Stress-induced Switch in Numb Isoforms Enhances Notch-dependent Expression of Subtype-specific Transient Receptor Potential Channel

Abstract

The Notch signaling pathway plays an essential role in the regulation of cell specification by controlling differentiation, proliferation, and apoptosis. Numb is an intrinsic regulator of the Notch pathway and exists in four alternative splice variants that differ in the length of their phosphotyrosine-binding domain (PTB) and proline-rich region domains. The physiological relevance of the existence of the Numb splice variants and their exact regulation are still poorly understood. We previously reported that Numb switches from isoforms containing the insertion in PTB to isoforms lacking this insertion in neuronal cells subjected to trophic factor withdrawal (TFW). The functional relevance of the TFW-induced switch in Numb isoforms is not known. Here we provide evidence that the TFW-induced switch in Numb isoforms regulates Notch signaling strength and Notch target gene expression. PC12 cells stably overexpressing Numb isoforms lacking the PTB insertion exhibited higher basal Notch activity and Notch-dependent transcription of the transient receptor potential channel 6 (TRPC6) when compared with those overexpressing Numb isoforms with the PTB insertion. The differential regulation of TRPC6 expression is correlated with perturbed calcium signaling and increased neuronal vulnerability to TFW-induced death. Pharmacological inhibition of the Notch pathway or knockdown of TRPC6 function ameliorates the adverse effects caused by the TFW-induced switch in Numb isoforms. Taken together, our results indicate that Notch and Numb interaction may influence the sensitivity of neuronal cells to injurious stimuli by modulating calcium-dependent apoptotic signaling cascades.The Notch pathway is required for the embryonic and postnatal development of many organs by regulating cell proliferation, differentiation, and apoptosis through direct cell to cell contact (1). Notch consists of a family of single-pass transmembrane receptors (Notch1-4), which can be activated by interaction with membrane-tethered ligands of the Delta/Serrate/ Lag2 family expressed on neighboring cells (2). Upon activation, Notch is cleaved by ␥-secretase (3), releasing from the membrane the Notch intracellular domain (NICD) 3 that translocates to the nucleus. NICD associates with CSL (an acronym for the mammalian CBF-1, Drosophila Suppressor of Hairless and Caenorhabditis elegans Lag-1 transcription factors (4)) to control the expression of target genes, most notably the basic helix-loop-helix transcription factors belonging to the Hes (hairy and enhancer of split) (5) and Herp (Hes-related protein) family (6). These transcription factors in turn regulate the ability of the cells to respond to environmental clues. In the absence of Notch signaling, CSL represses transcription of Notch target genes, and following activation by Notch, CSL is converted into a transcriptional activator and activates transcription of the same genes (7). Disruption of the Notch gene in mice results in severe developmental defects and embryonic lethality, supporting a major rol...

Notch Activation of Ca2+ Signaling in the Development of Hypoxic Pulmonary Vasoconstriction and Pulmonary Hypertension

“…Previous studies in neuronal cells have demonstrated NICD-dependent transcription of TRPC6 (23) and that TRPC6 is a key mediator of Notch-driven growth and invasiveness of glioblastoma cells (22). We show in this study that blockade of the hypoxia-induced increase in TRPC6 expression and function inhibits acute HPV and the development of HPH in mice.…”

“…Recently, two studies have demonstrated Notch-dependent expression of TRPC6, raising the possibility that Notch signaling may transcriptionally regulate TRPC6 and may thereby play a critical role in HPH (22,23).…”

Notch Signaling Is Involved in Neurogenic Commitment of Human Periodontal Ligament-Derived Mesenchymal Stem Cells

“…Different culture medium supplementation influenced other intracellular signaling, which further controlled and interacted with Notch signaling [50]. Moreover, a withdrawal of trophic factors resulted in the reversible change of Numb isoforms that further controlled the activation of Notch signaling target genes [51]. Together, these results suggest that culture environment has considerable influences on the different regulation of Notch signaling target gene expression and cell's behaviors.…”

Notch signaling in the brain: In good and bad times

“…There are, however, several reports showing that bath application of soluble ligands, containing the DSL binding site for Notch, can lead to Notch activation in neurons, neuronal stem cells and other mammalian cell lines (Androutsellis-Theotokis et al, 2006;Kyriazis et al, 2010;Wang et al, 2004). The presence of a soluble ligand has been confirmed in a recent paper indicating constitutive ligand shedding by ADAM-17 (Parr-Sturgess et al, 2010).…”

“…Nevertheless, it has been recently proposed that the interaction between Numb and Notch can be reverted upon stress conditions. This study showed that trophic factor withdrawal induces a switch in Numb isoform to a shorter phosphotyrosine-binding domain (Numb-SPTB) that favors endocytic sorting/processing of Notch (Kyriazis et al, 2010) (Fig. 3).…”