![]() |
ARTICLE |
---|
![]() ![]() ![]() |
---|
-ADRENERGIC AGONISTS,
besides their cardiac and bronchial muscle relaxing effects, have
been shown to possess anti-inflammatory functions. These functions are
most notable during systemic inflammatory response syndrome when
lipopolysaccharide (LPS) induces the production of proinflammatory
cytokines and, at the same time, stimulates the release of
catecholamines. A number of published studies demonstrated that
-adrenergic agonists suppress LPS-induced production of tumor
necrosis factor (TNF)-
(24, 31) and, in some cases, interleukin (IL)-1
(11) and IL-6 (33). It
is believed that accumulation of intracellular cAMP after activation of
adenylyl cyclase by Gs
proteins that couple to
-adrenergic receptors is responsible for the suppressive effect of
-adrenergic agonists. Other agents that elevate intracellular cAMP,
e.g., forskolin (30, 34), prostaglandin (PG)
E2 (28), and theophylline (31), have anti-inflammatory properties similar to those of
-adrenergic agonists. Although these findings were made years ago, the mechanism by
which cAMP negatively regulates cytokine production remains incompletely understood.
TNF- and IL-1
are produced primarily by monocytes in response to
LPS stimulation. Like other cytokine proteins, TNF-
and IL-1
production are regulated at transcriptional and posttranscriptional levels. It was reported that elevation of cAMP has no effect on IL-1
mRNA accumulation (31). mRNA obtained from cells treated with PGs, theophylline, cholera toxin, or dibutyryl cAMP was able to
produce IL-1
when injected into frog oocytes similar to the production from untreated cells (10). Cells
treated with cAMP-elevating agents have unchanged levels of
cell-associated IL-1
but have reduced IL-1
secretion
(34). Unlike IL-1
, the production of TNF-
in
monocytes appears to be regulated primarily at the transcriptional level. Agents that increase intracellular cAMP level have been shown to
reduce TNF-
messages (31) but have no effect on its stability (30). Transcription of the TNF-
gene is
regulated by nuclear factor (NF)-
B. Therefore, factors that
influence the translocation and activation of NF-
B proteins as well
as the phosphorylation and degradation of the inhibitory protein for NF-
B (I
B) are targets of regulation for TNF-
gene expression.
NF-B is a ubiquitously expressed and highly regulated dimeric
transcription factor (2, 3). Although initially found constitutively activated in plasma cells (23), NF-
B
remains dormant in most other cell types due to association with I
B
proteins (1). A large number of extracellular stimuli,
including LPS, TNF-
, IL-1
, phorbol ester, viral infection,
ionizing irradiation, and selective agonists for G protein-coupled
receptors, can induce rapid phosphorylation of I
B proteins
(9), which subsequently degrade by a ubiquitin-mediated
process (5). The released p50/p65 (Rel A) proteins then
migrate to the nucleus, bind the
B sequence, and induce
transcriptional activation. Protein kinases play an important role in
NF-
B activation through phosphorylation of I
B-
and I
B-
,
which is necessary for I
B degradation (9), as well as
through phosphorylation of p65, which is required for the effective
recruitment of coactivators such as proteins that bind the
cAMP-responsive element-binding protein (CREB) (15, 16,
37).
A paper published in this issue by Farmer and Pugin (6a) examined
a collection of -adrenergic agonists and antagonists for their
effects on LPS-induced TNF-
and IL-8 production in the THP-1 human
monocytic cell line. Results obtained from that study indicate a
long-term (>1-h and up to 8-h) effect of cAMP-elevating agents in the
inhibition of NF-
B activation. Treatment of the cells with
isoproterenol did not have an immediate effect on nuclear translocation
and DNA binding of NF-
B proteins as measured by electrophoresis
mobility shift assay. However, in 3 h, a decrease in DNA binding
by NF-
B proteins was observed. Isoproterenol did not affect the
degradation of I
B-
, which occurred within 30 min after LPS
stimulation. However, a significant increase in I
B-
protein level
was observed 3 h after treatment with LPS in the presence of
isoproterenol. This increase became more prominent 8 h after the
cells were stimulated. PGE2, which also induces elevation
of intracellular cAMP, produced an effect similar to that of
isoproterenol. H-89, a potent inhibitor of cAMP-dependent protein
kinase [protein kinase A (PKA)], blocked this effect of isoproterenol. These results suggest that elevated cAMP is responsible for the increased cellular I
B-
level.
