Terminal Differentiation Markers Research Articles

Myogenic differentiation depends on the interplay of Grb2 and N-WASP

Myogenesis requires a well-coordinated withdrawal from cell cycle, morphological changes and cell fusion mediated by actin cytoskeleton. Grb2 is an adaptor protein whose central SH2 domain binds to phosphorylated tyrosine residues of activated receptors and activates intracellular signaling pathway, while its N-terminal and C-terminal SH3 domains bind to proline rich proteins such as N-WASP (Neural-Wiskott Aldrich Syndrome Protein). We found that the expression of Grb2 was increased at the beginning of differentiation and remained constant during differentiation in C2C12 myoblasts. Knocking down endogenous Grb2 expression caused a significant increase in the fusion index and expression of MyHC, a terminal differentiation marker when compared with the control. Over expression of Grb2 in C2C12 (C2C12Grb2-Myc) reduced myotube formation and expression of MyHC. Similarly over expression of Grb2P49L-Myc (N-terminal SH3 domain mutant) or Grb2R86K-Myc (SH2 domain mutant) inhibited myogenic differentiation of C2C12 cells. However, the expression of Grb2P206L-Myc (C-terminal SH3 domain mutant) did not inhibit myotube formation and expression of MyHC. This suggests that the C-terminal SH3 domain of Grb2 is critical for the inhibition of myogenic differentiation. The C2C12Grb2-Myc cells have reduced phalloidin staining at late stages of differentiation. Expression of N-WASP in C2C12Grb2-Myc cells rescued the myogenic defect and increased phalloidin staining (increased F-actin) in these cells. Thus our results suggest that Grb2 is a negative regulator of myogenesis and reduces myogenic differentiation by inhibiting actin polymerization/remodeling through its C-terminal SH3 domain.

An expanded population of pathogenic regulatory T cells in giant cell arteritis is abrogated by IL-6 blockade therapy

ObjectivesRandomised-controlled trials have recently proven the efficacy of the interleukin (IL)-6 receptor antagonist tocilizumab (TCZ) in giant cell arteritis (GCA). However, the mechanism of action of IL-6 blockade in this...

Anti-CD19 ARTEMISTM Therapy Drastically Reduces Cytokine Release without Compromising Efficacy Against Preclinical Lymphoma Models

Anti-CD19 chimeric antigen receptor (CAR) T cell therapies for B cell malignancies have demonstrated the remarkable curative potential of T cell immunotherapies. However, in clinical trials anti-CD19-CAR T cells continue to trigger life threatening adverse events that are often associated with excessive cytokine release and excessive T-cell proliferation. We reasoned that the activation pathway of current CAR T cells could be altered to better regulate proliferation and cytokine secretion, and thus disentangle the correlation between cytokine release syndrome (CRS) and efficacy of T cell-based therapies.Through protein engineering, we developed the ARTEMISTM (1) signaling platform which when expressed on primary T-cells results in a dramatic reduction of cytokine release during tumor cell lysis, without sacrificing efficacy. Using a human phage display library, we also identified several human CD19 antibodies with improved specificity and affinity that will be less immunogenic as compared to the murine-derived anti-CD19 antibodies that are currently used in most trials. Our lead antibody clone CD19(7) was then engineered into both CD28z-CAR and ARTEMISTM platforms for comparison.When tested in vitro, both CD19(7)-ARTEMISTM T cells and CD19(7)-CD28z-CAR T cells specifically lysed multiple CD19+ leukemia and lymphoma cell lines with similar potencies. However, during the 16 hour killing assays, ARTEMIS™ T cells secreted over 1000-fold less IL-2 and dramatically lower levels of IFN-γ, GM-CSF, IL-10 and IL-6. ARTEMISTM T cells also accumulated less PD-1, LAG3, and TIM3 on their surface during culturing and following in vitro killing, indicating a diminished propensity for exhaustion. Furthermore, during in vitro T cell expansion, ARTEMISTM cells were enriched for naïve/central memory subpopulations, had lower expression of granzyme B, a marker of terminal differentiation, and had reduced rates of receptor internalization upon antigen engagement. These characteristics suggest that T-cells activated through the ARTEMISTM receptor will have improved persistence and long-term proliferation potential, as well as a safer, more controlled cytokine release when used for T-cell therapies.When tested in vivo against CD19+ Raji systematic lymphoma xenografts, intravenous administration of CD19(7)-ARTEMISTM T cells caused rapid, complete, and lasting tumor regression that was better than that achieved with an equal dose of CD19(7)-CD28z-CAR T cells (Figure 1). In agreement with our in vitro data, mice treated with ARTEMISTM T cells had nearly undetectable levels of cytokines in their blood at 24 hours post dosing, a time in which CD19(7)-CAR-treated mice had markedly elevated levels of human IFN-γ, IL-2, TNFα, and IL-10.While flow cytometry analysis of the peripheral blood showed that CD19(7)-CAR T cells expanded more rapidly in mice, CD19(7)-ARTEMISTM T cells better controlled Raji tumor growth and were negative for PD-1 expression which was high on circulating CAR T cells. At 7 weeks post dosing, a time when all ARTEMISTM T cell-treated mice had no detectable tumors, they were re-challenged with Raji lymphoma. While tumors grew rapidly in control mice, ARTEMISTM T cell-treated mice resisted the Raji lymphoma re-challenge, indicating that ARTEMISTM T cells persisted in these mice despite the absence of tumors and remained antigen-responsive (Figure 2).Our data demonstrates that CD19(7)-ARTEMISTM T cells are highly potent against lymphoma preclinical models while releasing drastically lower levels of cytokines. Thus we have developed and pre-clinically validated a novel fully human anti-CD19 T cell therapy that has the potential to persist longer in patients and, importantly, presents a lower risk of cytokine-related toxicities without compromising efficacy. A clinical trial testing CD19(7)-ARTEMISTM T cell therapy in humans is expected to begin in 2017. [Display omitted] [Display omitted] DisclosuresLiu:Eureka Therapeutics: Employment, Equity Ownership, Patents & Royalties. Long:Eureka Therapeutics: Employment, Equity Ownership. Green:Eureka Therapeutics: Employment. Horan:Eureka Therapeutics: Employment. Zimdahl:Eureka Therapeutics: Employment. Liu:Eureka Therapeutics: Employment, Equity Ownership, Patents & Royalties.

