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Critical Regulators (critical + regulator)
Selected AbstractsDecoding epithelial signals: critical role for the epidermal growth factor receptor in controlling intestinal transport functionACTA PHYSIOLOGICA, Issue 1 2009D. F. McCole Abstract The intestinal epithelium engages in bidirectional transport of fluid and electrolytes to subserve the physiological processes of nutrient digestion and absorption, as well as the elimination of wastes, without excessive losses of bodily fluids that would lead to dehydration. The overall processes of intestinal ion transport, which in turn drive the secretion or absorption of water, are accordingly carefully regulated. We and others have identified the epidermal growth factor receptor (EGFr) as a critical regulator of mammalian intestinal ion transport. In this article, we focus on our studies that have uncovered the intricate signalling mechanisms downstream of EGFr that regulate both chloride secretion and sodium absorption by colonocytes. Emphasis will be placed on the EGFr-associated regulatory pathways that dictate the precise outcome to receptor activation in response to signals that may seem, on their face, to be quite similar if not identical. The concepts to be discussed underlie the ability of the intestinal epithelium to utilize a limited set of signalling effectors to produce a variety of outcomes suitable for varying physiological and pathophysiological demands. Our findings therefore are relevant not only to basic biological principles, but also may ultimately point to new therapeutic targets in intestinal diseases where ion transport is abnormal. [source] Role of histone and transcription factor acetylation in diabetes pathogenesisDIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 5 2005Steven G. Gray Abstract Globally, diabetes (and, in particular, type 2 diabetes) represents a major challenge to world health. Currently in the United States, the costs of treating diabetes and its associated complications exceed $100 billion annually, and this figure is expected to soar in the near future. Despite decades of intense research efforts, the genetic basis of the events involved in the pathogenesis of diabetes is still poorly understood. Diabetes is a complex multigenic syndrome primarily due to beta-cell dysfunction associated with a variable degree of insulin resistance. Recent advances have led to exciting new developments with regard to our understanding of the mechanisms that regulate insulin transcription. These include data that implicate chromatin as a critical regulator of this event. The ,Histone Code' is a widely accepted hypothesis, whereby sequential modifications to the histones in chromatin lead to regulated transcription of genes. One of the modifications used in the histone code is acetylation. This is probably the best characterized modification of histones, which is carried out under the control of histone acetyltransferases (HATs) and histone deacetylases (HDACs). These enzymes also regulate the activity of a number of transcription factors through acetylation. Increasing evidence links possible dysregulation of these mechanisms in the pathogenesis of diabetes, with important therapeutic implications. Copyright © 2005 John Wiley & Sons, Ltd. [source] p38 MAPK is a critical regulator of the constitutive and the ,4,integrin-regulated expression of IL-6 in human normal thymic epithelial cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2003Fabrizio Mainiero Abstract Cytokines and adhesion receptors are key mediators in the dialog occurring between thymic epithelial cells (TEC) and thymocytes and regulating T,cell maturation and epithelial embryonic differentiation. Among cytokines, IL-6 can be critical in the thymus, fostering proliferation, differentiation and/or survival of both TEC and thymocytes. We have previously reported in human normal TEC that clustering of the laminin receptor ,6,4 integrin induced by thymocyte contact or monoclonal antibody-mediated cross-linking regulates IL-6 gene expression via activation of NF-,B and NF-IL6 transactivators. Here we show that ,6,4 integrin activates p38 mitogen-activated protein kinase (MAPK) and that p38 is essential for IL-6 gene expression. In fact, ,4 cross-linking activated p38 and extracellular signal-regulated kinase (ERK) MAPK, Rac1, p21-activated protein kinase,1 (PAK1) and MAPK kinases (MKK),3/MKK6. However, pharmacological blockade of p38 or ERK demonstrated that p38 inhibition abrogated both basal and ,4,integrin-induced production of IL-6 preventing NF-,B and NF-IL6 activation, whereas ERK inhibition reduced IL-6 production, hampering only NF-,B activation. Overall, our results indicate that p38 MAPK and ,6,4,integrin, expressed by TEC throughout their life, are critical regulators of the intrathymic availability of a cytokine controlling fate and functions of cells governing development and maintenance of thymic architecture and immune responses. [source] Regulation of NMDA receptor trafficking and function by striatal-enriched tyrosine phosphatase (STEP)EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2006Steven P. Braithwaite Abstract Regulation of N -methyl- d -aspartate (NMDA) receptors is critical for the normal functioning of the central nervous system. There must be precise mechanisms to allow for changes in receptor function required for learning and normal synaptic transmission, but within tight constraints to prevent pathology. Tyrosine phosphorylation is a major means by which NMDA receptors are regulated through the equilibrium between activity of Src family kinases and tyrosine phosphatases. Identification of NMDA receptor phosphatases has been difficult, the best candidate being striatal-enriched tyrosine phosphatase (STEP). Here we demonstrate that STEP is a critical regulator of NMDA receptors and reveal that the action of this tyrosine phosphatase controls the constitutive trafficking of NMDA receptors and leads to changes in NMDA receptor activity at the neuronal surface. We show that STEP binds directly to NMDA receptors in the absence of other synaptic proteins. The activity of STEP selectively affects the expression of NMDA receptors at the neuronal plasma membrane. The result of STEP's