Role Of Numb Research Articles

Numb is required for optimal contraction of skeletal muscle.

BackgroundThe role of Numb, a protein that is important for cell fate and development and that, in human muscle, is expressed at reduced levels with advanced age, was investigated; adult mice skeletal muscle and its localization and function within myofibres were determined.MethodsNumb expression was evaluated by western blot. Numb localization was determined by confocal microscopy. The effects of conditional knock out (cKO) of Numb and the closely related gene Numb‐like in skeletal muscle fibres were evaluated by in situ physiology, transmission and focused ion beam scanning electron microscopy, three‐dimensional reconstruction of mitochondria, lipidomics, and bulk RNA sequencing. Additional studies using primary mouse myotubes investigated the effects of Numb knockdown on cell fusion, mitochondrial function, and calcium transients.ResultsNumb protein expression was reduced by ~70% (P < 0.01) at 24 as compared with 3 months of age in gastrocnemius and tibialis anterior muscle. Numb was localized within muscle fibres as bands traversing fibres at regularly spaced intervals in close proximity to dihydropyridine receptors. The cKO of Numb and Numb‐like reduced specific tetanic force by 36% (P < 0.01), altered mitochondrial spatial relationships to sarcomeric structures, increased Z‐line spacing by 30% (P < 0.0001), perturbed sarcoplasmic reticulum organization and reduced mitochondrial volume by over 80% (P < 0.01). Only six genes were differentially expressed in cKO mice: Itga4, Sema7a, Irgm2, Vezf1, Mib1, and Tmem132a. Several lipid mediators derived from polyunsaturated fatty acids through lipoxygenases were up‐regulated in Numb cKO skeletal muscle: 12‐HEPE was increased by ~250% (P < 0.05) and 17,18‐EpETE by ~240% (P < 0.05). In mouse primary myotubes, Numb knockdown reduced cell fusion (~20%, P < 0.01) and delayed the caffeine‐induced rise in cytosolic calcium concentrations by more than 100% (P < 0.01).ConclusionsThese findings implicate Numb as a critical factor in skeletal muscle structure and function and suggest that Numb is critical for calcium release. We therefore speculate that Numb plays critical roles in excitation–contraction coupling, one of the putative targets of aged skeletal muscles. These findings provide new insights into the molecular underpinnings of the loss of muscle function observed with sarcopenia.

Loss of Numb promotes hepatic progenitor expansion and intrahepatic cholangiocarcinoma by enhancing Notch signaling

Numb, a stem cell fate determinant, acts as a tumor suppressor and is closely related to a wide variety of malignancies. Intrahepatic cholangiocarcinoma (iCCA) originates from hepatic progenitors (HPCs); however, the role of Numb in HPC malignant transformation and iCCA development is still unclear. A retrospective cohort study indicated that Numb was frequently decreased in tumor tissues and suggests poor prognosis in iCCA patients. Consistently, in a chemically induced iCCA mouse model, Numb was downregulated in tumor cells compared to normal cholangiocytes. In diet-induced chronic liver injury mouse models, Numb ablation significantly promoted histological impairment, HPC expansion, and tumorigenesis. Similarly, Numb silencing in cultured iCCA cells enhanced cell spheroid growth, invasion, metastasis, and the expression of stem cell markers. Mechanistically, Numb was found to bind to the Notch intracellular domain (NICD), and Numb ablation promoted Notch signaling; this effect was reversed when Notch signaling was blocked by γ-secretase inhibitor treatment. Our results suggested that loss of Numb plays an important role in promoting HPC expansion, HPC malignant transformation, and, ultimately, iCCA development in chronically injured livers. Therapies targeting suppressed Numb are promising for the treatment of iCCA.

