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Epithelial-to-mesenchymal transitions (EMTs) are phenotypic plasticity processes that confer migratory and invasive properties to epithelial cells during development, wound-healing, fibrosis and cancer.sup.1-4. EMTs are driven by SNAIL, ZEB and TWIST transcription factors.sup.5,6 together with microRNAs that balance this regulatory network.sup.7,8. Transforming growth factor [beta] (TGF-[beta]) is a potent inducer of developmental and fibrogenic EMTs.sup.4,9,10. Aberrant TGF-[beta] signalling and EMT are implicated in the pathogenesis of renal fibrosis, alcoholic liver disease, non-alcoholic steatohepatitis, pulmonary fibrosis and cancer.sup.4,11. TGF-[beta] depends on RAS and mitogen-activated protein kinase (MAPK) pathway inputs for the induction of EMTs.sup.12-19. Here we show how these signals coordinately trigger EMTs and integrate them with broader pathophysiological processes. We identify RAS-responsive element binding protein 1 (RREB1), a RAS transcriptional effector.sup.20,21, as a key partner of TGF-[beta]-activated SMAD transcription factors in EMT. MAPK-activated RREB1 recruits TGF-[beta]-activated SMAD factors to SNAIL. Context-dependent chromatin accessibility dictates the ability of RREB1 and SMAD to activate additional genes that determine the nature of the resulting EMT. In carcinoma cells, TGF-[beta]-SMAD and RREB1 directly drive expression of SNAIL and fibrogenic factors stimulating myofibroblasts, promoting intratumoral fibrosis and supporting tumour growth. In mouse epiblast progenitors, Nodal-SMAD and RREB1 combine to induce expression of SNAIL and mesendoderm-differentiation genes that drive gastrulation. Thus, RREB1 provides a molecular link between RAS and TGF-[beta] pathways for coordinated induction of developmental and fibrogenic EMTs. These insights increase our understanding of the regulation of epithelial plasticity and its pathophysiological consequences in development, fibrosis and cancer. RAS and TGF-[beta] pathways regulate distinct modes of epithelial-to-mesenchymal transition via RAS-responsive element binding protein 1., Author(s): Jie Su [sup.1] , Sophie M. Morgani [sup.2] [sup.3] , Charles J. David [sup.1] [sup.6] , Qiong Wang [sup.1] [sup.7] , Ekrem Emrah Er [sup.1] , Yun-Han Huang [sup.1] [...]

Activated CD4 T cells proliferate rapidly and remodel epigenetically before exiting the cell cycle and engaging acquired effector functions. Metabolic reprogramming from the naive state is required throughout these phases of activation.sup.1. In CD4 T cells, T-cell-receptor ligation--along with co-stimulatory and cytokine signals--induces a glycolytic anabolic program that is required for biomass generation, rapid proliferation and effector function.sup.2. CD4 T cell differentiation (proliferation and epigenetic remodelling) and function are orchestrated coordinately by signal transduction and transcriptional remodelling. However, it remains unclear whether these processes are regulated independently of one another by cellular biochemical composition. Here we demonstrate that distinct modes of mitochondrial metabolism support differentiation and effector functions of mouse T helper 1 (T.sub.H1) cells by biochemically uncoupling these two processes. We find that the tricarboxylic acid cycle is required for the terminal effector function of T.sub.H1 cells through succinate dehydrogenase (complex II), but that the activity of succinate dehydrogenase suppresses T.sub.H1 cell proliferation and histone acetylation. By contrast, we show that complex I of the electron transport chain, the malate-aspartate shuttle and mitochondrial citrate export are required to maintain synthesis of aspartate, which is necessary for the proliferation of T helper cells. Furthermore, we find that mitochondrial citrate export and the malate-aspartate shuttle promote histone acetylation, and specifically regulate the expression of genes involved in T cell activation. Combining genetic, pharmacological and metabolomics approaches, we demonstrate that the differentiation and terminal effector functions of T helper cells are biochemically uncoupled. These findings support a model in which the malate-aspartate shuttle, mitochondrial citrate export and complex I supply the substrates needed for proliferation and epigenetic remodelling early during T cell activation, whereas complex II consumes the substrates of these pathways, which antagonizes differentiation and enforces terminal effector function. Our data suggest that transcriptional programming acts together with a parallel biochemical network to enforce cell state. Genetic, pharmacological and metabolomics experiments reveal that the malate-aspartate shuttle and mitochondrial citrate export support the differentiation of mouse T helper 1 cells, whereas succinate dehydrogenase enforces their terminal effector function., Author(s): Will Bailis [sup.1] [sup.2] , Justin A. Shyer [sup.1] , Jun Zhao [sup.1] [sup.3] [sup.4] , Juan Carlos Garcia Canaveras [sup.5] [sup.6] [sup.7] , Fatimah J. Al Khazal [sup.8] [...]

