Notch signaling regulates late-stage epidermal differentiation and maintains postnatal hair cycle homeostasis

Background

Notch signaling involves ligand-receptor interactions through direct cell-cell contact. Multiple Notch receptors and ligands are expressed in the epidermis and hair follicles during embryonic development and the adult stage. Although Notch signaling plays an important role in regulating differentiation of the epidermis and hair follicles, it remains unclear how Notch signaling participates in late-stage epidermal differentiation and postnatal hair cycle homeostasis.

Methodology and Principal Findings

We applied Cre/loxP system to generate conditional gene targeted mice that allow inactivation of critical components of Notch signaling pathway in the skin. Rbpj, the core component of all four Notch receptors, and Pofut1, an essential factor for ligand-receptor interactions, were inactivated in hair follicle lineages and suprabasal layer of the epidermis using the Tgfb3-Cre mouse line. Rbpj conditional inactivation resulted in granular parakeratosis and reactive epidermal hyperplasia. Pofut1 conditional inactivation led to ultrastructural abnormalities in the granular layer and altered filaggrin processing in the epidermis, suggesting a perturbation of the granular layer differentiation. Disruption of Pofut1 in hair follicle lineages resulted in aberrant telogen morphology, a decrease of bulge stem cell markers, and a concomitant increase of K14-positive keratinocytes in the isthmus of mutant hair follicles. Pofut1-deficent hair follicles displayed a delay in anagen re-entry and dysregulation of proliferation and apoptosis during the hair cycle transition. Moreover, increased DNA double stand breaks were detected in Pofut1-deficent hair follicles, and real time PCR analyses on bulge keratinocytes isolated by FACS revealed an induction of DNA damage response and a paucity of DNA repair machinery in mutant bulge keratinocytes.

Significance

our data reveal a role for Notch signaling in regulating late-stage epidermal differentiation. Notch signaling is required for postnatal hair cycle homeostasis by maintaining proper proliferation and differentiation of hair follicle stem cells.     

Identification of epidermal Pdx1 expression discloses different roles of Notch1 and Notch2 in murine Kras(G12D)-induced skin carcinogenesis in vivo

Background

The Ras and Notch signaling pathways are frequently activated during development to control many diverse cellular processes and are often dysregulated during tumorigenesis. To study the role of Notch and oncogenic Kras signaling in a progenitor cell population, Pdx1-Cre mice were utilized to generate conditional oncogenic Kras^G12D mice with ablation of Notch1 and/or Notch2.

Methodology/Principal Findings

Surprisingly, mice with activated Kras^G12D and Notch1 but not Notch2 ablation developed skin papillomas progressing to squamous cell carcinoma providing evidence for Pdx1 expression in the skin. Immunostaining and lineage tracing experiments indicate that PDX1 is present predominantly in the suprabasal layers of the epidermis and rarely in the basal layer. Further analysis of keratinocytes in vitro revealed differentiation-dependent expression of PDX1 in terminally differentiated keratinocytes. PDX1 expression was also increased during wound healing. Further analysis revealed that loss of Notch1 but not Notch2 is critical for skin tumor development. Reasons for this include distinct Notch expression with Notch1 in all layers and Notch2 in the suprabasal layer as well as distinctive p21 and β-catenin signaling inhibition capabilities.

Conclusions/Significance

Our results provide strong evidence for epidermal expression of Pdx1 as of yet not identified function. In addition, this finding may be relevant for research using Pdx1-Cre transgenic strains. Additionally, our study confirms distinctive expression and functions of Notch1 and Notch2 in the skin supporting the importance of careful dissection of the contribution of individual Notch receptors.     

An obligatory role of mind bomb-1 in notch signaling of mammalian development

Background

The Notch signaling pathway is an evolutionarily conserved intercellular signaling module essential for cell fate specification that requires endocytosis of Notch ligands. Structurally distinct E3 ubiquitin ligases, Neuralized (Neur) and Mind bomb (Mib), cooperatively regulate the endocytosis of Notch ligands in Drosophila. However, the respective roles of the mammalian E3 ubiquitin ligases, Neur1, Neur2, Mib1, and Mib2, in mammalian development are poorly understood.

