scholarly journals Ceramide inhibition of chondrocyte proliferation and bone growth is IGF-I independent

2006 ◽  
Vol 191 (2) ◽  
pp. 369-377 ◽  
Author(s):  
V E MacRae ◽  
T Burdon ◽  
S F Ahmed ◽  
C Farquharson
Keyword(s):  
Insulin Receptor ◽  
Growth Plate ◽  
Bone Growth ◽  
Inflammatory Diseases ◽  
Thymidine Uptake ◽  
Growth Plate Chondrocytes ◽  
Igf I ◽  
Presence And Absence ◽  
Underlying Mechanisms ◽  
Induced Apoptosis

Proinflammatory cytokines inhibit growth plate development. However, their underlying mechanisms of action are unclear. These effects may be mediated by ceramide, a sphingosine-based lipid second messenger, which is elevated in a number of chronic inflammatory diseases. To test this hypothesis, we determined the effects of C2-ceramide, a cell permeable ceramide analogue, on the growth of the ATDC5 chondrogenic cell line and on cultured fetal mice metatarsals. In ATDC5 cells, C2-ceramide significantly induced apoptosis at both 40 (82%; P < 0.05) and 25 μM (53%; P < 0.05). At 40 μM, C2-ceramide significantly reduced proliferation ([3H]-thymidine uptake/mg protein) (62%; P < 0.05). C2-ceramide did not markedly alter the differentiation state of the cells as judged by the expression of markers of chondrogenesis and differentiation (sox 9, collagen II and collagen X). The IGF-I signalling pathway is the major autocrine/paracrine regulator of bone growth. Both in the presence and absence of IGF-I, C2-ceramide (25 μM) induced an equivalent reduction in proliferation (60%; P < 0.001). Similarly, C2-ceramide (40 μM) induced a 31% reduction in fetal metatarsal growth both in the presence and absence of IGF-I (both P < 0.001). Furthermore, C2-ceramide reduced ADCT5 proliferation in the presence of AG1024, an IGF-I and insulin receptor blocker. Therefore, C2-ceramide-dependent inhibition appears to be independent of IGF-mediated stimulation of bone growth. Indeed, biochemical studies demonstrated that C2-ceramide (25 μM) pretreatment did not alter IGF-I-stimulated phosphorylation of insulin receptor substrate-1, Akt or P44/42 MAP kinase. In conclusion, C2-ceramide inhibits proliferation and induces apoptosis in growth plate chondrocytes through an IGF-I independent mechanism.

scholarly journals Prostacyclin Regulates Bone Growth via the Epac/Rap1 Pathway

Endocrinology ◽  
2014 ◽  
Vol 156 (2) ◽  
pp. 499-510 ◽  
Author(s):  
Michele R. Hutchison ◽  
Perrin C. White
Keyword(s):  
Growth Plate ◽  
Bone Growth ◽  
Articular Chondrocytes ◽  
Prostaglandin D2 ◽  
Growth Plate Chondrocytes ◽  
Inhibition Of Proliferation ◽  
Erk Activation ◽  
Growth Plate Cartilage ◽  
Igf I ◽  
Cox 2

Prostaglandins, particularly PGE2, are important to adult bone and joint health, but how prostaglandins act on growth plate cartilage to affect bone growth is unclear. We show that growth plate cartilage is distinct from articular cartilage with respect to cyclooxygenase (COX)-2 mRNA expression; although articular chondrocytes express very little COX-2, COX-2 expression is high in growth plate chondrocytes and is increased by IGF-I. In bovine primary growth plate chondrocytes, ATDC5 cells, and human metatarsal explants, inhibition of COX activity with nonsteroidal antiinflammatory drugs (NSAIDs) inhibits chondrocyte proliferation and ERK activation by IGF-I. This inhibition is reversed by prostaglandin E2 and prostacyclin (PGI2) but not by prostaglandin D2 or thromboxane B2. Inhibition of COX activity in young mice by ip injections of NSAIDs causes dwarfism. In growth plate chondrocytes, inhibition of proliferation and ERK activation by NSAIDs is reversed by forskolin, 8-bromoadenosine, 3′,5′-cAMP and a prostacyclin analog, iloprost. The inhibition of proliferation and ERK activation by celecoxib is also reversed by 8CPT-2Me-cAMP, an activator of Epac, implicating the small G protein Rap1 in the pathway activated by iloprost. These results imply that prostacyclin is required for proper growth plate development and bone growth.

