Epithelial Stem Cell Maintenance Research Articles

Cbl and Cbl-b ubiquitin ligases are essential for intestinal epithelial stem cell maintenance

Receptor tyrosine kinases (RTKs) control stem cell maintenance vs. differentiation decisions. Casitas B-lineage lymphoma (CBL) family ubiquitin ligases are negative regulators of RTKs, but their stem cell regulatory roles remain unclear. Here, we show that Lgr5+ intestinal stem cell (ISC)-specific inducible Cbl-knockout (KO) on a Cblb null mouse background (iDKO) induced rapid loss of the Lgr5 Hi ISCs with transient expansion of the Lgr5 Lo transit-amplifying population. LacZ-based lineage tracing revealed increased ISC commitment toward enterocyte and goblet cell fate at the expense of Paneth cells. Functionally, Cbl/Cblb iDKO impaired the recovery from radiation-induced intestinal epithelial injury. Invitro, Cbl/Cblb iDKO led to inability to maintain intestinal organoids. Single-cell RNA sequencing in organoids identified Akt-mTOR (mammalian target of rapamycin) pathway hyperactivation upon iDKO, and pharmacological Akt-mTOR axis inhibition rescued the iDKO defects. Our results demonstrate a requirement for Cbl/Cblb in the maintenance of ISCs by fine-tuning the Akt-mTOR axis to balance stem cell maintenance vs. commitment to differentiation.

Design of functional decellularized matrix for conjunctival epithelial stem cell maintenance and ocular surface reconstruction

Conjunctival defects severely impair ocular surface homeostasis and vision.However, the existing conjunctival reconstruction methods are still unable to achieve satisfactory functional replacement of conjunctiva. Scaffold materials that provide a more stem-cell-friendly microenvironment are in urgent need of improvement. Herein we reported a functional conjunctival reconstruction scaffold constructed from decellularized matrix derived from rabbit subconjunctival fibroblasts (DM-SCF) loaded with conjunctival epithelial stem cells (CjESCs) for ocular surface repair. The DM-SCF had a thickness and mechanical strength close to that of normal conjunctiva and showed excellent biocompatibility. CjESCs inoculated on DM-SCF maintained good stem cell properties, which showed long-term maintenance of low differentiation and good proliferation activity.DM-SCF transplantation with CjESCs showed satisfactory results in rabbit models of a large conjunctival defect.The conjunctiva was reconstructed with abundant goblet cell expression and regular fiber arrangement. Protein mass spectrometry revealed the active extracellular matrix components of DM-SCF, regulating the activation of Wnt/β-catenin and Notch pathways for stem cell maintenance. Overall, our study developed a promising new strategy for ocular surface reconstruction by optimizing bioactive scaffold and providing functional stem cell niche, so as to achieve long-standing and superior functional repair.

Diverse tumorigenic consequences of human papillomavirus integration in primary oropharyngeal cancers.

Human papillomavirus (HPV) causes 5% of all cancers and frequently integrates into host chromosomes. The HPV oncoproteins E6 and E7 are necessary but insufficient for cancer formation, indicating that additional secondary genetic events are required. Here, we investigate potential oncogenic impacts of virus integration. Analysis of 105 HPV-positive oropharyngeal cancers by whole-genome sequencing detects virus integration in 77%, revealing five statistically significant sites of recurrent integration near genes that regulate epithelial stem cell maintenance (i.e., SOX2, TP63, FGFR, MYC) and immune evasion (i.e., CD274). Genomic copy number hyperamplification is enriched 16-fold near HPV integrants, and the extent of focal host genomic instability increases with their local density. The frequency of genes expressed at extreme outlier levels is increased 86-fold within ±150 kb of integrants. Across 95% of tumors with integration, host gene transcription is disrupted via intragenic integrants, chimeric transcription, outlier expression, gene breaking, and/or de novo expression of noncoding or imprinted genes. We conclude that virus integration can contribute to carcinogenesis in a large majority of HPV-positive oropharyngeal cancers by inducing extensive disruption of host genome structure and gene expression.