Methodology/Principal Findings

Through extensive use of mammalian genetics, here we show that Neur1 and Neur2 double mutants and Mib2^−/− mice were viable and grossly normal. In contrast, conditional inactivation of Mib1 in various tissues revealed the representative Notch phenotypes: defects of arterial specification as deltalike4 mutants, abnormal cerebellum and skin development as jagged1 conditional mutants, and syndactylism as jagged2 mutants.

Conclusions/Significance

Our data provide the first evidence that Mib1 is essential for Jagged as well as Deltalike ligand-mediated Notch signaling in mammalian development, while Neur1, Neur2, and Mib2 are dispensable.     

The pro-inflammatory cytokine IL-22 up-regulates keratin 17 expression in keratinocytes via STAT3 and ERK1/2

Background

To investigate the regulation of K17 expression by the pro-inflammatory cytokine IL-22 in keratinocytes and its important role in our previously hypothesized “K17/T cell/cytokine autoimmune loop” in psoriasis.

Materials and Methods

K17 expression was examined in the IL-22-treated keratinocytes by real-time quantitative PCR, ELISA, Western blot and Immunofluorescence. In addition, the signaling pathways involved in K17 regulation were investigated with related inhibitors and siRNAs. In addition, K17 expression was examined in the epidermis of IL-22-injected mouse skin.

Results

IL-22-induced K17 expression was confirmed in keratinocytes and the epidermis of IL-22-injected mouse skin at both mRNA and protein levels, which is an important complement to the autoimmune loop. We further investigated the regulatory mechanisms and found that both STAT3 and ERK1/2 were involved in the up-regulation of K17 expression induced by IL-22.

Conclusion

IL-22 up-regulates K17 expression in keratinocytes in a dose-dependent manner through STAT3- and ERK1/2-dependent mechanisms. These findings indicated that IL-22 was also involved in the K17/T cell/cytokine autoimmune loop and may play an important role in the progression of psoriasis.     

Uif, a large transmembrane protein with EGF-like repeats, can antagonize Notch signaling in Drosophila

Background

Notch signaling is a highly conserved pathway in multi-cellular organisms ranging from flies to humans. It controls a variety of developmental processes by stimulating the expression of its target genes in a highly specific manner both spatially and temporally. The diversity, specificity and sensitivity of the Notch signaling output are regulated at distinct levels, particularly at the level of ligand-receptor interactions.

Methodology/Principal Findings

Here, we report that the Drosophila gene uninflatable (uif), which encodes a large transmembrane protein with eighteen EGF-like repeats in its extracellular domain, can antagonize the canonical Notch signaling pathway. Overexpression of Uif or ectopic expression of a neomorphic form of Uif, Uif*, causes Notch signaling defects in both the wing and the sensory organ precursors. Further experiments suggest that ectopic expression of Uif* inhibits Notch signaling in cis and acts at a step that is dependent on the extracellular domain of Notch. Our results suggest that Uif can alter the accessibility of the Notch extracellular domain to its ligands during Notch activation.

Conclusions/Significance

Our study shows that Uif can modulate Notch activity, illustrating the importance of a delicate regulation of this signaling pathway for normal patterning.     

Notch signaling activation suppresses v-Src-induced transformation of neural cells by restoring TGF-β-mediated differentiation

Background

We have been investigating how interruption of differentiation contributes to the oncogenic process and the possibility to reverse the transformed phenotype by restoring differentiation. In a previous report, we correlated the capacity of intracellular Notch (ICN) to suppress v-Src-mediated transformation of quail neuroretina (QNR/v-src^ts) cells with the acquisition by these undifferentiated cells of glial differentiation markers.

Methodology/Principal Findings

In this work, we have identified autocrine TGF-β3 signaling activation as a major effector of Notch-induced phenotypic changes, sufficient to induce transition in differentiation markers expression, suppress morphological transformation and significantly inhibit anchorage-independent growth. We also show that this signaling is constitutive of and contributes to ex-vivo autonomous QNR cell differentiation and that its down-regulation is essential to achieve v-Src-induced transformation.

Conclusions/Significance

These results support the possibility that Notch signaling induces differentiation and suppresses transformation by a novel mechanism, involving secreted proteins. They also underline the importance of extracellular signals in controlling the balance between normal and transformed phenotypes.     