A defining feature of adaptive immunity is the development of long-lived memory T cells to curtail infection. Recent studies have identified a unique stem-like T-cell subset amongst exhausted CD8-positive T cells in chronic infection.sup.1-3, but it remains unclear whether CD4-positive T-cell subsets with similar features exist in chronic inflammatory conditions. Amongst helper T cells, T.sub.H17 cells have prominent roles in autoimmunity and tissue inflammation and are characterized by inherent plasticity.sup.4-7, although how such plasticity is regulated is poorly understood. Here we demonstrate that T.sub.H17 cells in a mouse model of autoimmune disease are functionally and metabolically heterogeneous; they contain a subset with stemness-associated features but lower anabolic metabolism, and a reciprocal subset with higher metabolic activity that supports transdifferentiation into T.sub.H1-like cells. These two T.sub.H17-cell subsets are defined by selective expression of the transcription factors TCF-1 and T-bet, and by discrete levels of CD27 expression. We also identify signalling via the kinase complex mTORC1 as a central regulator of T.sub.H17-cell fate decisions by coordinating metabolic and transcriptional programmes. T.sub.H17 cells with disrupted mTORC1 signalling or anabolic metabolism fail to induce autoimmune neuroinflammation or to develop into T.sub.H1-like cells, but instead upregulate TCF-1 expression and acquire stemness-associated features. Single-cell RNA sequencing and experimental validation reveal heterogeneity in fate-mapped T.sub.H17 cells, and a developmental arrest in the T.sub.H1 transdifferentiation trajectory upon loss of mTORC1 activity or metabolic perturbation. Our results establish that the dichotomy of stemness and effector function underlies the heterogeneous T.sub.H17 responses and autoimmune pathogenesis, and point to previously unappreciated metabolic control of plasticity in helper T cells.Phenotypically, transcriptionally and metabolically diverse subsets of T.sub.H17 cells develop in a chronic autoimmune disease: one subset has inferred stemness features and low anabolic metabolism, while a reciprocal subset has higher metabolic activity that supports transdifferentiation into T.sub.H1 cells., Author(s): Peer W. F. Karmaus [sup.1] , Xiang Chen [sup.2] , Seon Ah Lim [sup.1] , Andrés A. Herrada [sup.1] , Thanh-Long M. Nguyen [sup.1] , Beisi Xu [sup.2] , [...]

The mammalian cochlea contains two types of mechanosensory hair cell that have different and critical functions in hearing. Inner hair cells (IHCs), which have an elaborate presynaptic apparatus, signal to cochlear neurons and communicate sound information to the brain. Outer hair cells (OHCs) mechanically amplify sound-induced vibrations, providing enhanced sensitivity to sound and sharp tuning. Cochlear hair cells are solely generated during development, and hair cell death--most often of OHCs--is the most common cause of deafness. OHCs and IHCs, together with supporting cells, originate in embryos from the prosensory region of the otocyst, but how hair cells differentiate into two different types is unknown.sup.1-3. Here we show that Insm1, which encodes a zinc finger protein that is transiently expressed in nascent OHCs, consolidates their fate by preventing trans-differentiation into IHCs. In the absence of INSM1, many hair cells that are born as OHCs switch fates to become mature IHCs. To identify the genetic mechanisms by which Insm1 operates, we compared the transcriptomes of immature IHCs and OHCs, and of OHCs with and without INSM1. In OHCs that lack INSM1, a set of genes is upregulated, most of which are normally preferentially expressed by IHCs. The homeotic cell transformation of OHCs without INSM1 into IHCs reveals a mechanism by which these neighbouring mechanosensory cells begin to differ: INSM1 represses a core set of early IHC-enriched genes in embryonic OHCs and makes them unresponsive to an IHC-inducing gradient, so that they proceed to mature as OHCs. Without INSM1, some of the OHCs in which these few IHC-enriched transcripts are upregulated trans-differentiate into IHCs, identifying candidate genes for IHC-specific differentiation.Conditional deletion of Insm1 in mice demonstrates that INSM1 is the key switch that causes the maturation of outer hair cells in the cochlea, with its absence resulting in an increase in inner hair cells instead., Author(s): Teerawat Wiwatpanit [sup.1] [sup.2] , Sarah M. Lorenzen [sup.1] [sup.3] , Jorge A. Cantú [sup.1] , Chuan Zhi Foo [sup.1] [sup.2] , Ann K. Hogan [sup.1] [sup.2] , Freddie [...]

In quantitative PCR (qPCR), replicates can minimize the impact of intra-assay variation; however, inter-assay variations must be minimized to obtain a robust quantification method. The method proposed in this study uses Savitzky--Golay smoothing and differentiation (SGSD) to identify a derivative-maximum-based cycle of quantification. It does not rely on curve modeling, as is the case with many existing techniques. PCR fluorescence data sets challenged for inter-assay variations (different thermocycler units, different reagents batches, different operators, different standard curves, and different labs) were used for the evaluation. The algorithm was compared with a four-parameter logistic model (4PLM) method, the [C.sub.y]0 method, and the threshold method. The SGSD method compared favourably with all methods in terms of inter-assay variation. SGSD was statistically different from the 4PLM (P = 0.03), [C.sub.y]0 (P = 0.05), and threshold (P = 0.004) methods on relative error comparison basis. For intra-assay variations, SGSD outperformed the threshold method (P = 0.005) and equalled the 4PLM and [C.sub.y]0 methods (P > 0.05) on relative error basis. Our results demonstrate that the SGSD method could potentially be an alternative to sigmoid modeling based methods (4PLM and [C.sub.y]0) when PCR data are challenged for inter-assay variations. Key words: gene quantification, PCR quantification, quantification cycle, qPCR, Savitzky--Golay. Dans le domaine du PCR quantitatif (qPCR), l'utilisation de replicas permet de minimiser l'impact des variations intra-essais; toutefois, les variations inter-essais doivent egalement etre minimisees dans le but d'obtenir une methode de quantification robuste. La methode proposee dans cette etude utilise la technique de lissage et differentiation de Savitzky--Golay (SGSD) pour determiner le cycle de quantification d'une amplification de PCR en se basant sur le maximum d'une derivee. Contrairement a plusieurs methodes, celle-ci permet d'eviter de passer par une etape de modelisation de la courbe sigmoide. Des jeux de donnees mis a l'epreuve au niveau des variations inter-essais (differents thermocycleurs, differents lots de reactifs, differents operateurs, differentes courbes standards, et differents laboratoires) ont ete utilises pour 1'evaluation. L'algorithme a ete compare a une methode basee sur un modele logistique a quatre parametres (4PLM), a la methode du [C.sub.y]0 et a la methode du seuil ([C.sub.t]). La methode SGSD s'est comparee favorablement par rapport aux autres methodes au niveau des variations inter-essais. Au niveau de l'erreur relative, SGSD differe statistiquement de 4PLM (P = 0,03), de [C.sub.y]0 (P = 0,05) et de [C.sub.t] (P = 0,004). Quant aux variations intra-essais, SGSD surpasse la methode du seuil (P = 0,005) et equivaut aux methodes 4PLM et Cy0 (P > 0,05) par rapport a l'erreur relative. Nos resultats demontrent que la methode SGSD peut etre une alternative potentielle aux methodes basees sur la modelisation de la courbe sigmoide (4PLM et [C.sub.y]0) lorsque les donnees sont mises a l'epreuve par des variations inter-essais. Mots-cles: quantification genique, quantification par PCR, cycle de quantification, qPCR, Savitzky--Golay., Introduction Since its development by Kary Mullis in 1983 (Mullis 1993), DNA amplification by polymerase chain reaction (PCR) and its variant, quantitative PCR (qPCR), have been applied in diverse fields [...]