action upon the NMDA receptor affects the functional properties of the receptor and its downstream signaling. These effects are evident when STEP levels are chronically reduced, indicating that there is no redundancy amongst phosphatases to compensate for altered STEP function in the CNS. STEP may have evolved specifically to fill a pivotal role as the NMDA receptor phosphatase, having a distinct and restricted localization and compartmentalization, and unique activity towards the NMDA receptor and its signaling pathway. [source] Cell culture analysis of the regulatory frameshift event required for the expression of mammalian antizymesGENES TO CELLS, Issue 11 2001Michael T. Howard Background Antizyme is a critical regulator of cellular polyamine levels due to its effect on polyamine transport and its ability to target ornithine decarboxylase for degradation. Antizyme expression is autoregulatory, through dependence on an unusual +1 translational frameshift mechanism that responds to polyamine levels. Results HEK293 cells were depleted of polyamines by treatment with an ornithine decarboxylase inhibitor, difluoromethylornithine (DFMO), and grown in the presence or absence of exogenous polyamines prior to the analysis of ribosomal frameshifting levels. Results obtained using an optimized dual luciferase assay system reveal a 10-fold dynamic range of frameshifting, which correlates positively with polyamine addition. Polyamine addition to cells, which have not been pre-treated with DFMO, also resulted in an increase in antizyme frameshifting but to a lesser degree (1.3 to 1.5-fold). In addition, the constructs with the 3, deletion were more responsive to stimulation by polyamine addition than those with the 5, deletion. Conclusions The observed regulation of antizyme frameshifting demonstrates the efficiency of a polyamine homeostatic mechanism, and illustrates the utility of a quantifiable cell-based assay for the analysis of polyamines or their analogues on translational frameshifting. [source] PRDX4, a member of the peroxiredoxin family, is fused to AML1 (RUNX1) in an acute myeloid leukemia patient with a t(X;21)(p22;q22)GENES, CHROMOSOMES AND CANCER, Issue 4 2004Yanming Zhang The AML1 gene (also known as RUNX1) at 21q22 codes for core binding factor (CBF) ,, which forms a heterodimer with CBF , that acts as a transcriptional activating factor. CBF is a critical regulator in the generation and differentiation of definitive hematopoietic stem cells and is frequently disrupted in leukemia through chromosome translocations. We cloned a novel AML1 partner gene, PRDX4, in an X;21 translocation in a 74-year-old male patient diagnosed with acute myeloid leukemia,M2. Chromosome analysis detected a t(X;21)(p22;q22) as the sole abnormality in bone marrow samples. The involvement of AML1 was confirmed by fluorescence in situ hybridization studies. Using 3, RACE-PCR, we cloned a fusion between exon 5 of AML1 and exon 2 of PRDX4. RT-PCR confirmed the fusion and detected another fusion between exon 6 of AML1 and exon 2 of PRDX4, indicating alternative splicing of exon 6 of AML1 in the fusion transcripts. PRDX4 is one of six peroxiredoxin-family genes that are highly conserved in eukaryotes and prokaryotes and are ubiquitously expressed. Peroxiredoxin genes exhibit thioredoxin-dependent peroxidase activity and have been implicated in a number of other cellular functions such as cell proliferation and differentiation. PRDX4 plays a regulatory role in the activation of the transcription factor NF-,B and is significantly down-regulated in acute promyelocytic leukemia. This is the first example of antioxidant enzyme involvement in a chromosome translocation in leukemia. © 2004 Wiley-Liss, Inc. [source] Associations between carbon isotope ratios of ecosystem respiration, water availability and canopy conductanceGLOBAL CHANGE BIOLOGY, Issue 10 2004N. G. McDowell Abstract We tested the hypothesis that the stable carbon isotope signature of ecosystem respiration (,13CR) was regulated by canopy conductance (Gc) using weekly Keeling plots (n=51) from a semiarid old-growth ponderosa pine (Pinus ponderosa) forest in Oregon, USA. For a comparison of forests in two contrasting climates we also evaluated trends in ,13CR from a wet 20-year-old Douglas-fir (Pseudotsuga menziesii) plantation located near the Pacific Ocean. Intraannual variability in ,13CR was greater than 8.0, at both sites, was highest during autumn, winter, and spring when rainfall was abundant, and lowest during summer drought. The ,13CR of the dry pine forest was consistently more positive than the wetter Douglas-fir forest (mean annual ,13CR: ,25.41, vs. ,26.23,, respectively, P=0.07). At the Douglas-fir forest, ,13CR,climate relationships were consistent with predictions based on stomatal regulation of carbon isotope discrimination (,). Soil water content (SWC) and vapor pressure deficit (vpd) were the most important factors governing ,13CR in this forest throughout the year. In contrast, ,13CR at the pine forest was relatively insensitive to SWC or vpd, and exhibited a smaller drought-related enrichment (,2,) than the enrichment observed during drought at the Douglas-fir forest (,5,). Groundwater access at the pine forest may buffer canopy,gas exchange from drought. Despite this potential buffering, ,13CR at the pine forest was significantly but weakly related to canopy conductance (Gc), suggesting that ,13CR remains coupled to canopy,gas exchange despite groundwater access. During drought, ,13CR was strongly correlated with soil temperature at both forests. The hypothesis that canopy-level physiology is a critical regulator of ,13CR was supported; however, belowground respiration may become more important during rain-free periods. [source] NKX2-1 mutations leading to surfactant protein promoter dysregulation cause interstitial lung disease in "Brain-Lung-Thyroid Syndrome",HUMAN MUTATION, Issue 2 2010Loïc Guillot Abstract NKX2-1 (NK2 homeobox 1) is a critical regulator of transcription for the surfactant protein (SP)-B and -C genes (SFTPB and SFTPC, respectively). We identified and functionally characterized two new de novo NKX2-1 mutations c.493C>T (p.R165W) and c.786_787del2 (p.L263fs) in infants with closely similar severe interstitial lung disease (ILD), hypotonia, and congenital hypothyroidism. Functional analyses using A549 and HeLa cells revealed that NKX2-1-p.L263fs induced neither SFTPB nor SFTPC promoter activation and had a dominant negative effect on wild-type (WT) NKX2-1. In contrast,NKX2-1-p.R165W activated SFTPC, to a significantly greater extent than did WTNKX2-1,whileSFTPB activation was only significantly reduced in HeLa cells. In accordance with our in vitro data, we found decreased amounts of SP-B and SP-C by western blot in bronchoalveolar lavage fluid (patient with p.L263fs) and features of altered surfactant protein metabolism on lung histology (patient with NKX2-1-p.R165W). In conclusion, ILD in patients with NKX2-1 mutations was associated with altered surfactant protein metabolism, and both gain and loss of function of the mutated NKX2-1 genes on surfactant protein promoters were associated with ILD in "Brain-Lung-Thyroid syndrome". © 2009 Wiley-Liss, Inc. [source] Transcription factors that regulate memory in humoral responsesIMMUNOLOGICAL REVIEWS, Issue 1 2006Kathryn Calame Summary:, At least three types of B lymphocytes are important for providing memory in a humoral immune response: ,classical' memory cells that do not secrete immunoglobulin (Ig), long-lived plasma cells (LLPCs) in the bone marrow, and ,innate-like' B-1 cells. In this review, our work on B-lymphocyte-induced maturation protein-1 (Blimp-1), a critical regulator of terminal B-cell differentiation, is discussed in the context of current knowledge of all transcriptional controls that regulate these three types of B cells. Blimp-1 is not required for formation of memory cells, but it is required for them to progress toward becoming plasma cells. Blimp-1 is required for Ig secretion in plasma cells and in B-1 cells. Induction of the activator X-box-binding protein-1 and formation of µ-secreted mRNA depend on Blimp-1 in both cell types. Finally, even after their formation, LLPCs in the bone marrow continue to require Blimp-1 for their maintenance. [source] Osteoclast Differentiation by RANKL Requires NF-,B-Mediated Downregulation of Cyclin-Dependent Kinase 6 (Cdk6),JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2004Toru Ogasawara Abstract This study investigated the involvement of cell cycle factors in RANKL-induced osteoclast differentiation. Among the G1 cell cycle factors, Cdk6 was found to be a key molecule in determining the differentiation rate of osteoclasts as a downstream effector of the NF-,B signaling. Introduction: A temporal arrest in the G1 phase of the cell cycle is a prerequisite for cell differentiation, making it possible that cell cycle factors regulate not only the proliferation but also the differentiation of cells. This study investigated cell cycle factors that critically influence differentiation of the murine monocytic RAW264.7 cells to osteoclasts induced by RANKL. Materials and Methods: Growth-arrested RAW cells were stimulated with serum in the presence or absence of soluble RANKL (100 ng/ml). Expressions of the G1 cell cycle factors cyclin D1, D2, D3, E, cyclin-dependent kinase (Cdk) 2, 4, 6, and Cdk inhibitors (p18 and p27) were determined by Western blot analysis. Involvement of NF-,B and c- jun N-terminal kinase (JNK) pathways was examined by overexpressing dominant negative mutants of the I,B kinase 2 (IKKDN) gene and mitogen-activated protein kinase kinase 7 (MKK7DN) gene, respectively, using the adenovirus vectors. To determine the direct effect of Cdk6 on osteoclast differentiation, stable clones of RAW cells transfected with Cdk6 cDNA were established. Osteoclast differentiation was determined by TRACP staining, and cell cycle regulation was determined by BrdU uptake and flow cytometric analysis. Results and Conclusion: Among the cell cycle factors examined, the Cdk6 level was downregulated by RANKL synchronously with the appearance of multinucleated osteoclasts. Inhibition of the NF-,B pathway by IKKDN overexpression, but not that of the JNK pathway by MKK7DN overexpression, caused the decreases in both Cdk6 downregulation and osteoclastogenesis by RANKL. RAW cells overexpressing Cdk6 resist RANKL-induced osteoclastogenesis; however, cell cycle regulation was not affected by the levels of Cdk6 overexpression, suggesting that the inhibitory effect of Cdk6 on osteoclast differentiation was not exerted through cell cycle regulation. These results indicate that Cdk6 is a critical regulator of RANKL-induced osteoclast differentiation and that its NF-,B-mediated downregulation is essential for efficient osteoclast differentiation. [source] The neuronal MAP kinase cascade: a biochemical signal integration system subserving synaptic plasticity and memoryJOURNAL OF NEUROCHEMISTRY, Issue 1 2001J. David Sweatt The mitogen-activated protein kinase (MAP kinase, MAPK) cascade, as the name implies, was originally discovered as a critical regulator of cell division and differentiation. As further details of this signaling cascade were worked out, it became clear that the MAPK cascade is in fact a prototype for a family of signaling cascades that share the motif of three serially linked kinases regulating each other by sequential phosphorylation. Thus, a revised nomenclature arose that uses the term MAPK to refer to the entire superfamily of signaling cascades (comprising the erks, the JNKs and the p38 stress activated protein kinases), and specifies the prototype MAPK as the extracellular signal-regulated kinase (erk). The two erk MAPK isoforms, p44 MAPK and p42 MAPK, are referred to as erk1 and erk2, respectively. The erks are abundantly expressed in neurons in the mature central nervous system, raising the question of why the prototype molecular regulators of cell division and differentiation are present in these non-dividing, terminally differentiated neurons. This review will describe the beginnings of an answer to this question. Interestingly, the general model has begun to emerge that the erk signaling system has been co-opted in mature neurons to function in synaptic plasticity and memory. Moreover, recent insights have led to the intriguing prospect that these molecules serve as biochemical signal integrators and molecular coincidence detectors for coordinating responses to extracellular signals in neurons. In this review I will