The finding that isoproterenol used together with LPS increased the
level of IB-
raises the question of whether and how isoproterenol
induces I
B-
production. Farmer and Pugin (6a) conducted two
experiments that provided some clues. They found that isoproterenol
alone did not induce I
B-
. Therefore, LPS supplies at least part
of the signals. It has been known that the I
B-
gene contains a
B binding site. Therefore, signals that induce NF-
B activation
may also stimulate I
B-
gene expression (4, 6, 29).
This autoregulation mechanism contributes to the synthesis of I
B-
after its degradation. Farmer and Pugin (6a) then showed an increased
half-life of I
B-
protein in isoproterenol-treated cells. These
results when combined suggest that isoproterenol stabilizes newly
synthesized I
B-
, although how the
-adrenergic agonist affects
the half-life of I
B-
protein remains unclear.
The above findings appear to be in agreement with a previous
report (28) that dibutyryl cAMP when added 1.5-3 h
post-LPS stimulation was still able to reduce TNF- production.
Another study (32) demonstrated an early inhibitory effect
of cAMP on TNF-
production that occurs shortly after treatment of
the cells and peaks within 4 h after LPS stimulation. These
studies suggest the presence of multiple mechanisms for NF-
B
suppression by cAMP. Using THP-1 cells and human umbilical vein
endothelial cells (HUVECs), Ollivier et al. (17) and Parry
and Mackman (19) found that elevated cAMP
inhibited NF-
B-mediated transcription of the tissue factor gene that
contains a
B binding site. These studies were conducted within
1 h after LPS stimulation and demonstrated unaffected p65
translocation to the nucleus in cells treated with forskolin. In either
LPS-stimulated THP-1 cells or TNF-
-stimulated HUVECs, a gel mobility
shift assay showed unaltered NF-
B binding pattern, and
phosphorylation of p65 was not noticeably changed in the presence or
absence of forskolin (17). Using a GAL4-p65 chimeric
reporter, these investigators subsequently found that in THP-1 cells,
PKA-mediated phosphorylation of CREB leads to recruitment of
CREB-binding protein (CBP), which is a limiting factor for effective
transcription by NF-
B. Thus phosphorylated CREB competes with p65
for CBP, resulting in reduced NF-
B activation (19).
Gerritsen et al. (8), Sheppard et al.
(26), and Wadgaonkar et al. (35) provided
additional data that demonstrated the importance of CBP in NF-B
activation. Nuclear microinjection of antibodies against CBP or the
CBP-associated factor p/CAF prevented NF-
B activation
(27). Gerritsen et al. (8) also
demonstrated a direct interaction of CBP and p300 (a protein related to
CBP) with p65. These authors showed that inhibition of NF-
B
activation by the adenovirus E1A 12S protein, which complexes with CBP
and p300, could be reversed by overexpression of CBP or p300. That CBP
is a limiting factor for NF-
B activation was also suggested by a
study in which the glucocorticoid receptor was shown to compete with
p65 for CBP and SRC-1, the steroid receptor coactivator-1 that is
required for maximal NF-
B activity (26). Ultraviolet light-induced p53 activity represses p65-mediated transcription, apparently by competition for the limited CBP (35).
PKA is an immediate effector of elevated cytosolic cAMP. It is
therefore reasonable to speculate that the NF-B-suppressive effect
of cAMP-elevating agents is mediated through PKA. Indeed, the potent
PKA inhibitor H-89 has been shown to abrogate suppression of NF-
B by
elevated cAMP (6a). Activation of PKA, however, does not always lead to
the suppression of NF-
B. Zhong et al. (36) provided
extensive experimental data demonstrating that NF-
B activation can
be enhanced by increased PKA activity, an effect independent of cAMP.
