REGULATION OF CHILDHOOD GROWTH
, Head, Section on Growth and Development
Kevin Barnes, PhD, Senior Research Assistant
Gabriella Finkielstain, MD, Postdoctoral Fellow
Julian Lui, PhD, Postdoctoral Fellow
Maria Chang, BS, Predoctoral Fellow
Patricia Forcinito, BS, Special Volunteer
Rachel Gafni, MD, Special Volunteer
We investigate the cellular and molecular mechanisms governing childhood growth and development, focusing particularly on the skeletal system. One goal of our work is to improve treatment of childhood growth disorders. Given that the cellular processes underlying bone growth, such as cell proliferation, terminal differentiation, angiogenesis, and cell migration, are also essential for the development of other tissues, we seek to uncover general principles of developmental biology. We have most recently focused on the regulation of gene expression that is responsible for both the complex spatial organization of the growth plate and temporal changes during growth plate senescence. Combined with previous functional studies, the current findings indicate a complex system involving many members of the BMP, FGF, and IGF families of genes, their receptors, and interacting proteins.
Spatial organization of the growth plate
Longitudinal bone growth occurs at the growth plate, which consists of three principal layers: the resting zone, the proliferative zone, and the hypertrophic zone. Studies in our laboratory indicate that stem-like cells in the resting zone differentiate into the rapidly dividing chondrocytes of the proliferative zone. The proliferative chondrocytes then terminally differentiate into the nondividing chondrocytes of the hypertrophic zone.
To explore the molecular switches responsible for this two-step differentiation program, we developed a microdissection method to isolate RNA from the resting, proliferative, and hypertrophic zones of growing rats. Microarray analysis followed by real-time PCR analysis identified genes whose expression changed dramatically during the differentiation program, including several genes functionally related to bone morphogenetic proteins (BMPs). We found that BMP-2 and BMP-6 were upregulated in the hypertrophic zone as compared with the resting and proliferative zones. In contrast, BMP signaling inhibitors, including BMP-3, gremlin, and growth differentiation factor-10, were expressed early in the differentiation pathway, that is, in the resting and proliferative zones. Our findings suggest that a BMP signaling gradient across the growth plate is established by differential expression of several BMPs and BMP inhibitors in specific zones. We have previously shown that BMPs can stimulate both proliferation and hypertrophic differentiation of growth plate chondrocytes. Taken together, our findings suggest that low levels of BMP signaling in the resting zone may help maintain these cells in a quiescent state. In the lower resting zone, greater BMP signaling may help induce differentiation to proliferative chondrocytes. Farther down the growth plate, even greater BMP signaling may help induce hypertrophic differentiation. Thus, BMP signaling gradients may be an important mechanism responsible for spatial regulation of chondrocyte proliferation and differentiation in the growth plate.
Fibroblast growth factor (FGF) signaling is also essential for endochondral bone formation. Mutations in the FGF signaling pathway cause skeletal dysplasias, including achondroplasia, the most common human skeletal dysplasia. To explore the role of FGF signaling in the postnatal growth plate, we measured expression of FGFs and FGF receptors (FGFRs). We dissected rat proximal tibial growth plates and surrounding tissues and quantitated specific mRNAs with real-time RT-PCR. To assess the FGF system without bias, we first screened for expression of all known FGFs and major FGFR isoforms. The perichondrium expressed FGFs 1, 2, 6, 7, 9, and 18 and, at lower levels, FGFs 21 and 22. The growth plate expressed FGFs 2, 7, 18, and 22. Perichondrial expression was generally greater than growth plate expression, supporting the concept that perichondrial FGFs regulate growth plate chondrogenesis. Nevertheless, FGFs synthesized by growth plate chondrocytes may be physiologically important because FGFs are released in proximity to target receptors. In the growth plate, we found expression of FGFRs 1, 2, and 3. For each of these receptors, the c isoform was the predominant splice variant expressed. Thought to regulate chondrogenesis negatively, FGFRs 1 and 3 were expressed at greater levels and at later stages of chondrocyte differentiation, with FGFR1 upregulated in the hypertrophic zone and FGFR3 upregulated in both the proliferative and hypertrophic zones. In contrast, the putative positive regulators FGFR 2 and 4 were expressed at earlier stages of differentiation, with FGFR2 upregulated in the resting zone and FGFR4 in the resting and proliferative zones. Our analysis thus identified ligands and receptors not previously known to be expressed in the growth plate and revealed a complex pattern of spatial regulation of FGFs and FGFRs in the different zones of the growth plate, a pattern that may establish the growth plate’s spatial organization.
