Medicine (St Vincent's) - Theses
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ItemPhysiological role of osteocytic parathyroid hormone-related protein (PTHrP)( 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.
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ItemOsteoblastic EphrinB2 in normal and parathyroid hormone (PTH)-stimulated bone formation.( 2016)Bone remodelling renews the skeleton through cycles of bone resorption by osteoclasts and formation by osteoblasts. Two interacting members of the Eph tyrosine kinase family, EphrinB2 and EphB4, are expressed by osteoblasts and their expression of EphrinB2 is stimulated by parathyroid hormone (PTH), the only approved anabolic therapy for osteoporosis. I investigated the roles of the ephrinB2/EphB4 interaction in osteoblast lineage cells in bone remodelling and mediating anabolic actions of PTH. I began by assessing the effects of EphB4 overexpression in mouse osteoblasts in vivo and how this affected anabolic PTH. While PTH induced increases in osteoblast and osteoid parameters in both wild-type and EphB4-overexpressing mice, these changes were not significantly different between the two groups. However, we observed some evidence of a suppressed osteoclast response to PTH, and a slight augmentation of the anabolic action of PTH on bone volume in female mice only. Lack of consistent overexpression of EphB4 in these mice limited our ability to gain further conclusions. Next, I investigated the effects of pharmacological blockade of ephrinB2/EphB4 interaction in vivo in the presence and absence of anabolic PTH. A previously described pharmacologic inhibitor of this interaction, sEphB4, was used alone or in combination with anabolic PTH. In both settings, sEphB4 treatment increased osteoblast formation and mRNA levels of early osteoblast markers (Runx2, alkaline phosphatase, Collagen 1a1, and PTH receptor [PTHR1]) without causing significantly higher bone formation rate or late markers of osteoblast/osteocyte differentiation. In the presence of PTH, sEphB4 treatment significantly increased osteoclast formation and converted the anabolic effect of PTH to a catabolic effect. This effect on osteoclasts was recapitulated in vitro in co-cultures, suggesting that it is a cell-autonomous effect. This indicates a key role for the ephrinB2/EphB4 interaction within the osteoblast lineage in osteoblast differentiation and support of osteoclastogenesis. To assess the role of ephrinB2 signalling in the osteoblast lineage in normal bone formation, I analyzed bone remodelling in mice that lacked osteoblastic ephrinB2. For this purpose, an osteoblast-specific genetic deletion mouse model was used. These mice showed a delay in bone mineralisation along with lower mRNA levels of late osteoblast differentiation markers and greater levels of osteoblast and osteocyte apoptosis. Additionally, osteoblastic support of osteoclast formation was defective in these mice and in osteoblasts cultured from these mice. Finally, I assessed how lack of osteoblastic ephrinB2 signalling affected anabolic PTH effects by treating the above-mentioned mice with anabolic PTH. This experiment showed that osteoblastic ephrinB2 is required, not only for physiological bone remodelling, but also for effects of anabolic PTH in causing higher osteoblast number, surface and osteoid production. This requirement appears to be due to the control ephrinB2 may have over osteoid deposition and mineralisation in trabecular and cortical bone. I conclude that ephrinB2, through its binding partner EphB4, promotes the late stages of osteoblast maturation that are required for mineralisation, and supports osteoclast formation. These functions are essential for the full anabolic action of PTH, such that blockade of signalling events downstream of the ephrinB2:EphB4 interaction turns anabolic action of PTH into a catabolic effect. Given the current interest in targeting the ephrinB2/EphB4 interaction in antiangiogenic and anticancer therapies, my findings show that targeting EphB4 may have less skeletal side effects than blocking both sides of this interaction. Enhancing ephrinB2 signalling may be a viable strategy for conditions where more bone formation is desired such as osteoporosis, osteogenesis imperfecta, and fracture healing.