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regulation of skeletal growth
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Kevin Barnes, PhD,
Senior Research Assistant Rose Marino, MD, Postdoctoral
Fellow Ola Nilsson, MD, Postdoctoral
Fellow Anita Hegde, BS,
Predoctoral Fellow Lenneke Schrier, BS, Predoctoral Fellow Joyce Emons, MD,
Special Volunteer Rachel Gafni, MD, Special
Volunteer Ellen Leschek, MD, Special Volunteer Benjamin Nwosu, MD, Special Volunteer |
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We investigate the cellular and molecular
mechanisms governing bone growth and development. One goal of our work is to
improve medical treatment of growth disorders and childhood metabolic bone
diseases. In addition, we seek to uncover general principles of developmental
biology, given that the cellular processes underlying bone growth, such as
cell proliferation, terminal differentiation, angiogenesis, and cell
migration, are also essential for development in other tissues. Longitudinal bone growth: cellular and
molecular mechanisms Baron,
Barnes, Nilsson, Marino, Schrier, Hegde, Emons, Gafni, Nwosu Longitudinal bone growth occurs at the growth
plate, a thin layer of cartilage that lies near the ends of long bones and
vertebrae. The growth plate consists of three principal layers: the resting
zone, the proliferative zone, and the hypertrophic zone. Studies in our
laboratory indicate that the resting zone contains stem-like cells that are
capable of generating new clones of proliferative chondrocytes. These
proliferative cells undergo clonal expansion followed by cellular
hypertrophy. The hypertrophic cartilage is then remodeled into bone tissue.
The net effect is that new bone tissue is progressively created at the bottom
of the growth plate, resulting in bone elongation. With age, growth plate chondrocyte
proliferation slows down, causing longitudinal bone growth to slow and
eventually stop. Also with increasing age, the growth plate undergoes
structural changes. These functional and structural changes appear not to be
attributable to a systemic mechanism but instead to a mechanism intrinsic to
the growth plate. We termed this intrinsic program growth plate senescence
and found evidence that it is a function not of time per se but rather of the
number of replications that the growth plate chondrocytes have undergone. In
particular, our previous studies suggest that stem-like cells, located in the
resting zone of the growth plate, have a finite proliferative capacity, which
is gradually exhausted, thus producing the growth deceleration and other
senescent changes. Similar replicative senescence occurs when
many types of animal cells are placed in primary cell culture, an effect
known as the Hayflick phenomenon. We therefore hypothesized that the same
mechanisms were responsible for growth plate senescence. However, our recent
findings do not support that hypothesis. We found that the number of
population doublings of rabbit resting zone chondrocytes in culture did not
depend on the age of the animal from which the cells were harvested. Thus,
previous proliferation in vivo had no effect on subsequent
proliferation in vitro, suggesting that the mechanisms limiting
replicative capacity of growth plate chondrocytes in vivo are distinct
from those responsible for limiting replication in vitro. One mechanism that has been proposed to
explain replicative senescence involves epigenetic changes, including
methylation of genomic DNA. Some CG sequences in mammalian genomic DNA are
methylated on the cytosine moiety. When DNA is replicated, the new strand is
initially not methylated. However, DNA methyltransferase 1 recognizes the
hemimethylated CGs and adds the missing methyl groups. If maintenance
methylation is incomplete, methylation levels may gradually decrease with
repeated cell replication. Thus, the level of DNA methylation could serve as
a cell-cycle counter. Evidence suggests that such epigenetic changes may
contribute to replicative senescence and terminal differentiation in some
cell types. In particular, growth plate chondrocytes exposed to a
demethylating agent in vitro undergo hypertrophic differentiation.