first outline the essential components of this signal transduction cascade, and briefly describe recent results implicating the erks in mammalian synaptic plasticity and learning. I will then proceed to outline recent results implicating the erks as molecular signal integrators and, potentially, coincidence detectors. Finally, I will speculate on what the critical downstream effectors of the erks are in neurons, and how they might provide a readout of the integrated signal. [source] Immunohistochemical study of receptor activator of nuclear factor kappa-B ligand (RANK-L) in human osteolytic bone tumorsJOURNAL OF SURGICAL ONCOLOGY, Issue 3 2002Christopher R. Good BA Abstract Background and Objectives Osteolytic bone tumors produce intercellular signaling proteins that regulate bone remodeling by altering the rates of osteoclast and osteoblast differentiation and activity. This report examines osteolytic bone tumor expression of receptor activator of nuclear factor B-ligand (RANK-L), a cytokine that is arguably the most critical regulator of osteoclast differentiation and activation. Methods This prospective immunohistochemical study examined RANK-L expression in frozen tissues from sixteen surgical specimens of patients who underwent surgery for the treatment of osteolytic bone tumors between 1999 and 2000. Results RANK-L was positive in 13 of the 16 cases. Primary benign bone tumors, primary malignant bone tumors, and metastasis to bone were positive for RANK-L. Conclusions The cells in some, but not all, osteolytic tumors produce the cytokine RANK-L. Further study is necessary to determine in which specific tumors RANK-L is the cytokine responsible for increased osteoclastic activity, and to develop possible therapeutic use of RANK-L antagonists such as osteoprotegerin (OPG). J. Surg. Oncol. 2002;79:174,179. © 2002 Wiley,Liss, Inc. [source] Analysis of Testosterone Effects on Sonic Hedgehog Signaling in Juvenile, Adolescent and Adult Sprague Dawley Rat PenisTHE JOURNAL OF SEXUAL MEDICINE, Issue 3 2010Christopher W. Bond MS ABSTRACT Introduction., Smooth muscle apoptosis is a major contributing factor to erectile dysfunction (ED) development in prostatectomy and diabetic patients and animal models. A critical regulator of penile smooth muscle and apoptosis is Sonic hedgehog (SHH). The SHH protein is decreased in ED models and SHH treatment of cavernous nerve (CN) injured rats prevents smooth muscle apoptosis. A close association between androgen deficiency and ED has been suggested in the literature, but few studies have examined the molecular effects on penile smooth muscle and on known signaling mechanisms that regulate morphology. Aim., Examine testosterone and SHH interaction in eugonadal adult, adolescent and juvenile rats by performing castration studies and treatment with supraphysiological testosterone. Methods., The eugonadal adult Sprague Dawley rats were either treated with testosterone for 7 or 14 days (N = 14) or were castrated for 4 or 7 days (N = 12). The juvenile rats were treated with testosterone for 8 days (N = 7). The adolescent rats were castrated and sacrificed at P88 (N = 8). The control rats had empty vehicle (N = 22) or sham surgery (N = 20). Main Outcome Measures., The active form of SHH protein and mRNA were quantified by semi-quantitative immunohistochemical analysis and real-time reverse transcriptase polymerase chain reaction (RT-PCR). Results., Testosterone treatment did not alter SHH signaling in juvenile rats. Shh mRNA increased 3.2-fold and SHH protein increased 1.2-fold in rats castrated during puberty. In adult rats, castration decreased Shh mRNA 3.2-fold but did not alter SHH protein. Testosterone supplement in adult rats increased Shh mRNA 2.3-fold and decreased SHH protein 1.3-fold. Conclusions., SHH signaling is independent of testosterone in normal juvenile rats and is sensitive to testosterone during adolescence, while testosterone supplement in the adult adversely impacts SHH signaling in a very similar manner to that observed with CN injury. Bond CW, Angeloni NL, and Podlasek CA. Analysis of testosterone effects on sonic hedgehog signaling in juvenile, adolescent and adult Sprague Dawley rat penis. J Sex Med 2010;7:1116,1125. [source] Ex vivo Inhibition of NF-,B Signaling in Alloreactive T-cells Prevents Graft-Versus-Host DiseaseAMERICAN JOURNAL OF TRANSPLANTATION, Issue 3 2009M. J. O'Shaughnessy The ex vivo induction of alloantigen-specific hyporesponsiveness by costimulatory pathway blockade or exposure to immunoregulatory cytokines has been shown to inhibit proliferation, IL-2 production, and the graft-versus-host disease (GVHD) capacity of adoptively transferred T-cells. We hypothesized that inhibition of the intracellular NF-,B pathway in alloreactive T-cells, which is critical for T-cell activation events including IL-2 transcription, could lead to alloantigen hyporesponsiveness and loss of GVHD capacity. We demonstrate that treatment of mixed lymphocyte reaction (MLR) cultures with PS1145, a potent inhibitor of NF-,B activation, can induce T-cell hyporesponsiveness to alloantigen in primary and secondary responses while preserving in vitro responses to potent mitogenic stimulation. GVHD lethality in recipients of ex vivo PS1145-treated cells was profoundly inhibited. Parking of control or PS1145-treated MLR cells in syngeneic Rag,/, recipients resulted in intact contact hypersensitivity (CHS) responses. However, GVHD lethality capacity also was restored, suggesting that lymphopenic expansion uncoupled alloantigen hyporesponsiveness. These results indicate that the NF-,B pathway is a critical regulator of alloresponses and provide a novel small molecule inhibitor based approach that is effective in preventing early posttransplant GVHD lethality but that also permits donor T-cell responses to recover after a period of lymphopenic expansion. [source] Role of AML1/Runx1 in the pathogenesis of hematological malignanciesCANCER SCIENCE, Issue 10 2003Mineo Kurokawa AML1/Runx1, originally identified as a gene located at the breakpoint of the t(8;21) translocation, encodes one of the two subunits forming a heterodimeric transcription factor. AML1 contains a highly evolutionally conserved domain called the Runt domain, responsible for both DNA binding and