Calpain Drives Megakaryopoiesis by Targeting the 7SK snRNP Complex: A Connection to Down Syndrome Megakaryocytic Neoplasia
Notice bibliographique
Résumé
Abstract Abstract 552 Megakaryocytic differentiation uniquely relies on a hypertrophic program involving massive cellular enlargement and nuclear polyploidization. A critical mechanism driving this program consists of global P-TEFb activation, a process involving the release of an active Cdk9-cyclin T kinase module from a larger, inactive 7SK snRNP complex. We previously implicated the protease complex calpain 2/S1 in P-TEFb activation during megakaryopoiesis and demonstrated a direct contribution of calpain activity to megakaryocytic differentiation (Elagib et al., ASH 2009). The clinical relevance of this pathway was suggested by findings in patient samples and knockin mice of defective calpain 2 upregulation in megakaryocytes bearing the leukemogenic GATA-1s mutant. Current studies address: 1) the mechanism by which calpain 2/S1 activates P-TEFb during normal megakaryopoiesis, 2) the genetic program controlled by P-TEFb during megakaryopoiesis, and 3) the relevance of calpain 2 deficiency to abnormal megakaryopoiesis associated with GATA-1s. Regarding the mechanism of P-TEFb activation by calpain, analysis of a P-TEFb interactome database identified an interaction of calpain S1 with the protein BCDIN3 (Jeronimo CD. et. al. Mol Cell 2007). BCDIN3 stabilizes the 7SK snRNA through capping activity, but also exerts enzyme-independent stabilization by forming a core complex with 7SK snRNA and the protein LARP7. This core complex provides the foundation for assembly of the 7SK snRNP involved in restraining P-TEFb activity. Using adult human CD34+ cells cultured in unilineage megakaryocytic medium, both BCDIN3 and LARP7 proteins were found to undergo dramatic downregulation during differentiation, leading to global destabilization of 7SK snRNA. By contrast, neither BCDIN3, LARP7, nor 7SK snRNA underwent downregulation during erythroid differentiation. Megakaryocytic downregulation of BCDIN3, but not LARP7, required calpain activity. Calpain inhibitors as well as shRNA knockdown of calpain 2 prevented BCDIN3 downregulation during megakaryocytic cultures. In vitro reactions with recombinant purified factors confirmed direct cleavage of BCDIN3 by calpain 2/S1. The role of BCDIN3 downregulation in megakaryopoiesis was assessed by shRNA knockdown experiments. Notably, enforcing downregulation of BCDIN3 in CD34+ cells in erythroid medium reprogrammed differentiation away from the erythroid lineage and toward the megakaryocytic lineage. Thus BCDIN3 is a critical target of calpain 2/S1 during megakaryocytic activation of P-TEFb. To identify P-TEFb target genes, a bioinformatic approach was used to screen megakaryocytic expression databases for genes coregulated with known P-TEFb targets, e.g. HEXIM1. This approach yielded a cohort of coregulated genes encoding actin associated factors: ACTN1, FLNA, TGFB1I1, and MKL1. Immunoblot analsysis validated that all four factors underwent concomitant upregulation during megakaryocytic but not erythroid differentiation. shRNA knockdowns of calpain 2 and Cdk9 confirmed that upregulation of these factors depended on calpain and P-TEFb activation. Previous studies have indicated a requirement for ACTN1 downregulation in cytokinesis. Thus, shRNA knockdowns determined whether ACTN1 upregulation contributed to megakaryocytic polyploidization. Strikingly, ACTN1 knockdown blocked the polyploidization of human CD34+ cells but allowed other features of megakaryocytic differentiation, e.g. CD41 upregulation. Thus, a key aspect of the genetic program controlled by P-TEFb in megakaryopoiesis consists of the upregulation of a cohort of actin-associated factors which contribute to a hypertrophic program of cellular enlargement and polyploidization. To address the role of calpain in the aberrant megakaryopoiesis associated with mutant GATA-1s, fetal liver (fl) cells from GATA-1s knockin (G1s ki) mice were studied. Previous studies have shown a hyperproliferative phenotype in G1s ki fl but not adult megakaryocytes. In the current study, enforced calpain 2 expression by lentiviral transduction reversed this phenotype and enhanced terminal differentiation, reflected by polyploidization and CD42 expression. Thus, the defective upregulation of calpain 2 in megakaryocytes with GATA-1s most likely plays a critical role in the abnormal megakaryopoiesis seen in Down syndrome-associated megakaryoctyic proliferative disorders. Disclosures: No relevant conflicts of interest to declare.
Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.
Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,002 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».