Osterix (Osx) is a transcription factor required for osteoblast differentiation and bone formation. We previously demonstrated that continuous parathyroid hormone (PTH) treatment inhibited Osx expression in murine calvaria and osteoblastic UMR106-01 cells through the regulation of two regions on the Osx promoter. Mutational analysis of transcription factor elements within these regions revealed two "Sp-sites" were vital for Osx promoter activity. Blockage of these Sp-sites with Mithramycin A demonstrated their importance for Osx expression. Osx bound to its own promoter at these sites, while PTH treatment inhibited this association. PTH regulation of Osx expression in vivo was investigated in mice by: daily injection of PTH for 3 days, continuous infusion of PTH from osmotic pumps for 14 days, or mice fed a calcium-deficient diet for 21 days. Osx expression was decreased by daily injection, while Osx expression was stimulated in mice receiving continuous PTH infusion and mice fed a calcium-deficient diet.

Osterix (Osx) is a transcription factor required for osteoblast differentiation and bone formation. We previously demonstrated that continuous parathyroid hormone (PTH) treatment inhibited Osx expression in murine calvaria and osteoblastic UMR106-01 cells through the regulation of two regions on the Osx promoter. Mutational analysis of transcription factor elements within these regions revealed two "Sp-sites" were vital for Osx promoter activity. Blockage of these Sp-sites with Mithramycin A demonstrated their importance for Osx expression. Osx bound to its own promoter at these sites, while PTH treatment inhibited this association. PTH regulation of Osx expression in vivo was investigated in mice by: daily injection of PTH for 3 days, continuous infusion of PTH from osmotic pumps for 14 days, or mice fed a calcium-deficient diet for 21 days. Osx expression was decreased by daily injection, while Osx expression was stimulated in mice receiving continuous PTH infusion and mice fed a calcium-deficient diet.

Indiana University-Purdue University Indianapolis (IUPUI) / Many factors are present in the development of skeletal tissue. Some factors lead to an increase in bone mass while some lead to a decrease. One factor that is known to have an influence on skeletal development is Signal Transducer and Activator of Transcription 3 (Stat3). This knowledge arose because of a mutation in the Stat3 gene in humans causing a disease called Hyper-IgE Syndrome. This mutation leads to a variety of issues, including decreased bone mass. Because of this, our lab has sought to study Stat3 in its relation to bone. Many studies have already been conducted that discern how Stat3 influences skeletal biology by observing its role in osteoclasts, osteoblasts, and other bone cells. Its role is still unclear, and many studies have provided seemingly contradictory results in how it works on bone tissue. Our lab set up several different studies in order to further elucidate what role Stat3 plays in skeletal development by looking at its effects on osteoblasts and osteoclasts, the bone-forming and bone-destroying cells of the body, respectively. We conditionally knocked out Stat3 in the osteoblasts of mice and compared several different bone parameters to their wild type counterparts at 8 weeks of age. Differences were noted in bone phenotype, including decreased femur length, weight, bone mineral density, and bone mineral content in the cKO compared to their WT counterparts. While no significant difference in trabecular integrity was noted, several differences were observed in cortical bone. These differences indicate that Stat3 has a positive role in osteoblast differentiation, leading to an overall positive effect on bone mass. To observe the role of Stat3 in osteoclasts, in vitro experiments were set up in which pre-osteoclast RAW 264.7 cells were manipulated with Stat3 siRNA or a Stat3 overexpression construct and RANKL to induce differentiation. Using qPCR and western blot assays, it was determined that when Stat3 is knocked down, several important genes in osteoclastogenesis and osteoclast function are more highly expressed than in the control groups. When Stat3 is overexpressed, a similar pattern is observed where these same genes are downregulated in the presence of higher Stat3 levels. These results indicate that Stat3 has an overall inhibitory effect on osteoclastogenesis and osteoclast function, indicating it has a positive effect on bone mass. Future studies could be performed to further elucidate the effects of Stat3 on skeletal development. Isolating the osteoblasts from cKO and WT mice and performing qPCR and western blot assays could be useful in finding out how Stat3 is influencing these cells. Further studies could also be done on the RAW 264.7 cells to find where Stat3 is interacting with the RANKL pathway. A resorption assay could be done with these cells to better understand how function might be influenced by Stat3.

