Medicine (St Vincent's) - Theses

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    Physiological role of osteocytic parathyroid hormone-related protein (PTHrP)
    Ansari, Niloufar ( 2018)
    Parathyroid hormone-related protein (PTHrP) is an autocrine/paracrine regulator in many tissues, including bone and mammary glands. Mice with conditional deletion of PTHrP in the osteoblast lineage showed an osteopenic phenotype, and low bone formation levels by the age of 6 weeks, suggesting a paracrine role for PTHrP in promoting bone formation. Since PTHrP is also expressed in osteocytes, I sought to determine the role of osteocytic PTHrP in bone. For this purpose, I have developed an in vitro model of PTHrP (gene: Pthlh) knockdown by using shRNA in a new osteocytic cell line, Ocy454 cells. Pthlh mRNA levels were knocked down by 80% (compared to luciferase) on 3D scaffolds. Pthlh knockdown increased osteocyte markers, Sost (>2-fold greater than luciferase control at day 7), Mepe and Dmp1 (3-4-fold higher than luciferase control at day 14), and reduced osteoblast markers Alpl and Bglap. Undifferentiated Pthlh knockdown cells also had lower levels of cAMP compared to vector control cells. These results showed that PTHrP acts as an autocrine/paracrine factor in osteocytes to regulate mineralisation and bone formation. Next, I generated 10kb-Dmp1Cre.Pthlhf/f mice, which had conditional deletion of PTHrP in osteocytes, from heterozygous breeders. My results demonstrating that Dmp1Cre.Pthlhf/f mice showed no significant differences in cortical or trabecular bone structure compared to Dmp1Cre.Pthlhw/w littermates at 6 weeks of age, indicate that osteocytic PTHrP is not essential for skeletal development in growing mice. However, trabecular bone mass was lower in male and female Dmp1Cre.Pthlhf/f mice compared to controls at 12 weeks of age (the peak of trabecular bone mass in adults). Dmp1Cre.Pthlhf/f mice had lower bone formation, with no difference in bone resorption, compared to controls. Although there was no significant alteration in cortical dimensions, three-point bending tests revealed that Dmp1Cre.Pthlhf/f femora had significantly lower fracture tolerance, with lower ultimate force and deformation, and yield point and fracture occurred at a lower strain compared to controls. These findings indicate that osteocyte-derived PTHrP is required to maintain trabecular bone mass and material strength of adult bones. Dmp1Cre.Pthlhf/f mice from homozygous breeders (Dmp1Cre.Pthlhf/f(hom)) showed a phenotype different from the above heterozygous-bred mice. To our surprise, Dmp1Cre.Pthlhf/f(hom) mice showed higher trabecular bone mass and wider long bones compared to Dmp1Cre.Pthlhw/w(hom) controls. These changes were initially observed in adult male mice, however, I confirmed that this wider cortical bone was detected at 12 days of age in both male and female Dmp1Cre.Pthlhf/f(hom) mice, and retained throughout life in male Dmp1Cre.Pthlhf/f(hom) mice. PTHrP is produced by mammary glands and is secreted into milk. Our results showed that Pthlh DNA recombination directed by Dmp1Cre occurred not only in bones, but also in mammary tissues. To determine the cause of this phenotype I assessed milk content during lactation and fetal bone development during embryogenesis. My results showed recombination of Pthlh in mammary tissues, however there was no difference in milk PTHrP content of Dmp1Cre.Pthlhf/f mothers, compared to controls. Studying the embryonic skeletons at E18.5 showed that Dmp1Cre.Pthlhf/f(hom) fetuses have wider femora, indicating accelerated fetal skeletal development. Finally, I studied the effect of osteocytic PTHrP on the anabolic action of parathyroid hormone (PTH), the only currently approved anabolic therapy for osteoporosis. These studies were initially carried out on homozygous-bred mice. Since these mice exhibited a different basal phenotype to heterozygous-bred mice, we repeated the experiments on the latter to exclude any effect of parental genotype. These two sets of experiment showed that deletion of endogenous osteocytic PTHrP (in both heterozygous- and homozygous-bred Dmp1Cre.Pthlhf/f mice) had no impact on PTH-induced bone formation. I conclude that PTHrP has both local and systemic functions driven by cells that express Dmp1Cre, that influence the skeleton. Osteocytic PTHrP, acting in an autocrine/paracrine manner, is required for normal gene expression by osteocytes and maintains trabecular bone mass and strength at the peak of bone mass in adults. Maternal PTHrP limits fetal skeletal development and radial growth; this effect of maternal PTHrP is important, not only in neonatal mice, but also influences the bone mass of adult male mice.