Murine RAW Macrophages Are a Suitable Model to Study the CD3 Signaling in Myeloid Cells.

In recent years, a growing body of evidence has shown the presence of a subpopulation of macrophages that express CD3, especially in the context of mycobacterial infections. Despite these findings, the function of these cells has been poorly understood. Furthermore, the low frequency of CD3+ macrophages in humans limits the study of this subpopulation. This work aimed to evaluate the expression of CD3 in a murine macrophage cell line and its potential for the study of CD3 signaling. The murine macrophage cell line RAW was used to evaluate CD3 expression at the transcriptional and protein levels and the effect of in vitro infection with the Mycobacterium bovis Bacillus Calmette-Guérin (BCG) on these. Our data showed that RAW macrophages express CD3, both the ε and ζ chains, and it is further increased at the transcriptional level after BCG infection. Furthermore, our data suggest that CD3 can be found on the cell surface and intracellularly. However, this molecule is internalized constantly, mainly after activation with anti-CD3 stimulus, but interestingly, it is stably maintained at the transcriptional level. Finally, signaling proteins such as NFAT1, c-Jun, and IKK-α are highly expressed in RAW macrophages. They may play a role in the CD3-controlled signaling pathway to deliver inflammatory cytokines such as TNF and IL-6. Our study provides evidence to support that RAW cells are a suitable model to study the function and signaling of the CD3 complex in myeloid cells.

Ubiquitination and the proteasome rather than caspase-3-mediated C-terminal cleavage are involved in the EAAT2 degradation by staurosporine-induced cellular stress.

Diminished glutamate (Glu) uptake via the excitatory amino acid transporter EAAT2, which normally accounts for ~90% of total forebrain EAAT activity, may contribute to neurodegeneration via Glu-mediated excitotoxicity. C-terminal cleavage by caspase-3 (C3) was reported to mediate EAAT2 inactivation and down-regulation in the context of neurodegeneration. For a detailed analysis of C3-dependent EAAT2 degradation, we employed A172 glioblastoma as well as hippocampal HT22 cells and murine astrocytes over-expressing VSV-G-tagged EAAT2 constructs. C3 activation was induced by staurosporine (STR). In HT22 cells, STR-induced C3 activation-induced rapid EAAT2 protein degradation. The mutation of asparagine 504 to aspartate (D504N), which should inactivate the putative C3 cleavage site, increased EAAT2 activity in A172 cells. In contrast, the D504N mutation did not protect EAAT2 protein against STR-induced degradation in HT22 cells, whereas inhibition of caspases, ubiquitination and the proteasome did. Similar results were obtained in astrocytes. Phylogenetic analysis showed that C-terminal ubiquitin acceptor sites-but not the putative C3 cleavage site-exhibit a high degree of conservation. Moreover, C-terminal truncation mimicking C3 cleavage increased rather than decreased EAAT2 activity and stability as well as protected EAAT2 against STR-induced ubiquitination-dependent degradation. We conclude that cellular stress associated with endogenous C3 activation degrades EAAT2 via a pathway involving ubiquitination and the proteasome but not direct C3-mediated cleavage. In addition, C3 cleavage of EAAT2, described to occur in other models, is unlikely to inactivate EAAT2. However, mutation of the highly conserved D504 within the putative C3 cleavage site increases EAAT2 activity via an unknown mechanism.

Bcl-xL knockout attenuates mitochondrial respiration and causes oxidative stress that is compensated by pentose phosphate pathway activity.

Bcl-xL is an anti-apoptotic protein that localizes to the outer mitochondrial membrane and influences mitochondrial bioenergetics by controlling Ca2+ influx into mitochondria. Here, we analyzed the effect of mitochondrial Bcl-xL on mitochondrial shape and function in knockout (KO), wild type and rescued mouse embryonic fibroblast cell lines. Mitochondria of KO cells were more fragmented, exhibited a reduced ATP concentration, and reduced oxidative phosphorylation (OXPHOS) suggesting an increased importance of ATP generation by other means. Under steady-state conditions, acidification of the growth medium as a readout for glycolysis was similar, but upon inhibition of ATP synthase with oligomycin, KO cells displayed an instant increase in glycolysis. In addition, forced energy production through OXPHOS by replacing glucose with galactose in the growth medium rendered KO cells more susceptible to mitochondrial toxins. KO cells had increased cellular reactive oxygen species and were more susceptible to oxidative stress, but had higher glutathione levels, which were however more rapidly consumed under conditions of oxidative stress. This coincided with an increased activity and protein abundance of the pentose phosphate pathway protein glucose-6-phosphate dehydrogenase, which generates NADPH necessary to regenerate reduced glutathione. KO cells were also less susceptible to pharmacological inhibition of the pentose phosphate pathway. We conclude that mitochondrial Bcl-xL is involved in maintaining mitochondrial respiratory capacity. Its deficiency causes oxidative stress, which is associated with an increased glycolytic capacity and balanced by an increased activity of the pentose phosphate pathway.