Glucocorticoid effects on chondrogenesis, differentiation and apoptosis in the murine ATDC5 chondrocyte cell line

Mushtaq, Talat; Farquharson, Colin; Seawright, Elaine; Ahmed, S. Faisal

doi:10.1677/joe.0.1750705

Cited by 70 publications

(56 citation statements)

References 47 publications

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“…Cells were differentiated at 37°C in a humidified atmosphere containing 5% CO 2 for 8 -15 days, and Dex (final concentration 10 Ϫ6 M) was added to the cells at the indicated times. We and others have previously shown that, from 8 days of differentiation, ATDC5 cells express the chondrocyte marker gene collagen type II, and by day 15 the cells display a terminally differentiating phenotype with increased levels of collagen type X and aggrecan expression and the formation of nodules (49,16). To test lipocalin 2 expression following exposure to other antiproliferative treatments, ATDC5 cells were cultured in the presence of ceramide (40 M), TNF␣ (10 ng/ml), or serum-free medium (SFM) for 48 h. The protein synthesis blocker cycloheximide (CHX; 8 g/ml), and the GC receptor antagonist RU-486 (10 Ϫ5 M) were added to ATDC5 cells to investigate the mechanisms involved in Dex-induced lipocalin 2 expression.…”

Section: Methodsmentioning

confidence: 82%

Dexamethasone-induced expression of the glucocorticoid response gene lipocalin 2 in chondrocytes

Owen¹,

Roberts²,

Ahmed³

et al. 2008

American Journal of Physiology-Endocrinology and Metabolism

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Owen HC, Roberts SJ, Ahmed SF, Farquharson C. Dexamethasone-induced expression of the glucocorticoid response gene lipocalin 2 in chondrocytes. Am J Physiol Endocrinol Metab 294: E1023-E1034, 2008. First published April 1, 2008 doi:10.1152/ajpendo.00586.2007 are commonly used anti-inflammatory drugs, but long-term use can result in marked growth retardation in children due to their actions on growth plate chondrocytes. To gain an insight into the mechanisms involved in GC-induced growth retardation, we performed Affymetrix microarray analysis of the murine chondrogenic cell line ATDC5, incubated with 10 Ϫ6 M dexamethasone (Dex) for 24 h. Downregulated genes included secreted frizzled-related protein and IGF-I, and upregulated genes included serum/GC-regulated kinase, connective-tissue growth factor, and lipocalin 2. Lipocalin 2 expression increased 40-fold after 24-h Dex treatment. Expression increased further after 48-h (75-fold) and 96-h (84-fold) Dex treatment, and this response was Dex concentration dependent. Lipocalin 2 was immunolocalized to both proliferating and hypertrophic growth plate zones, and its expression was increased by Dex in primary chondrocytes at 6 h (3-fold, P Ͻ 0.05). The lipocalin 2 response was blocked by the GC-receptor antagonist RU-486 and was increased further by the protein synthesis blocker cycloheximide. Proliferation in lipocalin 2-overexpressing cells was less than in control cells (49%, P Ͻ 0.05), and overexpression caused an increase in collagen type X expression (4-fold, P Ͻ 0.05). The effects of lipocalin 2 overexpression on chondrocyte proliferation (64%, P Ͻ 0.05) and collagen type X expression (8-fold, P Ͻ 0.05) were further exacerbated with the addition of 10 Ϫ6

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Section: Methodsmentioning

confidence: 82%

Dexamethasone-induced expression of the glucocorticoid response gene lipocalin 2 in chondrocytes

Owen¹,

Roberts²,

Ahmed³

et al. 2008

American Journal of Physiology-Endocrinology and Metabolism

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“…To mechanistically investigate the role of HPSE in endochondral bone formation, we used the well accepted ATDC5 cell in vitro model system, because it permits study of both chondrogenesis and osteogenesis within the same cell culture system [76]. ATDC5 also can mineralize spontaneously without the addition of an exogenous substrate such as β-glycerophosphate [77].…”

Section: Discussionmentioning

confidence: 99%

Heparanase expression and activity influences chondrogenic and osteogenic processes during endochondral bone formation