Phenotype changes of oral epithelial stem cells after in vitro culture.

The aim of our study was to isolate populations of keratinocyte stem cells based on the expression of cell surface markers and to investigate whether the culture could affect their phenotype. keratinocytes from human oral mucosa were sorted based on the expression of the epithelial stem cell markers p75NTR and CD71. We also examined the co-expression of other epithelial stem markers such as integrins β1 and α6 and their stem cell-like proprieties in in vitro assays. Three passages after being sorted by MACS, more than 93% of the p75NTR+ve cells lost the expression of p75NTR, while 5.46% of the p75NTR-ve gained it. Within the small population of the p75NTR+ve cells, 88% co-expressed other epithelial stem cell markers such as integrins β1 and α6, while only 28% of p75NTR-ve cells co-expressed these markers. These results were confirmed by sorting cells by FACS. Additionally, when double staining was used for sorting cells, 99% of the p75NTR+veCD71-ve and 33% of the p75NTR-veCD71+ve cells expressed both integrins, but just one week after culture, only 1.74% of the p75NTR+veCD71-ve cells still expressed p75NTR and only 0.32% still expressed CD71. Similar results were obtained when co-culturing p75NTR+ve and p75NTR-ve populations before analysis. Our results suggest that phenotype changes may be part of an intrinsic cellular mechanism to conserve levels of protein expression as they may found in the human body. In addition, in vitro culture may not offer ideal conditions for epithelial stem cell maintenance due to phenotype changes under standard culture conditions.

Functionally Distinctive Ptch Receptors Establish Multimodal Hedgehog Signaling in the Tooth Epithelial Stem Cell Niche.

Continuous growth of the mouse incisor teeth is due to the life-long maintenance of epithelial stem cells (SCs) in their niche called cervical loop (CL). Several signaling factors regulate SC maintenance and/or their differentiation to achieve organ homeostasis. Previous studies indicated that Hedgehog signaling is crucial for both the maintenance of the SCs in the niche, as well as for their differentiation. How Hedgehog signaling regulates these two opposing cellular behaviors within the confinement of the CL remains elusive. In this study, we used in vitro organ and cell cultures to pharmacologically attenuate Hedgehog signaling. We analyzed expression of various genes expressed in the SC niche to determine the effect of altered Hedgehog signaling on the cellular hierarchy within the niche. These genes include markers of SCs (Sox2 and Lgr5) and transit-amplifying cells (P-cadherin, Sonic Hedgehog, and Yap). Our results show that Hedgehog signaling is a critical survival factor for SCs in the niche, and that the architecture and the diversity of the SC niche are regulated by multiple Hedgehog ligands. We demonstrated the presence of an additional Hedgehog ligand, nerve-derived Desert Hedgehog, secreted in the proximity of the CL. In addition, we provide evidence that Hedgehog receptors Ptch1 and Ptch2 elicit independent responses, which enable multimodal Hedgehog signaling to simultaneously regulate SC maintenance and differentiation. Our study indicates that the cellular hierarchy in the continuously growing incisor is a result of complex interplay of two Hedgehog ligands with functionally distinct Ptch receptors. Stem Cells 2019;37:1238-1248.

Assessment of corneal substrate biomechanics and its effect on epithelial stem cell maintenance and differentiation

Whilst demonstrated extensively in vitro, the control of cell behaviour via modulation of substrate compliance in live tissues has not been accomplished to date. Here we propose that stem cells can be regulated solely through in situ modulation of tissue biomechanics. By first establishing, via high-resolution Brillouin spectro-microscopy, that the outer edge (limbus) of live human corneas has a substantially lower bulk modulus compared to their centre, we then demonstrate that this difference is associated with limbal epithelial stem cell (LESC) residence and YAP-dependent mechanotransduction. This phenotype-through-biomechanics correlation is further explored in vivo using a rabbit alkali burn model. Specifically, we show that treating the burnt surface of the cornea with collagenase effectively restores the tissue’s mechanical properties and its capacity to support LESCs through mechanisms involving YAP suppression. Overall, these findings have extended implications for understanding stem cell niche biomechanics and its impact on tissue regeneration.

