Cullin 4B-RING E3 ligase complex in immune cell differentiation and function

The Cullin 4B-RING E3 ligase (CRL4B) complex comprises a scaffold protein CUL4B (cullin 4B), a RING-finger protein ROC (RING of cullin), an adaptor protein DDB1 (damaged DNA-binding protein 1), and a substrate recognition protein DCAF (DDB1 and cullin associated factor) (Fig. 1A). As an E3 ubiquitin ligase complex, the CRL4B complex catalyzes the polyubiquitination of protein substrates, targeting them for proteasomal degradation [1]. This complex can also monoubiquitinate H2A at K119, resulting in recruitment of the polycomb repressive complex 2 (PRC2) complex and repression of transcription (Fig. 1A) [2]. CRL4B has been shown to regulate various biological processes, including embryogenesis, spermatogenesis, adipogenesis, and tumor progression [1]. In recent years, increasing evidence has demonstrated that CRL4B is important for the differentiation and functions of multiple types of immune cells [3–9].

Fig. 1.

CRL4B regulation of immune cells. A Structure of CRL4B. B Mechanisms underlying CRL4B regulation of macrophages, MDSCs and T cells

CUL4B, which encodes the scaffold protein in the CRL4B complex, is a commonly mutated gene in X-linked intellectual disability [10, 11]. In addition to intellectual disability, patients with CUL4B mutations present with an elevated number of monocytes in peripheral blood [10], implying a role for CUL4B in regulating monocytes/macrophages. Monocytes/macrophages are critical players in innate immunity. Hung et al. found that depletion of CUL4B in myeloid cells aggravated lipopolysaccharide (LPS)-induced acute peritonitis [3]. They further showed that after LPS stimulation, Cul4b-deficient macrophages secreted more chemokines than control macrophages, which might have accounted for the elevated infiltration of immune cells into the peritoneum [3]. Our previous work demonstrated that myeloid-specific Cul4b-knockout mice exhibited reduced survival after LPS injection or Salmonella typhimurium infection compared to control mice [6]. In contrast to work from Hung et al., which showed that Cul4b-deficient macrophages secreted less TNFα and IL-6 after LPS stimulation [3], our works showed that deletion of CUL4B in macrophages increased the production of proinflammatory cytokines and decreased the expression of the anti-inflammatory cytokine IL-10 in response to the activation of Toll-like receptor (TLR). CUL4B regulation of macrophages relies on the ubiquitination activity of the CRL4B complex. The CRL4B complex catalyzes monoubiquitination of H2AK119 at the promoter of Pten, resulting in the repression of Pten transcription and subsequent activation of AKT and AKT-dependent inhibition of GSK3β, which suppresses TLR-triggered proinflammatory responses (Fig. 1B) [6]. Consistent with our work, another study showed that macrophages lacking DCAF1, a substrate recognition protein in the CRL4B complex, exhibited elevated production of proinflammatory cytokines such as TNFα, IL-6 and IL-1β after LPS stimulation. Dcaf1 deficiency in myeloid cells exacerbated Staphylococcus aureus-induced osteomyelitis [12]. These studies indicate that the CRL4B complex in macrophages prevents acute inflammation. However, our most recent work on diabetic kidney disease, in which macrophage-mediated chronic inflammation was found to be critical for disease progression, revealed a different role for the CRL4B complex in chronic inflammatory disease. In db/db mice or mice with streptozotocin-induced diabetes, Cul4b deficiency in myeloid cells dramatically ameliorated diabetes-induced renal injury and fibrosis and suppressed the infiltration of macrophages from the circulatory system into diabetic kidneys. The CRL4B complex represses the expression of miR-194-5p in cooperation with the PRC2 complex and histone deacetylases (HDACs). miR-194-5p inhibits integrin α9 (ITGA9), a protein required for macrophage migration and adhesion (Fig. 1B) [9]. The reason that CRL4B in macrophages functions differently under acute and chronic inflammation conditions remains unclear.

