Casitas B-lineage lymphoma (CBL) proteins constitute a conserved family of ubiquitin ligases, including c-Cbl (also known as RNF55), Cbl-b (also known as RNF56), and Cbl-c (also known as Cbl-3, RNF57) [1]. Structurally, CBL proteins feature highly conserved and family-specific domains including an N-terminal tyrosine kinase binding (TKB) domain, linker region, really interesting new gene (RING) finger catalytic domains, proline-rich region, C-terminal phosphorylation sites, and ubiquitin-associated domains (Figure 1) [2]. Within the TKB domain, the Src homology region 2 domain interacts with phosphorylated tyrosine residues of proteins, while the RING domain serves as the E3 ubiquitin ligase activity region of CBL proteins, interacting with E2 enzymes. In 1977, Goldknopf et al. discovered the first substrate of the ubiquitin pathway mediated by the Polycomb repressive complex, involving efficient mono-ubiquitination of histone H2A [3]. Ubiquitination requires the coordinated action of E1 ubiquitin-activating enzymes (E1), E2 ubiquitin-conjugating enzymes (E2), and E3 ubiquitin ligases (E3). E1 activates ubiquitin which is then transferred to E2, and E3 facilitates the specific recognition of substrates, enabling the attachment of ubiquitin from E2 to target proteins [4]. Ubiquitination, as a post-translational process, regulates the function, degradation, cellular localization, and activity of target proteins by attaching ubiquitin peptides to lysine residues, thereby influencing various key biological processes such as signal transduction, cell cycle progression, and immune responses [5]. The primary function of CBL proteins involves the ubiquitination and degradation of both receptor and non-receptor tyrosine kinase proteins, predominantly affecting signal transduction in immune cells such as T cells and B cells.
The Physiology and Pathophysiology of CBL-Associated Cardiovascular Diseases are Illustrated in the Figure. C-Cbl, Cbl-b and Cbl-c proteins belong to the E3 ubiquitin ligase CBL family. The ubiquitination pathway is also depicted in the Figure.
Abbreviations: 4H, four-helix bundle; EF, EF hand; SH2, Src homology region 2; L, Linker region; RF, Ring Finger domain; PRR, Proline-rich region; UBA, Ubiquitin-association domain.
In this issue of the Journal of the Cardiovascular Innovations and Applications, Chen et al. performed an update study on one public mRNA and miRNA expression dataset (GEO accession number: GSE98770), including intima-media layers from six dissected aorta samples and five donor aorta samples. It employs a novel bioinformatics analysis, such as differential analysis, establishment of a mRNA-microRNA regulatory network, Comparative Toxicogenomics Database (CTD), and transcription factor (TF) prediction. Specifically, differentially expressed mRNAs (DE-mRNAs) and microRNAs (DE-microRNAs) from the dataset were screened, the regulatory network was established, the hub genes were identified, the relationships between hub genes and aortic dissection (AD) were confirmed, and the TFs were discovered. CBL among DE-mRNAs and miR-1321 among DE-microRNAs are both linked to the most genes and interrelate with each other. Homeobox b13 (HOXB13), as a TF, is the only DE-mRNA in the dataset that regulates CBL on the transcriptional level. So far, a TF-mRNA-microRNA regulatory network has been established and HOXB13-CBL-miR1321 was identified as a key regulatory axis in the mechanism of AD. This article indicates that CBL mediates tyrosine kinase ubiquitination and plays an essential role in multiple cell signaling pathways, such as cell differentiation [6]. Inhibition of CBL may be a potential therapeutic option for AD due to its effects on the differentiation and phenotypic switching of vascular smooth muscle cells (VSMC) [7].