Abstract P6-08-02: Disruption of the estradiol-regulated NTN1-UNC5A dependence receptor signaling axis causes a hybrid basal/luminal molecular phenotype in estrogen receptor-positive breast cancer cells

Abstract Luminal subtype of breast cancers that express the estrogen receptor alpha (ERα) represents approximately two-thirds of all breast cancer cases. ER+ tumors tend to have the most favorable prognoses when treated with endocrine therapy. However, a relapse or endocrine therapy resistance is often seen in ER+ breast tumors. UNC5A belongs to the dependence receptor family which can mediate two different intracellular signals: cell survival, differentiation or migration when engaged with its ligand (such as Netrin-1; NTN1) or cell death/apoptosis in the absence of the ligand. Here we demonstrate that, depending upon the cell type, UNC5A and NTN1 are estradiol (E2)-inducible genes. Using shRNA or CRISPR knockdown strategies, we show that the disruption of the NTN1-UNC5A signaling axis in ER+ (MCF7 and T-47D) cells generates a mixed basal-like/luminal phenotype with stem cell-like characteristics. RNA-seq of UNC5A knockdown cells showed deregulated expression of several E2-target genes in both cell lines. Moreover, knockdown of UNC5A resulted in increased cell proliferation, and elevated expression of the E2-inducible anti-apoptotic, BCL2. Furthermore, the expression of ΔNp63 was enhanced in UNC5A knockdown cells. ΔNp63 is a TP53 family transcription factor that promotes breast epithelial stem cell maintenance and basal-like breast cancer. Accordingly, UNC5A knockdown cells displayed cancer stem cell phenotype as evident from ~3-fold increase in the number of CD44+/CD24+, CD44+/EPCAM+ and ITGA6+/EPCAM+ subpopulation compared with control cells. In addition, the expression of NTN4, a pro-angiogenic and lymphangiogenic factor, was increased upon UNC5A knockdown. In vivo, UNC5A knockdown cells implanted in nude mice were able to form tumors in the mammary fat pad independent of E2 supplementation and were able to colonize and develop into overt metastasis in multiple organs such as lungs, ovaries and adrenal glands. Consequently, analysis of mammary fat pad tumors from animals that received UNC5A knockdown cells revealed an increased expression of PECAM1 (CD31), a marker for endothelial cells used to evaluate tumor angiogenesis. In contrast to UNC5A, knocking down NTN1, decreased the expression of BCL2 and TP63 in both cell lines. Thus, knockdown of UNC5A resulted in deregulated expression of E2-regulated genes, E2-independent and anti-estrogen-resistant growth in vitro, and E2-independent tumor formation in xenograft models. Consistent with results of in vitro studies, analysis of tissue samples from breast cancer patients (n=196) revealed that lower expression of UNC5A is associated with lower overall survival (P < 0.05). Thus, loss or mutational inactivation of UNC5A could lead to unrestricted E2:ERα signaling and anti-estrogen resistant growth while simultaneously enabling ERα-positive luminal breast cancer cells to acquire basal-like and cancer stem cell-like features. Citation Format: Padua MB, Bhat-Nakshatri P, Anjanappa M, Hao Y, Liu Y, McElyea K, Sandusky G, Althouse S, Perkins S, Nakshatri H. Disruption of the estradiol-regulated NTN1-UNC5A dependence receptor signaling axis causes a hybrid basal/luminal molecular phenotype in estrogen receptor-positive breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-08-02.

An essential role of CBL and CBL-B ubiquitin ligases in mammary stem cell maintenance.

