Differentiation through inhibition of notch signaling

Numb's primary function in cell differentiation is as an inhibitor of Notch signaling which is essential for maintaining self-renewal potential in stem and progenitor cells. Notch is a transmembrane signaling receptor that is activated by DSL family ligands. Notch binds the ligands Delta and Serrate in Drosophila. The human ligands are Delta-like and Jagged, respectively. These ligands are themselves integral membrane proteins. Following ligand binding of the Notch receptor, the intracellular fragment of Notch (NICD, or notch intracellular domain) is released into the cytoplasm and transported to the nucleus, where it can form a complex with binding partners such as EP300 and histone acetyltransferase and act as a transcription factor for Notch target genes.^[12] Among the Notch target genes are members of the HES and HEY gene families whose protein products can act as transcriptional repressors for tissue-specific transcription factors, thus maintaining the cell's potential for self-renewal.

Inhibition of notch signaling through the ubiquitination pathway

Numb exerts its functional role on cell fate decisions by antagonizing Notch signaling activities. The molecular mechanisms underlying this relationship appear to rely on the ubiquitination of the membrane bound Notch1 receptor and the subsequent degradation of its NICD following receptor activation.^[13] In support of this, Numb's ability to ubiquinate Notch1 directly correlated with its functional inhibition of Notch1 signaling activities. The ubiquitination pathway directs protein recycling by directly tagging specific proteins for proteasome degradation. Through a multi-step process, free ubiquitin is first attached to an activating enzyme (E1) and then transferred to a conjugating enzyme (E2) which partners with a ligase (E3) which functions as an adaptor to selectively transfer the ubiquitin to specific protein substrates. Numb expression was found to selectively tag the membrane Notch1 receptor for ubiquitination through the interaction of its Phosphotyrosine-binding domain with the E3 ubiquitin ligase Itch. Numb and Itch work in concert to promote the ubiquitination of the full-length membrane-tethered Notch receptor prior to activation. However, Numb only appears to promote the degradation of the NICD cleavage product following receptor activation, targeting it for proteasome degradation and preventing its translocation to the nucleus.

Inhibition of notch signaling through sanpodo

Numb acts as an antagonist for Notch by causing its selective endocytosis and degradation.^[14] Another mechanism proposed for how this is accomplished in Drosophila involves a protein called Sanpodo. Sanpodo is a protein that associates with both Notch and Numb. It is located at the plasma membrane and is necessary for Notch activation, promoting Notch cleavage and NICD signaling in the nucleus.^[9] Numb converts Sanpodo from an activator to an inhibitor of Notch signaling, magnifying the differences in Notch signaling between different daughter cells. In daughter cells containing Numb, Sanpodo allows Numb to inhibit Notch. In daughter cells with no Numb, Sanpodo potentiates Notch signaling. Sanpodo therefore allows cells to maintain Notch signaling at a below- or above-threshold level.^[6]

External sensory organ development

The Drosophila external sensory organ is a sensory structure in the peripheral nervous system that consists of four cells; a neuron, a sheath cell that surrounds the dendrite, and hair and socket cells, which are considered the “outer” support cells. All four cell fates are descendants of the sensory organ precursor (SOP) cell. In response to the proper cues, SOPs first divide into two secondary precursor cells. The posterior daughter cell is called the pIIa cell and the anterior daughter cell is called the pIIb. The pIIa cell divides to produce a bristle cell and a socket cell, while the pIIb cell divides to produce a neuron and a glial cell. The asymmetric division of the SOP into daughter cells with distinct fates is dependent upon the distribution of Numb. Numb is distributed uniformly in the cytoplasm until mitotic division, when it is selectively localized to the anterior pole of the cell. Thus, Numb is selectively segregated into the pIIb daughter cell upon division of the SOP.^[15]

