Identification of the NF-κB inhibitor A20 as a key regulator for human adipogenesis.

The zinc-finger protein A20 is a key player in the negative feedback regulation of the nuclear factor kappa-light-chain-enhancer of activated B-cell (NF-κB) pathway in response to multiple stimuli. Tumor necrosis factor alpha (TNFα), a cytokine with pleiotropic effects on cellular proliferation and differentiation, dramatically increases A20 expression in all tissues. As TNFα inhibits adipocyte differentiation, we have determined the contribution of A20 to the adipogenic capacity of human mesenchymal stromal cells (MSCs). Here we show that A20 is constitutively expressed in MSCs, which previously has been observed only in cells that are either tumor or immune cells (T/B lymphocytes). TNFα stimulation induced a rapid degradation of A20 protein mediated exclusively by the proteasome in MSCs and not by caspases. This degradation is concomitant to the induction of its own mRNA, which suggests that a tight regulation of NF-κB signaling in MSCs is fundamental. On one hand, we demonstrate that the knockdown of A20-mediated transcript dramatically decreases the adipogenic capacity of MSCs, which correlates with the phenotype observed in the presence of TNFα. On the other hand, A20 overexpression blocks NF-κB activation and drives to increased adipogenesis, even in the presence of TNFα treatment. In conclusion, our data demonstrate that the presence of A20 allows MSCs to differentiate into adipocytes by maintaining NF-κB signaling at a basal state.

Accelerated DNA replication in E2F1- and E2F2-deficient macrophages leads to induction of the DNA damage response and p21(CIP1)-dependent senescence.

E2F1-3 proteins appear to have distinct roles in progenitor cells and in differentiating cells undergoing cell cycle exit. However, the function of these proteins in paradigms of terminal differentiation that involve continued cell division has not been examined. Using compound E2F1/E2F2-deficient mice, we have examined the effects of E2F1 and E2F2 loss on the differentiation and simultaneous proliferation of bone-marrow-derived cells toward the macrophage lineage. We show that E2F1/E2F2 deficiency results in accelerated DNA replication and cellular division during the initial cell division cycles of bone-marrow-derived cells, arguing that E2F1/E2F2 are required to restrain proliferation of pro-monocyte progenitors during their differentiation into macrophages, without promoting their cell cycle exit. Accelerated proliferation is accompanied by early expression of DNA replication and cell cycle regulators. Remarkably, rapid proliferation of E2F1/E2F2 compound mutant cultures is temporally followed by induction of a DNA damage response and the implementation of a p21(CIP1)-dependent senescence. We further show that differentiating E2F1/E2F2-knockout macrophages do not trigger a DNA damage response pathway in the absence of DNA replication. These findings underscore the relevance of E2F1 and E2F2 as suppressors of hematopoietic progenitor expansion. Our data indicate that their absence in differentiating macrophages initiates a senescence program that results from enforcement of a DNA damage response triggered by DNA hyper-replication.