They showed association of the 42-kDa catalytic subunit of PKA with
I
B proteins in a manner similar to that with the regulatory subunit
of PKA. Signals (e.g., LPS) that induce degradation of I
B proteins
release and therefore activate the catalytic subunit of PKA, which then
phosphorylates the nearby p65 protein at Ser276. The p65
protein contains a number of potential phosphorylation sites for PKA. Phosphorylation of
Ser276 by PKA presumably leads to a
change in p65 conformation, exposing a binding site for CBP and p300
(36). Association of p65 with CBP/p300 creates a
"transcriptional synergy" (15), thereby promoting NF-
B activation. This mechanism is supported by data published in
another study (12) demonstrating a transient increase in NF-
B binding activity in splenocytes after stimulation with
forskolin. However, PKA activity was sustained for 2 h, whereas
B binding activity peaked at 30 min and returned to basal level by
2 h.
An obvious question to the aforementioned effect of PKA is whether
activation of PKA invariably phosphorylates p65 and therefore enhances
NF-B activation. Zhong et al. (36) showed that
dibutyryl cAMP-induced PKA activation does not result in a significant
increase in p65 phosphorylation (36). They suggested that
efficient p65 phosphorylation occurs only when the catalytic subunit of
PKA is activated as a result of I
B degradation, leading to
phosphorylation of the closely associated p65. Indeed, an independent
study demonstrated that treatment of HUVECs with forskolin did not
increase p65 phosphorylation or alter TNF-
-induced p65
phosphorylation (17). Thus it appears that elevated cAMP
induces PKA-dependent phosphorylation of CREB (19) but not
of p65.
The above studies when combined suggest that cAMP imposes both
short-term and long-term effects on NF-B activation. In the short
term (within 1 h after LPS stimulation), cAMP-stimulated activation of PKA results in phosphorylation of CREB, which then competes with p65 for recruitment of CBP and p300. The long-term effects include stabilization of I
B proteins and may involve other
secondary functions of PKA. It was reported that elevated cAMP
potentiates LPS-induced production of IL-10 (20, 32), a
cytokine generally considered to be anti-inflammatory, and has recently
been shown to inhibit I
B kinases and DNA binding by NF-
B
(22). Finally, it is noted that although both the
short-term and long-term NF-
B-inhibitory effects of cAMP appear to
be mediated through PKA, activation of PKA does not always lead to
suppression of NF-
B.
Inhibition of NF-B activation provides an effective
anti-inflammatory mechanism for
-adrenergic agonists, which act on
heptahelical receptors coupled to Gs
proteins. It is
interesting to note that many other G protein-coupled receptors
transduce signals that lead to activation of NF-
B. These receptors
bind a broad spectrum of agonists including thrombin (14,
21), platelet-activating factor (13),
endothelin (7), bradykinin (18),
and lysophosphatidic acid (25). The actions of these
agonists likely are mediated through other G
and G
proteins.
Thus G protein-coupled heptahelical receptors possess the ability to
regulate transcription events by selective activation of one or another
class of G
proteins.
![]() |
REFERENCES |
---|
![]() ![]() ![]() |
---|
1.
Baeuerle, PA,
and
Baltimore D.
IB: a specific inhibitor of the NF-
B transcription factor.
Science
242:
540-546,
1988[ISI][Medline].
2.
Baeuerle, PA,
and
Baltimore D.
NF-B: ten years after.
Cell
87:
13-20,
1996[ISI][Medline].
3.
Baldwin, ASJ
The NF-B and I
B proteins: new discoveries and insights.
Annu Rev Immunol
14:
649-681,
1996[ISI][Medline].
4.
Brown, K,
Park S,
Kanno T,
Franzoso G,
and
Siebenlist U.
Mutual regulation of the transcriptional activator NF-B and its inhibitor, I
B-
.
Proc Natl Acad Sci USA
90:
2532-2536,
1993[Abstract].
5.
Chen, ZJ,
Parent L,
and
Maniatis T.
Site-specific phosphorylation of IB-
by a novel ubiquitination-dependent protein kinase activity.
Cell
84:
853-862,
1996[ISI][Medline].
6.
Chiao, PJ,
Miyamoto S,
and
Verma IM.
Autoregulation of IB-
activity.
Proc Natl Acad Sci USA
91:
28-32,
1994[Abstract].
6a.
Farmer, P,
and
Pugin J.
-Adrenergic agonists exert their "anti-inflammatory" effects in monocytic cells through the I
B/NF-
B pathway.
Am J Physiol Lung Cell Mol Physiol
279:
L675-L682,
2000
7.
Gallois, C,
Habib A,
Tao J,
Moulin S,
Maclouf J,
Mallat A,
and
Lotersztajn S.
