ARM-1 mRNA was found to be differentially expressed in endothelial cell lines of various tissue origin and lymphocyte cell lines.. Overexpression of ARM-1 in skin endothelial cells incre
Trang 1protein on endothelial cells
Nathalie Lamerant and Claudine Kieda
Centre de Biophysique Mole´culaire, CNRS UPR, Orle´ans Cedex, France
To fight infection, lymphocytes must continuously
cir-culate through the body to maximize the opportunity
to recognize their cognate antigen Therefore they
cir-culate from the blood into tissues Unlike naive cells
which circulate through secondary lymphoid organs
(e.g spleen, lymph nodes and Peyer’s patches),
activa-ted lymphocytes also circulate in nonlymphoid tissues
and show remarkable selectivity in their homing [1–3]
Homing is a highly regulated, tissue-specific
mechan-ism A multistep model has been proposed for this
pro-cess [4,5], and numerous adhesion molecules involved
in this cascade have been identified, such as selectins,
integrins and, more recently, chemokines [6–8] The
molecular mechanisms behind the selectivity are
start-ing to be characterized Differential expression of
chemokines probably plays a key role in this selectivity
[9–12], but we hypothesize the existence of additional
adhesion molecules involved in the first steps of the cascade, which confer specificity of recognition between lymphocytes and endothelial cells [13,14]
As a tool to determine the molecular basis of endo-thelial selectivity, microvascular endoendo-thelial cell lines
of distinct tissue origin were established [13–15] Endothelial cells isolated from lymphoid tissues (lymph nodes and appendix) and from nonlymphoid immune sites were immortalized Their general endothelial char-acteristics, such as the presence of von Willebrand fac-tor, angiotensin-converting enzyme, VE-cadherin and the intracellular E-selectin, were preserved These cell lines display phenotypic characteristics related to their tissue of origin, as the expression of mucosal or peripheral lymph nodes addressins [15] They also showed specific expression of sugar receptors depend-ing on their tissue of origin [13,14] These cell lines are
Keywords
adhesion-regulating molecule-1 (ARM-1); cell
adhesion; endothelium; organospecificity
Correspondence
C Kieda, Centre de Biophysique
Mole´culaire, CNRS UPR, 4301 Rue Charles
Sadron, 45071 Orle´ans Cedex 02, France
Tel ⁄ Fax: +33 2 38 25 55 61
E-mail: kieda@cnrs-orleans.fr
(Received 21 October 2004, revised 1
February 2005, accepted 14 February 2005)
doi:10.1111/j.1742-4658.2005.04613.x
Numerous adhesion molecules have been described, and the molecular mechanisms of lymphocyte trafficking across the endothelium is starting to
be elucidated Identification of the molecules involved in the organoselec-tivity of this process would help in the targeting of drug therapy to specific tissues Adhesion-regulating molecule-1 (ARM-1) is an adhesion-regulating molecule previously identified on T cells It does not belong to any known families of adhesion molecules In this study, we show the presence of ARM-1 in endothelial cells, the adhesion partners of lymphocytes ARM-1 mRNA was found to be differentially expressed in endothelial cell lines of various tissue origin and lymphocyte cell lines Interestingly, ARM-1 is absent from skin endothelial cells In our assay, skin endothelial cells dis-play a distinct capacity to mediate adhesion of activated T lymphocytes Overexpression of ARM-1 in skin endothelial cells increased adhesion of CEMT4 and NK lymphocytes, confirming that ARM-1 also regulates adhesion in endothelial cells We also show that ARM-1 is a cytosolic protein associated with the plasma membrane However, no cell surface expression of the protein was observed These results suggest an indirect role of ARM-1 in adhesion rather than a direct role as an adhesion mole-cule itself
Abbreviations
ARM-1, adhesion-regulating molecule-1; HEC, high endothelial cell; HSkMEC, human skin microvascular endothelial cell; PBSc, phosphate-buffered saline, supplemented with 1 mm CaCl 2 and 0.5 mm MgCl 2
Trang 2therefore a good model for studying endothelium
or-ganospecificity
To better characterize the molecules responsible for
endothelial cell specificity, we used the differential
dis-play method [16] to compare gene expression between
two endothelial cell lines from lymphoid organs:
per-ipheral lymph nodes and mucosal (Peyer’s patches)
tis-sues In this way, we highlighted adhesion-regulating
molecule-1 (ARM-1) protein, an adhesion-regulating
molecule previously identified on T cells [17] We
found that ARM-1 was widely expressed in endothelial
cells from various tissues except skin This was
inter-esting, as skin endothelial cells, in our assay, showed
a small capacity to mediate adhesion of activated
T lymphocytes (CEMT4 cells) ARM-1 was also found
differentially expressed in various lymphocyte cell lines,
independently of their T or B lineage In this study, we
also attempted to elucidate the role of ARM-1 in the
lymphocyte homing mechanism We found that
ARM-1 is a secreted, probably unglycosylated protein, which
may be associated with the cell membrane We also
