared to MCP-1.