Hepatic stem cell Numb gene is a potential target of Huang Qi Decoction against cholestatic liver fibrosis

Numb is a negative regulator of Notch signal pathway. Previous study has demonstrated that Notch signal pathway activation is required for hepatic progenitor cell (HPC) differentiating into cholangiocytes in cholestatic liver fibrosis (CLF), and Huang Qi Decoction (HQD) could prevent CLF through inhibition of the Notch signal pathway. However, the role of Numb in HQD against CLF is yet unclear. Thus, CLF rats transplanted into rat bone marrow-derived mesenchymal stem cells with knocked down Numb gene (BMSCNumb-KD) were treated with HQD. Simultaneously, Numb gene knockdown was also performed in WB-F344 cell line and then treated with refined HQD in vitro. In vivo study revealed that liver fibrosis was inhibited by HQD plus BMSCNumb-KD treatment, while Hyp content in liver tissue, the gene and protein expression of α-SMA, gene expression of Col I, TNF-α, and TGF-β1 were increased compared to that in HQD group. Furthermore, Notch signal pathway was inhibited by HQD plus BMSCNumb-KD, while the protein expression of Numb was decreased and RBP-Jκ and Hes1 was increased compared to that in HQD group. In vitro, HQD reduced the differentiation of WB-F344 cells into cholangiocyte phenotype, while this effect was attenuated after Numb-knockdown. This study highlights that the absence of hepatic stem cell Numb gene decreases effect of HQD against CLF, which give rise the conclusion that Numb might be a potential target for HQD against CLF.

Numb ameliorates necrosis and inflammation in acute kidney injury induced by cisplatin

Cisplatin induces acute renal failure in humans and mice.Tubular apoptosis, necrosis and inflammation are the primary pathogenesis of cisplatin-induced acute kidney injury(AKI). We previously reported that the depletion of Numb from proximal tubules exacerbates tubular cells apoptosis in cisplatin-induced AKI, however, the role of Numb in tubular necrosis and renal inflammation in cisplatin-induced AKI remains unclear. A mouse model of AKI was produced by cisplatin intraperitoneally injection in mice from proximal tubule-specific depletion of Numb (PT-Nb-KO) and their wild-type littermates (PT-Nb-WT) respectively. Renal Numb expression was determined by Western blotting. Renal morphological damage was examined by hematoxylin and eosin staining (H&E staining). Tubular necrosis was evaluated by histological study and the protein level of renal Mixed lineage kinase domain-like protein (MLKL) which is a molecular marker of necrosis. Leukocyte infiltration and pro-inflammatory cytokines was determined by immunostaining and quantitative real-time PCR (qRT-PCR) respectively.The protein level of Numb was dramatically decreased in kidneys of PT-Nb-KO mice compared with PT-Nb-WT mice. After cisplatin injection, a significant increase of tubular injury score and the protein level of renal MLKL were detected in PT-Nb-KO mice compared with those in PT-Nb-WT. In addition, the number of F4/80-positve and CD3-positive cells, markers for macrophages and neutraphils respectively, showed significantly increased in kidneys from PT-Nb-KO mice compared with those in PT-Nb-WT mice. Consistently, the gene expression of pro-inflammatory cytokines including TNF-α and MCP-1 in the kidneys was higher in PT-Nb-KO mice than those in PT-Nb-WT mice. Numb play additional protective role in cisplatin-induced AKI through ameliorating tubular necrosis and renal inflammation besides attenuating cisplatin-induced tubular apoptosis.

A role for Numb in Protein kinase M (PKM)-mediated increase in surface AMPA receptors during facilitation in Aplysia.

There is considerable evidence from both vertebrates and invertebrates that persistently active protein kinases maintain changes in synaptic strength that underlie memory. In the hermaphrodite marine mollusk, Aplysia californica, truncated forms of protein kinase C (PKC) termed protein kinase Ms have been implicated in both intermediate- and long-term facilitation, an increase in synaptic strength between sensory neurons and motor neurons thought to underlie behavioural sensitization in the animal. However, few substrates have been identified as candidates that could mediate this increase in synaptic strength. PKMs have been proposed to maintain synaptic strength through preventing endocytosis of AMPA receptors. Numb is a conserved regulator of endocytosis that is modulated by phosphorylation. We have identified and cloned Aplysia Numb (ApNumb). ApNumb contains three conserved PKC phosphorylation sites and PKMs generated from classical and atypical Aplysia PKCs can phosphorylate ApNumb in vitro and in cells. Over-expression of ApNumb that lacks the conserved PKC phosphorylation sites blocks increases in surface levels of a pHluorin-tagged Aplysia glutamate receptor measured using live imaging after intermediate- or long-term facilitation. Over-expression of this form of ApNumb did not block increases in synaptic strength seen during intermediate-term facilitation, but did block increases in synaptic strength seen during long-term facilitation. There was no effect of over-expression of this form ofApNumb on other putative Numb targets as measured using increases in calcium downstream of neurotrophins or agonists of metabotropic glutamate receptors. These results suggest that in Aplysia neurons, Numb specifically regulates AMPA receptor trafficking and is an attractive candidate for a target of PKMs in long-term maintenance of synaptic strength. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.

