Vascular cell adhesion molecule-1 expression in human intestinal
microvascular endothelial cells is regulated by PI 3-kinase/Akt/MAPK/NF-?B:
inhibitory role of curcumin
David G. Binion,1Jan Heidemann,2Mona S. Li,3Victoria M. Nelson,4Mary F. Otterson,3
and Parvaneh Rafiee3
1Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania;2Department of Medicine B,
University of Muenster, Muenster, Germany; and Departments of3Surgery and4Medicine, Medical College of Wisconsin,
Submitted 9 March 2009; accepted in final form 2 June 2009
Binion DG, Heidemann J, Li MS, Nelson VM, Otterson MF,
Rafiee P. Vascular cell adhesion molecule-1 expression in human
intestinal microvascular endothelial cells is regulated by PI 3-kinase/
Akt/MAPK/NF-?B: inhibitory role of curcumin. Am J Physiol Gas-
trointest Liver Physiol 297: G259–G268, 2009. First published June
11, 2009; doi:10.1152/ajpgi.00087.2009.—Endothelial activation and
surface expression of cell adhesion molecules (CAMs) is critical for
binding and recruitment of circulating leukocytes in tissues during the
inflammatory response. Endothelial CAM expression plays a critical
role in the intestinal microvasculature in inflammatory bowel disease
(IBD), as blockade of leukocyte ?4-integrin binding by gut endothelial
CAM ligands has therapeutic benefit in IBD. Mechanisms underlying
expression of vascular cell adhesion molecule (VCAM)-1, a ligand for
?4-integrin in primary cultures of human intestinal microvascular
endothelial cells (HIMEC) has not been defined. We investigated the
effect of curcumin, phosphatidylinositol 3-kinase (PI 3-kinase)/pro-
tein kinase B (Akt), and mitogen-activated protein kinase (MAPK)
inhibitors on VCAM-1 expression and function in HIMEC. CAM
expression was assessed and HIMEC-leukocyte adhesion was visual-
ized under static and flow conditions. Western blotting and in vitro
kinase assays were used to assess Akt and MAPK activation. Nuclear
factor-?B (NF-?B) activation and nuclear translocation of its p65
subunit were determined. Tumor necrosis factor (TNF)-?/lipopoly-
saccharide (LPS)-induced VCAM-1 expression in HIMEC was sup-
pressed by Akt small-interfering RNA, curcumin, and inhibitors of
NF-?B (SN-50), p38 MAPK (SB-203580) and PI 3-kinase/Akt (LY-
294002). VCAM-1 induction was partially suppressed by p44/42
MAPK (PD-098059) but unaffected by c-Jun NH2-terminal kinase
(SP-600125) inhibition. Curcumin inhibited Akt/MAPK/NF-?B activ-
ity and prevented nuclear translocation of the p65 NF-?B subunit
following TNF-?/LPS. At physiological shear stress, curcumin atten-
uated leukocyte adhesion to TNF-?/LPS-activated HIMEC monolay-
ers. In conclusion, curcumin inhibited the expression of VCAM-1 in
HIMECs through blockade of Akt, p38 MAPK, and NF-?B. Cur-
cumin may represent a novel therapeutic agent targeting endothelial
activation in IBD.
phosphatidylinositol 3-kinase; protein kinase B; mitogen-activated
protein kinase; nuclear factor-?B
IN THE DYSREGULATED, CHRONIC inflammation that characterizes
human inflammatory bowel disease [IBD; Crohn’s disease
(CD) and ulcerative colitis (UC)], alterations in intestinal
microvascular endothelium have been identified at the morpho-
logical and functional levels, including increased expression of
cell adhesion molecules (CAM) and enhanced leukocyte re-
cruitment (4). To further characterize microvascular endothe-
lial changes in gut inflammation, human intestinal microvas-
cular endothelial cells (HIMEC) cultures have been generated,
demonstrating inducible expression of the molecules E-selec-
tin, intercellular adhesion molecule (ICAM)-1, MAdCAM-1,
and vascular cell adhesion molecule (VCAM)-1 following
activation with inflammatory cytokines [i.e., tumor necrosis
factor (TNF)-?, interleukin-1?] and lipopolysaccharide (LPS)
(4). Characterizing mechanisms underlying gut microvascular
endothelial activation and CAM expression represents an es-
sential area for defining mechanisms of IBD pathogenesis as
well as developing novel targets for therapy.