Author(s): Achim Werner [1, 2]; Shintaro Iwasaki [2]; Colleen A. McGourty [2]; Sofia Medina-Ruiz [2]; Nia Teerikorpi [1, 2]; Indro Fedrigo [2]; Nicholas T. Ingolia [2]; Michael Rape (corresponding author) [...]

There is an ever-growing demand for transplantable organs to replace acute and chronically dam-aged tissues. This demand cannot be met by the currently available donor organs. Efforts to pro-vide an alternative source have led to the development of organ engineering, a discipline that com-bines cell biology, tissue engineering, and cell/organ transplantation. Over the last several years, engineered organs have been implanted into rodent recipients and have shown modest function. In this article, we summarize the most recent advances in this field and provide a perspective on the challenges of translating this promising new technology into a proven regenerative therapy., Introduction Solid organ transplantation is the therapy of choice for nonreversible acute and chronic organ failure from almost any cause. The success of transplantation, which is based on advances in [...]

It is becoming increasingly clear that cholesterol-independent effects of statins also contribute to the cardioprotective effects, but these mechanisms remain poorly understood. We investigated the effects of lovastatin on vascular smooth muscle phenotype. We have previously shown that mammalian target of rapamycin complex 1 (mTORCI) inhibition with rapamycin induces vascular smooth muscle cell (VSMC) differentiation. We found that lovastatin also inhibits mTORC1 signaling and that this inhibition is required for VSMC differentiation. Lovastatin inhibition of mTORC1 was farnesylation dependent, suggesting the farnesylated G protein Rheb (Ras homologue enriched in brain), a known upstream activator of mTORC1. Rheb overexpression induced mTORC1 activity and repressed contractile protein expression, but a farnesylation-deficient mutant (C18S) elicited the opposite effect. Rheb knockdown with small interfering RNA was also sufficient to inhibit mTORC1 and induce contractile protein expression, and it prevented statin-induced VSMC differentiation. Notably, mTORC1 activity was elevated in VSMC isolated from an intimal hyperplastic patient lesion compared with normal media, and lovastatin treatment inhibited mTORC1 activity in these cultures. Furthermore, lovastatin inhibited mTORC1 activity and prevented the downregulation of contractile protein expression in an ex vivo angioplasty model. In conclusion, these findings illustrate a mechanism for the cardioprotective effects of lovastatin through inhibition of Rheb and mTORC1 and promotion of a differentiated VSMC phenotype. vascular smooth muscle cell differentiation; statin; mammalian target of rapamycin doi: 10.1152/ajpcell.00429.2009.

Estrogen receptors (ERs) play a pivotal role in adipogenesis; therefore, compounds targeting ERs may also affect fat formation. Recent studies have shown that the Dioscorea plant (commonly called yam) exhibits an antiobesity effect on rodents. However, the active compounds and underlying mechanisms responsible for this effect are not yet fully understood. We evaluated the effects of pseudoprotodiocsin (PPD), a steroid saponin from Dioscorea nipponica Makino (a type of Dioscorea), on adipogenesis and the mechanisms underlying this effect. Treatment with PPD at the onset of adipogenic differentiation resulted in significantly decreased adipogenesis in both in vitro and in vivo experimental systems. An increased amount of ERa mRNA, protein, and the accumulation of ERa in the nucleus were also observed. However, the expression pattern of ER[beta] was not altered. Furthermore, the antiadipogenic effect of PPD was found to be ER dependent. It was also accompanied by the decreased expression of several genes involved in adipogenesis, including lipoprotein lipase (LPL), leptin, CCAAT/ enhancer-binding-protein-[alpha] (C/EBP[alpha]), and peroxisome proliferator-activated receptor--[gamma] (PPAR[gamma]), as well as the increased expression of some negative factors of adipogenesis, including preadipocyte factor 1 (Pre-1), GATA-binding protein 2 (GATA-2), GC-induced leucinezipper protein (GILZ), and C/EBP homologous protein (CHOP-10). In addition to its estrogenic action, PPD also abolished the p38 mitogen-activated protein kinase (p38 MAPK) activation. Our results suggest that PPD inhibits adipogenesis in an ER-dependent manner and induces the expression of ERa. These findings may provide a lead toward a novel agent that can be used to treat obesity. obesity; Dioscorea nipponica Makino; steroid saponin doi: 10.1152/ajpcell.00538.2009.