show that ARM-1 overexpression in skin endothelial
cells increases lymphocyte adhesion
Results
Differential display
To identify new molecules responsible for high
endo-thelial cell (HEC) specificity, a differential display
method was used to compare two immortalized HEC
lines, one from mouse peripheral lymph nodes
(HECa10) and the other from mouse Peyer’s patches
(HECpp) Analysis of differentially expressed mRNAs
in HECa10 compared with the HECpp cell line, using
12 different combinations of primers, revealed six
HECpp-specific cDNA fragments and four
HECa10-specific cDNA fragments The cDNA fragments were
cloned, sequenced, and compared with database listed
sequences using the blastn program Two cDNA
frag-ments exclusively present in Peyer’s patch HECs
shared the same sequence and had 100% homology
with the ARM-1 gene Interestingly, ARM-1 is
involved in cell adhesion but has no homologous
sequence with previously known families of adhesion
molecules It was originally discovered on T cells [17],
whereas we identified this molecule in endothelial cells
Differential expression of ARM-1, analyzed
by semiquantitative RT-PCR
To study the expression of ARM-1 mRNA in various
endothelial and lymphocyte cell lines, semiquantitative
RT-PCR was used The cDNA of interest was coam-plified with an actin cDNA fragment as an internal control ARM-1 is differentially expressed in endothel-ial cells from various organs according to their tissue
of origin (Fig 1) We could not confirm the results from differential display, as ARM-1 mRNA was also observed in mouse peripheral lymph nodes HECs (HECa10) We noticed the absence of ARM-1 mRNA from endothelial cells from skin [human skin micro-vascular endothelial cells (HSkMECs)] To confirm this result, primary endothelial cells from human skin were isolated as described previously [13] No ARM-1 mRNA was detected (Fig 2A)
Expression of ARM-1 mRNA was also studied in different mouse and human lymphocyte cell lines (Fig 2B) The ARM-1 expression pattern was very dif-ferent according to the cell line It seems there is no link with T or B lineage of the cells, as ARM-1 mRNA was present in NKL1, EL4 and EL4-IL2
T cells and Raw 8.1 B cells but in neither CEMT4 nor NKL2 T cells
Skin endothelial cells showed a small capacity to mediate adhesion of the CEMT4 lymphocyte cell line
ARM-1 Actin
HSkMECHBrMECHUVEC HIMECHPLNEC B3 Mark
er HMLNEC Negati
ve control HSpMEC HLMECHECa10HECpp
0.8 0.6 0.4 0.2 0
mRNA units ARM-1/Actin
Endothelial cell lines
HSkMEC HBrMEC HUVEC HIMEC HPLNEC B3 HMLNEC HSpMEC HLMEC HECa10 HECpp
A
B
Fig 1 Differential expression of ARM-1 mRNA in endothelial cell lines from various tissues, analyzed by semiquantitative RT-PCR ARM-1 cDNA was coamplified by RT-PCR with an actin cDNA frag-ment as an internal control Reaction products were resolved on 1% agarose gel (A) and quantified using the IMAGEQUANT 5.1 pro-gram (Molecular Dynamics) The mRNA units represent signal intensity as assessed by densitometric analysis after normalization against actin (B).
Trang 3(Fig 3) We suggest that there is a correlation between
the absence of ARM-1 in skin endothelial cells and their
weak adhesive activity for CEMT4 lymphocytes We
know that ARM-1 promotes adhesion when it is
over-expressed in the endothelial cell partners (the
lympho-cytes) [17] However, we do not know if ARM-1 is able
to play the same role in endothelial cells
ARM-1 promotes lymphocyte adhesion
The potential role of ARM-1 in lymphocyte adhesion
was studied by comparing adhesion properties of
ARM-1-nonexpressing cells before and after
transfec-tion with ARM-1 cDNA The assays were carried out
with transiently transfected COS cells, which do not
possess the mRNA for ARM-1 (data not shown), and
transfected HSkMECs after sorting by flow cytometry
The adhesion assays were quantified by flow
cytomet-ric analysis The lymphocytes used for the adhesion
assays were T lymphocytes (CEMT4) and NK cells
(NKL1 and NKL2) which display characteristic
recruitment during the primary as well as secondary
immune responses
Western blot analysis of HSkMECs and COS cells transiently transfected with pcDNA-ARM-1 and pIRES-hrGFP-ARM-1 vectors, respectively, showed a single protein band at 50 kDa (Fig 4), which is comparable to the 54 kDa reported by Simins et al [17] Just below this band was observed another wea-ker protein band, which corresponds to the predicted size (42 kDa) of ARM-1 protein before post-transla-tional modifications
Static adhesion assays on transiently transfected COS cells were carried out at various temperatures, incubation times and lymphocyte⁄ adherent cell ratios Results are shown in Fig 5 Whatever the conditions,
ARM-1
Actin
Primary
ski
n EC
Mar
ker HPL NEC B3
ARM-1
Actin
EL4 EL4-I
L2
A
B
Fig 2 ARM-1 mRNA expression in primary skin endothelial cells
(A) and in various lymphocyte cell lines (B), analyzed by
semiquanti-tative RT-PCR (A) HPLNEC B3 was used as a positive control for
the PCR amplification of ARM-1 in human primary skin endothelial
cells (B) EL4 and EL4-IL2 are mouse activated T lymphocytes,
NKL1 and NKL2 are human natural killer cells, CEMT4 are human
CD4+ T-cell line and Raw 8.1 are mouse B lymphocytes.