The New Immortalized Uroepithelial Cell Line HBLAK Contains Defined Genetic Aberrations Typical of Early Stage Urothelial Tumors.

Background: Cell culture models of normal urothelial cells are important for studying differentiation, disease mechanisms and anticancer drug development. Beyond primary cultures with their limitations in lifespan, interindividual heterogeneity and supply, few conditionally immortalized cell lines with limited applicability due to partial transformation or impaired differentiation capacity are available. We describe characteristics of the new spontaneously immortalized cell line HBLAK derived from a primary culture of uroepithelial cells.Objective: To characterize utility and limitations of HBLAK cells as an urothelial cell culture model.Methods: Differentiation markers were investigated by immunofluorescence and RT-PCR, genetic changes by standard karyotyping, array-CGH, PCR, RT-PCR and exome sequencing; expression of p53 and p21 by Western blotting.Results: HBLAK cells proliferated for >50 passages without senescing. They expressed cytokeratins of basal urothelial cells. Terminal differentiation markers appeared only after induction of differentiation by specific protocols. The karyotype was stable, with few chromosomal changes, especially gains of chromosomes 5 and 20 and a chromosome 9p21 deletion resulting in p16INK4A loss. A C228T TERT promoter mutation was present, but no other mutation typical of urothelial carcinoma. TP53 was wild-type and the cell cycle was arrested in response to genomic stress.Conclusions: HBLAK cells retain some differentiation potential and respond to cytotoxic agents similar to normal urothelial cells, but contain genetic changes contributing to immortalization in urothelial tumors. HBLAK may be valuable for evaluating the tumor specificity of novel cancer drugs, but may also be applied as an urothelial in vitro carcinogenesis model.

Absence of collagen IX accelerates hypertrophic differentiation in the embryonic mouse spine through a disturbance of the Ihh-PTHrP feedback loop

Collagen IX (Col IX) is a component of the cartilage extracellular matrix and contributes to its structural integrity. Polymorphisms in the genes encoding the Col IX ɑ2- and ɑ3-chains are associated with early onset of disc degeneration. Col IX-deficient mice already display changes in the spine at the newborn stage and premature disc degeneration starting at 6months of age. To determine the role of Col IX in early spine development and to identify molecular mechanisms underlying disc degeneration, the embryonic development of the spine was analyzed in Col IX -/- mice. Histological staining was used to show tissue morphology at different time points. Localization of extracellular matrix proteins as well as components of signaling pathways were analyzed by immunohistochemistry. Developing vertebral bodies of Col IX -/- mice were smaller and already appeared more compact at E12.5. At E15.5, vertebral bodies of Col IX -/- mice revealed an increased number of hypertrophic chondrocytes as well as enhanced staining for the terminal differentiation markers alkaline phosphatase and collagen X. This correlates with an imbalance in the Ihh-PTHrP signaling pathway at this time point, reflected by an increase of Ihh and a concomitant decrease of PTHrP expression. An accelerated hypertrophic differentiation caused by a disturbed Ihh-PTHrP signaling pathway may lead to a higher bone mineral density in the vertebral bodies of newborn Col IX -/- mice and, as a result, to the early onset of disc degeneration.

