Our findings show that like cytokines such as IL-3 and IL- 7, which promote survival of hematopoietic cells, BAFF supports B cell survival by increasing metabolic fitness. Treatment of B cells with BAFF induces transcription of mRNAs that encode components of carbohydrate metabolism. The BAFF-induced metabolic bias toward glycolysis might be especially important for B cell survival in lymphoid organs and at inflammatory sites where oxygen tension is low compared with arterial blood (45
). In this environment, activation of glycolysis will provide energy to sustain the active protein synthesis and cell growth caused by BAFF. Indeed, prolonged BAFF treatment led to expression of the hypoxia-inducible factor α (unpublished data).
Normally, accumulation of proteins, particular those controlling cell cycle, and increase in cell volume precedes cell division. However, the lack of BrdU incorporation in BAFF-treated cells shows that BAFF does not induce DNA-replication, thus precluding cell division. Previous findings show persistent expression of Cdk inhibitor proteins p18 and p27 in BAFF-treated cells (47
). Therefore, it is likely that high expression of these and possibly other cell cycle inhibitors prevents the proliferation of BAFF-treated B cells in the absence of an antigenic signal.
BAFF-stimulated cells enter BCR-induced proliferation more readily than untreated cells. This result suggests that the accumulation of cell cycle–controlling proteins in response to BAFF prepares resting B cells for an immediate immune response upon antigenic challenge. It is also attractive to speculate that, in response to BAFF, B cells might establish a storage pool of certain cell cycle–controlling mRNAs or proteins, which could be used for several rounds of cell division. Such a mechanism would help explain the astonishingly short replication time (~7 h) of B cells at the height of the germinal center response (48
Akt activation requires two potentially independent pathways. The PI3K-dependent pathway is shared both by BAFF-R and BCR. This feature of the pathway makes it particularly important in the regulation of B cell immunity and explains the poor survival of B cells in the absence of the p85 regulatory subunit of PI3K (50
). The pathway that involves PKCβ appears to be BAFF specific. Thus, PKCβ deficiency impairs phosphorylation of Akt on activating serine 473 in response to BAFF but not BCR stimulation. The residual BAFF-induced Akt S473 phosphorylation, which we observe in the absence of PKCβ, indicates an ability of other kinases to partially adopt this role. This would be expected, as a plethora of kinases has previously been implicated in mediating Akt S473 phosphorylation (52
Increased translocation of PKCβ to the plasma membrane and PKCβ association with Akt in response to BAFF suggest a direct involvement of PKCβ in BAFF signaling. Furthermore, PKCβ dependency of Akt phosphorylation at S473 but not T308 agrees with in vitro data on Akt phosphorylation by PKCβ (38
) and suggests a direct action of PKCβ on S473 of Akt upon BAFF stimulation.
Involvement of PKCβ in BAFF and BCR signaling provides a mechanistic explanation for the poor survival and altered peripheral maturation of PKCβ-deficient B cells in vitro and in vivo. However, the relatively mild reduction of B cell numbers in the absence of PKCβ argues in favor of additional PKCβ-independent signaling pathways initiated by BAFF-R and/or BCR. Signaling from both receptors induces activation of NF-κB, albeit through distinct mechanisms. Although PKCβ has an important function in canonical NF-κB activation upon BCR-triggering, BAFF-induced NF-κB2 processing is independent of PKCβ. Another PKC family member, PKCδ, also plays an important role in the regulation of B cell survival, but it promotes cell death rather than survival. The proapoptotic potential of PKCδ is contained by BAFF, which prevents its accumulation in the nucleus. This BAFF-dependent survival mechanism can also function in the absence of PKCβ.
BAFF-mediated B cell survival likely represents the collective outcome of several BAFF-induced signaling features, including NF-κB activation, cytoplasmic retention of PKCδ, and Akt activation. It is thus conceivable that the loss of a single signaling branch can to some degree be compensated for. In this case, the loss of PKCβ would be expected to cause some damage to B cell survival, but it would be less severe than the complete abrogation of BAFF signaling. This could explain the more dramatic changes to the B cell compartment in BAFF- or BAFF-R–deficient mice than in PKCβ knockouts. Although PKCβ-deficient B cells were partially responsive to the survival action of BAFF, they appeared to be largely refractive to BAFF-mediated cell growth. This cellular process is closely associated with Akt- and mammalian target of rapamycin–dependent signaling pathways and represents an important distinction between growth factor–mediated cellular fitness on the one hand and mere cell survival on the other (19
). The latter can also be achieved through an altered ratio in the expression of pro- and antiapoptotic proteins, but, in this context, cells become progressively smaller (54
). It appears that, perhaps in contrast to BAFF-induced NF-κB activation and cytoplasmic retention of PKCδ, which mediate B cell survival (55
), PKCβ-dependent Akt activation preferentially regulates the fitness facet of BAFF-mediated cellular responses.
Our findings may have practical implications. There is increasing evidence that BAFF plays an important role in the control of autoreactive B cells (8
). Therefore, identification of Akt and PKCβ as components of BAFF signaling may suggest novel ways of pharmacological intervention in B cell–mediated autoimmune disorders.