Adipose tissue controls body lipid and energy metabolism, as well as food intake, and abnormalities in adipose function play a central role in diseases such as obesity and type-2 diabetes. Adipocyte differentiation is controlled by a transcriptional cascade involving PPAR[gamma] and members of the C/EBP family of transcription factors. Here, we demonstrate that C/EBP[alpha] is targeted for degradation by the ubiquitin ligase Fbxw7 in a phosphorylation-dependent manner. Importantly, inactivation of Fbxw7 is sufficient to convert mouse preadipocytes into mature adipocytes in a manner dependent on C/EBP[alpha]. In addition, inactivation of Fbxw7 promotes adipocyte differentiation of human adult stem cells. Taken together, our results suggest that Fbxw7 is a negative regulator of adipogenesis by targeting C/EBP[alpha] for degradation. This notion is supported by the observation that the expression of Fbxw7 is down-regulated during adipocyte differentiation, resulting in the accumulation of proadipogenic proteins such as C/EBP[alpha]. Thus, Fbxw7 could be an important regulator of energy and lipid metabolism. adipogenesis | CCAAT/enhancer-binding protein doi/ 10.1073/pnas.0913367107

Multiple sclerosis is a neurodegenerative disease characterized by episodes of autoimmune attack of oligodendrocytes leading to demyelination and progressive functional deficits. Because many patients exhibit functional recovery in between demyelinating episodes, understanding mechanisms responsible for repair of damaged myelin is critical for developing therapies that promote remyelination and prevent disease progression. The chemokine CXCL12 is a developmental molecule known to orchestrate the migration, proliferation, and differentiation of neuronal precursor cells within the developing CNS. Although studies suggest a role for CXCL12 in oligodendroglia ontogeny in vitro, no studies have investigated the role of CXCL12 in remyelination in vivo in the adult CNS. Using an experimental murine model of demyelination mediated by the copper chelator cuprizone, we evaluated the expression of CXCL12 and its receptor, CXCR4, within the demyelinating and remyelinating corpus callosum (CC). CXCL12 was significantly up-regulated within activated astrocytes and microgila in the CC during demyelination, as were numbers of CXCR4+ NG2+ oligodendrocyte precursorcells (OPCs). Loss of CXCR4 signaling via either pharmacological blockade or in vivo RNA silencing led to decreased OPCs maturation and failure to remyelinate. These data indicate that CXCR4 activation, by promoting the differentiation of OPCs into oligodendrocytes, is critical for remyelination of the injured adult CNS. chemokine | cuprizone | CXCL12 | central nervous system doi/ 10.1073/pnas.1006301107

Lipopolysaccharide (LPS) of Gram-negative bacteria is a common pathogen-associated molecular pattern (PAMP) that induces potent innate immune responses. The host immune response against LPS is triggered by myeloid differentiation factor 2 (MD-2) in association with Toll-like receptor 4 (TLR4) on the cell surface. The MD-2/ TLR4-mediated LPS response is regulated by the evolutionarily related complex of MD-1 and Toll-like receptor homoIog RP105. Here, we report crystallographic and biophysical data that demonstrate a previously unidentified direct interaction of MD-1 with LPS. The crystal structure of chicken MD-1 (cMD-1) at 2.0 A resolution exhibits a [beta]-cup-like fold, similar to M D-2, that encloses a hydrophobic cavity between the two [beta]-sheets. A lipid-like moiety was observed inside the cavity, suggesting the possibility of a direct MD-1/ LPS interaction. LPS was subsequently identified as an MD-1 ligand by native gel electrophoresis and gel filtration analyses. The crystal structure of CMD-1 with lipid IVa, an LPS precursor, at 2.4 A resolution revealed that the lipid inserts into the deep hydrophobic cavity of the [beta]-cup-like structure, but with some important differences compared with MD-2. These findings suggest that soluble MD-1 alone, in addition to its complex with RP105, can regulate host LPS sensitivity. lipopolysaccharide | MD-2 | RP105 | TLR4 | innate immunity doi/ 10.1073/pnas.1004153107

Raman microspectroscopy is an attractive approach for chemical imaging of biological specimens, including live cells, without the need for chemi-selective stains. Using a microspectrometer, near-infrared Raman spectra throughout the range 663 [cm.sup.-1] to 1220 [cm.sup.-1] were obtained from colonies of CA1 human embryonic stem cells (hESCs) and CA1 cells that had been stimulated to differentiate for 3 weeks by 10% fetal bovine serum on gelatin. Distributions and intensities of spectral bands attributed to proteins varied significantly between undifferentiated and differentiated cells. Importantly, compared to proteins and lipids, the band intensities of nucleic acids were dominant in undifferentiated cells with a dominance-reversal in differentiated cells. Thus, we could identify intensity ratios of particular protein-related bands (e.g., 757 [cm.sup.-1] tryptophan) to nucleic acid bands (784 [cm.sup.-1] DNA/RNA composite) that were effective in discriminating between spectra of undifferentiated and differentiated cells. We observed no discernible negative effects due to the laser exposure in terms of morphology, proliferation, or pluripotency of the stem cells. We conclude that Raman microscopy and complementary data processing procedures provide a rapid, noninvasive approach that can distinguish hESCs from differentiated cells. This is the first report to identify specific Raman markers for the diferentiation status of hESCs. 10.1021/ac9O2697q

Flatworms called planarians can break off fragments of themselves that regenerate to form new, complete worms. The molecular cues that regulate the frequency of such fission events have been revealed. Cues that control tissue patterning regulate fission in flatworms., Author(s): Thomas W. Holstein Author Affiliations: What makes flatworms go to pieces Understanding how tissues and organs can regenerate requires an appreciation of the mechanisms and factors that organize cells [...]

