Published as doi: 10.1096/fj.06-6527com.
(The FASEB Journal. 2007;21:1376-1382.)
© 2007 FASEB
Bruton’s tyrosine kinase (BTK) is a binding partner for hypoxia induced mitogenic factor (HIMF/FIZZ1) and mediates myeloid cell chemotaxis
Qingning Su,
Yifu Zhou and
Roger A. Johns1
Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
1Correspondence: Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University, Ross 361, 720 Rutland Ave., Baltimore, MD 21205, USA. E-mail: rajohns{at}jhmi.edu
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ABSTRACT
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Hypoxia induced mitogenic factor (HIMF) is a member of the FIZZ/resistin/RELM family of proteins that we have shown to have potent mitogenic, angiogenic, and vasoconstrictive effects in the lung vasculature. In the current report, we identified Bruton’s tyrosine kinase (BTK) as a functional HIMF binding partner through glutathione S-transferase (GST)-HIMF pull-down studies and mass spectrometry. Using primary cultured HIMF-stimulated murine bone marrow cells, we demonstrated that HIMF causes redistribution of BTK to the leading edge of the cells. HIMF stimulation induced BTK autophosphorylation, which peaked at 2.5 min. A transwell migration assay showed that treatment with recombinant murine HIMF induced migration of primary cultured bone marrow cells that was completely blocked by the BTK inhibitor, LFM-A13. Our results demonstrate BTK as the first known functional binding partner of the HIMF/FIZZ family of proteins and that HIMF acts as a chemotatic molecule in stimulating the migration of myeloid cells through activation of the BTK pathway.—Su, Q., Zhou, Y., and Johns, R. A. Bruton’s tyrosine kinase (BTK) is a binding partner for hypoxia induced mitogenic factor (HIMF/FIZZ1) and mediates myeloid cell chemotaxis.
Key Words: resistin • LFM-A13 • migration
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INTRODUCTION
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HYPOXIA-INDUCED MITOGENIC FACTOR (HIMF), also called FIZZ1 or RELM alpha, is a resistin-like molecule that is up-regulated in the lungs in rodent models of chronic hypoxia induced pulmonary hypertension (1
, 2)
. We have shown HIMF to be expressed in the remodeling hyperplastic vascular smooth muscle and endothelium and to stimulate pulmonary microvascular mitogenesis. HIMF was found to have angiogenic properties and to increase pulmonary artery pressure and pulmonary vascular resistance more potently than endothelin, angiotensin (ANG), or serotonin (1)
. The expression of HIMF was also markedly up-regulated in hypertrophic, hyperplastic bronchial epithelium during allergic pulmonary inflammation in mouse models of acute pulmonary inflammation (3)
and in the lymph nodes (4)
, with the highest expression in B cells and macrophages.
As an inflammatory and ischemic tissue marker, the physiological function of HIMF remains unclear. XCP1, another member of the FIZZ/resistin/RELM family, has been reported as a secreted protein that is chemotactic to myeloid cells from C/EBP-epsilon-null mice and interacts with alpha-defensin (4)
. We therefore investigated whether HIMF is a targeting molecule for bone marrow cells and which molecule is the HIMF-binding partner. By using GST-pulldown and mass spectrometry techniques, we isolated a HIMF-binding molecule identified as BTK, a molecule known to be crucial in regulation of B-cell maturation and involved in cell migration. Mutations in BTK are responsible for X-linked agamma globulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice (5
, 6)
.
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MATERIALS AND METHODS
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Constructs and reagents
Flag-tagged HIMF was prepared as described previously (1)
. For GST-HIMF construction, mouse HIMF cDNA was first amplified by polymerase chain reaction (PCR) from a mouse lung cDNA library using primers 5'- GAATTCATGGCGTATAAAAGCATCTCA-3' and 5'- CTCGAGTTAGGACAGTTGGCAGCAGCG-3' and cloned into TA vector. Then the insert was cut from TA vector by EcoR I and XhoI, and cloned into pGEX-5X-1 with the sites of EcoRI and XhoI.