Indiana University-Purdue University Indianapolis (IUPUI) / Intervertebral discs (IVD) degenerate earlier than many other musculoskeletal tissues and will continue to degenerate with aging. IVD degeneration affects up to 80 percent of the adult population and is a major contributing factor to low back pain. Anti-sclerostin antibody is an FDA-approved treatment for osteoporosis in postmenopausal women at high-risk for fracture and, as a systemic stimulant of the Wnt/LRP5/β-Catenin signaling pathway, may impact the IVD. Stabilization of β-Catenin in the IVD increases Wnt signaling and is anabolic to the extracellular matrix (ECM), while deletion of β-catenin or LRP5 decreases Wnt signaling and is catabolic to the ECM. Here, we hypothesized that a reduction of Sost would stimulate ECM anabolism. Lumbar and caudal (tail) IVD and vertebrae of Sost KO and WT (wildtype) mice (n=8 each) were harvested at 16 weeks of age and tested by MRI, histology, immunohistochemistry, Western Blot, qPCR, and microCT. Compared to WT, Sost KO reduced sclerostin protein and Sost gene expression. Next, Sost KO increased the hydration of the IVD and the proteoglycan stain in the nucleus pulposus and decreased the expression of genes associated with IVD degeneration, e.g., heat shock proteins. However, deletion of Sost was compensated by less unphosphorylated (active) β-Catenin protein in the cell nucleus, upregulation of Wnt signaling inhibitors Dkk1 and sFRP4, and catabolic ECM gene expression. Consequently, notochordal and early chondrocyte-like cells (CLCs) were replaced by mature CLCs. Overall, Sost deletion increased hydration and proteoglycan protein content, but activated a compensatory suppression of Wnt signaling that may trigger chondrogenesis and may potentially be iatrogenic to the IVD in the long-term.

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Tissue engineering and ex vivo gene therapy can be used synergically as tool to regenerate bone, which overcome the problems of currently available bone replacements. Recently, a new source of mesenchymal stromal cells (MSCs) has been found in the umbilical cord; human umbilical cord perivascular cells (HUCPVCs) provide an alternative to bone marrow derived MSCs and due to their easy harvest, fast expansion, and non-immunogeneic and immunomodulatory phenotype we hypothesized that HUCPVCs are a putative candidate cell source for osteogenic ex vivo gene therapy. This work proposes the generation of cocktails of genetically modified HUCPVCs and their cryopreservation as an “off the shelf” therapeutic. This approach involves the engineering of osteogenic cell populations, by genetically modifying HUCPVCs using recombinant adenoviruses to deliver four fundamental genes for bone formation: bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (Runx2), Osterix (OSX/SP7) transcription factor and vascular endothelial growth factor (VEGF). Our results show that HUCPVCs can be efficiently modified by adenoviruses and can be cryopreserved without affecting the production efficiency and bioactivity of proteins of interest produced by the cells. Moreover, overexpression of BMP2, Runx2 and SP7 enhances ALP activity levels in HUCPVCs and upregulates ALP, OPN, COL1A1 and OCN gene expression; data that provides the first evidence of the effects of combinational expression of BMP2, Runx2 and SP7. Furthermore, we report for the first time the genetic modification of human BMSCs to express SP7 and Runx2, which enhances their ALP activity and matrix mineralization capacity.

Tissue engineering and ex vivo gene therapy can be used synergically as tool to regenerate bone, which overcome the problems of currently available bone replacements. Recently, a new source of mesenchymal stromal cells (MSCs) has been found in the umbilical cord; human umbilical cord perivascular cells (HUCPVCs) provide an alternative to bone marrow derived MSCs and due to their easy harvest, fast expansion, and non-immunogeneic and immunomodulatory phenotype we hypothesized that HUCPVCs are a putative candidate cell source for osteogenic ex vivo gene therapy. This work proposes the generation of cocktails of genetically modified HUCPVCs and their cryopreservation as an “off the shelf” therapeutic. This approach involves the engineering of osteogenic cell populations, by genetically modifying HUCPVCs using recombinant adenoviruses to deliver four fundamental genes for bone formation: bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (Runx2), Osterix (OSX/SP7) transcription factor and vascular endothelial growth factor (VEGF). Our results show that HUCPVCs can be efficiently modified by adenoviruses and can be cryopreserved without affecting the production efficiency and bioactivity of proteins of interest produced by the cells. Moreover, overexpression of BMP2, Runx2 and SP7 enhances ALP activity levels in HUCPVCs and upregulates ALP, OPN, COL1A1 and OCN gene expression; data that provides the first evidence of the effects of combinational expression of BMP2, Runx2 and SP7. Furthermore, we report for the first time the genetic modification of human BMSCs to express SP7 and Runx2, which enhances their ALP activity and matrix mineralization capacity.