NUMB knockdown enhanced the anti-tumor role of cisplatin on ovarian cancer cells by inhibiting cell proliferation and epithelial-mesenchymal transition

Background: NUMB is an inhibitory regulator of NOTCH signaling, which is critical for the induction of epithelial-mesenchymal transition (EMT). Loss of NUMB expression is correlated with the genesis and development of multiple tumors. Recent studies reported that NUMB expression was upregulated in human ovarian cancer. However, the role of NUMB in ovarian cancer is still unclear. Here, we invested the effect of NUMB knockdown on the proliferation and EMT in ovarian cancer cells and explored the role of NUMB in the effect of cisplatin. Methods: Two ovarian cancer cells (OVCAR-3 and SK-OV-3) were used in the experiments. The proliferation and apoptosis of ovarian cancer cells was examined using methyl thiazolyl tetrazolium (MTT) test and flow cytometry assays. The invasion and migration of ovarian cancer cells were examined using Transwell assays. The expression of EMT markers were examined using Simple Western analysis. Results: NUMB knockdown inhibited cell proliferation, invasion, and migration in both ovarian cancer cells. NUMB knockdown enhanced cisplatin-induced cell growth inhibiting and apoptosis in both ovarian cancer cells. NUMB knockdown enhanced cisplatin-induced cell invasion in SK-OV-3 cells. NUMB knockdown also decreased the expression of N-cadherin and Vimentin in SK-OV-3 cells. Conclusions: NUMB acted as an oncogene in ovarian cancer and NUMB knockdown enhanced the anti-tumor role of cisplatin on ovarian carcinoma cells by inhibiting cell proliferation and EMT.

Advances in research on cell fate determinant Numb regulating liver cancer

Liver cancer is one of the most common malignant tumors in China, ranking fifth in malignant tumors and the third in tumor-related deaths. As a membrane-related protein, the asymmetric distribution of cell fate determinant Numb plays a key role in cell differentiation. Research reports that Numb may be closely associated to the occurrence and development of tumors. Recently, scholars have gradually valued its important role in liver cancer. This article briefly reviews the structure of Numb molecule, relationship between Numb and tumorigenesis, the molecular mechanism of Numb-regulated tumors, and the role of Numb in the development of liver cancer.

Loss of nuclear NOTCH1, but not its negative regulator NUMB, is an independent predictor of cervical malignancy.

The participation of NOTCH signaling in invasive cervical cancer (ICC) remains controversial since both tumor suppressive and oncogenic properties have been described. Additionally, the role of NUMB, a negative regulator of NOTCH, remains unclear in ICC. We aimed to investigate the role of NOTCH1 and NUMB expression and their localization in cervical intraepithelial neoplasia (CIN) and ICC samples. A total of 144 biopsies were obtained from the Instituto Nacional de Cancerología, México from 2004 to 2017, and were subjected to immunohistochemistry for NOTCH1 and NUMB. We found that nuclear NOTCH1 expression was more frequently found in CIN samples compared with ICC (77.55% vs. 15.79%, p = 0.001). NUMB was almost exclusively found in the nucleus of CIN samples (32.65% vs. 6.32%, p = 0.001). Cytoplasmic expression of NOTCH1 (44.21%) and NUMB (35.79%) was the most frequent localization in ICC. Multivariable-adjusted analysis showed that the loss of nuclear NOTCH1 expression was an independent predictor of malignancy (β = –3.428, 95% confidence interval [95% CI] = –5.127, –1.728, p = 0.001). In contrast, the association between cytoplasmic NUMB expression and cervical cancer was lost after adjusting for nuclear NOTCH1 expression (β = 2.074, 95% [CI] = –0.358, 4.506, P = 0.094). Additionally, patients with cytoplasmic NOTCH1 expression showed a borderline association with longer overall survival (OS) than those with nuclear NOTCH1 expression (P = 0.08). Our data suggest that the loss of nuclear NOTCH1 but not NUMB might be an independent predictor of malignancy in cervical cancer.