Antibodies and inhibitor
Anti-actin and anti-Fyn rabbit polyclonal antibodies were purchased from Sigma. Anti-BTK monoclonal antibody (mAb), Rabbit anti-BTK phosphorylated (Y223), and phospho-Src family (Y416) polyclonal antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA) Anti-focal adhesion kinase F-actin (FAK) rabbit polyclonal antibody (pAb) was purchased from Upstate (Lake Placid, NY, USA). Monoclonal anti-fyn (Y528) phospho-specific antibody was purchased from BD Transduction Laboratories (Franklin Lakes, NJ, USA). Antihis mAb and anti-GST mAb were purchased from Novagen (Madison, WI, USA). Anti-BTK rabbit pAb was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). FITC and rhodamine-labeled secondary antibodies were purchased from Jackson ImmunoResearch. BTK inhibitor LFM-A13 was purchased from Calbiochem (San Diego, CA, USA).
Cell culture and transfections
Mouse bone marrow cells were maintained in Dulbecco’s modified Eagle’s high glucose medium (DMEM; GIBCO, Gaithersburg, MD, USA) containing 10% FBS at 37°C and 5% CO2. Cells were transfected with plasmids, as mentioned, using LipofectAMINE 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocols. The transfected cells were then fixed in precooled methanol for immunocytochemistry.
GST and GST-HIMF fusion protein expression
BL21 cells harboring GST or GST-HIMF constructs were grown overnight in a 50 ml tube with LB medium containing 50 µg/ml ampicillin and then transferred to a 500 ml flask and grown until the optical density (OD) was 0.6 at 600 nm. The cultures were then induced with isopropyl-ß-D-thiogalactopyranoside (IPTG) for an additional 4 h. Cell lysates were prepared in TBST buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride).
GST pull-down assay
Bone marrow cells were cultured in ten 150 x 25 mm plates until confluence and collected for lysate using TBST buffer. Lysate from 10 plates of bone marrow cells was used for a HIMF-binding partner screening assay. Hypoxia tissue homogenate was prepared as described above. BL21 bacterial lysates for GST and GST-HIMF were first incubated with glutathione agarose in 0.1% TBST buffer for 3 h and then washed three times by 0.1% TBST buffer. The GST and GST-HIMF binding glutathione agaroses were incubated with bone marrow cell lysate or hypoxia tissue homogenate for 3 h and then washed three to five times before SDS-PAGE.
Mass spectrometry
Glutathione-Sepharose beads that bind to GST-HIMF fusion protein and GST were incubated with bone marrow cell lysate in TBST buffer at 4°C for 3 h. The beads were washed five times with TBST, and loading buffer was added to the samples. After electrophoresis, the gel was stained by Coomassie blue. The band that was pulled-down by GST-HIMF was cut from the Coomassie blue stained gel and another band in the unstained area was cut from the same gel as the control. The bands were washed with 50% methanol twice and stored in 1.7 ml ultraClear tubes with 50% methanol. Then, the samples were washed twice with deionized water and dehydrated in 80 µl acetonitrile (ACN) twice. The samples were then swollen in a digestion buffer containing 20 mmol/l NH4HCO3 and 12.5 ng/µl trypsin at 4°C for 30 min and then 37°C for 12 h. Peptides were then extracted twice using 0.1% TFA in 50% ACN at room temperature. The extracts were dried under the protection of N2, and the peptides were eluted onto the target with 0.7 µl matrix solution (
-cyano-4-hydroxy-cinnamic acid in 0.1% TFA, 50% ACN). Samples were allowed to air-dry before being inserted into them into the mass spectrometer. Mass spectrum data from MALDI-TOF-MS were analyzed by searching against an NCBInr database using MASCOT (Matrix Science, London, UK) search software.
Protein phosphorylation assays
Bone marrow cells were cultured in 100 x 20 mm culture plates and treated with 50 nm BSA or HIMF for different time serials, washed quickly by PBS, and lysed in TBST buffer containing 1 mM sodium vanadate. The samples were incubated on ice for 20 min, mixed several times during the incubation, and then centrifuged. The supernatants of the samples were quantified for protein concentration and subjected to electrophoresis on a 4–15% SDS-polyacrylamide gel (Bio-Rad, Hercules, CA, USA). Rabbit anti-BTK phosphorylation (Y-223) pAb and anti-BTK mAb were used for immunoblotting. The same samples were used for the detection of Fyn phosphorylation. Rabbit anti-phospho-Src family (Y416) pAb, monoclonal anti-fyn (Y528) phospho-specific antibody, and rabbit anti-Fyn poloyclonal antibody were used for the blotting.
