The Hedgehog (Hh) pathway is essential for the embryonic development and homeostatic maintenance of many adult tissues and organs. It has also been associated with some functions of the innate and ...adaptive immune system. However, its involvement in the immune response has not been well determined. Here we study the role of Hh signalling in the modulation of the immune response by using the Ptch-1-LacZ
mouse model (hereinafter referred to as
), in which the hemizygous inactivation of Patched-1, the Hh receptor gene, causes the constitutive activation of Hh response genes. The in vitro TCR stimulation of spleen and lymph node (LN) T cells showed increased levels of Th2 cytokines (IL-4 and IL-10) in
cells compared to control cells from wild-type (wt) littermates, suggesting that the Th2 phenotype is favoured by Hh pathway activation. In addition, CD4
cells secreted less IL-17, and the establishment of the Th1 phenotype was impaired in
mice. Consistently, in response to an inflammatory challenge by the induction of experimental autoimmune encephalomyelitis (EAE),
mice showed milder clinical scores and more minor spinal cord damage than wt mice. These results demonstrate a role for the Hh/ptch pathway in immune response modulation and highlight the usefulness of the
mouse model for the study of T-cell-mediated diseases and for the search for new therapeutic strategies in inflammatory diseases.
Spry2 is a molecular modulator of tyrosine kinase receptor signaling pathways that has cancer-type-specific effects. Mammalian Spry2 protein undergoes tyrosine and serine phosphorylation in response ...to growth factor stimulation. Spry2 expression is distinctly altered in various cancer types. Inhibition of the proteasome functionality results in reduced intracellular Spry2 degradation. Using in vitro and in vivo assays, we show that protein kinase D (PKD) phosphorylates Spry2 at serine 112 and interacts in vivo with the C-terminal half of this protein. Importantly, missense mutation of Ser112 decreases the rate of Spry2 intracellular protein degradation. Either knocking down the expression of all three mammalian PKD isoforms or blocking their kinase activity with a specific inhibitor contributes to the stabilization of Spry2 wild-type protein. Downregulation of CSN3, a component of the COP9/Signalosome that binds PKD, significantly increases the half-life of Spry2 wild-type protein but does not affect the stability of a Spry2 after mutating Ser112 to the non-phosphorylatable residue alanine. Our data demonstrate that both PKD and the COP9/Signalosome play a significant role in control of Spry2 intracellular stability and support the consideration of the PKD/COP9 complex as a potential therapeutic target in tumors where Spry2 expression is reduced.
Se pretende valorar el estado psicológico de 127 mujeres que denuncian maltrato por parte de su expareja, y de 55 hombres denunciados, a través del MCMI-III, analizando asimismo la influencia de las ...variables sociodemográficas y del maltrato sobre las puntuaciones del MCMI-III. Las mujeres presentan edad media de 36.25 años (DT = 10.48), y los varones 42.54 años (DT = 12.93). Ambos grupos fueron remitidos al Instituto de Medicina Legal de una provincia española por parte del Juzgado de Violencia sobre la Mujer. Se les administró el MCMI-III, obteniendo perfiles característicos en función de la edad, duración del maltrato y años de convivencia. Las mujeres obtienen puntuaciones altas en deseabilidad social, personalidad compulsiva, ansiedad, distimia, somatomorfo y depresión. Los varones presentan rasgos narcisistas, depresión y dependencia de sustancias. A mayor duración del maltrato aumentan en las mujeres las puntuaciones en personalidad esquizoide, depresiva y autodestructiva, junto a distimia y estrés postraumático, con puntuaciones más bajas en deseabilidad social e histrionismo.
In this study, psychological assessment of 127 women presenting charges of gender-based violence (GVB) and 55 male suspects, were analyzed by the MCMI-III, as well as the influence of sociodemographic data and GVB features on these MCMI-III scores. Average age of women was 36.25 (SD = 10.48) and average age of men was 42.54 years old (SD = 12.93). Both groups were referred to the Legal Medicine Institute by the Woman Violence Court and all of them were administered the Millon Clinical Multiaxial Inventory III (MCMI-III). Differential profiles according to age, maltreatment duration and years of cohabitation were obtained. Women presented high scores in Social desirability, Compulsive personality, Anxiety, Dysthymia, Somatization and Depression. Men presented Narcissistic features, Depression and Substance abuse. As maltreatment extended in time, Schizoid, Depressive and Self-destructive features increased in women, as well as Dysthymia and PTSD, obtaining lower scores on Social desirability and Histrionic personality.
