To study the regulation of VEGF production in macrophages we show that stimulation of mono-cyte-macrophage-like RAW-264.7 cells by lipopolysaccharide LPS increa-ses expression of VEGF mR
Trang 1in macrophages
Min Du1, Kristen M Roy1, Lihui Zhong1, Zheng Shen1, Hannah E Meyers1and Ralph C Nichols1,2,3
1 Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, NH, USA
2 Veterans Administration Research Service, White River Junction, VT, USA
3 Department of Medicine, Dartmouth Medical School, Hanover, NH, USA
The angiogenic cytokine vascular endothelial growth
factor (VEGF) is associated with macrophage cell
infil-tration in inflammation Both VEGF and macrophage
cells play a critical role in inflammatory processes
including synovial joint inflammation [1–3],
athero-sclerosis [4], tumorigenesis [5], nephritis [6], corneal [7]
and diabetic [8] neovascularization The relationship
between macrophages and VEGF protein is important
to the inflammatory response for several reasons:
(1) macrophages produce VEGF protein [9,10];
(2) neo-vascularization induced by VEGF contributes
to disease in the inflamed joint [11,12] and other inflammatory diseases [13]; (3) macrophages express VEGFR-1 (Flt-1) and respond to VEGF protein [14]
To better understand the contribution of VEGF to inflammatory disease we investigated regulation of VEGF gene expression in macrophages
The relationship between macrophage activation and VEGF has been poorly explored in part because the macrophage cell expresses the VEGFR-1 receptor but
Keywords
VEGF; mRNA stability; 3¢ UTR; AURE;
macrophage
Correspondence
R.C Nichols, Mailstop 151, Veterans
Administration Research Service, 215 North
Main Street, White River Junction,
VT 05009-0001, USA
Fax: +1 802 296 6308
Tel: +1 802 295 9363 extn 5891
E-mail: ralph.c.nichols@dartmouth.edu
(Received 4 April 2005, revised 12 December
2005, accepted 16 December 2005)
doi:10.1111/j.1742-4658.2006.05106.x
The macrophage is critical to the innate immune response and contributes
to human diseases, including inflammatory arthritis and plaque formation
in atherosclerosis Vascular endothelial growth factor (VEGF) is an angio-genic cytokine that is produced by macrophages To study the regulation
of VEGF production in macrophages we show that stimulation of mono-cyte-macrophage-like RAW-264.7 cells by lipopolysaccharide (LPS) increa-ses expression of VEGF mRNA and protein Three alternative splicing VEGF mRNA isoforms are produced, and the stability of VEGF mRNA increases following cellular activation To study post-transcriptional regula-tion of the VEGF gene the 3¢-untranslated region (3¢ UTR) was introduced into the 3¢ UTR of the luciferase gene in a reporter construct In both RAW-264.7 cells and thioglycollate-elicited macrophages, the 3¢ UTR sequence dramatically reduces reporter expression Treatment with activa-tors of macrophages, including LPS, lipoteichoic acid, and VEGF protein, stimulates expression of 3¢ UTR reporters Finally, mapping studies of the 3¢ UTR of VEGF mRNA show that deletion of the heterogeneous nuclear ribonucleoprotein l binding site affects basal reporter expression in RAW-264.7 cells, but does not affect reporter activation with LPS Together these results demonstrate that a post-transcriptional mechanism contributes to VEGF gene expression in activated macrophage cells
Abbreviations
AURE, adenosine-uridine-rich element; CAURE, cytidine-adenosine-uridine-rich element; CSD ⁄ PTB, cold shock domain ⁄ polypyrimidine tract binding protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLUT1, glucose transporter-1; hnRNP, heterogeneous nuclear ribonucleoprotein; HuR, ELAV protein HuA; hVEGF, human vascular endothelial growth factor; LPS, lipopolysaccharide; LTA, lipoteichoic acid; mVEGF, mouse vascular endothelial growth factor; TG-Mac, thioglycollate-elicited macrophages; TNFa, tumor necrosis factor-alpha; UTR, untranslated region.
