scholarly journals Πρόληψη της ανάπτυξης εξάρτησης από τη μορφίνη έπειτα από υπερέκφραση της πρωτεΐνης RGS9 στον εγκέφαλο

2011 ◽  
Author(s):  
Μαρία-Μάρθα Παπαχατζάκη
Keyword(s):  
Dominant Negative ◽  
Adeno Associated Virus

Μέχρι σήμερα τα οπιοειδή παραμένουν τα φάρμακα με την ισχυρότερη αναλγητική ανταπόκριση σε αρκετές μορφές πόνου. Ένα από τα μεγαλύτερα κλινικά αλλά και κοινωνικά προβλήματα στην αντιμετώπιση του χρόνιου πόνου είναι η ανάπτυξη εξάρτησης από τα οπιοειδή σε ασθενείς που χρειάζονται χρόνια αναλγητική θεραπεία και η εμφάνιση στερητικού συνδρόμου μετά την απότομη διακοπή χορήγησης τους. Πέραν όμως από το πρόβλημα της εξάρτησης στην χρόνια λήψη οπιοειδών, οι ασθενείς παρουσιάζουν και μείωση της αναλγητικής τους ανταπόκρισης, λόγω ανάπτυξης ανοχής στην χορηγούμενη δόση μορφίνης. Η ανοχή οδηγεί σε συνεχή αύξηση της δοσολογίας του φαρμάκου, προκειμένου να επιτευχθεί το αναμενόμενο αναλγητικό αποτέλεσμα, με συνέπεια, την ανάπτυξη εξάρτησης στην ουσία και την εμφάνιση σοβαρών παρενεργειών τόσο στο ΚΝΣ, όσο και σε περιφερικούς ιστούς. Όλα αυτά, καθιστούν την χορήγηση της μορφίνης, καθώς και άλλων οπιοειδών φαρμάκων προβληματική, σε ασθενείς που θα την είχαν ανάγκη για μεγάλο χρονικό διάστημα καθώς και περιορισμένη κυρίως σε κλινικές οντότητες με χαμηλό προσδόκιμο ζωής ή τελικού σταδίου νόσου (πχ καρκινοπαθείς). Οι αλλαγές αυτές της δράσης της μορφίνης στον οργανισμό είναι αποτέλεσμα προσαρμοστικών μεταβολών που συμβαίνουν σε κυτταρικό επίπεδο και σε συγκεκριμένα νευρωνικά υποστρώματα. Οι ακριβείς μηχανισμοί αυτών των μεταβολών δεν είναι απολύτως διευκρινισμένοι και πιθανώς συμπεριλαμβάνουν τροποποιήσεις σε πολλαπλά νευρωνικά δίκτυα και στην έκφραση μεγάλου αριθμού γονιδίων, άρα και πρωτεϊνών. Για τον λόγο αυτό, είναι σημαντική η ταυτοποίηση των πρωτεϊνών που συμμετέχουν στους προσαρμοστικούς μηχανισμούς που οδηγούν στην ανάπτυξη ανοχής και εξάρτησης του οργανισμού στην μορφίνη. Μία ομάδα πρωτεϊνών που συμβάλει στους προσαρμοστικούς μηχανισμούς της χρόνιας χορήγησης μορφίνης στον οργανισμό είναι η οικογένεια των RGS πρωτεϊνών. Οι πρωτεΐνες αυτές ρυθμίζουν την ένταση και διάρκεια της δραστικότητας των G πρωτεϊνών, ρυθμίζοντας έτσι έμμεσα το εύρος σηματοδότησης των υποδοχέων που είναι προσδεδεμένοι με τις G πρωτεΐνες. Οι RGS πρωτεΐνες παίζουν ρόλο σε πολλές χρόνιες ασθένειες, όπως η σχιζοφρένεια, η κατάθλιψη και η εξάρτηση