Fig 3 Adhesion of CEMT4 lymphocytes to endothelial cell lines from various tissues CEMT4 lymphocyte adhesion to endothelial cells was analyzed after a 20 min incubation at room temperature with a 5 : 1 lymphocyte ⁄ endothelial cell ratio Lymphocyte adhe-sion was determined as described in Experimental procedures Val-ues are the mean of triplicate measurements, and error bars were calculated from one representative experiment out of three.
Fig 4 Expression of ARM-1 protein in transfected COS (A) and skin endothelial (B) cells COS cells (lane 3) and skin endothelial cells (lane 5) were transfected by the pIRES-hrGFP-ARM-1 vector.
As a negative control, COS cells (lane 1) and skin endothelial cells (lane 4) were transfected by the empty vector ARM-1 was immu-noprecipitated 48 h after transfection and detected by western blot-ting using Flag antibodies and the Western blue stabilized substrate for alkaline phosphatase (Promega) A size marker is shown on lanes 2 and 6.
Trang 4we observed an increase in CEMT4 lymphocyte
adhe-sion on transfected COS cells The largest relative
increase was obtained after a 40 min incubation of
lymphocytes and transfected COS cells (10 : 1 ratio) at
4C It is remarkable that efficiently transfected COS
cells represented 10% of the total population
Conse-quently, the increase in adhesion reaches 92% relative
to basic adhesion to COS cells The increase in
adhe-sion obtained at 37C was not as large as for mock
transfected COS cells, which bound CEMT4
lympho-cytes more efficiently than at 4C Indeed, at 37 C,
various adhesion molecules are induced, thus
increas-ing the background level
After transfection of skin endothelial cells with the
pIRES-hrGFP-ARM-1 vector, nontransfected and
transfected HSkMECs were sorted by FACS Diva
cytometer Static adhesion assays with various lympho-cyte cell lines were carried out on the sorted skin endothelial cell populations The results are shown in Fig 6 An RT-PCR analysis confirmed the absence
of ARM-1 mRNA in the subpopulation of nontrans-fected HSkMECs and its presence in the different sub-populations of transfected HSkMECs (Fig 6A) A slight increase in CEMT4 lymphocyte adhesion was observed on transfected cells compared with nontrans-fected cells (Fig 6B) Overexpression of ARM-1 in HSkMECs significantly increases adhesion of NKL1 lymphocytes (Fig 6C) but not of NKL2 lymphocytes, the adhesion level of which did not change (Fig 6D) These results are interesting as NKL1 lymphocytes constitutively express ARM-1 mRNA in contrast with CEMT4 or NKL2 lymphocytes (Fig 2B)
The static adhesion assay was also performed with human primary peripheral leukocytes from normal donors, on ARM-1-transfected or mock-transfected skin endothelial cells (Fig 7) As shown, leukocyte adhesion to ARM-1-transfected HSkMECs was greatly increased compared with the controls This large increase clearly shows the adhesion-regulating proper-ties of ARM-1
ARM-1 is a secreted and cell-associated protein
As ARM-1 protein has a putative signal peptide at the N-terminus, we investigated whether it was a secreted protein Sorted skin endothelial cells expressing Flag-tagged ARM-1 protein were used Twenty four hours after cell seeding, the medium was removed and fresh medium added to the cells After 3 days, the culture supernatant was collected and the cells were detached from dishes by scraping The cells were growing expo-nentially and no dead cells were detected Samples collected from these two fractions were subjected to immunoprecipitation followed by western blot analysis using Flag antibodies ARM-1 was detected in cells (total cell lysate) and in the conditioned cell culture medium (medium) but not in fractions from the mock vector transfected cells (Fig 8A) This shows that ARM-1 is a cell-associated protein that can be secreted
ARM-1 is a membrane-associated protein
As the majority of expressed ARM-1 protein appears
to be cell-associated (Fig 8A), we next determined its subcellular distribution by biochemical fractionation Sorted skin endothelial cells expressing Flag-tagged ARM-1 proteins were lysed in hypotonic buffer, and low and high speed centrifugation were performed
to obtain a membrane fraction and a cytoplasmic
A
B
Fig 5 CEMT4 lymphocyte adhesion induced by ARM-1 expression
in COS cells COS cells were transiently transfected with the
pcDNA-ARM-1 vector (gray bars) or with the pcDNA3.1 ⁄ Myc-His
empty vector (black bars) CEMT4 lymphocyte adhesion to
trans-fected COS cells was analyzed at 4 C (A) or 37 C (B) at two
dif-ferent lymphocyte ⁄ COS cell ratios (5 : 1 and 10 : 1) and two
different incubation times (20 and 40 min) Lymphocyte adhesion
was determined as described in Experimental procedures, 48 h
after transfection Values are the mean of triplicate measurements,
and error bars were calculated from one representative experiment
out of two.
