A screen for transcription factor targets of glycogen synthase kinase-3 highlights an inverse correlation of NFκB and androgen receptor signaling in prostate cancer

This is an open-access article distributed under the terms of the Creative Commons Attribution License.Expression of Glycogen Synthase Kinase-3 (GSK-3) is elevated in prostate cancer and its inhibition reduces prostate cancer cell proliferation, in part by reducing androgen receptor (AR) signaling. However, GSK-3 inhibition can also activate signals that promote cell proliferation and survival, which may preclude the use of GSK-3 inhibitors in the clinic. To identify such signals in prostate cancer, we screened for changes in transcription factor target DNA binding activity in GSK-3-silenced cells. Among the alterations was a reduction in AR DNA target binding, as predicted from previous studies, and an increase in NFκB DNA target binding. Consistent with the latter, gene silencing of GSK-3 or inhibition using the GSK-3 inhibitor CHIR99021 increased basal NFκB transcriptional activity. Activation of NFκB was accompanied by an increase in the level of the NFκB family member RelB. Conversely, silencing RelB reduced activation of NFκB by CHIR99021. Furthermore, the reduction of prostate cancer cell proliferation by CHIR99021 was potentiated by inhibition of NFκB signaling using the IKK inhibitor PS1145. Finally, stratification of human prostate tumor gene expression data for GSK3 revealed an inverse correlation between NFκB-dependent and androgen-dependent gene expression, consistent with the results from the transcription factor target DNA binding screen. In addition, there was a correlation between expression of androgen-repressed NFκB target genes and reduced survival of patients with metastatic prostate cancer. These findings highlight an association between GSK-3/AR and NFκB signaling and its potential clinical importance in metastatic prostate cancer.This study was funded by grants from the Spanish Ministry of Science and Innovation (SAF 2008-00768; SAF2011-30494), the Prostate Cancer Research Foundation (now PCUK) and the Department of Industry, Tourism and Trade (Etortek) and Department of Innovation Technology of the Government of the Autonomous Community of the Basque Country.Peer Reviewe

A screen for transcription factor targets of glycogen synthase kinase-3 highlights an inverse correlation of NFκB and androgen receptor signaling in prostate cancer

Expression of Glycogen Synthase Kinase-3 (GSK-3) is elevated in prostate cancer and its inhibition reduces prostate cancer cell proliferation, in part by reducing androgen receptor (AR) signaling. However, GSK-3 inhibition can also activate signals that promote cell proliferation and survival, which may preclude the use of GSK-3 inhibitors in the clinic. To identify such signals in prostate cancer, we screened for changes in transcription factor target DNA binding activity in GSK-3-silenced cells. Among the alterations was a reduction in AR DNA target binding, as predicted from previous studies, and an increase in NFκB DNA target binding. Consistent with the latter, gene silencing of GSK-3 or inhibition using the GSK-3 inhibitor CHIR99021 increased basal NFκB transcriptional activity. Activation of NFκB was accompanied by an increase in the level of the NFκB family member RelB. Conversely, silencing RelB reduced activation of NFκB by CHIR99021. Furthermore, the reduction of prostate cancer cell proliferation by CHIR99021 was potentiated by inhibition of NFκB signaling using the IKK inhibitor PS1145. Finally, stratification of human prostate tumor gene expression data for GSK3 revealed an inverse correlation between NFκB-dependent and androgen-dependent gene expression, consistent with the results from the transcription factor target DNA binding screen. In addition, there was a correlation between expression of androgen-repressed NFκB target genes and reduced survival of patients with metastatic prostate cancer. These findings highlight an association between GSK-3/AR and NFκB signaling and its potential clinical importance in metastatic prostate cancer

Notch activates cell cycle reentry and progression in quiescent cardiomyocytes

The inability of heart muscle to regenerate by replication of existing cardiomyocytes has engendered considerable interest in identifying developmental or other stimuli capable of sustaining the proliferative capacity of immature cardiomyocytes or stimulating division of postmitotic cardiomyocytes. Here, we demonstrate that reactivation of Notch signaling causes embryonic stem cell–derived and neonatal ventricular cardiomyocytes to enter the cell cycle. The proliferative response of neonatal ventricular cardiomyocytes declines as they mature, such that late activation of Notch triggers the DNA damage checkpoint and G2/M interphase arrest. Notch induces recombination signal-binding protein 1 for Jκ (RBP-Jκ)-dependent expression of cyclin D1 but, unlike other inducers, also shifts its subcellular distribution from the cytosol to the nucleus. Nuclear localization of cyclin D1 is independent of RBP-Jκ. Thus, the influence of Notch on nucleocytoplasmic localization of cyclin D1 is an unanticipated property of the Notch intracellular domain that is likely to regulate the cell cycle in multiple contexts, including tumorigenesis as well as cardiogenesis

Endocytosis as a biological response in receptor pharmacology: evaluation by fluorescence microscopy