Using immunohistochemistry and in situ hybridization, previous studies of IGF (insulin-like growth factor) system gene expression in the growth plate yielded conflicting results. We therefore measured the spatial patterns of mRNA expression of the IGF system in the rat proximal tibial growth plate. We demonstrated that IGF-I mRNA expression was minimal in the growth plate compared with the perichondrium, metaphyseal bone, muscle, and liver, whereas IGF-II mRNA was expressed at higher levels than in bone and liver. IGF-II expression was higher in the proliferative and resting zones than in the hypertrophic zone. Growth hormone receptor and type 1 and 2 IGF receptors were expressed throughout the growth plate. Expression of the mRNA encoding IGF-binding proteins (IGFBPs) 1 through 6 was low throughout the growth plate compared with the perichondrium and bone. The data suggest that regulation depends primarily on IGF-II produced by chondrocytes and IGF-I produced by surrounding structures.
Andrade AC, Nilsson O, Barnes KM, Baron J. Wnt gene expression in the postnatal growth plate: regulation with chondrocyte differentiation. Bone 2007;40:1361-9.
Lazarus JE, Hegde A, Andrade AC, Nilsson O, Baron J. Fibroblast growth factor expression in the postnatal growth plate. Bone 2007;40:577-86.
Nilsson O, Parker EA, Hegde A, Chau M, Barnes KM, Baron J. Gradients in bone morphogenetic protein-related gene expression across the growth plate. J Endocrinol 2007;193:75-84.
Temporal regulation of growth
With age, growth plate chondrocyte proliferation slows down, causing longitudinal bone growth to slow and eventually stop. This functional change in the growth plate is accompanied by structural changes; with age, the number of resting, proliferative, and hypertrophic chondrocytes declines, as does the size of individual hypertrophic cells. Furthermore, the chondrocyte columns become more widely spaced. We have termed this developmental program growth plate senescence and report that it appears to be caused by a mechanism intrinsic to the growth plate. To explore the molecular mechanisms responsible for growth plate senescence, we analyzed how gene expression patterns change in the growth plate during postnatal life while the rate of longitudinal bone growth decreases.
In particular, we analyzed the IGF system because IGFs are capable of potently regulating growth plate chondrocyte proliferation and differentiation. With increasing age (3-, 6-, 9-, and 12-week rats), IGF-I mRNA levels increased in the proliferative zone but remained at least 10-fold lower than levels in perichondrium and bone. IGF-II mRNA decreased dramatically—780-fold—in the proliferative zone, whereas type 2 IGF receptor and IGFBP-1, IGFBP-2, IGFBP- 3, and IGFBP-4 increased significantly with age in the growth plate and/or surrounding perichondrium and bone. These findings suggest that the decrease in growth velocity that occurs with age may be caused, in part, by decreasing expression of IGF-II and increasing expression of type 2 IGF receptor and several IGFBPs.
We also analyzed temporal changes in FGF expression in the growth plate. We identified several changes in FGF and FGFR expression that may contribute to growth plate senescence. In the growth plate, expression of FGFRs 2 and 4, both implicated as positive regulators of growth, decline with age. In the perichondrium, we observed increases in FGF 1, 7, 18, and 22 mRNA with age. Increasing levels of these ligands, interacting in the growth plate with constant levels of FGFR3, a negative regulator of chondrogenesis, might also contribute to growth plate senescence.
Emons JA, Marino R, Nilsson O, Barnes KM, Even-Zohar N, Andrade AC, Chatterjee NA, Wit JM, Karperien M, Baron J. The role of p27 kip1 in the regulation of growth plate chondrocyte proliferation in mice. Pediatr Res 2006;60:288-93.
Emons JAM, Boersma B, Baron J, Wit JM. Catch-up growth: testing the hypothesis of delayed growth plate senescence in humans. J Pediatr 2005;147:843-6.
Nilsson O, Mitchum RD, Schrier L, Barnes KM, Troendle JF, Baron J. Growth plate senescence is associated with loss of DNA methylation. J Endocrinol 2005;186:241-9.
Parker EA, Hegde A, Buckley M, Barnes KM, Baron J, Nilsson O. Spatial and temporal regulation of GH-IGF-related gene expression in growth plate cartilage. J Endocrinol 2007;194:31-40.
Schrier L, Ferns SP, Barnes KM, Emons JAM, Newman E, Nilsson O, Baron J. Depletion of resting zone chondrocytes during growth plate senescence. J Endocrinol 2006;189:27-36.
COLLABORATOR
Ola Nilsson, MD, PhD, Karolinska Universitetssjukhuset, Stockholm, Sweden
Jan-Maarten Wit, MD, Leides Universitair Medisch Centrum, Leiden, The Netherlands
For further information, contact jeffrey_baron@nih.gov.
