Comparison of the Action of Transient and Continuous PTH on Primary Osteoblast Cultures Expressing Differentiation Stage-Specific GFP

Wang, Yu‐Hsiung; Liu, Yaling; Buhl, Kathy; Rowe, David W.

doi:10.1359/jbmr.041016

Cited by 81 publications

(51 citation statements)

References 46 publications

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“…It has been shown that vascular smooth muscle cells and skeletal osteoblasts respond oppositely to the same stimuli, such as oxidized phospholipids and continuous activation of the PKA pathway (as in hyperparathyroidism) (3,24,(33)(34)(35)(36)(37), i.e. these factors promote osteoblastic differentiation of vascular cells but inhibit osteoblastic differentiation of bone cells.…”

Section: Table 2 Effect Of Pki On Tnf-␣ Induction Of Ranklmentioning

confidence: 99%

PKA-induced Receptor Activator of NF-κB Ligand (RANKL) Expression in Vascular Cells Mediates Osteoclastogenesis but Not Matrix Calcification

Tseng

Graham

Geng

et al. 2010

Journal of Biological Chemistry

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Vascular calcification is a predictor of cardiovascular mortality and is prevalent in patients with atherosclerosis and chronic renal disease. It resembles skeletal osteogenesis, and many bone cells as well as bone-related factors involved in both formation and resorption have been localized in calcified arteries. Previously, we showed that aortic medial cells undergo osteoblastic differentiation and matrix calcification both spontaneously and in response to PKA agonists. The PKA signaling pathway is also involved in regulating bone resorption in skeletal tissue by stimulating osteoblast-production of osteoclast regulating cytokines, including receptoractivator of nuclear B ligand (RANKL) and interleukins. Therefore, we investigated whether PKA activators regulate osteoclastogenesis in aortic smooth muscle cells (SMC). Treatment of murine SMC with the PKA agonist forskolin stimulated RANKL expression at both mRNA and protein levels. Forskolin also stimulated expression of interleukin-6 but not osteoprotegerin (OPG), an inhibitor of RANKL. Consistent with these results, osteoclastic differentiation was induced when monocytic preosteoclasts (RAW264.7) were cocultured with forskolin-treated aortic SMC. Oxidized phospholipids also slightly induced RANKL expression in T lymphocytes, another potential source of RANKL in the vasculature. Because previous studies have shown that RANKL treatment alone induces matrix calcification of valvular and vascular cells, we next examined whether RANKL mediates forskolin-induced matrix calcification by aortic SMC. RANKL inhibition with OPG had little or no effect on osteoblastic differentiation and matrix calcification of aortic SMC. These findings suggest that, as in skeletal tissues, PKA activation induces bone resorptive factors in the vasculature and that aortic SMC calcification specifically induced by PKA, is not mediated by RANKL.

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Section: Table 2 Effect Of Pki On Tnf-␣ Induction Of Ranklmentioning

confidence: 99%

PKA-induced Receptor Activator of NF-κB Ligand (RANKL) Expression in Vascular Cells Mediates Osteoclastogenesis but Not Matrix Calcification

Tseng

Graham

Geng

et al. 2010

Journal of Biological Chemistry

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“…The Col2.3 promoter has been well characterized and specifically targets gene expression to mature osteoblasts (7)(8)(9)(10). Green fluorescent protein (GFP) transgenic mice using the same fragment of the type I collagen promoter to drive GFP expression (2.3Col-GFP) demonstrated that only cells with features of mature osteoblasts show fluorescence in vivo and in vitro (7,9). Microarray analysis data on this population of GFP-positive cells show it to be highly enriched for expression of mature osteoblast-specific genes such as osteocalcin, bone sialoprotein (BSP), and dentin matrix protein-1 (8).…”

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confidence: 99%

Osteoblasts Directly Control Lineage Commitment of Mesenchymal Progenitor Cells through Wnt Signaling