hSos1 is a Ras guanine-nucleotide exchange factor. It was suggested that the carboxyl-terminal region of hSos1 down-regulates hSos1 functionality and that the intrinsic guanine-nucleotide exchange ...activity of this protein may be different before and after stimulation of tyrosine kinase receptors. Using different myristoylated hSos1 full-length and carboxyl-terminal truncated mutants, we show that Grb2 function accounts not only for recruitment of hSos1 to the plasma membrane but also for modulation of hSos1 activity. Our results demonstrate that the first two canonical Grb2 binding sites, inside the carboxyl-terminal region of hSos1, are responsible for this regulation. Following different approaches, such as displacement of Grb2 from the hSos1-Grb2 complex or depletion of Grb2 levels by small interfering RNA, we found that the full-length Grb2 proteins mediate negative regulation of the intrinsic Ras guanine-nucleotide exchange activity of hSos1.
Se pretende valorar el estado psicológico de 127 mujeres que denuncian maltrato por parte de su expareja, y de 55 hombres denunciados, a través del MCMI-III, analizando asimismo la influencia de las ...variables sociodemográficas y del maltrato sobre las puntuaciones del MCMI-III. Las mujeres presentan edad media de 36.25 años (DT = 10.48), y los varones 42.54 años (DT = 12.93). Ambos grupos fueron remitidos al Instituto de Medicina Legal de una provincia española por parte del Juzgado de Violencia sobre la Mujer. Se les administró el MCMI-III, obteniendo perfiles característicos en función de la edad, duración del maltrato y años de convivencia. Las mujeres obtienen puntuaciones altas en deseabilidad social, personalidad compulsiva, ansiedad, distimia, somatomorfo y depresión. Los varones presentan rasgos narcisistas, depresión y dependencia de sustancias. A mayor duración del maltrato aumentan en las mujeres las puntuaciones en personalidad esquizoide, depresiva y autodestructiva, junto a distimia y estrés postraumático, con puntuaciones más bajas en deseabilidad social e histrionismo.
Multiple sclerosis is a widespread inflammatory demyelinating disease. Several immunomodulatory therapies are available, including interferon-β, glatiramer acetate, natalizumab, fingolimod, and ...mitoxantrone. Although useful to delay disease progression, they do not provide a definitive cure and are associated with some undesirable side-effects. Accordingly, the search for new therapeutic methods constitutes an active investigation field. The use of mesenchymal stem cells (MSCs) to modify the disease course is currently the subject of intense interest. Decidua-derived MSCs (DMSCs) are a cell population obtained from human placental extraembryonic membranes able to differentiate into the three germ layers. This study explores the therapeutic potential of DMSCs.
We used the experimental autoimmune encephalomyelitis (EAE) animal model to evaluate the effect of DMSCs on clinical signs of the disease and on the presence of inflammatory infiltrates in the central nervous system. We also compared the inflammatory profile of spleen T cells from DMSC-treated mice with that of EAE control animals, and the influence of DMSCs on the in vitro definition of the Th17 phenotype. Furthermore, we analyzed the effects on the presence of some critical cell types in central nervous system infiltrates.
Preventive intraperitoneal injection of DMSCs resulted in a significant delay of external signs of EAE. In addition, treatment of animals already presenting with moderate symptoms resulted in mild EAE with reduced disease scores. Besides decreased inflammatory infiltration, diminished percentages of CD4(+)IL17(+), CD11b(+)Ly6G(+) and CD11b(+)Ly6C(+) cells were found in infiltrates of treated animals. Early immune response was mitigated, with spleen cells of DMSC-treated mice displaying low proliferative response to antigen, decreased production of interleukin (IL)-17, and increased production of the anti-inflammatory cytokines IL-4 and IL-10. Moreover, lower RORγT and higher GATA-3 expression levels were detected in DMSC-treated mice. DMSCs also showed a detrimental influence on the in vitro definition of the Th17 phenotype.
DMSCs modulated the clinical course of EAE, modified the frequency and cell composition of the central nervous system infiltrates during the disease, and mediated an impairment of Th17 phenotype establishment in favor of the Th2 subtype. These results suggest that DMSCs might provide a new cell-based therapy for the control of multiple sclerosis.