heterodimerization with the partner protein, CBF,. AML1 is widely expressed in all hematopoietic lineages, and regulates the expression of a variety of hematopoietic genes. Numerous studies have shown that AML is a critical regulator of hematopoietic development. In addition, AML1 and CBF, are frequent targets for chromosomal translocation in human leukemia. Translocations lead to the generation of fusion proteins, which play a causative role for the development of leukemia, primarily by inhibiting AML1 function. Point mutations that impair AML1 function are also associated with familial and sporadic leukemias. Loss of AML1 function is thus implicated in a number of leukemias through multiple pathogenic mechanisms. However, AML1-related translocations or haploinsufficiency of AML1 are not immediately leukemogenic in animal models, suggesting that additional genetic events are required for the development of full-blown leukemia. [source] Tankyrase is necessary for canonical Wnt signaling during kidney developmentDEVELOPMENTAL DYNAMICS, Issue 7 2010Courtney M. Karner Abstract Recent studies using small molecule antagonists have revealed that the poly(ADP-ribose) polymerases (PARPs) Tankyrase 1 and 2 are critical regulators of canonical Wnt signaling in some cellular contexts. However, the absence of any activity during zebrafish embryogenesis suggested that the tankyrases may not be general/core components of the Wnt pathway. Here, we show that Tnks1 and 2 are broadly expressed during mouse development and are essential during kidney and lung development. In the kidney, blockage of tankyrase activity phenocopies the effect of blocking production of all Wnt ligands. Tankyrase inhibition can be rescued by activation of ,-catenin demonstrating its specificity for the Wnt pathway. In addition, treatment with tankyrase inhibitors appears to be completely reversible in some cell types. These studies suggest that the tankyrases are core components of the canonical Wnt pathway and their inhibitors should enjoy broad usage as antagonists of Wnt signaling. Developmental Dynamics 239:2014,2023, 2010 © 2010 Wiley-Liss, Inc. [source] PDGFR-, signaling is critical for tooth cusp and palate morphogenesisDEVELOPMENTAL DYNAMICS, Issue 1 2005Xun Xu Abstract Platelet-derived growth factor receptor alpha (PDGFR-,) and PDGF ligands are key regulators for embryonic development. Although Pdgfr, is spatially expressed in the cranial neural crest (CNC)-derived odontogenic mesenchyme, mice deficient for Pdgfr, are embryonic lethal, making it impossible to investigate the functional significance of PDGF signaling in regulating the fate of CNC cells during tooth morphogenesis. Taking advantage of the kidney capsule assay, we investigated the biological function of PDGF signaling in regulating tooth morphogenesis. Pdgfr, and Pdgfa are specifically and consistently expressed in the CNC-derived odontogenic mesenchyme and the dental epithelium, respectively, throughout all stages of tooth development, suggesting a paracrine function of PDGF signaling in regulating tooth morphogenesis. Highly concentrated expression patterns of Pdgfr, and Pdgfa are associated with the developing dental cusp, suggesting possible functional importance of PDGF signaling in regulating cusp formation. Loss of the Pdgfr, gene does not affect proper odontoblasts proliferation and differentiation in the CNC-derived odontogenic mesenchyme but perturbs the formation of extracellular matrix and the organization of odontoblast cells at the forming cusp area, resulting in dental cusp growth defect. Pdgfr,,/, mice have complete cleft palate. We show that the cleft palate in Pdgfr, mutant mice results from an extracellular matrix defect within the CNC-derived palatal mesenchyme. The midline epithelium of the mutant palatal shelf remains functionally competent to mediate palatal fusion once the palatal shelves are placed in close contact in vitro. Collectively, our data suggests that PDGFR, and PDGFA are critical regulators for the continued epithelial,mesenchymal interaction during tooth and palate morphogenesis. Disruption of PDGFR, signaling disturbs the growth of dental cusp and interferes with the critical extension of palatal shelf during craniofacial development. Developmental Dynamics 232:75,84, 2005. © 2004 Wiley-Liss, Inc. [source] Ovariectomy increases vascular calcification via the OPG/RANKL cytokine signalling pathwayEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 4 2008B. G. Choi ABSTRACT Background, Observational studies suggest a strong relationship between menopause and vascular calcification. Receptor activator of nuclear factor-,, ligand (RANKL) and osteoprotegerin (OPG) are critical regulators of bone remodelling and modulate vascular calcification. We assessed the hypothesis that ovariectomy increases vascular calcification via the OPG/RANKL axis. Materials and methods, Age-matched sexually mature rabbits were randomized to ovariectomy (OVX, n = 12) or sham procedure (SHAM, n = 12). One month post-procedure, atherosclerosis was induced by 15 months 0·2%-cholesterol diet and endothelial balloon denudations (at months 1 and 3). Aortic atherosclerosis was assessed in vivo by magnetic resonance imaging (MRI) at months 9 and 15. At sacrifice, aortas were harvested for ex vivo microcomputed tomography (µCT) and molecular analysis of the vascular tissue. Results, Vascular calcification density and calcific particle number were significantly greater in OVX than SHAM (8·4 ± 2·8 vs. 1·9 ± 0·6 mg cm,3, P = 0·042, and 94 ± 26 vs. 33 ± 7 particles cm,3, P = 0·046, respectively). Calcification morphology, as assessed by the arc angle subtended by the largest calcific particle, showed no difference between groups (OVX 33 ± 7° vs. SHAM 33 ± 5°, P = 0·99). By Western blot analysis, OVX increased the vascular OPG:RANKL ratio by 66%, P = 0·029, primarily by decreasing RANKL (P = 0·019). At month 9, MRI demonstrated no difference in atheroma volume between OVX and SHAM, and no significant change was seen by the end of the study. Conclusions, In contrast to bone, vascular OPG:RANKL ratio increased in response to ovariectomy with a corresponding fourfold increase in arterial calcification. This diametrical organ-specific response may explain the comorbid association