scholarly journals Catch-Up Growth after Hypothyroidism Is Caused by Delayed Growth Plate Senescence

Endocrinology ◽  
2008 ◽  
Vol 149 (4) ◽  
pp. 1820-1828 ◽  
Author(s):  
Rose Marino ◽  
Anita Hegde ◽  
Kevin M. Barnes ◽  
Lenneke Schrier ◽  
Joyce A. Emons ◽  
...  
Keyword(s):  
Growth Rate ◽  
Growth Inhibition ◽  
Growth Plate ◽  
Bone Growth ◽  
Structural Characteristics ◽  
Linear Growth Rate ◽  
Growth Plate Chondrocytes ◽  
Growth Plates ◽  
Catch Up ◽  
Growth Inhibiting

Catch-up growth is defined as a linear growth rate greater than expected for age after a period of growth inhibition. We hypothesized that catch-up growth occurs because growth-inhibiting conditions conserve the limited proliferative capacity of growth plate chondrocytes, thus slowing the normal process of growth plate senescence. When the growth-inhibiting condition resolves, the growth plates are less senescent and therefore grow more rapidly than normal for age. To test this hypothesis, we administered propylthiouracil to newborn rats for 8 wk to induce hypothyroidism and then stopped the propylthiouracil to allow catch-up growth. In untreated controls, the growth plates underwent progressive, senescent changes in multiple functional and structural characteristics. We also identified genes that showed large changes in mRNA expression in growth plate and used these changes as molecular markers of senescence. In treated animals, after stopping propylthiouracil, these functional, structural, and molecular senescent changes were delayed, compared with controls. This delayed senescence included a delayed decline in longitudinal growth rate, resulting in catch-up growth. The findings demonstrate that growth inhibition due to hypothyroidism slows the developmental program of growth plate senescence, including the normal decline in the rate of longitudinal bone growth, thus accounting for catch-up growth.

Pi transport regulation by chicken growth plate chondrocytes

1994 ◽  
Vol 267 (1) ◽  
pp. E24-E31
Author(s):  
C. Montessuit ◽  
J. P. Bonjour ◽  
J. Caverzasio
Keyword(s):  
Growth Plate ◽  
Primary Cultures ◽  
Growth Plate Chondrocytes ◽  
Phosphonoformic Acid ◽  
Pi Transport ◽  
Igf I ◽  
Sodium Dependent ◽  
Half Maximal Effective Concentration ◽  
Cartilage Cells ◽  
Extracellular Sodium

Inorganic phosphate (Pi) is a key element for the growth and mineralization of the epiphyseal cartilage. In this study, the characteristics of the transport of Pi in growth plate chondrocytes have been determined using primary cultures of chicken growth plate cartilage cells. The uptake of Pi was significantly increased in the presence of extracellular sodium. The kinetic parameters of the saturable sodium-dependent Pi transport (NaPiT) were determined. The Michaelis constant for Pi was 0.443 +/- 0.095 mM, and the concentration of sodium with which half-maximal Pi transport was observed was 48.0 +/- 8.7 mM. Stoichiometric analysis suggested that more than one sodium ion was cotransported with each Pi molecule. NaPiT was sensitive to inhibition by Pi analogues such as phosphonoformic acid and arsenate. These data strongly suggest that Pi uptake by chicken growth plate chondrocytes is a carrier-mediated process driven by the transmembrane electrochemical gradient of sodium. Two important regulators of biosynthetic activities of growth plate chondrocytes, insulin-like growth factor I (IGF-I) and parathyroid hormone (PTH), selectively regulated Pi transport. With IGF-I, maximal stimulation (117 +/- 7% above control) was observed at doses > 5 nM, with an half-maximal effective concentration of 0.46 +/- 0.18 nM. A significant effect was observed after 1 h of exposure and was maintained for up to 24 h. PTH increased Pi transport with a biphasic dose-response curve. The change in NaPiT was transient, being maximally observed after 8 h (58 +/- 8%) and unexpressed after 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Nuclear Factor-κB p65 Facilitates Longitudinal Bone Growth by Inducing Growth Plate Chondrocyte Proliferation and Differentiation and by Preventing Apoptosis