Increased Numb protein expression predicts poor clinical outcomes in esophageal squamous cell carcinoma patients

ABSTRACTNumb is a protein whose asymmetric segregation during cell division determines cell fate and has numerous functions relevant to multiple fields of study, including developmental neurobiology and cancer biology. Little is known about the role of Numb in esophageal squamous cell carcinoma (ESCC), the predominant histological esophageal carcinoma in Asian populations. In this study, we focused on the expression and biologic functions of Numb in the context of ESCC. From analysis of tissue microarrays with 212 patients, it was found that Numb was significantly upregulated in ESCC tissues compared with corresponding non-cancerous tissues. Kaplan-Meier survival analysis suggests that higher expression of Numb was significantly associated with a high tumor recurrence (p = 0.015) and poor overall post-surgical survival (p = 0.016). Using multiple Cox regression, the expression of Numb was determined to be an independent predictor of poor prognosis. When siRNA was used to knockdown Numb in ESCC cell lines, there was a consistent increase in caspase-3 dependent apoptosis and inhibition of cellular proliferation, as well as downregulation of expression of the cancer stem cell markers Oct-4, SOX-2 and Nanog. In addition, downregulated Numb expression was not significantly associated with the migration of ESCC cells. These results indicate that Numb acts as an oncoprotein and has potential as a novel prognostic biomarker and therapeutic target in ESCC patients.

P28 Nrf2 antioxidant pathway suppresses epithelial-mesenchymal transition through up-regulating Numb during pulmonary fibrosis

Epithelial mesenchymal transition(EMT) is a key process in the development of pulmonary fibrosis (PF). Our previous studies have shown that nuclear factor erythroid 2 related factor 2 (Nrf2), an important regulator of antioxidant defence, alleviates the EMT in BLM-induced mouse models of PF. In recent work, Numb, a cell fate determinant, has been shown to appear even more frequently in PF or emphysema, but the relationship between Nrf2 and Numb is still unknown. To further explore the role of Numb in PF, bleomycin (BLM) was intratracheally injected into both Nrf2-knockout (Nrf2 −/− ) and wild-type mice to enable the assessment of the relationship between the Nrf2 antioxidant pathway, Numb and EMT. Rat type II alveolar epithelial cells (RLE-6TN) and human epithelial cells (A549) were treated with the Nrf2 activator sulforaphane, or transfected with Nrf2 and Numb siRNAs to determine the level of influence of Numb on EMT induced by TGF-β1. This study revealed that BLM-induced EMT and lung fibrosis were more severe in Nrf2 −/− mice compared to wild-type mice and interestingly Numb expression was upregulated at day 7 in the fibrosis model with also a protective increase in the level of Nrf2. In vitro , sulforaphane treatment attenuated TGF-β1-induced EMT accompanied by the further up-regulation of Numb. However, when Numb was silenced, the therapeutic effect of sulforaphane on the progression of EMT in RLE-6TN and A549 cells was attenuated in vitro . These findings suggest that the Nrf2 antioxidant pathway suppresses epithelial-mesenchymal transition through regulating Numb during pulmonary fibrosis.

CD3ε recruits Numb to promote TCR degradation.

Modulation of TCR signaling upon ligand binding is achieved by changes in the equilibrium between TCR degradation, recycling and synthesis; surprisingly, the molecular mechanism of such an important process is not fully understood. Here, we describe the role of a new player in the mediation of TCR degradation: the endocytic adaptor Numb. Our data show that Numb inhibition leads to abnormal intracellular distribution and defective TCR degradation in mature T lymphocytes. In addition, we find that Numb simultaneously binds to both Cbl and a site within CD3ε that overlaps with the Nck binding site. As a result, Cbl couples specifically to the CD3ε chain to mediate TCR degradation. The present study unveils a novel role of Numb that lies at the heart of TCR signaling initiation and termination.

