Cardiac-specific blockade of NF-kappaB in cardiac pathophysiology: differences between acute and chronic stimuli in vivo.
ABSTRACT The role of NF-kappaB in cardiac physiology and pathophysiology has been difficult to delineate due to the inability to specifically block NF-kappaB signaling in the heart. Cardiac-specific transgenic models have recently been developed that repress NF-kappaB activation by preventing phosphorylation at specific serine residues of the inhibitory kappaB (IkappaB) protein isoform IkappaBalpha. However, these models are unable to completely block NF-kappaB because of a second signaling pathway that regulates NF-kappaB function via Tyr42 phosphorylation of IkappaBalpha. We report the development of transgenic (3M) mouse lines that express the mutant IkappaBalpha(S32A,S36A,Y42F) in a cardiac-specific manner. NF-kappaB activation in cardiomyopathic TNF-1.6 mice is completely blocked by the 3M transgene but only partially blocked (70-80%) by the previously described double-mutant 2M [IkappaBalpha(S32A,S36A)] transgene, which demonstrates the action of two proximal pathways for NF-kappaB activation in TNF-alpha-induced cardiomyopathy. In contrast, after acute stimuli including administration of TNF-alpha and ischemia-reperfusion (I/R), NF-kappaB activation is blocked in both 2M and 3M transgenic mice. This result suggests that phosphorylation of the regulatory Ser32 and Ser36 predominantly mediates NF-kappaB activation in these situations. We show that infarct size after I/R is reduced by 70% in 3M transgenic mice, which conclusively demonstrates that NF-kappaB is involved in I/R injury. In summary, we have engineered novel transgenic mice that allow us to distinguish two major proximal pathways for NF-kappaB activation. Our results demonstrate that the serine and tyrosine phosphorylation pathways are differentially activated during different pathophysiological processes (cardiomyopathy and I/R injury) and that NF-kappaB contributes to infarct development after I/R.
- SourceAvailable from: Yiru Guo[show abstract] [hide abstract]
ABSTRACT: The goal of this study was to interrogate the role of inducible NO synthase (iNOS) in the late phase of ischemic preconditioning (PC) in vivo. A total of 321 mice were used. Wild-type mice preconditioned 24 h earlier with six cycles of 4-min coronary occlusion/4-min reperfusion exhibited a significant (P < 0.05) increase in myocardial iNOS protein content, iNOS activity (assessed as calcium-independent L-citrulline formation), and nitrite + nitrate tissue levels. In contrast, endothelial NOS protein content and calcium-dependent NOS activity remained unchanged. No immunoreactive neuronal NOS was detected. When wild-type mice were preconditioned 24 h earlier with six 4-min occlusion/4-min reperfusion cycles, the size of the infarcts produced by a 30-min coronary occlusion followed by 24 h of reperfusion was reduced markedly (by 67%; P < 0.05) compared with sham-preconditioned controls, indicating a late PC effect. In contrast, when mice homozygous for a null iNOS allele were preconditioned 24 h earlier with the same protocol, infarct size was not reduced. Disruption of the iNOS gene had no effect on early PC or on infarct size in the absence of PC. These results demonstrate that (i) the late phase of ischemic PC is associated with selective up-regulation of iNOS, and (ii) targeted disruption of the iNOS gene completely abrogates the infarct-sparing effect of late PC (but not of early PC), providing unequivocal molecular genetic evidence for an obligatory role of iNOS in the cardioprotection afforded by the late phase of ischemic PC. Thus, this study identifies a specific protein that mediates late PC in vivo.Proceedings of the National Academy of Sciences 10/1999; 96(20):11507-12. · 9.74 Impact Factor
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ABSTRACT: Dithiocarbamates and iron chelators were recently considered for the treatment of AIDS and neurodegenerative diseases. In this study, we show that dithiocarbamates and metal chelators can potently block the activation of nuclear factor kappa B (NF-kappa B), a transcription factor involved in human immunodeficiency virus type 1 (HIV-1) expression, signaling, and immediate early gene activation during inflammatory processes. Using cell cultures, the pyrrolidine derivative of dithiocarbamate (PDTC) was investigated in detail. Micromolar amounts of PDTC reversibly suppressed the release of the inhibitory subunit I kappa B from the latent cytoplasmic form of NF-kappa B in cells treated with phorbol ester, interleukin 1, and tumor necrosis factor alpha. Other DNA binding activities and the induction of AP-1 by phorbol ester were not affected. The antioxidant PDTC also blocked the activation of NF-kappa B by bacterial lipopolysaccharide (LPS), suggesting a role of oxygen radicals in the intracellular signaling of LPS. This idea was supported by demonstrating that treatment of pre-B and B cells with LPS induced the production of O2- and H2O2. PDTC prevented specifically the kappa B-dependent transactivation of reporter genes under the control of the HIV-1 long terminal repeat and simian virus 40 enhancer. The results from this study lend further support to the idea that oxygen radicals play an important role in the activation of NF-kappa B and HIV-1.Journal of Experimental Medicine 06/1992; 175(5):1181-94. · 13.21 Impact Factor
