Constitutive degradation of IκBα in human T lymphocytes is mediated by calpain
© Ponnappan et al; licensee BioMed Central Ltd. 2005
Received: 11 August 2005
Accepted: 04 November 2005
Published: 04 November 2005
Activation-induced induction of transcription factor NFκB in T lymphocytes is regulated by its inhibitor IκBα. NFκB activation has been demonstrated to occur either by phosphorylation on serine residues 32 and 36 of the inhibitor, IκBα, followed by ubiquitination and degradation of the inhibitor by the 26S proteasome, or by a proteasome-independent mechanism involving tyrosine phosphorylation, but not degradation. However, the mechanism underlying constitutive regulation of the levels of the inhibitor, IκB, in primary human T lymphocytes, remains to be fully delineated.
We demonstrate here, the involvement of a proteasome-independent pathway for constitutive regulation of IκBα levels in primary human T lymphocytes. Pretreatment with a cell permeable calpain inhibitor, E64D, but not with a proteasome specific inhibitor, lactacystin, blocks stimulus-independent IκBα degradation in primary human T cells. However, E64D pre-treatment fails to impact on IκBα levels following stimulation with either TNFα or pervanadate. Other isoforms of the inhibitor, IκBβ, and IκBγ, appear not to be subject to a similar ligand-independent regulation. Unlike the previously reported decline in ligand-induced degradation of IκBα in T cells from the elderly, constitutive degradation does not exhibit an age-associated decline, demonstrating proteasome-independent regulation of the activity.
Our studies support a role for an E64D sensitive protease in regulating constitutive levels of IκBα in T cells, independent of the involvement of the 26S proteasome, and suggests a biological role for constitutive degradation of IκBα in T cells.
Transcription factor NFκB exists as homo-or-hetero-dimeric complexes, consisting of the Rel family of proteins . These dimers operate as transcriptional regulators essential for a variety of cellular processes ranging from cell cycle progression to immune response gene induction . In human T lymphocytes, like most other cells, NFκB exists in the cytoplasm coupled to its inhibitor IκBα or IκBβ, predominant members of IκB family of proteins . A high affinity for RelA and c-Rel molecules enables these inhibitory proteins to associate with and thus restrict nuclear localization of the NFκB molecules. Most stimuli responsible for NFκB induction have been demonstrated to either invoke serine phosphorylation of the inhibitory proteins followed by ubiquitination and degradation via the 26S proteasome pathway, or involve the activation of tyrosine phosphorylation, as in the case of oxidative stress mediated stimuli, which is independent of proteasomal degradation mechanism [4, 5]. While stimulation-induced modification of IκB has been studied extensively, little is known about the constitutive regulation of IκB protein in T cells under resting conditions.
Recent studies in B cell lines have demonstrated that IκBα, but not IκBβ, is constitutively degraded and is important for the induction of constitutive NFκB activity [6, 7]. These studies indicate that constitutive degradation of IκB is mediated by a proteasome independent pathway. Studies also suggest that a calcium-dependent protease, calpain, may be important in regulating levels of IκBα [8–10]. These studies prompted us to investigate whether a similar regulation of "constitutive" i.e., stimulus-independent levels of IκBα occurs in primary T lymphocytes. Unlike B cells, NFκB induction and IκB regulation reported in T cells is clearly mediated by exogenous activating stimuli, with little or no constitutive nuclear NFκB present under basal condition.
Further, as NFκB regulation is significantly altered during aging in human T cells, we examined whether abnormal constitutive regulation may underlie lowered activation-mediated induction. Employing primary T cells obtained from human donors, we evaluated whether aging affects the regulation of constitutive levels of IκBα. We hypothesized that, as constitutive levels of IκBα were relatively unaffected by age in primary T cells, this may reflect minimal effect of age on calpain activity in T cells. We now report that E64D sensitive protease, calpain, is indeed responsible for regulating constitutive levels of IκBα, but not IκBβ or IκBγ, in human T cells. Further, calcium-ionophore mediated increase in calpain activity induced in T cells from young donors showed consistently higher activity at early time points after activation, when compared to the elderly. However, total calpain activity measured at the end of 60 minutes demonstrated no significant modulation based on the age of the donor. Thus, while the kinetics of calpain activation appears to be altered in T cells from the elderly, cumulative activity over a period of time remains unaffected. Additionally, we demonstrate that aging does not significantly affect stimulus-independent degradation of IκBα mediated by calpain, demonstrating proteasome-independent regulation. Thus, the calpain system is involved in the constitutive regulation of IκBα, and hence the NFκB signaling pathway, under resting conditions, in primary human T lymphocytes.
