Thalidomide‐induced teratogenesis: History and mechanisms

Nearly 60 years ago thalidomide was prescribed to treat morning sickness in pregnant women. What followed was the biggest man‐made medical disaster ever, where over 10,000 children were born with a range of severe and debilitating malformations. Despite this, the drug is now used successfully to treat a range of adult conditions, including multiple myeloma and complications of leprosy. Tragically, a new generation of thalidomide damaged children has been identified in Brazil. Yet, how thalidomide caused its devastating effects in the forming embryo remains unclear. However, studies in the past few years have greatly enhanced our understanding of the molecular mechanisms the drug. This review will look at the history of the drug, and the range and type of damage the drug caused, and outline the mechanisms of action the drug uses including recent molecular advances and new findings. Some of the remaining challenges facing thalidomide biologists are also discussed. Birth Defects Research (Part C) 105:140–156, 2015. © 2015 The Authors Birth Defects Research Part C: Embryo Today: Reviews Published by Wiley Periodicals, Inc.     

Cereblon is expressed in the retina and binds to voltage-gated chloride channels

The function of the central nervous system depends on the correct regulation of ion channels by interacting proteins. Here, we identified cereblon as a new interactor of the voltage-gated chloride channel ClC-2. A distal region of the ClC-2 C-terminus interacts with the Lon domain of cereblon. Cereblon is expressed in several brain regions including the retina. There, we detected cereblon in nuclear and synaptic layers and localized the protein in the same subcellular region of bipolar cell bodies previously reported to express ClC-2. Our data suggest that cereblon might be associated with voltage-gated chloride channels in the central nervous system.

Structured summary

MINT-6823070: CIC-2 (uniprotkb:O54822) physically interacts (MI:0218) with CRBN (uniprotkb:Q0P564) by two hybrid (MI:0018) MINT-6823160, MINT-6823197: CIC-2 (uniprotkb:O54822) physically interacts (MI:0218) with CRBN (uniprotkb:Q56AP7) by pull down (MI:0096) MINT-6823105: CIC-2 (uniprotkb:O54822) physically interacts (MI:0218) with IK (uniprotkb:A4FUY8) by two hybrid (MI:0018)     

Identification and functional characterization of cereblon as a binding protein for large-conductance calcium-activated potassium channel in rat brain

Large-conductance Ca2+-activated K+ (BK(Ca)) channels are activated by membrane depolarization and modulated by intracellular Ca2+. Here, we report the direct interaction of cereblon (CRBN) with the cytosolic carboxy-terminus of the BK(Ca) channel alpha subunit (Slo). Rat CRBN contained the N-terminal domain of the Lon protease, a 'regulators of G protein-signaling' (RGS)-like domain, a leucine zipper (LZ) motif, and four putative protein kinase C (PKC) phosphorylation sites. RNA messages of rat cereblon (rCRBN) were widely distributed in different tissues with especially high-levels of expression in the brain. Direct association of rCRBN with the BK(Ca) channel was confirmed by immunoprecipitation in brain lysate, and the two proteins were co-localized in cultured rat hippocampal neurons. Ionic currents evoked by the rSlo channel were dramatically suppressed upon coexpression of rCRBN. rCRBN decreased the formation of the tetrameric rSlo complex thus reducing the surface expression of functional channels. Therefore, we suggest that CRBN may play an important role in assembly and surface expression of functional BK(Ca) channels by direct interaction with the cytosolic C-terminus of its alpha-subunit.     

Partial monosomy 21 (q11.2→q21.3) combined with 3p25.3→pter monosomy due to an unbalanced translocation in a patient presenting dysmorphic features and developmental delay

We describe a female patient of 1 year and 5 months-old, referred for genetic evaluation due to neuropsychomotor delay, hearing impairment and dysmorphic features. The patient presents a partial chromosome 21 monosomy (q11.2→q21.3) in combination with a chromosome 3p terminal monosomy (p25.3→pter) due to an unbalanced de novo translocation. The translocation was confirmed by fluorescence in situ hybridization (FISH) and the breakpoints were mapped with high resolution array. After the combined analyses with these techniques the final karyotype was defined as 45,XX,der(3)t(3;21)(p25.3;q21.3)dn,-21.ish der(3)t(3;21)(RP11-329A2-,RP11-439F4-,RP11-95E11-,CTB-63H24 +).arr 3p26.3p25.3(35,333-10,888,738)) × 1,21q11.2q21.3(13,354,643-27,357,765) × 1. Analysis of microsatellite DNA markers pointed to a paternal origin for the chromosome rearrangement. This is the first case described with a partial proximal monosomy 21 combined with a 3p terminal monosomy due to a de novo unbalanced translocation.     

Ablation of cereblon attenuates myocardial ischemia–reperfusion injury

Cereblon (CRBN) was originally identified as a target protein for a mild type of mental retardation in humans. However, recent studies showed that CRBN acts as a negative regulator of AMP-activated protein kinase (AMPK) by binding directly to the AMPK catalytic subunit. Because AMPK is implicated in myocardial ischemia–reperfusion (I–R) injury, we reasoned that CRBN might play a role in the pathology of myocardial I–R through regulation of AMPK activity. To test this hypothesis, wild-type (WT) and crbn knockout (KO) mice were subjected to I–R (complete ligation of the coronary artery for 30 min followed by 24 h of reperfusion). We found significantly smaller infarct sizes and less fibrosis in the hearts of KO mice than in those of WT mice. Apoptosis was also significantly reduced in the KO mice compared with that in WT mice, as shown by the reduced numbers of TUNEL-positive cells. In parallel, AMPK activity remained at normal levels in KO mice undergoing I–R, whereas it was significantly reduced in WT mice under the same conditions. In rat neonatal cardiomyocytes, overexpression of CRBN significantly reduced AMPK activity, as demonstrated by reductions in both phosphorylation levels of AMPK and the expression of its downstream target genes. Collectively, these data demonstrate that CRBN plays an important role in myocardial I–R injury through modulation of AMPK activity.     