Mouse bone marrow-derived mesenchymal stem cell preparation and culture
Six C57BL/6 mice (7 wk old) were anesthetized with intramuscularly injection of 1 mg ketamine plus 0.5 mg xylazine per animal. Tibiae and femurs were isolated using sterile techniques. The mouse bone marrow cells were prepared by flushing the tibiae and femurs with serum-free DMEM (low glucose, supplemented with 1x penicillin-streptomycin and 1 mM EDTA) using 25 G needles. Pooled marrow from three animals was first dispersed by gentle pipetting and then separated by gradient centrifugation with lymphocyte separation liquid (Sigma, density: 1.083 g/ml) as follows: 6 ml of the medium containing the marrow cells was layered on top of 3 ml of separation liquid and centrifuged at room temperature at 2800 rpm for 20 min. The mononuclear cells in the middle layer were collected and then washed with serum-free DMEM three times by centrifugation, first at 2000 rpm for 15 min and then two times at 700 rpm for 10 min. The cells collected after the last wash (2–3x108) were resuspended in 10 ml DMEM supplemented with 10% FBS and 1x penicillin/streptomycin and then cultured at 37°C with 5% CO2 in one 10-cm culture dish (uncoated plastic). Three days later, nonadherent cells were removed by changing medium and the adherent cells were grown for 2 wk.
Reverse transcriptase-polymerase chain reaction (RT-PCR)
Fresh bone marrow cells from three mice were prepared as described above. Mouse RAW 309 Cr.1 monocyte cell line was cultured in a 60cm plate with 10% FBS D-MEM medium until confluent and collected by cell scraper. Both bone marrow cells and RAW cells were used for RNA purification using an RNeasy mini kit (Qiagen, Valencia, CA, USA). Two micrograms of total RNA for each sample were used in the cDNA synthesis by Amersham first-strand cDNA synthesis kit. Primers for mouse HIMF coding region (310 bp), ß-actin C-terminal coding region (500 bp), and BTK C-terminal coding region (800 bp) were used for the PCR (95°C 2 min, 35 cycles of 95°C 30 s, 60°C 30 s and 72°C 90 s, final extension was set for 72°C 7 min).
Coimmunoprecipitation
Three microliters of rabbit IgG and rabbit anti-BTK pAb were mixed with 300 µl TBST solution and 20 µl agarose-protein A/G mixture and incubated at 4°C for 3 h. After being washed by TBST three times, 300 µl of TBST and 30 µl of bone marrow cell lysate were mixed with the IgG bound agarose-protein A/G and incubated at 4°C for 3 h. The samples were washed by TBST three times and run on 4–20% SDS-PAGE gel. Rabbit anti-HIMF antibody was used for the blotting.
Mouse hind limb ischemic model
Animals were subjected to left femoral artery ligation and excision to create unilateral hind limb ischemia. For each animal, 25 mg/kg ketamine plus 10 mg/kg xylazine were injected subcutaneously. Skin incisions were performed at the middle portion of the left hind limb overlying the femoral artery. The femoral artery was gently isolated. First the proximal portion and then the distal portion of the femoral artery were ligated, and then other arterial branches as well as veins were dissected free and excised. The overlying skin was closed using two surgical staples. Tissue in the hypoxia area was removed and homogenized in TBST buffer 2 wk after the operation.
Cell migration assay
Bone marrow cells were detached with trypsin-EDTA, washed in serum-free medium, and then counted and adjusted to106 cells/ml. Five hundred microliters of the cell suspension was placed in the Transwell membranes and allowed to migrate to the underside for 6 h or overnight at 37°C in the presence of 50 nM BSA, HIMF, or HIMF plus 25 µM of the BTK inhibitor, LFM-A13. The cells were fixed in precooled methanol and stained with Coomassie blue solution for 10 min. The cells on the top chamber were removed with a cotton swab, and the cells migrating to the underside of the filter were visualized and photographed randomly using a Nikon Eclipse microscope. Migration cells were counted under the microscope or from the pictures.
Immunofluorescence microscopy
Bone marrow cells were cultured on coverslips in DMEM containing 10% FBS and fixed in precooled methanol for 5 min. The cells were then permeabilized with 0.2% Triton X-100 in PBS and blocked with 0.5% BSA in PBS followed by incubation with the indicated antibodies. FITC-donkey anti-rabbit IgG or FITC-donkey anti-rat IgG and rhodamine-donkey anti-mouse IgG were used as second antibodies. For transfection experiments, cells were cultured overnight and transfected with indicated constructs in serum-free medium for 4 h and then changed into DMEM containing 10% FBS overnight. Cells were fixed and stained as above. A 510 confocal microscope was used for the imaging.