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ABSTRACT: The eukaryotic transcription factor NF-kappa B plays a central role in the induced expression of human immunodeficiency virus type 1 and in many aspects of the genetic program mediating normal T-cell activation and growth. The nuclear activity of NF-kappa B is tightly regulated from the cytoplasmic compartment by an inhibitory subunit called I kappa B alpha. This cytoplasmic inhibitor is rapidly phosphorylated and degraded in response to a diverse set of NF-kappa B-inducing agents, including T-cell mitogens, proinflammatory cytokines, and viral transactivators such as the Tax protein of human T-cell leukemia virus type 1. To explore these I kappa B alpha-dependent mechanisms for NF-kappa B induction, we identified novel mutants of I kappa B alpha that uncouple its inhibitory and signal-transducing functions in human T lymphocytes. Specifically, removal of the N-terminal 36 amino acids of I kappa B alpha failed to disrupt its ability to form latent complexes with NF-kappa B in the cytoplasm. However, this deletion mutation prevented the induced phosphorylation, degradative loss, and functional release of I kappa B alpha from NF-kappa B in Tax-expressing cells. Alanine substitutions introduced at two serine residues positioned within this N-terminal regulatory region of I kappa B alpha also yielded constitutive repressors that escaped from Tax-induced turnover and that potently inhibited immune activation pathways for NF-kappa B induction, including those initiated from antigen and cytokine receptors. In contrast, introduction of a phosphoserine mimetic at these sites rectified this functional defect, a finding consistent with a causal linkage between the phosphorylation status and proteolytic stability of this cytoplasmic inhibitor. Together, these in vivo studies define a critical signal response domain in I kappa B alpha that coordinately controls the biologic activities of I kappa B alpha and NF-kappa B in response to viral and immune stimuli.Molecular and Cellular Biology 06/1995; 15(5):2809-18. · 5.37 Impact Factor
CARDIAC-SPECIFIC BLOCKADE OF NF-kappaB IN CARDIAC PATHOPHYSIOLOGY:
DIFFERENCES BETWEEN ACUTE AND CHRONIC STIMULI IN VIVO.
Maria A. Brown#, Michael McGuinness#, Terry Wright, Xiaoping Ren, Yang Wang, Gregory P. Boivin*,
Harvey S. Hahn†, Arthur M. Feldman¶ and W. Keith Jones
Department of Pharmacology and Cell Biophysics, Department of Pathology and Laboratory
Medicine*, Division of Cardiology, Department of Medicine,† University of Cincinnati, Cincinnati, Ohio
45267, Department of Medicine¶, Jefferson Medical College, Philadelphia, PA 19107
# These authors contributed equally to the study.
Running Title: NF-κB blockade in Cardiac Pathophysiology
Address for correspondence: W. Keith Jones
Department of Pharmacology and Cell Biophysics
231 Albert Sabin Way ML0575
University of Cincinnati
Cincinnati, OH 45267-0575
Telephone: (513) 558-2330
Fax: (513) 558-1169
Articles in PresS. Am J Physiol Heart Circ Physiol (February 4, 2005). doi:10.1152/ajpheart.00170.2004
Copyright © 2005 by the American Physiological Society.
The role of NF-κB in cardiac physiology and pathophysiology has been difficult to delineate due to
the inability to specifically block NF-κB signaling in the heart. Cardiac specific transgenic models have
recently been developed that repress NF-κB activation by preventing phosphorylation at specific
serine residues of IκBα. However, these models are unable to completely block NF-κB because of a
second signaling pathway that regulates NF-κB function via Tyr42 phosphorylation of IκBα. We
report the development of transgenic (3M) mouse lines that express a mutant IκBαS32A,S36A,Y42F in a
cardiac specific manner. NF-κB activation in cardiomyopathic TNF1.6 mice is completely blocked by
the 3M transgene, but only partially blocked (70-80%) by the previously described double mutant 2M
(IκBαS32A,S36A) transgene, demonstrating the action of two proximal pathways for NF-κB activation in
TNF-α-induced cardiomyopathy. In contrast, after acute stimuli, including administration of TNF-α and
ischemia/reperfusion (I/R) in vivo, NF-κB activation is blocked in both the 2M and 3M transgenic mice.