στις ουσίες. Συγκεκριμένα, μια σειρά πρόσφατων μελετών, συνδέει την πρωτεΐνη RGS9-2 με την εξάρτηση. Η RGS9-2, η οποία παρουσιάζει πλούσια έκφραση στο κέντρο εθισμού, ρυθμίζει την διάρκεια ενεργοποίησης της άλφα υπομονάδας των G πρωτεϊνών, μέσω κατάλυσης της επανασύνδεσης των α και βγ υπομονάδων της, άρα και την λήξη σηματοδότησης του υποδοχέα. Πολλαπλές in vitro και in vivo μελέτες έδειξαν ότι η RGS9-2 ρυθμίζει δυναμικά την ανταπόκριση των μ-οπιοειδών υποδοχέων (MOR) και παίζει σημαντικό ρόλο στις δράσεις της μορφίνης ενώ επίσης προάγει την ευαισθητοποίηση στις ανταμοιβικές ιδιότητες των οπιοειδών και ψυχοδιεγερτικών ουσιών (Burns&Wensel 2003, Rahman et al 2003, Zachariou et al 2003, Psifogeorgou et al. 2007& 2010). Πιο συγκεκριμένα, προηγούμενες μελέτες του εργαστηρίου μας σε διαγονιδιακά ζώα, με καθολική απαλοιφή του γονιδίου της RGS9, έδειξαν ότι τα ζώα αυτά εμφανίζουν αυξημένη συμπεριφορική ευαισθησία στην ανταμοιβική δράση της μορφίνης, σε σχέση με τα αγρίου τύπου ζώα, αναλγητική ανταπόκριση σε πολύ χαμηλότερες δόσεις από αυτές που χρειάζονταν τα αγρίου τύπου, καθώς και καθυστέρηση στην ανάπτυξη ανοχής (Zachariou et al 2003). Τα δεδομένα αυτά δείχνουν ότι επέμβαση στη δράση της RGS9-2, επηρεάζει δυναμικά τις δράσεις της μορφίνης στο ΚΝΣ και καθιστούν το μόριο αυτό, σημαντικό φαρμακολογικό στόχο. Στόχος της παρούσας διατριβής είναι να διερευνηθούν οι επιδράσεις της στοχευμένης τροποποίησης των επίπεδων της RGS9-2 σε ειδικούς πυρήνες του εγκεφάλου, όπου εμφανίζει πλούσια κατανομή. Το ραχιαίο ραβδωτό και ιδίως ο επικλινής πυρήνας (κοιλιακό ραβδωτό), αποτελούν τους σημαντικότερους πυρήνες στο παθοφυσιολογικό και φαρμακολογικό μονοπάτι του εθισμού, αφού ρυθμίζουν τις ενισχυτικές και εξαρτησιογόνες δράσεις των οπιοειδών έπειτα απο διέγερση των μ-οπιοειδών υποδοχέων και εμφανίζουν τα υψηλότερα επίπεδα έκφρασης της πρωτείνης στον εγκέφαλο. Πέραν των ανωτέρω περιοχών όμως, αρκετά υψηλά επίπεδα έκφρασης της RGS9-2 πρωτεΐνης έχουν ανιχνευθεί και στα οπίσθια κέρατα του νωτιαίου μυελού, που όπως είναι γνωστό, αποτελούν σημαντικές περιοχές νευροδιαβίβασης των επώδυνων ερεθισμάτων, καθώς και στόχο αρκετών αναλγητικών φαρμάκων όπως τα οπιοειδή. Όλες οι προαναφερόμενες περιοχές εκφράζουν σε υψηλά επίπεδα την RGS9-2. Με στόχο την διερεύνηση του ρόλου της RGS9-2 στις συγκεκριμένες περιοχές του ΚΝΣ, δημιουργήθηκαν γενετικά μοντέλα υπερέκφρασης