Statistical analysis
All the results are expressed as mean ± SE. Differences between groups were analyzed by the Student-Newman-Keul’s method with P < 0.05 considered to be significant.
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RESULTS
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BTK was pulled down by GST-HIMF
To search for HIMF binding partners, we first constructed GST-HIMF plasmids and prepared bacterial lysate for GST and GST-HIMF protein from BL21. We then conducted an experiment to pull down the binding protein from the lysate of bone marrow primary cultured cells. As shown in Fig. 1
, GST-HIMF pulled down a protein of 
70 kDa. To identify the amino acid sequence for the HIMF-binding protein, the candidate band was cut from the gel and sent for mass spectrometry analysis. Peptide mass fingerprinting (PMF), and tandem mass spectrum data from MALDI-TOF-MS were analyzed by searching against an NCBInr database using MASCOT (Matrix Science, London, UK) search software. With the use of this approach, a protein corresponding to BTK that is involved in B cell maturation was found.
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Figure 1. BTK is a HIMF-binding molecule. GST-HIMF fusion protein and GST were incubated with glutathione-Sepharose beads and then incubated with bone marrow cell lysate, respectively, in TBST buffer at 4°C for 3 h. Beads were washed 5 times by TBST, and loading buffer was added to samples. After electrophoresis, gel was stained by Coomassie blue. Arrow shows a HIMF-binding candidate protein (A). GST-HIMF and GST were first bonded to glutathione-Sepharose and then incubated with lysate of bone marrow cells (left) or homogenate of ischemic tissue (right) at 4°C for 3 h and then washed by TBST solution 3 times. After SDS-PAGE and transfer to membrane, samples were detected by anti-BTK and anti-GST antibodies (B). Agarose-protein A/G bound rabbit IgG and rabbit anti-BTK antibody were incubated with bone marrow cell lysate respectively in TBST buffer at 4°C for 3 h and then washed by TBST 3 times before SDS-PAGE. Rabbit anti-HIMF antibody was used for immunoblot (C). RT-PCR was conducted by using cDNA prepared from bone marrow cells and from RAW cells as described in Materials and Methods. ß-actin primers were used as a control (D).
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To confirm our finding, we conducted two additional binding experiments. First, bone marrow lysate and bacteria lysates of GST and GST-HIMF were used in pull-down assays. GST-HIMF pulled down BTK from bone marrow cell lysate but GST did not. When homogenate of mouse hind limb hypoxic tissue was used instead of bone marrow cell lysate in the GST-HIMF pull-down assay, BTK was again shown to bind to GST-HIMF but not to GST. These results further indicate that BTK is a HIMF binding partner. Next, we used BTK antibody to test whether HIMF can be coprecipitated with BTK from endogenous proteins. Again, BTK antibody can pull down HIMF from bone marrow cell lysate but rabbit IgG cannot.
Colocalization between BTK and HIMF
To demonstrate that BTK acts as a HIMF binding partner, we conducted experiments to show whether BTK and HIMF colocalize together in bone marrow cells. Bone marrow cells were cultured on cover glasses and cotransfected with enhanced GFP (EGFP)-BTK and his-HIMF plasmids. The transfected cells were then fixed by methanol and used for immunofluorescence by anti-his mAb and rhodamine-labeled donkey anti-mouse IgG. As shown in Fig. 2
, BTK and HIMF colocalized in transfected bone marrow cells.
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Figure 2. Colocalization between BTK and HIMF. EGFP-BTK and His-HIMF plasmids were cotransfected into bone marrow cells. Cells were fixed by precooled methanol and stained by antihis mAb and rhodamine-labeled donkey anti-mouse IgG second antibody. EGFP-BTK and his-HIMF colocalized very well in transfected cells.