Activin B promotes epithelial wound healing in vivo through RhoA-JNK signaling pathway

Background

Activin B has been reported to promote the proliferation and migration of keratinocytes in vitro via the RhoA-JNK signaling pathway, whereas its in vivo role and mechanism in wound healing process has not yet been elucidated.

Principal Findings

In this study, we explored the potential mechanism by which activin B induces epithelial wound healing in mice. Recombinant lentiviral plasmids, with RhoA (N19) and RhoA (L63) were used to infect wounded KM mice. The wound healing process was monitored after different treatments. Activin B-induced cell proliferation on the wounded skin was visualized by electron microscopy and analyzed by 5′-bromodeoxyuridine (BrdU) incorporation assay. Protein expression of p-JNK or p-cJun was determined by immunohistochemical staining and immunoblotting analysis. Activin B efficiently stimulated the proliferation of keratinocytes and hair follicle cells at the wound area and promoted wound closure. RhoA positively regulated activin B-induced wound healing by up-regulating the expression of p-JNK and p-cJun. Moreover, suppression of RhoA activation delayed activin B-induced wound healing, while JNK inhibition recapitulated phenotypes of RhoA inhibition on wound healing.

Conclusion

These results demonstrate that activin B promotes epithelial wound closure in vivo through the RhoA-Rock-JNK-cJun signaling pathway, providing novel insight into the essential role of activin B in the therapy of wound repair.     

The notch and TGF-β signaling pathways contribute to the aggressiveness of clear cell renal cell carcinoma

Background

Despite recent progress, therapy for metastatic clear cell renal cell carcinoma (CCRCC) is still inadequate. Dysregulated Notch signaling in CCRCC contributes to tumor growth, but the full spectrum of downstream processes regulated by Notch in this tumor form is unknown.

Methodology/Principal Findings

We show that inhibition of endogenous Notch signaling modulates TGF-β dependent gene regulation in CCRCC cells. Analysis of gene expression data representing 176 CCRCCs showed that elevated TGF-β pathway activity correlated significantly with shortened disease specific survival (log-rank test, p = 0.006) and patients with metastatic disease showed a significantly elevated TGF-β signaling activity (two-sided Student''s t-test, p = 0.044). Inhibition of Notch signaling led to attenuation of both basal and TGF-β1 induced TGF-β signaling in CCRCC cells, including an extensive set of genes known to be involved in migration and invasion. Functional analyses revealed that Notch inhibition decreased the migratory and invasive capacity of CCRCC cells.

Conclusion

An extensive cross-talk between the Notch and TGF-β signaling cascades is present in CCRCC and the functional properties of these two pathways are associated with the aggressiveness of this disease.     

Vaccinia Virus Induces Rapid Necrosis in Keratinocytes by a STAT3-Dependent Mechanism

Rationale

Humans with a dominant negative mutation in STAT3 are susceptible to severe skin infections, suggesting an essential role for STAT3 signaling in defense against cutaneous pathogens.

Methods

To focus on innate antiviral defenses in keratinocytes, we used a standard model of cutaneous infection of severe combined immunodeficient mice with the current smallpox vaccine, ACAM-2000. In parallel, early events post-infection with the smallpox vaccine ACAM-2000 were investigated in cultured keratinocytes of human and mouse origin.

Results

Mice treated topically with a STAT3 inhibitor (Stattic) developed larger vaccinia lesions with higher virus titers and died more rapidly than untreated controls. Cultured human and murine keratinocytes infected with ACAM-2000 underwent rapid necrosis, but when treated with Stattic or with inhibitors of RIP1 kinase or caspase-1, they survived longer, produced higher titers of virus, and showed reduced activation of type I interferon responses and inflammatory cytokines release. Treatment with inhibitors of RIP1 kinase and STAT3, but not caspase-1, also reduced the inflammatory response of keratinocytes to TLR ligands. Vaccinia growth properties in Vero cells, which are known to be defective in some antiviral responses, were unaffected by inhibition of RIP1K, caspase-1, or STAT3.

Conclusions

Our findings indicate that keratinocytes suppress the replication and spread of vaccinia virus by undergoing rapid programmed cell death, in a process requiring STAT3. These data offer a new framework for understanding susceptibility to skin infection in patients with STAT3 mutations. Interventions which promote prompt necroptosis/pyroptosis of infected keratinocytes may reduce risks associated with vaccination with live vaccinia virus.     

