title: Cell cycle and apoptosis regulatory protein (CARP)-1 is expressed in osteoblasts and regulated by PTH.
abstract: Bone mass is dependent on osteoblast proliferation, differentiation and life-span of osteoblasts. Parathyroid hormone (PTH) controls osteoblast cell cycle regulatory proteins and suppresses mature osteoblasts apoptosis. Intermittent administration of PTH increases bone mass but the mechanism of action are complex and incompletely understood. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 (aka CCAR1) is a novel transducer of signaling by diverse agents including cell growth and differentiation factors. To gain further insight into the molecular mechanism, we investigated involvement of CARP-1 in PTH signaling in osteoblasts. Immunostaining studies revealed presence of CARP-1 in osteoblasts and osteocytes, while a minimal to absent levels were noted in the chondrocytes of femora from 10 to 12-week old mice. Treatment of 7-day differentiated MC3T3-E1 clone-4 (MC-4) mouse osteoblastic cells and primary calvarial osteoblasts with PTH for 30min to 5h followed by Western blot analysis showed 2- to 3-fold down-regulation of CARP-1 protein expression in a dose- and time-dependent manner compared to the respective vehicle treated control cells. H-89, a Protein Kinase A (PKA) inhibitor, suppressed PTH action on CARP-1 protein expression indicating PKA-dependent mechanism. PMA, a Protein Kinase C (PKC) agonist, mimicked PTH action, and the PKC inhibitor, GF109203X, partially blocked PTH-dependent downregulation of CARP-1, implying involvement of PKC. U0126, a Mitogen-Activated Protein Kinase (MAPK) Kinase (MEK) inhibitor, failed to interfere with CARP-1 suppression by PTH. In contrast, SB203580, p38 inhibitor, attenuated PTH down-regulation of CARP-1 suggesting that PTH utilized an Extracellular Signal Regulated Kinase (ERK)-independent but p38 dependent pathway to regulate CARP-1 protein expression in osteoblasts. Immunofluorescence staining of differentiated osteoblasts further revealed nuclear to cytoplasmic translocation of CARP-1 protein following PTH treatment. Collectively, our studies identified CARP-1 for the first time in osteoblasts and suggest its potential role in PTH signaling and bone anabolic action.
authors.affiliation: Department of Internal Medicine/Endocrinology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
authors.forename: Sonali
authors.initials: S
authors.lastname: Sharma
authors.forename: Chandrika D
authors.initials: CD
authors.lastname: Mahalingam
authors.forename: Varsha
authors.initials: V
authors.lastname: Das
authors.forename: Shazia
authors.initials: S
authors.lastname: Jamal
authors.forename: Edi
authors.initials: E
authors.lastname: Levi
authors.forename: Arun K
authors.initials: AK
authors.lastname: Rishi
authors.forename: Nabanita S
authors.initials: NS
authors.lastname: Datta
mesh_terms: D016475:3T3 Cells; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D005260:Female; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010006:Osteoblasts / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000502:physiology*
chemical_list: D051017:Apoptosis Regulatory Proteins; C540324:CCAR1 protein, mouse; D018797:Cell Cycle Proteins; D010281:Parathyroid Hormone
keywords: CARP-1; Differentiation; EDTA; ERK; MAPK; MAPK kinase; MC-4; MC3T3-E1 clone 4; MEK; Osteoblast; PKA; PKC; PMA; PTH; PTH receptor-1; PTHR1; cell cycle and apoptosis regulatory protein -1; ethylenediamine tetraacetic acid; extracellular signal regulated kinase; mitogen-activated protein kinase; parathyroid hormone; phorbol-12-myristate-13 acetate; protein kinase A; protein kinase C