της. Συγκεκριμένα χρησιμοποιήθηκαν AAV ιϊκά οχήματα αδενοσυσχετιζόμενων ιών (adeno associated virus) τα οποία εγχύθηκαν μέσω στερεοταξικής επέμβασης σε επιλεγμένες περιοχές του εγκεφάλου. Οι ιοί που χρησιμοποιούνται στην συγκεκριμένη διατριβή υπερεκφράζουν την RGS9-2 ή την μεταλλαγμένη μορφή της πρωτεΐνης η οποία έχει dominant negative δράση. Συγκεκριμένα, οι ιοί που κατασκευάστηκαν, φέρουν είτε την RGS9-2 σεσημασμένη με GFP, είτε την μεταλλαγμένη μορφή της RGS9-2 DEPless (η RGS9-2 πρωτεΐνη χωρίς το DEP domain της, το οποίο της προσδίδει την ικανότητα πρόσδεσης της στην κυτταρική μεμβράνη, άρα και την δημιουργία σταθερών συμπλόκων κοντά σ' αυτήν) σεσημασμένη με GFP, με στόχο την υπερέκφραση των πρωτεϊνών αυτών στις περιοχές του εγκεφάλου που διοχετεύεται ο ιός. Προ έναρξης ευρείας χρήσης των AAV οχημάτων σε συμπεριφορικές μελέτες έγινε αρχικά ταυτοποίηση του ποσοστού υπερέκφρασης και της κατανομής του ιού στην περιοχή έγχυσης καθώς και στα νευρικά κύτταρα στα οποία εκφράζεται ο ιός υπερέκφρασης της RGS9-2. Μελέτες που έγιναν σε πρωτογενείς κυτταρικές σειρές, δείχνουν ότι η παρουσία της μεταλλαγμένης RGS9-2 (RGS9-2-DEPless) παίζει dominant negative ρόλο (Psifogeorgou et al. 2007), ανταγωνίζεται δηλαδή την δράση της ενδογενούς RGS9-2 πρωτεΐνης σχηματίζοντας σύμπλοκα με τα σηματοδοτικά της μόρια μακριά από την κυτταρική μεμβράνη - λόγω έλλειψης του DEP τμήματος από την κυτταρική δομή της RGS9-2 πρωτεΐνης (Kassi Psifogeorgiou 2007). Επίσης κατασκευάστηκε ιός ο οποίος έφερε μόνο το γονίδιο της EGFP για την παραγωγή της ομάδας των ποντικών μαρτύρων. Στην συνέχεια μελετήθηκαν οι επιπτώσεις της υπερέκφρασης ή ανταγωνισμού δράσης της RGS9-2 στις συγκεκριμένες περιοχές του εγκεφάλου, στην συμπεριφορά των ζώων μετά από εφάπαξ ή χρόνια χορήγηση μορφίνης, μέσω ευρέως γνωστών συμπεριφορικών παραδειγμάτων αξιολόγησης της εξάρτησης, ανταμοιβής, ανοχής και αναλγησίας στην μορφίνη. Μέσω αυτών διαπιστώθηκε: 1) η σπουδαιότητα της δράσης της RGS9-2 στον επικλινή πυρήνα και στους μηχανισμούς ανάπτυξης εθισμού, αναλγησίας και ανοχής στα οπιοειδή, 2) η σπουδαιότητα της δράσης της RGS9-2 στο ραχιαίο ραβδωτό και στους μηχανισμούς εθισμού, αναλγησίας και ανοχής στα οπιοειδή, 3) η περαιτέρω σχέση των πυρήνων αυτών με τον εθισμό και την αναλγησία, καθώς και 4) ο ρόλος της RGS9-2 στα οπίσθια κέρατα του νωτιαίου μυελού στις αναλγητικές δράσεις της μορφίνης.

