Regulation of Bim in Health and Disease

Abstract

The BH3-only Bim protein is a major determinant for initiating the intrinsic apoptotic pathway under both physiological and pathophysiological conditions. Tight regulation of its expression and activity at the transcriptional, translational and post-translational levels together with the induction of alternatively spliced isoforms with different pro-apoptotic potential, ensure timely activation of Bim. Under physiological conditions, Bim is essential for shaping immune responses where its absence promotes autoimmunity, while too early Bim induction eliminates cytotoxic T cells prematurely, resulting in chronic inflammation and tumor progression. Enhanced Bim induction in neurons causes neurodegenerative disorders including Alzheimer's, Parkinson's and Huntington's diseases. Moreover, type I diabetes is promoted by genetically predisposed elevation of Bim in β-cells. On the contrary, cancer cells have developed mechanisms that suppress Bim expression necessary for tumor progression and metastasis. This review focuses on the intricate network regulating Bim activity and its involvement in physiological and pathophysiological processes.

Keywords: Bim; apoptosis; autoimmunity; cancer; neurodegenerative diseases.

Figure 1. The gene structure of Bim and its major isoform transcripts

A. Presentation of the Bim (Bcl-2L11) gene structure according to the nomenclature of U et al. [28]. There has been much confusion in the literature concerning the nomenclature of the exon numbers where some research groups have denoted exons E2A, E2B and E2C as exons 2, 3 and 4 respectively, and exons E3, E4 and E5 as exons 5, 6 and 7, respectively. As there is no intron between E2A and E2B, and E2B and E2C, these regions are part of one exon, where intraexonal alternative splicing gives raise to the inclusion or exclusion of the E2B and/or E2C region. DBD - Dynein-binding domain. The BH3 domain is located in exon E4. The numbers beneath the exons refer to the amount of coding nucleotides in each exon. B. Presentation of various Bim isoform transcripts formed by alternative splicing. Bim_EL, Bim_L and Bim_S are the major classical isoforms, but also other isoforms have been identified as described in Section 1.2. Bimα1-2 and Bimβ1-4 were described by U et al. [28], while Bimγ by Ng et al. [31] that was coined Bimγ2 by Anczukow et al. [229]. The latter research group characterized an additional Bimγ isoform (Bimγ1) that also retained the E2B exon [229]. Although Ng et al. [31] claim for a mutual exclusion of exon E3 and E4, the Bimβ3 isoform described by U et al. [28] does contain both exons. As the E3 exon contains a stop codon, its inclusion leads to a truncated protein lacking the pro-apoptotic BH3 domain. The Bim-ABCD, Bim-ACD and Bim-AD described by Marani et al. [9] corresponds to Bimα1, Bimα2 and Bimα3. Bimα3 resembles Bimα2, but lacks E2C.

Figure 2. Bim-induced apoptosis

Following exposure to a pro-apoptotic stimulus, a sudden intracellular rise in free activated Bim molecules (e.g., by increased transcription and/or translation, increased alternative splicing in favor of Bim_S, and/or release of Bim from sequestered intracellular storages as a result of phosphorylation) initiates the intrinsic mitochondrial apoptotic pathway. Bim induces apoptosis by directly activating Bax and Bak, or indirectly by interacting with the anti-apoptotic proteins Bcl-2, Bcl-xL and Mcl-1, leading to the release and mitochondrial transfer of Bax and Bak. Under normal conditions, Bak is hold in check by Mcl-1, VDAC2 and Bcl-xL. Bax/Bak oligomerization in the mitochondrial outer membrane results in dissipation of the mitochondrial outer membrane potential (Δψ_m) and release of the apoptogenic proteins cytochrome C (CytoC), Smac/DIABLO and HtrA2 into the cytosol. Cytochrome C activates Apoptotic protease activating factor 1 (Apaf-1) that facilitates the formation of the apoptosome where caspase 9 is activated to initiate the apoptotic cascade concluded with the activation of caspase 3. Smac/DIABLO antagonizes the anti-apoptotic function of Inhibitors of apoptosis protein (IAPs) such as XIAP, cIAP1 and cIAP2, thereby enhancing apoptosis induction by cytochrome C.