Trang 5fraction Subcellular distribution of ARM-1 protein
was monitored by anti-Flag immunoprecipitation and
immunoblotting As shown in Fig 8B, ARM-1 protein
was partitioned into the membrane and the cytosolic
fractions
ARM-1 distribution was analysed by
immuno-fluorescence microscopy Skin endothelial cells were
transiently transfected with the pires-hrGFP-ARM-1 vector ARM-1 expression was followed 48 h after cell transfection, by immunofluorescence detection using Flag antibodies (Fig 9)
Fluorescence confocal microscopy analysis of perme-abilized transfected cells revealed ARM-1 to be a cyto-solic protein (Fig 9B) However, sometimes it was found beneath the plasma membrane (Fig 9C), and was therefore probably membrane associated In non-activating conditions, no ARM-1 molecules were expressed on the plasma membrane surface, as
observ-ed with nonpermeabilizobserv-ed transfectobserv-ed cells (Fig 9D) The latter was confirmed by a cell surface biotinylation experiment and FACS analyses Activation with tumor necrosis factor a, interferon c, lipopolysaccharide or histamin did not result in any noticeable change in the
Fig 7 Leukocyte adhesion induced by ARM-1 expression in skin endothelial cells HSkMECs were transfected with the pIRES-hrGFP-ARM-1 vector or the pIRES-hrGFP empty vector Leukocyte adhesion to FACS-sorted transfected HSkMECs was analyzed at
37 C with a 5 : 1 leukocyte ⁄ endothelial cell ratio and a 30 min incubation Leukocyte adhesion was determined as described in Experimental procedures Values are the mean of duplicate meas-urements, and error bars were calculated from one experiment.
ARM-1
Actin
NT sub popTr sub pop 1Tr sub pop 2Tr sub pop 3Mark
er
A
B
C
D
Fig 6 Lymphocyte adhesion induced by ARM-1 expression in skin endothelial cells Skin endothelial cells (HSkMECs) were transiently transfected with the pIRES-hrGFP-ARM-1 vector After transfection, nontransfected and transfected HSkMECs were sorted by FACS Diva cytometer Expression of ARM-1 mRNA in the sorted popula-tions was tested by semiquantitative RT-PCR (A) (NT sub pop, non-transfected sorted subpopulation; Tr sub pop, non-transfected sorted subpopulation) NT cells (black bars) and Tr cells (gray bars) were submitted to static adhesion assays with CEMT4 (B), NKL1 (C) or NKL2 (D) cells Lymphocyte adhesion was analyzed at 37 C for
30 min at a 5 : 1 lymphocyte ⁄ endothelial cell ratio Adhesion rate was determined as described in Experimental procedures Values for adhesion to transfected cells were normalized against the value for nontransfected cells Values are the mean of triplicate measure-ments, and error bars were calculated from one representative experiment out of two.
Trang 6subcellular localization of ARM-1 in transfected skin
endothelial cells (data not shown) The absence of
ARM-1 expression on the cell surface was also
con-firmed by transiently transfected COS cells with the
pires-hrGFP-ARM-1 or the pCMV-ARM-1 vector
encoding the ARM-1 protein fused to a Flag tag at the
C-terminus and a Myc tag at the N-terminus,
respect-ively In the same way, ARM-1 was not observed on
the plasma membrane surface of COS cells transfected
with the C-terminus Flag tag or the N-terminus Myc
tag plasmid (data not shown)
ARM-1 is not N-glycosylated ARM-1 expressed in skin endothelial cells appears to
be 50 kDa, slightly larger than the 42 kDa predicted size of full-length ARM-1 Because ARM-1 possesses two putative N-linked glycosylation motifs and several putative O-linked glycosylation motifs [17], we hypo-thesized that it was subject to post-translational glyco-sylation Thus, we investigated whether cell treatment with tunicamycin, an inhibitor of N-glycosylation, or a-benzyl-GalNAc, an inhibitor of O-glycosylation, would affect the molecular size of the protein (Fig 10A) Tunicamycin treatment did not modify the molecular size, indicating that ARM-1 is not N-glycosylated a-Benzyl-GalNAc treatment also did not affect the molecular size, but we cannot conclude the absence of O-glycosylated motifs, as a-benzyl-GalNAc is not a total inhibitor of O-glycosylation Furthermore, a-ben-zyl-GalNAc was highly toxic to the endothelial cell culture, preventing long-term culture
Direct enzymatic deglycosylating treatment was applied to the immunoprecipitated ARM-1 protein, using N-glycanase, sialidase A, b-1,4-galactosidase, b-N-acetylglucosaminidase and O-glycanase These enzymes remove the most common N-linked and O-linked oligosaccharides Global treatment of ARM-1 with these enzymes did not affect its molecular size on migration in polyacrylamide gel (Fig 10B) N-Glyca-nase removes almost all N-linked oligosaccharides so
we can conclude the probable absence of N-glycosyla-tion of ARM-1, confirming the result of tunicamycin treatment Enzymatic treatments to remove O-glycosyl-ated structures are less global, and several enzymes
pIRES-hrGFP
pIRES-hrGFP ARM-1
pIRES-hrGFP ARM-1
Permeabilized cells
Permeabilized cells
30 µm
30 µm
30 µm
30 µm
Non permeabilized cells
neutral GFP ARM-1 superposition
A
B
C
D
Fig 9 ARM-1 is a cytosolic protein that can be associated with the plasma membrane Skin endothelial cells were transiently transfected with the pIRES-hrGFP (A) or the pIRES-hrGFP ARM-1 (B, C, D) vector Then 48 h after transfection, expression of ARM-1 protein was ana-lyzed by immunofluorescence microscopy using mouse anti-Flag Igs revealed in red fluorescence by anti-mouse tetramethylrhodamine iso-thiocyanate-conjugated secondary IgG The green fluorescence observed was due to the green fluorescent protein coexpressed with ARM-1 protein in the transfected cells ARM-1 expression studies were carried out on permeabilized (A, B, C) and nonpermeabilized (D) cells.