Trang 2not the VEGFR-2 (Flk-1) receptor [14] The VEGFR-1
receptor has been considered a decoy receptor on
endothelial cells where VEGFR-1 is considerably less
active (low kinase activity) than VEGFR-2 [15]
However, critical new reports show that the blockade
of VEGFR-1, but not VEGFR-2, reverses joint
destruction in the K⁄ BxN mouse model of arthritis
[16,17] In separate studies we show that the VEGFR-1
receptor is upregulated in activated macrophages
(K Roy, R Fava, R.C Nichols, unpublished data),
suggesting that macrophages both produce and respond
to VEGF in inflamed tissues Although an autocrine
mechanism of macrophage activation is suggested,
VEGFR-1 is also the target of placental growth factor
[17], and the role of VEGF in macrophage stimulation
is not fully understood
Regulation of VEGF gene expression is controlled
by both transcriptional and post-transcriptional
mech-anisms [18], and post-transcriptional regulation is
responsible for a major increase in VEGF production
under hypoxia [18–25] Post-transcriptional regulation
is mediated by mRNA binding proteins that act on
defined cis-acting elements, usually found in the
3¢-un-translated region (3¢ UTR) of the targeted mRNA
[26,27] Several reports have identified cis elements in
the 3¢ UTR of VEGF mRNA that are recognized by
heterogeneous nuclear ribonucleoprotein (hnRNP) L
[25], ELAV protein HuA (HuR) [23] and the cold
shock domain⁄ polypyrimidine tract binding protein
(CSD⁄ PTB) complex [28] To extend these studies, we
now show that the 3¢ UTR of VEGF mRNA plays a
role in VEGF gene expression in mouse macrophages
under inflammatory conditions We have introduced
the 3¢ UTR of mouse VEGF (mVEGF) into the 3¢
UTR of the luciferase reporter gene and show that
reporter activity increases when cells are treated with
lipopolysaccharide (LPS), lipoteichoic acid (LTA) or
VEGF protein Finally, mapping studies of the 3¢
UTR suggests that the proximal region of the 3¢ UTR
contains cis elements important in activated
macro-phage cells Together, these findings demonstrate that
VEGF gene expression in macrophages is controlled
by a post-transcriptional mechanism
Results
Macrophage-like RAW-264.7 cells produce three
VEGF isoforms
Alternative splicing of VEGF mRNA produces
mul-tiple VEGF isoforms [29] To determine which
iso-forms are produced in macrophage cells we designed
PCR primers homologous to sequences in exons 5 and
8 Three PCR products are produced from cDNA iso-lated from RAW-264.7 cells (Fig 1A) These PCR products were sequenced and show that RAW-264.7 cells produce three VEGF isoforms, VEGF188, VEGF164 (exon 6 skipped), and VEGF120 (exons 6 and 7 skipped) The same isoforms were expressed
by thioglycollate-elicited macrophages (TG-mac) from C3H⁄ HeN cells (data not shown) The VEGF120 isoform is the most abundant PCR product The PCR reaction is more efficient for smaller PCR products and the abundance of the smallest VEGF isoform may reflect this In separate experiments we found that immunoblotting of whole-cell RAW-264.7 lysates with anti-VEGF antibody did not detect VEGF pro-tein (data not shown), suggesting that most of the VEGF protein is not cell associated and is exported from macrophages Finally, in experiments where RAW-264.7 or thioglycollate-elicited macrophages were stimulated, the VEGF mRNA levels of all three isoforms increased but the relative amount of each isoform did not change, suggesting that alternative splicing of VEGF mRNA is not affected by cellular activation
Effects of stimulation of RAW-264.7 cells on VEGF protein
Rheumatoid tissue is populated by macrophages, and these cells produce VEGF mRNA and protein [1] To show that stimulated RAW-264.7 cells increase pro-duction of VEGF we plated cells overnight, and then treated cells with the Toll-like receptor-4 activator LPS (100 ngÆmL)1) in fresh media for 4 h Levels of VEGF protein in culture media were measured by ELISA As shown in Fig 1B, VEGF production increased by 60% with LPS treatment Treatment of RAW-264.7 cells with tumour necrosis factor-a (TNFa) also increased VEGF production (3.1 ± 0.6-fold increase with
10 ngÆmL)1TNFa for 4 h; mean ± SD for four experi-ments) Finally we measured the effects of human VEGF (hVEGF) on VEGF production by RAW-264.7 cells Human VEGF is cross-reactive with mouse anti-bodies (R & D Systems, Minneapolis, MN) used for ELISA measurement To correct for contaminating hVEGF, parallel wells of were treated with cyclohexi-mide (CHX), as described in Experimental procedures Cells were treated overnight without or with VEGF protein (2 lgÆmL)1), media was replaced with media not containing hVEGF protein, and mVEGF was measured in the media after 4 h As shown in Fig 1C, treatment with hVEGF (NCI Clinical Repository, Frederick, MD) protein stimulated de novo protein synthesis fivefold