scholarly journals N6-methyladenosine demethylase FTO impairs hepatic ischemia–reperfusion injury via inhibiting Drp1-mediated mitochondrial fragmentation

2021 ◽  
Vol 12 (5) ◽  
Author(s):  
Ying Dong Du ◽  
Wen Yuan Guo ◽  
Cong Hui Han ◽  
Ying Wang ◽  
Xiao Song Chen ◽  
...  
Keyword(s):  
Reperfusion Injury ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Mitochondrial Fragmentation ◽  
Hepatic Ischemia ◽  
Adeno Associated Virus ◽  
Downstream Target ◽  
Hepatic Ischemia Reperfusion

AbstractDespite N6-methyladenosine (m6A) is functionally important in various biological processes, its role and the underlying regulatory mechanism in the liver remain largely unexplored. In the present study, we showed that fat mass and obesity-associated protein (FTO, an m6A demethylase) was involved in mitochondrial function during hepatic ischemia–reperfusion injury (HIRI). We found that the expression of m6A demethylase FTO was decreased during HIRI. In contrast, the level of m6A methylated RNA was enhanced. Adeno-associated virus-mediated liver-specific overexpression of FTO (AAV8-TBG-FTO) ameliorated the HIRI, repressed the elevated level of m6A methylated RNA, and alleviated liver oxidative stress and mitochondrial fragmentation in vivo and in vitro. Moreover, dynamin-related protein 1 (Drp1) was a downstream target of FTO in the progression of HIRI. FTO contributed to the hepatic protective effect via demethylating the mRNA of Drp1 and impairing the Drp1-mediated mitochondrial fragmentation. Collectively, our findings demonstrated the functional importance of FTO-dependent hepatic m6A methylation during HIRI and provided valuable insights into the therapeutic mechanisms of FTO.

scholarly journals The Diagnostic Journey of a Patient with Prader–Willi-Like Syndrome and a Unique Homozygous SNURF-SNRPN Variant; Bio-Molecular Analysis and Review of the Literature

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 875
Author(s):  
Karlijn Pellikaan ◽  
Geeske M. van Woerden ◽  
Lotte Kleinendorst ◽  
Anna G. W. Rosenberg ◽  
Bernhard Horsthemke ◽  
...  
Keyword(s):  
Intellectual Disability ◽  
Single Nucleotide Polymorphism Array ◽  
Genetic Defect ◽  
Missense Variant ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Pituitary Hormone ◽  
Negative Effect

Prader–Willi syndrome (PWS) is a rare genetic condition characterized by hypotonia, intellectual disability, and hypothalamic dysfunction, causing pituitary hormone deficiencies and hyperphagia, ultimately leading to obesity. PWS is most often caused by the loss of expression of a cluster of genes on chromosome 15q11.2-13. Patients with Prader–Willi-like syndrome (PWLS) display features of the PWS phenotype without a classical PWS genetic defect. We describe a 46-year-old patient with PWLS, including hypotonia, intellectual disability, hyperphagia, and pituitary hormone deficiencies. Routine genetic tests for PWS were normal, but a homozygous missense variant NM_003097.3(SNRPN):c.193C>T, p.(Arg65Trp) was identified. Single nucleotide polymorphism array showed several large regions of homozygosity, caused by high-grade consanguinity between the parents. Our functional analysis, the ‘Pipeline for Rapid in silico, in vivo, in vitro Screening of Mutations’ (PRiSM) screen, showed that overexpression of SNRPN-p.Arg65Trp had a dominant negative effect, strongly suggesting pathogenicity. However, it could not be confirmed that the variant was responsible for the phenotype of the patient. In conclusion, we present a unique homozygous missense variant in SNURF-SNRPN in a patient with PWLS. We describe the diagnostic trajectory of this patient and the possible contributors to her phenotype in light of the current literature on the genotype–phenotype relationship in PWS.

scholarly journals Mirtron-mediated RNA knockdown/replacement therapy for the treatment of dominant retinitis pigmentosa

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Harry O. Orlans ◽  
Michelle E. McClements ◽  
Alun R. Barnard ◽  
Cristina Martinez-Fernandez de la Camara ◽  
Robert E. MacLaren
Keyword(s):  
Retinitis Pigmentosa ◽  
Autosomal Dominant ◽  
Gene Mutations ◽  
Adeno Associated Virus ◽  
Treatment Development ◽  
Common Cause ◽  
Toxic Protein ◽  
Rna Knockdown