A Novel Role of Numb as A Regulator of Pro-inflammatory Cytokine Production in Macrophages in Response to Toll-like Receptor 4.

Activation of macrophages triggers the release of pro-inflammatory cytokines leading to inflammation. Numb is a negative regulator of Notch signaling, but the role of Numb in macrophages is not fully understood. In this study, the role of Numb as a regulator of inflammatory responses in macrophages was investigated. Murine bone marrow-derived macrophages, in which expression of Numb was silenced, secreted significantly less TNFα, IL-6 and IL-12 and more IL-10 upon activation by lipopolysaccharide (LPS), a ligand for Toll-like receptor 4 (TLR4), despite increased Notch signaling. The Tnfα mRNA levels both in Numb-deficient and wild-type macrophages were not significantly different, unlike those of Il6 and Il12-p40. In Numb-deficient macrophages, the Tnfα mRNAs were degraded at faster rate, compared to those in control macrophages. Activation of p38 MAPK and NF-κΒ p65 were compromised in activated Numb deficient macrophages. Numb was found to interact with the E3 ubiquitin ligase, Itch, which reportedly regulates p38 MAPK. In addition, blocking the Notch signaling pathway in activated, Numb-deficient macrophages did not further reduce TNFα levels, suggesting a Notch-independent role for Numb. A proteomics approach revealed a novel funciton for Numb in regulating complex signaling cascades downstream of TLRs, partially involving Akt/NF-κB p65/p38 MAPK in macrophages.

Abstract 178: Targeting of multiple oncogenic miRNAs on NUMB gene to mediate head and neck carcinogenesis

Abstract Head and neck squamous cell carcinoma (HNSCC) is the 6th leading malignancy in the world. MicroRNAs (miRNAs) are small, non-coding RNAs, which are associated with the genesis and progression of cancers. NUMB is a multi-functional adaptor protein, which can activate E3 ubiquitin ligase and degrade target proteins to modulate pathogenesis. In silico modules predict that miR-31, miR-96, miR-182 and other miRNAs might target NUMB. This study investigates the concordant targeting of multiple oncogenic miRNAs to NUMB, and to verify the roles of NUMB in HNSCC. miR-31, miR-96 and miR-182 were found up-regulated in the vast majority of HNSCC tissues. On the contrary, NUMB expression was largely down-regulated in the tumors. A reverse correlation was noted between miR-31 expression and NUMB expression. Reporter assays using wild type and mutant constructs specified that these oncogenic miRNAs directly target the sequences in the 3′UTR of NUMB gene. However, miR-31 was unable to target a predicted site in the coding sequence of NUMB gene to mediate regulation. Exogenous expression of these miRNAs decreased NUMB expression and increased the migration of HNSCC cells. Knockdown of NUMB expression enhanced oncogenicity of HNSCC cells. However, the exogenous NUMB expression decreased the migration and anchorage-independent growth of HNSCC cells. The oncogenicity induced by these oncogenic miRNAs was attenuated by NUMB expression. This study provides in vitro and in vivo clues demonstrating that miR-31, miR-96 and miR-182 co-target NUMB gene in HNSCC. The coordinative abrogation of these miRNAs together with NUMB enhancement may be contributive to new therapeutic intervention against head and neck cancers. Citation Format: Chung-Shan Chou, Kuo-Wei Chang, Shu-Chun Lin. Targeting of multiple oncogenic miRNAs on NUMB gene to mediate head and neck carcinogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 178. doi:10.1158/1538-7445.AM2015-178

Role of Numb expression and nuclear translocation in endometrial cancer.