2007 ◽  
Vol 282 (46) ◽  
pp. 33698-33706 ◽  
Author(s):  
Shufang Wu ◽  
Janna K. Flint ◽  
Geoffrey Rezvani ◽  
Francesco De Luca
Keyword(s):  
Growth Plate ◽  
Bone Growth ◽  
Chondrocyte Apoptosis ◽  
Pyrrolidine Dithiocarbamate ◽  
Growth Plate Chondrocytes ◽  
Chondrocyte Proliferation ◽  
Longitudinal Bone Growth ◽  
Metatarsal Bones ◽  
Proliferation And Differentiation ◽  
Cultured Chondrocytes

NF-κB is a group of transcription factors involved in cell proliferation, differentiation, and apoptosis. Mice deficient in the NF-κB subunits p50 and p52 have retarded growth, suggesting that NF-κB is involved in bone growth. Yet, it is not clear whether the reduced bone growth of these mice depends on the lack of NF-κB activity in growth plate chondrocytes. Using cultured rat metatarsal bones and isolated growth plate chondrocytes, we studied the effects of two NF-κB inhibitors (pyrrolidine dithiocarbamate (PDTC) or BAY11-7082 (BAY)), p65 short interference RNA (siRNA), and of the overexpression of p65 on chondrocyte proliferation, differentiation, and apoptosis. To further define the underlying mechanisms, we studied the functional interaction between NF-κB p65 and BMP-2 in chondrocytes. PDTC and BAY suppressed metatarsal linear growth. Such growth inhibition resulted from decreased chondrocyte proliferation and differentiation and from increased chondrocyte apoptosis. In cultured chondrocytes, the inhibition of NF-κB p65 activation (by PDTC and BAY) and expression (by p65 siRNA) led to the same findings observed in cultured metatarsal bones. In contrast, overexpression of p65 in cultured chondrocytes induced chondrocyte proliferation and differentiation and prevented apoptosis. Although PDTC, BAY, and p65 siRNA reduced the expression of BMP-2 in cultured growth plate chondrocytes, the overexpression of p65 increased it. The addition of Noggin, a BMP-2 antagonist, neutralized the stimulatory effects of p65 on chondrocyte proliferation and differentiation, as well as its anti-apoptotic effect. In conclusion, our findings indicate that NF-κB p65 expressed in growth plate chondrocytes facilitates growth plate chondrogenesis and longitudinal bone growth by inducing BMP-2 expression and activity.

scholarly journals Inhibition of IGF-I–related intracellular signaling pathways by proinflammatory cytokines in growth plate chondrocytes

2014 ◽  
Vol 76 (3) ◽  
pp. 245-251 ◽  
Author(s):  
Daniela Choukair ◽  
Ulrike Hügel ◽  
Anja Sander ◽  
Lorenz Uhlmann ◽  
Burkhard Tönshoff
Keyword(s):  
Signaling Pathways ◽  
Growth Plate ◽  
Proinflammatory Cytokines ◽  
Intracellular Signaling ◽  
Growth Plate Chondrocytes ◽  
Intracellular Signaling Pathways ◽  
Igf I

scholarly journals Apoptosis of Growth Plate Chondrocytes Occurs through a Mitochondrial Pathway

2007 ◽  
Vol 77 (1) ◽  
pp. 129-134 ◽  
Author(s):  
Cristina C. Teixeira ◽  
Aida P. Padron Costas ◽  
Yelena Nemelivsky
Keyword(s):  
Cytochrome C ◽  
Growth Plate ◽  
Caspase 3 ◽  
Mitochondrial Pathway ◽  
Chondrocyte Apoptosis ◽  
Cytochrome C Release ◽  
Growth Plate Chondrocytes ◽  
Chondrocyte Viability ◽  
Induced Apoptosis ◽  
Free Media