Trang 3Effects of stimulation of RAW-264.7 cells on
VEGF mRNA
We next measured the effects of cellular activation on
levels of endogenous VEGF mRNA and on the
stabil-ity of VEGF mRNA Lipopolysaccharide activates
Toll-4 receptor; RAW-264.7 cells were untreated or
treated with LPS (100 ngÆmL)1 for 4 h) and mRNA
levels were measured by RT⁄ PCR As shown in
Fig 2A, levels of endogenous VEGF mRNA increased
by 45% in cells treated with LPS To evaluate whether
VEGF mRNA was stabilized following LPS treatment,
RAW-264.7 cells were treated with LPS for 4 h fol-lowed by no treatment or treatment with Actinomycin
D for 1 or 2 h The levels of VEGF mRNA were determined by RT⁄ PCR and normalized to 18S rRNA
We found that the half-life of VEGF mRNA was increased in activated cells (Fig 2B) The estimated mRNA half-lives at 2 h were 0.86 h (untreated) and 1.6 h (LPS treated) This finding is comparable to the
45 min half-life of VEGF mRNA reported in unstimu-lated monocyte-derived macrophages [30] This result shows that mRNA stabilization is one mechanism that increases VEGF production in stimulated macro-phages The stability of VEGF mRNA is affected by cis elements in the 3¢ UTR in other cell types [19,23,25,28,31] and we next tested the activity of AU-rich elements in macrophage cells
Effects of AU-rich sequences on reporter activity
in macrophages Post-transcriptional regulation is mediated by mRNA binding proteins that act on cis elements in the 3¢ UTR [26–28,32] Previous studies have shown that both AU-rich elements (AURE) and non-AURE ele-ments are active in the regulation of VEGF [19,23, 25,31] To study AURE we created the AUUUA-luc luciferase reporter that contains a 30-nt sequence with multiple tandem repeats of the AUUUA pentamer and includes six overlapping UUAUUUAUU nonamers Four hours after transfection in RAW-264.7 cells, the activity of the AUUUA-luc reporter was 75% less than the parent reporter pGL3 (Fig 3) The activity of a control reporter (AUGUA-luc), in which guanosine
A
B
C
Fig 1 Production of VEGF by RAW-264.7 cells (A) VEGF isoforms Primers specific to exons 5 and 8 were designed to amplify all known mouse VEGF alternative splice isoforms from cDNA The PCR products for three isoforms (VEGF188, VEGF164, VEGF120) were found Bars show DNA size standards (B) Effects of LPS on VEGF protein production To measure VEGF production, RAW-264.7 were untreated or treated in fresh media for 4 h with LPS (100 ngÆmL)1) Levels of VEGF protein (mean ± SD; P < 0.03) were measured in the media from triplicate wells by ELISA Results are from one of two experiments with similar results (C) Effects of human VEGF protein on de novo VEGF protein production To measure the effects of human VEGF on VEGF production, RAW-264.7 were untreated or treated with human VEGF (2 lgÆmL)1) overnight Medium was replaced with fresh media without VEGF, and levels of VEGF protein (mean ± SD; P < 0.02) were measured
in the media from triplicate wells by ELISA To correct for contami-nating hVEGF, parallel wells were treated with CHX (20 l M ), as described in Experimental procedures Results are from one of two experiments with similar results.
Trang 4residues were substituted for uridine residues in each
pentamer, was only 10% less than the parent
con-struct The 30-nt AURE from glucose transporter-1
(AURE⁄ GLUT1-luc) [33], which contains no AUUUA
pentamers, was 85% less than the parent reporter
These results show that both AUUUA and
non-AUUUA AURE elements are active in RAW-264.7
cells The 3¢ UTR of VEGF mRNA contains AURE
and we next tested the activity of the full-length 3¢
UTR using reporter constructs
The effects of the 3¢-untranslated region of mouse VEGF mRNA on gene expression in a heterologous reporter
We evaluated the role of the 3¢ UTR in gene exp-ression by introducing the SmaI⁄ XbaI fragment (nt 209–1747) of the mouse 3¢ UTR into the 3¢ UTR of the luciferase gene The structure of the full-length reporter (VEGF-FL-luc) and related 3¢ UTR reporters
is shown in Fig 4A The VEGF-FL-luc reporter con-tains all regulatory elements (or homologous regions) reported in rat, mouse, and hVEGF 3¢ UTR The native polyadenylation signal identified by Dibbens
et al [34] was removed, and we used the more efficient bovine growth hormone poly(A) signal [35] present
in the pcDNA-3.1 reporter construct The poly(A) sequence and poly(A) binding protein mediate transla-tion of mRNA, and are not thought to affect mRNA stability [36–38] Further studies are needed to deter-mine if the native poly(A) signal affects VEGF mRNA stability We transfected the parental reporter (3.1-luc)