This result suggests that phosphorylation of the regulatory Ser32,36 predominantly mediates NF-κB
activation in these situations. We show that infarct size after I/R is reduced by 70% in 3M transgenic
mice, conclusively demonstrating that NF-κB is involved in I/R injury. In summary, we have
engineered novel transgenic mice that allow us to distinguish two major proximal pathways for NF-κB
activation. Our results demonstrate that the serine and tyrosine phosphorylation pathways are
differentially activated during different pathophysiological processes (cardiomyopathy and I/R injury)
and that NF-κB contributes to infarct development after I/R.
Keywords, NF-kappaB, TNF-alpha, signal transduction, ischemia/reperfusion
Since nuclear factor kappa-B (NF-κB) was discovered in 1986 (57), considerable research has been
directed towards its characterization. NF-κB exists as a dimer of related proteins classified as Rel
family members due to the presence of a rel-homology domain. There are five mammalian family
members, p50/p105 (NF-κB1), p52/p100 (NF-κB2), p65 (RelA), c-rel and RelB. NF-κB dimers typically
consist of a p50/p105 or p52/p100 subunit complexed with another member of the Rel protein family,
with the most common form being p65/p50 (2). Generally, NF-κB dimers associate with an inhibitory
κB (IκB) protein that acts to inhibit NF-κB function. Although there are seven known members of the
IκB protein family, regulation of NF-κB activity is most frequently mediated by the two most common
isoforms, IκBα and IκBβ.
A diverse range of stimuli can induce either phosphorylation of two regulatory serines (Ser32,36) or
phosphorylation of a single regulatory tyrosine (Tyr42) within the amino terminus of the IκB protein. In
addition to the regulatory phosphorylation sites (Ser32,36 and Tyr42), IκBα also contains eleven
additional serines and seven carboxy-terminal tyrosines, several of which are known to be
phosphorylated. Phosphorylation of IκB at Ser32,36 by the IκB kinaseβ (IKKβ) is the predominant
mechanism by which NF-κB becomes activated and is operative during ischemia (31,47). Tumor
necrosis factor-α (TNF-α) as well as other signaling pathways activate the IκB kinase complex (IKK),
which in turn phosphorylates Ser32 and Ser36 of IκBα, leading to ubiquitination and immediate
degradation of IκB (55). It is less well known that Tyr42 of IκBα is subject to phosphorylation in
response to several stimuli (nerve growth factor, interferon-γ (INF-γ) and, in macrophages, TNF-α) (7;
25). Phosphorylation of IκB at Tyr42 may involve the protein tyrosine kinases (PTKs) c-Src, p56 (lck)
and ZAP-70, and causes IκB to dissociate from NF-κB without immediate proteolysis (1,11,34,35).
Degradation or dissociation of NF-κB from IκB via either the serine or tyrosine phosphorylation
pathway enhances nuclear localization of NF-κB and is necessary for activation of NF-κB-dependent
genes. Although phosphorylation of other Ser, Thr and Tyr moieties within IκBα is known to occur,
both constitutively and inducibly, these sites are thought not to affect NF-κB activation directly
(reviewed in 2, 25).
More than 200 genes are known to be regulated by NF-κB. Thus, it is not surprising that this
transcription factor affects a multitude of biological processes in response to diverse stimuli and
pathophysiological states. Genes of cardiovascular relevance that are regulated, at least in part, by
NF-κB include those that function in nitric oxide (NO) production, prostaglandin biosynthesis, calcium
handling, cardiomyocyte function, cell death/survival, stress responses, natriuretic factors, growth
factors, extracellular matrix (ECM), remodeling/cell adhesion, and antioxidant proteins (2,4,22,23).