Histone deacetylases (HDAC) 1 and 2 are highly similar enzymes that help regulate chromatin structure as the core catalytic components of corepressor complexes. Although tissue-specific deletion of HDAC1 and HDAC2 has demonstrated functional redundancy, germ-line deletion of HDAC1 in the mouse causes early embryonic lethality, whereas HDAC2 does not. To address the unique requirement for HDAC1 in early embryogenesis we have generated conditional knockout embryonic stem (ES) cells in which HDAC1 or HDAC2 genes can be inactivated. Deletion of HDAC1, but not HDAC2, causes a significant reduction in the HDAC activity of Sin3A, NuRD, and CoREST corepressor complexes. This reduced corepressor activity results in a specific 1.6-fold increase in histone H3 K56 acetylation (H3K56Ac), thus providing genetic evidence that H3K56Ac is a substrate of HDAC1. In culture, ES cell proliferation was unaffected by loss of either HDAC1 or HDAC2. Rather, we find that loss of HDAC1 affects ES cell differentiation. ES ceils lacking either HDAC1 or HDAC2 were capable of forming embryoid bodies (EBs), which stimulates differentiation into the three primary germ layers. However, HDAC1-deficient EBs were significantly smaller, showed spontaneous rhythmic contraction, and increased expression of both cardiomyocyte and neuronal markers. In summary, our genetic study of HDAC1 and HDAC2 in ES cells, which mimic the embryonic epiblast, has identified a unique requirement for HDAC1 in the optimal activity of HDAC1/2 corepressor complexes and cell fate determination during differentiation. corepressor | acetylation | deacetylation | chromatin doi/ 10.1073/pnas.1000478107

Stem cells support tissue maintenance by balancing self-renewal and differentiation. In mice, it is believed that a homogeneous stem cell population of single spermatogonia supports spermatogenesis, and that differentiation, which is accompanied by the formation of connected cells (cysts) of increasing length, is linear and nonreversible. We evaluated this model with the use of lineage analysis and live imaging, and found that this putative stem cell population is not homogeneous. Instead, the stem cell pool that supports steady-state spermatogenesis is contained within a subpopulation of single spermatogonia. We also found that cysts are not committed to differentiation and appear to recover stem cell potential by fragmentation, and that the fate of individual spermatogonial populations was markedly altered during regeneration after damage. Thus, there are multiple and reversible paths from stem cells to differentiation, and these may also occur in other systems. 10.1126/science.1182868

The role of lung epithelial stem cells in maintenance and repair of the adult lung is ill-defined, and their identity remains contentious because of the lack of definitive markers for their prospective isolation and the absence of clonogenic assays able to measure their stem/progenitor cell potential. In this study, we show that replication of epithelial-mesenchymal interactions in a previously undescribed matrigel-based clonogenic assay enables the identification of lung epithelial stem/progenitor cells by their colonyforming potential in vitro. We describe a population of [EpCAM.sup.hi] [CD49f.sup.pos] [CD104.sup.pos] [CD24.sup.Iow] epithelial cfus that generate colonies comprising airway, alveolar, or mixed lung epithelial cell lineages when cocultured with [EpCAM.sup.neg] [Sca-1.sup.pos] lung mesenchymal cells. We show that soluble fibroblast growth factor-10 and hepatocyte growth factor partially replace the requirement for mesenchymal support of epithelial colony formation, allowing clonal passaging and demonstration of their capacity for self-renewal. These data support a model in which the adult mouse lung contains a minor population of multipotent epithelial stem/progenitor cells with the capacity for self-renewal and whose descendants give rise to airway and alveolar epithelial cell lineages in vitro. colony-forming assay | lung epithelium | lineage specificity | differentiation | EpCAM doi/ 10.1073/pnas.0909207107

Mucous hypersecretion is a serious feature of chronic airway diseases such as asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. Although mucins are produced via activation of an EGF receptor (EGFR) signaling cascade, the mechanisms leading to exaggerated mucin production in mucous hypersecretory diseases are unknown. Because expression of ICAM-1 and of the ICAM-1 ligand fibrinogen is increased in the airways of subjects with mucous hyper-secretory diseases, we hypothesized that fibrinogen binding to ICAM-1 could increase EGFR-dependent mucin production in human airway (NCI-H292) epithelial cells. Consistent with this hypothesis, we found that an ICAM-1 neutralizing antibody and an ICAM-1(8-22) peptide that binds fibrinogen decreased mucin production induced by the EGFR ligand transforming growth factor (TGF)-[alpha] dose-dependently. Exogenous fibrinogen and a fibrinogen(117-133) peptide that binds ICAM-1 rescued mucin production in cells treated with the ICAM-1(8-22) peptide. Surprisingly, the ICAM-1(8-22) peptide increased EGFR phosphotyrosine and phospho-ERK1/2 in cells treated with TGF-[alpha]. The ICAM-1 (8-22) peptide-induced increases in EGFR phosphotyrosine and phospho-ERK1/2 were prevented by exogenous fibrinogen, by the fibrinogen(117-133) peptide, and by selective inhibitors of phospholipase C (PLC), protein kinase C (PKC)-[alpha]/[beta], and metalloproteases. These results suggest that fibrinogen binding to ICAM-1 promotes mucin production by decreasing TGF-[alpha]-induced EGFR and ERK 1/2 activation and that the fibrinogen-ICAM-1-dependent decrease in EGFR and ERK1/2 activation occurs via inhibition of an early positive feedback pathway involving PLC-and PKC-[alpha]/[beta]-dependent metalloprotease activation and subsequent metalloprotease-dependent EGFR reactivation. cell surface molecules; innate immunity; mucous hypersecretion; cell differentiation

