J C LUI and others Molecular pathways in 53:1 T1–T9 Thematic Review growth plate RECENT RESEARCH ON THE GROWTH PLATE Recent insights into the regulation of the growth plate Julian C Lui1, Ola Nilsson1,2 and Jeffrey Baron1 1Program in Developmental Endocrinology and Genetics, Eunice Kennedy Shriver National Institute of Child Health Correspondence and Human Development, National Institutes of Health, CRC, Room 1-3330, 10 Center Drive, MSC 1103, should be addressed Bethesda, Maryland 20892-1103, USA to J C Lui 2Center for Molecular Medicine and Pediatric Endocrinology Unit, Department of Women’s and Children’s Health, Email Karolinska Institutet and Karolinska University Hospital, SE-171 76 Stockholm, Sweden [email protected] Abstract For most bones, elongation is driven primarily by chondrogenesis at the growth plates. Key Words This process results from chondrocyte proliferation, hypertrophy, and extracellular matrix " growth hormone secretion, and it is carefully orchestrated by complex networks of local paracrine factors and " signal transduction modulated by endocrine factors. We review here recent advances in the understanding of " IGF growth plate physiology. These advances include new approaches to study expression " microarray patterns of large numbers of genes in the growth plate, using microdissection followed by " skeletal microarray. This approach has been combined with genome-wide association studies to provide insights into the regulation of the human growth plate. We also review recent studies elucidating the roles of bone morphogenetic proteins, fibroblast growth factors, C-type natriuretic peptide, and suppressor of cytokine signaling in the local regulation of Journal of Molecular Journal of Molecular Endocrinology growth plate chondrogenesis and longitudinal bone growth. Endocrinology (2014) 53, T1–T9 Introduction In the postnatal mammal, elongation of tubular bones chondrocytes secrete proteins and proteoglycans that occurs at the growth plate. This cartilaginous structure form the cartilage extracellular matrix. In the resting and comprises three zones that contain chondrocytes at proliferative zones, collagen II represents a major com- different stages of differentiation (Kronenberg 2003). The ponent of this matrix, whereas in the hypertrophic zone, zone closest to the epiphysis is termed the resting zone. there is a shift to production of collagen X (Kronenberg The resting zone is thought to contain chondrocytes that 2003). The hypertrophic chondrocytes farthest from the serve as progenitor cells, which can generate new clones of epiphysis undergo cell death. This cell death has been rapidly proliferating chondrocytes (Abad et al. 2002). Each attributed to apoptosis, but more recent evidence chal- derivative clone forms a cell column aligned parallel to lenges this conclusion (Emons et al. 2009). This region is the long axis of the bone. As these cells replicate, the two then invaded from the metaphyseal bone by blood vessels daughters line up parallel to the long axis to maintain the and differentiating osteoblasts and osteoclasts, which columnar organization. The chondrocytes farther from remodel the cartilage into bone tissue. The net result of the epiphysis undergo termination differentiation, in this chondrogenesis and ossification is the formation of which they cease proliferating and enlarge to form the new bone underneath the growth plate and therefore hypertrophic zone. Throughout the growth plate, bone elongation. http://jme.endocrinology-journals.org Ñ 2014 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JME-14-0022 Printed in Great Britain This paper is one of 4 papers in a thematic review section on Recent Research on the Growth Plate: Regulation, Injury andDownloaded Regeneration. from Bioscientifica.com The guest editor forat 09/25/2021 this section 11:37:59AM was Cory Xian, University of South Australia, Adelaide, Australia via free access Thematic Review J C LUI and others Molecular pathways in 53:1 T2 growth plate The integrated processes of chondrocyte differen- analyzed using bioinformatic algorithms (Lui et al. 2010). tiation, proliferation, cartilage matrix secretion, cell Expression in the resting and the proliferative zones death, and vascular and bone cell invasion are regulated were compared to identify pathways involved in the and coordinated by a complex array of paracrine signaling differentiation of resting zone to proliferative zone molecules, which includes insulin-like growth factors chondrocytes. This analysis implicated vitamin D recep- (IGFs), fibroblast growth factors (FGFs), Indian hedgehog tor/retinoid X receptor (VDR/RXR) activation, platelet- (IHH) and parathyroid hormone-related protein (PTHrP), derived growth factor (PDGF) signaling, BMP