A
B
Fig 8 ARM-1 is a secreted protein and can be associated with the
membrane Skin endothelial cells were transiently transfected with
the pIRES-hrGFP or the pIRES-hrGFP ARM-1 vector Then 48 h
after transfection, ARM-1 protein was immunoprecipitated using
mouse antibodies to Flag, and its expression was analyzed by
western blotting in the conditioned culture mediums compared
with the total cell lysates (A) and in the different subcellular
frac-tions (B) M, Size marker.
Trang 7need to be used However, sialidase A,
b-1,4-galacto-sidase, b-N-acetylglucosaminidase and O-glycanase
treatment did not modify the molecular size of
ARM-1 Certain O-linked structures are resistant to
these enzymes, so we cannot confirm that ARM-1 is
not O-glycosylated
Discussion
Lymphocyte trafficking is a highly regulated and
tis-sue-specific mechanism in which endothelium plays a
critical role Identification of the molecules involved in
endothelium organoselectivity would help us to target
drug treatments to specific tissues, particularly
anti-tumor treatments
To identify new molecules involved in endothelial
cell specificity, we used the differential display method
of gene expression to compare two immortalized HEC
lines, one from mouse peripheral lymph nodes and the
other from mouse Peyer’s patches In this way, we
highlighted the ARM-1 protein Simins et al [17]
des-cribed ARM-1 as a novel cell adhesion-promoting
receptor expressed on lymphocytes, the expression of
which is up-regulated in metastatic cancer cells This
protein does not belong to any of the known families
of cell adhesion molecules Homologous proteins are
present in species as different as human (110-kDa anti-gen, isolated from gastric carcinoma cells) [18,19], rat [20], chicken, Xenopus laevis [21,22], Drosophilia melanogaster, Arabidopsis thaliana and Caenorhabditis elegans
In this study, we show for the first time the presence
of ARM-1 in endothelial cells It was found to be differ-entially expressed in endothelial cell lines according to their tissue of origin Interestingly, ARM-1 is absent in endothelial cells from skin This result was confirmed by the same analysis on primary skin endothelial cells Skin endothelial cells, in our assay, showed a weak capacity to mediate adhesion of CEMT4 lymphocytes
To study the potential link between the absence of ARM-1 in skin endothelial cells and their weak adhe-sion activity for CEMT4 lymphocytes, ARM-1 was expressed in COS cells (which do not express this protein) and in skin endothelial cells CEMT4 lympho-cyte adhesion to ARM-1-transfected COS cells was increased by up to a factor two Overexpression of ARM-1 in skin endothelial cells significantly increased NKL1 lymphocyte adhesion and more weakly CEMT4 lymphocyte adhesion On the other hand, no change in NKL2 adhesion was observed Simins et al [17] showed that ARM-1 promoted cell adhesion when overexpressed in lymphocytes Here, we show that ARM-1 promoted cell adhesion when overexpressed in the lymphocyte adhesion partners, the endothelial cells, and moreover in a selective way The latter observa-tion and the specific expression pattern of ARM-1 sug-gest a very selective role for this protein We show in particular the presence of ARM-1 in NKL1 cells and its absence in NKL2 cells NKL1 and NKL2 cells were established from the peripheral blood of two different patients with large granular lymphocyte (LGL) leuke-mia NKL2 cells, as opposed to NKL1 cells, require interleukin-2 (IL2) to grow, but IL2 treatment did not influence ARM-1 expression (data not shown) The differences between the two NK clones in terms of sus-ceptibility to IL2 activation and IL2 dependency for growth and killing activity [23] reflect the differences in gene expression during tumor clonal selection and pro-gression In the same way, Simins et al [17] showed overexpression of ARM-1 in metastatic cancer cells compared with nonmetastatic ones, leading us to hypo-thesize that ARM-1 expression could be related to tumor dissemination The direct demonstration of ARM-1 as an adhesion-regulating molecule was pro-vided by the human peripheral leukocyte adhesion studies Indeed, the data clearly indicate that, when the cells expressed ARM-1, leukocyte adhesion was increased by 70%, which is a large difference com-pared with the increase observed with some cell lines
Transfected Transfected
No treatment
Tunicamycin
α-benzyl-GalNAc
ARM-1
+
A
B
Fig 10 ARM-1 is not a N-glycosylated protein (A) COS cells and
skin endothelial cells were transiently transfected with the
pIRES-hrGFP ARM-1 vector and cultured for 48 h in the presence of
10 lgÆmL)1tunicamycin as N-glycosylation inhibitor or 3 m M
a-ben-zyl-GalNAc as O-glycosylation inhibitor Glycosylation inhibitors
were added to the cells 6 h after transfection ARM-1 was then
immunoprecipitated and analyzed by western blotting (B)
Enzymat-ic deglycosylation treatment was performed on the ARM-1 protein,
immunoprecipitated from transiently transfected skin endothelial
cells Bovine fetuin was used as a positive control for the
enzy-matic treatment.