Brown

Alicknavitch

D'Souza

et al. 2008

Bone

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Endochondral bone formation is a highly orchestrated process involving coordination among cellcell, cell-matrix and growth factor signaling that eventually results in the production of mineralized bone from a cartilage template. Chondrogenic and osteogenic differentiation occur in sequence during this process, and the temporospatial patterning clearly requires the activities of heparin binding growth factors and their receptors. Heparanase (HPSE) plays a role in osteogenesis, but the mechanism by which it does so is incompletely understood. We used a combination of ex vivo and in vitro approaches and a well described HPSE inhibitor, PI-88 to study HPSE in endochondral bone formation. In situ hybridization and immunolocalization with HPSE antibodies revealed that HPSE is expressed in the peri-chondrium, peri-osteum, and at the chondro-osseous junction, all sites of key signaling events and tissue morphogenesis. Transcripts encoding Hpse also were observed in the prehypertrophic zone. Addition of PI-88 to metatarsals in organ culture reduced growth and suggested that HPSE activity aids the transition from chondrogenic to osteogenic processes in growth of long bones. To study this, we used high density cultures of ATDC5 pre-chondrogenic cells grown under conditions favoring chondrogenesis or osteogenesis. Under chondrogenic conditions, HPSE/Hpse was expressed at high levels during the mid-culture period, at the onset of terminal chondrogenesis. PI-88 addition reduced chondrogenesis and accelerated osteogenesis, including a dramatic upregulation of osteocalcin levels. In normal growth medium, addition of PI-88 reduced migration of ATDC-5 cells, suggesting that HPSE facilitates cartilage replacement by bone at the chondro-osseous junction by removing the HS component of proteoglycans, such as perlecan/HSPG2, that otherwise prevent osteogenic cells from remodeling hypertrophic cartilage. †Corresponding Author:

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“…(43) The bones were washed in PBS (3 Â 15 minutes) and unbound thymidine extracted using 5% TCA (2 Â 30 minutes). (45,46) Experiments requiring serum deprivation and GH or IGF-1 challenge were treated as described above for primary cells.…”

Section: Metatarsal [ 3 H]-thymidine Proliferation Assaymentioning

confidence: 99%

SOCS2 is the critical regulator of GH action in murine growth plate chondrogenesis

Pass

MacRae

Huesa

et al. 2012

Journal of Bone and Mineral Research

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Suppressor of Cytokine Signaling-2 (SOCS2) is a negative regulator of growth hormone (GH) signaling and bone growth via inhibition of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway. This has been classically demonstrated by the overgrowth phenotype of SOCS2 À/À mice, which has normal systemic insulin-like growth factor 1 (IGF-1) levels. The local effects of GH on bone growth are equivocal, and therefore this study aimed to understand better the SOCS2 signaling mechanisms mediating the local actions of GH on epiphyseal chondrocytes and bone growth. SOCS2, in contrast to SOCS1 and SOCS3 expression, was increased in cultured chondrocytes after GH challenge. Gain-and loss-of-function studies indicated that GH-stimulated chondrocyte STATs-1, -3, and -5 phosphorylation was increased in SOCS2 À/À chondrocytes but not in cells overexpressing SOCS2. This increased chondrocyte STAT signaling in the absence of SOCS2 is likely to explain the observed GH stimulation of longitudinal growth of cultured SOCS2À/À embryonic metatarsals and the proliferation of chondrocytes within. Consistent with this metatarsal data, bone growth rates, growth plate widths, and chondrocyte proliferation were all increased in SOCS2 À/À 6-week-old mice as was the number of phosphorylated STAT-5-positive hypertrophic chondrocytes. The SOCS2 À/À mouse represents a valid model for studying the local effects of GH on bone growth. ß

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Glucocorticoid effects on chondrogenesis, differentiation and apoptosis in the murine ATDC5 chondrocyte cell line

Cited by 70 publications

References 47 publications

Dexamethasone-induced expression of the glucocorticoid response gene lipocalin 2 in chondrocytes

Dexamethasone-induced expression of the glucocorticoid response gene lipocalin 2 in chondrocytes

Heparanase expression and activity influences chondrogenic and osteogenic processes during endochondral bone formation

SOCS2 is the critical regulator of GH action in murine growth plate chondrogenesis

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