Treatment with a cell permeable cysteine protease inhibitor, E64D, inhibits constitutive degradation of IκBα but not IκBβ or IκBγ in T lymphocytes from young and elderly donors
As IκBα degradation induced by exogenous signals has been reported to be differentially regulated during aging [8, 9], we next assessed the effect of treatment with E64D on IκB-α in T cells obtained from young and elderly donors. Results presented in fig. 1, demonstrate that, irrespective of the age of the T cell donor, E64D pretreatment significantly protected IκBα levels from constitutive degradation, and had little impact on isoforms, IκBβ (fig. 2) and IκBγ (fig. 3). Thus, aging does not influence cysteine protease-sensitive constitutive degradation of IκBα in T cells.
Inhibition of cysteine protease activity by E64D does not affect TNFα-induced degradation of IκBα in T cells
Pretreatment with E64D does not interfere with activation-induced modification mediated by pervanadate treatment in T cells
As a next step in the analyses, we examined the effect of pretreatment with E64D on modes of activation that does not involve signal-induced degradation of IκBα, such as those mediated by pervanadate. Similar to the observation with TNFα, pretreatment with E64D failed to impact on activation-induced IκBα modification in cells pre-treated with pervanadate, irrespective of the age of the donor (fig. 4 and 5B). It is important to note that IκBα in cells treated with pervanadate clearly demonstrate a slower mobility band representing tyrosine-phosphorylated IκBα. Thus, irrespective of the activation stimuli (TNFa or Pervanadate), E64D appeared not to impact on the activation-induced modulation of IκBα. NFκB dependent luciferase activity was also assayed following pervanadate treatment, in the presence or absence of E64D. In keeping with the data obtained with IκBα, E64D pretreatment, failed to impact on NFκB dependent luciferase activity, (data not shown).
Treatment of T cells with proteasome inhibitor, Lactacystin, does not influence constitutive IκB-α levels, unlike that mediated by E64D
Treatment of T cell lysates with purified calpain, mimics constitutive degradation of IκBα
Kinetics of induction of calpain activity following calcium ionophore treatment, is modulated by age, but has no impact on overall effective calpain activity in T cells
Calpain system has been demonstrated to be the main protease involved in constitutive degradation of IκBα [7–9]. Delineation of the exact region of IκBα necessary for degradation by calpain resulted in the identification of the C-terminal 39 amino acid sequence containing the PEST sequence to be critical for degradation in vitro by Shumway and Miyamoto . However, their studies, demonstrated that calpain was not responsible for the degradation of IκBα in primary B cells . Unlike B cells, that express constitutive nuclear NFκB, significant nuclear expression of NFκB in T cells occurs predominantly following stimulus-induced activation. Few studies, to date, have delved into constitutive regulation of NFκB in resting T cells. In our current studies, employing E64D, we demonstrate specificity of constitutive degradation of IκBα mediated by calpain in human primary T lymphocytes. The inhibition of this degradation in the presence of E64D, a cell permeable, cysteine protease inhibitor, supports the potential involvement of calpain activity in this process. Given the central role of NFκB in cell survival and signaling , constitutive degradation of the inhibitor IκBα is vital in understanding steady state kinetics of T cell regulation in the context of immune activation.
Our studies also demonstrate that degradation of IκBα under resting condition is refractory to proteasome inhibitor, Lactacystin, but not to calpain inhibitor, E64D. Therefore, unlike that reported for activation-induced degradation , constitutive levels of IκBα appear not to be subject to proteosomal regulation. This is particularly important given that our previous findings clearly showed that inducible degradation of IκBα is subject to an "age-effect" due to the inhibitory action of aging on proteasome- associated proteolytic activity [13, 14].
Calpain-dependent degradation of IκBα has been demonstrated to occur in other cell types, which are refractory to proteasome activity . Thus, it appears, that the degradation of IκBα can occur through two mutually exclusive pathways, dependent on the state of the cells, i.e., resting versus activated. Calpain system plays a role in constitutive, but not induced IκBα degradation, while proteasome degradation dictates induced levels in T cells [6, 12]. Calpain activity has been demonstrated to be involved in the degradation of IκBα under certain conditions of viral infection . It is therefore likely that this ability of constitutive degradation may be exploited by certain pathogens.
