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Endometriosis is a common female reproductive disease characterized by invasion of endometrial cells into other organs, frequently causing pelvic pain and infertility. Alterations of the vitamin D system have been linked to endometriosis incidence and severity. To shed light on the potential mechanism for these associations, we examined the effects of 1,25(OH)2D3 on gene expression in endometriosis cells. Stromal cell lines derived from endometriosis tissue were treated with 1,25(OH)2D3, and RNA-seq was used to identify genes differentially expressed between treated and untreated cells. Gene ontology and pathway analyses were carried out using Partek Flow and Ingenuity software suites, respectively. We identified 1627 genes that were differentially expressed (886 down-regulated and 741 up-regulated) by 1,25(OH)2D3. Only one gene, CYP24A1, was strongly up-regulated (369-fold). Many genes were strongly down-regulated. 1,25(OH)2D3 treatment down-regulated several genetic pathways related to neuroangiogenesis, cellular motility, and invasion, including pathways for axonal guidance, Rho GDP signaling, and matrix metalloprotease inhibition. These findings support a role for vitamin D in the pathophysiology of endometriosis, and provide new targets for investigation into possible causes and treatments.

It has been suggested that multipotent stem cells with neural crest (NC) origin persist into adulthood in oral mucosa. However their exact localization and role in normal homeostasis is unknown. In this study, we discovered that Lgr5 is expressed in NC cells during embryonic development, which give rise to the dormant stem cells in the adult tongue and oral mucosa. Those Lgr5 positive oral stromal stem cells display properties of NC stem cells including clonal growth and multipotent differentiation. RNA sequencing revealed that adult Lgr5+ oral stromal stem cells express high number of neural crest related markers like Sox9, Twist1, Snai1, Myc, Ets1, Crabp1, Epha2, and Itgb1. Using lineage-tracing experiments, we show that these cells persist more than a year in the ventral tongue and some areas of the oral mucosa and give rise to stromal progeny. In vivo transplantation demonstrated that these cells reconstitute the stroma. Our studies show for the first time that Lgr5 is expressed in the NC cells at embryonic day 9.5 (E9.5) and is maintained during embryonic development and postnataly in the stroma of the ventral tongue, and some areas of the oral mucosa and that Lgr5+ cells participate in the maintenance of the stroma.

There is currently no FDA approved therapeutic agent for ARS mitigation post radiation exposure. Here we report that the small molecule YH250, which specifically antagonizes p300/catenin interaction, stimulates hematopoiesis in lethally or sublethally irradiated mice. A single administration of YH250 24 hours post irradiation can significantly stimulate HSC proliferation, improve survival and accelerate peripheral blood count recovery. Our studies suggest that promotion of the expansion of the remaining HSC population via stimulation of symmetric non-differentiative proliferation is at least part of the mechanism of action.

Abnormal stem cell function makes a known contribution to many malignant tumors, but the role of stem cells in benign tumors is not well understood. Here, we show that ossifying fibroma (OF) contains a stem cell population that resembles mesenchymal stem cells (OFMSCs) and is capable of generating OF-like tumor xenografts. Mechanistically, OFMSCs show enhanced TGF-β signaling that induces aberrant proliferation and deficient osteogenesis via Notch and BMP signaling pathways, respectively. The elevated TGF-β activity is tightly regulated by JHDM1D-mediated epigenetic regulation of thrombospondin-1 (TSP1), forming a JHDM1D/TSP1/TGF-β/SMAD3 autocrine loop. Inhibition of TGF-β signaling in OFMSCs can rescue their abnormal osteogenic differentiation and elevated proliferation rate. Furthermore, chronic activation of TGF-β can convert normal MSCs into OF-like MSCs via establishment of this JHDM1D/TSP1/TGF-β/SMAD3 autocrine loop. These results reveal that epigenetic regulation of TGF-β signaling in MSCs governs the benign tumor phenotype in OF and highlight TGF-β signaling as a candidate therapeutic target.