Trang 8and comparable to the NKL1 behavior This suggests
that ARM-1 may select a subpopulation of human
peripheral blood leukocytes
In this study, we also determined the cellular
local-ization of ARM-1 Analysis of the ARM-1 amino-acid
sequence with separate algorithms did not reveal any
transmembrane region However, subcellular
fraction-ation analysis showed its presence in both the cytosolic
and membrane fractions The same observation was
made for Xoom, the homologous protein of ARM-1 in
Xenopus [22] ARM-1 can probably be associated with
the plasma membrane We also showed that ARM-1
can be secreted However, our data, as well as those of
Simins et al [17] using C-terminal tagging of ARM-1,
did not allow us to make firm conclusions about the
presence of the protein on the outer membrane, unlike
the human and Xenopus ARM-1 homologous proteins
This behavior may be due to a loose association of the
secreted protein with the outer membrane Even
though the only means of detecting external ARM-1
was by using beads coated with Tag antibodies to label
cells growing as a monolayer, the literature that
des-cribes ARM-1 homologous proteins as membrane
pro-teins deals with either transformed (cancerous) [18] or
embryonic [21] cells, thus representing very particular
contexts
Tunicamycin treatment of cell culture and
N-glyca-nase treatment of ARM-1 failed to show any
N-glycos-ylated oligosacharides on ARM-1, despite the presence
of two potential N-glycosylation sites in its sequence
In most cases, cytosolic proteins, as ARM-1 was
mainly observed to be, are not N-glycosylated but can
be O-glycosylated [24] Enzymatic treatment did not
reveal any O-glycans on ARM-1, despite numerous
potential O-glycosylation sites, particularly in the
cen-tre of its sequence However, we cannot confirm their
absence, as they are more difficult to remove than
N-glycans ARM-1 may also only have O-linked
b-N-acetylglucosamine motifs, which are very
abun-dant modifications of cytosolic proteins [25–26] which
do not change the molecular mass of proteins as much
as complex glycans Interestingly, the human
homolog-ous protein of ARM-1 has a molecular mass of
110 kDa, which is very much higher than the predicted
42 kDa [18,19] The expression of this protein was
studied in human gastric carcinoma cells Abnormal
glycosylation is often observed in the pathological
state, in particular in cancer [27] If the glycosylation
state of ARM-1 is different in tumors, this again
suggests an important role for ARM-1 in disease
pro-gression
To summarize, these results give us new insights into
ARM-1 function The fact that ARM-1 is present in
some cell lines and absent from others and that its overexpression in endothelial cells mediates lympho-cyte adhesion with preferential activity for some lym-phocyte cell lines and⁄ or leukocyte subpopulations indicates a specific role for this protein in lymphocyte homing At this time, the mechanism by which
ARM-1 mediates adhesion in lymphocytes and endothelial cells is not known ARM-1 is mainly expressed in cyto-sol but also appears as a membrane-associated protein This suggests an indirect role in adhesion as a signal-transducing molecule rather than a direct role as an adhesion molecule itself
It is certain that ARM-1 plays an important role in cell adhesion, as confirmed by its up-regulation in metastatic mammary tumors [17] To determine its pre-cise function, it would be interesting to know whether
it is involved in the classic adhesion cascade [4,5]
Experimental procedures
Cell culture and RNA isolation
All organospecific endothelial cell lines were established in the laboratory from tissue biopsy specimens (Kieda et al [15]; CNRS patent No 99–16169) and were the following: HECa10 (mouse peripheral lymph nodes HEC clone a10), HECpp (mouse Peyer’s patch HECs), HSkMEC (human skin microvascular endothelial cells), HBrMEC (human brain microvascular endothelial cells), HUVEC (human umbilical vein endothelial cells), HIMEC (human intestine mucosal endothelial cells), HPLNEC B3 (human peripheral lymph nodes endothelial cells clone B3), HMLNEC (human mesenteric lymph nodes endothelial cells), HSpMEC (human
(human lung microvascular endothelial cells), HAPEC (human appendix endothelial cells), HOMEC (human ovary microvascular endothelial cells)