沙利度胺抑制肿瘤细胞分泌VEGF/bFGF机制的探讨*

目的: 探讨Cereblon(CRBN)对沙利度胺抑制人肺癌A549细胞及人肝癌HepG2细胞分泌VEGF/bFGF的影响。方法: 采用慢病毒介导的短发夹RNA(shRNA)干扰技术建立稳定敲低CRBN的A549细胞系(A549_CRBN)及HepG2细胞系(HepG2_CRBN)并通过实时定量PCR(Real-time PCR)和蛋白质印记(Western blot)实验验证。将A549细胞分为阴性对照组(A549_luciferase)、CRBN低表达组(A549_CRBN);HepG2细胞分为阴性对照组(HepG2_luciferase)、CRBN低表达组(HepG2_CRBN),以上细胞按照 3×10⁵ cells/well接种到6孔板中,放入37℃,5%CO2的培养箱中培养24 h,分别加入1 ml含100 μmol/L沙利度胺(thalidomide组)和1 ml 1‰ DMSO(control组)的培养液,继续培养24 h再行后续实验,每组设计3个复孔。MTS法检测沙利度胺对细胞增殖的影响;Real-time PCR检测VEGF、bFGF、c-jun mRNA表达,ELISA法检测VEGF、bFGF蛋白表达。结果: 与对照组比较,沙利度胺在浓度为1、10、50、100 μmol/L 时对A549 及HepG2细胞的增殖能力无显著影响(P＞0.05)。与A549_CRBN或HepG2_CRBN组比较,A549_luciferase及HepG2_luciferase组分泌的VEGF及bFGF均显著降低(P＜0.05)。与A549_luciferase或HepG2_luciferase细胞的对照组比较,沙利度胺可抑制A549_luciferase和HepG2_luciferase细胞的VEGF和bFGF的表达(P＜0.05),而对A549_CRBN和HepG2_CRBN细胞中VEGF和bFGF的表达无显著抑制作用;与HepG2_luciferase细胞的对照组比较,沙利度胺可抑制HepG2_luciferase细胞的c-Jun表达(P＜0.01),而对HepG2_CRBN细胞的c-Jun表达无显著抑制作用。结论: 沙利度胺对A549和HepG2细胞VEGF和bFGF表达的抑制作用可能是通过CRBN介导的,而c-Jun可能是抑制作用的关键转录因子之一。     

Mechanism of immunomodulatory drugs＇ action in the treatment of multiple myeloma

Although immunomodulatory drugs （IMiDs）, such as thal- idomide, lenalidomide, and pomalidomide, are widely used in the treatment of multiple myeloma （MM）, the molecular mechanism of IMiDs＇ action is largely unknown. In this review, we will summarize recent advances in the application of IMiDs in MM cancer treatment as well as their effects on immunomodulatory activities, anti-angiogenic activities, intervention of cell surface adhesion molecules between myeloma cells and bone marrow stromal cells, anti-inflam- matory activities, anti-proliferation, pro-apoptotic effects, cell cycle arrest, and inhibition of cell migration and metastasis. In addition, the potential IMiDs＇ target protein, IMiDs＇ target protein＇s functional role, and the potential molecular mechanisms of IMiDs resistance will be discussed. We wish, by presentation of our naive discussion, that this review article will facilitate further investigation in these fields.     

Comparative analysis of dermal collagen and lipids in cereblon ablated mice using a multimodal nonlinear optical system

By utilizing a multimodal nonlinear optical system that combines coherent anti-Stokes Raman scattering and second harmonic generation to investigate biological characteristics of dermal tissues ex vivo, we demonstrate the potential feasibility of using this optical approach as a powerful new investigative tool for future biomedical research. For this study, our optical system was utilized for the first time to analyze lipid and collagen profiles in cereblon knockout (KO) mouse skin, and we were able to discover significant alterations in the number of carbon–carbon double bonds (wild-type vs. cereblon KO; N_CC: 0.75 vs. 0.85) of skin fatty acids in triacylglycerides as well as changes in dermal collagen fibers (25% reduction in cereblon KO). By adopting our optical system to biological studies, we provide researchers with another diagnostic approach to validate their experimental results, which will significantly advance the state of biomedical research.     

The Ubiquitination-Dependent and -Independent Functions of Cereblon in Cancer and Neurological Diseases

Cereblon (CRBN) mediates the teratogenic effect of thalidomide in zebrafish, chickens, and humans. It additionally modulates the anti-myeloma effect of the immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide. IMiDs bind to CRBN and recruit neo-substrates for their ubiquitination and proteasome-mediated degradation, which significantly expands the application of proteolysis-targeting chimeras (PROTACs) for targeted drug discovery. However, the underlying molecular mechanisms by which CRBN mediates the teratogenicity and anti-myeloma effect of IMiDs have not been fully elucidated. Furthermore, the normal physiological functions of endogenous CRBN have not been extensively studied, which prevents the thorough assessment of side effects of the CRBN ligand-based PROTACs in the treatment of cancer and neurological diseases. To advance our understanding of the diverse functions of CRBN, in this review, we will survey the ubiquitination-dependent and -independent functions of CRBN, summarize recent advances in the discovery of constitutive substrates and neo-substrates of CRBN, and explore the molecular functions of CRBN in cancer treatment and in the development of neurological diseases. We will also discuss the potential future directions toward the identification of CRBN substrates/interacting proteins and CRBN ligand-based drug discovery in the treatment of cancer and neurological diseases.