Unlike the most predominant inhibitor IκBα; IκBβ and IκBγ isoforms, appear not to be susceptible to this calpain-mediated degradation. Recent elegant experiments by Miyamoto et al implicate similar degradation kinetics for IκBα isoform in B cell lines . Drawing upon the significance of such degradation events in the constitutive induction of NFκB in B cells, the role for constitutive regulation of NFκB by the calpain pathway in primary T cells was examined here. Results from these experiments clearly provide a biological basis for stimulus-independent degradation and its importance in the maintenance of NFκB in cell survival, which is not evident, unless challenged by stimuli capable of inducing apoptosis (data not shown).
Our studies on the effect of advancing age on constitutive degradation of IκBα, clearly implicate absence of any effect of donor age on the maintenance of E64D protease sensitive/calpain activity responsible for this degradation. Experiments conducted to determine the impact of aging on calpain activity clearly indicate that the effective activity is unaltered during aging. This is also reflected in the levels of IκBα in resting T cells from young and elderly donors, which are unaffected by age. Thus, despite loss in proteasome activity accompanying aging, calpain-mediated degradation of IκBα remains unaltered, demonstrating little or no role for the proteasomal regulation in calpain-mediated pathway that regulates IκBα levels. This observation is in keeping with earlier reports from our laboratory that demonstrated minimal effect of age on overall cellular proteolytic activity, especially, T cell chymotryptic activity . It is also interesting to note that reports on calpain activity as a function of advancing age have been conflicting, with some demonstrating increased activity, [16, 17] and others, decreased activity [18, 19], however, these studies either used other cell types, employed exogenous substrates or cell lysates for the evaluation of the activity. Using a fluorogenic model substrate that is cell permeable, we now demonstrate that, ionomycin-inducible specific calpain activity, inhibitable by E64D, is unaffected; however, proteolytic activity observed in T cell lysates appeared to follow different kinetics in cells from the young than those observed in the elderly. Importantly, while 90% of the activity in cells from the young was clearly inhibitable by treatment with E64D, only 50% of the activity was inhibitable in cells from the elderly (data not shown).
While constitutive degradation of IκBα is clearly regulated by E64D sensitive calpain in T cells, activation-induced degradation, appears unaffected by pretreatment with E64D. Similarly, while activation-induced degradation of IκBα is sensitive to proteasome inhibition, constitutive degradation is unaffected by pretreatment with lactacystin, a proteasome inhibitor. Clearly, susceptibility of IκBα to degradation is not only dependent on the state of activation but also on the specificity of the protease. The physiologic significance of the degradation of the inhibitor clearly dictates induction of NFκB levels, and thus anti-apoptotic or survival ability, under uninduced conditions. Thus, regulation of IκBα levels in basal state of a cell is crucial and sets the stage for activation-induced survival signals. These results also indicate that the calpain pathway works independently of phosphorylation, since neither TNF nor pervanadate that induce serine and tyrosine phosphorylation, respectively, were affected by the inhibitor. Further, while proteasome dependent activation-induced degradation pathway, as well as proteasome pathway has been demonstrated to be compromised in T cells during aging, calpain activity clearly appears to be still functional, and is minimally affected by advancing age.
In summary, we have demonstrated that basal levels of IκBα, but not IκBβ or IκBγ are subject to regulation by E64D sensitive protease, and can be mimicked by pretreatment with calpain. The regulation of IκBα levels by cysteine protease appears to have no effect on activation-induced IκBα or on other isoforms of IκB, irrespective of the stimuli employed. Additionally, it appears that interference with this decrease in basal degradation of IκBα does not impact on cell survival under resting conditions.
Fluorochrome labeled anti-CD3, and FITC- and PE-labeled isotype controls were obtained from Sigma Chemical Co. (St. Louis, MO). Anti IgG coupled to horseradish peroxidase was obtained from BD-Transduction Laboratories (Lexington, KY). All other antibodies were from Santa Cruz Biotech (Carlsbad,CA). Enhanced Chemi-luminescence reagents were from Amersham (Arlington Heights, IL). All fine chemicals unless otherwise mentioned were obtained from Sigma Chemical Company, (St. Louis, MO.), Electrophoresis supplies and Molecular weight standards were from BioRad (Richmond, CA.). E64D and lactacystin were from Calbiochem (CA). Substrate for Calpain was from Molecular probes, (Eugene, OR).
Blood was obtained from healthy individuals living in the community. Young donors were between 21 and 30 years and old donors were between 65 and 85 years of age. A minimum of at least four donor pairs were used in each experiment. Both young and elderly donors were in good physical and mental health, had no apparent illness as suggested by an elaborate screening history and were not on any medication directly impacting the immune system during the course of this study.