signaling, bone morphogenetic proteins (BMPs), WNTs, and vascular and notch signaling. Similar analyses of the proliferative endothelial growth factors (VEGFs). In addition, the rate to hypertrophic differentiation step implicated p53 of endochondral bone formation at the growth plate signaling, ephrin receptor signaling, oncostatin M signal- is regulated by an array of endocrine signals, including ing, and BMP signaling (Lui et al. 2010). growth hormone (GH), IGF1, thyroid hormone, gluco- corticoids, androgens, and estrogens. One of the principal Evidence for a BMP signaling gradient across apparent functions of this endocrine system is to allow the growth plate rapid growth only when the organism is able to consume abundant nutrients. As noted above, microarray analysis implicated BMP Because the growth plate requires so many paracrine signaling in both the differentiation of resting zone and endocrine signaling pathways to function normally, chondrocytes to proliferative zone chondrocytes and of mutations in many genes involved in these signaling proliferative zone chondrocytes to hypertrophic zone pathways lead to bones that are short, which in humans chondrocytes. More extensive analysis of the BMP signal- presents as short stature, and often malformed, which ing pathway using microdissection followed by real-time presents as a skeletal dysplasia. Thus, mutations in more PCR has shown evidence for a BMP signaling gradient than 200 genes cause distinct skeletal dysplasias (Warman across the growth plate with the greatest BMP signaling et al. 2011). occurring in the hypertrophic zone and the least in the Although there has been remarkable progress recently resting zone (Nilsson et al. 2007). Consistent with this in our understanding of these signaling pathways that concept, immunolocalization of phosphorylated SMAD1, regulate the postnatal growth plate, much remains to be SMAD5, and SMAD8 in the growth plate increases with learned. In this review, we present some recent studies increasing distance from the epiphysis (Yoon et al. 2006). giving new insights into these control systems. The number These patterns indicate that a BMP signaling gradient Journal of Molecular Endocrinology of studies to be reviewed had to be limited, and therefore across the growth plate may contribute to the progressive not all important areas of progress could be included. differentiation of resting to proliferative to hypertrophic chondrocytes (Fig. 1). Low levels of BMP signaling in the Delineating gene expression patterns in the resting zone may help maintain the progenitor cell state. mammalian postnatal growth plate Farther from the epiphysis, greater BMP signaling may induce differentiation to proliferative chondrocytes and, In the past, gene expression within the growth plate even farther from the epiphysis, yet greater BMP signaling has typically been studied by in situ hybridization, which may induce terminal differentiation to hypertrophic provides much useful information but necessarily involves chondrocytes. Functional studies support this model. studying one candidate gene at a time. However, recently, BMP2 stimulates resting zone chondrocytes to proliferate methods have been developed to study expression and stimulates proliferative zone chondrocytes to hyper- patterns of large numbers of genes in the growth plate, trophy in an organ culture model (De Luca et al. 2001). using microdissection, followed by microarray analysis In vivo overexpression of constitutively active Bmpr1a (Nilsson et al. 2007). Frozen sections of the growth plate in mice has no effect on proliferation but accelerates are first microdissected into their constituent zones hypertrophic differentiation (Kobayashi et al. 2005). after which RNA is isolated and mRNA patterns are Recent evidence specifically implicates Bmp2 in this assessed by microarray analysis. Presumably, the method process. In mice, conditional targeted ablation of Bmp2 could readily be modified to use RNA sequencing in place causes severe defects in chondrocyte proliferation and of microarray analysis. differentiation through a mechanism involving RUNX2 This approach was applied to the proximal tibiae of protein levels (Shu et al. 2011). The effects of BMPs on the 1-week-old rats and the resulting expression data were growth plate appear to involve the canonical BMP http://jme.endocrinology-journals.org Ñ 2014 Society for Endocrinology Published by Bioscientifica Ltd DOI: 10.1530/JME-14-0022 Printed in Great Britain Downloaded from Bioscientifica.com at 09/25/2021 11:37:59AM via free access Thematic Review J C LUI and others Molecular pathways in 53:1 T3 growth plate differentiation. More recent evidence has indicated that the IHH–PTHrP system is maintained in the postnatal