Oct4 and Nanog are two embryonic stem (ES) cell-specific transcription factors that play critical roles in the maintenance of ES cell pluripotency. In this study, we investigated the effects of Oct4 and Nanog expression on the differentiation state of myogenic cells, which is sustained by a strong positive feedback loop. Oct4 and Nanog, either independently or simultaneously, were overexpressed in C2C 12 myoblasts, and the expression of myogenic lineage-specific genes and terminal differentiation was observed by RT-PCR. Overexpression of Oct4 in C2C12 cultures repressed, while exogenous Nanog did not significantly alter C2C12 terminal differentiation. The expression of Pax7 was reduced in all Oct4-overexpressing myoblasts, and we identified a major Oct4-binding site in the Pax7 promoter. Simultaneous expression of Oct4 and Nanog in myoblasts inhibited the formation of myotubes, concomitant with a reduction in the endogenous levels of hallmark myogenic markers. Furthermore, overexpression of Oct4 and Nanog induced the expression of their endogenous counterparts along with the expression of Sox2. Using mammalian two-hybrid assays, we confirmed that Oct4 functions as a transcriptional repressor whereas Nanog functions as a transcriptional activator during muscle terminal differentiation. Importantly, in nonobese diabetic (NOD) severe combined immunodeficiency (SCID) mice, the pluripotency of C2C12 cells was conferred by overexpression of Oct4 and Nanog. These results suggest that Oct4 in cooperation with Nanog strongly suppresses the myogenic differentiation program and promotes pluripotency in myoblasts. Pax7; pluripotency; differentiation; stem cell

The sarcoplasmic reticulum (SR) of skeletal muscle cells is a complex network of tubules and cisternae that share a common lumen delimited by a single continuous membrane. The SR contains longitudinal and junctional domains characterized by distinctive patterns of protein localization, but how SR proteins reach and/or are retained at these sites is not known. Here, we report that the organization of longitudinal SR proteins is a slow process characterized by temporally distinct patterns of protein localization. In contrast, junctional SR proteins rapidly and synchronously assembled into clusters which, however, merged into mature triadic junctions only after completion of longitudinal SR protein organization. Fluorescence recovery after photobleaching experiments indicated that SR organization was accompanied by significant changes in the dynamic properties of longitudinal and junctional proteins. The decrease in mobility that accompanied organization of the longitudinal SR proteins ank1.5-GFP and GFP-InsP3R1 was abrogated by deletion of specific binding sites for myofibrillar or cytoskeletal proteins, respectively. Assembly of junctional SR domains was accompanied by a strong decrease in mobility of junctional proteins that in triadin appeared to be mediated by its intraluminal region. Together, the data suggest that the organization of specific SR domains results from a process of membrane reorganization accompanied by the establishment of multiple protein-protein interactions with intrinsic and extrinsic cues. calcium store | excitation-contraction coupling | muscle differentiation | protein dynamics

Epithelial-mesenchymal transition (EMT) is required for mesodermal differentiation during development. The zinc-finger transcription factor, Snail1, can trigger EMT and is sufficient to transcriptionally reprogram epithelial cells toward a mesenchymal phenotype during neoplasia and fibrosis. Whether Snail1 also regulates the behavior of terminally differentiated mesenchymal cells remains unexplored. Using a Snail conditional knockout model, we now identify Snail1 as a regulator of normal mesenchymal cell function. Snail1 expression in normal fibroblasts can be induced by agonists known to promote proliferation and invasion in vivo. When challenged within a tissue-like, three-dimensional extracellular matrix, Snail1-deficient fibroblasts exhibit global alterations in gene expression, which include defects in membrane type-1 matrix metalloproteinase (MT1-MMP)-dependent invasive activity. Snail1-deficient fibroblasts explanted atop the live chick chorioallantoic membrane lack tissue-invasive potential and fail to induce angiogenesis. These findings establish key functions for the EMT regulator Snail1 after terminal differentiation of mesenchymal cells.

Mycelial extracts have a wide range of biological activities that modulate functions of mammalian cells. In this report, we sought to identify antiadipogenic mycelia with the use of 3T3-L1 cells and found that the extract of Cordyceps militaris exclusively suppressed differentiation of 3T3-L1 preadipocytes into mature adipocytes without affecting cell viability. This inhibitory effect was dose dependent, reversible, and associated with 1) a decrease in lipid accumulation, 2) blunted induction of adipocyte markers including adiponectin, peroxisome proliferator-activated receptor-[gamma], and CCAAT/enhancer binding protein-[alpha], and 3) sustained expression of a preadipocyte marker, monocyte chemoattractant protein-1. C. militaris also significantly decreased accumulation of lipid and hypertrophy in mature adipocytes and preserved their response to insulin (phosphorylation of Akt) during prolonged culture. Subsequent experiments revealed that C. militaris has the potential to activate the aryl hydrocarbon receptor (AhR). In 3T3-L1 cells, treatment with AhR agonists including benzo[a]pyrene and 3-methylcholanthrene reproduced the antiadipogenic effect of C. militaris. Furthermore, dominant-negative inhibition of AhR abrogated the suppressive effect of C. militaris on adipocyte differentiation. These results suggest that C. militaris has the potential to interfere with adipocyte differentiation through activation of AhR. mycelium; adipogenesis; 3T3-L1 cells