Hair follicles facilitate the study of stem cell behavior because stem cells in progressive activation stages, ordered within the follicle architecture, are capable of cyclic regeneration. To study the gene network governing the homeostasis of hair bulge stem cells, we developed a Keratin 15-driven genetic model to directly perturb molecular signaling in the stem cells. We visualize the behavior of these modified stem cells, evaluating their hair-regenerating ability and profile their molecular expression. Bone morphogenetic protein (BMP)-inactivated stem cells exhibit molecular profiles resembling those of hair germs, yet still possess multipotentiality in vivo. These cells also exhibit up-regulation of Wnt7a, Wnt7b, and Wnt16 ligands and Frizzled (Fzd) 10 receptor. We demonstrate direct transcriptional modulation of the Wnt7a promoter. These results highlight a previously unknown intra-stem cell antagonistic competition, between BMP and Wnt signaling, to balance stem cell activity. Reduced BMP signaling and increased Wnt signaling tilts each stem cell toward a hair germ fate and, vice versa, based on a continuous scale dependent on the ratio of BMP/Wnt activity. This work reveals one more hierarchical layer regulating stem cell homeostasis beneath the stem cell–dermal papilla-based epithelial–mesenchymal interaction layer and the hair follicle–intradermal adipocyte-based tissue interaction layer. Although hierarchical layers are all based on BMP/Wnt signaling, the multilayered control ensures that all information is taken into consideration and allows hair stem cells to sum up the total activators/inhibitors involved in making the decision of activation.

Enamel maturation is a dynamic process that involves high rates of mineral acquisition, associated fluctuations in extracellular pH, and resorption of extracellular enamel proteins. During maturation, ameloblasts change from having a tall, thin, and highly polarized organization, characteristic of the secretory stage, to having a low columnar and widened morphology in the maturation stage. To identify potential differences in gene expression throughout maturation, we obtained enamel organ epithelial cells derived from the early- and late-maturation stages of rat incisor and analyzed the global gene-expression profiles at each stage. Sixty-three candidate genes were identified as having potential roles in the maturation process. Quantitative PCR was used to confirm the results of this genome-wide analysis in a subset of genes. Transcripts enriched during late maturation (n = 38) included those associated with lysosomal activity, solute carrier transport, and calcium signaling. Also up-regulated were transcripts involved in cellular responses to oxidative stress, proton transport, cell death, and the immune system. Transcripts down-regulated during the late maturation stage (n =25) included those with functions related to cell adhesion, cell signaling, and T-cell activation. These results indicate that ameloblasts undergo widespread molecular changes during the maturation stage of amelogenesis and hence provide a basis for future functional investigations into the mechanistic basis of enamel mineralization.

We identify four novel DNA-binding complexes in the nuclear-encoded Lhcb1 promoter of the chlorophyte alga Dunaliella tertiolecta that are regulated by photosynthetic pathways in the plastid. The binding activities of three of the complexes were positively correlated with time-dependent changes in Lhcb1 transcript abundance, implicating their roles as transcriptional enhancers in a retrograde signal transduction pathway. Using a combination of inhibitors, uncouplers, and antimycin A, and by following the kinetic pattern of gene regulation, we infer two different sensors in the signal transduction pathway. On short time scales of 0.5 to about 4 h, the transthylakoid membrane potential appears to be a critical determinant of gene expression, whereas on time scales of 8 h or longer, the redox state of the plastoquinone pool becomes increasingly more important. The differentiation of these two types of signals was observed in parallel effects on gene transcription and on the patterns of DNAbinding activities in the Lhcb1 promoter. These signals appear to be transduced at the nuclear level via a coordinated ensemble of DNA-binding complexes located between 2367 and 2188 bp from the start codon of the gene. The regulation of these elements allows the cell to up- or down-regulate the expression on Lhcb1 in response to changes in irradiance. All photosynthetic organisms can adjust the rate of photon absorption to optimize photosynthetic electron transport (PET) in relation to changes in spectral irradiance. Long and persistent shifts in photon flux density (PFD) often involve changes in gene expression that modify the activity, concentration, and structure of the photosynthetic machinery (Falkowski and LaRoche, 1991; Anderson et al., 1995; Durnford and Falkowski, 1997). In comparison with higher plants, photoacclimation is far more pronounced in unicellular algae, where not only the magnitude and rates of the responses are extraordinary, but also the response can easily be distinguished from developmental processes (Falkowski and Chen, 2003). In particular, species in the marine unicellular chlorophyte genusDunaliella can undergo an almost 10-fold change in the abundance of antenna chlorophyll (Chl)-binding proteins on a per cell basis within a single generation (Sukenik et al., 1988). In eukaryotic algae, these changes involve transcription and translation of both nuclear and chloroplast genes. Consequently, tight interorganellar regulatory networks are required to coordinate gene expression in these two compartments. In this paper, we examine how changes in the abundance of the nuclear-encoded Lhcb

