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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 15, 2001 as doi:10.1096/fj.01-0359fje. |
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Laboratory of Molecular Immunoregulation, National Cancer Institute at Frederick, Frederick, Maryland 21702, USA
2Correspondence: Bldg. 559, Rm. 9, NCI-Frederick, Frederick, MD 21702, USA. E-mail: yoshimur{at}mail.ncifcrf.gov
SPECIFIC AIMS
Discoidin domain receptor (DDR) 1 is a receptor tyrosine kinase whose ligand is collagen. In our search for genes expressed by activated human neutrophils using cDNA arrays, we detected considerable up-regulation of DDR1 mRNA expression after in vitro activation of neutrophils, leading us to hypothesize that tissue-infiltrating leukocytes interact with collagen of the extracellular matrix (ECM) through DDR1; the subsequent activation of the receptor results in enhanced migration, activation, and differentiation of leukocytes during inflammatory responses. The original aim of our study was to clarify the inducible expression of DDR1 in leukocytes and identify the biological roles of this receptor in leukocyte functions, especially migration, in a 3-dimensional tissue microenvironment.
PRINCIPAL FINDINGS
1. Expression of DDR1 is inducible in leukocytes
Low level DDR1 mRNA expression was detected in freshly isolated peripheral blood mononuclear cells (PBMC) and freshly isolated neutrophils by Northern blotting. The level of DDR1 mRNA in PBMC progressively increased during incubation in RPMI1640 containing 10% FCS (complete medium) and peaked by 16 h. The level of DDR1 mRNA in neutrophils was also markedly increased after 8 h incubation in the complete medium. Addition of tumor necrosis factor 
, interleukin 1ß, granulocyte-macrophage colony-stimulating factor (GM-CSF), lipopolysaccharides, or PHA further augmented expression levels of DDR1 mRNA in PBMC.
There was no detectable DDR1 protein in freshly isolated PBMC by Western blotting. However, DDR1 protein was detectable after 12 h incubation in the complete medium. The expression level reached a peak by 3 days and was still detectable after 6 days in culture. Activation of monocytes with GM-CSF or lymphocytes with PHA increased DDR1 protein expression by each cell type.
In vivo, DDR1 mRNA was readily detected in the cytoplasm of tissue infiltrating mononuclear cells, probably macrophages, by in situ hybridization. Since we did not detect DDR1 mRNA in freshly isolated PBMC by the same method, the DDR1 mRNA expression appeared to be up-regulated in cells that had emigrated into the extravascular space.
Finally, we used RT-PCR to evaluate the expression levels of DDR1 mRNA isoforms in leukocytes. The DDR1a isoform was found to be the major isoform in leukocytes.
2. Overexpression of DDR1a or DDR1b in THP-1 cells enhances adherence of the cells to collagen-coated plates
To determine the biological role of the DDR1a isoform and compare it with that of the DDR1b isoform in leukocytes, we overexpressed DDR1a or DDR1b in the human monocytic leukemia cell line THP-1, using a retrovirus system. None of the cell lines showed significant adherence to BSA-coated plates after 30 min incubation at 37°C. Small numbers of parental and mock-infected THP-1 cells adhered to collagen-coated plates. They could be largely inhibited by an anti-ß1 integrin blocking antibody. In contrast, greater numbers of either DDR1a- or DDR1b-overexpressing THP-1 cells (
62% of total cells) adhered to collagen-coated plates after 30 min; adhesion of these cells was not inhibited by the anti-ß1 integrin blocking antibody. At 2 h, all four cell lines exhibited increased adherence to collagen-coated plates. However, adherence of parental or mock-infected cells was largely inhibited by the anti-ß1 integrin blocking antibody, which had much less effect on the adherence of DDR1a- or DDR1b-overexpressing cells.
3. Overexpression of DDR1a, but not DDR1b, induces morphological change in THP-1 cells on collagen-coated plates
There was a dramatic morphological difference between DDR1a- and DDR1b-overexpressing cells after 1 h incubation on collagen-coated plates. DDR1a-expressing cells extended long pseudopods (Fig. 1
C, E). In contrast, DDR1b-overexpressing cells, as well as parental or mock-infected cells, did not show similar pseudopod extension (Fig. 1A
, B
, D). An identical morphological change was observed when DDR1a-overexpressing cells were seeded in 3-dimensional collagen lattices. In contrast to the effects of collagen, coating the plates with fibronectin had no effect on the adherence or shape change of DDR1a- or DDR1b-overexpressing cells.