Foxc1 Ablated Mice Are Anhidrotic and Recapitulate Features of Human Miliaria Sweat Retention Disorder

Sweat glands are critical for thermoregulation. The single tubular structure of sweat glands has a lower secretory portion and an upper reabsorptive duct leading to the secretory pore in the skin. Genes that determine sweat gland structure and function are largely unidentified. Here we report that a Fox family transcription factor, Foxc1, is obligate for appreciable sweat duct activity in mice. When Foxc1 was specifically ablated in skin, sweat glands appeared mature, but the mice were severely hypohidrotic. Morphologic analysis revealed that sweat ducts were blocked by hyperkeratotic or parakeratotic plugs. Consequently, lumens in ducts and secretory portions were dilated, and blisters and papules formed on the skin surface in the knockout mice. The phenotype was strikingly similar to the human sweat retention disorder miliaria. We further show that Foxc1 deficiency ectopically induces the expression of keratinocyte terminal differentiation markers in the duct luminal cells, which most likely contribute to keratotic plug formation. Among those differentiation markers, we show that Sprr2a transcription is directly repressed by overexpressed Foxc1 in keratinocytes. In summary, Foxc1 regulates sweat duct luminal cell differentiation, and mutant mice mimic miliaria and provide a possible animal model for its study.

Aryl hydrocarbon receptor negatively regulates lipid synthesis and involves in cell differentiation of SZ95 sebocytes in vitro

The aryl hydrocarbon receptor (AhR) is the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), benzo(a)pyrene (BaP) and other exogenous compounds. In human sebocytes, TCDD and BaP were found to activate the expression of multiple genes, including cytochrome P450 1A1 (CYP1A1), and inhibit lipid synthesis via AhR, while little is known about endogenous functions of the AhR. In order to expand this knowledge, we analyzed the impact of AhR knockdown on lipid synthesis as well as on cell differentiation of SZ95 sebocytes in vitro and observed that lipid synthesis was significantly induced in AhR silenced SZ95 sebocytes. In line with this result, expression of lipogenesis-associated genes, such as peroxisome proliferator activated receptor (PPAR) δ and PPARγ, was increased. Morphological changes with smaller cells in size but more abundant cytoplasmic lipids were observed in AhR silenced SZ95 sebocytes compared with the AhR activated cells. Besides, the expression of keratin 7, an early sebaceous differentiation marker, was increased, while the expression of the terminal sebocyte differentiation marker epithelial membrane antigen (EMA) was reduced. Moreover, the terminal keratinocyte differentiation markers keratin 10 and involucrin, and the AhR downstream protein CYP1A1 were reduced after AhR silencing. To the best of our knowledge, we provide evidence that in the absence of exogenous ligands, the AhR inhibits lipid synthesis and involves in cell differentiation of human SZ95 sebocytes, which indicates the physiological function of this receptor in human sebocytes.

TLR3 activation induces S100A7 to regulate keratinocyte differentiation after skin injury

Human S100A7 (psoriasin) is highly expressed in psoriasis and other inflammatory diseases; however, the function of S100A7 in wound repair remains largely unknown. Here we demonstrated that skin injury increased the expression of S100A7. Damaged cells from wounded skin induced the expression of S100A7 via the activation of Toll-like receptor 3 (TLR3) followed by the activation of p38 MAPK. S100A7, in turn, acted on keratinocytes to induce the expression of terminal differentiation marker gene loricrin through the activation of p38 MAPK and caspase-1. The differentiation of keratinocytes induced by S100A7 resulted in skin stratification, thus efficiently promoting wound closure. Taken together, our results demonstrate that the activation of TLR3 accelerates wound closure via the induction of S100A7 to induce keratinocyte differentiation. These findings also provide new insights into the development of different forms of treatment with skin wounds.

Deficient Neutrophil Extracellular Trap Formation in Patients Undergoing Bone Marrow Transplantation.

