Insulin-Like Growth Factor-I Augments Chondrocyte Hypertrophy and Reverses Glucocorticoid-Mediated Growth Retardation in Fetal Mice Metatarsal Cultures

Abstract The study aims were to improve our understanding of the mechanisms of glucocorticoid-induced growth retardation at the growth plate and determine whether IGF-I could ameliorate the effects. Fetal mouse metatarsals were cultured for up to 10 d with dexamethasone (Dex; 10–6m) and/or IGF-I and GH (both at 100 ng/ml). Both continuous and alternate-day Dex treatment inhibited bone growth to a similar degree, whereas IGF-I alone or together with Dex caused an increase in bone growth. GH had no effects. These observations may be explained at the cellular level; cell proliferation within the growing bone was decreased by Dex and increased by IGF-I and these effects were more marked in the cells of the perichondrium than those in the growth plate. However, the most prominent observation was noted in the hypertrophic zone where all treatments containing IGF-I significantly increased (3-fold) the length of this zone, whereas Dex alone had no significant effect. In conclusion, Dex impaired longitudinal growth by inhibiting chondrocyte proliferation, whereas IGF-I stimulated chondrocyte hypertrophy and reversed the growth-inhibitory Dex effects. However, the IGF-I-mediated improvement in growth was at the expense of altering the balance between proliferating and hypertrophic chondrocytes within the metatarsal.

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Evidence supporting dual, IGF-I-independent and IGF-I-dependent, roles for GH in promoting longitudinal bone growth

Journal of Endocrinology ◽

10.1677/joe.0.1800247 ◽

2004 ◽

Vol 180 (2) ◽

pp. 247-255 ◽

Cited By ~ 122

Author(s):

J Wang ◽

J Zhou ◽

CM Cheng ◽

JJ Kopchick ◽

CA Bondy

Keyword(s):

Growth Hormone ◽

Growth Plate ◽

Bone Growth ◽

Long Bone ◽

Null Mouse ◽

Chondrocyte Proliferation ◽

Chondrocyte Hypertrophy ◽

Longitudinal Bone Growth ◽

Germinal Zone ◽

Igf I

The possibility that growth hormone (GH) has effects on long bone growth independent of insulin-like growth factor-I (IGF-I) has long been debated. If this is true, then long bone growth should be more profoundly affected by the absence of GH (since both GH and GH-stimulated IGF-I effects are absent) than by the absence of IGF-I alone (since GH is still present and actually elevated). To test this hypothesis, we compared long bone growth in mice with targeted deletions of Igf1 vs growth hormone receptor (Ghr). Tibial linear growth rate was reduced by approximately 35% in Igf1 null mice and by about 65% in Ghr null mice between postnatal days 20 and 40, a time of peak GH effect during normal longitudinal growth. The Igf1 null mouse growth plate demonstrated significant enlargement of the germinal zone; chondrocyte proliferation and numbers were normal but chondrocyte hypertrophy was significantly reduced. In contrast, the Ghr null mouse germinal zone was hypoplastic, chondrocyte proliferation and numbers were significantly reduced, and chondrocyte hypertrophy was also reduced. We have previously demonstrated that IGF-II is highly expressed in growth plate germinal and proliferative zones, so we considered the possibility that GH-stimulated IGF-II production might promote germinal zone expansion and maintain normal proliferation in the Igf1 null mouse growth plate. Supporting this view, IGF-II mRNA was increased in the Igf1 null mouse and decreased in the Ghr null mouse growth plate.Thus, in the complete absence of IGF-I but in the presence of elevated GH in the Igf1 null mouse, reduction in chondrocyte hypertrophy appears to be the major defect in longitudinal bone growth. In the complete absence of a GH effect in the Ghr null mouse, however, both chondrocyte generation and hypertrophy are compromised, leading to a compound deficit in long bone growth. These observations support dual roles for GH in promoting longitudinal bone growth: an IGF-I-independent role in growth plate chondrocyte generation and an IGF-I-dependent role in promoting chondrocyte hypertrophy. The question of whether GH has direct effects on chondrocyte generation is still not settled, however, since it now appears that IGF-II may medicate some of these effects on the growth plate.