of osteoporosis with calcifying atherosclerosis in post-menopausal women. [source] The key regulators of adult T helper cell responses, STAT6 and T-bet, are established in early life in miceEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2006Shawn Rose Abstract Murine neonatal immunity is typically Th2 biased. This is characterized by high-level IL-4 production at all phases of the immune response and poor IFN-, memory responses. The differential expression of Th1/Th2 cytokines by neonates and adults could arise if the critical regulators of Th differentiation and function, STAT6 and T-bet, operate differently during the neonatal period. To test this idea, the Th cell responses of wild-type, T-bet-deficient, or STAT6-deficient mice were compared in vitro and in vivo. The absence of these factors had similar qualitative effects on the development of effector function in neonates and adults, i.e., if a Th lineage was inhibited or enhanced in adult animals, a similar phenomenon was observed in neonates. However, there was a striking difference observed in the in vivo Th1 memory responses of STAT6-deficient mice initially immunized as neonates. Antigen-specific IFN-, production was increased 50,100-fold in STAT6-deficient neonates, achieving levels similar to those of STAT6-deficient adults. These findings demonstrate that STAT6 and T-bet signals are central in shaping Th responses in wild-type neonates, as in adult mice, and that the master regulators of Th cell development and function are already firmly established in early life. [source] p38 MAPK is a critical regulator of the constitutive and the ,4,integrin-regulated expression of IL-6 in human normal thymic epithelial cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2003Fabrizio Mainiero Abstract Cytokines and adhesion receptors are key mediators in the dialog occurring between thymic epithelial cells (TEC) and thymocytes and regulating T,cell maturation and epithelial embryonic differentiation. Among cytokines, IL-6 can be critical in the thymus, fostering proliferation, differentiation and/or survival of both TEC and thymocytes. We have previously reported in human normal TEC that clustering of the laminin receptor ,6,4 integrin induced by thymocyte contact or monoclonal antibody-mediated cross-linking regulates IL-6 gene expression via activation of NF-,B and NF-IL6 transactivators. Here we show that ,6,4 integrin activates p38 mitogen-activated protein kinase (MAPK) and that p38 is essential for IL-6 gene expression. In fact, ,4 cross-linking activated p38 and extracellular signal-regulated kinase (ERK) MAPK, Rac1, p21-activated protein kinase,1 (PAK1) and MAPK kinases (MKK),3/MKK6. However, pharmacological blockade of p38 or ERK demonstrated that p38 inhibition abrogated both basal and ,4,integrin-induced production of IL-6 preventing NF-,B and NF-IL6 activation, whereas ERK inhibition reduced IL-6 production, hampering only NF-,B activation. Overall, our results indicate that p38 MAPK and ,6,4,integrin, expressed by TEC throughout their life, are critical regulators of the intrathymic availability of a cytokine controlling fate and functions of cells governing development and maintenance of thymic architecture and immune responses. [source] Cross-species divergence of the major recognition pathways of ubiquitylated substrates for ubiquitin/26S proteasome-mediated proteolysisFEBS JOURNAL, Issue 3 2010Antony S. Fatimababy The recognition of ubiquitylated substrates is an essential element of ubiquitin/26S proteasome-mediated proteolysis (UPP), which is mediated directly by the proteasome subunit RPN10 and/or RPN13, or indirectly by ubiquitin receptors containing ubiquitin-like and ubiquitin-associated domains. By pull-down and mutagenesis assays, we detected cross-species divergence of the major recognition pathways. RPN10 plays a major role in direct recognition in Arabidopsis and yeast based on the strong affinity for the long and K48-linked ubiquitin chains. In contrast, both the RPN10 and RPN13 homologs play major roles in humans. For indirect recognition, the RAD23 and DSK2 homologs (except for the human DSK2 homolog) are major receptors. The human RAD23 homolog is targeted to the 26S proteasome by the RPN10 and RPN13 homologs. In comparison, Arabidopsis uses UIM1 and UIM3 of RPN10 to bind DSK2 and RAD23, respectively. Yeast uses UIM in RPN10 and LRR in RPN1. Overall, multiple proteasome subunits are responsible for the direct and/or indirect recognition of ubiquitylated substrates in yeast and humans. In contrast, a single proteasome subunit, RPN10, is critical for both the direct and indirect recognition pathways in Arabidopsis. In agreement with these results, the accumulation of ubiquitylated substrates and severe pleiotropic phenotypes of vegetative and reproductive growth are associated with the loss of RPN10 function in an Arabidopsis T-DNA insertion mutant. This implies that the targeting and proteolysis of the critical regulators involved are affected. These results support a cross-species mechanistic and functional divergence of the major recognition pathways for ubiquitylated substrates of UPP. Structured digital abstract ,,A list of the large number of protein-protein interactions described in this article is available via the MINT article ID MINT-7307429 [source] A novel function of WAVE in lamellipodia: WAVE1 is required for stabilization of lamellipodial protrusions during cell spreadingGENES TO CELLS, Issue 5 2005Daisuke Yamazaki When a cell spreads and moves, reorganization of the actin cytoskeleton pushes the cell membrane, and the resulting membrane protrusions create new points of contact with the substrate and generate the locomotive force. Membrane extension and adhesion to a substrate must be tightly coordinated for effective cell movement, but little is known about the mechanisms underlying these processes. WAVEs are critical regulators of Rac-induced actin reorganization. WAVE2 is essential for formation of lamellipodial structures at the cell periphery stimulated by growth factors, but it is thought that WAVE1 is dispensable for such processes in mouse embryonic fibroblasts (MEFs). Here we show a novel function of WAVE in lamellipodial protrusions during cell spreading. During spreading on