VCAM-1 is involved in firm adhesion and transmigration of
leukocytes expressing the ?4-integrin ligand. ?4-Integrin-ex-
pressing leukocytes play a critical role in the pathogenesis of
rodent models of IBD and a spontaneous form of chronic
colitis that occurs in primates (i.e., cotton-top tamarin). Recent
clinical trials have confirmed the importance of ?4-leukocyte
trafficking in the intestine in human IBD, as natalizumab and
MLN02 (10, 30) have demonstrated benefit in CD and UC
respectively. The clinical efficacy of ?4-integrin inhibition
is believed to result from interruption of interaction with
MAdCAM-1, but at present it is not known whether an
alternative ligand, VCAM-1, may also be playing a role in
IBD chronic inflammation.
VCAM-1 upregulation by proinflammatory cytokines has
been reported in various cells (6, 7, 35). Induction of VCAM-1
in endothelial cells by TNF-? requires nuclear factor (NF)-?B,
activator protein-1, phospholipase C, and phosphatidylinositol
3-kinase (PI 3-kinase) activation (16, 23). Downregulation of
VCAM-1 expression with subsequent impairment of endothe-
lial-leukocyte adhesion in rodents was linked to inhibition of PI
3-kinase/protein kinase B (Akt) signaling (19–21, 25, 32).
Endothelial expression of VCAM-1 in both human and animal
models of IBD has been difficult to confirm at a histological
level. Smooth muscle and mesenchymal expression of
VCAM-1 has been readily demonstrated, but endothelial ex-
pression in tissue histology has been less definitive. However,
specific targeting of VCAM-1 in rodent models of IBD has
demonstrated clear evidence of benefit, suggesting that this
molecule is playing an important role in IBD chronic gut
Curcuma longa Linn, Zingiberaceae (curcumin), the major
yellow coloring pigment found in the household spice tur-
meric, has been used for centuries in food preparation and
Address for reprint requests and other correspondence: P. Rafiee, Dept. of
Surgery, Medical College of Wisconsin, 8701 Watertown Plank Road, Mil-
waukee, WI 53226 (e-mail: email@example.com).
Am J Physiol Gastrointest Liver Physiol 297: G259–G268, 2009.
First published June 11, 2009; doi:10.1152/ajpgi.00087.2009.
0193-1857/09 $8.00 Copyright © 2009 the American Physiological Societyhttp://www.ajpgi.org G259
Ayurvedic traditional medicine to treat inflammatory disorders
(34). Curcumin has low toxicity, has treated chronic gut in-
flammation in animal models, and has shown benefit in a
randomized, multicenter crossover trial in the treatment of UC
(12). Mechanistically, curcumin inhibits: 1) production of reactive
oxygen species; 2) nitric oxide synthase activity; 3) lipoxygenase
5, 17, 29).
Prior work by our group has shown that expression of the
?4-integrin ligand MAdCAM-1 in HIMECs following activa-
tion with TNF-?/LPS is regulated through the PI 3-kinase/Akt
pathway but neither E-selectin nor ICAM-1 expression was
affected by this signaling mechanism (24). In this study, we
demonstrate that, in addition to activation of p38 mitogen-
activated protein kinase (MAPK) and NF-?B, VCAM-1 ex-
pression in TNF-?/LPS-stimulated HIMEC also requires sig-
naling through PI 3-kinase/Akt. Moreover, VCAM-1 expres-
sion and leukocyte adhesion in TNF-?/LPS-activated HIMEC
were inhibited by curcumin, LY-294002 (PI 3-kinase/Akt),
SN-50 (NF-?B), and SB-203580 (p38 MAPK) pretreatment.
The present data further define the intracellular mechanisms of
PI 3-kinase/Akt-mediated activation in gut microvascular en-
dothelium and leukocyte recruitment through ?4-integrin bind-
ing, identifying potential endothelial targets for IBD therapy.
