Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 is Expressed inOsteoblasts and Regulated by PTH

doi:10.1016/j.bbrc.2013.05.136

Biochemical and Biophysical Research Communications

Volume 436, Issue 4, 12 July 2013, Pages 607-612

https://doi.org/10.1016/j.bbrc.2013.05.136 Get rights and content

Highlights

•
CARP-1 is identified for the first time in bone cells.
•
PTH downregulates CARP-1 expression in differentiated osteoblasts.
•
PTH displaces CARP-1 from nucleus to the cytoplasm in differentiated osteoblasts.
•
Downregulation of CARP-1 by PTH involves PKA, PKC and P-p38 MAPK pathways.

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 30 min to 5 h 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.

Introduction

Osteoblasts are bone forming cells that synthesize and mineralize the skeleton [1]. Bone mass is dependent on proliferation, differentiation and life-span of mature osteoblasts. Parathyroid hormone (PTH), secreted from parathyroid glands, is involved in calcium homeostasis, and is a critical mediator of skeletal development and remodeling [2]. Daily injection of PTH increases bone mass and reduces fracture incidence in osteoporotic patients [3]. PTH mediates multiple signals that coordinate distinct cellular functions in bone including osteoblast proliferation, differentiation and apoptosis [4], [5], [6], [7]. While PTH is critical for maintenance of bone homeostasis, the intracellular mechanisms of PTH receptor-1 (PTHR1) signaling remains unclear and continued to be explored. The therapeutic use of PTH is limited by the principal side effects of disruption of calcium homeostasis [8] and possible bone cancer concerns [9]. Therefore, elucidating the molecular mechanisms underlying the anabolic action of PTH is essential for understanding the pathophysiology of bone loss, optimizing patient care and yielding novel therapeutic strategies to promote bone formation.

Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 (aka CCAR1) is a novel transducer of cell growth and apoptosis signaling by diverse agents including cell growth and differentiation factors [10]. CARP-1 was previously characterized as a peri-nuclear protein that functions to regulate chemotherapy-dependent apoptosis signaling in breast cancer cells [11], or demonstrated as a nuclear protein following UV-C irradiation in mouse embryonic fibroblasts [12]. Recent studies revealed CARP-1 interaction with cell cycle regulatory Anaphase-Promoting Complex/Cyclosome (APC/C) subunit APC-2 that regulates cell growth and apoptosis [13]. CARP-1 binding with APC-2 causes G₂M cell cycle arrest. Depletion of CARP-1, however, interferes with agonist dependent cell growth inhibition [13]. The fact that PTH prevents osteoblast apoptosis [6] and induces growth arrest in differentiated osteoblasts by modulating cell cycle associated proteins [14], [15], [16] suggests that CARP-1 could be important in the PTH regulation of osteoblast growth and differentiation. We tested this hypothesis and revealed that CARP-1 is expressed in osteoblasts and osteocytes, and involved in PTH/PTHR1 regulation of osteoblast growth and differentiation.

Section snippets

Experimental animals

The experiments in this study were performed with 10–12 week old male or female 129J/C57BL6 mice, fed with rodent chow (Lab diet, Bentwood, MD). All animals were maintained in facilities operated by Wayne State University, and all animal experimental procedures were approved by the Institutional Animal Care and Use Committee for the Use and Care of Animals.

Antibodies and reagents

Generation of anti CARP-1 (α2) rabbit polyclonal antibodies was described previously [11]. Anti Glyceraldehyde 3-phospho Dehydrogenase

Results and discussion

PTH has both anabolic and catabolic action in bone, but the downstream mediators of the intracellular signaling pathways are not well defined relative to specific actions. Current views of the anabolic action of PTH indicate that it acts on osteoblasts to promote their proliferation or differentiation, while inhibiting osteoblast and osteocyte apoptosis [14], [15], [17]. Despite the reported role of CARP-1 as a key intracellular transducer of apoptosis and cell proliferation of normal as well

Acknowledgments

This work was supported by funding from WSU OVPR and NIH DK087848 to N.S.D.; and the Medical Research Services of the Department of Veteran Affairs Merit Review grant to A.K.R.