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Translocation of BTK in bone marrow cells in response to the stimulation of HIMF
BTK family tyrosine kinases have been shown to regulate actin cytoskeleton and to mediate cell mobility in response to stimulation (7
, 8)
. The involvement of BTK in thrombin-stimulated platelets (9
, 10)
suggested that BTK may be a mediator of cytoskeleton reorganization. The activation of BTK family tyrosine kinases will result in their stimulated translocation to membrane fractions (11)
. As a partner of BTK, HIMF may be involved in BTK signaling pathways and play a role in regulation of BTK activity. We therefore conducted an assay to examine whether the distribution of BTK in bone marrow cells was altered in response to the stimulation of HIMF. Bone marrow cells were cultured on coverslips in 12-well plates for 2 days and then treated with HIMF (50 nM) or BSA (50 nM) for 5 min. The cells were fixed in precooled methanol at –20°C for 10 min and used for immunocytochemistry. As the results show in Fig. 3
and Fig. 4
, HIMF was found to induce a rapid redistribution of BTK to cell processes, the leading edge of cells (Fig. 3D-L
), where BTK colocalizes with actin (Fig. 4A
) and FAK (Fig. 4B
). Fyn, a binding protein of BTK, was also found colocalized with BTK in the cell processes.
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Figure 3. BTK redistribution in response to the stimulation of HIMF in primary cultured bone marrow. Bone marrow cells were cultured on cover glass and then treated with 50 nM BSA (A–C) or HIMF (D–L) for 5 min. Cells were fixed by precooled methanol before indirect immunofluorescence. BTK or FYN, a binding partner for BTK, was redistributed in bone marrow cells after the treatment of HIMF. Outline of cell (D, E) was shown in I. DIC approach was also used to show the leading edge of cell (J–K).
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Figure 4. BTK colocalizes with FAK and actin in HIMF stimulating cells. Bone marrow cells were cultured on cover glass in 12-well plates in DMEM culture medium containing 1% FBS and then treated with 50nM HIMF for 5 min. Cells were fixed by precooled methanol at –20°C for 5–10 min. Monoclonal anti-BTK antibody and rabbit polyclonal anti-FAK (A) or antiactin (B) antibody were used for indirect immunofluorescence. Signals of BTK overlap with those of FAK and actin in cells.
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BTK was autophosphorylated by HIMF stimulation
To address whether HIMF can activate BTK, we tested the autophosphorylation of BTK in response to HIMF. Bone marrow cells expanded from mouse were stimulated with HIMF (50 nM). HIMF induced BTK autophosphorylation at site Y223 (Fig. 5
A). The tyrosine phosphorylation of BTK returned to normal after 30 min of treatment. To find out the more exact process of BTK phosphorylation, we treated the cells with HIMF in time series of 2.5, 5, 10, and 20 min. The phosphorylation of BTK reached a peak at or before 2.5 min and gradually decreased after 10 min with the treatment of HIMF. To verify if HIMF specifically activates BTK, the same cell lysates used in BTK phosphorylation experiments were used for the phosphorylation study of Fyn. As shown in Fig. 5C
, phosphorylation sites of both the activated and inactivated state of Fyn remained the same in response to the stimulation of HIMF. Therefore, HIMF is a specific stimulator for the activation of BTK. Fyn was heavily phosphorylated in bone marrow cells without any stimulation, suggesting nonspecificity.
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Figure 5. BTK self-phosphorylation in response to treatment of HIMF. Three plates of primary cultured bone marrow cells that were passed from same plate of cells were cultured to confluence and then treated with HIMF (5 and 30 min) or without HIMF. Cell lysates were used for the Western blot. Membrane was first probed by rabbit polyclonal anti-BTK phosphorylation (Y223) antibody and then was probed by anti-BTK mAb after stripping (A). Five plates of primary cultured bone marrow cells that were passed from the same plates of cells were cultured to confluence and then treated with HIMF (2.5, 5, 10, and 20 min respectively) or without HIMF. Cell lysates were used for the Western blot. The membrane was first detected by Rabbit polyclonal anti-BTK phosphorylation (Y223) antibody and then by anti-BTK mAb after stripping (B). Same samples were used for the detection of Fyn phosphorylation (C). Rabbit antiphospho-Src family (Y416) pAb or monoclonal Anti-Fyn (Y528) phospho-specific antibody were used for the first blot. Then membranes were probed by rabbit anti-Fyn poloyclonal antibody to show equal loading after stripping. Phosphorylation of Y416 up-regulates enzyme activity. Phosphorylation of Y528 negatively regulates enzyme activity.