Figure 3. Bim at the cross-road between apoptosis and autophagy

In certain cell types such as epithelial and neuronal cells, Bim_EL and Bim_L are sequestered to the dynein motor complex through interaction with the dynein light chain DYNLL1. Bim also binds Beclin-1, thus preventing autophagy. Upon exposure to microtubule-disrupting agents such as paclitaxel, or stress-stimuli such as starvation or UV radiation that activate JNK, Bim_EL becomes phosphorylated at Ser65 and Thr112 and Bim_L at Thr56, leading to their release from the microtubule and mitochondrial translocation where the intrinsic apoptotic pathway is activated. Simultaneously, Beclin-1 is released from Bim resulting in induction of autophagy. Beclin-1 also competes with Mcl-1 for the USP9X deubiquitinase resulting in enhanced Mcl-1 degradation, thereby tipping the Bim/Mcl-1 balance towards Bim-induced apoptosis. In addition, the p53- and BRCA1-regulated stress protein GADD45a promotes Bim dissociation from the microtubules. GADD45a also inhibits the function of the microtubule-severing protein EF-1α.

Figure 4. Transcriptional activation of Bim

Bim transcription can be upregulated by a whole range of transcription factors (outlined in green) whose activities are tightly regulated. Some of these pathways are cell-type specific and depend on the stimulus. Those proteins positively regulating the transcription factors are presented in red, while those inhibiting in blue. H2AX is a histone whose phosphorylation at Ser139 by p38 is important for Bim transcription. The different pathways are described in more detail in Section 3.1.1.

Figure 5. Transcriptional repression of Bim

Several transcription factors prevent Bim transcription. Some of them, e.g., NFκB and E2F-1, play a dual role by regulating both Bim transactivation (Figure 4) and transrepression. The RelA component of NFκB acts as a transactivator in the absence of YY-1, while becomes a transrepressor in its presence. E2F1 may directly or indirectly transactivate Bim by inducing c-Myc and B-Myb expression (Figure 4), but, in addition, it induces the polycomb histone methyl transferase EZH2 that promotes trimethylation of Histone 3 on Lys27 (H3K27me3), leading to the repression of Bim expression. Spi-1/PU.1 and Trim33 cooperate with EZH2 to repress Bim transcription. Furthermore, E2F1 directly or through c-Myc induces miR106∼25 that prevents Bim translation (Figure 6). The multiple effects of E2F1 may ensure fine tuning of Bim expression. HIF-1α that is, among others, induced by hypoxic conditions within the tumor microenvironment, prevents Bim expression, thus providing a growth advantage to the tumor cells. Description of the other repressors can be found in Section 3.1.2.

Figure 6. Translational regulation of Bim

A. Various Bim isoforms can be formed by alternative splicing, where upregulation of Bim_S is especially efficient in inducing apoptosis, while formation of BH3-deficient Bimγ lacks pro-apoptotic activity. The splicing factor SRSF1 promotes Bimγ formation, while activation of SRp55 by the B-Raf inhibitor PLX4720 or Zn²⁺ favors Bim_S. PTBP1, Brm and hnRNP2 favor the inclusion of the BH3-containing E4 exon over the E3 exon. B. Various RNA-binding proteins such as Hsp27 and Hsc70 prevent Bim translation. C. A range of microRNAs can target Bim, among them miR-17∼92, miR-106∼25, miR-181, miR-148 and miR-221/222 are considered as oncomiRs that can promote tumor progression.