Furthermore, disruption of the SNF2-like gene PASG, which is required for
normal maintenance of DNA methylation, results in growth retardation and
premature aging. These observations suggest that replicative senescence of
growth plate chondrocytes involves changes in DNA methylation and other
related epigenetic modifications. To investigate this possible mechanism, we
measured the overall level of genomic DNA methylation in growth plate
cartilage from rabbits of different ages. Consistent with our hypothesis, we
found that growth plate senescence is associated with a loss of DNA
methylation in resting zone chondrocytes. We observed a similar loss of
methylation with age in the proliferative and hypertrophic zone chondrocytes,
which are thought to be progeny of the resting zone chondrocytes. However,
within each age, we observed no significant difference in the level of DNA
methylation between the different zones of the growth plate. Therefore, loss
of methylation appears to occur specifically during replication of resting
zone chondrocytes but not during the more rapid replication of proliferative
zone chondrocytes. Thus, complete maintenance of methylation may occur in the
proliferative zone, but not in the resting zone, suggesting that loss of
methylation might be responsible for the temporal limits that cause
chondrocyte replication to slow with age but not for the spatial limits that
cause chondrocyte proliferation to slow as the cells descend further down the
chondrocyte columns. Abad V, Meyers JL, Weise M, Gafni RI, Barnes
KM, Nilsson O, Bacher JD, Baron J. The role of the resting zone in growth
plate chondrogenesis. Endocrinology 2002;143:1851-1857. Cadet ER, Nilsson O, Abad V, Chrysis D, Ritzen EM,
Savendahl L, Baron J. Estrogen
receptor-alpha and -beta are expressed throughout postnatal development in
the rat and rabbit growth plate. J Endocrinol 2002;173:407-414. Nilsson O, Falk J, Ritzen EM, Baron J,
Savendahl L. Raloxifene acts as an estrogen agonist on the rabbit growth
plate. Endocrinology 2003;144:1481-1485. Human growth and postnatal development:
clinical studies Marino,
Gafni, Leschek, Barnes, Emons, Nwosu, Baron The primary mechanism that initiates puberty
is unknown. One clue is that pubertal maturation often parallels skeletal
maturation. Conditions that delay skeletal maturation also tend to delay the
onset of puberty, whereas conditions that accelerate skeletal maturation tend
to hasten the onset of puberty. To examine this relationship, we studied boys
with congenital adrenal hyperplasia and familial male-limited precocious
puberty, two conditions that accelerate maturation, and boys with idiopathic
short stature in which maturation is sometimes delayed. In all three conditions, the onset of central
puberty generally occurs at an abnormal chronological age but a normal bone
age. Boys with the greatest skeletal advancement begin central puberty at the
earliest age while boys with the greatest skeletal delay begin puberty at the
latest age. Furthermore, the magnitude of the skeletal advancement or delay
matched the magnitude of the pubertal advancement or delay. We observed this
synchrony between skeletal maturation and hypothalamic-pituitary-gonadal axis
maturation among patients within each condition and between conditions. In
contrast, the maturation of the hypothalamic-pituitary-gonadal axis did not
remain synchronous with other maturational processes, including increases in
weight, height, or body mass index (BMI). We conclude that, in boys with abnormal
developmental tempo, maturation of the skeleton and
hypothalamic-pituitary-gonadal axis remains synchronous. The synchrony is
consistent with the hypothesis that skeletal maturation influences
hypothalamic-pituitary-gonadal axis maturation. Flor A, Leschek EW, Merke DP, Barnes KM, Leschek EW, Rose SR, Yanovski JA, Troendle JF,
Quigley CA, Chipman JJ, Crowe BJ, Ross JL, Cassorla FG, Cutler GB, Baron J.
Effect of growth hormone treatment on adult height in peripubertal children
with idiopathic short stature: a randomized, double-blind, placebo-controlled
trial. J Clin Endocrinol Metab 2004;89:3140-3148. Nwosu BU, Coco M, Jones J, Barnes KM, Yanovski
JA, Baron J. Short stature with normal growth hormone stimulation testing:
lack of evidence for partial growth hormone deficiency or insensitivity. Horm
Res 2004;62:97-102. Weise M, Flor A, Barnes KM, Cutler GB, Baron
J. Determinants of growth during gonadotropin-releasing hormone analog
therapy for precocious puberty. J Clin Endocrinol Metab
2004;89:103-107. COLLABORATORS John Chipman, MD, Eli Lilly
and Company, James Troendle, PhD, Biometry
and Statistics Branch, NICHD, Jan-Maarten Wit, MD, For
further information, contact jbaron@mail.nih.gov |
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