A S CHAGIN and H M KRONENBERG G-proteins in chondrocyte 53:2 R39–R45 Review differentiation Role of G-proteins in the differentiation of epiphyseal chondrocytes Andrei S Chagin and Henry M Kronenberg1 Correspondence should be addressed Department of Physiology and Pharmacology, Karolinska Institutet, Nanna Svartz Vagen 2, Stockholm 17177, to A S Chagin Sweden Email 1Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114-2696, USA [email protected] Abstract Herein, we review the regulation of differentiation of the growth plate chondrocytes by Key Words G-proteins. In connection with this, we summarize the current knowledge regarding each " bone family of G-protein a subunit, specifically, Gas,Gaq/11,Ga12/13, and Gai/o. We discuss different " G proteins mechanisms involved in chondrocyte differentiation downstream of G-proteins and " intracellular signaling different G-protein-coupled receptors (GPCRs) activating G-proteins in the epiphyseal " parathyroid and bone chondrocytes. We conclude that among all G-proteins and GPCRs expressed by chondrocytes, " skeletal Gas has the most important role and prevents premature chondrocyte differentiation. Receptor for parathyroid hormone (PTHR1) appears to be the major activator of Gas in chondrocytes and ablation of either one leads to accelerated chondrocyte differentiation, Journal of Molecular premature fusion of the postnatal growth plate, and ultimately short stature. Endocrinology (2014) 53, R39–R45 Journal of Molecular Endocrinology The growth plate from the surrounding perichondrium (a thin layer of flat undifferentiated cells surrounding every cartilage The majority of bones in the skeleton are formed via element) into the center of the element (Maes et al. the process of endochondral bone formation. During this 2010). Thus, true bone tissue starts to be formed by process, a cartilage template is first formed and then osteoblasts in the center of cartilage anlagen, creating the replaced by true bone tissue. Formation of skeletal primary ossification center. In the case of long bones, elements starts with condensation of mesenchymal cells the secondary ossification centers are subsequently with their subsequent differentiation into chondrocytes formed in a similar manner at the ends of the growing (Hall & Miyake 1995). The chondrocytes proliferate cartilage template. The secondary ossification center thereby increasing the size of the primary skeletal element. develops into the epiphysis and primary ossification The cells in the center of the element stop proliferating, center develops into the metaphysis and diaphysis. The change their genetic program, and enlarge (hypertrophic cartilage remaining between the epiphysis and metaphysis differentiation), further facilitating growth of the skeletal forms the growth plate, a disc with spatially organized element. The cartilage elements are avascular and reach chondrocytes. The growth plate can be morphologically a substantial size, triggering hypoxia in the middle of divided into three zones: a resting zone with round stem- the skeletal element. The hypertrophic chondrocytes like chondrocytes located toward the epiphysis, a flat cell secrete vascular endothelial growth factor that attracts zone with proliferating chondrocytes in the middle, and blood vessels (Zelzer et al. 2004). Invasion by blood vessels a hypertrophic zone containing differentiated enlarged is accompanied by closely associated pre-osteoblasts chondrocytes located toward the metaphysis (Fig. 1). The http://jme.endocrinology-journals.org Ñ 2014 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JME-14-0093 Printed in Great Britain Downloaded from Bioscientifica.com at 09/27/2021 08:06:09PM via free access Review A S CHAGIN and H M KRONENBERG G-proteins in chondrocyte 53:2 R40 differentiation plate and decrease in IHH levels, in turn, leads to a decrease in PTHRP expression and a decrease in the distance Epiphysis between round and pre-hypertrophic cells. Thus, together Secondary IHH and PTHRP form a feedback loop, which controls the ossification center height of the growth plate (reviewed by Kronenberg (2003)). Subsequently, chondrocyte differentiation is associated with dramatic cell enlargement (hypertrophy); this cellular enlargement accounts for 59% of bone Epiphyseal growth lengthening (up to 73% with associated matrix (Wilsman plate et al. 1996)). Eventually, hypertrophic chondrocytes die, allowing new bone to be formed on the cartilage template. The zone of cartilage to bone transition is called the Metaphyseal bone chondro-osseous junction and the entire process is called endochondral bone formation. Thus, the rate of bone growth depends on the Zone of resting following steps: recruitment of stem-like quiescent