Certain cell types escape the strict mechanisms imposed on the majority of somatic cells to ensure the faithful inheritance
of parental DNA content. This is the case in many embryonic tissues and ...certain adult cells such as mammalian hepatocytes
and megakaryocytes. Megakaryocytic endomitosis is characterized by repeated S phases followed by abortive mitoses, resulting
in mononucleated polyploid cells. Several cell cycle regulators have been proposed to play an active role in megakaryocytic
polyploidization; however, little is known about upstream factors that could control endomitosis. Here we show that ectopic
expression of the transcriptional repressor escargot interferes with the establishment of megakaryocytic endomitosis. Phorbol ester-induced polyploidization was inhibited in
stably transfected megakaryoblastic HEL cells constitutively expressing escargot . Analysis of the expression and activity of different cell cycle factors revealed that Escargot affects the G 1 /S transition by influencing Cdk2 activity and cyclin A transcription. Nuclear proteins that specifically bind the Escargot-binding
element were detected in endomitotic and non-endomitotic megakaryoblastic cells, but down-regulation occurred only during
differentiation of cells that become polyploid. As Escargot was originally implicated in ploidy maintenance of Drosophila embryonic and larval cells, our results suggest that polyploidization in megakaryocytes might respond to mechanisms conserved
from early development to adult cells that need to escape normal control of the diploid state.
The Hedgehog signaling pathway regulates embryo patterning and progenitor cell homeostasis in adult tissues, including epidermal appendages. A role for the Hh pathway in mammary biology and breast ...cancer has also been suggested. The aim of this study was to analyze Hh signaling in the mouse mammary gland through the generation of transgenic mice that express Sonic Hedgehog (Shh) under the control of the mammary-specific WAP promoter (WAP-Shh mice). To identify mammary cells capable of activating the Hh pathway we bred WAP-Shh mice to Ptch1-lacZ knock-in mice, in which the expression of a nuclear-targeted β-galactosidase reporter protein (β-gal) is driven by the endogenous Patched 1 gene regulatory region. After two cycles of induction of transgenic Shh expression, we detected areas of X-gal reactivity. Immunohistochemical analysis showed nuclear β-gal staining in clusters of mammary cells in WAP-Shh/Ptch1-lacZ bitransgenic mice. These were epithelial cells present in a basal location of displastic ducts and alveoli, adjacent to Shh-expressing luminal cells, and overexpressed epithelial basal markers keratin 5, 14 and 17 and transcription factor p63. Absence of smooth muscle actin expression and a cuboidal morphology differentiated Hh-responding cells from flat-shaped mature myoepithelial cells. Groups of cells expressing stem cell markers integrin β3 and keratins 6 and 15 were also detected within Hh-responding areas. In addition, we found that Hh-responding cells in the mammary glands of WAP-Shh/Ptch1-lacZ mice were ciliated and exhibited a low proliferation rate. Our data show the paracrine nature of hedgehog signaling in the epithelial compartment of the mouse mammary gland, where a subset of basal cells that express mammary progenitor cell markers and exhibit primary cilia is expanded in response to secretory epithelium-derived Shh.
► Mammary secretory epithelium-derived Shh activates the Hh pathway in a paracrine intraepithelial fashion. ► Hedgehog signaling induces the expansion of Hh-responsive cells in the basal epithelium. ► Hedgehog-responsive cells are ciliated, cubical and express progenitor cell markers.
Sos1 is an universal, widely expressed Ras guanine nucleotide-exchange factor (RasGEF) in eukaryotic cells. Its N-terminal HD motif is known to be involved in allosteric regulation of Sos1 GEF ...activity through intramolecular interaction with the neighboring PH domain. Here, we searched for other cellular proteins also able to interact productively with the Sos1 HD domain. Using a yeast two-hybrid system, we identified the interaction between the Sos1 HD region and CSN3, the third component of the COP9 signalosome, a conserved, multi-subunit protein complex that functions in the ubiquitin-proteasome pathway to control degradation of many cellular proteins. The interaction of CSN3 with the HD of Sos1 was confirmed in vitro by GST pull-down assays using truncated mutants and reproduced in vivo by co-immunoprecipitation with the endogenous, full-length cellular Sos1 protein. In vitro kinase assays showed that PKD, a COP9 signalosome-associated-kinase, is able to phosphorylate Sos1. The intracellular levels of Sos1 protein were clearly diminished following CSN3 or PKD knockdown. A sizable fraction of the endogenous Sos1 protein was found ubiquitinated in different mammalian cell types. A significant reduction of RasGTP formation upon growth factor stimulation was also observed in CSN3-silenced as compared with control cells. Our data suggest that the interaction of Sos1 with the COP9 signalosome and PKD plays a significant role in maintenance of cellular Sos1 protein stability and homeostasis under physiological conditions and raises the possibility of considering the CSN/PKD complex as a potential target for design of novel therapeutic drugs.
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