Role of NF-B in the antiproliferative effect of endothelin-1 and tumor necrosis factor-
in human hepatic stellate cells. Involvement of cyclooxygenase-2.
J Biol Chem
273:
23183-23190,
1998
8.
Gerritsen, ME,
Williams AJ,
Neish AS,
Moore S,
Shi Y,
and
Collins T.
CREB-binding protein/p300 are transcriptional coactivators of p65.
Proc Natl Acad Sci USA
94:
2927-2932,
1997
9.
Ghosh, S,
and
Baltimore D.
Activation in vitro of NF-B by phosphorylation of its inhibitor I
B.
Nature
344:
678-682,
1990[ISI][Medline].
10.
Knudsen, PJ,
Dinarello CA,
and
Strom TB.
Prostaglandins posttranscriptionally inhibit monocyte expression of interleukin 1 activity by increasing intracellular cyclic adenosine monophosphate.
J Immunol
137:
3189-3194,
1986
11.
Koff, WC,
Fann AV,
Dunegan MA,
and
Lachman LB.
Catecholamine-induced suppression of interleukin-1 production.
Lymphokine Res
5:
239-247,
1986[ISI][Medline].
12.
Koh, WS,
Jeon YJ,
Herring AC,
and
Kaminski NE.
Transient CRE- and B site-binding is cross-regulated by cAMP-dependent protein kinase and a protein phosphatase in mouse splenocytes.
Life Sci
60:
425-432,
1997[ISI][Medline].
13.
Kravchenko, VV,
Pan Z,
Han J,
Herbert JM,
Ulevitch RJ,
and
Ye RD.
Platelet-activating factor induces NF-kappa B activation through a G protein-coupled pathway.
J Biol Chem
270:
14928-14934,
1995
14.
Maruyama, I,
Shigeta K,
Miyahara H,
Nakajima T,
Shin H,
Ide S,
and
Kitajima I.
Thrombin activates NF-kappa B through thrombin receptor and results in proliferation of vascular smooth muscle cells: role of thrombin in atherosclerosis and restenosis.
Ann NY Acad Sci
811:
429-436,
1997[Abstract].
15.
Merika, M,
Williams AJ,
Chen G,
Collins T,
and
Thanos D.
Recruitment of CBP/p300 by the IFN enhanceosome is required for synergistic activation of transcription.
Mol Cell
1:
277-287,
1998[ISI][Medline].
16.
Naumann, M,
and
Scheidereit C.
Activation of NF-B in vivo is regulated by multiple phosphorylations.
EMBO J
13:
4597-4607,
1994[Abstract].
17.
Ollivier, V,
Parry GCN,
Cobb RR,
de Prost D,
and
Mackman N.
Elevated cyclic AMP inhibits NF-B-mediated transcription in human monocytic cells and endothelial cells.
J Biol Chem
271:
20828-20835,
1996
18.
Pan, ZK,
Zuraw BL,
Lung CC,
Prossnitz ER,
Browning DD,
and
Ye RD.
Bradykinin stimulates NF-B activation and interleukin-1
gene expression in cultured human fibroblasts.
J Clin Invest
98:
2042-2049,
1996
19.
Parry, GC,
and
Mackman N.
Role of cyclic AMP response element-binding protein in cyclic AMP inhibition of NF-B-mediated transcription.
J Immunol
159:
5450-5456,
1997[Abstract].
20.
Platzer, C,
Meisel C,
Vogt K,
Platzer M,
and
Volk HD.
Up-regulation of monocytic IL-10 by tumor necrosis factor- and cAMP elevating drugs.
Int Immunol
7:
517-523,
1995[Abstract].
21.
Rahman, A,
Anwar KN,
True AL,
and
Malik AB.
Thrombin-induced p65 homodimer binding to downstream NF-B site of the promoter mediates endothelial ICAM-1 expression and neutrophil adhesion.
J Immunol
162:
5466-5476,
1999
22.
Schottelius, AJ,
Mayo MW,
Sartor RB,
and
Baldwin AS, Jr.
Interleukin-10 signaling blocks inhibitor of B kinase activity and nuclear factor
B DNA binding.
J Biol Chem
274:
31868-31874,
1999
23.
Sen, R,
and
Baltimore D.
Multiple nuclear factors interact with the immunoglobulin enhancer sequences.