fibronectin (FN), MEFs with knockouts (KOs) of WAVE1 and WAVE2 showed different membrane dynamics, suggesting that these molecules have distinct roles in lamellipodium formation. Formation of lamellipodial structures on FN was inhibited in WAVE2 KO MEFs. In contrast, WAVE1 is not essential for extension of lamellipodial protrusions but is required for stabilization of such structures. WAVE1-deficiency decreased the density of actin filaments and increased the speed of membrane extension, causing deformation of focal complex at the tip of spreading edges. Thus, at the tip of the lamellipodial protrusion, WAVE2 generates the membrane protrusive structures containing actin filaments, and modification by WAVE1 stabilizes these structures through cell-substrate adhesion. Coordination of WAVE1 and WAVE2 activities appears to be necessary for formation of proper actin structures in stable lamellipodia. [source] Synergistic induction of cyclin D1 in oligodendrocyte progenitor cells by IGF-I and FGF-2 requires differential stimulation of multiple signaling pathwaysGLIA, Issue 10 2007Terra J. Frederick Abstract D-type cyclins are direct targets of extracellular signals and critical regulators of G1 progression. Our previous data demonstrated that IGF-I and FGF-2 synergize to enhance cyclin D1 expression, cyclin E/cdk2 complex activation, and S-phase entry in OP cells. Here, we provide a mechanistic explanation for how two growth factor signaling pathways converge on a major cell cycle regulator. IGF-I and FGF-2 differentially activate signaling pathways to coordinately promote cyclin D1 expression. We show that the p44/p42 MAPK signaling pathway is essential for FGF-2 induction of cyclin D1 mRNA. In contrast, blocking the PI3-Kinase pathway results in loss of IGF-I/FGF-2 synergistic induction of cyclin D1 protein levels. Moreover, the presence of IGF-I significantly enhances nuclear localization of cyclin D1, which also requires PI3K signaling. GSK-3,, a downstream target of the PI3K/Akt pathway, is phosphorylated in the presence of IGF-I in OPs. Consistent with a known role for GSK-3, in cyclin D1 degradation, we show that proteasome inhibition in OPs exposed to FGF-2 increased cyclin D1 levels, equivalent to levels seen in IGF-I/FGF-2 treated cells. Thus, we provide a model for cyclin D1 coordinate regulation where FGF-2 stimulation of the MAPK pathway promotes cyclin D1 mRNA expression while IGF-I activation of the PI3K pathway inhibits proteasome degradation of cyclin D1 and enhances nuclear localization of cyclin D1. © 2007 Wiley-Liss, Inc. [source] MicroRNAs control hepatocyte proliferation during liver regeneration,HEPATOLOGY, Issue 5 2010Guisheng Song MicroRNAs (miRNAs) constitute a new class of regulators of gene expression. Among other actions, miRNAs have been shown to control cell proliferation in development and cancer. However, whether miRNAs regulate hepatocyte proliferation during liver regeneration is unknown. We addressed this question by performing 2/3 partial hepatectomy (2/3 PH) on mice with hepatocyte-specific inactivation of DiGeorge syndrome critical region gene 8 (DGCR8), an essential component of the miRNA processing pathway. Hepatocytes of these mice were miRNA-deficient and exhibited a delay in cell cycle progression involving the G1 to S phase transition. Examination of livers of wildtype mice after 2/3 PH revealed differential expression of a subset of miRNAs, notably an induction of miR-21 and repression of miR-378. We further discovered that miR-21 directly inhibits Btg2, a cell cycle inhibitor that prevents activation of forkhead box M1 (FoxM1), which is essential for DNA synthesis in hepatocytes after 2/3 PH. In addition, we found that miR-378 directly inhibits ornithine decarboxylase (Odc1), which is known to promote DNA synthesis in hepatocytes after 2/3 PH. Conclusion: Our results show that miRNAs are critical regulators of hepatocyte proliferation during liver regeneration. Because these miRNAs and target gene interactions are conserved, our findings may also be relevant to human liver regeneration. (HEPATOLOGY 2010) [source] Hypoxia-inducible factor-dependent production of profibrotic mediators by hypoxic Kupffer cellsHEPATOLOGY RESEARCH, Issue 5 2010Bryan L. Copple Aim:, Liver fibrosis develops when chronic liver injury stimulates cells in the liver to produce mediators that activate hepatic stellate cells and stimulate them to secrete collagen. Recent studies suggest that the hypoxia-regulated transcription factor, hypoxia-inducible factor-1,, is essential for upregulation of profibrotic mediators, such as platelet-derived growth factor, in the liver during the development of liver fibrosis. What remains unknown, however, is the cell type-specific regulation of profibrotic mediators by hypoxia-inducible factors. Accordingly, in the present study the hypothesis tested was that hypoxia-inducible factors regulate production of profibrotic mediators by hypoxic Kupffer cells. Methods:, Kupffer cells were isolated from control mice and hypoxia-inducible factor-1,-deficient mice and exposed to room air or 1% oxygen (i.e. hypoxia). Levels of profibrotic mediators were quantified by real-time polymerase chain reaction. Results:, Exposure of Kupffer cells isolated from control mice to 1% oxygen activated hypoxia-inducible factor-1,, and increased mRNA levels of platelet-derived growth factor-B, vascular endothelial growth factor, angiopoietin-1 and monocyte chemotactic protein-1. Upregulation of all of these mediators by hypoxia was prevented in Kupffer cells isolated from hypoxia-inducible factor-1,-deficient mice. Conclusion:, Results from these studies suggest that hypoxia-inducible factors are critical regulators of profibrotic mediator production by hypoxic Kupffer cells. [source] Insulin-like 3 signalling in testicular descentINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 5 2004Ibrahim M. Adham Summary Undescended testis is one of the most common congenital defects in the newborn boys and the common cause of cryptorchidism. If left untreated, this condition is strongly associated with infertility and drastically increased risk of testicular cancer in adulthood. Testis position in developing males is defined by sexual dimorphic differentiation of two gonadal