The cell fate determinant Numb orchestrates numerous cell physiological and pathological processes and previous evidence has indicated that Numb expression is associated with tumorigenesis. The present study investigated the potential role of Numb in endometrial cancer (EC). Numb expression was compared between the normal endometrium and EC tissue by immunohistochemistry, and the protein levels were assessed by western blotting and confocal microscopy in the human endometrial HEC-1B cancer cell line and normal epithelial cells. The intracellular localization of Numb in HEC-1B cells was examined by immunofluorescence. Numb was found to be expressed at higher levels in endometrial cancer compared with the normal endometrium. Although Numb localizes to the cytoplasm and plasma membrane in the normal epithelium, the present study found that Numb accumulated in the nucleus of HEC-1B cells. The present data reveals the upregulation of Numb expression in EC tissues and indicates that Numb plays a role in the occurrence of EC, which may be mediated by its translocation into the nucleus. The role of Numb in cancer development requires additional investigation.

Numb-dependent integration of pre-TCR and p53 function in T-cell precursor development

Numb asymmetrically segregates at mitosis to control cell fate choices during development. Numb inheritance specifies progenitor over differentiated cell fates, and, paradoxically, also promotes neuronal differentiation, thus indicating that the role of Numb may change during development. Here we report that Numb nuclear localization is restricted to early thymocyte precursors, whereas timed appearance of pre-T-cell receptor (pre-TCR) and activation of protein kinase Cθ promote phosphorylation-dependent Numb nuclear exclusion. Notably, nuclear localization of Numb in early thymocyte precursors favors p53 nuclear stabilization, whereas pre-TCR-dependent Numb nuclear exclusion promotes the p53 downmodulation essential for further differentiation. Accordingly, the persistence of Numb in the nucleus impairs the differentiation and promotes precursor cell death. This study reveals a novel regulatory mechanism for Numb function based on its nucleus–cytosol shuttling, coupling the different roles of Numb with different stages of T-cell development.

Sustained vs. oscillating expressions of Ngn2, Dll1 and Hes1: A model of neural differentiation of embryonic telencephalon

Neural progenitor cells show oscillatory expression of the Notch ligand Delta-like1 (Dll1), the Notch target Hes1 and the proneural gene Neurogenin 2 (Ngn2) during embryonic development of the mammalian telencephalon. On the other hand, expression of these genes is sustained in postmitotic neurons (upregulated for Ngn2 and Dll1, down regulated for Hes1). These facts suggest that a switch from oscillatory to sustained expression of proneural and other genes is critical in neural fate decisions. Moreover, despite controversies over the role of Numb in determining the neural fate in mammals, there is evidence that inheritance of Numb during neurogenic cell division is involved in neural differentiation. It is also known that mNumb activates Notch1 receptor degradation. The arrest of oscillations in a given cell may be due to increasing degradation of Notch1 brought about by mNumb during neurogenic division. We introduce a modification in a previous model of the gene network for two cells coupled by the Delta-Notch pathway (Wang et al., 2011). We analyze the consequences of an asymmetry between two neighbor cells in the rate of degradation of Notch (mimicking the effect of asymmetric inheritance of mNumb during the neurogenic division). The results show that a slight difference in Notch degradation between the two cells keeps oscillation going in one of them while oscillation stops in the other. Moreover, when Delta-Notch coupling is canceled, both cells show sustained expression (upregulated levels for Ngn2 and Dll1, downregulated for Hes1). We show that the model is stable against parameter variations. Moreover, to take into account the possible influence of the environment on both cells, neighboring cells are included in a mean field approximation. Both, parameter fluctuations and effects of the environment lead to asynchronous oscillations of Hes1/Ngn2 in different progenitor cells.

Numb/Notch Signaling Plays an Important Role in Cerebral Ischemia-induced Apoptosis

Numb has been shown to play diverse roles in the central nervous system of adult mammals, and accumulating evidence indicates a role for Numb in apoptosis. In this study, we characterize the role of Numb in ischemia-induced apoptosis, and investigate the underlying pathway involved in this process. In vivo, exposure of pheochromocytoma (PC12) cells to glucose deprivation (GD) resulted in caspase-3-dependent apoptosis. Numb expression was upregulated by GD in a time-dependent manner, while Notch expression was down regulated. Knocking down endogenous Numb expression via siRNA protected PC12 cells from GD-induced apoptosis, whereas Numb overexpression sensitized PC12 cells to GD-induced apoptosis. In vivo, significantly increased Numb expression levels, together with activation of apoptosis, can be observed in the ischemic penumbra following cerebral ischemia. Taken together, our data show that Numb promotes ischemia-induced apoptosis. Based on these results, we conclude that inhibition of Numb could be a novel therapeutic approach for inhibiting apoptosis in the ischemic penumbra.