The ubiquitin ligases CBL and CBL-B are negative regulators of tyrosine kinase signaling with established roles in the immune system. However, their physiological roles in epithelial tissues are unknown. Here, we used MMTV-Cre-mediated Cbl gene deletion on a Cbl-b null background, as well as a tamoxifen-inducible mammary stem cell (MaSC)-specific Cbl and Cbl-b double knockout (Cbl/Cbl-b DKO) using Lgr5-EGFP-IRES-CreERT2, to demonstrate a mammary epithelial cell-autonomous requirement of CBL and CBL-B in the maintenance of MaSCs. Using a newly engineered tamoxifen-inducible Cbl and Cbl-b deletion model with a dual fluorescent reporter (Cblflox/flox; Cbl-bflox/flox; Rosa26-CreERT; mT/mG), we show that Cbl/Cbl-b DKO in mammary organoids leads to hyperactivation of AKT-mTOR signaling with depletion of MaSCs. Chemical inhibition of AKT or mTOR rescued MaSCs from Cbl/Cbl-b DKO-induced depletion. Our studies reveal a novel, cell-autonomous requirement of CBL and CBL-B in epithelial stem cell maintenance during organ development and remodeling through modulation of mTOR signaling.

Native thymic extracellular matrix improves invivo thymic organoid T cell output, and drives invitro thymic epithelial cell differentiation.

Although the thymus is a primary lymphoid organ, its function is compromised by an age-induced loss of resident epithelial cells, which results in reduced naïve T cell output. This has important implications for immune recovery in aged and elderly patients following damage from cytoablative therapies. As thymic architecture plays a crucial role in naïve T cell development, a tissue specific scaffold that provides essential supporting matrix may assist in stem cell-based thymus regeneration to recreate complex organoids. Here we investigate thymus decellularization approaches that preserve major extracellular matrix components and support thymic epithelial cells for the generation of a functional thymic microenvironment with improved T cell output. We also established an invitro, serum-free culture system that both maintains a progenitor thymic epithelial cell pool and drives their differentiation in the presence of decellularized thymic matrix. This approach enables further dissection of key cellular and niche components involved in thymic epithelial stem cell maintenance and T cell production.

Abstract 1851: Estradiol-inducible dependence receptor UNC5a restricts estrogen receptor activity and imparts estradiol dependence to breast cancer cells

Abstract Signaling by Dependence Receptors (DRs) is a new paradigm in cell signaling and recently has been recognized to play a major role in cancers. DRs are cell surface receptors that function as tumor suppressors or induce apoptosis when disengaged from their ligands. In the presence of ligands, these receptors transmit proliferation, migratory, and anti-apoptotic signals thus creating a cellular state that is dependent on ligands for survival. This study was focused on regulation of their expression in breast cancer. We observed that UNC5a, a DR, and its ligand Netrin-1 (NTN1) are estradiol (E2)-inducible depending on cell types. Since E2 and its receptor estrogen receptor alpha (ERα) are major signaling hubs in ERα-positive breast cancers, we examined the function of UNC5a in ERα-positive breast cancer cells. Knockdown of UNC5a resulted in deregulated expression of E2-regulated genes, E2-independent and anti-estrogen-resistant growth in vitro, and E2-indpendent tumor formation in xenograft models. RNA-seq analyses of parental and UNC5a knockdown cells with or without E2 treatment revealed ∼ 10-fold increase in number of E2-regulated genes in UNC5a knockdown cells compared with parental cells. The basal expression of E2-inducible anti-apoptotic BCL-2 was elevated >30-fold at mRNA and/or protein levels in UNC5a knockdown cells compared with vector control cells. In addition, UNC5a knockdown resulted in aberrant NF-κB signaling, which is typically under negative regulation by ERα:E2. Furthermore, UNC5a knockdown cells showed elevated expression of p63, a p53 family transcription factor that promotes breast epithelial stem cell maintenance and basal-like breast cancer. Consistent with the known role of NF-κB and p63 in cancer stem cells, UNC5a-knockdown cells displayed cancer stem cell phenotype as evident from ∼3-fold increase in the number of CD44+/CD24+ and CD44+/EpCAM+ subpopulation compared with parental cells. Overall, our results suggest that E2 induces UNC5a expression as a negative regulatory loop to restrict or fine tune ERα:E2 signaling and maintain luminal phenotype. Loss or mutation of UNC5a, as observed frequently in cancer, could lead to unrestricted E2:ERα signaling and anti-estrogen resistant growth while simultaneously enabling ERα-positive luminal breast cancer cells to acquire basal-like and cancer stem cell-like features. This work is supported by grants from the Susan G. Komen for the Cure (SAC110025 to HN) Citation Format: Poornima Bhat-Nakshatri, Manjushree Anjanappa, Yangyang Hao, Howard Edenberg, Yunlong Liu, Harikrishna Nakshatri. Estradiol-inducible dependence receptor UNC5a restricts estrogen receptor activity and imparts estradiol dependence to breast cancer cells. [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 1851. doi:10.1158/1538-7445.AM2015-1851