Overwhelming infection causes significant morbidity and mortality among patients treated with bone marrow transplantation (BMT) for primary immune deficiencies, syndromes of bone marrow failure, or cancer. The polymorphonuclear leukocyte (PMN; neutrophil) is the first responder to microbial invasion and acts within the innate immune system to contain and clear infections. PMNs contain, and possibly clear, infections in part by forming neutrophil extracellular traps (NETs). NETs are extensive lattices of extracellular DNA and decondensed chromatin decorated with antimicrobial proteins and degradative enzymes, such as histones, myeloperoxidase, and neutrophil elastase. They trap and contain microbes, including bacteria and fungi, and may directly affect extracellular microbial killing. Whether or not deficient NET formation contributes to the increased risk for overwhelming infection in patients undergoing BMT remains incompletely characterized, especially in the pediatric population. We examined NET formation in vitro in PMNs isolated from 24 patients who had undergone BMT for 13 different clinical indications. For these 24 study participants, the median age was 7 years. For 6 of the 24 patients, we examined NET formation by PMNs isolated from serial, peripheral blood samples drawn at three different clinical time points: pre-BMT, pre-engraftment, and post-engraftment. We found decreased NET formation by PMNs isolated from patients prior to BMT and during the pre-engraftment and post-engraftment phases, with decreased NET formation compared with healthy control PMNs detected even out to 199 days after their BMT. This decrease in NET formation after BMT did not result from neutrophil developmental immaturity as we demonstrated that >80% of the PMNs tested using flow cytometry expressed both CD10 and CD16 as markers of terminal differentiation along the neutrophilic lineage. These pilot study results mandate further exploration regarding the mechanisms or factors regulating NET formation by PMNs in patients at risk for overwhelming infection following BMT.

Dysfunctional phenotypes of CD4+ and CD8+ T cells are comparable in patients initiating ART during early or chronic HIV-1 infection.

Early initiation of antiretroviral therapy (ART) is becoming a common clinical practice according to current guidelines recommending treatment to all HIV-1-infected patients. However, it is not known whether ART initiated during the early phase of infection prevents the establishment of abnormal phenotypic features previously reported in CD4+ and CD8+T cells during chronic HIV-1 infection. In this cross-sectional study, blood specimens were obtained from 17 HIV-1-infected patients who began ART treatment shortly after infection (early ART [EA]), 17 age-matched HIV-1-infected patients who started ART during chronic phase of infection (late ART [LA]), and 25 age-matched non-HIV-1-infected controls. At collection of specimens, patients in EA and LA groups had received ART for comparable periods of time. Total HIV-1 DNA was measured in white blood cells by quantitative PCR. The concentration of 9 inflammatory parameters and 1 marker of fibrosis, including sCD14 and β-2 microglobulin, was measured in plasma. Furthermore, expression of markers of abnormal immune activation (human leukocyte antigen - antigen D related [HLA-DR] and CD38), exhaustion (programmed death 1, CD28, CD57) and terminal differentiation (CD127) was measured on CD4+ and CD8+T cells. T-cell proliferation was measured through Ki67 expression. The copies of total HIV-1 DNA in blood were significantly lower (P = 0.009) in EA compared with that in LA group. Only the expression of HLA-DR on naïve CD4+ T cells distinguished EA from LA, whereas expression of 3 surface markers distinguished T-cell populations of HIV-1-infected patients from controls. These included HLA-DR distinguishing CD4+ T cells from EA compared with controls, and also CD38 and CD127 on CD4+ and CD8+ T cells, respectively, distinguishing both groups of patients from controls. The sCD14 levels were significantly higher in EA patients, and β-2 microglobulin levels were higher in LA group compared with that in controls. Our results demonstrate an equivalent abnormal expression of activation (HLA-DR and CD38 on CD4+ T cells) and terminal differentiation (CD127 on CD8+ T cells) markers in T cells from both EA and LA patients. The size of total HIV-1 DNA copies in blood of EA was lower compared with LA patients. These findings suggest that some abnormalities taking place in the T-cell compartment during primary HIV-1 infection may not be corrected by early ART.

682 Foxc1 ablated mice are anhidrotic and recapitulate features of a sweat retention syndrome, miliaria

Sweat glands play a critical role in thermoregulation. The single tubular structure of sweat glands has a lower secretory portion and an upper reabsorptive duct leading to the secretory pore in the skin. The transcription factors that determine sweat gland structure and function are largely unidentified. Here we report that a Fox family transcription factor, Foxc1, is obligate for appreciable sweat duct activity in mice. When Foxc1 was specifically ablated in skin, sweat glands appeared mature, but the mice were severely hypohidrotic. Morphological analysis revealed that sweat ducts were blocked by hyperkeratotic or parakeratotic plugs. Consequently, lumens in ducts and secretory portions were dilated, and blisters and papules formed on the skin surface in the knockout mice. The phenotype was strikingly similar to the human sweat retention syndrome miliaria. We further found that Foxc1 deficiency ectopically induced the expression of keratinocyte terminal differentiation markers in the luminal duct cells, which most likely contributed to keratotic plug formation. In summary, Foxc1 regulates sweat duct luminal cell differentiation, and mutant mice may provide a model for miliaria.