Zhou

Mak

Zheng

et al. 2008

Journal of Biological Chemistry

139

103

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Lineage commitment of mesenchymal progenitor cells is still poorly understood. Here we demonstrate that Wnt signaling by osteoblasts is essential for mesenchymal progenitor cells to differentiate away from a default adipogenic into an osteoblastic lineage. Dominant adipogenesis and reduced osteoblastogenesis were observed in calvarial cell cultures from transgenic mice characterized by osteoblast-targeted disruption of glucocorticoid signaling. This phenotypic shift in mesenchymal progenitor cell commitment was associated with reciprocal regulation of early adipogenic and osteoblastogenic transcription factors and with a reduction in Wnt7b and Wnt10b mRNA and ␤-catenin protein levels in transgenic versus non-transgenic cultures. Transwell co-culture of transgenic mesenchymal progenitor cells with wild type osteoblasts restored commitment to the osteoblast lineage. This effect was blocked by adding sFRP1, a Wnt inhibitor, to the co-culture. Treatment of transgenic cultures with Wnt3a resulted in stimulation of osteoblastogenesis and suppression of adipogenesis. Our findings suggest a novel cellular mechanism in bone cell biology in which osteoblasts exert direct control over the lineage commitment of their mesenchymalprogenitorthroughWntsignaling.Thisglucocorticoiddependent forward control function indicates a central role for osteoblasts in the regulation of early osteoblastogenesis.Mesenchymal stem cells are able to differentiate to multiple connective tissue and musculoskeletal cell types under the control of specific transcription factors directing their lineage commitment. For example, osteoblasts and adipocytes share a common mesenchymal cell progenitor that can be isolated from neonatal mouse or fetal rat calvaria (1). Primary cell cultures derived from these tissues can differentiate into osteoblasts or adipocytes. The differentiation of these mesenchymal progenitors into osteoblasts or adipocytes is governed by the expression of the master transcription factors runt-related gene 2 (Runx2) (2) or peroxisome proliferator-activated receptor ␥ (PPAR␥) 2 (3), respectively. However, the cellular interactions and mechanisms that must drive the expression of these transcription factors are poorly understood. Glucocorticoid (GC) signaling through its cognate receptor is known to influence osteoblast and adipocyte lineage commitment both in vitro and in vivo and is, thus, likely to have a role in regulating cellular interactions driving cell commitment. In addition, specific enzymes modulate GC metabolism within the cell at the pre-receptor level (4, 5). Within certain tissues, two isoforms of 11␤-hydroxysteroid dehydrogenase (11␤HSD) vary intracellular GC concentrations independent of circulating GC levels. 11␤HSD type 1 (11␤HSD1) predominantly converts inactive cortisone to active cortisol to increase intracellular GC concentrations; in contrast, 11␤HSD type 2 (11␤HSD2) unidirectionally catalyzes the conversion of active GC to their inactive metabolites (4).Kream and co-workers (6) generated a Col2.3-11␤HSD2 tra...

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“…Previously, we have shown that primary calvarial osteoblast cultures derived from mice transgenic for promoter-driven stage-specific green fluorescent protein (GFP) marker genes pOBCol3.6GFP and pOBCol2.3GFP (24) respond to an early transient PTH exposure (from day 1 to 7) and, consequently, after the removal of PTH, acquire intensified expression of fluorescent markers and increased formation of mineralized bone nodules by day 21 (43). However, at day 21, the final DNA content in PTH-treated cultures was never higher than that of control cultures, making it unlikely that the early transient PTH exposure stimulates progenitor cell proliferation.…”

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confidence: 99%

Effects of transient PTH on early proliferation, apoptosis, and subsequent differentiation of osteoblast in primary osteoblast cultures

Wang¹,

Liu²,

Rowe³

2007

American Journal of Physiology-Endocrinology and Metabolism

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In primary calvarial osteoblast cultures derived from transgenic mice expressing green fluorescent protein (GFP) under the control of 3.6-kb Col1a1 promoter, the emergence of GFP signal marks the transition of multipotential osteoprogenitors into preosteoblasts. Early transient treatment (days 1-7) of these cultures with parathyroid hormone (PTH) has an anabolic effect that is not associated with an increase in total DNA content or cell number in day 21 cultures. In the present study, the effect of early PTH treatment on cell proliferation and apoptosis was examined in greater detail in GFP(+) and GFP(-) cells using flow cytometry. In preconfluent cultures, PTH significantly reduced the proportion of cells in S phase but increased those in G(0)/G(1) and G(2)+M phases in both GFP(+) and GFP(-) subpopulations. PTH decreased apoptosis only in GFP(-) but not GFP(+) cells, indicating an increased survival of GFP(-) cells. In contrast, PTH did not change the amounts of cell proliferation and apoptosis seen in either compartment after these cultures reached confluence. To further assess the effect of early PTH treatment on osteogenic differentiation, secondary cultures of sorted GFP(+) or GFP(-) cells were obtained from day 7 primary cultures that had been treated for 1 wk with PTH. This treatment resulted in larger areas of GFP expression accompanied by increased xylenol orange/von Kossa staining in the secondary cultures of GFP fractions. Early transient PTH treatment appears to enhance the commitment of progenitor cells to an osteogenic fate and results in a higher proportion of cells that achieve full osteoblast differentiation.

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Comparison of the Action of Transient and Continuous PTH on Primary Osteoblast Cultures Expressing Differentiation Stage-Specific GFP

Cited by 81 publications

References 46 publications

PKA-induced Receptor Activator of NF-κB Ligand (RANKL) Expression in Vascular Cells Mediates Osteoclastogenesis but Not Matrix Calcification

PKA-induced Receptor Activator of NF-κB Ligand (RANKL) Expression in Vascular Cells Mediates Osteoclastogenesis but Not Matrix Calcification

Osteoblasts Directly Control Lineage Commitment of Mesenchymal Progenitor Cells through Wnt Signaling

Effects of transient PTH on early proliferation, apoptosis, and subsequent differentiation of osteoblast in primary osteoblast cultures

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