Meis1 Is Required for the Maintenance of Postnatal Thymic Epithelial Cells

Most epithelial tissues retain stem/progenitor cells to maintain homeostasis of the adult tissues; however, the existence of a thymic epithelial cell (TEC) progenitor capable of maintaining homeostasis of the postnatal thymus remains unclear. Here, we show that a cell population expressing high levels of Meis1, a homeodomain transcription factor, is enriched in TECs with an immature cellular phenotype. These TECs selectively express genes involved in embryonic thymic organogenesis and epithelial stem cell maintenance, and also have the potential to proliferate and differentiate into mature TEC populations. Furthermore, postnatal inactivation of Meis1 in TECs caused disorganization of the thymic architecture, which ultimately leads to premature disappearance of the thymus. There was an age-associated reduction in the proportion of the TEC population expressing high levels of Meis1, which may also be related to thymic involution. These findings indicate that Meis1 is potentially involved in the maintenance of postnatal TECs with progenitor activity that is required for homeostasis of the postnatal thymus.

P63 is a prosurvival factor in the adult mammary gland during post-lactational involution, affecting PI-MECs and ErbB2 tumorigenesis

In embryogenesis, p63 is essential to develop mammary glands. In the adult mammary gland, p63 is highly expressed in the basal cell layer that comprises myoepithelial and interspersed stem/progenitor cells, and has limited expression in luminal epithelial cells. In adult skin, p63 has a crucial role in the maintenance of epithelial stem cells. However, it is unclear whether p63 also has an equivalent role as a stem/progenitor cell factor in adult mammary epithelium. We show that p63 is essential in vivo for the survival and maintenance of parity-identified mammary epithelial cells (PI-MECs), a pregnancy-induced heterogeneous population that survives post-lactational involution and contain multipotent progenitors that give rise to alveoli and ducts in subsequent pregnancies. p63+/- glands are normal in virgin, pregnant and lactating states. Importantly, however, during the apoptotic phase of post-lactational involution p63+/- glands show a threefold increase in epithelial cell death, concomitant with increased activation of the oncostatin M/Stat3 and p53 pro-apoptotic pathways, which are responsible for this phase. Thus, p63 is a physiologic antagonist of these pathways specifically in this regressive stage. After the restructuring phase when involution is complete, mammary glands of p63+/- mice again exhibit normal epithelial architecture by conventional histology. However, using Rosa(LSL-LacZ);WAP-Cre transgenics (LSL-LacZ, lox-stop-lox β-galactosidase), a genetic in vivo labeling system for PI-MECs, we find that p63+/- glands have a 30% reduction in the number of PI-MEC progenitors and their derivatives. Importantly, PI-MECs are also cellular targets of pregnancy-promoted ErbB2 tumorigenesis. Consistent with their PI-MEC pool reduction, one-time pregnant p63+/- ErbB2 mice are partially protected from breast tumorigenesis, exhibiting extended tumor-free and overall survival, and reduced tumor multiplicity compared with their p63+/+ ErbB2 littermates. Conversely, in virgin ErbB2 mice p63 heterozygosity provides no survival advantage. In sum, our data establish that p63 is an important survival factor for pregnancy-identified PI-MEC progenitors in breast tissue in vivo.

