Martin Klempt

Novel insights into structure and function of MRP8 (S100A8) and MRP14 (S100A9)

The two migration inhibitory factor- (MIF)-related protein-8 (MRP8; S100A8) and MRP14 (S100A9) are two calcium-binding proteins of the S100 family. These proteins are expressed during myeloid differentiation, are abundant in granulocytes and monocytes, and form a heterodimeric complex in a Ca2+-dependent manner. Phagocytes expressing MRP8 and MRP14 belong to the early infiltrating cells and dominate acute inflammatory lesions. In addition, elevated serum levels of MRP8 and MRP14 have been found in patients suffering from a number of inflammatory disorders including cystic fibrosis, rheumatoid arthritis, and chronic bronchitis, suggesting conceivable extracellular roles for these proteins. Although a number of possible functions for MRP8/14 have been proposed, the biological function still remains unclear. This review addresses recent developments regarding the MRP14-mediated promotion of leukocyte-endothelial cell-interactions and the characterization of MRP8/14 heterodimers as a fatty acid binding protein complex. In view of the current knowledge, the authors will hypothesize that MRP8 and MRP14 play an important role in leukocyte trafficking, but do not affect neutrophil effector functions.

The Two Calcium-binding Proteins, S100A8 and S100A9, Are Involved in the Metabolism of Arachidonic acid in Human Neutrophils

Recently, we identified the two myeloid related protein-8 (MRP8) (S100A8) and MRP14 (S100A9) as fatty acid-binding proteins (Klempt, M., Melkonyan, H., Nacken, W., Wiesmann, D., Holtkemper, U., and Sorg, C. (1997) FEBS Lett. 408, 81–84). Here we present data that the S100A8/A9 protein complex represents the exclusive arachidonic acid-binding proteins in human neutrophils. Binding and competition studies revealed evidence that (i) fatty acid binding was dependent on the calcium concentration; (ii) fatty acid binding was specific for the protein complex formed by S100A8 and S100A9, whereas the individual components were unable to bind fatty acids; (iii) exclusively polyunsaturated fatty acids were bound by S100A8/A9, whereas saturated (palmitic acid, stearic acid) and monounsaturated fatty acids (oleic acid) as well as arachidonic acid-derived eicosanoids (15-hydroxyeicosatetraenoic acid, prosta- glandin E2, thromboxane B2, leukotriene B4) were poor competitors. Stimulation of neutrophil-like HL-60 cells with phorbol 12-myristate 13-acetate led to the secretion of S100A8/A9 protein complex, which carried the released arachidonic acid. When elevation of intracellular calcium level was induced by A23187, release of arachidonic acid occurred without secretion of S100A8/A9. In view of the unusual abundance in neutrophilic cytosol (approximately 40% of cytosolic protein) our findings assign an important role for S100A8/A9 as mediator between calcium signaling and arachidonic acid effects. Further investigations have to explore the exact function of the S100A8/A9-arachidonic acid complex both inside and outside of neutrophils.

The heterodimer of the Ca2+-binding proteins MRP8 and MRP14 binds arachidonic acid

The S100 proteins MRP8 and MRP14 have been shown to be expressed by myeloid cells during inflammatory reactions. Since the majority of S100 proteins exhibit their biological activity when associated as complex it was investigated whether murine MRP8 and MRP14 form heterodimers and whether this complex may bind lipids of the cell membrane. This is of particular importance since their anchoring into the plasma membrane is unclear although upon calcium binding the proteins translocate from the cytoplasma to the cytoskeleton and the plasma membrane. Using recombinant proteins we could show that not the monomers but only the heterodimers specifically bind arachidonic acid. This finding opens new perspectives for the role of MRP8 and MRP14 in acute and chronic inflammatory processes.

Binding of Two Nuclear Complexes to a Novel Regulatory Element within the Human S100A9 Promoter Drives the S100A9 Gene Expression

S100A9, also referred to as MRP14, is a calcium-binding protein whose expression is tightly regulated during differentiation of myeloid cells. The present study was performed to study the cell type- and differentiation-specific transcriptional regulation of the S100A9 gene. Analysis of the S100A9 promoter in MonoMac-6 cells revealed evidence for a novel regulatory region from position −400 to −374 bp, termed myeloid-related protein regulatory element (MRE). MRE deletion resulted in a 5.2-fold reduction of promoter activity. By electrophoretic mobility shift analysis two nuclear complexes binding to this region were identified and referred to as MRE-binding complex A (MbcA) and MRE-binding complex B (MbcB). By mutagenesis the MRE-binding motif could be narrowed to a 12-bp region. The relevance of MRE is deduced from the observations that the formation of either MRE-binding complex A or MRE-binding complex B strongly correlated with S100A9 gene expression in a cell type-specific, activation- and differentiation-dependent manner. Moreover, DNA affinity chromatography and Western blot studies indicate that a Kruppel-related zinc finger protein and the transcriptional intermediary factor 1β (TIF1β) are involved in an MRE-binding complex, thereby regulating the S100A9 gene expression.

