Regulator of calcineurin 1 (RCAN1), a gene identified from the critical region of Down syndrome, has been implied in pathogenesis of Alzheimer's disease (AD). RCAN1 expression was shown to be increased in AD brains;however, the mechanism of RCAN1 gene regulation is not well defined. The present study was designed to investigate the molecular mechanism of RCAN1 protein degradation. In addition to being degraded through the ubiquitin proteasome pathway, we found that lysosomal inhibition markedly increased RCAN1 protein expression in a time‐ and dosage‐dependent manner. Inhibition of macroautophagy reduced RCAN1 expression, indicating that RCAN1 degradation is not through a macroautophagy pathway. However, disruption of chaperone‐mediated autophagy (CMA) increased RCAN1 expression. Two CMA recognition motifs were indentified in RCAN1 protein to mediate its degradation through a CMA‐lysosome pathway. A promoter assay further demonstrated that inhibition of RCAN1 degradation in cells reduced calcineurin‐NFAT activity. Dysfunctions of ubiquitin‐proteasome and autophagy‐lysosome pathways have been implicated in neurodegenerative diseases. Therefore, elucidation of RCAN1 degradation by a ubiquitin proteasome pathway and CMA‐lysosome pathway in the present study may greatly advance our understanding of AD pathogenesis.—Liu, H., Wang, P., Song, W., Sun, X. Degradation of regulator of calcineurin 1 (RCAN1) is mediated by both chaperone‐mediated autophagy and ubiquitin proteasome pathways. FASEB J . 23, 3383–3392 (2009). www.fasebj.org

Background: Notch1 is a potent regulator known to play an oncogenic role in many malignancies including T-cell acute lymphoblastic leukemia (T-ALL). Tumor hypoxia and increased hypoxia-inducible factor-1α (HIF-1α) activity can act as major stimuli for tumor aggressiveness and progression. Although hypoxia-mediated activation of the Notch1 pathway plays an important role in tumor cell survival and invasiveness, the interaction between HIF-1α and Notch1 has not yet been identified in T-ALL. This study was designed to investigate whether hypoxia activates Notch1 signalling through HIF-1α stabilization and to determine the contribution of hypoxia and HIF-1α to proliferation, invasion and chemoresistance in T-ALL. Methods: T-ALL cell lines (Jurkat, Sup-T1) transfected with HIF-1α or Notch1 small interference RNA (siRNA) were incubated in normoxic or hypoxic conditions. Their potential for proliferation and invasion was measured by WST-8 and transwell assays. Flow cytometry was used to detect apoptosis and assess cell cycle regulation. Expression and regulation of components of the HIF-1α and Notch1 pathways and of genes related to proliferation, invasion and apoptosis were assessed by quantitative real-time PCR or Western blot. Results: Hypoxia potentiated Notch1 signalling via stabilization and activation of the transcription factor HIF-1α. Hypoxia/HIF-1α-activated Notch1 signalling altered expression of cell cycle regulatory proteins and accelerated cell proliferation. Hypoxia-induced Notch1 activation increased the expression of matrix metalloproteinase-2 (MMP2) and MMP9, which increased invasiveness. Of greater clinical significance, knockdown of Notch1 prevented the protective effect of hypoxia/HIF-1α against dexamethasone-induced apoptosis. This sensitization correlated with losing the effect of hypoxia/HIF-1α on Bcl-2 and Bcl-xL expression. Conclusions: Notch1 signalling is required for hypoxia/HIF-1α-induced proliferation, invasion and chemoresistance in T-ALL. Pharmacological inhibitors of HIF-1α or Notch1 signalling may be attractive interventions for T-ALL treatment.

Acute myeloid leukemia (AML), caused by abnormal proliferation and accumulation of hematopoietic progenitor cells, is one of the most common malignancies in adults. We reported here DYRK1A expression level was reduced in the bone marrow of adult AML patients, comparing to normal controls. Overexpression of DYRK1A inhibited the proliferation of AML cell lines by increasing the proportion of cells undergoing G0/G1 phase. We reasoned that the proliferative inhibition was due to downregulation of c-Myc by DYRK1A, through mediating its degradation. Moreover, overexpression of c-Myc markedly reversed AML cell growth inhibition induced by DYRK1A. DYRK1A also had significantly lower expression in relapsed/refractory AML patients, comparing to newly-diagnosed AML patients, which indicated the role of DYRK1A in chemoresistance of AML. Our study provided functional evidences for DYRK1A as a potential tumor suppressor in AML.

DYRK1A (dual specificity tyrosine phosphorylation-regulated kinase 1A) has been shown to be involved in learning and memory impairments in Alzheimer disease and Down syndrome. As a homolog of Drosophila minibrain gene, DYRK1A also plays important roles in neurodevelopment; however, the function and regulatory mechanism of DYRK1A in neurodevelopment remain elusive. REST (RE1 silencing transcription factor) plays vital roles in neuronal differentiation. Here, we found that REST can activate DYRK1A transcription via a neuron-restrictive silencer element at bp −833 to −815 of human DYRK1A promoter. The coordinated expression of DYRK1A and REST in mouse brain further supports the cross-interaction of DYRK1A and REST during neurodevelopment. Moreover, we showed that DYRK1A dosage imbalance reduced REST protein stability and transcriptional activity through facilitating ubiquitination and subsequent degradation of REST protein. Therefore, the regulation of DYRK1A by REST in a negative feedback loop suggests that DYRK1A and REST are closely related in neurodevelopment.

Mitochondria are intracellular organelles involved in cell survival and death, and dysfunctions of mitochondria are related to neurodegenerative diseases. As the most abundant protein in the mitochondrial inner membrane, adenine nucleotide translocator 1 (ANT1) plays a critical role in mitochondrial function, including the exchange of adenosine triphosphate/adenosine diphosphate (ATP/ADP) in mitochondria, basal proton leak and mitochondrial permeability transition pore (mPTP). Here, we show that ANT1 transcription is regulated by transcription factor NF-kappa B (NF-κB). NF-κB is bound to two NF-κB responsive elements (NREs) located at +1 to +20 bp and +41 to +61 bp in the ANT1 promoter. An NF-κB signalling stimulator, tumour necrosis factor alpha (TNFα), suppresses ANT1 mRNA and protein expression. Activation of NF-κB by TNFα impairs ATP/ADP exchange and decreases ATP production in mitochondria. Activation of NF-κB by TNFα decreases calcium induced mPTP opening, elevates mitochondrial potential and increases reactive oxygen species (ROS) production in both T98G human glioblastoma cells and rat cortical neurons. These results demonstrate that NF-κB signalling may repress ANT1 gene transcription and impair mitochondrial functions.