Normal Bone Growth Requires Optimal Estrogen Levels: Negative Effects of Both High and Low Dose Estrogen on the Number of Growth Plate Chondrocytes

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.

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

Journal of Biological Chemistry ◽

10.1074/jbc.m702991200 ◽

2007 ◽

Vol 282 (46) ◽

pp. 33698-33706 ◽

Cited By ~ 46

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.

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

Journal of Endocrinology ◽

10.1677/joe.0.1490277 ◽

1996 ◽

Vol 149 (2) ◽

pp. 277-285 ◽

Cited By ~ 15

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

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

Journal of Endocrinology ◽

10.1530/joe-11-0353 ◽

2012 ◽

Vol 213 (2) ◽

pp. 163-172 ◽

Cited By ~ 9

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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Liver X Receptor activation regulates genes involved in lipid homeostasis in developing chondrocytes

10.1101/705269 ◽

2019 ◽

Author(s):

Margaret Man-Ger Sun ◽

Frank Beier

Keyword(s):

Lipid Metabolism ◽

Growth Plate ◽

Cholesterol Efflux ◽

Molecular Mechanisms ◽

Bone Growth ◽

Articular Chondrocytes ◽

Protein Localization ◽

Receptor Activation ◽

Lipid Homeostasis ◽

Growth Plate Chondrocytes

AbstractObjectiveOsteoarthritis (OA) is the most common type of arthritis and causes debilitating symptoms and decreased quality of life. Currently available treatment options target symptoms but do not address the underlying issue of joint tissue degeneration. As such, a better understanding of the molecular mechanisms maintaining cartilage health is needed for developing novel therapeutic strategies. Liver X Receptors (LXRs) are nuclear receptors that have been previously shown to offer protection against OA. This is potentially due to suppression of chondrocyte hypertrophy in endochondral bone growth in response to LXR activation. In order to better understand the regulatory mechanisms behind this effect, we aimed to systematically examine LXR’s effects on growth plate chondrocyte gene expression.MethodsPrimary chondrocytes isolated from the long bones of E15.5 mice were treated with the specific LXR agonist, GW3965, and RNA was isolated for Affymetrix microarrays followed by real time qPCR validation. Bioinformatics analyses were performed using Gene Ontology (GO) and KEGG pathway analysis. Immunohistochemistry was conducted to examine protein localization of LXR and identified targets in GW3965-treated E15.5 tibiae compared to control.ResultsActivation of LXR in primary growth plate chondrocytes resulted in differential regulations of various genes involved in lipid metabolism, including several genes involved in cholesterol efflux. This pattern was compared to LXR activation in immature murine articular chondrocytes (IMACs), which revealed similar roles in lipid homeostasis. Immunohistochemical analysis of LXR and its identified targets Abca1 and Srebf1 revealed preferential protein localization to pre-hypertrophic and resting chondrocytes in GW3965-treated tibial growth plates compared to controls.ConclusionOur findings show for the first time that LXR activation alters expression of lipid metabolism genes in growth plate chondrocytes, in part through activation of molecules responsible for cellular cholesterol efflux. This provides insight into potential mechanisms through which LXR regulates cellular metabolism to alter chondrocyte behavior and phenotype.

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Cartilage Ablation of Sirt1 Causes Inhibition of Growth Plate Chondrogenesis by Hyperactivation of mTORC1 Signaling

Endocrinology ◽

10.1210/en.2019-00427 ◽

2019 ◽

Vol 160 (12) ◽

pp. 3001-3017 ◽

Cited By ~ 5

Author(s):

Xinxin Jin ◽

Xiaomin Kang ◽

Liting Zhao ◽

Mao Xu ◽

Tianping Xie ◽

...

Keyword(s):

Growth Plate ◽

Growth Retardation ◽

Bone Growth ◽

Bone Development ◽

Conditional Knockout ◽

Negative Regulator ◽

Sirtuin 1 ◽

Growth Plate Chondrocytes ◽

Mtorc1 Signaling

Abstract A growing body of evidence implies a pivotal role of sirtuin-1 (Sirt1) in chondrocyte function and homeostasis; however, its underlying mechanisms mediating chondrogenesis, which is an essential process for physiological skeletal growth, are still poorly understood. In the current study, we generated TamCartSirt1−/− [Sirt1 conditional knockout (cKO)] mice to explore the role of Sirt1 during postnatal endochondral ossification. Compared with control mice, cKO mice exhibited growth retardation associated with inhibited chondrocyte proliferation and hypertrophy, as well as activated apoptosis. These effects were regulated by hyperactivation of mammalian target of rapamycin complex 1 (mTORC1) signaling, and thereby inhibition of autophagy and induction of endoplasmic reticulum stress in growth plate chondrocytes. IP injection of the mTORC1 inhibitor rapamycin to mice with Sirt1 deletion partially neutralized such inhibitory effects of Sirt1 ablation on longitudinal bone growth, indicating the causative link between SIRT1 and mTORC1 signaling in the growth plate. Mechanistically, SIRT1 interacted with tuberous sclerosis complex 2 (TSC2), a key upstream negative regulator of mTORC1 signaling, and loss of Sirt1 inhibited TSC2 expression, resulting in hyperactivated mTORC1 signaling in chondrocytes. In conclusion, our findings suggest that loss of Sirt1 may trigger mTORC1 signaling in growth plate chondrocytes and contributes to growth retardation, thus indicating that SIRT1 is an important regulator during chondrogenesis and providing new insights into the clinical potential of SIRT1 in bone development.