or the VEGF-FL-luc reporter into nonactivated cells and measured luciferase levels after 4 h The basal level of expression of the full-length 3¢ UTR reporter was dramatically less than that of the parent vector
in both RAW-264.7 cells (80% reduction) and in TG-Mac cells (65% reduction) (Fig 4B and C) Well-to-well variation was large in primary TG-Mac cells Therefore, we cotransfected TG-Mac cells with sea pansy luciferase (renilla) and we express these results as ‘normalized luciferase units.’ Together, these results demonstrate that the 3¢ UTR of VEGF mRNA inhibits expression of a heterologous reporter gene in macrophages
Effects of the 3¢ UTR of VEGF mRNA on luciferase reporter mRNA levels
Regulation by cis elements in the 3¢ UTR can affect mRNA levels or may affect translational efficiency [39] To determine how the introduction of the 3¢ UTR of VEGF mRNA affected luciferase mRNA lev-els, we transfected RAW-264.7 cells with either the parental reporter, 3.1-luc, or the VEGF-FL-luc repor-ter and measured luciferase mRNA levels by
RT⁄ PCR As shown in Fig 4D, luciferase mRNA was reduced by 40% in cells expressing the VEGF-FL-luc
as compared to the parental luciferase mRNA It is important to note that the parental 3.1-luc and VEGF-FL-luc constructs are driven by the same pro-moter, and any differences between the luciferase mRNA levels are due to actions on the 3¢ UTR sequence of VEGF mRNA
A
B
Fig 2 Effects of macrophage activation on VEGF mRNA (A) VEGF
mRNA levels RAW-264.7 cells were not treated or treated with
LPS for 4 h and VEGF mRNA levels determined by RT ⁄ PCR The
levels of the three VEGF isoform PCR products from each sample
were combined, and the sum was normalized to the level of 18S
rRNA PCR product from the same sample Results are relative
val-ues, three samples per condition (mean ± SD; P < 0.04) and are
representative of two experiments (B) Half-life determination of
VEGF mRNA Decay of VEGF mRNA was measured in cells
not-treated or not-treated with actinomycin D for 1 or 2 h Levels of VEGF
PCR products were normalized to levels of 18S rRNA from the
same sample Results are set to unity for the zero time point, and
the mean ± SD of triplicate determinations are shown VEGF
mRNA levels at 2 h were significantly less than at zero hour
(untreated, P < 0.005; LPS treated, P < 0.002) VEGF mRNA levels
were significantly greater in LPS-treated samples than in untreated
samples at 2 h (P < 0.015).
Trang 5Effects of agents that activate macrophages on
VEGF-FL-luc reporter activity
We show in Fig 1 that stimulated macrophages
increase production of VEGF mRNA and protein We
next tested the effects of macrophage activating agents
on VEGF-FL-luc reporter activity in RAW-264.7 cells
Dose–response treatment with LPS showed that
repor-ter activity was maximal with 100 ngÆmL)1 (1.82 ±
0.25-fold greater than untreated, mean ± SD)
Reporter activity increased for up to 6 h and did not
increase further after 24 h (data not shown) Because
LPS acts on the Toll-4 receptor, we next determined if
the Toll-2 receptor ligand, LTA [40], could affect the
VEGF-FL-luc reporter activity When RAW-264.7
cells were treated with LTA (1 lgÆmL)1, 24 h),
VEGF-FL-luc reporter activity increased 1.69 ± 0.17-fold
over untreated controls Next, we tested the effects of
VEGF protein on activation of VEGF-FL-luc reporter activity
Macrophage cells express the VEGFR-1 (Flt-1) receptor [41] and macrophages migrate in response to VEGF protein at levels as low as 12 ngÆmL)1[14] To determine if VEGF protein affects RAW-264.7 cells,
we first measured production of a primary marker
of macrophage activation, TNFa Cells treated with
100 ngÆmL)1 of recombinant hVEGF for 24 h increased TNFa production by 9.3 fold (untreated,
1359 ± 186 pgÆmL)1: hVEGF treated, 12 604 ±
3461 pgÆmL)1; mean ± SD) We next measured the effects of hVEGF on VEGF-FL-luc reporter expres-sion in RAW-264.7 cells Treatments with hVEGF as low as 3.3 ngÆmL)1 increased reporter activity with maximal activity seen at 100 ngÆmL)1 (2.2 ± 0.2 fold increase over untreated; mean ± SD) Effects of acti-vating agents on VEGF-FL-luc reporters are
normal-Fig 3 AU-rich sequences reduce reporter activity Sequences were introduced into the 3¢ UTR of the luciferase gene of the eukaryotic expression vector pGL3-Control (Promega) Luciferase activity was measured in cell lysates 4 h after transfection of RAW-264.7 cells On the right are diagrams of the reporter constructs showing the nucleotide sequence of an artificial AU-rich element (AUUUA), a negative con-trol (AUGUA) and the non-AUUUA AURE identified in the 3¢ UTR of GLUT1 mRNA (AURE ⁄ GLUT1) [33] Error bars are SD Results shown are representative of five experiments Results from cells transfected for 24 h were similar (data not shown).