ligaments, gubernaculum and cranial suspensory ligament. Recent transgenic mouse studies identified testicular hormone insulin-like 3 (INSL3), and its receptor, GREAT/LGR8, as the critical regulators of the gubernacular differentiation. Mutation analysis of the two genes in patients with undescended testis revealed functionally deleterious mutations, which may be responsible for the abnormal phenotype in some of the patients. [source] From JNK to Pay Dirt: Jun Kinases, their Biochemistry, Physiology and Clinical ImportanceIUBMB LIFE, Issue 4-5 2005Michael Karin Abstract The c-Jun N-terminal kinases (JNKs) were originally identified by their ability to phosphorylate c-Jun in response to UV-irradiation, but now are recognized as critical regulators of various aspects of mammalian physiology, including: cell proliferation, cell survival, cell death, DNA repair and metabolism. JNK-mediated phosphorylation enhances the ability of c-Jun, a component of the AP-1 transcription factor, to activate transcription, in response to a plethora of extracellular stimuli. The JNK activation leads to induction of AP-1-dependent target genes involved in cell proliferation, cell death, inflammation, and DNA repair. The JNKs, which are encoded by three different Jnk loci, are now known to be regulated by many other stimuli, from pro-inflammatory cytokines to obesity, in addition to UV-irradiation. Targeted disruption of the Jnk loci in mice has proved to be a critical tool in better understanding their physiological functions. Such studies revealed that the JNKs play important roles in numerous cellular processes, including: programmed cell death, T cell differentiation, negative regulation of insulin signaling, control of fat deposition, and epithelial sheet migration. Importantly, the JNKs have become prime targets for drug development in several important clinical areas, including: inflammation, diabetes, and cancer. IUBMB Life, 57: 283-295, 2005 [source] Insulin-Like Growth Factor I Production Is Essential for Anabolic Effects of Thyroid Hormone in Osteoblasts,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 2 2000Bill K. Huang Abstract Thyroid hormone (T3) and insulin-like growth factor I (IGF-I) are critical regulators of skeletal function. T3 increases IGF-I production in bone. To assess the potential role of IGF-I as a mediator of T3 actions, we characterized phenotypic markers of osteoblast activity in two osteoblast models, normal mouse osteoblasts and MC3T3-E1 cells, exposed to T3 alone or under conditions that interfere with IGF-I actions. T3 significantly increased osteoblast 3H-proline incorporation, alkaline phosphatase (ALP), and osteocalcin. Both ,IR3, a neutralizing monoclonal antibody to the IGF-I receptor, and JB1, an IGF-I analogue antagonist, attenuated the stimulatory effects of T3. T3 effects also were decreased in cells transfected with antisense oligonucleotide (AS-ODN) to the IGF-I receptor gene. Both IGF-I and T3 had mitogenic effects that were inhibited by the antagonists. IGF-I by itself did not stimulate 3H-proline incorporation, ALP, and osteocalcin in the models used, revealing that although IGF-I is essential for the anabolic effects of T3, it acts in concert with other factors to elicit these phenotypic responses. (J Bone Miner Res 2000;15:188,197) [source] Molecular regulation of androgen action in prostate cancer,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2006Scott M. Dehm Abstract Androgens are critical regulators of prostate differentiation and function, as well as prostate cancer growth and survival. Therefore, androgen ablation is the preferred systemic treatment for disseminated prostate cancer. Androgen action is exerted in target tissues via binding the androgen receptor (AR), a nuclear receptor transcription factor. Historically, the gene expression program mediated by the AR has been poorly understood. However, recent gene expression profiling and more traditional single-gene characterization studies have revealed many androgen-regulated genes that are important mediators of androgen action in both normal and malignant prostate tissue. This review will focus on the androgen-regulated gene expression program, and examine how recently identified androgen-regulated genes are likely to contribute to the development and progression of prostate cancer. We will also summarize several recent studies that have attempted to unravel how these genes are deregulated in androgen depletion independent prostate cancer. J. Cell. Biochem. 99: 333,344, 2006. © 2006 Wiley-Liss, Inc. [source] Potential roles of the nucleotide exchange factor ECT2 and Cdc42 GTPase in spindle assembly in Xenopus egg cell-free extracts,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2003Takashi Tatsumoto Abstract The ECT2 protooncogene encodes a guanine nucleotide exchange factor for the Rho family of small GTPases. ECT2 contains motifs of cell cycle regulators at its N-terminal domain. We previously showed that ECT2 plays a critical role in cytokinesis. Here, we report a potential role of XECT2, the Xenopus homologue of the human ECT2, in spindle assembly in cell-free Xenopus egg extracts. Cloned XECT2 cDNA encodes a 100 kDa protein closely related to human ECT2. XECT2 is specifically phosphorylated in M phase extracts. Affinity-purified anti-XECT2 antibody strongly inhibited mitosis in Xenopus cell-free extracts. Instead of bipolar spindles, where chromosomes are aligned at the metaphase plane in control extracts, the addition of anti-XECT2 resulted in the appearance of abnormal spindles including monopolar and multipolar spindles as well as bipolar spindles with misaligned chromosomes. In these in vitro synthesized spindle structures, XECT2 was found to tightly associate with mitotic spindles. The N-terminal half of XECT2 lacking the catalytic domain also strongly inhibited spindle assembly in vitro, resulting in the formation of mitotic spindles with a low density. Among the representative Rho GTPases, a dominant-negative form of Cdc42 strongly inhibited spindle assembly in vitro. These results suggest that the Rho family GTPase Cdc42 and its exchange factor XECT2 are critical regulators of spindle assembly in Xenopus egg extracts. Published 2003 Wiley-Liss, Inc., [source] | ||||||||||||||||||||||||||||