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Interaction of discoidin domain receptor 1 isoform b (DDR1b) with collagen activates p38 mitogen-activated protein kinase and promotes differentiation of macrophages

WATARU MATSUYAMA^*, HIDENOBU KAMOHARA^*, CAROLE GALLIGAN^*, MICHEL FAURE and TEIZO YOSHIMURA^*,2

^* Laboratory of Molecular Immunoregulation, National Cancer Institute at Frederick, Frederick, Maryland, USA; and
SUGEN, Inc., South San Francisco, California, USA

²Correspondence: Bldg. 559, Rm. 9, NCI-Frederick, Frederick, MD 21702, USA. E-mail:yoshimur{at}mail.ncifcrf.gov

SPECIFIC AIMS

The aim of this study was to evaluate whether DDR1–collagen interaction could play a role in the differentiation of THP-1 cells.

PRINCIPAL FINDINGS

1. Activation of DDR1b, but not DDR1a, with collagen promotes differentiation of phorbol 12-myristate 13-acetate (PMA)-treated THP-1 cells
Activation of DDR1a or DDR1b with collagen had no effect on the differentiation of DDR1a- or DDR1b-overexpressing THP-1 cells. However, when the cells were induced to differentiate with PMA, activation of DDR1b (but not DDR1a) with collagen or 513 agonistic anti-DDR1 antibody (Ab) promoted the differentiation of THP-1 cells, as determined by reduced cell proliferation, and up-regulated cell surface HLA-DR, CD11c, CD14, and CD40 expression. The HLA-DR expression was accompanied by the expression of transcripts for HLA-DR and its regulator MHC class II transactivator.

2. Activation of DDR1b with collagen causes recruitment of Shc and phosphorylation of p38 mitogen-activated protein (MAP) kinase
Autophosphorylation of DDR1b was highly detectable as early as 30 min after collagen activation, reached a peak at 60 min, and returned to a basal level by 120 min; autophosphorylation of DDR1a was first detected 90 min after collagen activation, reached a peak at 4 h, and became undetectable by 12 h. By immunoprecipitation of DDR1 and subsequent Western blot, we detected the recruitment of Shc to DDR1b, but not to DDR1a. Since phosphorylated Shc was coimmunoprecipitated with DDR1b only when DDR1b was phosphorylated, recruitment and phosphorylation of Shc appeared to depend on the activation of DDR1b by collagen.

Several proteins were found to be tyrosine-phosphorylated after collagen activation of each cell line (Fig. 1A ). The band indicated by arrow a, which migrated to the estimated molecular mass of 38 kDa, was detected only in DDR1b-overexpressing cells (Fig. 1A , lane 4). We identified this 38 kDa protein to be p38 MAP kinase (Fig. 1B ). The transcription factor ATF2, a substrate of p38 MAP kinase, was phosphorylated in these cells with delayed kinetics (Fig. 1C ). Treatment of DDR1b-overexpressing cells with cycloheximide (CHX) before PMA treatment inhibited the phosphorylation of p38 MAP kinase in response to collagen (Fig. 1E ). Substitution of Y⁵¹⁴ in the LXNPXY motif of the juxtamembrane domain of DDR1b (Y514F) abrogated collagen-induced recruitment of Shc, and collagen activation did not induce the phosphorylation of p38 MAP kinase in Y514F-transfected cells. Finally, SB203580, an inhibitor of p38 MAP kinase, dose-dependently inhibited the expression of HLA-DR at 24 h.

View larger version (38K): [in this window] [in a new window]   Figure 1. Phosphorylation of p38 MAP kinase after collagen activation of PMA-treated, DDR1b-overexpressing THP-1 cells. DDR1a- or DDR1b-overexpressing THP-1 cells were plated at 5 x 106 cells/mL, stimulated with PMA (10 nM) for 24 h, then starved in RPMI 1640 without FCS for 10 h. 50 µg/mL of collagen type I was added and the cells were incubated for additional times. Ten million cells from each culture were lysed in 1 mL of lysis buffer. 20 µL of cell lysates was mixed directly with 20 µL of sample buffer, then subjected to Western blot. A) Membranes were blotted with anti-phosphotyrosine Ab. B) Membranes were blotted with Abs specific for phospho p38 MAP kinase or p38 MAP kinase. C) Membranes were blotted with Abs specific for phospho ATF2 or ATF2. D) The lysates were subjected to immunoprecipitation with anti-p38 MAP kinase and bound proteins were analyzed by Western blot with anti-phospho p38 MAP kinase or anti-p38 MAP kinase Ab. E) Ten million DDR1b-overexpressing cells were pretreated with 10 µg/mL of CHX for 30 min, washed three times, incubated with 10 nM PMA for 24 h, and activated with 50 µg/mL collagen for 60 min. Cell lysates were prepared and Western blot was performed. Membranes were blotted with Abs specific for phosphorylated p38 MAP kinase or p38 MAP kinase.