Regulation of adult stem cells (SCs) is fundamental for organ maintenance and tissue regeneration. On the body surface, different ectodermal organs exhibit distinctive modes of regeneration and the dynamics of their SC homeostasis remain to be unraveled. A slow cycling characteristic has been used to identify SCs in hair follicles and sweat glands; however, whether a quiescent population exists in continuously growing nails remains unknown. Using an in vivo label retaining cells (LRCs) system, we detected an unreported population of quiescent cells within the basal layer of the nail proximal fold, organized in a ring-like configuration around the nail root. These nail LRCs express the hair stem cell marker, keratin 15 (K15), and lineage tracing show that these K15-derived cells can contribute to both the nail structure and peri-nail epidermis, and more toward the latter. Thus, this stem cell population is bifunctional. Upon nail plucking injury, the homeostasis is tilted with these SCs dominantly delivering progeny to the nail matrix and differentiated nail plate, demonstrating their plasticity to adapt to wounding stimuli. Moreover, in vivo engraftment experiments established that transplanted nail LRCs can actively participate in functional nail regeneration. Transcriptional profiling of isolated nail LRCs revealed bone morphogenetic protein signaling favors nail differentiation over epidermal fate. Taken together, we have found a previously unidentified ring-configured population of bifunctional SCs, located at the interface between the nail appendage organ and adjacent epidermis, which physiologically display coordinated homeostatic dynamics but are capable of rediverting stem cell flow in response to injury.

SummaryRegulatory T (Treg) cells are essential for maintenance of immune homeostasis. Here we found that hydrogen sulfide (H2S) was required for Foxp3+ Treg cell differentiation and function and that H2S deficiency led to systemic autoimmune disease. H2S maintained expression of methylcytosine dioxygenases Tet1 and Tet2 by sulfhydrating nuclear transcription factor Y subunit beta (NFYB) to facilitate its binding to Tet1 and Tet2 promoters. Transforming growth factor-β (TGF-β)-activated Smad3 and interleukin-2 (IL-2)-activated Stat5 facilitated Tet1 and Tet2 binding to Foxp3. Tet1 and Tet2 catalyzed conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in Foxp3 to establish a Treg-cell-specific hypomethylation pattern and stable Foxp3 expression. Consequently, Tet1 and Tet2 deletion led to Foxp3 hypermethylation, impaired Treg cell differentiation and function, and autoimmune disease. Thus, H2S promotes Tet1 and Tet2 expression, which are recruited to Foxp3 by TGF-β and IL-2 signaling to maintain Foxp3 demethylation and Treg-cell-associated immune homeostasis.

Adult stem cells (SCs) are important to maintain homeostasis of tissues including several mini-organs like hair follicles and sweat glands. However, the existence of stem cells in minor salivary glands (SGs) is largely unexplored. In vivo histone2B green fluorescent protein pulse chase strategy has allowed us to identify slow-cycling, label-retaining cells (LRCs) of minor SGs that preferentially localize in the basal layer of the lower excretory duct with a few in the acini. Engraftment of isolated SG LRC in vivo demonstrated their potential to differentiate into keratin 5 (basal layer marker) and keratin 8 (luminal layer marker)-positive structures. Transcriptional analysis revealed activation of TGFβ1 target genes in SG LRC and BMP signaling in SG progenitors. We also provide evidence that minor SGSCs are sensitive to tobacco-derived tumor-inducing agent and give rise to tumors resembling low grade adenoma. Our data highlight for the first time the existence of minor SG LRCs with stem cells characteristic and emphasize the role of transforming growth factor beta (TGFβ) pathway in their maintenance. Stem Cells 2014;32:2267–2277

