Role of E2F transcription factors in the regulation of childhood growth The human fetus grows at an enormous rate, increasing in mass more than 100 fold between the end of embryogenesis and birth. Somatic growth then slows progressively in postnatal life and ceases by the end of the second decade. The deceleration in body growth is due primarily to a progressive decline in cell proliferation, but the underlying mechanisms are largely unknown. We recently identified a juvenile multi-organ genetic program, which involves the downregulation of a large set of growth-promoting genes in mice and rats, and showed evidence that this program helps explain the rapid body growth of early life and the subsequent slowing of growth with age. We next sought to determine the molecular mechanisms that orchestrate the downregulation of this large gene set. Bioinformatic analysis indicated that E2f transcription factor binding sites are strongly overrepresented among genes participating in this growth-regulating genetic program. We also found that transcription factors E2f1, E2f3a, and E2f3b are downregulated with age in multiple mouse organs. Furthermore, restoration of high activator E2f expression in late juvenile murine hepatocytes helped restore high expression levels of multiple genes in the program. Thus, the findings suggest that E2f transcription factors act as a molecular switch that helps orchestrate the juvenile growth-limiting program. We next focused on one important gene in this program, Igf2, which serves as a potent fetal growth factor. Mice with targeted Igf2 disruption exhibit severe growth retardation at birth. Similarly, in humans, decreased IGF2 expression is associated with poor fetal growth in Silver-Russell syndrome, whereas increased IGF2 expression is associated with excessive fetal growth and predisposition to tumors in Beckwith-Wiedemann syndrome. Postnatally, Igf2 mRNA levels are downregulated in multiple organs. Thus prior studies suggest that IGF2 is one of the growth-promoting genes that are highly expressed in early life, contributing to rapid body growth, but then are downregulated, causing growth to slow. We investigated the mechanisms responsible for the downregulation of IGF2 with age. Chromatin immunoprecipitation indicated that E2f3 specifically binds to the Igf2 P2 promoter region and that this binding decreases with age. Overexpression of E2f3 strongly induced Igf2 expression and could also activate reporter constructs containing the mouse Igf2 P2 promoter. Analysis of existing expression microarray data suggested that similar declines with age in E2F3 and IGF2 expression occur also in the human. Taken together, these findings suggest that downregulation of E2F3 with age contributes to the normal downregulation of IGF2, a key growth factor that controls the rate of body growth. Overexpression of IGF2 is commonly observed in childhood malignancies, as well as many cancers of adulthood. Thus, the high-level expression of IGF2 that occurs in fetal tissues and supports normal rapid body growth is often re-established in many malignant cells, perhaps contributing to their rapid proliferation. Based on our studies linking E2F and IGF2 expression in normal tissues, we hypothesized that the overexpression of IGF2 in some malignancies is due to overexpression of E2Fs. Using microarray expression data, we found that, in E2F3-overexpressing prostate and bladder cancers, IGF2 expression is also elevated and positively correlated with E2F3 expression, providing evidence that the regulation of IGF2 by E2F3 may be an important mechanism for supporting rapid proliferation in cancer cells. Identification of chondrocyte-binding peptides by phage display There are currently no effective treatments for skeletal dysplasias. However, recent studies have identified many paracrine factors that positively regulate growth plate chondrogenesis and therefore might be used to treat these disorders, including bone morphogenetic proteins, C-type natriuretic peptide, and Indian hedgehog. We reasoned that these locally-acting molecules could be used therapeutically by administering them systemically but directing them to growth plate cartilage using cartilage-targeting peptides. In addition, cartilage-targeting peptides could be used to direct endocrine therapy specifically to growth plate cartilage. For example, in achondroplasia, growth hormone increases bone length only modestly and dose is limited by effects on other tissues. Therefore, growth hormone linked to a cartilage-targeting peptide might augment the therapeutic growth-promoting effect and minimize adverse effects due to actions on other tissues, including the potential for promoting malignancies. As an initial step toward targeting cartilage tissue for potential therapeutic application, we sought cartilage-binding peptides using phage display, a powerful technology for selection of peptides that bind to molecules of interest. A library of phage displaying random12-amino acid peptides was iteratively incubated with cultured chondrocytes to select phage that bind cartilage. The resulting phage clones demonstrated increased affinity to chondrocytes by ELISA, when compared to a wild-type, insertless phage. Furthermore, the selected phage showed little preferential binding to other cell types, including primary skin fibroblast, myocyte and hepatocyte cultures, suggesting a tissue-specific interaction. Immunohistochemical staining revealed that the selected phage bound chondrocytes themselves and the surrounding extracellular matrix. FITC-tagged peptides were synthesized based on the sequence of cartilage-binding phage clones. These peptides, but not a random peptide, bound cultured chondrocytes and extracelluar matrix. In conclusion, using phage display, we identified peptide sequences that specifically target chondrocytes. We anticipate that such peptides may be coupled to therapeutic molecules to provide targeted treatment for cartilage disorders.

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Lui, Julian C; Chen, Weiping; Cheung, Crystal S F et al. (2014) Broad shifts in gene expression during early postnatal life are associated with shifts in histone methylation patterns. PLoS One 9:e86957
Delaney, Angela; Padmanabhan, Vasantha; Rezvani, Geoffrey et al. (2014) Evolutionary conservation and modulation of a juvenile growth-regulating genetic program. J Mol Endocrinol 52:269-77
Chau, Michael; Lui, Julian C; Landman, Ellie B M et al. (2014) Gene Expression Profiling Reveals Similarities between the Spatial Architectures of Postnatal Articular and Growth Plate Cartilage. PLoS One 9:e103061
Nilsson, Ola; Guo, Michael H; Dunbar, Nancy et al. (2014) Short stature, accelerated bone maturation, and early growth cessation due to heterozygous aggrecan mutations. J Clin Endocrinol Metab 99:E1510-8
Lui, Julian C; Nilsson, Ola; Baron, Jeffrey (2014) Recent research on the growth plate: Recent insights into the regulation of the growth plate. J Mol Endocrinol 53:T1-9
Nilsson, Ola; Weise, Martina; Landman, Ellie B M et al. (2014) Evidence that estrogen hastens epiphyseal fusion and cessation of longitudinal bone growth by irreversibly depleting the number of resting zone progenitor cells in female rabbits. Endocrinology 155:2892-9
Jee, Youn Hee; Baron, Jeffrey; Phillip, Moshe et al. (2014) Malnutrition and catch-up growth during childhood and puberty. World Rev Nutr Diet 109:89-100
van Duyvenvoorde, Hermine A; Lui, Julian C; Kant, Sarina G et al. (2014) Copy number variants in patients with short stature. Eur J Hum Genet 22:602-9
Finkielstain, G P; Lui, J C; Baron, J (2013) Catch-up growth: cellular and molecular mechanisms. World Rev Nutr Diet 106:100-4
Guo, Michael H; Stoler, Joan; Lui, Julian et al. (2013) Redefining the progeroid form of Ehlers-Danlos syndrome: report of the fourth patient with B4GALT7 deficiency and review of the literature. Am J Med Genet A 161A:2519-27

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