The Jagged-2/Notch-1/Hes-1 Pathway Is Involved in Intestinal Epithelium Regeneration after Intestinal Ischemia-Reperfusion Injury

Background

Notch signaling plays a critical role in the maintenance of intestinal crypt epithelial cell proliferation. The aim of this study was to investigate the role of Notch signaling in the proliferation and regeneration of intestinal epithelium after intestinal ischemia reperfusion (I/R) injury.

Methods

Male Sprague-Dawley rats were subjected to sham operation or I/R by occlusion of the superior mesenteric artery (SMA) for 20 min. Intestinal tissue samples were collected at 0, 1, 2, 4, and 6 h after reperfusion. Proliferation of the intestinal epithelium was evaluated by immunohistochemical staining of proliferating nuclear antigen (PCNA). The mRNA and protein expression levels of Notch signaling components were examined using Real-time PCR and Western blot analyses. Immunofluorescence was also performed to detect the expression and location of Jagged-2, cleaved Notch-1, and Hes-1 in the intestine. Finally, the γ-secretase inhibitor DAPT and the siRNA for Jagged-2 and Hes-1 were applied to investigate the functional role of Notch signaling in the proliferation of intestinal epithelial cells in an in vitro IEC-6 culture system.

Results

I/R injury caused increased intestinal crypt epithelial cell proliferation and increased mRNA and protein expression of Jagged-2, Notch-1, and Hes-1. The immunofluorescence results further confirmed increased protein expression of Jagged-2, cleaved Notch-1, and Hes-1 in the intestinal crypts. The inhibition of Notch signaling with DAPT and the suppression of Jagged-2 and Hes-1 expression using siRNA both significantly inhibited the proliferation of IEC-6 cells.

Conclusion

The Jagged-2/Notch-1/Hes-1 signaling pathway is involved in intestinal epithelium regeneration early after I/R injury by increasing crypt epithelial cell proliferation.     

Notch Signaling in Descending Thoracic Aortic Aneurysm and Dissection

Background

Descending thoracic aortic aneurysm and dissection (DTAAD) is characterized by progressive medial degeneration, which may result from excessive tissue destruction and insufficient repair. Resistance to tissue destruction and aortic self-repair are critical in preventing medial degeneration. The signaling pathways that control these processes in DTAAD are poorly understood. Because Notch signaling is a critical pathway for cell survival, proliferation, and tissue repair, we examined its activation in DTAAD.

Methods

We studied descending thoracic aortic tissue from patients with sporadic thoracic aortic aneurysm (TAA; n = 14) or chronic thoracic aortic dissection (TAD; n = 16) and from age-matched organ donors (n = 12). Using western blot, real-time RT-PCR, and immunofluorescence staining, we examined aortic tissue samples for the Notch ligands Delta-like 1, Delta-like 4 (DLL1/4), and Jagged1; the Notch receptor 1 (Notch1); the Notch1 intracellular domain (NICD); and Hes1, a downstream target of Notch signaling.

Results

Western blots and RT-PCR showed higher levels of the Notch1 protein and mRNA and the NICD and Hes1 proteins in both TAA and TAD tissues than in control tissue. However, immunofluorescence staining showed a complex pattern of Notch signaling in the diseased tissue. The ligand DLL1/4 and Notch1 were significantly decreased and NICD and Hes1 were rarely detected in medial vascular smooth muscle cells (VSMCs) in both TAA and TAD tissues, indicating downregulation of Notch signaling in aortic VSMCs. Interestingly Jagged1, NICD, and Hes1 were highly present in CD34+ stem cells and Stro-1+ stem cells in aortas from TAA and TAD patients. NICD and Hes1 were also detected in most fibroblasts and macrophages that accumulated in the aortic wall of DTAAD patients.

Conclusions

Notch signaling exhibits a complex pattern in DTAAD. The Notch pathway is impaired in medial VSMCs but activated in stem cells, fibroblasts, and macrophages.     

Forced notch signaling inhibits commissural axon outgrowth in the developing chick central nerve system

Background

A collection of in vitro evidence has demonstrated that Notch signaling plays a key role in the growth of neurites in differentiated neurons. However, the effects of Notch signaling on axon outgrowth in an in vivo condition remain largely unknown.