Abstract Objective: To determine the role of mitochondria in chondrocyte apoptosis induced by inorganic phosphate (Pi). Materials and Methods: Chondrocytes isolated from the growth plates of chick embryo tibia were treated with Pi in serum-free media; chondrocyte viability, mitochondrial membrane potential, cytochrome c release from mitochondria, caspase 3 activity, endonuclease activity, and DNA fragmentation were investigated. Results: Exposure to Pi for 24 hours induced apoptosis in growth plate chondrocytes through a pathway that involved loss of mitochondrial function, release of cytochrome c into the cytoplasm, increases in caspase 3 and endonuclease activities, and fragmentation of DNA. Conclusions: This study suggests that mitochondria are important players in Pi-induced apoptosis.

The expression of transforming growth factor-β by cultured chick growth plate chondrocytes: differential regulation by 1,25-dihydroxyvitamin D3

1996 ◽  
Vol 149 (2) ◽  
pp. 277-285 ◽  
Author(s):  
C Farquharson ◽  
A S Law ◽  
E Seawright ◽  
D W Burt ◽  
C C Whitehead
Keyword(s):  
Growth Factor ◽  
Conditioned Medium ◽  
Growth Plate ◽  
Bone Growth ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Mrna Levels ◽  
Chick Growth ◽  
Growth Plate Chondrocytes ◽  
Tgf Β1

Abstract 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) and transforming growth factor-β (TGF-β) are both important regulators of chondrocyte growth and differentiation. We report here that 1,25(OH)2D3 differentially regulates the expression of the genes for TGF-β1 to -β3 and the secretion of the corresponding proteins in cultured chick chondrocytes. Confluent growth plate chondrocytes were serum-deprived and cultured in varying concentrations of 1,25(OH)2D3. Cells were assayed for TGF-β mRNA and conditioned medium was assayed for TGF-β activity and isoform composition. Active TGF-β was only detected in 10−8m 1,25(OH)2D3-treated cultures (8·37 ng active TGF-β/mg protein). There was a significant decrease in total (latent+active) TGF-β activity in conditioned medium of 10−12 m (23·4%; P<0·05) and 10−10 m (20·7%; P<0·05) 1,25(OH)2D3-treated cultures but 10−8 m 1,25(OH)2D3 significantly increased (30·9%; P<0·01) TGF-β activity. The amounts of TGF-β1, -β2 and -β3 isoforms produced were similar in control, 10−10 or 10−12m 1,25(OH)2D3-treated cultures but the conditioned medium of 10−8 m 1,25(OH)2D3-treated cultures contained significantly higher amounts of all three isoforms. Quantification of TGF-β mRNA demonstrated differential control of TGF-β gene expression with TGF-β1 and -β3 mRNA levels reduced by all concentrations of 1,25(OH)2D3 examined (10−8, 10−10 and 10−12 m) whilst TGF-β2 mRNA concentrations were elevated. Our results indicated that 1,25(OH)2D3 regulates chick growth plate chondrocyte TGF-β secretion and mRNA expression in a concentration-dependent and isoform-specific manner. This interaction may be important in the regulation of chondrocyte metabolism and endochondral bone growth. Journal of Endocrinology (1996) 149, 277–285

scholarly journals Imaging IGF-I uptake in growth plate cartilage using in vivo multiphoton microscopy

2017 ◽  
Vol 123 (5) ◽  
pp. 1101-1109 ◽  
Author(s):  
Maria A. Serrat ◽  
Gabriela Ion
Keyword(s):  
Real Time ◽  
Growth Plate ◽  
Multiphoton Microscopy ◽  
Biologically Active ◽  
Growth Plate Chondrocytes ◽  
Growth Plates ◽  
Metatarsal Bones ◽  
Wide Range ◽  
Igf I