MATERIALS AND METHODS
Reagents. TNF-? and antibodies to ICAM-1, VCAM-1, E-selectin,
and the Tissue-Cell-Staining kit were purchased from R&D Systems
(Minneapolis, MN). Nonradioactive In vitro MAPK Kinase Assay
Kits and MAPK antibodies were from Cell Signaling Technology
(Danvers, MA). The nonradioactive in vitro Akt Kinase Assay Kit
was obtained from Upstate Cell Signaling (Lake Placid, NY). PD-
098059, SB-203580, SP-600125, SN-50, and LY-294002 inhibitors
were obtained from Calbiochem (La Jolla, CA). RNA Cell Protect
reagent and RNeasy Plus Mini Kits were obtained from Qiagen
(Valencia, CA). Akt primers catalog no. PPH0008A were obtained
from SA Biosciences (Frederick, MD). Akt small-interfering RNA
(siRNA) (target siRNA), glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) siRNA (positive control), and nontargeting siRNA (nega-
Fig. 1. Effect of tumor necrosis factor (TNF)-?/lipopolysaccharide (LPS) on vascular cell adhesion molecule (VCAM)-1 mRNA and protein expression in human
intestinal microvascular endothelial cells (HIMEC). The effect of TNF-?/LPS on VCAM-1 mRNA and protein expression was determined. A: detection of
VCAM-1 mRNA in HIMEC by semiquantitative reverse transcriptase-PCR using specific primers. HIMEC does not constitutively express mRNA for VCAM-1.
Stimulation of HIMEC with a combination of TNF-? (100 IU/ml) and LPS (1 ?g/ml) led to marked upregulation of VCAM-1. VCAM-1 mRNA expression in
HIMEC following TNF-?/LPS activation was time dependent; the maximum response was achieved by 4 h, which was lasted for 12 h and then returned to the
basal level by 24 h (left). ?-Actin served as an internal loading control. B: TNF-?/LPS enhanced VCAM-1 protein expression in HIMEC. VCAM-1 protein
expression in HIMEC following TNF-?/LPS activation was assessed using Western blot analysis. Total cell lysates from cultured HIMEC stimulated with TNF-?
(100 IU/ml) and LPS (1 ? g/ml) were subjected to SDS-PAGE and immunoblotted with VCAM-1 antibody. TNF-?/LPS activation of HIMEC significantly
increased VCAM-1 protein expression by 12 h, was sustained through 24 h, decreased at 36 h, and returned to baseline by 48 h (left). Time-matched controls
for VCAM-1 mRNA and protein are shown on right. The same blot was stripped and reprobed with an anti-actin antibody to ensure equivalent protein loading.
Data shown are from one of three independent experiments.
Fig. 2. Effect of TNF-?/LPS on protein kinase B (Akt) phosphorylation (p) in
HIMEC. A: TNF-?/LPS induces Akt phosphorylation in HIMEC. Activation
of Akt in HIMEC by TNF-?/LPS was assessed using Western blot analysis.
Total cell lysates from cultured HIMEC stimulated with TNF-? (100 IU/ml)
and LPS (1 ?g/ml) were subjected to SDS-PAGE and immunoblotted with
total and phospho-(Ser473)-Akt antibodies. TNF-?/LPS induced phosphoryla-
tion of Akt in a time-dependent fashion. TNF-?/LPS stimulation of HIMEC
resulted in a strong increase in Akt phosphorylation at 30 min that lasted up to
60 min and declined by 120 min compared with total Akt. Time-matched
controls did not show any changes in Akt phosphorylation at any given time
points (data not shown). B: pretreatment of HIMEC for 30 min with either
curcumin (Cu, 10 ?M) or PI 3-kinase inhibitor LY-294002 (LY, 10 ?M)
before TNF-?/LPS stimulation abolished the Akt phosphorylation, whereas
SN-50 (SN, NF-?B inhibitor) did not. Stripping and reprobing of the blot with
total Akt antibody demonstrates equivalent protein loading. GSK, glycogen
synthase kinase. Data shown are from one of three independent experiments.
INHIBITION OF VCAM-1 EXPRESSION BY CURCUMIN
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