Abstract 1430: The role of IL27 as a driver of skin carcinogenesis.

Abstract Although the genetic components that drive carcinogenesis and the stem cell involvement in cancer initiation have been established, the mechanism through which the immune response/cytokine milieu within the tumor microenvironment promotes cancer formation and stem cell behavior is an exciting yet understudied area of research. This statement is specifically true with an important regulator of the immune response, IL27, which is known to have both pro- and anti-inflammatory properties. During an inflammatory response, IL27 has been shown to regulate two canonical cytokines IL10 and IL17, and to affect multiple inflammatory and infectious diseases. Yet, the function of IL27 at physiological levels during the instigation of carcinogenesis remains unknown. To determine the role of IL27 during the early stages of cancer formation, the well-established two step skin carcinogenesis model (DMBA followed by the promoter benzoyl peroxide (BP)) was used. Interestingly, mice that lack IL27 signaling (IL27RA-/-) are protected during the initiation of carcinogenesis as they have lower incidence of papilloma formation over time when compared to wildtype mice. Additionally, wildtype mice treated with IL27 via gene therapy have a higher incidence and develop more papillomas when compared to control counterparts. Bone marrow transfer studies showed that IL27 signaling in both hematopoietic and non-hematopoietic cells is needed to drive skin carcinogenesis. Additionally, IL27 signaling induces loss of epithelial stem cell homeostasis/maintenance. In mice treated with DMBA/BP, 23% of the wildtype mice treated had ulcerations that failed to heal over time, while none of IL27RA-/- showed any defects in the skin. In mice that went under bone marrow transfer followed by DMBA/BP, 40% of wildtype ones developed severe skin ulcerations, while none of the IL27RA-/- developed any skin abnormalities. And lastly, mice undergoing the tumorigenesis protocol that were treated with IL27 developed hair loss, which was mirrored by loss of hair follicles. These phenotypes are like due to the loss of epithelial stem cell maintenance and/or homeostasis by IL27. These finding propose an unrecognized role of IL27 in promoting papilloma formation and disrupting epithelial cell maintenance and/or homeostasis. Citation Format: Denada Dibra, Melissa Newman, Jeffry Cutrera, Shulin Li. The role of IL27 as a driver of skin carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1430. doi:10.1158/1538-7445.AM2013-1430

Analysis of the oligomeric state and transactivation potential of TAp73α

The proteins p73 and p63 are members of the p53 protein family and are involved in important developmental processes. Their high sequence identity with the tumor suppressor p53 has suggested that they act as tumor suppressors as well. While p63 has a crucial role in the maintenance of epithelial stem cells and in the quality control of oocytes without a clear role as a tumor suppressor, p73's tumor suppressor activity is well documented. In a recent study we have shown that the transcriptional activity of TAp63α, the isoform responsible for the quality control in oocytes, is regulated by its oligomeric state. The protein forms an inactive, dimeric and compact conformation in resting oocytes, while the detection of DNA damage leads to the formation of an active, tetrameric and open conformation. p73 shows a high sequence identity to p63, including those domains that are crucial in stabilizing its inactive state, thus suggesting that p73's activity might be regulated by its oligomeric state as well. Here, we have investigated the oligomeric state of TAp73α by size exclusion chromatography and detailed domain interaction mapping, and show that in contrast to p63, TAp73α is a constitutive open tetramer. However, its transactivation potential depends on the cellular background and the promoter context. These results imply that the regulation of p73's transcriptional activity might be more closely related to p53 than to p63.