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BTK stimulated the migration of bone marrow cells
To test whether HIMF stimulates the migration of bone marrow cells, bone marrow cells were cultured in transwells in the presence of 50 µM HIMF or BSA. The number of migrated cells was significantly increased in the membrane treated with HIMF for the overnight culture (Fig. 6
A). To demonstrate whether BTK is involved in HIMF-stimulated cell migration, cells were cultured in transwell as above in the presence of 50 µM BSA, 50 µM HIMF, or 50 µM HIMF plus 25 µM BTK inhibitor LFM-A13 for 6 h. Significantly more bone marrow cells were stimulated to migrate out of the HIMF stimulated transwell than from the transwell treated with BSA (Fig. 6B
). The migration stimulated by HIMF was blocked by the BTK inhibitor LFM-A13 (Fig. 6B
). Statistical analysis using Student-Newman-Keuls method showed a significant change (P<0.001) in the number of migrated cells after treatment with HIMF (Fig. 6C
).
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Figure 6. HIMF stimulated bone marrow cell migration; 5 x 105 bone marrow cells were cultured in transwell plates in the presence of 50 nM BSA or HIMF. Cells were cultured overnight, fixed in precooled methanol and stained by Coomassie blue solution. Cells growing on the surface of the membrane were removed after staining (A). A Nikon Eclipse microscope was used for the imaging. 5 x 105 bone marrow cells were cultured as above in the presence of 50 nM BSA, HIMF, or HIMF plus 25 µM LFM-A13, respectively. Cells were cultured for 6 h, fixed in precooled methanol, and stained by Coomasie Blue solution. Cells growing on the surface of membrane were removed after staining. Bone marrow cell migration was induced by HIMF, and this induction was inhibited by BTK inhibitor LFM-A13 (B). Migration cells as described in B were counted under a microscope and quantitated as in C (n=4). Differences between groups were analyzed by Student-Newman-Keul’s method with P < 0.001 (P<0.05 is considered to be significant).
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DISCUSSION
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BTK has been shown to be crucial in the regulation of B-cell maturation, and defects in BTK lead to X-linked agamma globulinemia in humans and X-linked immunodeficiency defect in mice (5
, 6)
. BTK family kinases play diverse roles in various cellular processes including growth, differentiation, apoptosis, cytoskeletal reorganization, and cell motility (7
8
9
10
, 12)
. The mutation of BTK resulting in immunodeficiency diseases is a good example to demonstrate the physiological importance of this kinase. In the regulation of differentiation of bone marrow hematopoietic cells into B cells, the BTK pathway is obviously necessary. Other members of the BTK family kinase have been shown to be involved in the signaling pathway of integrins that are key molecules regulating actin cytoskeleton and cell mobility (13)
.
By using bone marrow cells as the starting material in our study, we demonstrated that BTK is a HIMF binding molecule. As an inflammatory marker molecule, HIMF may likely be involved in the regulation of the immune system in response to inflammatory stimulation (3)
. A large number of studies have shown that leukocytes promote angiogenesis in inflammatory tissues by delivering vascular endothelial growth factor (VEGF) to the target sites (14)
, where vascular remodeling is important for the tissue regeneration. The activation of BTK will induce the differentiation and migration of bone marrow-derived leukocytes that may be involved in inflammatory responses in hypoxic tissues (15)
. HIMF, acting as a chemotactic molecule, may stimulate the migration of bone marrow derived cells to targeted tissue in response to tissue inflammation or hypoxia.
Pleckstrin homology domains (PH) are commonly found in eukaryotic signaling proteins. They are often involved in protein-protein interactions and target proteins to the plasma membrane. Mutations in BTK within its PH domain cause XLA in patients (5
, 6)
. HIMF was up-regulated in inflammatory or hypoxic tissues and stimulated the phosphorylation of AKT, a kinase with a PH domain, in cultured cells (1)
. Interestingly, BTK is also a PH domain containing molecule. HIMF can also stimulate the autophosphorylation of BTK in bone marrow cells (Fig. 5)
, indicating that BTK is a HIMF-targeted molecule that is activated in response to the stimulation of hypoxia or inflammatory reactions. Fyn, another soluble tyrosine kinase of src family members, was not changed in activity by the stimulation of HIMF, although Fyn was reported as a BTK-binding protein (16)
and shared common distribution with BTK in the cells (Fig. 3)
. But the redistribution of Fyn and colocalization with BTK after treatment with HIMF suggested that Fyn may be involved in the BTK signaling pathway. BTK was found to colocalize with actin and FAK at the cell process (Fig. 4)
. These results indicate that BTK is active in the mediation of cell migration in response to HIMF stimulation. The heavy phosphorylation of Fyn with or without HIMF simulation indicates that Fyn may be a common intermediate regulator in the cell signaling process. Consequently, BTK is a specific targeted molecule for HIMF binding and interaction. Cells cotransfected with GFP-BTK and His-HIMF plasmids also showed a clear colocalization of BTK and HIMF (Fig. 2)
.