We previously described a critical role for a fibroblast precursor population in the development of a murine fibrotic cardiomyopathy model (I/RC). These precursors arose from circulating bone marrow-derived cells of monocytic origin. Administration of serum amyloid P (SAP) prevented the presence of this cell population in the heart and the cardiomyopathy. Because SAP binds to Fc receptors (FcRs) expressed on monocytes, we investigated the involvement of FcR signaling. We chose mice lacking the FcR [gamma] chain protein (FcR[[gamma].sup.-/-]), a common membrane-signaling component of activating FcRs. Like wild-type mice, FcR[[gamma].sup.-/-] mice developed fibrosis and cardiac dysfunction when subjected to I/RC. However, unlike wild-type mice, SAP in FcR[[gamma].sup.-/-] mice failed to inhibit the development of fibrosis and cardiac dysfunction and did not diminish the numbers of u-smooth muscle [actin.sup.+] and [CD34.sup.+], [CD45.sup.+] fibroblasts that were typical for I/RC. To further examine the role of SAP in monocyte-to-fibroblast transition, we performed in vitro assays in which human peripheral blood mononuclear cells (PBMCs) migrated through human umbilical vein endothelial cells (HUVECs). We found that MCP-l-dependent transendothelial migration of monocytes markedly accelerated their differentiation into fibroblasts. This monocyte differentiation to fibroblasts was eliminated when SAP was added to the PBMC suspension before endothelial transmigration. Adding SAP to cells after successful migration did not inhibit fibroblast maturation. These data indicate that SAP inhibits the differentiation of a blood-borne, myeloid cell population into fibroblasts by signaling through activating FcRs before transendothelial migration has occurred. We suggest that FcR activation of circulating precursor cells may represent a new treatment target for adverse remodeling and cardiac fibrosis. endothelial transmigration | fibrosis heart | monocytes | serum amyloid P

Dicer, an enzyme involved in microRNA (miRNA) maturation, is required for proper cell differentiation and embryogenesis in mammals. Recent evidence indicates that Dicer and miRNA may also regulate tumorigenesis. To better characterize the role of miRNA in primary cell growth, we generated Dicer-conditional mice. Ablation of Dicer and loss of mature miRNAs in embryonic fibroblasts up-regulated [p19.sup.Arf] and p53 levels, inhibited cell proliferation, and induced a premature senescence phenotype that was also observed in vivo after Dicer ablation in the developing limb and in adult skin. Furthermore, deletion of the Ink4a/Arf or p53 locus could rescue fibroblasts from premature senescence induced by Dicer ablation. Although levels of Ras and Myc oncoproteins appeared unaltered, loss of Dicer resulted in increased DNA damage and p53 activity in these cells. These results reveal that loss of miRNA biogenesis activates a DNA damage checkpoint, up-regulates [p19.sup.Arf]-p53 signaling, and induces senescence in primary cells.

The pituitary gland adapts the proportion of each of its endocrine cell types to meet differing hormonal demands throughout life. There is circumstantial evidence that multipotent adult progenitor cells contribute to this plasticity, but these cells have not been identified. Here, we describe a small ( stem cell | folliculostellate cells

Human adipose tissue-derived stem cells (hASCs) represent a potentially valuable cell source for clinical therapeutic applications. The present study was designed to investigate properties of ionic channel currents present in undifferentiated hASCs and their impact on hASCs proliferation. The functional ion channels in hASCs were analyzed by whole-cell patch-clamp recording and their mRNA expression levels detected by RT-PCR. Four types of ion channels were found to be present in hASCs: most of the hASCs (73%) showed a delayed rectifier-like [K.sup.+] current ([I.sub.KDR]); [Ca.sup.2+]-activated [K.sup.+] current ([I.sub.KCa]) was detected in examined cells; a transient outward [K.sub.+] current ([I.sub.to]) was recorded in 19% of the cells; a small percentage of cells (8%) displayed a TTX-sensitive transient inward sodium current ([I.sub.Na.TTX]). RT-PCR results confirmed the presence of ion channels at the mRNA level: Kv1.1, Kv2.1, Kv1.5, Kv7.3, Kv11.1, and hEAG1, possibly encoding [I.sub.KDR]; MaxiK, KCNN3, and KCNN4 for [I.sub.KCa]; Kv1.4, Kv4.1, Kv4.2, and Kv4.3 for [I.sub.to] and hNE-Na for [I.sub.Na.TTX]. The [I.sub.KDR] was inhibited by tetraethyl ammonium (TEA) and 4-aminopyridine (4-AP), which significantly reduced the proliferation of hASCs in a dose-dependent manner (P < 0.05), as suggested by bromode-oxyurindine (BrdU) incorporation. Other selective potassium channel blockers, including linopiridine, iberiotoxin, clotrimazole, and apamin also significantly inhibited [I.sub.KDR]. TTX completely abolished [I.sub.Na.TTX]. This study demonstrates for the first time that multiple functional ion channel currents such as [I.sub.KDR], [I.sub.KCa], [I.sub.to], and [I.sub.Na.TTX] are present in undifferentiated hASCs and their potential physiological function in these cells as a basic understanding for future in vitro experiments and in vivo clinical investigations. proliferation; differentiation; delayed rectifier [K.sub.+] current; voltage-dependent [Ca.sup.2+] current; [Ca.sup.2+]-activated [K.sup.+] current; tetrodotoxin-sensitive [Na.sup.+] current; transient outward [K.sup.+] current

Cell differentiation in embryogenesis involves extensive changes in gene expression structural reorganization within the nucleus, including chromatin condensation and nucleoprotein immobilization. We hypothesized that nuclei in naive stem cells would therefore prove to be physically plastic and also more pliable than nuclei in differentiated cells. Micromanipulation methods indeed show that nuclei in human embryonic stem cells are highly deformable and stiffen 6-fold through terminal differentiation, and that nuclei in human adult stem cells possess an intermediate stiffness and deform irreversibly. Because the nucleo-skeletal component Lamin A/C is not expressed in either type of stem cell, we knocked down Lamin A/C in human epithelial cells and measured a deformability similar to that of adult hematopoietic stem cells. Rheologically, lamin-deficient states prove to be the most fluid-like, especially within the first [approximately equal to] 10 sec of deformation. Nuclear distortions that persist longer than this are irreversible, and fluorescence-imaged microdeformation with photobleaching confirms that chromatin indeed flows, distends, and reorganizes while the lamina stretches. The rheological character of the nucleus is thus set largely by nucleoplasm/chromatin, whereas the extent of deformation is modulated by the lamina. chromatin remodeling | cell mechanics | nuclear plasticity