The CRL4B complex regulates myeloid-derived suppressor cells (MDSCs), which are critical to the establishment of an immunosuppressive microenvironment for cancer. Specific knockout of Cul4b in the hematopoietic system or the myeloid lineage results in the accumulation of MDSCs and enhances their immunosuppressive activity, which promotes the growth and metastasis of melanoma cells [4, 5]. CRL4B regulates the abundance of MDSCs and their tumor suppressive function through different mechanisms. The aberrant accumulation of MDSCs in Cul4b-knockout mice was a result of the inactivation of AKT and activation of GSK3β. In MDSCs, CRL4B activates AKT by repressing the transcription of two phosphatases that dephosphorylate and inactivate AKT, PP2A and PHLPP1/2 (Fig. 1B) [4]. Moreover, Cul4b-deficient MDSCs express and secrete more IL-6 than is expressed or secreted from control MDSCs, resulting in hyperactivation of STAT3 and acquisition of stem cell-like properties in cancer cells. In contrast to macrophages, in which loss of CUL4B induces elevated IL-6 production through upregulated TLR signaling, MDSCs present with the CRL4B complex directly binding to the promoter of IL-6 to repress IL-6 transcription because of the monoubiquitination of H2AK119 and CRL4B complex interaction with the PRC2 complex and HDACs (Fig. 1B) [5]. CUL4B has been shown to be an oncogene in many types of solid tumors [1]. Identification of its regulatory function in MDSC-mediated immunosuppression revealed the dichotomous role it plays in cancer progression.

After exposure to pathogens, T cells are rapidly activated and undergo clonal expansion, an important step in adaptive immunity. CUL4B is upregulated after T-cell activation. Loss of CUL4B reduced the proliferation and survival of CD4⁺ T cells after activation of T-cell receptors both in vitro and in Rag1^-/- mice. Cul4b-deficient CD4⁺ T cells exhibited increased DNA damage and cell cycle arrest (Fig. 1B) [7]. Loss of DDB1, the adaptor protein in the CRL4B complex, in activated CD4⁺ T cells induced DNA damage and suppressed the generation and expansion of CD4⁺ T helper (Th) cells [13]. Hence, the CRL4B complex allows the expansion of activated T cells by promoting the repair of damaged DNA. CRL4B also regulates the differentiation and plasticity of Th cells [8]. Deletion of Cul4b in CD4⁺ T cells increases the differentiation and plasticity of both Th1 and Th2 cells. Differentiation of T cells into Th1 cells is regulated by TBX21, while T-cell differentiation into Th2 cells involves GATA3 and MAF. The CRL4B complex and PRC2 complex cooperate to repress transcription of the Tbx21 and Maf loci during T-cell differentiation into Th cells (Fig. 1B) [8]. Deletion of the SET domain in EZH2, the core component in the PRC2 complex, in CD4⁺ T cells led to increased differentiation and plasticity of Th1 and Th2 cells, effects similar to those after the loss of CUL4B [14]. Ezh2 mutation causes upregulation of Gata3 [14], whereas loss of CUL4B does not affect Gata3 expression [8], suggesting that the PRC2 complex may prohibit T-cell differentiation into Th cells by interacting with different molecules. Taken together, these findings help us understand the mechanisms by which the CRL4B complex regulates the differentiation and function of CD4⁺ T cells.

In summary, the CRL4B complex regulates macrophages, MDSCs and CD4⁺ T cells through its ubiquitination activity and plays important roles in innate and adaptive immunity. However, studies on CRL4B in immunology are still rare. Additional efforts are required to investigate the role of CRL4B in other types of immune cells and the molecular mechanism underlying CRL4B regulation of inflammatory diseases and cancer immunology.

Author contributions

YS drafted the manuscript. SJ drew the figure. GS and YG revised the manuscript.

Competing interests

The authors declare no competing interests.