References (34)

R.L. Jilka
Molecular and cellular mechanisms of the anabolic effect of intermittent PTH
Bone
(2007)
N.S. Datta et al.
PTH and PTHrP signaling in osteoblasts
Cell. Signal.
(2009)
Y. Jiang et al.
A novel mechanism of cell growth regulation by cell cycle and apoptosis regulatory protein (CARP)-1
J. Mol. Signal.
(2010)
A.K. Rishi et al.
Identification and characterization of a cell cycle and apoptosis regulatory protein-1 as a novel mediator of apoptosis signaling by retinoid CD437
J. Biol. Chem.
(2003)
V.T. Puliyappadamba et al.
Antagonists of anaphase-promoting complex (APC)-2–cell cycle and apoptosis regulatory protein (CARP)-1 interaction are novel regulators of cell growth and apoptosis
J. Biol. Chem.
(2011)
N.S. Datta et al.
Distinct roles for mitogen-activated protein kinase phosphatase-1 (MKP-1) and ERK-MAPK in PTH1R signaling during osteoblast proliferation and differentiation
Cell. Signal.
(2010)
R.L. Jilka et al.
Intermittent PTH stimulates periosteal bone formation by actions on post-mitotic preosteoblasts
Bone
(2009)
A.K. Rishi et al.
Cell cycle- and apoptosis-regulatory protein-1 is involved in apoptosis signaling by epidermal growth factor receptor
J. Biol. Chem.
(2006)
J.H. Kim et al.
CCAR1, a key regulator of mediator complex recruitment to nuclear receptor transcription complexes
Mol. Cell
(2008)
C.Y. Ou et al.
Requirement of cell cycle and apoptosis regulator 1 for target gene activation by Wnt and beta-catenin and for anchorage-independent growth of human colon carcinoma cells
J. Biol. Chem.
(2009)

M. Almeida et al.

Wnt proteins prevent apoptosis of both uncommitted osteoblast progenitors and differentiated osteoblasts by beta-catenin-dependent and -independent signaling cascades involving Src/ERK and phosphatidylinositol 3-kinase/AKT

J. Biol. Chem.

(2005)

J.B. Lian et al.

Bone formation: maturation and functional activities of osteoblast lineage cells

J.M. Hock et al.

Actions of parathyroid hormone

R.M. Neer et al.

Effect of parathyroid hormone (1–34) on fractures and bone mineral density in postmenopausal women with osteoporosis

N. Engl. J. Med.

(2001)

R.L. Jilka et al.

Increased bone formation by prevention of osteoblast apoptosis with parathyroid hormone

J. Clin. Invest.

(1999)

Y.H. Wang et al.

Effects of transient PTH on early proliferation, apoptosis, and subsequent differentiation of osteoblast in primary osteoblast cultures

Am. J. Physiol. Endocrinol. Metab.

(2007)

P.D. Miller et al.

Occurrence of hypercalciuria in patients with osteoporosis treated with teriparatide

J. Clin. Endocrinol. Metab.

(2007)

Cited by (12)

