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Hailey‐Hailey disease (HHD) is a rare autosomal dominantly inherited disorder caused by mutations in the ATP2C1 gene that encodes an adenosine triphosphate (ATP)‐powered calcium channel pump. HHD is characterized by impaired epidermal cell‐to‐cell adhesion and defective keratinocyte growth/differentiation. The mechanism by which mutant ATP2C1 causes HHD is unknown and current treatments for affected individuals do not address the underlying defects and are ineffective. Notch signalling is a direct determinant of keratinocyte growth and differentiation. We found that loss of ATP2C1 leads to impaired Notch1 signalling, thus deregulation of the Notch signalling response is therefore likely to contribute to HHD manifestation. NOTCH1 is a transmembrane receptor and upon ligand binding, the intracellular domain (NICD) translocates to the nucleus activating its target genes. In the context of HHD, we found that loss of ATP2C1 function promotes upregulation of the active NOTCH1 protein (NICD‐Val1744). Here, deeply exploring this aspect, we observed that NOTCH1 activation is not associated with the transcriptional enhancement of its targets. Moreover, in agreement with these results, we found a cytoplasmic localization of NICD‐Val1744. We have also observed that ATP2C1‐loss is associated with the degradation of NICD‐Val1744 through the lysosomal/proteasome pathway. These results show that ATP2C1‐loss could promote a mechanism by which NOTCH1 is endocytosed and degraded by the cell membrane. The deregulation of this phenomenon, finely regulated in physiological conditions, could in HHD lead to the deregulation of NOTCH1 with alteration of skin homeostasis and disease manifestation. [ABSTRACT FROM AUTHOR]

Bone regeneration strategies in dentistry utilize biodegradable scaffolds seeded with stem cells able to induce bone formation. However, data on regeneration capacity of these tissue engineering constructs are still deficient. In this study micro-Computed tomography (micro-CT) and positron emission tomography (PET) analyses were used to investigate bone regeneration induced by two scaffolds [Granular deproteinized bovine bone (GDPB) and Beta-tricalcium phosphate (β-TCP)] used alone or in combination with dental pulp stem cells (DPSC) in a tissue engineered construct implanted in a rat critical calvarial defect. Bone mineral density (BMD) and standard uptake value (SUV) of tracer incorporation were measured after 2, 4, 8, and 12 weeks post-implant. The results showed that: (1) GDPB implants were mostly well positioned, as compared to ß-TCP; (2) GDPB induced higher BMD and SUV values within the cranial defect as compared to ß-TCP, either alone or in combination with stem cells; (3) addition of DPSC to the grafts did not significantly induce an increase in BMD and SUV values as compared to the scaffolds grafted alone, although a small tendency to increase was observed. Thus our study demonstrates that GDPB, when used to fill critical calvarial defects, induces a greater percentage of bone formation as compared to ß-TCP. Moreover, this study shows that addition of DPSC to pre-wetted scaffolds has the potential to ameliorate bone regeneration process, although the set of optimal conditions requires further investigation. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 102B: 815-825, 2014. [ABSTRACT FROM AUTHOR]

Constitutive activation of the transmembrane receptor, Notch3, and loss of function of the hematopoietic transcription repressor, Ikaros (IK), play direct roles in T-cell differentiation and leukemogenesis that are dependent on pre-T-cell receptor (pre-TCR) signaling. We demonstrate the occurrence of crosstalk between Notch3 and IK that results in transcriptional regulation of the gene encoding the pTα chain of the pre-TCR. We also show that, in the presence of the pre-TCR, constitutive activation of Notch3 in thymocytes causes increased expression of dominantnegative non-DNA-binding IK isoforms, which are able to restrain the IK inhibition of Notch3's transcriptional activation of pTα. This effect appears to be mediated by Notch3's pre-TCR-dependent upregulation of the RNA-binding protein, HuD. Notch3 signaling thus appears to play a critical role in the diminished IK activity described in several lymphoid leukemias. By exerting transcription-activating and transcription-repressing effects on the pTα promoter, Notch3 and IK cooperate in the fine-tuning of pre-TCR expression and function, which has important implications for the regulation of thymocyte differentiation and proliferation. [ABSTRACT FROM AUTHOR]

Notch and basic helix-loop-helix Exa pathways specify cell fate and regulate neoplastic transformation in a variety of cell types. Whereas Notch enhances tumorigenesis, E2A suppresses it. However, whether and how Notch and E2A interact functionally in an integrative mechanism for regulating neoplastic transformation remains to be understood. It has been shown that Notch3-induced T-cell leukaernia is abrogated by the inactivation of pTα/pre-T-cell antigen receptor (pre-TCR). We report here that Notch3-induced transcriptional activation of pTα/pre-TCR is responsible for the down regulation of E2A DNA binding and transcriptional activity. Further, the E2A messenger RNA and protein levels remain unaltered but the E2A inhibitor ld1 expression is augmented in thymocytes and T lymphoma cells derived from Notch3 transgenic mice. The increase in ld1 expression is achieved by pre-TCR-induced extracellular-signalling-regulated kinase 1/2. These observations support a model in which the up regulation of pre-TCR signalling seems to be the prerequisite for Notch3-induced inhibition of E2A, thus leading to the development of lymphoma in Notch3 transgenic mice. [ABSTRACT FROM AUTHOR]

The multiplicity of Notch receptors raises the question of the contribution of specific isoforms to T-cell development. Notch3 is expressed in CD4-8-thymocytes and is down-regulated across the CD4-8- to CD4+8+ transition, controlled by pre-T-cell receptor signaling. To determine the effects of Notch3 on thymocyte development, transgenic mice were generated, expressing lck promoter-driven intracellular Notch3. Thymuses of young transgenics showed an increased number of thymocytes, particularly late CD4-8- cells, a failure to down-regulate CD25 in post-CD4-8- subsets and sustained activity of NF-ΚB. Subsequently, aggressive multicentric T-cell lymphomas developed with high penetrance. Tumors sustained charactaristics of immature thymocytes, including expression of CD25, pTα and activated NF-ΚB via IKKα-dependent degradation of IΚBα and enhancement of NF-ΚB-dependent anti-apoptotic and proliferative pathways. Together, these data identify activated Notch3 as a link between signals leading to NF-ΚB activation and T-cell tumorigenesis. The phenotypes of pre-malignant thymocytes and of lymphomas indicate a novel and particular role for Notch3 in co-ordinating growth and differentiation of thymocytes, across the pre-T/T cell transition, consistent with the normal expression pattern of Notch3. [ABSTRACT FROM AUTHOR]