Characterization and role of NUMB in the human extravillous trophopblast

NUMB is a multifunctional protein involved in asymmetric cell differentiation, proliferation and maintenance. Four mammalian NUMB isoforms have been identified, which utilize the phosphotyrosine binding (PTB) domain and the proline rich region (PRR) domain to regulate cell growth and differentiation in the developing nervous system. The observation that a decrease in spongiotrophoblast number and thickness of placentae of null ( Numb −/− ) mouse embryos, which died at E10.5, suggests NUMB may play a role in placental development. In this study, we demonstrated for the first time, that NUMB isoforms 1, 2, 3, and 4 are present in the human placenta and the human extravillous trophoblast (EVT) cell line HTR8/SVneo. We report three novel isoforms, NUMB 7, 8, and 9, identified by cloning of RT-PCR products and sequencing. Corresponding sequences of novel isoforms were submitted to genebank (accession numbers for each new isoform: NUMB 7- EU265736, NUMB 8- EU265737 and NUMB 9- EU265738). Western blot analysis confirmed the presence of all NUMB isofoms in human placental samples in all trimesters and in EVT cells. NUMB immunosignals were extensively localized in human extravillous trophoblasts and decidual cells at the maternal–fetal interface. NUMB 8 appeared to be the predominant isoform in placental villi. Furthermore, cell migration studies revealed NUMB isoform 1 to be involved in EVT cell migration and NUMB isoforms 2 and 4 to induce EVT apoptosis.

Numb regulates meiotic spindle organisation in mouse oocytes

Numb is an adaptor protein that controls the fate of cells in different species through asymmetrical inheritance by sibling cells during division. It has been investigated extensively in mitosis, mostly in neural progenitor cells, but its function in meiosis remains unknown. The present study was designed to investigate the expression, subcellular localisation and functional roles of Numb during mouse oocyte meiotic maturation. Using real-time polymerase chain reaction and western blotting, we found that the expression of Numb increased from the germinal vesicle (GV) to MII stages. Immunofluorescent staining revealed that Numb was mainly concentrated in the GV before meiosis resumption, aggregated in the vicinity of the chromosomes after GV breakdown and then localised to the spindle poles from prometaphase I to MII. Nocodazole treatment resulted in spindle destruction and Numb diffusion into the cytoplasm. However, Numb appeared at the spindle poles again once the spindles had formed when nocodazole-treated oocytes were washed and cultured for spindle recovery. Depletion of Numb by RNA interference resulted in chromosome misalignment, spindle deformation and even doubled spindle formation. Our results suggest that Numb is critical for spindle organisation during mouse oocytes meiosis. The present study provides evidence of a new function for Numb in addition to its action as a cell fate-determining factor.

Numb Regulates Post-endocytic Trafficking and Degradation of Notch1

Notch is a transmembrane receptor that controls cell fate decisions during development and tissue homeostasis. Both activation and attenuation of the Notch signal are tightly regulated by endocytosis. The adaptor protein Numb acts as an inhibitor of Notch and is known to function within the intracellular trafficking pathways. However, a role for Numb in regulating Notch trafficking has not been defined. Here we show that mammalian Notch1 is constitutively internalized and trafficked to both recycling and late endosomal compartments, and we demonstrate that changes in Numb expression alter the dynamics of Notch1 trafficking. Overexpression of Numb promotes sorting of Notch1 through late endosomes for degradation, whereas depletion of Numb facilitates Notch1 recycling. Numb mutants that do not interact with the ubiquitin-protein isopeptide ligase, Itch, or that lack motifs important for interaction with endocytic proteins fail to promote Notch1 degradation. Our data suggest that Numb inhibits Notch1 activity by regulating post-endocytic sorting events that lead to Notch1 degradation.