Fig 4 The effects of the 3¢ UTR of VEGF mRNA on reporter expression in macrophages (A) Diagram of mVEGF reporter constructs Upper-most bar shows the 3¢ UTR region studied Adenosine-uridine-rich (AU) and cytidine-adenosine-uridine-rich (CAU and CSD ⁄ PTD) regions are noted The parent construct, 3.1-luc, was created as described in Experimental procedures The full-length reporter (VEGF-FL-luc) construct contains nt-209–1747 of the 3¢ UTR of mVEGF mRNA, and the sequence is shown below The CAURE site recognized by hnRNP
L (nt 322–342), the CAURE recognized by HuR (nt 1622–1665) [23], and the CSD ⁄ PTB binding site (nt 1727–39) [28] are shown in bold ⁄ underlined type Additional AU-rich sequences, including all AUUUA pentamers and nonomers, are shown in bold type Engineering of reporter constructs is described in Experimental procedures (B, C) The 3¢ UTR of VEGF mRNA suppresses reporter activity in macrophages The effects on reporter activity of the 3¢ UTR of VEGF mRNA were measured in untreated macrophage cells by transfection of either the control reporter construct (3.1-luc) or the full-length VEGF-3¢ UTR reporter (VEGF-FL-luc) RAW-264.7 cells (B) or TG-Mac cells from C3H ⁄ HeN mice (C) were lysed 4 h after transfection, and luciferase activity was read by luminometry Similar results were seen after 24 h (data not shown) Diagrams on the right show the construct used to transfect cells Data are the mean ± SD, and are representative of at least three experiments (D) Luciferase mRNA levels RAW-264.7 cells were transfected with the parent vector (3.1-luc) or with VEGF-FL-luc for 24 h Total RNA was prepared from cell cytoplasm and RT ⁄ PCR was performed with luciferase primers or GAPDH primers as described
in Experimental procedures.
Trang 6ized to parent reporter levels in cells treated in an
iden-tical manner (see Experimental procedures, and
Fig 6) Treatment with hVEGF produced maximal
VEGF-FL-luc reporter activity in 4 h, with longer
times (up to 24 h) showing similar increases Together,
these results demonstrate that pro-inflammatory agents
of bacterial origin (LPS, LTA) and endogenous origin (VEGF) increase VEGF 3¢ UTR-dependent reporter expression In addition, these results show that the reporter construct is a useful tool for mapping the 3¢ UTR
A
C
B
D
Trang 7Mapping of the 3¢ UTR of VEGF mRNA
As shown in Fig 4A, the 3¢ UTR of mVEGF mRNA
is complex The region proximal to the coding region
contains a CAU-element [19,25] but no AUUUA
pen-tamers The distal region contains: (1) two active
CAU-rich elements [23,28]; and (2) six AUUUA
pen-tamers and two nonomers, some of which are active
[31] To isolate these regions we created luciferase
reporters that contain either the proximal region
(VEGF-209–750-luc) or the distal region (VEGF-751–
1747-luc) (see Fig 4A) When transfected into
RAW-264.7 cells, neither construct displayed the level of
inhibition found with the full-length construct (Fig 5)
Although we cannot rule out the possibility that a cis
element at nt 750 has been interrupted, this region
contains no AU- or CAU-rich sequence motifs The
reporter activity of the proximal VEGF-209–750-luc
construct was most similar to the full-length construct
In addition, the activity of the VEGF-209–750-luc
reporter increased in LPS-treated cells whereas the
VEGF-751–1747-luc was minimally affected by LPS
treatment (data not shown) For this reason we
exam-ined candidate elements in the proximal region of the
3¢ UTR
The hnRNP L element
Shih and Claffey identified a cis element in the
prox-imal region of hVEGF that was recognized by the
mRNA binding protein, hnRNP L [25] The hnRNP L
element is highly conserved between human and mouse
and we used site-directed mutagenesis to delete the
sequence (nt 322–42, CACCCACCCACAUACACA
CAU) from the full-length reporter As shown in Fig 6, deletion of the hnRNP L element (VEGF-dL-luc) reduced reporter activity by 25% in untreated RAW-264.7 cells (compare columns 1 and 3) In cells treated with LPS (100 ngÆmL)1), VEGF-FL-luc and VEGF-dL-luc responded with similar increases in reporter activity (VEGF-FL-luc increased 2.1-fold, VEGF-dL-luc increased 2.5-fold) Treatment of RAW-264.7 cells with hVEGF protein produced similar effects on these reporters (data not shown) We con-clude that the hnRNP L element affects basal levels of VEGF reporter activity but does not affect increases in VEGF reporter found in activated cells We are cur-rently extending this mapping analysis to identify addi-tional 3¢ UTR regulatory elements that are active in macrophages
Discussion
The VEGF cytokine plays a major role in cancer by controlling neo-vascularization in solid tumours [13]
In arthritis, VEGF stimulates vascularization that supports the ’tumour-like’ phenotype of the inflamed synovium [12] However, VEGF also plays a role
in vascular permeability and as a chemoattractant [11,12,14], suggesting that the role of VEGF in inflamed joints may be complex Macrophages in rheu-matoid joints produce VEGF [1], and we have found that VEGFR-1 is upregulated in activated macro-phages (K Roy, R Fava, R.C Nichols, unpublished data) Together these facts suggest that macrophages
at the site of inflammation both respond to VEGF and contribute to the inflammatory response by producing VEGF protein In this report we confirm that
macro-Fig 5 Mapping of the 3¢ UTR from VEGF mRNA Reporter constructs were created that separated the proximal and distal regions of the 3¢ UTR from VEGF mRNA (see Fig 4A) The reporter VEGF-209–750-luc contains the proximal region (nt 209–750) The reporter VEGF-751– 1747-luc contains the distal region (nt 751–1747) Reporter constructs were transfected into RAW-264.7 cells and luciferase activity meas-ured in cell lysates after 4 h Similar results were seen at 24 h (data not shown) Results are mean ± SD and are representative of five independent experiments.