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Mechanism of longitudinal bone growth and its regulation by growth plate chondrocytes

Microscopy Research and Technique ◽

10.1002/jemt.1070280606 ◽

1994 ◽

Vol 28 (6) ◽

pp. 505-519 ◽

Cited By ~ 289

Author(s):

Ernst B. Hunziker

Keyword(s):

Growth Plate ◽

Bone Growth ◽

Growth Plate Chondrocytes ◽

Longitudinal Bone Growth

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

Journal of Endocrinology ◽

10.1677/joe.1.06958 ◽

2006 ◽

Vol 191 (2) ◽

pp. 369-377 ◽

Cited By ~ 19

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.

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Chemotherapeutic agents used in the treatment of childhood malignancies have direct effects on growth plate chondrocyte proliferation

Journal of Endocrinology ◽

10.1677/joe.0.1570225 ◽

1998 ◽

Vol 157 (2) ◽

pp. 225-235 ◽

Cited By ~ 42

Author(s):

H Robson ◽

E Anderson ◽

OB Eden ◽

O Isaksson ◽

S Shalet

Keyword(s):

Suspension Culture ◽

Growth Plate ◽

Bone Growth ◽

Chemotherapeutic Agents ◽

Long Bone ◽

Direct Effects ◽

Growth Plate Chondrocytes ◽

Chondrocyte Proliferation ◽

Childhood Malignancies ◽

The Impact

Short stature is one of the most well recorded long term sequelae for adult survivors of childhood malignancies. It has become increasingly apparent that cytotoxic chemotherapy, as well as craniospinal irradiation, has a major impact on growth, but there are virtually no studies which explore the mechanisms by which these cytotoxic drugs affect growth. We have used an in vitro system to investigate the direct effects of a range of chemotherapeutic agents on the proliferative responses of rat tibial growth plate chondrocytes, both in suspension and monolayer culture. The glucocorticoids and purine anti-metabolites reduced chondrocyte proliferation both in monolayer and suspension cultures and this resulted from an increase in cell doubling times with a concomittant reduction in the numbers of S phase cells. DNA damaging agents (e.g. actinomycin-D) were also able to reduce chondrocyte proliferation, both in monolayer and suspension culture. This, however, was the result of a cell cycle arrest and subsequent cell death. In our studies, methotrexate had no significant effect on the proliferative responses of the chondrocytes either in monolayer or suspension culture. These results indicate direct effects of a range of chemotherapeutic agents on the proliferative responses of growth plate chondrocytes. Both cytostatic and cytotoxic effects were observed although the impact of either the potential loss of cells from the proliferative pool during chondrocyte differentiation, or the reduction in the rate of chondrocyte turnover on long bone growth remains to be elucidated.

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GnRHa/Stanozolol Combined Therapy Maintains Normal Bone Growth in Central Precocious Puberty

Frontiers in Endocrinology ◽

10.3389/fendo.2021.678797 ◽

2021 ◽

Vol 12 ◽

Author(s):

Shunye Zhu ◽

Lingli Long ◽

Yue Hu ◽

Ying Tuo ◽

Yubin Li ◽

...

Keyword(s):

Signaling Pathway ◽

Growth Plate ◽

Precocious Puberty ◽

Chondrogenic Differentiation ◽

Bone Growth ◽

Normal Growth ◽

Central Precocious Puberty ◽

Height Velocity ◽

Normal Bone ◽

Growth Deceleration

BackgroundGonadotropin-releasing hormone agonist (GnRHa) is the gold standard in the treatment of Central Precocious Puberty (CPP) with progressive puberty and accelerative growth. However, GnRHa treatment is reported to result in growth deceleration and prevents growth plate development which leads to a reduction in height velocity. Stanozolol (ST) has been used to stimulate growth in patients with delayed growth and puberty, nevertheless, the effects and mechanisms of ST on CPP with GnRHa treatment are currently unclear.Methods and ResultsIn the current study, we recorded the following vital observations that provided insights into ST induced chondrogenic differentiation and the maintenance of normal growth plate development: (1) ST efficiently prevented growth deceleration and maintained normal growth plate development in rats undergoing GnRHa treatment; (2) ST suppressed the inhibitory effect of GnRHa to promote chondrogenic differentiation; (3) ST induced chondrogenic differentiation through the activation of the JNK/c-Jun/Sox9 signaling pathway; (4) ST promoted chondrogenic differentiation and growth plate development through the JNK/Sox9 signaling pathway in vivo.ConclusionsST mitigated the inhibitory effects of GnRHa and promoted growth plate development in rats. ST induced the differentiation of chondrocytes and maintained normal growth plate development through the activation of JNK/c-Jun/Sox9 signaling. These novel findings indicated that ST could be a potential agent for maintaining normal bone growth in cases of CPP undergoing GnRHa treatment.

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