The Gene Encoding the Myeloid-related Protein 14 (MRP14), a Calcium-binding Protein Expressed in Granulocytes and Monocytes, Contains a Potent Enhancer Element in the First Intron

Myeloid-related proteins 8 and 14 (MRP8 and MRP14) are two Ca2+-binding proteins of the S-100 family highly abundant in myelomonocytic cells. The expression is not only dependent on the developmental status of the cell but also on the inflammatory situation in the tissue. In order to identify regulatory elements responsible for the high expression of MRP14 in myeloid cells, reporter gene constructs have been transfected into HL-60 cells, Mono Mac 6 cells, and L132 cells. We demonstrated that a DNA element in the first intron (positions 153–361) enhances the transcriptional activity of the homologous promoter and of the heterologous herpes simplex virus thymidine kinase promoter up to 37-fold. To further identify the functional site, the region between positions 153 and 192 was analyzed functionally using the thymidine kinase promoter. The region increased the expression in the same magnitude as the complete intron. This enhancer is highly conserved in the human and murine MRP genes, indicative of its involvement in the transcription of MRPs. Protein binding to the region is demonstrated using EMSA, DNA cross-linking, Southwestern blotting, and affinity purification. Affinity purification confirms that four proteins bind to the enhancer element.

The Transcription Factors c-myb and C/EBPα Regulate the Monocytic/Myeloic Gene MRP14

Abstract The entry of microorganisms into the body induces inflammatory processes.During this process a sequence of cellular, humoral, non-specific and specific actions are evoked to combat the infection.Macrophages and granulocytes, which are developed from a common progenitor cell, are the cellular components of the specific and non-specific immunoreaction.MRP14 (Macrophage migration inhibitory related protein) and MRP8, two S-100 proteins contained in high concentrations in these cells are obviously essential for adhesion and migration of monocytes and granulocytes.To investigate the transcriptional regulation of these genes we cotransfected constructs expressing CAT under control of the MRP14 promoter and expression constructs of C/EBPoc and v-myb, two transcription factors involved in myeloid/monocytic differentiation.Transfection with C/EBPoc revealed a massive enhancement of the MRP14 promoter in both, HL 60 cells (granulocytic differentiated) and L132 fibroblasts.In contrast, v-myb reduces MRP14 promoter activity.Northern blot analysis of L132 cells transfected with the C/EBPoc expression vector demonstrate that C/EBPcc is sufficient to enhance MRP14 expression in the context of the whole genome.

The mouse homologue of the HTLV-I tax responsive element binding protein TAXREB107 is a highly conserved gene which may regulate some basal cellular functions☆

We report the cDNA sequence of a mouse gene homologous to the HTLV-I tax responsive element binding protein TAXREB107 (M-TAXREB107). This gene is constitutively and ubiquitously expressed indicating a conserved biological function. We present evidence that its transcription is under strict control of a regulatory factor, which is rapidly metabolized.

Identification of epithelial and myeloid-specific DNA elements regulating MRP14 gene transcription.

Macrophage migration inhibition factor‐related protein 14 (MRP14) is a Ca2+‐binding protein of the S‐100 family highly abundant in myelomonocytic and epithelial cells. The expression pattern is restricted to myeloid and epithelial cells and therefore the MRP14 gene is ideally suited to study the regulation of gene expression in these cells. We characterized the human MRP14 promoter by using the chloramphenicol acetyltransferase reporter assay system. The analysis was performed in epithelial (TR146) and myeloid (HL‐60) cells, and we were successful in mapping positive and negative regulatory elements. The region ‐114/‐419 contains strong myeloid‐specific regulatory elements, whereas the domain ‐600/‐1000 enhances the MRP14 transcription in epithelial cells. The sequence ‐462/‐600 accommodates a regulatory element that enhances the promoter activity in both myeloid and epithelial cells. Regions ‐114/‐419 and ‐1000/‐4500 reduce the expression of MRP14 only in epithelial cells, whereas the domains ‐419/‐462 and ‐4500/‐6500 contain down‐regulating elements in both, epithelial and myeloid cells. The presented data demonstrate that transcription of the human MRP14 gene is regulated in a complex manner enabling the precise control of the MRP14 level in epithelial and myeloid cells. J. Cell. Biochem. 73:49–55, 1999.