Genetic determinants of bone growth
Sridevan, Sruthi M.
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Bone is a highly dynamic component of the skeleton that is constantly undergoing growth and remodeling. Growth proceeds via two mechanisms: intramembranous ossification (for craniofacial bones), or endochondral ossification (for long bones of the axial skeleton). The growth and stability of healthy bone is mediated by the specific cell types resident in bone, namely osteoblasts that form bone, osteoclasts that resorb bone, and osteocytes which act as calcium load sensors and coordinate a response to the microenvironment. The growth, mass, and strength of bone can also be influenced at the genetic level. Two examples of this are mutations altering transcription of the Igf2 gene, and post-translational modification of extracellular matrix proteins via citrullination. IGF-2 is a peptide hormone synthesized in the liver and found primarily in fetal tissues during fetal growth stages. It is responsible for proper growth of the body and skeleton and is usually never found in the adult skeleton. The effect of IGF-2 on the adult skeleton is not known. Knock out of one or both Igf2 genes results in a growth deficiency phenotype characterized by shorter bones with less bone mineral density in mice. Therefore, both copies of the gene are needed for complete genetic expression and normal growth. In contrast, dysregulated transcription of Igf2 in pigs, caused by a single nucleotide mutation in an intronic region, caused increased organ mass due to the loss of the binding site for the transcriptional repressor (ZBED6). Mice in which an analogous mutation was inserted by knock-in technology, referred to as Igf2G/A knock-ins, have been generated and similarly have increased organ mass. These mice allow us to study the effect of IGF-2 in the adult skeleton. Citrullination, on the other hand, is a post translational modification where peptidyl arginine deaminases (PADs) modify peptidyl arginine to peptidyl citrulline. This modification has been associated with altered protein function and loss of structural stability. To investigate the effects of IGF-2 on the adult skeleton, we analyzed the bone phenotype of 12-14 week-old mice where Igf2 transcription had been disinhibited via nucleotide substitution from G to A at the intronic binding site for ZBED6, an Igf2 transcriptional repressor. We hypothesized that mutant Igf2G/A mice will show a growth phenotype in terms of bone quantity measured via microCT, as well as quality measured via mechanical testing. Consistent with our hypothesis, we found that Igf2G/A mice have increased femur length, increased cortical area and periosteal perimeter, and increased resistance to torsion; the knock-in females have increased bone volume to trabecular volume (BV/TV), as well as increased trabecular number, decreased spacing between the trabeculae, and increased trabecular thickness. Initial mechanical testing shows no significant difference in work to fracture between WT and KI mice. To investigate the effects of citrullination on bone mechanical strength and phenotype, we used an existing murine model that was deficient in an inhibitor of PAD4 and citrullination, protein tyrosine phosphatase nonreceptor-22 (PTPN22). Citrullination of bone extracellular matrix proteins has been shown to promote bone loss in rheumatoid arthritis (RA). We therefore hypothesized that adult mice with increased citrullination resulting from PTPN22-deficiency will exhibit a bone loss phenotype. However, we observe no significant difference between the modified and wild type mice in either bone structural parameters measured via microCT, or in mechanical strength measured through three-point bending, although there was a slight decrease in femur length of female Ptpn22-/- mice compared to wild type controls.
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