Their general endothelial characteristics, such as the presence of von Willebrand factor, angiotensin-converting enzyme, VE-cadherin, and the intracellular E-selectin, were preserved Despite their immortalization, these cell lines dis-play phenotypic characteristics related to their tissue origin [13–15]
The murine and human endothelial cells were cultured at
with Glutamax-1 (Invitrogen, Cergy Pontoise, France) sup-plemented with 2% fetal bovine serum, 0.2% fungizone and 0.4% gentamicin
Human CEMT4, NKL1, NKL2 and mouse EL4 (ATCC TIB-39, Promochem, Molsheim, France), EL4-IL2 (ATCC TIB-181), and Raw 8.1 (ATCC TIB-50) lymphoid cell lines were cultured in the same conditions as the endothelial cells CEMT4 are human leukemic CD4+ T-cells, provided
by P Olivier, Institut Pasteur, Paris, France EL4 and
Trang 9EL4-IL2 are mouse activated T lymphocytes, NKL1 and
NKL2 are human natural killer cells, kindly provided by
S Chouaib, U487 INSERM IGR, Villejuif, France and
Raw 8.1 are mouse B lymphocytes
NKL1 and NKL2 cell lines were established from the
peripheral blood of two different patients with large
gran-ular lymphocyte (LGL) leukemia, as described elsewhere
[28] The NKL2 clone, but not the NKL1 clone, requires
Peripheral leukocytes were isolated from normal blood
sam-ples by Ficoll centrifugation and erythrocyte hypotonic lysis
COS-7 cells (ATCC CRL-1651) were grown in
Dul-becco’s modified Eagle’s medium (Invitrogen) supplemented
with 10% fetal bovine serum, 2 mm Glutamax-1, 1 mm
streptomycin
Total RNA was isolated using the RNeasy Mini Kit
from Qiagen To remove any trace of DNA, RNA was
treated with DNase I using the Message Clean Kit from
GenHunter (Nashville, TN, USA)
Differential display PCR
Analysis of differential mRNA expression was performed
using an RT-PCR with arbitrary primers For the reverse
transcriptase reaction, a 20-lL reaction mixture containing
0.2 lg total RNA from HECa10 or HECpp, 40 U RNase
inhibitor (Ambion, Huntingdon, UK), 10 mm dithiothreitol,
dNTPs, 0.2 lm oligo(dT) primers and 200 U Moloney
mu-rine leukemia virus reverse transcriptase (Invitrogen) was
then chilled on ice
The oligo(dT) primer was H-T11G (5¢-AAGCTTTTTTT
TTTTG-3¢), H-T11A (5¢-AAGCTTTTTTTTTTTA-3¢) or
H-T11C (5¢-AAGCTTTTTTTTTTTC-3¢) from GenHunter
(Nashville, TN, USA) To perform PCR, 1 lL of the cDNA
po-lymerase (Invitrogen) With the use of a thermal cycler, all
pri-mers included in the PCR were one of the three oligo(dT)
primers used for the RT reaction with one of the following
arbitrary primers from GenHunter: H-AP1 (5¢-AAGC
H-AP3 (5¢-AAGCTTTGGTCAG-3¢) or H-AP8 (5¢-AAGC
TTTTACCGC-3¢) So it represented 12 different
combina-tions of PCRs
The PCR products were separated by electrophoresis on
run for 2–3 h at 2000 V, transferred to filter paper, and
autoradiographed
Cloning and sequencing
DNA fragments from HECa10 and HECpp were then compared Bands unique to HECa10 or HECpp were gel purified, cloned using the TA Cloning Kit (Invitrogen), sequenced by the MWG Biotech Company (Germany), and compared in the database using the blastn pro-grams
Semiquantitative RT-PCR
Semiquantitative RT-PCR was performed with the Quan-tum RNA b-actin Internal Standards Kit (Ambion) accord-ing to the manufacturer’s instructions To amplify the control target (actin) at a level roughly similar to our gene
competim-ers was 2 : 8 The primer used for the RT reaction was an
PCR were PPDD1F (5¢-AGGAAGCTTTATATGGTGG
CGAGGCCTCATGGCCCTGCCGG-3¢) giving a PCR product of 801 bp Twenty amplification cycles were per-formed Reaction products were resolved on a 1% agarose gel and quantified using the ImageQuant 5.1 program (Mole-cular Dynamics, Amersham Biosciences, Orsay, France)
Plasmid construction
The full-length ARM-1 cDNA was obtained by RT-PCR from murine Peyer’s patch HEC RNA and introduced into
PCR was carried out with the following sense oligonucleo-tide carrying an HindIII site, 5¢-ATCAAGCTTATGA CGACTTCAGGCGCTCTG-3¢, and the following anti-sense oligonucleotide carrying a XhoI site, 5¢-ATGCTC GAGGTCTAGACTCATATCTTCTTCTTC-3¢
PCR product was sequenced by the MWG Biotech Com-pany (Germany) confirming that no error had been intro-duced
