Messenger RNA expression patterns

In mouse embryo, whole mount in situ hybridization showed that MMD was expressed in all tissues and brain structures, although at different expression levels (Figure 5.7). In contrast, MMD2 expression was highly restricted to certain structures of the central and peripheral nervous system such as the dorsal root ganglia and the trigeminal nerve (Figure 5.8). Dorsal root ganglia (DRG) along the spinal cord contain sensory neurons (Gilbert, 2000). The strong expression of MMD2 mRNA in these locations suggests its association with neurons.

Although both MMD and MMD2 transcripts are localized in the nervous system and brain region, MMD was absent from the spinal cord and DRG, and exhibited a rather diffused expression pattern. This finding suggests that MMD and MMD2 may be involved in different processes of the mouse development. In addition to the above, Menke et al. (Menke & Page, 2002) showed MMD2 expression in testis from E13.5 mouse embryos by whole mount in situ hybridization. In testicular cords MMD2 expression was attributed to Sertoli cells (Menke &

Page, 2002), that play a pivotal role in the regulation and maintenance of spermatogenesis (Russell & Griswold , 1993), suggesting a putative role of MMD2 in this process as well.

Northern blot analyses showed that the ubiquitous MMD and restricted MMD2 expression patterns were maintained during adult age, and conserved in mouse and human (Figure 5.6B-C and Figure 5.9C).

Taking into consideration the strong expression of MMD in macrophages, it is tempting to attribute its ubiquitous expression pattern partially to resident and recruited macrophages in tissues (see section 1.1.2). Tissue macrophages are responsible for immune surveillance and maintenance of tissue homeostasis (Burke B & Lewis Claire E., 2002). Moreover they are found in different parts of the developing embryo, including the brain, the central nervous system (Lichanska et al., 1999), and in areas of active tissue remodeling such as the branchial arches (giving rise to mandibular components) and developing limbs (Lichanska et al., 1999;

Hume et al., 1995). Thus the ubiquitous expression of MMD in the embryonic brain may be as well attributed to macrophages.

Nevertheless, Northern blot from different cell types showed that MMD expressions is not restricted to macrophages but is also found in other myeloid lineages (such as in T cells) and non-myeloid cell lines. The human skin (NHDFC) and dermal (HUVEC) fibroblasts, and the mouse embryonic fibroblast cell line, NIH3T3, expressed MMD as well, suggesting an involvement of this gene in connective tissues.

Moreover, CaCo-2, and HepG2, which are colon and hepatocyte carcinoma cell lines respectively, expressed strongly MMD as well. This finding is in agreement with the presence of human MMD (hMMD) transcript in colon and liver (Figure 5.6). According to the GNF SymATLAS database (Su et al., 2002), hMMD is strongly expressed in the hepatoma cell line, huh-7, various pancreatic cell lines such as Capan1 and Panc1 and in the myoblast cell line SKMC. This wide range of MMD expression emphasizes its role in several cell types.

Interestingly, multiple mouse MMD (mMMD) transcripts of different length were observed in the liver and heart, possibly resulting from the use of different polyadenylation (Poly-(A)) sites. Additionally, several ATTTA motifs are present in the 3’-untranslated region (UTR) of the mRNA. This sequence motif has been associated with mRNA stability and translational efficacity allowing fast changes in the proteins synthesis (Ross, 1995). It is therefore possible that these motifs may play a role in mMMD mRNA stability in the liver and heart.

In contrast to MMD, MMD2 transcript was not detected in any of the tested myeloid cell lines, nor in the non-myeloid fibroblasts cells (data not shown). Based on its restricted mRNA expression patterns in specific tissues, one can speculate that MMD2 may function in reproductive tissues (testis, ovary). However we cannot rule out the possibility that it may also be developmentally regulated or enriched in cell lines that have not been analysed in the present study.

Given the differential spatial expression of MMD and MMD2 despite high protein sequence homology (around 68% identical amino acids), one can speculate that these two genes arose from gene duplication of Hly-III. Subsequently a gene divergence occurred in regulatory elements leading to alteration of their expression pattern (Strachan T & Read A.P, 2004; Tang et al., 2005). The high degree of sequence homology shared between the orthologues results likely from a high selection pressure and suggests a conserved function among species.

As previously mentioned, mMMD was found strongly expressed in bone marrow macrophages (BMM) (Figure 5.9). Inflammatory stimuli activates macrophages and alter their gene expression pattern (see section 1.1.3). To investigate a possible regulation of mMMD expression in inflammation, mouse BMM were treated with bacterial lipopolysaccharides (LPS). LPS is a component of a Gram-negative bacteria, which binds to its receptor CD14 with the help of the soluble LPS-binding protein (LBP). The LPS-CD14 complex is then recognized by TLR4 which activates multiple signaling pathways leading to the activation of the transcription factor, NFκB (Abul K.Abbas & Andrew H.Lichtman, 2003). Consequently, a large number of genes are up- or downregulated, and their protein products provide macrophages with defense mechanisms against infection. Treatment of BMM with LPS induced a rapid increase of mMMD transcript, that reached the highest levels after 2 h, and was then downregulated 8 h after stimulation (Figure 5.10). This rapid response shows an association of mMMD with the macrophage innate activation (see section 1.1.3), which is

characterized by an increased phagocytotic capacity, and the production of cytokines and chemokines. These in turn can promote or inhibit inflammation (Burke B & Lewis Claire E., 2002). For instance IL-10, mainly produced by activated macrophages, functions as a feedback regulator, promoting their deactivation and thus protects against the damaging effects of persisting inflammation (Barsig et al., 1995; Abul K.Abbas & Andrew H.Lichtman, 2003). However, stimulation of BMM with IL-10 did not affect mMMD transcription levels, indicating that mMMD is probably not regulated by IL-10.

Cytokines, such as INFγ and IL-4, which prime macrophages towards a classical or an alternative activation phenotype respectively (see section 1.1.3), did not influence mMMD expression (Figure 5.11). These results lead to the conclusion, that mMMD expression is associated with the first phase of the macrophage activation by LPS, as it is regulated independently of IL-10, and not altered by INFγ or IL-4 priming.