Figure 7. Post-translational modifications of Bim

The amino acid sequences of human (H) and mouse (M) Bim_EL have been aligned, and important post-translational modification sites of Bim highlighted. ERK1/2 phosphorylates Bim_EL at three serine residues (Ser55, Ser65 and Ser100 in mouse corresponding to Ser59, Ser69 and Ser104 in human), which facilitate RSK1/2-mediated phosphorylation of Ser93/94/98 in human (corresponding to Ser89/90/94 in mouse), leading to ubiquitination at Lys3 and Lys108(M)/Lys112(H) by β-TrCP1 and proteasomal degradation. Aurora A phosphorylates the same residues as RSK1/2 during mitosis that occurs independently of previous ERK phosphorylation and leads to binding of APC^Cdc20 to the D-box consensus region, resulting in Bim degradation. PP2A dephosphorylates Ser93/94/98 at the exit from mitosis, thereby stabilizing Bim_EL. p38 and JNK also phosphorylate Bim_EL at Ser65 (M)/Ser69 (H), but, in addition, JNK phosphorylates Thr112(M)/Thr116(H) which lies within the dynein-binding domain (DBD). The latter phosphorylation leads to dissociation of Bim from the microtubules. JNK may also phosphorylate Bim_L at Thr56 (same residue as Thr112 in Bim_EL). p38 and JNK phosphorylation of Bim, at least in some cell types, lead to increased Bim activity. Also, Akt and PKA can phosphorylate Bim. The coding exons (E2A, E2B, E2C, E4 and E5) have been outlined, as well as the D-box consensus, DBD, BH3 domain and the hydrophobic C-terminal region.

Figure 8. Abberant Bim expression in diseases

Elevated Bim expression is associated with neuronal degenerative diseases, diabetes, fibrosis and liver damage, while too low or absent Bim expression is associated with cancer (Section 4). Absent Bim expression in the immune system may lead to autoimmune diseases due to lack of elimination of autoreactive immune cells, but is, in part, compensated by an increase in regulatory T cells. Treatment of neurodegenerative diseases and diabetes should aim in preventing Bim expression, while treatment of cancer should aim in increasing Bim expression. Too dramatic increase in Bim during cancer treatment might thus have adverse effects on the nerve system, liver and endocrine system, that needs to be taken into account. Targeting cancer-specific pro-survival pathways would reduce the adverse effects.

Figure 9. Growth factor-mediated cell survival versus apoptosis induction by growth factor deprivation

Growth factor receptor activation leads to activation of the Ras-Raf-MEK-ERK1/2 and PI3K-Akt pathways that co-operate in inhibiting Bim activity. ERK1/2-mediated phosphorylation of Bim_EL leads to its subsequent phosphorylation by RSK1/2 and ubiquitin-mediated proteasomal degradation. Akt-mediated phosphorylation of Bim leads to its sequestration to 14-3-3 proteins. Akt also inactivates Mst1 and FoxO3a. Following growth factor withdrawal, the lack of activation of the Ras-Raf-MEK-ERK1/2 and PI3K-Akt pathways leads to Mst1 and FoxO3a activation, resulting in the transcriptional upregulation of Bim. In addition, JNK phosphorylates Bim resulting in its activation and apoptosis induction. Inhibition of growth factor receptor signaling (e.g., by EGFR, ALK, c-Met and B-Raf^V600E inhibitors) is a major cancer-specific targeting strategy that aims to promote Bim-dependent apoptosis.

Figure 10. The decision between TGFβ-induced apoptosis and cell transformation

TGFβ may induce either apoptosis or cell transformation dependent on the cellular context (Section 4.11.5.1). TGFβ induces Bim expression by activating the Runx1/2/3, Smad3/4 and FoxO3a transcription factors. Also, BDH was found to be important for Bim induction. The simultaneous induction of DUSP4 that antagonizes ERK signaling, leads to stabilization of the Bim protein. TGFβ may also promote cell survival through Akt and MSK1-dependent mechanisms. Upon oncogenic switch, Bim expression is downregulated by reduced transcription, increased degradation and inhibition of Bim translation by the oncomiRs miR-106∼25 and miR-181. miR-106∼25 is upregulated by E2F1, c-Myc and Six1, while TGFβ itself may upregulate miR-181.