Lysyl oxidase plays a critical role in the formation of the extracellular matrix, and its activity is required for the normal maturation and cross-linking of collagen and elastin. An 18-kDa lysyl oxidase propeptide (LOPP) is generated from 50-kDa prolysyl oxidase by extracellular proteolytic cleavage during the biosynthesis of active 30-kDa lysyl oxidase enzyme. The fate and the functions of the LOPP are largely unknown, although intact LOPP was previously observed in osteoblast cultures. We investigated the spatial localization of molecular forms of lysyl oxidase, including LOPP in proliferating and differentiating osteoblasts, by using confocal immunofluorescence microscopy and Western blots of cytoplasmic and nuclear extracts. In the present study, a stage-dependent intracellular distribution of LOPP in the osteoblastic cell was observed. In proliferating osteoblasts, LOPP epitopes were principally associated with the Golgi and endoplasmic reticulum, and mature lysyl oxidase epitopes were found principally in the nucleus and perinuclear region. In differentiating cells, LOPP and mature lysyl oxidase immunostaining showed clear colocalization with the microtubule netwQrk. The subcellular distribution of LOPP and its temporal and physical association with microtubules were confirmed by Western blot and far Western blot studies. We also report that N-glycosylated and nonglycosylated LOPP are present in MC3T3-E1 cell cultures. We conclude that LOPP has a stage-dependent intracellular distribution in osteoblastic cells. Future studies are needed to investigate whether the LOPP associations with microtubules or the osteoblast nucleus have functional effects for osteoblast differentiation and bone formation. microtubules; confocal immunofiuorescence microscopy; extracellular matrix; osteoblast differentiation doi:10.1152/ajpcell.00613.2006

Development of the endocardial cushions in the heart involves cell migration and cell differentiation, which is known as epithelial--mesenchymal transformation (EMT). These processes are regulated by cell signaling systems. Yet, the roles of intracellular GTPases and their effectors on these cellular activities remain to be addressed. This study investigated the role Rho GTPase-associated kinases (ROCKs) in endocardial cushion development. Using reverse transcription (RT) and polymerase chain reaction (PCR), expression of the rock1 and rock2 genes was found in the endocardial cushions during development. To investigate the role of ROCKs in development, the ROCK inhibitor Y27632 and adenoviruses containing a dominant negative form of the rock gene were used to treat cultured endocardial cushions and cells. In monolayer cell culture and three-dimensional tissue culture, blockade of ROCK inhibited EMT development. Using three-dimensional collagen gel assays and confocal microscopy, we also observed inhibition of cell migration with ROCK inhibition. Examination of cell morphology and actin cytoskeleton revealed that inhibition of ROCK activity disturbed cytoskeletal organization and blocked the formation of lamellipodia and filopodia. Collectively, these data show that ROCKs play an essential role in endothelial cell differentiation and migration during endocardial cushion development. Keywords: ROCK: Rho kinase; Heart; Endocardial cushion: EMT: Cell migration

A Caenorhabditis elegans gene, ham-2, links Hox gene patterning to migration of the HSN motor neuron. The Caenorhabditis elegans HSN motor neurons make it possible to do genetic analysis of neuronal development at the single-cell level. A zinc finger protein encoded by ham-2 acts downstream of egl-5 to direct HSN cell migration, which takes place early in differentiation. The EGL-43 zinc finger protein is also necessary for HSN migration. It is expressed in the HSN specifically during its migration.

Author(s): Shree Ram Singh [1]; Xiankun Zeng [1]; Jiangsha Zhao [1]; Ying Liu [1]; Gerald Hou [1]; Hanhan Liu [1]; Steven X. Hou (corresponding author) [1] Cancer stem cells (CSCs) [...]

Segmentation of the paraxial mesoderm into parts lined up on the anteroposterior axis is an aspect of somitogenesis. Eph and ephrin are factors in signaling for patterning of presomitic mesoderm as well as in formating of somites. The role of certain proteins has been studied. Dominant negative forms of Eph receptors and ephrins were encoded. Eph signaling is essential for differentiation and segmentation of the somites.

Kss1, a mitogen-activated protein kinase (MAPK), has a double role in regulating filamentous, or invasive, growth of Saccharomyces cerevisiae yeast. The mechanism for the inhibiting of the invasive growth and how Ste7 function overcomes it have been examined. Unphosphorylated Kss1 binds to the transcription factor Ste12 directly. The binding is required for Kss1-mediated Ste12 repression. The stimulation function that Kss1 has requires its catalytic activity and activation by ste7, a MAPK/ERK kinase (MEK). Ste7-mediated phosphorylation of Kss1 cuts back on Kss1-Ste12 interaction; it also relieves Kss1-mediated repression. The Kss1 activation loop regulates binding to Ste12.

A regulatable retroviral vector in which the v-myc oncogene is driven by a tetracycline-controlled trans-activator and a human cytomegalovirus minimal promoter fused to a tet operator sequence was used for conditional immortalization of adult rat neuronal progenitor cells. A single clone, HC2S2, was isolated and characterized. Two days after the addition of tetracycline, the HC2S2 cells stopped proliferating, began to extend neurites, and expressed the neuronal markers tau, NeuN, neurofilament 200 kDa, and glutamic acid decarboxylase in accordance with the reduced production of the v-myc oncoprotein. Differentiated HC2S2 cells expressed large sodium and calcium currents and could fire regenerative action potentials. These results suggest that the suppression of the v-myc oncogene may be sufficient to make proliferating cells exit from cell cycles and induce terminal differentiation. The HC2S2 cells will be valuable for studying the differentiation process of neurons.