AbstractRhodopsin (RHO) gene mutations are a common cause of autosomal dominant retinitis pigmentosa (ADRP). The need to suppress toxic protein expression together with mutational heterogeneity pose challenges for treatment development. Mirtrons are atypical RNA interference effectors that are spliced from transcripts as short introns. Here, we develop a novel mirtron-based knockdown/replacement gene therapy for the mutation-independent treatment of RHO-related ADRP, and demonstrate efficacy in a relevant mammalian model. Splicing and potency of rhodopsin-targeting candidate mirtrons are initially determined, and a mirtron-resistant codon-modified version of the rhodopsin coding sequence is validated in vitro. These elements are then combined within a single adeno-associated virus (AAV) and delivered subretinally in a RhoP23H knock-in mouse model of ADRP. This results in significant mouse-to-human rhodopsin RNA replacement and is associated with a slowing of retinal degeneration. This provides proof of principle that synthetic mirtrons delivered by AAV are capable of reducing disease severity in vivo.

scholarly journals Extracellular Signal-Regulated Kinase Binds to TFII-I and Regulates Its Activation of the c-fosPromoter

2000 ◽  
Vol 20 (4) ◽  
pp. 1140-1148 ◽  
Author(s):  
Dae-Won Kim ◽  
Brent H. Cochran
Keyword(s):  
Map Kinase ◽  
Transcriptional Activation ◽  
Dominant Negative ◽  
Binding Motif ◽  
Dependent Manner ◽  
Consensus Map ◽  
Interaction Domain ◽  
Extracellular Signal

ABSTRACT We have previously shown that TFII-I enhances transcriptional activation of the c-fos promoter through interactions with upstream elements in a signal-dependent manner. Here we demonstrate that activated Ras and RhoA synergize with TFII-I for c-fospromoter activation, whereas dominant-negative Ras and RhoA inhibit these effects of TFII-I. The Mek1 inhibitor, PD98059 abrogates the enhancement of the c-fos promoter by TFII-I, indicating that TFII-I function is dependent on an active mitogen-activated protein (MAP) kinase pathway. Analysis of the TFII-I protein sequence revealed that TFII-I contains a consensus MAP kinase interaction domain (D box). Consistent with this, we have found that TFII-I forms an in vivo complex with extracellular signal-related kinase (ERK). Point mutations within the consensus MAP kinase binding motif of TFII-I inhibit its ability to bind ERK and its ability to enhance the c-fos promoter. Therefore, the D box of TFII-I is required for its activity on the c-fos promoter. Moreover, the interaction between TFII-I and ERK can be regulated. Serum stimulation enhances complex formation between TFII-I and ERK, and dominant-negative Ras abrogates this interaction. In addition, TFII-I can be phosphorylated in vitro by ERK and mutation of consensus MAP kinase substrate sites at serines 627 and 633 impairs the phosphorylation of TFII-I by ERK and its activity on the c-fos promoter. These results suggest that ERK regulates the activity of TFII-I by direct phosphorylation.

scholarly journals Dominant-Negative FADD Rescues the In Vivo Fitness of a Cytomegalovirus Lacking an Antiapoptotic Viral Gene

2007 ◽  
Vol 82 (5) ◽  
pp. 2056-2064 ◽  
Author(s):  
Luka Čičin-Šain ◽  
Zsolt Ruzsics ◽  
Juergen Podlech ◽  
Ivan Bubić ◽  
Carine Menard ◽  
...  
Keyword(s):  
Virus Replication ◽  
Control Cell ◽  
Death Receptor ◽  
Formal Proof ◽  
Dominant Negative ◽  
Viral Fitness ◽  
Viral Gene ◽  
Apoptosis Inhibition

ABSTRACT Genes that inhibit apoptosis have been described for many DNA viruses. Herpesviruses often contain even more than one gene to control cell death. Apoptosis inhibition by viral genes is postulated to contribute to viral fitness, although a formal proof is pending. To address this question, we studied the mouse cytomegalovirus (MCMV) protein M36, which binds to caspase-8 and blocks death receptor-induced apoptosis. The growth of MCMV recombinants lacking M36 (ΔM36) was attenuated in vitro and in vivo. In vitro, caspase inhibition by zVAD-fmk blocked apoptosis in ΔM36-infected macrophages and rescued the growth of the mutant. In vivo, ΔM36 infection foci in liver tissue contained significantly more apoptotic hepatocytes and Kupffer cells than did revertant virus foci, and apoptosis occurred during the early phase of virus replication prior to virion assembly. To further delineate the mode of M36 function, we replaced the M36 gene with a dominant-negative FADD (FADDDN) in an MCMV recombinant. FADDDN was expressed in cells infected with the recombinant and blocked the death-receptor pathway, replacing the antiapoptotic function of M36. Most importantly, FADDDN rescued ΔM36 virus replication, both in vitro and in vivo. These findings have identified the biological role of M36 and define apoptosis inhibition as a key determinant of viral fitness.