cells (stem-like) into flat zone, chondrocyte proliferation, differentiation chondrocytes from flat to hypertrophic chondrocytes, chondrocyte Zone of flat enlargement and, finally, replacement of hypertrophic (columnar or chondrocytes by bone tissue. The rates of all these proliferative) chondrocytes processes are tightly balanced; deregulation in the rate of any of them will lead to growth abnormalities, usually leading to short stature. Zone of hypertrophic chondrocytes G-proteins Chondro-osseous junction G-protein-coupled receptors (GPCRs), a large family of seven-transmembrane domain receptors, mediate their Journal of Molecular Endocrinology signaling via heterotrimeric G-protein complexes, which Figure 1 consists of a, b, and g subunits. Sixteen genes encode Structure of the growth plate. Tibial growth plate from 15-day-old mouse is shown. The section is stained with hematoxylin and eosin. Ga subunits, five encode Gb, and 12 genes encode Gg subunits. When inactive, guanosine diphosphate (GDP) is chondrocytes from the resting zone are recruited into bound to the a subunit, stabilizing the complex between the flat zone, where they go through several cycles of a, b, and g. Upon activation of the GPCR, the receptor proliferation, thereby substantially increasing in number functions as a nucleotide exchanger: GDP is replaced and thereafter further differentiate into hypertrophic by the more abundant GTP. This leads to the dissociation chondrocytes. As soon as the cells cease proliferation and of Ga–GTP from Gbg and allows Ga–GTP to activate become pre-hypertrophic (changing from flat to round downstream effectors. The Gbg dimer also may mediate appearance), they start expressing the genes for Indian signaling (reviewed in Gautam et al. (1998) and Oldham & hedgehog (IHH) and the receptor for parathyroid hormone Hamm (2008)), but the role of this signaling pathway (PTHR1). IHH is a secreted molecule that diffuses through- in chondrocytes is largely unknown. The Ga–GTP out the cartilage and stimulates the production of PTH- complex remains active until GTP is hydrolyzed. Gas has related peptide (PTHRP) by resting cells at the top of the an intrinsic hydrolytic activity (GTPase), which is growth plate. The PTHRP, in turn, diffuses back to pre- enhanced by the regulators of G protein signaling (or hypertrophic cells, binds to the PTHR1, and inhibits RGS). There are about 30 different RGSs and their role in chondrocyte differentiation. This inhibition increases the bone is comprehensively discussed in a recent review distance between the resting zone and the pre-hypertrophic (Keinan et al. 2014). chondrocytes and decreases the levels of IHH, which reach On the basis of similarity of their a-subunits, 16 cells of the resting zone. This expansion of the growth members of the Ga subunit family are divided into four http://jme.endocrinology-journals.org Ñ 2014 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JME-14-0093 Printed in Great Britain Downloaded from Bioscientifica.com at 09/27/2021 08:06:09PM via free access Review A S CHAGIN and H M KRONENBERG G-proteins in chondrocyte 53:2 R41 differentiation major classes: Gas,Gaq/11,Ga12/13, and Gai/o (Freissmuth Gas and chondrocyte differentiation et al. 1989, Simon et al. 1991, Neves et al. 2002). It appears that Gas is a major participant in chondrocyte The stimulatory a subunit (Gas) family consists of differentiation, and it inhibits the differentiation process. three members: Gas small and large splice isoforms of the In humans, mutation of the Gas-encoding gene, GNAS1, same GNAS1 gene and Gaolf. The active Gas activates leads to Albright’s hereditary osteodystrophy, which is adenylate cyclase, an enzyme that catalyzes the synthesis associated with growth abnormalities, short stature, of cAMP from ATP within cells. The GPCRs are activated brachydactyly, and premature fusion of the growth plates by many types of ligands, including glycoproteins, many (Steinbach et al. 1965). The mechanism underlying these peptide hormones, catecholamines, and neurotransmit- abnormalities has been explored using genetic mouse ters; all such types of ligands can activate Gas to raise the a et al concentration of intracellular cAMP in their target cells. models. Targeted ablation of G s in fetal (Sakamoto . et al cAMP works as a second messenger activating several 2005) or postnatal (Chagin . 2014) chondrocytes leads downstream targets including protein kinase A (PKA, also to dramatic acceleration of chondrocyte differentiation known as the cAMP-dependent protein kinase or A and cessation of longitudinal bone growth. Chimeric mice kinase), cAMP-regulated guanine nucleotide exchange with cells deficient in the Gnas1 gene demonstrate factors 1 and 2