Expression of the semicarbazide-sensitive amine oxidase in articular cartilage: its role in terminal differentiation of chondrocytes in rat and human

Semicarbazide-sensitive amine oxidase (SSAO) catalyzes the oxidation of primary amines into ammonia and reactive species (hydrogen peroxide, aldehydes). It is highly expressed in mammalian tissues, especially in vascular smooth muscle cells and adipocytes, where it plays a role in cell differentiation and glucose transport. The study aims at characterizing the expression and the activity of SSAO in rat and human articular cartilage of the knee, and to investigate its potential role in chondrocyte terminal differentiation. SSAO expression was examined by immunohistochemistry and western blot. Enzyme activity was measured using radiolabeled benzylamine as a substrate. Primary cell cultures of rat chondrocytes were treated for 21 days by a specific SSAO inhibitor, LJP 1586. Terminal chondrocyte differentiation markers were quantified by RT-qPCR. The basal and IL1β-stimulated glucose transport was monitored by the entrance of (3)[H]2-deoxyglucose in chondrocytes. SSAO was expressed in chondrocytes of rat and human articular cartilage. SSAO expression was significantly enhanced during the hypertrophic differentiation of chondrocytes characterized by an increase in MMP13 and in alkaline phosphatase (ALP) expressions. SSAO inhibition delayed the late stage of chondrocyte differentiation without cell survival alteration and diminished the basal and IL1β-stimulated glucose transport. Interestingly, SSAO activity was strongly increased in human osteoarthritic cartilage. SSAO was expressed as an active form in rat and human cartilage. The results suggest the involvement of SSAO in rat chondrocyte terminal differentiation via a modulation of the glucose transport. In man, the increased SSAO activity detected in osteoarthritic patients may trigger hypertrophy and cartilage degeneration.

Single Low-Dose Radiation Induced Regulation of Keratinocyte Differentiation in Calcium-Induced HaCaT Cells.

BackgroundWe are continually exposed to low-dose radiation (LDR) in the range 0.1 Gy from natural sources, medical devices, nuclear energy plants, and other industrial sources of ionizing radiation. There are three models for the biological mechanism of LDR: the linear no-threshold model, the hormetic model, and the threshold model.ObjectiveWe used keratinocytes as a model system to investigate the molecular genetic effects of LDR on epidermal cell differentiation.MethodsTo identify keratinocyte differentiation, we performed western blots using a specific antibody for involucrin, which is a precursor protein of the keratinocyte cornified envelope and a marker for keratinocyte terminal differentiation. We also performed quantitative polymerase chain reaction. We examined whether LDR induces changes in involucrin messenger RNA (mRNA) and protein levels in calcium-induced keratinocyte differentiation.ResultsExposure of HaCaT cells to LDR (0.1 Gy) induced p21 expression. p21 is a key regulator that induces growth arrest and represses stemness, which accelerates keratinocyte differentiation. We correlated involucrin expression with keratinocyte differentiation, and examined the effects of LDR on involucrin levels and keratinocyte development. LDR significantly increased involucrin mRNA and protein levels during calcium-induced keratinocyte differentiation.ConclusionThese studies provide new evidence for the biological role of LDR, and identify the potential to utilize LDR to regulate or induce keratinocyte differentiation.