scholarly journals Novel Caprine Adeno-Associated Virus (AAV) Capsid (AAV-Go.1) Is Closely Related to the Primate AAV-5 and Has Unique Tropism and Neutralization Properties

2005 ◽  
Vol 79 (24) ◽  
pp. 15238-15245 ◽  
Author(s):  
Alejandra E. Arbetman ◽  
Michael Lochrie ◽  
Shangzhen Zhou ◽  
Jennifer Wellman ◽  
Ciaran Scallan ◽  
...  
Keyword(s):  
Protein Expression ◽  
Neutralization Assay ◽  
Biological Properties ◽  
Scid Mice ◽  
Factor Ix ◽  
In Vivo Model ◽  
Adeno Associated Virus ◽  
Human Factor Ix

ABSTRACT Preexisting humoral immunity to adeno-associated virus (AAV) vectors may limit their clinical utility in gene delivery. We describe a novel caprine AAV (AAV-Go.1) capsid with unique biological properties. AAV-Go.1 capsid was cloned from goat-derived adenovirus preparations. Surprisingly, AAV-Go.1 capsid was 94% identical to the human AAV-5, with differences predicted to be largely on the surface and on or under the spike-like protrusions. In an in vitro neutralization assay using human immunoglobulin G (IgG) (intravenous immune globulin [IVIG]), AAV-Go.1 had higher resistance than AAV-5 (100-fold) and resistance similar to that of AAV-4 or AAV-8. In an in vivo model, SCID mice were pretreated with IVIG to generate normal human IgG plasma levels prior to the administration of AAV human factor IX vectors. Protein expression after intramuscular administration of AAV-Go.1 was unaffected in IVIG-pretreated mice, while it was reduced 5- and 10-fold after administration of AAV-1 and AAV-8, respectively. In contrast, protein expression after intravenous administration of AAV-Go.1 was reduced 7.1-fold, similar to the 3.8-fold reduction observed after AAV-8administration in IVIG-pretreated mice, and protein expression was essentially extinguished after AAV-2 administration in mice pretreated with much less IVIG (15-fold). AAV-Go.1 vectors also demonstrated a marked tropism for lung when administered intravenously in SCID mice. The pulmonary tropism and high neutralization resistance to human preexisting antibodies suggest novel therapeutic uses for AAV-Go.1 vectors, including targeting diseases such as cystic fibrosis. Nonprimate sources of AAVs may be useful to identify additional capsids with distinct tropisms and high resistance to neutralization by human preexisting antibodies.

scholarly journals Cleavage and Inactivation of ATM during Apoptosis

1999 ◽  
Vol 19 (9) ◽  
pp. 6076-6084 ◽  
Author(s):  
Graeme C. M. Smith ◽  
Fabrizio d’adda di Fagagna ◽  
Nicholas D. Lakin ◽  
Stephen P. Jackson
Keyword(s):  
Dna Damage ◽  
Dna Binding ◽  
Caspase 3 ◽  
Cysteine Proteases ◽  
Dominant Negative ◽  
Protein Kinase Activity ◽  
Dna Damage Signalling ◽  
In Cells