Computational model of the dual action of PTH — Application to a rat model of osteoporosis
2019, Journal of Theoretical Biology
This paper presents a pharmacokinetic/pharmacodynamic (PK/PD) model of the action of PTH(1-34) on bone modelling and remodelling, developed for quantitatively investigating the dose- and administration pattern-dependency of the bone tissue response to this drug. Firstly, a PK model of PTH(1-34) was developed, accounting for administration via subcutaneous injections. Subsequently, the PK model was coupled to a (mechanistic) bone cell population model of bone modelling and remodelling, taking into account the effects of PTH(1-34) on the differentiation of lining cells into active osteoblasts, on the apoptosis of active osteoblasts, and on proliferation of osteoblast precursors, as well as on the key regulatory pathways of bone cell activities. Numerical simulations show that the coupled PK/PD model is able to distinguish between continuous and intermittent administration patterns of PTH(1-34), in terms of yielding both catabolic bone responses (if drug administration is carried out continuously) and anabolic bone responses (if drug administration is carried out intermittently). The model also features a non-linear relation between bone gain and drug dose (as known from experiments); doubling the dose from 80  $μ$ g/kg/day to 160 $μ$ g/kg/day induced a 1.3-fold increase of the bone volume-to-total volume ratio. Furthermore, the model presented in this paper confirmed that bone modelling represents an essential mechanism of the anabolic response of bone to PTH(1–34) administration in rat models, and that the large amount of bone formation observed in such models cannot be explained via remodelling alone.
Parathyroid hormone: Anabolic and catabolic actions on the skeleton
2015, Current Opinion in Pharmacology
Citation Excerpt :
Additionally, PTH treatment of osteoblastic cells inhibits the pro-apoptotic effects of etoposide, dexamethasone, hydrogen peroxide induced oxidative stress, UV irradiation, serum withdrawal and nutrient deprivation [73,76,77]. The anti-apoptotic actions of PTH include phosphorylation and inactivation of the pro-apoptotic protein Bad, increased expression of survival genes like Bcl-2, increased expression of Runx2, downregulation of the apoptosis inducer CARP-1 (Cell Cycle and Apoptosis Regulatory Protein), and increase in DNA repair [76–78]. The differentiation of lining cells into active osteoblasts may also explain the PTH-induced increase in osteoblast number.
Parathyroid hormone (PTH) is essential for the maintenance of calcium homeostasis through, in part, its actions to regulate bone remodeling. While PTH stimulates both bone formation and bone resorption, the duration and periodicity of exposure to PTH governs the net effect on bone mass, that is whether it is catabolic or anabolic. PTH receptor signaling in osteoblasts and osteocytes can increase the RANKL/OPG ratio, increasing both osteoclast recruitment and osteoclast activity, and thereby stimulating bone resorption. In contrast, PTH-induced bone formation is explained, at least in part, by its ability to downregulate SOST/sclerostin expression in osteocytes, permitting the anabolic Wnt signaling pathway to proceed. The two modes of administration of PTH, that is, continuous vs. intermittent, can regulate, in bone cells, different sets of genes; alternatively, the same sets of genes exposed to PTH in sustained vs. transient way, will favor bone resorption or bone formation, respectively. This article reviews the effects of PTH on bone cells that lead to these dual catabolic and anabolic actions on the skeleton.
Cellular Actions of PTH: Osteoblasts, Osteoclasts, and Osteocytes
2015, The Parathyroids: Basic and Clinical Concepts: Third Edition
Parathyroid hormone (PTH) is essential for the maintenance of calcium homeostasis through direct actions on bone and kidney, and indirect actions on the gastrointestinal tract. In the skeleton, PTH directly affects osteoblasts and osteocytes, and indirectly affects osteoclasts through actions on osteoblasts and osteocytes. These PTH actions on bone cells promote both bone formation and bone resorption, and the final outcome on bone mass, either anabolic or catabolic, depends on the dose of PTH and the periodicity of the PTH signal during exposure. PTH favors bone formation by controlling osteoblast proliferation, differentiation, survival, and apoptosis. Moreover, it increases bone resorption by indirectly stimulating osteoclast formation and activity. The ability of PTH to increase bone remodeling is to a large extent mediated by its actions on osteocytes. This chapter reviews the effects of PTH on bone cells.
Anabolic and Catabolic Pathways of Parathyroid Hormone on the Skeleton
2015, The Parathyroids: Basic and Clinical Concepts: Third Edition
Parathyroid hormone (PTH) is essential for the maintenance of the serum calcium concentration within narrow, normal limits, a feat that is achieved, in part, by its ability to regulate bone resorption. In addition to this traditional catabolic role, the osteoanabolic actions of PTH are also well known. Clinically, the catabolic effect of PTH is best represented by primary hyperparathyroidism (PHPT), whereas its osteoanabolic effect is best seen when PTH or its biological amino-terminal fragment [PTH(1–34)] is used as a therapy for osteoporosis. At the cellular level, PTH favors bone resorption, mostly by affecting the RANKL–OPG–RANK system and ultimately increasing both osteoclast recruitment and osteoclast activity. On the osteoanabolic level, PTH increases bone formation, a feat mediated, in part, by control of sclerostin, a regulator of the osteoanabolic Wnt signaling pathway. This chapter focuses on the cellular bases by which PTH exerts these dual anabolic and catabolic actions on the skeleton.
PTH and the Regulation of Mesenchymal Cells within the Bone Marrow Niche
2024, Cells
Comparative transcriptome and proteome reveal synergistic functions of differentially expressed genes and proteins implicated in an over-dominant silkworm heterosis of increased silk yield
2022, Insect Molecular Biology

View all citing articles on Scopus

View full text

Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 is Expressed inOsteoblasts and Regulated by PTH

Highlights

Abstract

Introduction

Section snippets

Experimental animals

Antibodies and reagents

Results and discussion

Acknowledgments

Bone

Cell. Signal.

J. Mol. Signal.

J. Biol. Chem.

J. Biol. Chem.

Cell. Signal.

Bone

J. Biol. Chem.

Mol. Cell

J. Biol. Chem.

J. Biol. Chem.

Bone formation: maturation and functional activities of osteoblast lineage cells

Actions of parathyroid hormone

Effect of parathyroid hormone (1–34) on fractures and bone mineral density in postmenopausal women with osteoporosis

N. Engl. J. Med.

Increased bone formation by prevention of osteoblast apoptosis with parathyroid hormone

J. Clin. Invest.

Effects of transient PTH on early proliferation, apoptosis, and subsequent differentiation of osteoblast in primary osteoblast cultures

Am. J. Physiol. Endocrinol. Metab.

Occurrence of hypercalciuria in patients with osteoporosis treated with teriparatide

J. Clin. Endocrinol. Metab.