Methodology/Principal Findings

In this study, the neural tubes of HH10-11 chick embryos were in ovo electroporated with various Notch transgenes of activating or inhibiting Notch signaling, and then their effects on commissural axon outgrowth across the floor plate midline in the chick developing central nerve system were investigated. Our results showed that forced expression of Notch intracellular domain, constitutively active form of RBPJ, or full-length Hes1 in the rostral hindbrain, diencephalon and spinal cord at stage HH10-11 significantly inhibited commissural axon outgrowth. On the other hand, inhibition of Notch signaling by ectopically expressing a dominant-negative form of RBPJ promoted commissural axonal growth along the circumferential axis. Further results revealed that these Notch signaling-mediated axon outgrowth defects may be not due to the alteration of axon guidance since commissural axon marker TAG1 was present in the axons in floor plate midline, and also not result from the changes in cell fate determination of commissural neurons since the expression of postmitotic neuron marker Tuj1 and specific commissural markers TAG1 and Pax7 was unchanged.

Conclusions/Significance

We first used an in vivo system to provide evidence that forced Notch signaling negatively regulates commissural axon outgrowth.     

Activated Notch Causes Deafness by Promoting a Supporting Cell Phenotype in Developing Auditory Hair Cells

Purpose

To determine whether activated Notch can promote a supporting cell fate during sensory cell differentiation in the inner ear.

Methods

An activated form of the Notch1 receptor (NICD) was expressed in early differentiating hair cells using a Gfi1-Cre mouse allele. To determine the effects of activated Notch on developing hair cells, Gfi1-NICD animals and their littermate controls were assessed at 5 weeks for hearing by measuring auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs). The differentiation of NICD-expressing hair cells was assessed at postnatal day (P) 6, 11 and 20, using histological and molecular markers for hair cells, as well as supporting cells/progenitor cells. We also examined whether the effects of Notch were mediated by SOX2, a gene expressed in supporting cells and a likely downstream target of Notch, by crossing an inducible form of SOX2 to the Gfi1-Cre.

Results

Activation of Notch1 in developing auditory hair cells causes profound deafness. The NICD-expressing hair cells switch off a number of hair cell markers and lose their characteristic morphology. Instead, NICD-expressing hair cells adopt a morphology resembling supporting cells and upregulate a number of supporting cell markers. These effects do not appear to be mediated by SOX2, because although expression of SOX2 caused some hearing impairment, the SOX2-expressing hair cells did not downregulate hair cell markers nor exhibit a supporting cell-like phenotype.

Conclusions

Our data show that Notch signaling inhibits hair cell differentiation and promotes a supporting cell-like phenotype, and that these effects are unlikely to be mediated by SOX2.     

H-Ras Expression in Immortalized Keratinocytes Produces an Invasive Epithelium in Cultured Skin Equivalents

Background

Ras proteins affect both proliferation and expression of collagen-degrading enzymes, two important processes in cancer progression. Normal skin architecture is dependent both on the coordinated proliferation and stratification of keratinocytes, as well as the maintenance of a collagen-rich basement membrane. In the present studies we sought to determine whether expression of H-ras in skin keratinocytes would affect these parameters during the establishment and maintenance of an in vitro skin equivalent.

Methodology/Principal Findings

Previously described cdk4 and hTERT immortalized foreskin keratinocytes were engineered to express ectopically introduced H-ras. Skin equivalents, composed of normal fibroblast-contracted collagen gels overlaid with keratinocytes (immortal or immortal expressing H-ras), were prepared and incubated for 3 weeks. Harvested tissues were processed and sectioned for histology and antibody staining. Antigens specific to differentiation (involucrin, keratin-14, p63), basement-membrane formation (collagen IV, laminin-5), and epithelial to mesenchymal transition (EMT; e-cadherin, vimentin) were studied. Results showed that H-ras keratinocytes produced an invasive, disorganized epithelium most apparent in the lower strata while immortalized keratinocytes fully stratified without invasive properties. The superficial strata retained morphologically normal characteristics. Vimentin and p63 co-localization increased with H-ras overexpression, similar to basal wound-healing keratinocytes. In contrast, the cdk4 and hTERT immortalized keratinocytes differentiated similarly to normal unimmortalized keratinocytes.

Conclusions/Significance

The use of isogenic derivatives of stable immortalized keratinocytes with specified genetic alterations may be helpful in developing more robust in vitro models of cancer progression.