Cell
46:
705-716,
1986[ISI][Medline].
24.
Severn, A,
Rapson NT,
Hunter CA,
and
Liew FY.
Regulation of tumor necrosis factor production by adrenaline and -adrenergic agonists.
J Immunol
148:
3441-3445,
1992
25.
Shahrestanifar, M,
Fan X,
and
Manning DR.
Lysophosphatidic acid activates NF-B in fibroblasts. A requirement for multiple inputs.
J Biol Chem
274:
3828-3833,
1999
26.
Sheppard, KA,
Phelps KM,
Williams AJ,
Thanos D,
Glass CK,
Rosenfeld MG,
Gerritsen ME,
and
Collins T.
Nuclear integration of glucocorticoid receptor and nuclear factor-B signaling by CREB-binding protein and steroid receptor coactivator-1.
J Biol Chem
273:
29291-29294,
1998
27.
Sheppard, KA,
Rose DW,
Haque ZK,
Kurokawa R,
McInerney E,
Westin S,
Thanos D,
Rosenfeld MG,
Glass CK,
and
Collins T.
Transcriptional activation by NF-B requires multiple coactivators.
Mol Cell Biol
19:
6367-6378,
1999
28.
Spengler, RN,
Spengler ML,
Lincoln P,
Remick DG,
Strieter RM,
and
Kunkel SL.
Dynamics of dibutyryl cyclic AMP- and prostaglandin E2-mediated suppression of lipopolysaccharide-induced tumor necrosis factor- gene expression.
Infect Immun
57:
2837-2841,
1989[ISI][Medline].
29.
Sun, SC,
Ganchi PA,
Ballard DW,
and
Greene WC.
NF-B controls expression of inhibitor I
B-
: evidence for an inducible autoregulatory pathway.
Science
259:
1912-1915,
1993[ISI][Medline].
30.
Taffet, SM,
Singhel KJ,
Overholtzer JF,
and
Shurtleff SA.
Regulation of tumor necrosis factor expression in a macrophage-like cell line by lipopolysaccharide and cyclic AMP.
Cell Immunol
120:
291-300,
1989[ISI][Medline].
31.
Talmadge, J,
Scott R,
Castelli P,
Newman-Tarr T,
and
Lee J.
Molecular pharmacology of the -adrenergic receptor on THP-1 cells.
Int J Immunopharmacol
15:
219-228,
1993[ISI][Medline].
32.
Van der Poll, T,
Coyle SM,
Barbosa K,
Braxton CC,
and
Lowry SF.
Epinephrine inhibits tumor necrosis factor- and potentiates interleukin 10 production during human endotoxemia.
J Clin Invest
97:
713-719,
1996
33.
Van der Poll, T,
Jansen J,
Endert E,
Sauerwein HP,
and
van Deventer SJ.
Noradrenaline inhibits lipopolysaccharide-induced tumor necrosis factor and interleukin 6 production in human whole blood.
Infect Immun
62:
2046-2050,
1994[Abstract].
34.
Viherluoto, J,
Palkama T,
Silvennoinen O,
and
Hurme M.
Cyclic adenosine monophosphate decreases the secretion, but not the cell-associated levels, of interleukin-1 in lipopolysaccharide-activated human monocytes.
Scand J Immunol
34:
121-125,
1991[ISI][Medline].
35.
Wadgaonkar, R,
Phelps KM,
Haque Z,
Williams AJ,
Silverman ES,
and
Collins T.
CREB-binding protein is a nuclear integrator of nuclear factor-B and p53 signaling.
J Biol Chem
274:
1879-1882,
1999
36.
Zhong, H,
SuYang H,
Erdjument-Bromage H,
Tempst P,
and
Ghosh S.
The transcriptional activity of NF-B is regulated by the I
B-associated PKAc subunit through a cyclic AMP-independent mechanism.
Cell
89:
413-424,
1997[ISI][Medline].
37.
Zhong, H,
Voll RE,
and
Ghosh S.
Phosphorylation of NF-B p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300.
Mol Cell
1:
661-671,
1998[ISI][Medline].
Richard D. Ye, Associate Editor Department of Pharmacology, College of Medicine, University of Illinois, Chicago, Illinois 60612 American Journal of Physiology- Lung Cellular and Molecular Physiology October 2000, Volume 279 (23) |