Slow cycling is a common feature shared among several stem cells (SCs) identified in adult tissues including hair follicle and cornea. Recently, existence of unipotent SCs in basal and lumenal layers of sweat gland (SG) has been described and label retaining cells (LRCs) have also been localized in SGs; however, whether these LRCs possess SCs characteristic has not been investigated further. Here, we used a H2BGFP LRCs system for in vivo detection of infrequently dividing cells. This system allowed us to specifically localize and isolate SCs with label-retention and myoepithelial characteristics restricted to the SG proximal acinar region. Using an alternative genetic approach, we demonstrated that SG LRCs expressed keratin 15 (K15) in the acinar region and lineage tracing determined that K15 labeled cells contributed long term to the SG structure but not to epidermal homeostasis. Surprisingly, wound healing experiments did not activate proximal acinar SG cells to participate in epidermal healing. Instead, predominantly non-LRCs in the SG duct actively divided, whereas the majority of SG LRCs remained quiescent. However, when we further challenged the system under more favorable isolated wound healing conditions, we were able to trigger normally quiescent acinar LRCs to trans-differentiate into the epidermis and adopt its long term fate. In addition, dissociated SG cells were able to regenerate SGs and, surprisingly, hair follicles demonstrating their in vivo plasticity. By determining the gene expression profile of isolated SG LRCs and non-LRCs in vivo, we identified several Bone Morphogenetic Protein (BMP) pathway genes to be up-regulated and confirmed a functional requirement for BMP receptor 1A (BMPR1A)-mediated signaling in SG formation. Our data highlight the existence of SG stem cells (SGSCs) and their primary importance in SG homeostasis. It also emphasizes SGSCs as an alternative source of cells in wound healing and their plasticity for regenerating different skin appendages.

Recent studies suggested that the daily cycle of nitrogen fixation activity in the marine filamentous nonheterocystous cyanobacterium Trichodesmium sp. is controlled by a circadian rhythm. In this study, we evaluated the rhythm of nitrogen fixation in Trichodesmium sp. strain IMS 101 by using the three criteria for an endogenous rhythm. Nitrogenase transcript abundance oscillated with a period of approximately 24 h, and the cycle was maintained even under constant light conditions. The cyclic pattern of transcript abundance was maintained when the culture was grown at 24 and 28.5°C, although the period was slightly longer (26 h) at the higher temperature. The cycle of gene expression could be entrained with light-dark cues. Results of inhibitor experiments indicated that transcript abundance was regulated primarily by transcription initiation, rather than by degradation. The circadian rhythm, the first conclusively demonstrated endogenous rhythm in a filamentous cyanobacterium, was also reflected in nitrogenase MoFe protein abundance and patterns of Fe protein posttranslational modification-demodification.

In multicellular organisms, cell-cell contacts that are mediated by classical cadherins have essential roles in many fundamental processes, such as morphogenesis, maintenance of tissue integrity, wound healing and cell polarity. Furthermore, there is overwhelming evidence that the adherens junction (AJ) is also an important tumor and/or invasion suppressor and E-cadherin and α-catenin are very often down-regulated during tumor progression. As the primary cell-cell adhesion protein in epithelial cells, E-cadherin has long been studied in cancer progression. Similarly, additional roles for β-catenin in the Wnt signaling pathway have led to many studies of the role of β-catenin in cancer. Alpha-catenin, in contrast, has received less attention. However our recent data demonstrated that conditional loss of α-catenin in skin results in formation of squamous cell carcinoma like phenotype, accompanied by increased proliferation, motility and activation of NF-κB and its proinflammatory target genes, which account for many features of invasive epithelial cancer. The mechanism of this complex phenotype after α-catenin deletion is not well understood. Our new data show that the down-regulation of α-catenin is followed by the up-regulation of α-catenin homologue α-catulin. This protein was shown to interact directly with components of Rho GTPases and NF-κB signaling pathways, both crucial for tumor development and progression, and increase migration and survival of human squamous cell carcinoma cells. Therefore, our goal is to understand the role of α-catulin during normal development and tumor progression and how it influences the classical α-catenin function. Using a gene trap method, we have generated α-catulin KO mice. Straight α-catulin KO results in embryonic lethality, with embryos dieing around 10.5E with neural tube closure defects, craniofacial defects and hemorrhaging in the head. To study α-catulin expression pattern, we used a β-galactosidase reporter tag under endogenous α-catulin promoter, and found α-catulin to be expressed in the neural crest during development and maintained in some structures derived from neural crest cells during adulthood. Microarray analysis performed on α-catulin WT and KO embryos showed the most changes in axon guidance and Rho-mediated actin motility genes. These results led us to look at the role of α-catulin during cell migration. In vitro analyses of α-catulin WT and KO embryoid bodies and endodermal cells showed differential migration rates between the WT and KO cells. In vivo analyses by wound healing in heterozygous α-catulin mice showed an increase in α-catulin expression during wound healing. In addition, immunofluorescence staining showed α-catulin co-localized with actin cytoskeleton at the leading edge of the migrating cell. To summarize, α-catulin KO embryos have severe phenotypes and evidence suggests that α-catulin may play a role in migration during development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 999.