Trang 8phages respond to VEGF and other inflammatory
mediators by increasing VEGF production We also
show that macrophages produce multiple mRNA
VEGF isoforms, and the stability of VEGF mRNA
increases in activated cells We demonstrate that
macro-phage activation increases expression of VEGF
luci-ferase reporters containing the mouse VEGF 3¢ UTR
region, and we present mapping analysis of the VEGF
3¢ UTR Together, these results demonstrate that, as in
cancer models, VEGF expression is controlled at the
post-transcriptional level in macrophage-like cells
Increased production of VEGF has been shown to
act by both transcriptional and
post-transcript-ional mechanisms [13] Post-transcriptpost-transcript-ional regulation
involves mRNA binding proteins acting on cis
ele-ments to alter mRNA stability or the efficiency of
translation [32] Post-transcriptional regulation affects
VEGF production in a cancer model [18], but it is
unclear whether post-transcriptional regulation affects
VEGF gene expression in macrophage cells To
address this, we show here that under conditions in
which VEGF protein production increases: (a) VEGF
mRNA levels increased (Fig 2A); and (b) VEGF
mRNA stability increased (Fig 2B) However, these
increases in VEGF mRNA and protein production
may also result in part from increased transcription
We are currently assessing transcriptional regulation in
activated macrophages, and only report here studies of
3¢ UTR-mediated regulation
To study regulatory cis elements in VEGF mRNA
we introduced the 3¢ UTR of mVEGF mRNA into the 3¢ UTR of the luciferase gene in a reporter construct Using this method, we can segregate post-transcriptional regulation from both post-transcriptional regulation of the native VEGF promoter, and post-translational regulation acting on the VEGF pro-tein We show in both monocyte-macrophage-like RAW-264.7 cells and thioglycollate-elicited macro-phages that introduction of the full-length 3¢ UTR construct significantly reduced luciferase mRNA levels, and reduced basal reporter activity (Fig 4B, C, D) Next, we show that, under conditions in which VEGF mRNA is stabilized (LPS activation), luciferase repor-ter activity increased These results suggest that the 3¢ UTR in VEGF mRNA contributes to the increases in VEGF mRNA and protein found when macrophages are stimulated We are the first to show that the 3¢ UTR of VEGF mRNA affects gene expression
in macrophages
Post-transcriptional regulation of mRNA is medi-ated by mRNA binding proteins that recognize cis-act-ing elements, most frequently found in the 3¢ UTR region [26–28] The most studied cis-acting elements are AURE Analysis of the human genome estimates that 8% of human mRNAs contain AURE [42] Three classes of AURE have been identified [26,27], and the 3¢ UTR of VEGF mRNA contains two of these AURE classes: (a) Class I, nonoverlapping AUUUA
Fig 6 The effects of the hnRNP L element on reporter activity To test the activity of the cis element recognized by hnRNP L [25], the sequence (CACCCACCCACAUACACACAU) was deleted from the full-length reporter construct The deletant reporter, VEGF-dL-luc, produced less activity in untreated RAW-264.7 cells (compare columns 1 and 3) In macrophages treated with LPS (100 ngÆmL)1for 24 h), both the VEGF-FL-luc and VEGF-dL-luc reporters showed similar increases in reporter activity Treatment with LPS affects the promoter of the repor-ter construct To account for this effect, luciferase units (mean ± SD) are normalized to luciferase units of the 3.1-luc parental construct trea-ted in parallel wells For example, the reporter values without LPS treatment were: 3.1-luc, 693 000 ± 97 000 luc units; VEGF-FL-luc,
104 000 ± 4000 luc units Reporter values with LPS treatment were: 1157 000 ± 16 000 (3.1-luc) and 369 000 ± 24 000 (VEGF-FL-luc) The normalized value for VEGF-FL-luc reporter activity is set at unity (column 1).