Inducible deletion of epidermal Dicer and Drosha reveals multiple functions for miRNAs in postnatal skin

MicroRNAs (miRNAs) regulate the expression of many mammalian genes and play key roles in embryonic hair follicle development; however, little is known of their functions in postnatal hair growth. We compared the effects of deleting the essential miRNA biogenesis enzymes Drosha and Dicer in mouse skin epithelial cells at successive postnatal time points. Deletion of either Drosha or Dicer during an established growth phase (anagen) caused failure of hair follicles to enter a normal catagen regression phase, eventual follicular degradation and stem cell loss. Deletion of Drosha or Dicer in resting phase follicles did not affect follicular structure or epithelial stem cell maintenance, and stimulation of anagen by hair plucking caused follicular proliferation and formation of a primitive transient amplifying matrix population. However, mutant matrix cells exhibited apoptosis and DNA damage and hair follicles rapidly degraded. Hair follicle defects at early time points post-deletion occurred in the absence of inflammation, but a dermal inflammatory response and hyperproliferation of interfollicular epidermis accompanied subsequent hair follicle degradation. These data reveal multiple functions for Drosha and Dicer in suppressing DNA damage in rapidly proliferating follicular matrix cells, facilitating catagen and maintaining follicular structures and their associated stem cells. Although Drosha and Dicer each possess independent non-miRNA-related functions, the similarity in phenotypes of the inducible epidermal Drosha and Dicer mutants indicates that these defects result primarily from failure of miRNA processing. Consistent with this, Dicer deletion resulted in the upregulation of multiple direct targets of the highly expressed epithelial miRNA miR-205.

Inducible deletion of epidermal Dicer and Drosha reveals multiple functions for miRNAs in postnatal skin

MicroRNAs (miRNAs) regulate the expression of many mammalian genes and play key roles in embryonic hair follicle development; however, little is known of their functions in postnatal hair growth. We compared the effects of deleting the essential miRNA biogenesis enzymes Drosha and Dicer in mouse skin epithelial cells at successive postnatal time points. Deletion of either Drosha or Dicer during an established growth phase (anagen) caused failure of hair follicles to enter a normal catagen regression phase, eventual follicular degradation and stem cell loss. Deletion of Drosha or Dicer in resting phase follicles did not affect follicular structure or epithelial stem cell maintenance, and stimulation of anagen by hair plucking caused follicular proliferation and formation of a primitive transient amplifying matrix population. However, mutant matrix cells exhibited apoptosis and DNA damage and hair follicles rapidly degraded. Hair follicle defects at early time points post-deletion occurred in the absence of inflammation, but a dermal inflammatory response and hyperproliferation of interfollicular epidermis accompanied subsequent hair follicle degradation. These data reveal multiple functions for Drosha and Dicer in suppressing DNA damage in rapidly proliferating follicular matrix cells, facilitating catagen and maintaining follicular structures and their associated stem cells. Although Drosha and Dicer each possess independent non-miRNA-related functions, the similarity in phenotypes of the inducible epidermal Drosha and Dicer mutants indicates that these defects result primarily from failure of miRNA processing. Consistent with this, Dicer deletion resulted in the upregulation of multiple direct targets of the highly expressed epithelial miRNA miR-205.

Core Binding Factor Beta Functions in the Maintenance of Stem Cells and Orchestrates Continuous Proliferation and Differentiation in Mouse Incisors

Rodent incisors grow continuously throughout life, and epithelial progenitor cells are supplied from stem cells in the cervical loop. We report that epithelial Runx genes are involved in the maintenance of epithelial stem cells and their subsequent continuous differentiation and therefore growth of the incisors. Core binding factor β (Cbfb) acts as a binding partner for all Runx proteins, and targeted inactivation of this molecule abrogates the activity of all Runx complexes. Mice deficient in epithelial Cbfb produce short incisors and display marked underdevelopment of the cervical loop and suppressed epithelial Fgf9 expression and mesenchymal Fgf3 and Fgf10 expression in the cervical loop. In culture, FGF9 protein rescues these phenotypes. These findings indicate that epithelial Runx functions to maintain epithelial stem cells and that Fgf9 may be a target gene of Runx signaling. Cbfb mutants also lack enamel formation and display downregulated Shh mRNA expression in cells differentiating into ameloblasts. Furthermore, Fgf9 deficiency results in a proximal shift of the Shh expressing cell population and ectopic FGF9 protein suppresses Shh expression. These findings indicate that Shh as well as Fgf9 expression is maintained by Runx/Cbfb but that Fgf9 antagonizes Shh expression. The present results provide the first genetic evidence that Runx/Cbfb genes function in the maintenance of stem cells in developing incisors by activating Fgf signaling loops between the epithelium and mesenchyme. In addition, Runx genes also orchestrate continuous proliferation and differentiation by maintaining the expression of Fgf9 and Shh mRNA.

