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. ? ? 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. The developmental program of growth plate senescence could be a function of time per se. Thus, there could be a biological timing mechanism which drives the change in chondrocyte physiology, including the decline in proliferation rate. Alternatively, growth plate senescence could be a function of growth itself. In this case, instead of a biological timing mechanism, the system might be driven by a cell-cycle counter, that is a cellular mechanism that is progressively changed with each cell cycle.? ? To distinguish between the two alternative hypotheses, rats were treated with propylthiouracil to induce hypothyroidism and thus inhibit growth at the growth plate. Control animals showed the normal senescent decline in tibial growth rate, chondrocyte proliferation rate, growth plate height, resting zone height, number of resting zone chondrocytes, number of proliferative zone chondrocytes per column, number of hypertrophic chondrocytes per column, terminal hypertrophic cell height, and column density. In the animals that had previously been hypothyroid, the senescent decline in every one of these variables was delayed. We also studied molecular markers of growth plate senescence, genes whose mRNA expression in growth plate chondrocytes changed markedly during growth plate senescence. These genes included chondroadherin, osteoprotegerin, secreted frizzled-related protein 4, reelin, and nuclear protein 1. In control animals, mRNA levels of all of these genes increased markedly with age. Animals that had previously been hypothyroid expressed these genes at levels similar to younger control animals. Thus, molecular markers of senescence, like the structural and functional markers of senescence, also appeared to be delayed by the previous hypothyroidism. In the animals that had previously been hypothyroid, the observed delay in multiple functional, structural, and molecular markers of growth plate senescence strongly supports the hypothesis that hypothyroidism slows the developmental program of growth plate senescence. We previously showed evidence, though less extensive, that glucocorticoid excess in the rabbit also slows growth plate senescence. The combined finding, that growth plate senescence is slowed by two different growth-inhibiting conditions in two different species, supports the hypothesis that growth plate senescence is not a function of time per se, but rather of growth, and therefore inhibition of growth slows this developmental process. ? ? The rate of cell proliferation and consequent somatic growth slows with age and ceases not only in growth plate, but also in multiple other tissues. To investigate the underlying changes in cell-cycle kinetics, methyl-3H-thymidine and 5-bromo-2-deoxyuridine were used to double-label proliferating cells in mice at various ages. Proliferation of renal tubular epithelial cells and hepatocytes decreased with age. The average cell-cycle time did not increase in liver and increased only 1.7 fold in kidney. The fraction of cells in S-phase that will divide again declined approximately 10 fold with age. Concurrently, average cell area increased approximately 2 fold. The findings suggest that somatic growth deceleration primarily results not from an increase in cell-cycle time but from a decrease in growth fraction (fraction of cells that continue to proliferate). During the deceleration phase, cells appear to reach a proliferative limit and undergo their final cell divisions, staggered over time. Concomitantly, cells enlarge to a greater volume, perhaps because they are relieved of the size constraint imposed by cell division. In summary, a decline in growth fraction with age causes somatic growth deceleration and thus sets a fundamental limit on adult body size.? ? The rate of cell proliferation and somatic growth declines simultaneously in multiple organs yet appears not to be coordinated by a systemic mechanism. We therefore hypothesized that growth deceleration results from a growth-limiting genetic program that is common to multiple tissues. To test this hypothesis, we performed microarray analysis to identify changes in gene expression in mice during early postnatal life. We focused our attention on genes that were up- or down-regulated in multiple organs and thus are more likely to contribute to the putative common program of growth deceleration. We noticed that some of the genes that showed the greatest changes in expression with age are imprinted, that is, genes that show differential expression from the maternal and paternal alleles. It has previously been observed that some imprinted genes positively regulate fetal growth. Our microarray observations raised the possibility that expression of these genes persists into early postnatal life and that their subsequent down-regulation contributes to the dramatic decline in proliferation and somatic growth that determines adult body size. We therefore systematically assessed the age-related changes in expression of all known imprinted genes using microarray analysis for kidney, lung, and heart. We identified a set of 11 imprinted genes that show down-regulation of mRNA expression with age in multiple organs. For these genes, Igf2, H19, Plagl1, Mest, Peg3, Dlk1, Gtl2, Grb10, Ndn, Cdkn1c, and SLC38a4, the declines show a temporal pattern similar to the decline in growth rate. All 11 genes have been implicated in the control of cell proliferation or somatic growth. Thus, our findings suggest that the declining expression of these genes contributes to coordinate growth deceleration in multiple tissues. We next hypothesized that the coordinate decline in expression of these imprinted genes is caused by altered methylation and consequent silencing of the expressed allele. Contrary to this hypothesis, the methylation status of the promoter regions of Mest, Peg3 and Plagl1 did not change with age. In summary, our findings suggest that a set of growth-regulating imprinted genes is expressed at high levels in multiple tissues in early postnatal life, contributing to rapid somatic growth, but that these genes are subsequently down-regulated in multiple tissues simultaneously, contributing to coordinate growth deceleration and cessation, thus imposing a fundamental limit on adult body size.
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