Constitutive degradation of IκBα in human T lymphocytes is mediated by calpain

Transcription factor NFκB exists as homo-or-hetero-dimeric complexes, consisting of the Rel family of proteins [1]. These dimers operate as transcriptional regulators essential for a variety of cellular processes ranging from cell cycle progression to immune response gene induction [2]. 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 [3]. 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.

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 [6]. However, their studies, demonstrated that calpain was not responsible for the degradation of IκBα in primary B cells [6]. 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 [11], 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 [12], 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 [11]. 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 [8]. 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 [6]. 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 [15]. 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.