ABSTRACT The activation of the cysteine proteases with aspartate specificity, termed caspases, is of fundamental importance for the execution of programmed cell death. These proteases are highly specific in their action and activate or inhibit a variety of key protein molecules in the cell. Here, we study the effect of apoptosis on the integrity of two proteins that have critical roles in DNA damage signalling, cell cycle checkpoint controls, and genome maintenance—the product of the gene defective in ataxia telangiectasia, ATM, and the related protein ATR. We find that ATM but not ATR is specifically cleaved in cells induced to undergo apoptosis by a variety of stimuli. We establish that ATM cleavage in vivo is dependent on caspases, reveal that ATM is an efficient substrate for caspase 3 but not caspase 6 in vitro, and show that the in vitro caspase 3 cleavage pattern mirrors that in cells undergoing apoptosis. Strikingly, apoptotic cleavage of ATM in vivo abrogates its protein kinase activity against p53 but has no apparent effect on the DNA binding properties of ATM. These data suggest that the cleavage of ATM during apoptosis generates a kinase-inactive protein that acts, through its DNA binding ability, in a trans-dominant-negative fashion to prevent DNA repair and DNA damage signalling.

109 Dominant-negative TGFβ receptor 2 enhances GPC3-targeting CAR-T cell efficacy against hepatocellular carcinoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A121-A121
Author(s):  
Nina Chu ◽  
Michael Overstreet ◽  
Ryan Gilbreth ◽  
Lori Clarke ◽  
Christina Gesse ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Dominant Negative ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundChimeric antigen receptors (CARs) are engineered synthetic receptors that reprogram T cell specificity and function against a given antigen. Autologous CAR-T cell therapy has demonstrated potent efficacy against various hematological malignancies, but has yielded limited success against solid cancers. MEDI7028 is a CAR that targets oncofetal antigen glypican-3 (GPC3), which is expressed in 70–90% of hepatocellular carcinoma (HCC), but not in normal liver tissue. Transforming growth factor β (TGFβ) secretion is increased in advanced HCC, which creates an immunosuppressive milieu and facilitates cancer progression and poor prognosis. We tested whether the anti-tumor efficacy of a GPC3 CAR-T can be enhanced with the co-expression of dominant-negative TGFβRII (TGFβRIIDN).MethodsPrimary human T cells were lentivirally transduced to express GPC3 CAR both with and without TGFβRIIDN. Western blot and flow cytometry were performed on purified CAR-T cells to assess modulation of pathways and immune phenotypes driven by TGFβ in vitro. A xenograft model of human HCC cell line overexpressing TGFβ in immunodeficient mice was used to investigate the in vivo efficacy of TGFβRIIDN armored and unarmored CAR-T. Tumor infiltrating lymphocyte populations were analyzed by flow cytometry while serum cytokine levels were quantified with ELISA.ResultsArmoring GPC3 CAR-T with TGFβRIIDN nearly abolished phospho-SMAD2/3 expression upon exposure to recombinant human TGFβ in vitro, indicating that the TGFβ signaling axis was successfully blocked by expression of the dominant-negative receptor. Additionally, expression of TGFβRIIDN suppressed TGFβ-driven CD103 upregulation, further demonstrating attenuation of the pathway by this armoring strategy. In vivo, the TGFβRIIDN armored CAR-T achieved superior tumor regression and delayed tumor regrowth compared to the unarmored CAR-T. The armored CAR-T cells infiltrated HCC tumors more abundantly than their unarmored counterparts, and were phenotypically less exhausted and less differentiated. In line with these observations, we detected significantly more interferon gamma (IFNγ) at peak response and decreased alpha-fetoprotein in the serum of mice treated with armored cells compared to mice receiving unarmored CAR-T, demonstrating in vivo functional superiority of TGFβRIIDN armored CAR-T therapy.ConclusionsArmoring GPC3 CAR-T with TGFβRIIDN abrogates the signaling of TGFβ in vitro and enhances the anti-tumor efficacy of GPC3 CAR-T against TGFβ-expressing HCC tumors in vivo, proving TGFβRIIDN to be an effective armoring strategy against TGFβ-expressing solid malignancies in preclinical models.Ethics ApprovalThe study was approved by AstraZeneca’s Ethics Board and Institutional Animal Care and Use Committee (IACUC).

scholarly journals Critical role of the extracellular signal–regulated kinase–MAPK pathway in osteoblast differentiation and skeletal development