Trang 9pentamers and UUAUUUAA/UA/U nonamers; (b)
Class III, adenosine-uridine rich and uridine-rich
sequences that lack AURE pentamers Several reports
have analysed AU-rich elements in rat and human
VEGF 3¢ UTR [19,21–23,43–44] One Class III AURE
element that was identified in rat VEGF is not
con-served in mouse or human [20] Another type of cis
element, that we refer to as a CAU-rich element
(CAURE), contains adenosine, uridine and cytidine
residues, and CAURE have been identified in GLUT1
mRNA [45] (nt 2180–90) and in the 3¢ UTR of VEGF
mRNA [23,25,28,46] The hnRNP L binding site
(nt 322–342) [25], HuR binding site (nt 1622–1665)
[23,46], and CSD⁄ PTB complex binding site (nt 1727–
39) [28] in the 3¢ UTR of VEGF mRNA are
CAU-rich Here we present our analysis of 3¢ UTR-mediated
regulation in mouse macrophage cells
The 3¢ UTR of mVEGF mRNA contains cis
ele-ments in both the proximal and distal regions
[21,22,25,43,47] The distal two-thirds of the mouse 3¢
UTR contains six AURE pentamers, two nonomers,
two CAURE and seven additional regions of 10 or
more AU nucleotides that lack AUUUA pentamers
Several groups have shown that cis elements in the
distal region have strong affects on VEGF gene
expression under hypoxic stress [22,46] Under
norm-oxia these distal cis elements contribute to VEGF
mRNA instability It was surprising therefore to find
that when the distal region of the 3¢ UTR was
removed (to create VEGF-209–750-luc) reporter
activ-ity was similar to that of the full-length reporter
(Fig 5) In addition, cells stimulated with LPS or
with VEGF showed increased VEGF-209–750-luc
reporter activity In contrast, the VEGF-751–1747-luc
reporter responded poorly to cellular activation (data
not shown), suggesting that regulatory elements active
in macrophages reside in the proximal region of the
VEGF 3¢ UTR
The proximal region of the 3¢ UTR contains a
CAURE in hVEGF that is recognized by hnRNP L
under both normoxic and hypoxic conditions in M21
melanoma cells [25] The homologous mouse sequence
is nearly identical to the human CAURE, and deletion
of the hnRNP L element resulted in a decrease in
reporter activity in untreated RAW-264.7 cells
How-ever, the activity of the wild-type and VEGF-dL-luc
reporters both increased in cells treated with LPS
(Fig 6) Treatment with hVEGF produced similar
results (data not shown) Although there are no
AUUUA pentamers in the nt 209–750 region there is a
long AU-rich region (78-nt, 97% adenosine-uridine)
that is tandem to the hnRNP L element This AURE
is interesting because we found that the
GLUT1-AURE luciferase reporter, which is also a long non-AUUUA Class III, was very active in RAW-264.7 cells (Fig 3) Future studies will determine if this AURE or other noncanonical cis elements in the proximal region contribute to 3¢ UTR-dependent regulation in macro-phages
Three agents that stimulate macrophages (LPS, LTA and VEGF) increased VEGF 3¢ UTR-dependent repor-ter activity One mechanism we considered was that these agents act by initially stimulating production of TNFa, and then TNFa stimulates VEGF gene expres-sion To investigate this, we first showed that LPS sti-mulated production of TNFa (data not shown) Next
we determined whether TNFa affected VEGF 3¢ UTR-dependent reporter activity Although TNFa treatment increased VEGF protein production, we found that TNFa treatment decreased VEGF-FL-luc reporter activity modestly but significantly (10%; data not
shown) We conclude that post-transcriptional stimula-tion of VEGF gene expression by agents such as LPS and VEGF do not act through TNFa by an autocrine mechanism
Activation of VEGF gene expression in RAW-264.7 cells with LPS and VEGF was distinct from treatments with TNFa Treatment with TNFa decreased reporter activity and increased VEGF protein production Treatment with LPS or with VEGF protein increased both reporter activity and VEGF protein production However, the effect of LPS on VEGF protein produc-tion was modest compared to the effects of VEGF treatment These different profiles of VEGF gene regulation by TNFa, VEGF and LPS may result from different gene activation mechanisms Our results suggest that: (i) TNFa stimulates VEGF production
by a transcriptional mechanism, but inhibits the post-transcriptional pathway; (ii) treatment with VEGF stimulates both transcriptional and post-tran-scriptional pathways; (iii) treatment with LPS acts solely through a post-transcriptional mechanism, and does not affect VEGF transcription Studies of the effects by these agents on transcription are on going If LPS acts solely by a post-transcriptional mechanism, then long-term studies are needed to determine if the modest effects by LPS result in sufficient production of VEGF to initiate VEGF-driven autocrine production
of VEGF Importantly, our results suggest that post-transcriptional regulation of VEGF may be important under conditions in which TNFa is not active, inclu-ding under therapeutic conditions where TNFa action
is blocked
Originally identified as a permeability factor, VEGF
is now known to play an essential role in arterio-genesis [13], neo-vascularization in cancer [13], and
Trang 10inflammatory diseases [12] Our studies with