Exercise, Free Radical Metabolism, and Aging: Cellular and Molecular Processes

The human aging process is associated with a gradual and cumulative decline in the normal functioning of all major bodily systems. While life expectancy is increasing at an exponential rate, the length of time spent in good health across the lifespan (health-span) is in decline. Exercise and physical activity are widely regarded as important interventions to increase longevity and promote healthy aging and well-being. However, cell damage produced by acute and unaccustomed exercise, as a result of an enhanced production of free radical species, is a recognised phenomenon. While all physiological systems appear responsive to the beneficial effects of exercise, further research is required to understand the relationship between free radical metabolism in cellular and molecular processes affected by exercise in the context of human aging. This special issue contains both review and original research articles which, when combined together, provide an interesting insight into the current state of basic and applied research in free radical metabolism, exercise, and aging. In their review, O. F. Araneda et al. discuss how environmental factors, such as altitude and pollution, as well as different forms of exercise, intensity, and duration, affect pulmonary inflammation and oxidative imbalance. They review literature pertaining to antioxidants as well as prooxidants, in relation to oxidative damage, and they further discuss mediators of the inflammatory response to exercise. Importantly, they investigate the relationship between oxidative/inflammatory variables and the interaction between them. D. J. Flis et al. used a prolonged swimming model in rats to investigate mitochondrial swelling and changes in cholesterol in skeletal muscle and liver. A reduction in mitochondrial cholesterol following exercise is related to the inhibition of mitochondrial swelling, and it was also observed within this study that there was an increase in caveolin-1 concentration in muscle. Although markers of oxidative stress in mitochondria were enhanced, there were no similar changes apparent in liver. The work points to a possible adaptive role of caveolin-1 in rat muscle mitochondria, as a consequence of exercise stress. In the second review of this issue, M. Ross et al. discuss the increased risk of developing cardiovascular disease concurrent with increasing age, with respect to increased and prolonged exposure to oxidative stress. The positive and protective effect of regular exercise, in promoting endothelial homeostasis, which may offset the “vascular ageing” process, is further reviewed. It is suggested that acute strenuous exercise causes changes to the oxidant/antioxidant ratio and tissue inflammation and, in their contribution to this issue, H. Li et al. add to the literature in this area with their report on the acute effect of exercise and the inflammatory response in the myocardium of rats. The rats were exercised using a treadmill running protocol, and they observed a regulatory adaptation of mitochondrial function in rat myocardium in response to acute heavy exercise. Their work further demonstrates that myocardial injury in rats is minimized via a cascade activation of mitophagy, responding to myocardial inflammation, as a result of mitochondrial stress triggered during acute exercise. In the final research article of the issue, A. B. Bigley et al. focus on age, with regard to the effect of latent cytomegalovirus on natural killer cell phenotype and exercise responsiveness in the human model. In response to an acute bout of exercise, they show an increased mobilization of NK-cells, which is thought to be an important component of the “fight or flight” response. They also observed that this response is mediated by age, in reporting an increased proportion of NK-cells expressing the terminal differentiation marker CD57 with advancing years. Additionally, NK-cell mediated immunosurveillance following exercise was reported to be compromised by CMC serostatus irrespective of age. Although there is certainly great diversity in the papers presented in this special issue, we hope the reader will find the contributions both interesting and stimulating. We suggest that this collection of papers will serve to enrich our understanding and knowledge of the relationship between free radical metabolism in cellular and molecular processes affected by exercise in the context of aging. Geraint D. Florida-James Rickie Simpson Gareth Davison Graeme Close

Uroplakin 1b is critical in urinary tract development and urothelial differentiation and homeostasis

Proper development and maintenance of urothelium is critical to its function. Uroplakins are expressed in developing and mature urothelium where they establish plaques associated with the permeability barrier. Their precise functional role in development and disease is unknown. Here, we disrupted Upk1b invivo where its loss resulted in urothelial plaque disruption in the bladder and kidney. Upk1b(RFP/RFP) bladder urothelium appeared dysplastic with expansion of the progenitor cell markers, Krt14 and Krt5, increased Shh expression, and loss of terminal differentiation markers Krt20 and uroplakins. Upk1b(RFP/RFP) renal urothelium became stratified with altered cellular composition. Upk1b(RFP/RFP) mice developed age-dependent progressive hydronephrosis. Interestingly, 16% of Upk1b(RFP/RFP) mice possessed unilateral duplex kidneys. Our study expands the role of uroplakins, mechanistically links plaque formation to urinary tract development and function, and provides a tantalizing connection between congenital anomalies of the kidney and urinary tract along with functional deficits observed in a variety of urinary tract diseases. Thus, kidney and bladder urothelium are regionally distinct and remain highly plastic, capable of expansion through tissue-specific progenitor populations. Furthermore, Upk1b plays a previously unknown role in early kidney development representing a novel genetic target for congenital anomalies of the kidney and urinary tract.

Pseudo-immortalization of postnatal cochlear progenitor cells yields a scalable cell line capable of transcriptionally regulating mature hair cell genes.

