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. 2013 Jul 12;436(4):607-12.
doi: 10.1016/j.bbrc.2013.05.136. Epub 2013 Jun 11.

Cell cycle and apoptosis regulatory protein (CARP)-1 is expressed in osteoblasts and regulated by PTH

Sonali Sharma  1 , Chandrika D Mahalingam, Varsha Das, Shazia Jamal, Edi Levi, Arun K Rishi, Nabanita S Datta
Affiliations

Cell cycle and apoptosis regulatory protein (CARP)-1 is expressed in osteoblasts and regulated by PTH

Sonali Sharma et al. Biochem Biophys Res Commun. .

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.

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.

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Figures

Figure 1
Figure 1. CARP-1 expression and response to PTH in differentiated osteoblasts
(A) Representative immunostainings for CARP-1 protein expression are shown in osteocytes/osteoblasts in mouse femur (top row), growth plate chondrocytes (second row), differentiated primary calvarial osteoblasts (third row) and MC3TC-E1 osteoblastic cells (bottom row). NC, negative control. (B) MC3T3-E1 cells and (C) primary calvarial osteoblasts were induced to differentiate with ascorbic acid for 7 days and treated with 100nM PTH (P) or vehicle (V). Total cellular protein were harvested from proliferating or differentiated cells and subjected to SDS PAGE. Western blot analyses were performed with anti CARP-1 antibody. GAPDH was used as a protein loading control. Densitometric analyses were performed, normalized with GAPDH protein and plotted. Representative data (mean ± SEM) from at least three to four independent experiments are shown. *, p<0.05 vs V; Pro, proliferating; Diff, differentiated.
Figure 2
Figure 2. Immunofluorescence staining of CARP-1 translocation from the nuclear to the cytoplasm in differentiated osteoblasts
Left column: Treatment with vehicle shows nuclear localization of CARP-1 protein in differentiated MC3T3-E1 cells. Right column: Treatment with 100nM PTH for 2 hours translocates CARP-1 in the cytoplasm. NC, negative control.
Figure 3
Figure 3. Dose dependence and time course analysis of CARP-1 protein expression in response to PTH in differentiated osteoblasts
MC3T3-E1 cells were induced to differentiate with ascorbic acid for 7 days and treated with (A) various concentrations of PTH (P) or vehicle (V) for 2 hours, (B) with 100 nM P or V for 30 mins to 5 hours. Total cellular protein were harvested and subjected to SDS PAGE. Western blot analyses were performed, normalized and plotted as in Fig.1. Representative data (mean ± SEM) from at least three to four independent experiments are shown. *, p<0.05 vs no treatment (N) or V.
Figure 4
Figure 4. Downregulation of CARP-1 by PTH is PKA, PKC and P-p38 dependent
MC3T3-E1 cells were induced to differentiate with ascorbic acid for 7 days and were either pretreated with (A) the PKA inhibitor H-89 (H), the MEK inhibitor U0126 (U), the P-p38 inhibitor (SB), and (B) the PKC inhibitor (GF) followed by treatment with PTH (P) or vehicle (V), or treated with (B) the PKC agonist PMA alone as described in the Materials and Methods. Total cellular protein were harvested and subjected to SDS PAGE. Western blot analyses were performed, normalized and plotted as in Fig.1. Representative data (mean ± SEM) from three to four independent experiments are shown. *, p<0.05, **, p< 0.01 vs V.
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