The activation of G-protein coupled receptors by agonist compounds results in diverse biological responses in cells, such as the endocytosis process consisting in the translocation of receptors from the plasma membrane to the cytoplasm within internalizing vesicles or endosomes. In order to functionally evaluate endocytosis events resulted from pharmacological responses, we have developed an image analysis method -the Q-Endosomes algorithm- that specifically discriminates the fluorescent signal originated at endosomes from that one observed at the plasma membrane in images obtained from living cells by fluorescence microscopy. Mu opioid (MOP) receptor tagged at the carboxy-terminus with yellow fluorescent protein (YFP) and permanently expressed in HEK293 cells was used as experimental model to validate this methodology. Time-course experiments performed with several agonists resulted in different sigmoid curves depending on the drug used to initiate MOP receptor endocytosis. Thus, endocytosis resulting from the simultaneous activation of co-expressed MOP and serotonin 5-HT2C receptors by morphine plus serotonin was significantly different, in kinetics as well as in maximal response parameters, from the one caused by DAMGO, sufentanyl or methadone. Therefore, this analytical tool permits the pharmacological characterization of receptor endocytosis in living cells with functional and temporal resolution

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Presentació

Noise in gene expression limits the channel capacity of bacterial sensors

Trabajo presentado en EMBO Workshop Bacterial networks, celebrado en Sant Feliu de Guixols (España) del 04 al 09 de septiembre de 2022

El efecto sinérgico de dos mutaciones en una cianobacteria evolucionada experimentalmente genera un chasis autotrófico con tiempo de generación reducido

Trabajo presentado en el II Basque Microbiology Meeting MikrobioGUNE, celebrado en Bilbao (España) el 13 de diciembre de 2022

Estudio del crecimiento en cianobacterias a nivel unicelular

Trabajo presentado en el II Basque Microbiology Meeting MikrobioGUNE, celebrado en Bilbao (España) el 13 de diciembre de 2022

Dorso-ventral polarity is not required for digit tip regeneration

Trabajo presentado en The Company of Biologists Workshop Molecular mechanisms of developmental and regenerative biology (EMBO), celebrado en modalidad virtual del 26 al 29 de abril de 2022.The regeneration of the digit tip is an exceptional model of multi-tissue regeneration in mammals, with remarkable potential to advance regenerative medicine. Digit tip regeneration positively correlates with the presence of the nail organ, most likely because of the environment of the nail stem cells, but the underlying mechanisms are still unclear. The fact that the nail is a dorsal structure links regeneration with dorso-ventral (DV) polarity. Here, to evaluate the nail function more directly, and to disentangle it from DV positional information, we tested the regenerative ability of double-ventral digits and double-dorsal digits resulting from the limb-specific loss of Lmx1b and of En1 expression, respectively. Our results indicate that DV polarity is not required for digit tip regeneration but that an excessive inflammatory response prevents regeneration in the absence of Lmx1b. The possible causes of the exacerbated inflammation in the absence of Lmx1b are discussed

Absence of digit tip regeneration in Lmx1b-deficient nailless digits suggests a role for Lmx1b distinct from patterning

Trabajo presentado en el 19th International Congress of Developmental Biology, celebrado en Guia (Portugal) del 16 al 20 de octubre de 2022.Regeneration in mammals is limited to the digit tips. The amputation of the distal tip of the terminal phalanx triggers the formation of an undifferentiated pool of progenitor cells called a blastema that will regenerate the multiple tissues required to restore the missing part. Digit tip regeneration is linked to the presence of the dorsal nail organ, as only amputations that conserve the nail matrix regenerate. However, the regenerative potential of digits without nails has not yet been tested. To evaluate nailless digits, we used dLARM1/2 mutants that develop digits with no nails due to the limb-specific loss of the dorsal limb determinant Lmx1b. We report that dLARM1/2 mutant digits are unable to regenerate, although they form a blastema. To test whether the lack of regeneration was due to the absence of dorso-ventral (DV) polarity, a concept suggested in amphibia, we evaluated Del(27) mutants that also lack DV polarity with nails on both dorsal and ventral aspects of distal phalanges due to ventral overexpression of Lmx1b. Unlike dLARM1/2 mutants, Del(27) mutant digits regenerated indicating that DV polarity is dispensable for regeneration. Interestingly, Lmx1b expression (which continues in the nail dermis postnatally) is detected uniformly in the blastema of WT, but not dLARM1/2 mutant mice. In addition, comparison of dLARM1/2 vs WT blastemal transcriptomes revealed that the mutants had defective pro regenerative processes such as vasculogenesis and extracellular matrix remodeling, while inflammatory pathways were exacerbated. Gene expressions typical of the nail dermis were also lost in dLARM1/2 digit tips, suggesting regulation by Lmx1b. Collectively, our data suggest a direct role for Lmx1b in digit tip regeneration maintaining the nail dermis and promoting a competent blastema