Retinoic acid (RA) is the active ingredient of vitamin A. It exerts its canonical activity by binding to nuclear RA receptors (RARs) to regulate gene expression. Increasingly, RA is also known to elicit nongenomic RAR-independent activities, most widely detected in activating extracellular regulated kinase (ERK)1/2. This study validated the functional role of cellular retinoic acid-binding protein 1 (Crabp1) in mediating nongenomic activity in RA, specifically activating ERK1/2 to rapidly augment the cell cycle by expanding the growth 1 phase and slowing down embryonic stem cell and neural stem cell (NSC) proliferation. The study further uncovered the physiological activity of Crabp1 in modulating NSC proliferation and animal behavior. In the Crabp1 knockout mouse hippocampus, where Crabp1 is otherwise detected in the subgranular zone, neurogenesis and NSC proliferation increased and hippocampus-dependent brain functions such as learning and memory correspondingly improved. This study established the physiological role of Crabp1 in modulating stem cell proliferation and hippocampus-dependent brain activities such as learning and memory.
Cellular retinoic acid-binding protein 1 (CRABP1) is highly expressed in motor neurons. Degenerated motor neuron-like MN1 cells are engineered by introducing SODG93A or AR-65Q to model degenerated amyotrophic lateral sclerosis (ALS) or spinal bulbar muscular atrophy neurons. Retinoic acid (RA)/sonic hedgehog (Shh)-induced embryonic stem cells differentiation into motor neurons are employed to study up-regulation of Crabp1 by Shh. In SODG93A or AR-65Q MN1 neurons, CRABP1 level is reduced, revealing a correlation of motor neuron degeneration with Crabp1 down-regulation. Up-regulation of Crabp1 by Shh is mediated by glioma-associated oncogene homolog 1 (Gli1) that binds the Gli target sequence in Crabp1′s neuron-specific regulatory region upstream of minimal promoter. Gli1 binding triggers chromatin juxtaposition with minimal promoter, activating transcription. Motor neuron differentiation and Crabp1 up-regulation are both inhibited by blunting Shh with Gli inhibitor GANT61. Expression data mining of ALS and spinal muscular atrophy (SMA) motor neurons shows reduced CRABP1, coincided with reduction in Shh-Gli1 signaling components. This study reports motor neuron degeneration correlated with down-regulation in Crabp1 and Shh-Gli signaling. Shh-Gli up-regulation of Crabp1 involves specific chromatin remodeling. The physiological and pathological implication of this regulatory pathway in motor neuron degeneration is supported by gene expression data of ALS and SMA patients.
The rapidly accelerated fibrosarcoma (Raf) kinase is canonically activated by growth factors that regulate multiple cellular processes. In this kinase cascade Raf activation ultimately results in extracellular regulated kinase 1/2 (Erk1/2) activation, which requires Ras binding to the Ras binding domain (RBD) of Raf. We recently reported that all-trans retinoic acid (atRA) rapidly (within minutes) activates Erk1/2 to modulate cell cycle progression in stem cells, which is mediated by cellular retinoic acid binding protein 1 (Crabp1). But how atRA-bound Crabp1 regulated Erk1/2 activity remained unclear. We now report Raf kinase as the direct target of atRA-Crabp1. Molecularly, Crabp1 acts as a novel atRA-inducible scaffold protein for Raf/Mek/Erk in cells without growth factor stimulation. However, Crabp1 can also compete with Ras for direct interaction with the RBD of Raf, thereby negatively modulating growth factor-stimulated Raf activation, which can be enhanced by atRA binding to Crabp1. NMR heteronuclear single quantum coherence (HSQC) analyses reveal the 6-strand β-sheet face of Crabp1 as its Raf-interaction surface. We identify a new atRA-mimicking and Crabp1-selective compound, C3, that can also elicit such an activity. This study uncovers a new signal crosstalk between endocrine (atRA-Crabp1) and growth factor (Ras-Raf) pathways, providing evidence for atRA-Crabp1 as a novel modulator of cell growth. The study also suggests a new therapeutic strategy by employing Crabp1-selective compounds to dampen growth factor stimulation while circumventing RAR-mediated retinoid toxicity.
Cellular retinoic acid-binding protein 1 (CRABP1) binds retinoic acid (RA) specifically in the cytoplasm with unclear functions. CRABP1 is highly and specifically expressed in spinal motor neurons (MNs). Clinical and pre-clinical data reveal a potential link between CRABP1 and MN diseases, including the amyotrophic lateral sclerosis (ALS). We established a sequenced MN-muscle codifferentiation system to engineer an in vitro functional 3D NMJ model for molecular studies and demonstrated that CRABP1 in MNs contributes to NMJ formation and maintenance. Consistently, Crabp1 knockout (CKO) mice exhibited an adult-onset ALS-like phenotype with progressively deteriorated NMJs, characterized with behavioral, EchoMRI, electrophysiological, histological, and immunohistochemical studies at 2-20-months old. Mechanistically, CRABP1 suppresses CaMKII activation to regulate neural Agrn expression and downstream muscle LRP4-MuSK signaling, thereby maintaining NMJ. A proof-of-concept was provided by specific re-expression of CRABP1 to rescue Agrn expression and the phenotype. This study identifies CRABP1-CaMKII-Agrn signaling as a physiological pre-synaptic regulator in the NMJ. This study also highlights a potential protective role of CRABP1 in the progression of NMJ deficits in MN diseases.
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