The mammalian cochlea is a highly specialized organ within the inner ear. Sensory hair cells (HC) in the cochlea detect and transduce sound waves into electrical impulses that are sent to the brain. Studies of the molecular pathways regulating HC formation are hindered by the very sparse nature of HCs, where only ~3300 are found within an entire mouse cochlea. Current cell lines mimic certain aspects of HCs but lack terminal HC marker expression. Here we successfully “pseudo-immortalized” cochlear progenitor cells using the “conditional reprogramming” technique. These cells, termed “Conditionally Reprogrammed Otic Stem Cells” (CR-OSC), are able to bypass the senescence inherent to cochlear progenitor cells without genetic alterations, allowing for the generation of over 15 million cells from a single cochlea. These cells can be differentiated and up-regulate both early and terminal differentiation genes associated with HCs, including the terminal HC differentiation marker prestin. CR-OSCs also respond to known HC cues, including upregulation of HC genes in response to Atoh1 overexpression, and upregulation of prestin expression after thyroid hormone application. Overall, we describe the creation of a HC line capable of regulated expression of HC genes that can easily be recreated in any laboratory from any mouse of interest.

RASA3 Is Involved in Cell Cycle Progression, Hemoglobinization and Generation of Reactive Oxygen Species during Mammalian Erythropoiesis

RASA3, a Ras GTPase activating protein, is critical to vertebrate erythropoiesis and megakaryopoiesis. Defective RASA3 in zebrafish and mice results in severe anemia and thrombocytopenia. Indeed, in the mouse model scat (severe combined anemia and thrombocytopenia), a G125V mutation in Rasa3 leads to profound bone marrow failure with characteristics of aplastic anemia. The phenotype is cyclic, and mice alternate between periods of crisis and remission. Previous studies showed that this mutation in Rasa3 causes severely delayed erythroid differentiation at the polychromatophilic stage and decreased hemoglobinization due, at least in part, to mislocalization of RASA3 to the cytosol and resultant increased Ras activity. Here, we provide evidence that RASA3 plays a pivotal role in cell cycle progression and maintenance of reactive oxygen species (ROS) levels in erythropoiesis during crisis episodes. First, we analyzed the cell cycle progression in scat erythroblasts during crisis episodes. Using propidium iodide and flow cytometry, we found a significant increase in the G0/G1 phase (46.8% ± 3.1% in crisis vs 34.8% ± 2.5 in controls, p<0.001) while S phase was decreased (40.2% ± 2.9% vs. 49.6% ± 3.3% p<0.01) and G2 was not affected in scat proerythroblasts. These data suggest that Rasa3 is involved in the G1 checkpoint. In addition, we observed an increase in the ROS levels in scat throughout differentiation including proerythroblasts (1.03% ± 0.3% vs. 0.25% ± 0.1% in controls, p < 0.01), EryA (1.5% ± 0.21% vs. 0.25% ± 0.05%, p < 0.01), EryB (2.6% ± 1.41% vs. 0.25% ± 0.01%, p < 0.01), EryC (2.0% ± 0.45% vs. 0.25% ± 0.02%, p < 0.01) and mature red cells (1.15% ± 0.1% vs. 0.9% ± 0.03%, p < 0.05); the highest levels of ROS were observed in the basophilic-polychromatic erythroblast (i.e. EryB) containing populations. In addition, we consistently found increased levels of ROS in both reticulocytes and red blood cells from scat peripheral blood. Therefore, the cell cycle arrest and increased ROS likely contribute to the erythropoietic defect associated with scat. No mutations involving RASA3 have been involved in human bone marrow failure syndromes yet. However, in more than 30% of the cases, the etiology remains unexplained. To understand the putative role of RASA3 in the pathophysiology of hematopoiesis, and based on our data obtained in the mouse and the zebrafish, we investigated the role of RASA3 in human erythropoiesis using si and shRNA knockdowns in cord blood-derived CD34+ cells. Knockdown efficiencies were evaluated by western blot, and revealed that RASA3 protein levels were reduced by greater than 50% using two different shRNA constructs and siRNA. Cells were differentiated using an adapted 3-phase liquid culture system that fully recapitulates erythropoiesis. Similar to the scat mouse, we observed delayed differentiation by flow cytometry using glycophorin A, band3 and α4-integrin as markers of terminal differentiation with 62.0% of RASA3 knockdown erythroblasts α4-integrinhi (compared to 27.5% in controls) at day 16. In addition, the nucleus was substantially less pyknotic and the nucleocytoplasmic ratio remained elevated during differentiation, as compared to control cells. Indeed, 64 ± 7% of the cells had an uncondensed nucleus in the RASA3 knockdown samples, compared to 30 ± 3% in the scramble controls. Moreover as in scat, hemoglobinization was defective; qRT-PCR analyses revealed a 25% reduction in γ-globin and a 20% reduction in β-globin expression. These findings strengthen and emphasize the notion that RASA3 has a conserved function during vertebrate terminal erythropoiesis. Experiments investigating cell cycle progression, apoptosis and ROS production in human CD34+ cells are underway. Together, our studies demonstrate that RASA3 plays and important role in hemoglobinization, cell cycle progression and cell survival during terminal erythroid differentiation. DisclosuresNo relevant conflicts of interest to declare.