Indiana University-Purdue University Indianapolis (IUPUI) / Multiple myeloma (MM) is the most frequent cancer to involve the skeleton, with over 80% of myeloma patients developing lytic bone disease (MMBD). Importantly, MM-associated bone lesions rarely heal even when patients are in complete remission. Bone marrow stromal cells (BMSCs) isolated from MM patients have a distinct genetic profile and an impaired osteoblast (OB) differentiation capacity when compared to BMSCs from healthy donors. Utilizing an in vivo model of MMBD and patient samples, we showed that BMSCs from tumor-bearing bones failed to differentiate into OBs weeks after removal of MM cells. Both Runx2 and Osterix, the master transcription factors for OB differentiation, remained suppressed in these BMSCs. However, the molecular mechanisms for MM-induced long-term OB suppression are poorly understood. We characterized both Runx2 and Osterix promoters in murine pre-osteoblast MC4 cells by chromatin immunoprecipitation (ChIP). The transcriptional start sites (TSSs) of Runx2 and Osterix in untreated MC4 cells were co-occupied by transcriptionally active histone 3 lysine 4 tri-methylation (H3K4me3) and transcriptionally repressive histone 3 lysine 27 tri-methylation (H3K27me3), termed the “bivalent domain”. These bivalent domains became transcriptionally silent with increasing H3K27me3 levels when MC4 cells were co-cultured with MM cells or treated with TNF-α, an inflammatory cytokine increased in MM bone marrow microenvironment. The increasing H3K27me3 levels induced by MM cells or TNF-α were associated with the downregulation of the H3K27 demethylase JMJD3 in MC4 cells and murine BMSCs. Knockdown of JMJD3 in MC4 cells was sufficient to inhibit OB differentiation. Further, ectopic overexpression of JMJD3 in MC4 cells partially rescued the suppression of osteoblast differentiation induced by TNFa. We also found that pre-incubation of MC4 cells with the NF-kB inhibitor quinazoline (QNZ) before TNF-a treatment prevented the downregulation of JMJD3. In agreement with our in vitro findings, BMSCs from MM patients had persistently decreased JMJD3 expression compared to healthy BMSCs. Our findings together demonstrate that decreased JMJD3 expression in BMSCs contributes to the long-term OB suppression in MMBD by remodeling histone landscapes at the Runx2 and Osterix TSSs. Thus, developing strategies to restore JMJD3 expression in BMSCs should increase bone formation and possibly decrease tumor burden in MM.

Hintergrund: Knochenentwicklung und -umbau werden durch den Phosphoinositid-3-Kinase(PI3K)-Signalweg gesteuert. Es wurden die Auswirkungen der Herunterregulierung von Phosphatase and Tensin Homolog deleted on Chromosome 10 (PTEN), einem negativen Regulator des PI3K-Signalwegs, in Osteoprogenitorzellen untersucht. Methoden: Femora, Tibiae und Knochenmarkstromazellen (BMSCs) von Mäusen mit Cre-induzierbarem Pten-Knockout in Zellen, die den Transkriptionsfaktor Osterix/Sp7 exprimieren (Pten-cKO), wurden mit Cre-negativen Kontrollmäusen verglichen. Die Phänotypisierung des Knochens erfolgte mittels Mikro-Computertomographie (μCT) und Drei-Punkt-Biegeversuch. Die Anzahl der Osteoklasten und Osteoblasten wurde durch Färbung mittels Tartrat-resistenter saurer Phosphatase (TRAP) immunhistochemisch bestimmt. Nach der Isolation und Kultur der BMSCs wurde deren Proliferation durch Immunfluoreszenzfärbung des Proliferationsmarkers Ki-67 gemessen. In vitro erfolgte die adipogene und osteogene Differenzierung und Zellfärbung durch Ölrot-O- bzw. alkalische Phosphatase sowie die Bestimmung der Genexpression von osteogenen und adipogenen Markern. Zudem wurde der Knochenumsatz mittels ELISA im Mausserum zum Nachweis von C-terminalem Telopeptid (CTX) und N-terminalem Propeptid von Prokollagen Typ 1 (P1NP) sowie histomorphometrisch durch intraperitonealer Calcein-Injektionen zwei und fünf Tage vor der Tötung beurteilt. Ergebnisse: Pten-cKO-BMSCs und Cre-negative Kontrollen unterschieden sich funktionell voneinander. In vitro wurde ein Anstieg der Proliferationsrate von Pten-cKO-BMSCs festgestellt. Während in vitro kein signifikanter Unterschied hinsichtlich der adipogenen Differenzierung nachgewiesen werden konnte, war die osteogene Differenzierungskapazität in BMSCs von Pten-cKO-Mäusen signifikant erhöht. Die Knochenumsatzmarker P1NP und CTX zeigten sich im Serum von Pten-cKO-Mäusen im Vergleich zu Kontrollen erhöht. Zudem konnten in Pten-cKO-Knochen von männlichen Mäusen pro Knochenoberfläche weniger Osteoklasten und mehr Osteoblasten (p = 0.00095) und ein erhöhter Anteil mineralisierter Oberfläche sowie eine erhöhte Knochenbildungsrate im Vergleich zu Kontrollen nachgewiesen werden. Bei Pten-cKO-Mäusen wurden ein höheres trabekuläres Knochenvolumen (trab. BV/TV) und eine höhere Knochenmineraldichte (trab. BMD) als bei den Negativkontrollen festgestellt, während die BV/TV- und BMD-Werte bei kortikalen Knochen niedriger waren. Die biomechanische Analyse ergab eine signifikant höhere Maximalkraft (p = 0.00012) und Elastizität der Femora von Pten-cKO-Mäusen. Schlussfolgerung: Konditionaler Pten-Knockout in Osteoprogenitorzellen führt zu verstärkter Proliferation sowie osteogener Differenzierung von Knochenmarkstromazellen und erhöht die Knochenstabilität und -elastizität.

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