2007 ◽  
Vol 176 (5) ◽  
pp. 709-718 ◽  
Author(s):  
Chunxi Ge ◽  
Guozhi Xiao ◽  
Di Jiang ◽  
Renny T. Franceschi
Keyword(s):  
Transcriptional Activity ◽  
Mapk Pathway ◽  
Skeletal Development ◽  
Critical Role ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal

The extracellular signal–regulated kinase (ERK)–mitogen-activated protein kinase (MAPK) pathway provides a major link between the cell surface and nucleus to control proliferation and differentiation. However, its in vivo role in skeletal development is unknown. A transgenic approach was used to establish a role for this pathway in bone. MAPK stimulation achieved by selective expression of constitutively active MAPK/ERK1 (MEK-SP) in osteoblasts accelerated in vitro differentiation of calvarial cells, as well as in vivo bone development, whereas dominant-negative MEK1 was inhibitory. The involvement of the RUNX2 transcription factor in this response was established in two ways: (a) RUNX2 phosphorylation and transcriptional activity were elevated in calvarial osteoblasts from TgMek-sp mice and reduced in cells from TgMek-dn mice, and (b) crossing TgMek-sp mice with Runx2+/− animals partially rescued the hypomorphic clavicles and undemineralized calvaria associated with Runx2 haploinsufficiency, whereas TgMek-dn; Runx2+/− mice had a more severe skeletal phenotype. This work establishes an important in vivo function for the ERK–MAPK pathway in bone that involves stimulation of RUNX2 phosphorylation and transcriptional activity.

scholarly journals c-Jun N-Terminal Kinase Activation of Activator Protein-1 Underlies Homologous Regulation of the Gonadotropin-Releasing Hormone Receptor Gene in αT3-1 Cells

Endocrinology ◽  
2003 ◽  
Vol 144 (3) ◽  
pp. 839-849 ◽  
Author(s):  
Buffy S. Ellsworth ◽  
Brett R. White ◽  
Ann T. Burns ◽  
Brian D. Cherrington ◽  
Annette M. Otis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Cell Model ◽  
Receptor Gene ◽  
Critical Role ◽  
Dominant Negative ◽  
Activator Protein 1 ◽  
Activator Protein

Reproductive function is dependent on the interaction between GnRH and its cognate receptor found on gonadotrope cells of the anterior pituitary gland. GnRH activation of the GnRH receptor (GnRHR) is a potent stimulus for increased expression of multiple genes including the gene encoding the GnRHR itself. Thus, homologous regulation of the GnRHR is an important mechanism underlying gonadotrope sensitivity to GnRH. Previously, we have found that GnRH induction of GnRHR gene expression in αT3-1 cells is partially mediated by protein kinase C activation of a canonical activator protein-1 (AP-1) element. In contrast, protein kinase A and a cAMP response element-like element have been implicated in mediating the GnRH response of the GnRHR gene using a heterologous cell model (GGH3). Herein we find that selective removal of the canonical AP-1 site leads to a loss of GnRH regulation of the GnRHR promoter in transgenic mice. Thus, an intact AP-1 element is necessary for GnRH responsiveness of the GnRHR gene both in vitro and in vivo. Based on in vitro analyses, GnRH appeared to enhance the interaction of JunD, FosB, and c-Fos at the GnRHR AP-1 element. Although enhanced binding of cFos reflected an increase in gene expression, GnRH appeared to regulate both FosB and JunD at a posttranslational level. Neither overexpression of a constitutively active Raf-kinase nor pharmacological blockade of GnRH-induced ERK activation eliminated the GnRH response of the GnRHR promoter. GnRH responsiveness was, however, lost in αT3-1 cells that stably express a dominant-negative c-Jun N-terminal kinase (JNK) kinase, suggesting a critical role for JNK in mediating GnRH regulation of the GnRHR gene. Consistent with this possibility, we find that the ability of forskolin and membrane-permeable forms of cAMP to inhibit the GnRH response of the GnRHR promoter is associated with a loss of both JNK activation and GnRH-mediated recruitment of the primary AP-1-binding components.

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