CBL plays crucial roles in various cellular pathways, participating in the onset and progression of multiple diseases. Y371 was the most common mutation site in the CBL mutation, which affects the RF domain of CBL and E3-ubiquitin ligase function. The characteristics of juvenile myelomonocytic leukemia (JMML) with CBL mutations are defects in E3 ubiquitin ligase activity, low DNA methylation levels, and aberrant activation of the RAS pathway. Clinically, JMML leads to leukocytosis, anemia, thrombocytopenia, as well as the infiltration of monocytes and granulocytes in various organs [8]. Homozygous mutations in CBL are also common in myeloproliferative disorders, chronic myelomonocytic leukemia, and acute myeloid leukemia [1, 9, 10]. Knockout of c-Cbl induces endothelial cell proliferation and angiogenesis, including pathological angiogenesis in tumor-induced neovascularization in melanoma mouse models, and choroidal neovascularization exacerbating age-related macular degeneration [11]. CBL syndrome resembles Noonan syndrome and is caused by mutations in the RAS/MAPK cellular pathway genes [12]. Additionally, patients with CBL mutations may develop vascular pathologies such as Takayasu arteritis and pulmonary artery disease [13]. Loss of c-Cbl activity significantly enhances nuclear β-catenin expression and promotes colon cancer tumor growth [14]. Overall, CBL molecules play critical roles in multiple diseases and contribute to various important pathological processes.
In recent years, numerous studies have explored the role of CBL in cardiovascular diseases. Rafiq et al. found that in mouse myocardial ischemia-reperfusion injury, the absence of c-Cbl reduces cardiomyocyte apoptosis, promotes angiogenesis, improves cardiac function, and reduces the risk of death following chronic myocardial ischemia [15]. In human atherosclerotic plaques, the expression of Cbl-b is negatively correlated with necrotic core size [16]. In early-stage atherosclerosis models in ApoE−/− mice, deficiency of Cbl-b increases macrophage aggregation within plaques, facilitates lipid uptake mediated by CD36, promotes foam cell formation, and enhances inflammation termination induced by LPS and the production of reactive oxygen species, exacerbating atherosclerosis [16]. In late-stage atherosclerosis models in ApoE−/− mice, Cbl-b deficiency increases the number of CD8+ T cells in plaques and spleen, promotes macrophage death within plaques, and accelerates plaque inflammation and progression [16]. Mechanical (shear stress) and chemical (growth factors, such as VEGF) enhance CBL activity in aortic endothelial cells, tyrosine phosphorylation of the Cbl-bound Flk-1, as well as tyrosine phosphorylation of CBL through the FLK-1/CBL/AKT pathway activating IκB kinase (IKK), suggesting potential molecular mechanisms and therapeutic targets for vascular diseases, such as hypertension and neointima formation [17, 18]. Obesity is one of the major risk factors of atherosclerosis and cardiovascular disease. It may induce chronic low-grade inflammation in adipose tissue. Initially, Keane et al. found that the expression level of Cbl-b was upregulated during the differentiation of human leukemia cell lines HL60 and U937 towards the macrophage/monocyte lineage [19]. Later, Choi et al. reported that thioglycolate-induced peritonitis was exacerbated with increased recruitment of macrophages into the peritoneal cavity of Cbl-b−/− mice [20]. Han et al. further demonstrated that Cbl-b inhibited TLR4-induced macrophage activation in a CD11-dependent manner [21]. Therefore, Cbl-b negatively regulates macrophage activation and signal transduction. These findings highlight the role of Cbl-b in modulating macrophage function, its implications in immune responses and inflammatory conditions, as well as its importance in cardiovascular disease.
The ubiquitin-proteasome system is a fundamental cellular process increasingly considered for diagnostic and therapeutic strategies [22]. Current research on CBL primarily focuses on understanding its precise roles in the development of different diseases and exploring the feasibility of targeting CBL as a potential therapeutic target by inhibiting or enhancing its function. On the one hand, blocking the interaction between CBL and E2 by small molecules targeting the RING domain of CBL may enhance the activity of CBL-b and c-Cbl, potentially offering therapeutic value in cancer treatment. On the other hand, targeted deletion of the CBL gene in specific cells can suppress CBL protein expression levels. Thus, inhibiting the ubiquitination of CBL or enhancing its negative regulation of specific signaling pathways may provide novel targets for drug development in cardiovascular disease.