T Lymphocyte Isolation
Peripheral blood was obtained and T cells were purified and maintained in RPMI 1640 culture medium as previously described (20). Magnetic sorting by negative selection was used to isolate CD3+ T cells. Purity of the isolated T cells was determined by flow cytometry using anti-CD3 conjugated to FITC. Populations were 90–95% pure. Treatment of T cells (20 × 106cells/ml) with pervanadate (100 μM, freshly prepared before use) was carried out for indicated times at 37°C, before cell lysates were prepared. For experiments involving the use of inhibitor, cells were treated with E64D at 50 μM for 45 minutes.
Cytosolic extracts for Western blotting were prepared by homogenization of cells in lysis buffer (1 mM Hepes, 10 mM KCl, 1.5 mM MgCl2, and 1 mM sodium orthovanadate, and 0.5% NP-40) (20). The following reagents were added to all buffers prior to their use: 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 10 μg/ml each of aprotinin, leupeptin, and soybean trypsin inhibitor. Protein content of cytosolic extracts was determined using BioRad protein assay. Cell lysates equalized for protein (40 μg) were resolved by SDS-polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose, immuno-blotted with specific antibody/s, and detected using anti-IgG coupled to horseradish peroxidase followed by Enhanced Chemi-luminescence (ECL). Where possible samples from young and elderly donors were resolved on the same gel, but in experiments where different treatments were analyzed, samples from young and the elderly were resolved on different gels, but were run simultaneously, to avoid inter and intra experimental variability. Resolution of samples on different gels did not influence the outcome of the results.
Calpain activity assays
Using a cell permeable fluorescent substrate Boc-Met-AMC, calpain activity within live cells was measured using a spectrofluorometer. Fluorescence was measured in cell suspension (2.5 × 105cells/ml) following the addition of fluorophore, using the LS-50 model, Perkin-Elmer Spectrofluorometer. The fluorometer was equipped with a magnetic stirrer and warmed with recirculating water at 37°C using a pump. Fluorescence was measured using excitation and emission wavelengths of 380 and 460 nm, respectively. Values were obtained using a time drive mode, for up to 60 minutes.
Data were analyzed using student's t-test. Differences were considered significant, if p < 0.05.
This work was supported by Grants provided by NIH RO1 AG13081, MO1RR1288 and in part by the use of facilities at the VA Medical Center, Little Rock, Arkansas. We gratefully acknowledge the technical assistance of Mrs. Virginia Fitzhugh and assistance provided by the General Clinical Research Center, in sample collection.
- Hayden MS, Ghosh S: Signaling to NF-kappaB. Gene Dev. 2004, 18: 2195-2224. 10.1101/gad.1228704.View ArticlePubMedGoogle Scholar
- Hoffmann A, Levchenko A, Scott ML, Baltimore D: The IkappaB-NF-kappaB signaling module: temporal control and selective gene activation. Science. 2002, 298: 1241-1245. 10.1126/science.1071914.View ArticlePubMedGoogle Scholar
- Israel A: The IKK complex: an integrator of all signals that activate NF-kappaB?. Trends Cell Biol. 2000, 10: 129-133. 10.1016/S0962-8924(00)01729-3.View ArticlePubMedGoogle Scholar
- Imbert V, Rupec RA, Livolsi A, Pahl HL, Traenckner EB, Mueller-Dieckmann C, Farahifar D, Rossi B, Auberger P, Baeuerle PA, Peyron JF: Tyrosine phosphorylation of I kappa B-alpha activates NF-kappa B without proteolytic degradation of I kappa B-alpha. Cell. 1996, 86: 787-798. 10.1016/S0092-8674(00)80153-1.View ArticlePubMedGoogle Scholar
- Glickman MH, Ciechanover A: The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev. 2002, 82: 373-428.View ArticlePubMedGoogle Scholar