3. Activation of DDR1 induces phosphorylation of p38 MAP kinase and subsequent HLA-DR expression in granulocyte macrophage colony-stimulating factor-induced human monocyte-derived macrophages (GM macrophages)
Expression of DDR1a and DDR1b was induced during the differentiation of GM macrophages. The level of DDR1a peaked on days 3 and 4, and significantly decreased by day 5. In contrast, the level of DDR1b continued to increase, reached a peak on day 5, and plateaued. Autophosphorylation of DDR1 (DDR1a + DDR1b) in response to 513 Ab was first detected at 30 min and continued for up to 120 min. As in collagen-activated, PMA-treated DDR1b-overexpressing THP-1 cells, tyrosine-phosphorylated Shc was coimmunoprecipitated with DDR1 and phosphorylation of p38 MAP kinase was detected in response to 513 Ab. Although MKK3/6 were present in GM macrophages, phosphorylation of these kinases was not induced in response to 513 Ab.

We evaluated the expression of HLA-DR on GM macrophages. As shown in Fig. 2A, B , only 2% of freshly isolated monocytes expressed HLA-DR; 17% of 5 day GM macrophages expressed HLA-DR, and the percentage of HLA-positive cells increased to 39% when cells were further incubated with 513 Ab. Pretreatment with SB203580 completely inhibited the 513 Ab-induced increase of HLA-DR-positive cells. These results indicate that activation of DDR1, potentially DDR1b, up-regulates the expression of HLA-DR through activation of p38 MAP kinase in primary macrophages.

View larger version (18K): [in this window] [in a new window]   Figure 2. Effects of DDR1 activation on the expression of HLA-DR by GM macrophages. 5 day GM macrophages were incubated at the density of 5 x 106 cells/mL for an additional 2 days in the presence of 50 ng/mL GM-CSF with agonistic anti-DDR1 Ab or mouse monoclonal control IgM. To study the effect of SB 203580, GM macrophages were pretreated with SB203580 or DMSO for 30 min. The expression of HLA-DR was analyzed by flow cytometry. Statistical analysis was performed by Bonferroni/Dunn with 1-way factorial ANOVA.

CONCLUSIONS

Differentiation of monocytes into macrophages or dendritic cells (DCs) is a crucial step for the development of adaptive immunity. The capacity of collagen to induce differentiation of macrophages or DCs has been known for a decade, but without clear molecular mechanisms. We report here that collagen activation of DDR1b facilitates the differentiation of not only PMA-treated THP-1 cells but also primary macrophages.

Binding of Shc to DDR1b was previously demonstrated using the wild-type DDR1b and a recombinant Shc protein in vitro or chimeric receptors containing the juxtamembrane domain of DDR1b in vivo. Recruitment of Shc to DDR1b was detected in the human mammary tumor cell line T47D, which endogenously expresses a high level of DDR1b, but there was no activation of MAP kinases ERK1 or 2. Since a role for Shc in the activation of ERK1 and 2 has been established by studies using dominant negative Shc and mice lacking Shc, the lack of these MAP kinase activation after collagen activation of DDR1b-expressing tumor cell lines or DDR1b-transfected cells was puzzling. We detected the formation of a DDR1b:Shc complex in collagen-activated, PMA-treated DDR1b-overexpressing cells. We have detected, for the first time, activation of the downstream signaling molecule p38 MAP kinase after collagen activation of DDR1b. The phosphorylation of p38 MAP kinase occurred after phosphorylation of DDR1b and Shc; the abrogation of Shc recruitment resulted in the lack of collagen-induced phosphorylation of p38 MAP kinase, indicating that the activation of p38 MAP kinase was due to DDR1b autophosphorylation and subsequent Shc recruitment. Finally, SB203580 blocked collagen-induced HLA-DR expression. Thus, we have linked the DDR1b signaling to a p38 MAP kinase pathway. However, the specific upstream MAP kinase kinases for p38 MAP kinase, MKK3 and MKK6, were absent in PMA-treated DDR1b-overexpressing cells. Although MKK4 has been reported to activate p38 MAP kinase, it is unlikely that MKK4 was responsible for the activation of p38 MAP kinase because MKK4 activates JNK. An alternative MKK-independent pathway for p38 MAP kinase phosphorylation was reported, suggesting that the DDR1b signaling may use another pathway to activate p38 MAP kinase.