Loss of Numb function causes inappropriate differentiation of SOP cells into all pIIa cells, producing four outer support cells and no neurons or glia.^[16] In SOP loss of function Numb mutants, flies have a significant decrease in sensory neurons, leaving them “numb.” Gain of function Notch mutants express a similar phenotype.^[17] Ectopic expression of Numb during SOP division has the opposite effect, producing all pIIb cells and no outer support cells. In support of previous experiments demonstrating the role of Numb in Notch signaling inhibition, functional loss of Notch signaling components result in SOP division into two pIIb cells, suggesting Numb promotes acquisition of the pIIb cell fate through inhibition of Notch signaling.^[16] Thus, the asymmetric distribution of Numb into IIb secondary precursors during SOP division is necessary for daughter cells to acquire distinct cell fates.^[15]

Ganglion mother cell

A ganglion mother cell (GMC) is the cell derived from the division of a neuroblast in the Drosophila central nervous system. The neuroblast divides to produce two cells, a progenitor cell like the mother neuroblast and a GMC that will divide to produce neurons. The mother neuroblast divides along the apical-basal axis, with Numb localizing basally and ending up in the GMC.^[18]

Alternative splicing to support proliferation and differentiation

In mice embryos mutant for Numb, early neurons emerge in the expected spatial and temporal pattern but fail to maintain a sufficient pool of proliferating progenitors and nearly deplete the population of dividing cells shortly after the onset of neurogenesis.^[19] These embryos display precocious neuron production in the forebrain and defects in neural tube closure, dying around embryonic day 11.5.^[20] These studies suggest a functional role of mammalian Numb in promoting progenitor cell fate during neurogenesis, which directly opposes the proposed role of Numb in invertebrates. However, other studies have shown overexpression of Numb in the mammalian neural crest MONC-1 stem cell line biases neuronal differentiation, consistent with what is observed in drosophila.^[21]

Unlike the invertebrate Numb gene, the mammalian Numb gene undergoes alternative splicing to produce at least four functionally different Numb isoforms. While asymmetric divisions alone can produce sufficient neuron populations in Drosophila, mammalian brains are much more advanced and require larger populations of neurons that can't be established to asymmetric divisions alone.^[22] Thus, mammalian cortical progenitors must first need to undergo symmetric divisions to expand the progenitor pool before they can undergo later asymmetric divisions for neuronal generations. The mammalian brain has accounted for this by producing isoforms of Numb that maintain progenitor populations in addition to those which support neuronal differentiation.

Studies using the mouse embryonic P19 cell line have shown isoforms with the short proline rich region (PRR) domain promote neuronal differentiation, while those with the long PRR domain promote cell proliferation and prevent differentiation.^[23] The p71 and p72 isoforms, which contain the PRR insert, are primarily expressed in actively dividing tissues and are down regulated during differentiation suggesting these isoforms promote cell proliferation (Dho et al., 1999). In contrast, the Drosophila Numb gene encodes a 66 kDa protein.^[21] Consistent with the finding that Numb only supports differentiation and not proliferation in asymmetric division, the 66 kDa Drosophila protein is analogous to a shorter mammalian isoform lacking the PRR insert and thus promoting cell differentiation.^[21]

Role in mammary carcinomas

In approximately half of all human mammary carcinomas, Numb-mediated suppression of Notch signaling is lost due to Numb ubiquitination, tagging it for proteasomal degradation.^[24] Numb acts as an oncogene suppressor, inhibiting tumor cell proliferation through suppression of Notch signaling. Increased Notch signaling is observed in tumors where Numb activity has been lost and retrovirally mediated transient overexpression of Numb protein in these tumors restored basal levels of Notch signaling and significantly reduced their colony-forming abilities. Thus, the biological antagonism between Notch and Numb signaling that controls the proliferative/differentiative balance of many cell lineages appears to play a role in human breast carcinogenesis and perhaps other types of tumorigenesis. Pharmalogical inhibition of Notch signaling or enhancement of Numb signaling could be a source of treatment for cancer patients in the future.

Numb has been posited to have a role in tumor-suppression, through its ability to regulate Notch and TP53. Numb binds and inhibits the E3-ligase Mdm2 that is responsible for TP53 ubiquitination and degradation. The ablation of Numb in a cell leads to a decrease in TP53, causing impaired apoptosis and cell cycle checkpoint response. Restoring Numb levels also restores TP53 expression and tumor suppressant abilities.^[6]