- Shumway SD, Miyamoto S: A mechanistic insight into a proteasome-independent constitutive inhibitor kappaBalpha (IkappaBalpha) degradation and nuclear factor kappaB (NF-kappaB) activation pathway in WEHI-231 B-cells. Biochem J. 2004, 380: 173-180. 10.1042/BJ20031796.PubMed CentralView ArticlePubMedGoogle Scholar
- Huang TT, Kudo N, Yoshida M, Miyamoto S: A nuclear export signal in the N-terminal regulatory domain of IkappaBalpha controls cytoplasmic localization of inactive NF-kappaB/IkappaBalpha complexes. Proc Natl Acad Sci USA. 2000, 97: 1014-1019. 10.1073/pnas.97.3.1014.PubMed CentralView ArticlePubMedGoogle Scholar
- Waris G, Livolsi A, Imbert V, Peyron JF, Siddiqui A: Hepatitis C virus NS5A and subgenomic replicon activate NF-kappaB via tyrosine phosphorylation of IkappaBalpha and its degradation by calpain protease. J Biol Chem. 2003, 278: 40778-40787. 10.1074/jbc.M303248200.View ArticlePubMedGoogle Scholar
- Schaecher K, Goust JM, Banik NL: The effects of calpain inhibition on IkB alpha degradation after activation of PBMCs: identification of the calpain cleavage sites. Neurochem Res. 2004, 29: 1443-1451. 10.1023/B:NERE.0000026410.56000.dd.View ArticlePubMedGoogle Scholar
- Han Y, Weinman S, Boldogh I, Walker RK, Brasier AR: Tumor necrosis factor-alpha-inducible IkappaBalpha proteolysis mediated by cytosolic m-calpain. A mechanism parallel to the ubiquitin-proteasome pathway for nuclear factor-kappab activation. J Biol Chem. 1999, 274: 787-794. 10.1074/jbc.274.2.787.View ArticlePubMedGoogle Scholar
- Poulaki V, Mitsiades CS, Joussen AM, Lappas A, Kirchhof B, Mitsiades N: Constitutive nuclear factor-kappaB activity is crucial for human retinoblastoma cell viability. Am J Pathol. 2002, 161: 2229-2240.PubMed CentralView ArticlePubMedGoogle Scholar
- Alkalay I, Yaron A, Hatzubai A, Orian A, Ciechanover A, Ben-Neriah Y: Stimulation-dependent I kappa B alpha phosphorylation marks the NF-kappa B inhibitor for degradation via the ubiquitin-proteasome pathway. Proc Natl Acad Sci USA. 1995, 92: 10599-10603.PubMed CentralView ArticlePubMedGoogle Scholar
- Trebilcock GU, Ponnappan U: Evidence for lowered induction of nuclear factor kappa B in activated human T lymphocytes during aging. Gerontology. 1996, 42: 137-146.View ArticlePubMedGoogle Scholar
- Trebilcock GU, Ponnappan U: Nuclear factor-kappaB induction in CD45RO+ and CD45RA+ T cell subsets during aging. Mech Ageing Dev. 1998, 102: 149-163. 10.1016/S0047-6374(97)00160-7.View ArticlePubMedGoogle Scholar
- Ponnappan U: Ubiquitin-proteasome pathway is compromised in CD45RO+ and CD45RA+ T lymphocyte subsets during aging. Exp Gerontol. 2002, 37: 359-367. 10.1016/S0531-5565(01)00203-0.View ArticlePubMedGoogle Scholar
- Hinman JD, Duce JA, Siman RA, Hollander W, Abraham CR: Activation of calpain-1 in myelin and microglia in the white matter of the aged rhesus monkey. J Neurochem. 2004, 89: 430-441.View ArticlePubMedGoogle Scholar
- Averna M, De Tullio R, Salamino F, Minafra R, Pontremoli S, Melloni E: Age-dependent degradation of calpastatin in kidney of hypertensive rats. J Biol Chem. 2001, 276: 38426-38432. 10.1074/jbc.M101936200.View ArticlePubMedGoogle Scholar
- Guttmann RP, Johnson GV: Oxidative stress inhibits calpain activity in situ. J Biol Chem. 1998, 273: 13331-13338. 10.1074/jbc.273.21.13331.View ArticlePubMedGoogle Scholar
- Witkowski JM, Bryl E: Paradoxical age-related cell cycle quickening of human CD4 (+) lymphocytes: a role for cyclin D1 and calpain. Exp Gerontol. 2004, 39: 577-585. 10.1016/j.exger.2003.10.028.View ArticlePubMedGoogle Scholar
- Ponnappan U, Trebilcock GU, Zheng MZ: Studies into the effect of tyrosine phosphatase inhibitor phenylarsine oxide on NFkappaB activation in T lymphocytes during aging: evidence for altered IkappaB-alpha phosphorylation and degradation. Exp Gerontol. 1999, 34: 95-107. 10.1016/S0531-5565(98)00059-X.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.