Activation of p38 MAP kinase was detected only when DDR1b-overexpressing cells were treated with PMA to induce differentiation of THP-1 cells toward macrophages. Neither PMA nor collagen induced phosphorylation of p38 MAP kinase in undifferentiated THP-1 cells. Thus, changes that occur intracellularly during THP-1 cell differentiation are crucial for the transmission of signals generated by the interaction between DDR1b and collagen. In fact, pretreatment of DDR1b-overexpressing cells with CHX completely inhibited DDR1b-mediated p38 MAP kinase phosphorylation, suggesting that protein(s) synthesized during the differentiation of THP-1 cells may be critical for DDR1b signaling. High-level expression of DDR1b has been detected in malignant tumor cell lines, but downstream signaling pathways of DDR1 could not be detected in those tumor cells. In contrast, the DDR1b-expressing mouse skeletal muscle C2C12 cell line that undergoes terminal differentiation in response to growth factor withdrawal and immature mouse cerebellar granule cells that expressed DDR1b in a developing tissue exhibited further differentiation in response to collagen. These earlier observations and our own data presented here suggest that DDR1b functions in collaboration with other differentiation signals.

Since THP-1 cells are transformed cells, they may not reflect the state of normal macrophages or DCs. Therefore, we evaluated whether similar intracellular molecular events occur in normal GM macrophages. Although we previously reported that DDR1a was the major isoform expressed in leukocytes, both DDR1a and DDR1b were clearly detected in GM macrophages. DDR1a was mainly expressed at an early stage of differentiation whereas DDR1b expression became maximal at a late stage. Activation of endogenously expressed DDR1 with agonistic 513 Ab induced the recruitment of Shc and phosphorylation of p38 MAP kinase with delayed kinetics. Thus, the signaling events identified with DDR1b-overexpressing THP-1 cells are not artifacts. Our data suggest that macrophages at an early stage of differentiation can migrate through the ECM better than fully differentiated macrophages via DDR1a–collagen interaction, and DDR1b-mediated differentiation occurs in a later stage of differentiation.

A wide variety of inflammatory signals have the capacity to induce DC maturation. Recently, p38 MAP kinase was reported to play a critical role in the maturation of DCs induced by lipopolysaccharide, tumor necrosis factor-, and contact sensitizers such as dinitrochlorobenzene and NiSO₄. Serum-induced differentiation of monocytes into macrophages is reported to be dependent on the activation of p38 MAP kinase. Thus, activation of p38 MAP kinase appears to be a common event required for the maturation of macrophages and DCs. p38 MAP kinase regulates the expression of cytokines and chemokines in response to a wide variety of proinflammatory agents. It is not clear, however, whether the optimal concentrations of the proinflammatory agents capable of activating p38 MAP kinase are always available in tissues. Collagen is the major component of the ECM and readily available to activate DDR1b on infiltrating monocytes or on immature DCs either alone or in combination with other stimuli. Thus, DDR1b may serve as an important receptor that contributes to the development of immune responses by activating p38 MAP kinase in a tissue microenvironment.

Our study clarifies that DDR1b in response to collagen indeed transduces signals and activates a p38 MAP kinase pathway that is involved at least in part in the differentiation of PMA-primed THP-1 cells and GM macrophages (Fig. 3 ). However, several important questions remain to be answered. Why does DDR1b transduce signals only in PMA-treated THP-1 cells? What other signaling molecules are involved in the phosphorylation of p38 MAP kinase? Is there any other signaling pathway downstream of DDR1b? Does DDR1a transduce signals? More studies are needed to further dissect the signaling pathways of DDR1 and better understand the biological role of DDR1a and DDR1b expressed not only in leukocytes, but also in other types of cells, including normal epithelial and cancer cells.

View larger version (31K): [in this window] [in a new window]   Figure 3. DDR1b signaling in THP-1 cells. In untreated THP-1 cells, there is no signaling through DDR1b even though DDR1b is autophosphorylated in response to collagen. However, in PMA-treated THP-1 cells, collagen activation of DDR1b induces the recruitment of Shc and transduces signals that lead to the activation of p38 MAP kinase. Activated p38 MAP kinase further activates transcription factors such as ATF2, resulting in the promotion of differentiation. DDR1b signaling may lead to up-regulated transcription of other genes, such as cytokines and chemokines genes, through activation of p38 MAP kinase.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0320fje; doi: 10.1096/fj.02-0320fje

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