When bone marrow cells were treated with HIMF, BTK was recruited to the leading edge of the cells (Figs. 3
, 4)
. This result further indicated that HIMF stimulates the migration of bone marrow cells. By using the transwell migration assay, we found that HIMF markedly stimulated bone marrow cell migration. The chemotactic characteristic of HIMF was shown to be dependent on the activation of BTK because the BTK inhibitor completely inhibited the chemokine-like function of HIMF in the bone marrow cell migration assay. Hence, HIMF stimulates the migration of bone marrow cells through the activation of BTK and is a chemotatic factor for bone marrow derived cells. The recruitment of leukocytes to the target tissues may be one of the physiological functions of HIMF. Recently, we have found that intravenously tail vein injection of HIMF in mice caused a marked increase of CD68-positive inflammatory cells in the lungs (17)
. This in vivo result is fully consistent with our hypothesis that leukocytes may be recruited to hypoxia tissues in response to HIMF.
HIMF is a secreted protein, while BTK is reported to be a soluble cytoplasmic molecule. We know that HIMF is excreted because we produce it in eukaryotic cells, extracting it from the media in which they grow (1)
. BTK is a tyrosine kinase which makes it a potential receptor-like molecule. Although there is a region on the molecule that is a possible transmembrane domain according to some programs (such as TMpred and DAS program) for membrane prediction, BTK is mostly localized in cytoplasm. BTK indeed can localize to membrane as we and others have shown, but whether it becomes integrated with the membrane or just binds to the membrane is not known. HIMF is a small cysteine rich protein. Whether it functions as a ligand extracellularly or acts as a modulator intracellularly or both also remain a puzzle. Considering that many molecules like the thiorodoxin-like molecule (18)
, and EF hand molecules (19)
can work both inside and outside of the cells and function as chemokine-like proteins, HIMF may function in a similar manner.
Received for publication May 26, 2006.
Accepted for publication December 6, 2006.
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REFERENCES
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|---|
- Teng, X., Li, D., Champion, H. C., Johns, R. A. (2003) FIZZ1/RELM, a novel hypoxia-induced mitogenic factor in lung with vasoconstrictive and angiogenic properties. Circ. Res. 92,1065-1067[Abstract/Free Full Text]
- Wagner, K. F., Hellberg, A. K., Balenger, S., Depping, R., Dodd-O, J., Johns, R. A., Li, D. (2004) Hypoxia induced mitogenic factor has antiapoptotic action and is up-regulated in the developing lung: coexpression with hypoxia-inducible factor-2. Am. J. Respir. Cell Mol. Biol. 31,276-282[Abstract/Free Full Text]
- Holcomb, I. N., Kabakoff, R. C., Chan, B., Baker, T. W., Gurney, A., Henzel, W., Nelson, C., Lowman, H. B., Wright, B. D., Skelton, N. J., et al (2000) FIZZ1, a novel cysteine-rich secreted protein associated with pulmonary inflammation, defines a new gene family. EMBO J. 19,4046-4055[CrossRef][Medline]
- Chumakov, A. M., Kubota, T., Walter, S., Koeffler, H. P. (2004) Identification of murine and human XCP1 genes as C/EBP-epsilon-dependent members of FIZZ/Resistin gene family. Oncogene 23,3414-3425[CrossRef][Medline]
- Vetrie, D., Vorechovsky, I., Sideras, P., Holland, J., Davies, A., Flinter, F., Hammarstrom, L., Kinnon, C., Levinsky, R., Bobrow, M., et al (1993) The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases. Nature 361,226-233[CrossRef][Medline]
