 Here at the University of Michigan, Chad Novins, a recent PhD student, worked with colleagues in the medical school, the University of Florida, and the Ohio State University to investigate a novel PTH response gene. Proteoglycan 4 was identified in a microarray analysis of a tissue-engineered ossicle model, particularly responsive to anabolic actions of PTH. The proteoglycan 4 gene encodes four protein products implicated in the protection of particular joints, hematopoietic progenitor cells, and megakaryopoiesis. Our microarray data was confirmed via in vitro and in vivo quantitative real-time PCR studies, where PTH was found to maximally upregulate PRG4 expression in longbone, calvary and liver. Osteoblastic cells in vitro demonstrate the highest PRG4 levels in proliferative cells with decreasing levels as the cells differentiate and express osteocalcin. At all time points, PTH increases PRG4 with maximal fold increases found in the more differentiated cells. In analyzing skeletal actions of parathyroid hormone, we typically use mice at two different age groups. Our young model initiates parathyroid hormone administration at four days of age and goes for nearly three weeks. This is a model of skeletal modeling, and PTH is particularly responsive in this model and may have appropriate application for regenerative approaches. Our adult model initiates parathyroid hormone administration at 16 weeks of age and goes for six weeks, hence mice are evaluated at 22 weeks of age when the skeleton is mature. Notably, the proteoglycan 4 null mice showed an osteopenic phenotype at both age groups, both in the young and in the adult. The interesting finding was that they responded differently to parathyroid hormone, whereas the young mice had an anabolic response both in the knockouts and in the wild type litter mates of nearly 20 to 30%. In the adult skeleton, wild type mice responded with a nearly 50% increase, but the null mice responded with less than a 20%, and the difference was statistically significant, showing a diminished anabolic response of PTH in proteoglycan null mice. Nacre CT analyses provided three-dimensional scrutiny of the distal femur, and similar trends were found as in the histomorphometric analyses. The increase in trabecular bone volume in response to PTH was similar in young PRG4 null mice, but significantly restricted in adult PRG4 null versus wild type mice. To delve into the mechanisms for the diminished anabolic response in the proteoglycan 4 knockout mice, we performed a series of in vitro studies. We evaluated cell proliferation over time, looking at bone marrow stromal cells from proteoglycan 4 null and litter mates at 16 weeks of age. There was no difference in cell numbers over time, suggesting that there was not a proliferative effect of proteoglycan 4 for null mice. We also looked at osteoblast differentiation, measuring bone nodule formation and osteoblastic phenotypic gene expression under differentiation conditions, and saw no differences there as well. Hence, we concluded that there was no cell autonomous effects of proteoglycan 4 in cells of the osteoblast lineage. Valuable information was obtained relative to a role for FGF2. Adult null mice had lower FGF2 gene expression in the liver and protein levels in the serum. Interestingly, a single PTH injection increased marrow and liver FGF2 at one hour, and six weeks of treatment normalized serum FGF2 levels in null mice. This data suggests FGF2 is a candidate regulator of proteoglycan 4, and that the liver may be an important target organ. An important aspect of this study was the role of biomechanics. Spontaneous exploratory behavior, hind limb stands, and grid crossing showed PRG4 mice have reduced ambulation. These findings highlight a suggested role for the biomechanics of joint integrity and anabolic actions of PTH. To summarize our findings, in the developing skeleton, proteoglycan 4 supports endocondro bone formation with a role for FGF2. In the mature skeleton, proteoglycan 4 supports PTH anabolic actions by protecting joint function. Although I didn't have time to share with you today, we've also found that proteoglycan 4 supports PTH ability to expand hematopoietic cells in the bone marrow. Finally, the findings that PTH regulates gene expression in the bone and the liver in a similar manner suggest that the liver may be an important organ for skeletal actions of PTH. Thank you for your interest in our work. I hope you'll look into more details of this publication in the January issue of the JBMR.