FBXW7 protein has dual-role as tumor suppressor and inflammatory pathway inhibitor

There is a positive feedback loop between C/EBPδ and TLR4 and a negative feedback loop between C/EBPδ and FBXW7α, which together regulate TLR4 signaling and pro-inflammatory gene expression. Phosphorylation of C/EBPδ by GSK-3β is required for its degradation by FBXW7α. Therefore, inhibition of GSK-3β by TLR4 stabilizes C/EBPδ.

There is a positive feedback loop between C/EBPδ and TLR4 and a negative feedback loop between C/EBPδ and FBXW7α, which together regulate TLR4 signaling and pro-inflammatory gene expression. Phosphorylation of C/EBPδ by GSK-3β is required for its degradation by FBXW7α. Therefore, inhibition of GSK-3β by TLR4 stabilizes C/EBPδ.

Toll-like receptors (TLRs) are largely responsible for inducing innate immune responses to infection. TLR4 binds lipopolysaccharide (LPS) from Gram-negative bacteria and initiates a signaling pathway to activate inflammatory responses. TLR4 plays a role in diseases such as sepsis and chronic inflammatory disorders. In tumor cells, TLR4 is involved in dampening immune surveillance, and increasing proliferation, inflammatory cytokine production, and invasive migration. Determining how TLR4 expression and signaling is regulated may enable these adverse conditions to be better managed.

Previous studies have shown that the transcription factor CCAAT/enhancer binding protein delta (C/EBPδ) increases LPS signaling and is needed for LPS-induced gene expression and the clearance of Gram-negative bacterial infection. Data from experiments performed in mice lacking C/EBPδ suggest that the gene may have a role in systemic inflammatory diseases such as sepsis and multiple sclerosis. Esta Sterneck, Ph.D., in CCR’s Laboratory of Cell and Development Signaling, Kuppusamy Balamurugan, Ph.D., a Postdoctoral Fellow in her lab, and colleagues previously reported that C/EBPδ directly inhibits expression of the F-box and WD repeat domain containing protein 7 alpha (FBXW7α) in mammary tumor cells. FBXW7α is a tumor suppressor in solid cancers and targets several mammalian oncoproteins for degradation, such as Notch and mTOR. The lab has also shown that hypoxia-induced C/EBPδ inhibits FBXW7α, leading to an increase in mTOR, and the hypoxia-inducible factor 1 alpha (HIF-1α). HIF-1α is needed for cellular adaptation to hypoxia, and promotes macrophage activation and inflammatory responses.

Because C/EBPδ and HIF-1 were both known to be important for inflammatory signaling Sterneck and colleagues set out to test whether the C/EBPδ- FBXW7α-HIF-1 pathway is involved in macrophage activation. The researchers first analyzed FBXW7α gene expression in macrophages. Their data indicate that macrophages express FBXW7α and that this expression is downregulated by C/EBPδ and LPS. Human monocytes depleted of C/EBPδ were found to have increased FBXW7α expression and reduced HIF-1α accumulation. When FBXW7α was depleted, C/EBPδ and HIF-1α expression increased, demonstrating that FBXW7α suppresses C/EBPδ expression. In mouse macrophages lacking C/EBPδ, HIF-1α accumulation was rescued by knockdown of the elevated FBXW7α. These findings show that C/EBPδ promotes HIF-1α expression in activated macrophages by inhibiting FBXW7α expression.

The next step was to investigate how FBXW7α regulates C/EBPδ expression. Following a series of experiments performed in a macrophage cell line, they found that FBXW7α is required for ubiquitination and degradation of C/EBPδ, and that C/EBPδ binding to its own promoter increases when FBXW7α is silenced. These results demonstrate a negative feedback loop from FBXW7α to C/EBPδ. Further, the kinase GSK-3β, which is responsible for the phosphorylation of most FBXW7α substrates, was found to directly phosphorylate C/EBPδ, which led to a decrease in C/EBPδ levels in untreated macrophages. In activated macrophages, LPS was able to activate C/EBPδ expression partly by inhibition of the GSK-3β/FBXW7α pathway.

The researchers hypothesized that since FBXW7α targets C/EBPδ for degradation, FBXW7α may have an inhibitory role in pro-inflammatory signaling. They found that FBXW7α suppressed all tested responses of macrophages to LPS, such as iNOS, C/EBPδ, and COX-2 levels. Suppression of LPS responses by FBXW7α suggested that upstream factors in the LPS signaling pathway are downregulated by FBXW7α. Indeed, TLR4 expression was reduced along with C/EBPδ in macrophages. When FBXW7α was silenced, C/EBPδ depletion prevented TLR4 upregulation, indicating that FBXW7α downregulates TLR4 through inhibition of C/EBPδ expression. Indeed, C/EBPδ was found to bind to and directly activate the TLR4 promoter. In mice that had reduced FBXW7α levels due to RNA interference, plasma levels of the pro-inflammatory cytokine IL-6 increased, demonstrating that FBXW7α-depletion alone is enough to activate inflammatory signaling.

Previous studies have shown that TLR4 is expressed in both macrophages and tumor cells. After finding that C/EBPδ acts upstream of LPS signaling by directly activating TLR4 gene expression, the researchers investigated whether C/EBPδ modifies TLR4 expression in tumor cells. Depleting C/EBPδ in a mouse mammary tumor cell line or in human breast tumor cells led to reduced TLR4 protein expression and increased FBXW7α levels. In addition, many inflammatory markers were decreased in mammary tumors from C/EBPδ-deficient mice. Chemokines and their receptors, which have an important role in breast tumor progression and metastasis, had reduced expression in C/EBPδ-deficient tumors, which have decreased metastatic potential.

This paper identifies the tumor suppressor FBXW7α as an important attenuator of inflammatory signaling and may provide a new mechanistic link between inflammation and tumor progression. Interestingly, the GSK-3β enzyme, typically regarded as a pro-inflammatory agent, is implicated as an inhibitor of the inflammatory response. Drugs that inhibit GSK-3β’s activity are being developed for diseases such as Alzheimer’s, cancer, and diabetes. The anti-inflammatory role that is demonstrated in this work may complicate clinical application of such GSK-3β inhibitors or could be exploited by manipulation of FBXW7α expression. Furthermore, this work may lead toward a better understanding of the molecular events regulating acute versus chronic inflammation in cancer and other diseases.

Summary Posted: 04/2013

Reference

Balamurugan K, Sharan S, Klarmann KD, Zhang Y, Coppola V, Summers GH, Roger T, Morrison DK, Keller JR, Sterneck E. FBXW7α attenuates inflammatory signalling by downregulating C/EBPδ and its target gene Tlr4. Nat Commun. 2013 Apr 9 PubMed Link