- Thomas, J. D., Sideras, P., Smith, C. I., Vorechovsky, I., Chapman, V., Paul, W. E. (1993) Colocalization of X-linked agammaglobulinemia and X-linked immunodeficiency genes. Science 261,355-358[Abstract/Free Full Text]
- Mangla, A., Khare, A., Vineeth, V., Panday, N. N., Mukhopadhyay, A., Ravindran, B., Bal, V., George, A., Rath, S. (2004) Pleiotropic consequences of Bruton tyrosine kinase deficiency in myeloid lineages lead to poor inflammatory responses. Blood 104,1191-1197[Abstract/Free Full Text]
- Abassi, Y. A., Rehn, M., Ekman, N., Alitalo, K., Vuori, K. (2003) p130Cas Couples the tyrosine kinase Bmx/Etk with regulation of the actin cytoskeleton and cell migration. J. Biol. Chem. 278,35636-35643[Abstract/Free Full Text]
- Laffargue, M., Ragab-Thomas, J. M., Ragab, A., Tuech, J., Missy, K., Monnereau, L., Blank, U., Plantavid, M., Payrastre, B., Raynal, P., et al (1999) Phosphoinositide 3-kinase and integrin signalling are involved in activation of Bruton tyrosine kinase in thrombin-stimulated platelets. FEBS Lett. 443,66-70[CrossRef][Medline]
- Hamazaki, Y., Kojima, H., Mano, H., Nagata, Y., Todokoro, K., Abe, T., Nagasawa, T. (1998) Tec is involved in G protein-coupled receptor- and integrin-mediated signalings in Human blood platelets. Oncogene 16,2773-2779[CrossRef][Medline]
- Varnai, P., Rother, K. I., Balla, T. (1999) Phosphatidylinositol 3-kinase-dependent membrane association of the Bruton’s tyrosine kinase pleckstrin homology domain visualized in single living cells. J. Biol. Chem. 274,10983-10989[Abstract/Free Full Text]
- Hamazaki, Y., Kojima, H., Mano, H., Nagata, Y., Todokoro, K., Abe, T., Nagasawa, T. (1998) Tec is involved in G protein-coupled receptor- and integrin-mediated signalings in Human blood platelets. Oncogene 16,2773-2779[CrossRef][Medline]
- Chen, R., Kim, O., Li, M., Xiong, X., Guan, J. L., Kung, H. J., Chen, H., Shimizu, Y., Qiu, Y. (2001) Regulation of the PH-domain-containing tyrosine kinase Etk by focal adhesion kinase through the FERM domain. Nat. Cell Biol. 3,439-444[CrossRef][Medline]
- Scapini, P., Morini, M., Tecchio, C., Minghelli, S., Di Carlo, E., Tanghetti, E., Albini, A., Lowell, C., Berton, G., Noonan, D. M., et al (2004) CXCL1/macrophage inflammatory protein-2-induced angiogenesis in vivo is mediated by neutrophil-derived vascular endothelial growth actor-A. J. Immunol. 172,5034-5040[Abstract/Free Full Text]
- Jefferies, C. A., O’Neill, L. A. (2004) Bruton’s tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling?. Immunol. Lett. 92,15-22Review.[CrossRef][Medline]
- Alexandropoulos, K., Cheng, G., Baltimore, D. (1995) Proline-rich sequences that bind to Src homology 3 domains with individual specificities. Proc. Natl. Acad. Sci. U. S. A. 92,3110-3114[Abstract/Free Full Text]
- Yamaji-Kegan, K., Su, Q., Angelini, D. J, Champion, H. C., Johns, R. A. (2006 Aug 4) Hypoxia-induced mitogenic factor has pro-angiogenic and pro-inflammatory effects in the lung via VEGF and VEGF receptor-2. Am. J. Physiol. Lung Cell Mol Physiol. [Epub ahead of print]
- Bertini, R., Howard, OM., Dong, HF., Oppenheim, JJ., Bizzarri, C., Sergi, R., Caselli, G., Pagliei, S., Romines, B., Wilshire, JA., et al (1999) Thioredoxin, a redox enzyme released in infection and inflammation, is a unique chemoattractant for neutrophils, monocytes, and T cells. J. Exp. Med. 189,1783-1789[Abstract/Free Full Text]
- Xu, K., Geczy, C. L. (2000) IFN-gamma and TNF regulate macrophage expression of the chemotactic S100 protein S100A8. J. Immunol. 164,4916-4923[Abstract/Free Full Text]
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ABSTRACT
INTRODUCTION