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4. Overexpression of DDR1a, but not DDR1b, promotes random and directed migration of THP-1 cells in 3-dimensional collagen lattices
Using cell culture inserts, we next investigated the migration of DDR1a- or DDR1b-overexpressing THP-1 cells. As shown in Fig. 2
A, a small number of parental cells migrated without chemoattractant. Migration of mock-infected cells was slightly increased but not significant. Overexpression of DDR1a resulted in an threefold increase in the number of migrating cells, whereas overexpression of DDR1b resulted in approximately a twofold decrease. When monocyte chemoattractant protein-1 (MCP-1) was added to the outer wells as a chemoattractant, the migration of parental, mock-infected, and DDR1a-overexpressing THP-1 cells increased two- to threefold. Although the response of DDR1b-overexpressing cells to the complete medium was lower than that of parental or mock-infected cells, the response to MCP-1 was at the same level.
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The migration of DDR1a-overexpressing cells was increased even more when 3-dimensional collagen lattices were generated over the polycarbonate membranes (Fig. 2B
). Although the migration of parental or mock-infected cells in response to either the complete medium or MCP-1 was minimal in the presence of collagen lattices, the enhanced migratory response of DDR1a-overexpressing cells was still evident; the increase was 12-fold. Migration of DDR1b-overexpressing cells was completely inhibited in collagen lattices.
CONCLUSIONS
Migration of leukocytes in a tissue microenvironment requires a coordinated interaction with the ECM. Integrins play a major role in the interaction between leukocytes and endothelial cells during the transmigration process and in leukocyte migration on a 2-dimensional surface. However, integrin-independent mechanisms have been suggested to be involved in the migration of leukocytes in 3-dimensional collagen lattices. The present study indicates the presence of such a mechanism that regulates the migration of leukocytes through tightly meshed collagen fibers.
The expression of DDR1 mRNA was up-regulated not only in neutrophils, but also in monocytes and lymphocytes, just by in vitro incubation. Activation of PBMC with proinflammatory cytokines further increased the level of DDR1 mRNA and induced protein expression, suggesting that DDR1 expression is up-regulated at inflammatory sites. In fact, a high level of DDR1 mRNA was detected in vivo in infiltrating mononuclear cells. Since the ligand for this receptor is abundant in the ECM, tight regulation of DDR1 expression may be important to limit signaling between collagen and DDR1.
There are at least three isoforms of DDR1 mRNA. The DDR1b and DDR1c isoforms contain an in-frame insertion of 111 bp coding for an additional 37 amino acids in the proline-rich juxtamembrane region due to alternative splicing of this gene. The 37 amino acid insertion contains the LXNPXY motif that corresponds to the consensus binding motif for the Shc phosphotyrosine binding domain and interacts with Shc, whereas the juxtamembrane region of DDR1a interacts with another docking protein, FRS2. Thus, there is a biologically relevant structural difference between DDR1a and the others. In human leukocytes, the majority of DDR1 transcripts were DDR1a. It was previously shown that in the neonatal brain, DDR1a mRNA existed at greater frequency than in the adult brain, whereas DDR1b mRNA (including DDR1c) existed more frequently in the adult brain. Thus, the expression of each DDR1 isoform appears to be tightly regulated in normal tissues and each DDR1 isoform may have a different role.
DDR1b was previously found to play a role in the differentiation and myofibril formation in the myoblast cell line and in neurite outgrowth of cerebellar granule cells. A recent study of DDR1-null mice indicated an essential role of DDR1 in mammary gland development. Another study using aortic smooth muscle cells derived from the DDR1-null mice indicated a role of DDR1 in the adhesion, proliferation, and migration of aortic smooth muscle cells. Since both DDR1a and DDR1b are missing in the DDR1-null mice, the contribution of each isoform to the findings remains unclear. In our study, overexpression of DDR1a in THP-1 cells promoted the migration of THP-1 cells in 3-dimensional collagen lattices whereas overexpression of DDR1b appeared to function as a stop signal, which suggests a novel and distinct function of DDR1a and DDR1b in leukocyte migration.
In inflamed tissues, functions of inflammatory leukocytes can be regulated by up- or down-regulation of cell surface receptor expression and subsequent interaction of the receptors with their ligands, including a wide range of agents secreted at the sites and components of the ECM. In the present study, we have identified DDR1a as a novel leukocyte cell surface receptor involved in the interaction of leukocytes with collagen of a 3-dimensional ECM network (Fig. 3
). Circulating leukocytes do not express a significant level of DDR1a. By the time emigrating leukocytes reach an extravascular space, expression of DDR1a is likely to be up-regulated. A signal produced by the interaction of DDR1a with collagen induces a shape change necessary for the migration of the cells in tightly meshed 3-dimensional structures, enabling them to participate in host defense. Detection of DDR1 mRNA expression in tissue infiltrating leukocytes marks its in vivo relevance. Activation of DDR1a may also promote maturation or differentiation of leukocytes. Taken together, detection of DDR1 in leukocytes leads us to a new field of investigation involving a novel integrin-independent signaling pathway that is activated on interaction of cells with collagen of the ECM in a 3-dimensional tissue microenvironment.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0359fje; to cite this article, use FASEB J. (October 15, 2001) 10.1096/fj.01-0359fje 
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