title: 1α,25 dihydroxi-vitamin D₃ modulates CDK4 and CDK6 expression and localization.
abstract: We recently reported that the vitamin D receptor (VDR) and p38 MAPK participate in pro-differentiation events triggered by 1α,25(OH)₂-vitamin D₃ [1,25D] in skeletal muscle cells. Specifically, our studies demonstrated that 1,25D promotes G0/G1 arrest of cells inducing cyclin D3 and cyclin dependent kinases inhibitors (CKIs) p21(Waf1/Cip1) and p27(Kip1) expression in a VDR and p38 MAPK dependent manner. In this work we present data indicating that cyclin-dependent kinases (CDKs) 4 and 6 also play a role in the mechanism by which 1,25D stimulates myogenesis. To investigate VDR involvement in hormone regulation of CDKs 4 and 6, we significantly reduced its expression by the use of a shRNA against mouse VDR, generating the skeletal muscle cell line C2C12-VDR. Investigation of changes in cellular cycle regulating proteins by immunoblotting showed that the VDR is involved in the 1,25D -induced CDKs 4 and 6 protein levels at 6 h of hormone treatment. CDK4 levels remains high during S phase peak and G0/G1 arrest while CDK6 expression decreases at 12 h and increases again al 24 h. The up-regulation of CDKs 4 and 6 by 1,25D (6 h) was abolished in C2C12 cells pre-treated with the ERK1/2 inhibitor, UO126. Moreover, CDKs 4 and 6 expression induced by the hormone nor was detected when α and β isoforms of p38 MAPK were inhibited by compound SB203580. Confocal images show that there is not co-localization between VDR and CDKs at 6 h of hormone treatment, however CDK4 and VDR co-localizates in nucleus after 12 h of 1,25D exposure. Of relevance, at this time 1,25D promotes CDK6 localization in a peri-nuclear ring. Our data demonstrate that the VDR, ERK1/2 and p38 MAPK are involved in the control of CDKs 4 and 6 by 1,25D in skeletal muscle cells sustaining the operation of a VDR and MAPKs -dependent mechanism in hormone modulation of myogenesis.
authors.affiliation: INBIOSUR-CONICET y Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, San Juan 670, 8000 Bahía Blanca, Argentina.
authors.forename: Ana P
authors.initials: AP
authors.lastname: Irazoqui
authors.affiliation: INBIOSUR-CONICET y Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, San Juan 670, 8000 Bahía Blanca, Argentina.
authors.forename: Nadia B
authors.initials: NB
authors.lastname: Heim
authors.affiliation: INBIOSUR-CONICET y Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, San Juan 670, 8000 Bahía Blanca, Argentina.
authors.forename: Ricardo L
authors.initials: RL
authors.lastname: Boland
authors.affiliation: INBIOSUR-CONICET y Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, San Juan 670, 8000 Bahía Blanca, Argentina. Electronic address: cbuitrag@criba.edu.ar.
authors.forename: Claudia G
authors.initials: CG
authors.lastname: Buitrago
mesh_terms: D000818:Animals; D002117:Calcitriol / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4 / Q000378:metabolism*; D051361:Cyclin-Dependent Kinase 6 / Q000378:metabolism*; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018167:Receptors, Calcitriol / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D018167:Receptors, Calcitriol; C495902:Cdk4 protein, mouse; C495912:Cdk6 protein, mouse; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; D002117:Calcitriol
keywords: 1α,25 dihydroxi-vitamin D(3); Cyclin dependent kinases; Myogenesis; Skeletal muscle cells