TAp63 Is Important for Cardiac Differentiation of Embryonic Stem Cells and Heart Development

p63, a member of the p53 family, is essential for skin morphogenesis and epithelial stem cell maintenance. Here, we report an unexpected role of TAp63 in cardiogenesis. p63 null mice exhibit severe defects in embryonic cardiac development, including dilation of both ventricles, a defect in trabeculation and abnormal septation. This was accompanied by myofibrillar disarray, mitochondrial disorganization, and reduction in spontaneous calcium spikes. By the use of embryonic stem cells (ESCs), we show that TAp63 deficiency prevents expression of pivotal cardiac genes and production of cardiomyocytes. TAp63 is expressed by endodermal cells. Coculture of p63-knockdown ESCs with wild-type ESCs, supplementation with Activin A, or overexpression of GATA-6 rescue cardiogenesis. Therefore, TAp63 acts in a non-cell-autonomous manner by modulating expression of endodermal factors. Our findings uncover a critical role for p63 in cardiogenesis that could be related to human heart disease.

Cultured human corneal epithelial stem/progenitor cells derived from the corneal limbus

Stem/progenitor cells of the human corneal epithelium are present in the human corneal limbus, and several corneal epithelial stem/progenitor cell markers have been reported. Recently, the neurotrophin family receptors were reported to be useful markers of corneal epithelial stem/progenitor cells. Therefore, we examined an enzymatic separation method for obtaining corneal epithelial stem/progenitor cells and measuring the change in the expression of low-affinity neurotrophin receptor p75 (p75(NTR)), a receptor belonging to the neurotrophin family. As a result, it was found that our separation method preserved cell viability. Furthermore, p75(NTR) was mainly observed in epithelial basal cells as were the corneal epithelial stem/progenitor markers p63 and integrin β1. p75(NTR) was also observed in the cultured cells, but its frequency decreased with passage. In conclusion, we propose that our culture method will enable the culture of corneal stem cells and that it is a useful tool for elucidating the molecular basis of the niche that is necessary for the maintenance of epithelial stem cells in the corneal limbus. Furthermore, we conclude that p75(NTR) is a useful cell marker for evaluating the characteristics of stem/progenitor cells in culture.

NIR, an inhibitor of histone acetyltransferases, regulates transcription factor TAp63 and is controlled by the cell cycle

p63 is a sequence-specific transcription factor that regulates epithelial stem cell maintenance and epithelial differentiation. In addition, the TAp63 isoform with an N-terminal transactivation domain functions as an inducer of apoptosis during the development of sympathetic neurons. Previous work has indicated that the co-activator and histone acetyltransferase (HAT), p300, can bind to TAp63 and stimulate TAp63-dependent transcription of the p21Cip1 gene. Novel INHAT Repressor (NIR) is an inhibitor of HAT. Here, we report that the central portion of NIR binds to the transactivation domain and the C-terminal oligomerization domain of TAp63. NIR is highly expressed in G2/M phase of the cell cycle and only weakly expressed in G1/S. Furthermore, except during mitosis, NIR is predominantly localized in the nucleolus; only a small portion co-localizes with TAp63 in the nucleoplasm and at the p21 gene promoter. Consistent with NIR acting as a repressor, the induced translocation of NIR from the nucleolus into the nucleoplasm resulted in the inhibition of TAp63-dependent transactivation of p21. Conversely, knockdown of NIR by RNAi stimulated p21 transcription in the presence of TAp63. Thus, NIR is a cell-cycle-controlled, novel negative regulator of TAp63. The low levels of nucleoplasmic NIR might act as a buffer toward potentially toxic TAp63.