GM-CSF treatment prevents respiratory syncytial virus–induced pulmonary exacerbation responses in postallergic mice by stimulating alveolar macrophage maturation

Human respiratory syncytial virus (RSV) is a frequent cause of asthma exacerbations, yet the susceptibility of asthmatic patients to RSV is poorly understood. We sought to address the contribution of resident alveolar macrophages (rAMs) to susceptibility to RSV infection in mice that recovered from allergic airway eosinophilia. Mice were infected with RSV virus after clearance of allergic airway inflammation (AAI). The contribution of post-AAI rAMs was studied invivo by means of clodronate liposome-mediated depletion, adoptive transfer, and treatment with recombinant cytokines before RSV infection. After clearing the allergic bronchial inflammation, post-AAI mice had bronchial hyperreactivity and increased inflammatory cell influx when infected with RSV compared with nonallergic mice, whereas viral clearance was comparable in both mouse groups. Post-AAI rAMs were necessary and sufficient for mediating these proinflammatory effects. In post-AAI mice the residing CD11c(hi) autofluorescent rAM population did not upregulate the terminal differentiation marker sialic acid-binding immunoglobulin-like lectin F and overproduced TNF and IL-6 through increased nuclear factor κB nuclear translocation. In line with these results, post-AAI lungs had reduced levels of the rAM maturation cytokine GM-CSF. Intratracheal administration of GM-CSF induced final rAM maturation in post-AAI mice and prevented the increased susceptibility to RSV-induced hyperreactivity and inflammation. Defective production of GM-CSF leads to insufficient post-AAI rAM maturation in mice that recovered from an AAI, causing increased susceptibility to RSV-induced immunopathology. Promoting the differentiation of post-AAI rAMs might be a therapeutic option for preventing RSV-induced exacerbations in human asthmatic patients.

Reversal of Pathological Features of Graves' Orbitopathy by Activation of Forkhead Transcription Factors, FOXOs.

Graves' orbitopathy (GO) is a disfiguring/distressing, inflammatory autoimmune condition. This intractable problem is caused by expansion of the orbital contents around the eye by excessive fat generation (adipogenesis) and overproduction of extracellular matrix components, especially hyaluronan (HA) from preadipocytes/fibroblasts (PFs). Current immunosuppressive/antiinflammatory treatments are largely ineffective and have unpleasant side effects, and a better therapeutic strategy through understanding GO-associated pathological features is needed. Previously we identified depot-specific HA synthase 2 regulation (HAS2; major source of HA), which facilitates orbit-specific HA accumulation during adipogenesis, and targeting phosphatidylinositol-3-kinase/mechanistic target of rapamycin-complex-1 pathways blocked both pathological features. The current study revealed low expression levels of Forkhead box O (FOXOs; critical downstream effectors of phosphatidylinositol-3-kinase) in orbital PFs through adipogenesis compared with sc levels. We aimed to dissect the role of FOXOs in GO pathogenesis to identify nonimmunosuppressive targets for GO treatment. Human orbital and sc primary PFs were treated with small interfering RNA/chemical inhibitor (AS1842856) of FOXOs or FOXO enhancer trifluoperazine hydrochloride (TFP; Food and Drug Administration approved drug), in serum-free medium for 24 hours, or TFP treatment in adipogenic medium for 15 days. Quantitative PCR was used to measure HAS2 transcripts and the terminal adipogenesis differentiation marker lipoprotein lipase. HA accumulation in the medium was measured by an ELISA. Substantially increased or decreased HAS2/HA production was observed by inhibiting (small interfering RNA or chemical inhibitor) or enhancing (TFP) FOXO expression, respectively. TFP treatment is also sufficient to counteract thyrotropin receptor-activated HAS2/HA production and block adipogenesis in orbital PFs. FOXOs play a crucial repressor role in the regulation of HAS2/HA production and adipogenesis in orbital PFs. Our data reveal for the first time that resetting GO-associated pathological features through drug-targeted activation of FOXOs could provide a feasible nonimmunosuppressive therapeutic strategy for GO.

Imaging Inflammation in Cerebrovascular Disease.

Imaging inflammation in large intracranial artery pathology may play an important role in the diagnosis of and risk stratification for a variety of cerebrovascular diseases. Looking beyond the lumen has already generated widespread excitement in the stroke community, and the potential to unveil molecular processes in the vessel wall is a natural evolution to develop a more comprehensive understanding of the pathogenesis of diseases, such as ICAD and brain aneurysms.