macroph-ages demonstrate that the 3¢ UTR of VEGF
contri-butes to gene expression and provides a novel target to
treat active disease Post-transcriptional regulation of
VEGF in macrophages is mediated by the action of
VEGF on its cognate receptor, VEGFR-1 Regulation
of VEGF by an autocrine mechanism in cancer has
been reviewed [48] It is possible that VEGF and its
receptor interact intracellularly to regulate VEGF gene
expression [48] The mechanism of regulation of the
macrophage VEGF receptor, VEGFR-1, is now under
study to determine if VEGF protein and its receptor
are coordinately regulated
Experimental procedures
Cell culture, transfection and luciferase assay
The RAW-264.7 cell line was obtained from ATCC
(Manassas, VA) and maintained as described Media was
DMEM supplemented with 10% heat-inactivated fetal
bovine serum (Hyclone, Logan, VT) and
penicillin-strep-tomycin Cultured cells were plated 24 h prior to
trans-fection in six-well or 48-well plates Cells were adherent
and were transfected at 40% confluence in Opti-MEM
media (Invitrogen-Gibco, Carlsbad, CA) with luciferase
constructs (plasmid constructs are described below) For
cell transfection, plasmid DNA was complexed with
Lipofectamine-2000, as described by the manufacturer
(Invitrogen-Gibco) Cells in 48-well plates were lysed with
100 lL cell culture lysis reagent lysis buffer (Promega)
After 4 or 24 h, luciferase activity in 20 lL of lysate was
measured following addition of luciferin substrate
(Prome-ga, Madison, WI) in a Molecular Dynamics luminometer
Each transfection condition in 48-well plates was
per-formed in six wells Two wells were pooled (10 lLÆwell)1)
to give triplicate readings for each experimental
condi-tion Results are reported as the mean ± SD All
experi-ments are representative of two or more independent
experiments performed on different days The efficiency
of transfection in RAW-264.7 cells was evaluated by
co-transfection with renilla luciferase (pRL-SV40 Promega)
Cells in 48-well plates were transfected with 0.1 lg
luci-ferase and 0.01 lg renilla luciluci-feraseÆwell)1 and lysed in
Dual-Glo lysis buffer (Promega) The well-to-well
vari-ation of firefly luciferase activity in RAW-264.7 cells was
not improved by normalization to renilla luciferase
The effect of cell activation on 3¢ UTR reporter activity
was measured by adding the activating agent (LPS, LTA,
or human VEGF) 2 h after transfection and cells were lysed
4 or 24 h later In some experiments with macrophage
acti-vating agents the cell treatment affected the promoter of
the reporter construct In these experiments parallel wells
were transfecting with the parental (empty) reporter The
parallel wells were not treated⁄ treated with activating agent
in an identical manner as wells transfected with VEGF reporters To account for effects on the construct promoter, results were normalized to the parental luciferase values Thioglycollate-elicited macrophages (TG-Mac) were obtained from C3H⁄ HeN mice as previously described [49] Briefly, mice were injected with thioglycollate and after 3 days, mice were killed by cervical dislocation Macrophages were removed from the peritoneum and plated on 6- or 48-well tissue culture plates and main-tained in the same media used for RAW-264.7 cells Nonadherent cells were removed after 24 h The well-to-well efficiency of transfection of primary TG-Mac was monitored in reporter assays by cotransfection of pRL-SV40 renilla luciferase Reporter activity was normalized
to the value of renilla luciferase for each experimental condition
All experimental procedures were carried out in accor-dance with NIH guidelines regarding the care and use of experimental animals
Measurement of VEGF and TNFa production
by ELISA
The effects of LPS, TNFa and hVEGF on mVEGF pro-tein production was measured in cells plated 24 h before treatment After treatment, medium was collected and stored at )80 C Cells were treated with LPS (generous gift of H Yohe, Veterans Administration Research Service, White River Junction, VT) or human TNFa (gen-erous gift of R Fava, Veterans Administration Research Service, White River Junction, VT) Human VEGF was produced in baculovirus by R & D Systems, and obtained from the NCI Clinical Repository The activity of hVEGF was lost following boiling [50] Production of mVEGF
#MMV00, R & D Systems) Due to small but significant cross-reactivity between mVEGF and hVEGF, we could not directly measure mVEGF production by cells treated with hVEGF Therefore, production of mVEGF follow-ing stimulation with hVEGF was determined as follows RAW-264.7 cells were not treated or treated with
2 lgÆmL)1 hVEGF After 24 h, cells were rinsed and incu-bated with fresh media (without VEGF), and after 4 h media were collected To account for contaminating hVEGF, all treatments were performed in parallel, and these parallel wells were treated for 4 h with 20 lm CHX
to block de novo protein synthesis Levels of de novo VEGF protein was determined by subtracting VEGF lev-els in parallel CHX-treated wells Similar results were found with hVEGF from another source (R & D Sys-tems) Production of mouse TNFa was measured in media
by ELISA, according to the manufacturer’s directions (Mouse TNFa, #MTA00, R & D Systems)