The pcDNA-ARM-1 vector was used to introduce the ARM-1 cDNA into the pCMV Tag 3B vector (Stratagene, Amsterdam, the Netherlands), using the HindIII and XhoI restriction sites, in order to express the ARM-1 protein with an N-terminus Myc tag The pCMV-ARM-1 vector was used to introduce the ARM-1 cDNA in the pIRES-hrGFP-1a (Stratagene) by using the BamHI and XhoI restriction sites
Transfections and glycosylation inhibition experiments
Cells were plated 1 day before transfection into 24-well plates (Falcon; Becton-Dickinson, Grenoble, France) for adhesion assays, or on round glass slides in four-well
Trang 10plates for immunofluorescence microscopy Cells were
tran-siently transfected with the pCMV-ARM-1 or the
pIRES-hrGFP-ARM-1 expression vector using Lipofectamine Plus
(Invitrogen) for COS cells or Lipofectin (Invitrogen) for
endo-thelial cells, according to the manufacturer’s instructions
Adhesion assays and immunofluorescence detection were
performed 48 h after transfection
Skin endothelial cells (HSkMECs) transfected with the
pIRES-hrGFP-ARM-1 vector were sorted by a FACS Diva
cytometer (Becton-Dickinson)
For glycosylation inhibition experiments, transfected cells
tunicamycin (Sigma) as N-glycosylation inhibitor or 3 mm
a-benzyl-GalNAc (Sigma) as O-glycosylation inhibitor
Glycosylation inhibitors were added to the cells 6 h after
transfection Enzymatic deglycosylation treatment was
per-formed on the immunoprecipitated ARM-1 protein, by
using the enzymatic deglycosylation and the prO-LINK
according to the manufacturer’s instructions
Static adhesion assays
Quantitative adhesion assays were performed as follows
CEMT4, NK lymphocytes or peripheral leukocytes were
labeled by the PKH26 red fluorescent cell linker kit
(Sigma), according to the manufacturer’s instructions
PKH26 [29] is a nontoxic hydrophobic fluorescent dye,
which stably labels cell membranes ARM-1-transfected or
mock-transfected cells were washed once with PBSc
suspension was layered on to each transfected or
mock-transfected cell well at 5 or 10 lymphocytes to one adhered
gentle washes with PBSc Then, the cells were detached by
centri-fuged (5 min, 1000 g, at room temperature), and analyzed
by flow cytometry (FACSort apparatus; Becton Dickinson)
which allowed lymphoid cells (labeled) to be separated from
nonlymphoid cells (unlabeled) and to express the number
of lymphoid cells adhered per cell Each assay was
per-formed in triplicate
Immunoprecipitation and immunoblotting
Transfected cells with the pcDNA-ARM-1 or the
buf-fer, pH 8, containing 150 mm NaCl, 1% Triton X-100
phenyl-methanesulfonyl fluoride and 5 mm sodium tetrathionate)
were incubated with Protein G MicroBeads (Miltenyi
Biotec, Singapore) and antibodies to Myc (mouse
immunoprecipita-tion was carried out according to the manufacturer’s instructions
Protein samples were boiled for 5 min, separated by
Protran nitrocellulose membranes (Schleicher and Schuell, Dominique Dutscher, Brumath, France) Membranes were revealed with antibody to Myc or Flag and a secondary alkaline phosphatase-conjugated antibody (anti-mouse goat polyvalent immunoglobulins; Sigma) Proteins were detec-ted by Western blue stabilized substrate for alkaline phosphatase (Promega)
Immunofluorescence microscopy
All incubations were conducted at room temperature Forty eight hours after transfection, cells were washed twice with PBSc, pH 7.4, fixed with paraformaldehyde (2% in PBSc for 30 min for permeabilized cells and 1% in PBSc for
10 min for nonpermeabilized cells), washed twice with PBSc containing 20 mm glycine and, if necessary, permeabilized
20 mm glycine Then cells were washed once with PBSc, incubated for 45 min with the primary antibody, washed four times and incubated for 30 min with tetramethylrhod-amine isothiocyanate-conjugated goat anti-(mouse IgG) Igs (Sigma) After extensive washing, cells were mounted on a
anti-fading agent [30]
Fluorescence confocal microscopy analysis
Cells were observed with a fluorescence confocal imaging system MRC-1024 (Bio-Rad) equipped with a Nikon
laser Images were treated using Adobe photoshop software (Adobe Systems Inc., Mountain View, CA, USA)
Subcellular fractionation
Transfected cells were washed with PBSc and lysed in
phenylmethanesulfonyl fluoride and 5 mm sodium tetra-thionate) After incubation for 30 min on ice, cells were homogenized with 80 strokes in a tight fitting Dounce homogenizer The lysed cells were then centrifuged at
obtain the cytosolic and membrane fractions An