The effects of NF-κB on cell death and survival are complex, in that it can exert both anti-apoptotic
and pro-apoptotic effects; NF-κB can activate very different sets of genes depending upon the
specific kinetics of its activation (1). In the heart, NF-κB has been implicated in ischemia/reperfusion
(I/R) injury (8,40,45,52), preconditioning (19,41,71), unstable angina pectoris (50,65), myocarditis
(53), congestive heart failure (17), hypertrophy of isolated cardiomyocytes (17,20,21,48) and dilated
cardiomyopathy (6,28,59). Transgenic mice with cardiac-specific expression of TNF-α (TNF1.6 mice)
develop severe dilated cardiomyopathy with reduced contractile function and rapidly progress to
heart failure (28). NF-κB is activated (19,29) and several NF-κB dependent gene products, including
matrix metalloproteinases (mmps) and inducible nitric oxide synthase (iNOS) have been implicated in
the pathophysiology exhibited by these mice (14,29,33). Clinical studies show that NF-κB activation is
increased in human heart failure and there is an association between reduced NF-κB activity and
beneficial reverse remodeling in heart failure patients with left ventricular assist devices (15,51,68).
The transcriptional inhibitors pentoxifylline and thalidomide have yielded promising results in small
clinical trials for cardiomyopathy (38,60) and NF-κB inhibition is implicated in the mechanism of each
of these drugs (37,38,67). Unfortunately, all of the drugs that block NF-κB activity are relatively
nonspecific. Although pharmacological evidence supports that NF-κB is pro-cell death in I/R injury
(24,47,52), NF-κB suppresses apoptosis and is pro-cell survival in isolated rat ventricular myocytes
and after permanent coronary occlusion in vivo (43,46). Thus, NF-κB appears to mediate opposing
effects and the role of NF-κB in specific cardiac pathophysiology remains unclear.
We have engineered transgenic 3M mice expressing a dominant-negative IκBα with mutations of the
amino-terminal serines and the tyrosine that mediate NF-κB activation (IκBαS32A,S36A,Y42F). Like the
previously described 2M (IκBαS32A,S36A) transgenic mice (10), these mice exhibit normal cardiac
morphology, histopathology and physiology. Previous work with similar constructs in cultured cells has
validated that the mechanism of action of these dominant-negative non-phosphorylatable mutant
IκBα proteins is competitive titration of the endogenous IκB proteins from NF-κB. Furthermore, use of
the IκBαS32A,S36A and IκBαS32A,S36A,Y42F mutants has validated that the two proximal pathways that
mediate NF-κB activation, by IκB kinase (IKK) dependent phosphorylation of Ser32,36 or Tyr42, are
separable and have additive effects in the IκBαS32A,S36A,Y42F expressing human glioma cells (44).
Activation of NF-κB in response to cytokines, ischemia/reperfusion and TNF-induced cardiomyopathy
(i.e. TNF1.6 mice) is completely absent in 3M transgenic mice. Interestingly, the 2M transgene blocks
only 70-80% of NF-κB activation in TNF1.6 cardiomyopathic mice, despite the fact that the 2M line
expresses relatively more of the transgenic dominant-negative IκBα protein (10). This suggests that
the Tyr42 of the 2M IκBα protein is phosphorylated in TNF1.6/2M double transgenic mice, implicating
the two distinct pathways for NF-κB activation (e.g. Ser32,36 and Tyr42 phosphorylation) in this
cardiomyopathic model. Both the 2M and 3M transgenes completely block NF-κB activation after
acute TNF-α administration and after I/R injury, suggesting that phosphorylation of IκBα on Ser32,36
is the predominant pathway for NF-κB activation after these acute stimuli. Finally, we demonstrate
that NF-κB activation in the heart contributes to cell death and myocardial infarction in response to
I/R injury in vivo.
MATERIALS AND METHODS
Transgenic constructs and mice. The transgene used to construct the 3M mice was derived by in vitro
mutagenesis (see online supplement) from the plasmid pCMV4-FIκBα-Y42F, the kind gift of D. W.
Ballard (Vanderbilt University School of Medicine, Nashville, TN). The plasmid pCMV4-FIκBα-Y42F
containing the cDNA for human IκBα with a Tyr42Phe mutation was the kind gift of D. W. Ballard This
cDNA has been shown to block Tyr-42 phosphorylation-dependent activation of NF-κB (5). The cDNA
was used as PCR target in generating the IκBαS32A,S36A,Y42F cDNA, containing the Ser32Ala, Ser36Ala
and Tyr42Phe mutations using a forward PCR primer 5'- CGATGAGTCGACAATGTTCCAGGCGGCC-3'
and a mutagenic reverse primer 5'-
TCCAGGCCGGCGTC-3'. The PCR product was subcloned into the pαMyHC vector, behind the α-