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 rapidly dividing chondrocytes of the proliferative zone. The proliferative chondocytes 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 multiple genes functionally related to bone morphogenetic proteins (BMPs). BMP-2 and BMP-6 were found to be upregulated in hypertrophic zone compared with resting zone and proliferative zone. In contrast, BMP signaling inhibitors, including BMP-3, gremlin, and growth differentiation factor-10, were expressed early in the differentiation pathway, in the resting and proliferative zones. Our findings suggest a BMP signaling gradient across the growth plate, which is established by differential expression of multiple BMPs and BMP inhibitors in specific zones. We have previously shown evidence that BMPs can stimulate both proliferation and hypertrophic differentiation of growth plate chondrocytes. Therefore, 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 a key mechanism responsible for spatial regulation of chondrocyte proliferation and differentiation in growth plate cartilage. ? ? Fibroblast growth factor (FGF) signaling is also essential for endochondral bone formation. Mutations in FGF receptors cause skeletal dysplasias including achondroplasia, the most common human skeletal dysplasia. To explore the role of FGF signaling in the postnatal growth plate, we quantitatively analyzed expression of FGFs and FGF receptors (FGFRs). Rat proximal tibial growth plates and surrounding tissues were microdissected, and specific mRNAs were quantitated by real-time RT-PCR. To assess the FGF system without bias, we first screened for expression of all known FGFs and major FGFR isoforms. Perichondrium expressed FGFs 1, 2, 6, 7, 9, and 18 and, at lower levels, FGFs 21 and 22. 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 of their proximity to target receptors. In growth plate, we found expression of FGFRs 1, 2, and 3, primarily, but not exclusively, the c isoforms. FGFRs 1 and 3, thought to negatively regulate chondrogenesis, were expressed at greater levels and at later stages of chondrocyte differentiation, with FGFR1 upregulated in the hypertrophic zone and FGFR3 upregulated in both proliferative and hypertrophic zones. In contrast, FGFRs 2 and 4, putative positive regulators, were expressed at earlier stages of differentiation, with FGFR2 upregulated in the resting zone and FGFR4 in the resting and proliferative zones. Thus, this analysis identified ligands and receptors not previously known to be expressed in growth plate and revealed a complex pattern of spatial regulation of FGFs and FGFRs in the different zones of the growth plate.? ? Previous studies of the insulin-like growth factor (IGF) system gene expression in growth plate using immunohistochemistry and in situ hybridization have yielded conflicting results. We therefore studied the spatial patterns of mRNA expression of the IGF system in the rat proximal tibial growth plate quantitatively. IGF-I mRNA expression was minimal in growth plate compared with perichondrium, metaphyseal bone, muscle, and liver. In contrast, IGF-II mRNA was expressed at higher levels than in bone and liver. IGF-II expression was higher in the proliferative and resting zones compared with the hypertrophic zone. GH receptor and type 1 and 2 IGF receptors were expressed throughout the growth plate. Expression of IGF-binding proteins (IGFBPs) -1 through -6 mRNA was low throughout the growth plate compared with perichondrium and bone. These data suggest that regulation primarily depends on IGF-II produced by chondrocytes, and IGF-I produced by surrounding structures.? ? 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 decreases as does the size of the individual hypertrophic cells. The chondrocyte columns also become more widely spaced. We have termed this developmental program, growth plate senescence. Growth plate senescence 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, as the rate of longitudinal bone growth decreases. ? ? In particular we analyzed the insulin-like growth factor (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 tenfold lower than levels in perichondrium and bone. IGF-II mRNA decreased dramatically, 780-fold, in proliferative zone whereas, type 2 IGF receptor and IGF binding proteins (IGFBPs)-1, -2, - 3, and -4 increased significantly with age in growth plate and/or surrounding perichondrium and bone. These findings suggest that growth plate senescence, including 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 multiple IGFBPs. ? ? We also analyzed temporal changes in fibroblast growth factor (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, FGFRs 2 and 4, both implicated as positive regulators of growth, undergo a decline in expression with age. In perichondrium, we observed increases in FGFs 1, 7, 18, and 22 mRNA with age. Increasing levels of these ligands, interacting with constant levels of FGFR3 in growth plate might contribute to growth plate senescence.? ? These studies have begun to elucidate the regulation of gene expression that is responsible for the complex spatial organization of the growth plate and for the temporal changes of growth plate senescence. Combined with previous functional studies performed in our lab and by others, the findings indicate a highly complex system involving BMPs, FGFs, and IGFs, their receptors and other interacting proteins.
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