Bones elongate through endochondral ossification in cartilaginous growth plates located at ends of primary long bones. Linear growth ensues from a cascade of biochemical signals initiated by actions of systemic and local regulators on growth plate chondrocytes. Although cellular processes are well defined, there is a fundamental gap in understanding how growth regulators are physically transported from surrounding blood vessels into and through dense, avascular cartilage matrix. Intravital imaging using in vivo multiphoton microscopy is one promising strategy to overcome this barrier by quantitatively tracking molecular delivery to cartilage from the vasculature in real time. We previously used in vivo multiphoton imaging to show that hindlimb heating increases vascular access of large molecules to growth plates using 10-, 40-, and 70-kDa dextran tracers. To comparatively evaluate transport of similarly sized physiological regulators, we developed and validated methods for measuring uptake of biologically active IGF-I into proximal tibial growth plates of live 5-wk-old mice. We demonstrate that fluorescently labeled IGF-I (8.2 kDa) is readily taken up in the growth plate and localizes to chondrocytes. Bioactivity tests performed on cultured metatarsal bones confirmed that the labeled protein is functional, assessed by phosphorylation of its signaling kinase, Akt. This methodology, which can be broadly applied to many different proteins and tissues, is relevant for understanding factors that affect delivery of biologically relevant molecules to the skeleton in real time. Results may lead to the development of drug-targeting strategies to treat a wide range of bone and cartilage pathologies. NEW & NOTEWORTHY This paper describes and validates a novel method for imaging transport of biologically active, fluorescently labeled IGF-I into skeletal growth plates of live mice using multiphoton microscopy. Cellular patterns of fluorescence in the growth plate were completely distinct from our prior publications using biologically inert probes, demonstrating for the first time in vivo localization of IGF-I in chondrocytes and perichondrium. These results form important groundwork for future studies aimed at targeting therapeutics into growth plates.

scholarly journals Estrogen stimulates leptin receptor expression in ATDC5 cells via the estrogen receptor and extracellular signal-regulated kinase pathways

2012 ◽  
Vol 213 (2) ◽  
pp. 163-172 ◽  
Author(s):  
Shan-Jin Wang ◽  
Xin-Feng Li ◽  
Lei-Sheng Jiang ◽  
Li-Yang Dai
Keyword(s):  
Growth Plate ◽  
Cross Talk ◽  
Bone Growth ◽  
Leptin Receptor ◽  
Receptor Expression ◽  
Long Bone ◽  
Endochondral Bone Formation ◽  
Dependent Manner ◽  
Growth Plate Chondrocytes ◽  
Concentration Dependent Manner

Regulation of the physiological processes of endochondral bone formation during long bone growth is controlled by various factors including the hormones estrogen and leptin. The effects of estrogen are mediated not only through the direct activity of estrogen receptors (ERs) but also through cross talk with other signaling systems implicated in chondrogenesis. The receptors of both estrogen and leptin (OBR (LEPR)) are detectable in growth plate chondrocytes of all zones. In this study, the expression of mRNA and protein of OBR in chondrogenic ATDC5 cells and the effect of 17β-estradiol (E2) stimulation were assessed using quantitative PCR and western blotting. We have found that the mRNA of Obr was dynamically expressed during the differentiation of ATDC5 cells over 21 days. Application of E2 (10−7 M) at day 14 for 48 h significantly upregulated OBR mRNA and protein levels (P<0.05). The upregulation of Obr mRNA by E2 was shown to take place in a concentration-dependent manner, with a concentration of 10−7 M E2 having the greatest effect. Furthermore, we have confirmed that E2 affected the phosphorylation of ERK1/2 (MAPK1/MAPK3) in a time-dependent manner where a maximal fourfold change was observed at 10 min following application of E2. Finally, pretreatment of the cells with either U0126 (ERK1/2 inhibitor) or ICI 182 780 (ER antagonist) blocked the upregulation of OBR by E2 and prevented the E2-induced phosphorylation of ERK. These data demonstrate, for the first time, the existence of cross talk between estrogen and OBR in the regulation of bone growth whereby estrogen regulates the expression of Obr in growth plate chondrocytes via ERs and the activation of ERK1/2 signaling pathways.

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