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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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Dexamethasone-induced growth inhibition of porcine growth plate chondrocytes is accompanied by changes in levels of IGF axis components

Journal of Endocrinology ◽

10.1677/joe.0.1740343 ◽

2002 ◽

Vol 174 (2) ◽

pp. 343-352 ◽

Cited By ~ 33

Author(s):

JJ Smink ◽

JA Koedam ◽

JG Koster ◽

SC van Buul-Offers

Keyword(s):

Growth Inhibition ◽

Growth Plate ◽

Growth Retardation ◽

Culture Conditions ◽

Type I ◽

Mrna Levels ◽

Growth Plate Chondrocytes ◽

Type I Igf Receptor ◽

Igf I ◽

Igf Receptor

High (pharmacological) doses of glucocorticoids inhibit the proliferation of growth plate chondrocytes, which leads to one of the side-effects of these steroids, namely suppression of longitudinal growth. Growth inhibition by glucocorticoids is thought to be mediated in part by impaired action of components of the IGF axis, which are important for chondrocyte regulation and hence for longitudinal growth. The aim of the present study was to determine whether glucocorticoid-induced growth retardation involves changes in IGF axis components. Chondrocytes were isolated from epiphyseal growth plates of neonatal piglets and treated with pharmacological doses of dexamethasone (DXM) for 24 h to study glucocorticoid-induced growth retardation. Under IGF-I-supplemented (10 nM) culture conditions, IGF-binding proteins (IGFBPs)-2, -4 and -5 were secreted by the growth plate chondrocytes and IGFBP-2 protein and mRNA levels were decreased by the DXM treatment, whereas IGFBP-4 and -5 were not affected. Proliferation of the chondrocytes, as measured by [(3)H]thymidine incorporation, was 3.5-fold higher in serum-supplemented medium in contrast to IGF-I-supplemented (10 nM) medium. In the presence of serum, DNA synthesis was significantly inhibited by 50-63% when treated with 100 nM DXM, which was prevented by the glucocorticoid-receptor antagonist Org34116. mRNA levels of IGF axis components were determined using Northern blot analysis. IGFBP-2 to -6 were expressed in the chondrocytes, IGFBP-1 was absent and both IGF-I and IGF-II, and the type I and type II IGF receptors were expressed. Treatment with DXM (100 nM) resulted in a 2-fold increase in mRNA levels of both IGFBP-5 and the type I IGF receptor, whereas IGFBP-2 mRNA levels decreased by 55%, in concert with the decrease in protein level observed under IGF-I-supplemented culture conditions. The changes in mRNA levels due to the DXM treatment were prevented by the glucocorticoid receptor antagonist. Our data show that exposure to pharmacological doses of DXM results in inhibition of proliferation and changes in components of the IGF axis, IGFBP-2 and -5 and the type I IGF receptor, suggesting a role for these components in glucocorticoid-induced growth retardation at the local level of the growth plate.

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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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Increased Classical Endoplasmic Reticulum Stress Is Sufficient to Reduce Chondrocyte Proliferation Rate in the Growth Plate and Decrease Bone Growth

PLoS ONE ◽

10.1371/journal.pone.0117016 ◽

2015 ◽

Vol 10 (2) ◽

pp. e0117016 ◽

Cited By ~ 21

Author(s):

Louise H. W. Kung ◽

M. Helen Rajpar ◽

Richard Preziosi ◽

Michael D. Briggs ◽

Raymond P. Boot-Handford

Keyword(s):

Endoplasmic Reticulum ◽

Endoplasmic Reticulum Stress ◽

Growth Plate ◽

Bone Growth ◽

Proliferation Rate ◽

Chondrocyte Proliferation

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DHEA Suppresses Longitudinal Bone Growth by Acting Directly at Growth Plate through Estrogen Receptors

Endocrinology ◽

10.1210/en.2010-0920 ◽

2011 ◽

Vol 152 (4) ◽

pp. 1423-1433 ◽

Cited By ~ 8

Author(s):

Hongzhi Sun ◽

Weijin Zang ◽

Bo Zhou ◽

Lin Xu ◽

Shufang Wu

Keyword(s):

Estrogen Receptor ◽

Growth Plate ◽

Bone Growth ◽

Nuclear Factor Κb ◽

Binding Activity ◽

Skeletal Maturation ◽

Chondrocyte Apoptosis ◽

Chondrocyte Proliferation ◽

Longitudinal Bone Growth ◽

Growth Plate Chondrocyte

Abstract Dehydroepiandrosterone (DHEA) is produced by the adrenal cortex and is the most abundant steroid in humans. Although in some physiological and pathological conditions the increased secretion of DHEA and its sulfated form is associated with accelerated growth rate and skeletal maturation, it is unclear whether DHEA can affect longitudinal bone growth and skeletal maturation by acting directly at the growth plate. In our study, DHEA suppressed metatarsal growth, growth plate chondrocyte proliferation, and hypertrophy/differentiation. In addition, DHEA increased the number of apoptotic chondrocytes in the growth plate. In cultured chondrocytes, DHEA reduced chondrocyte proliferation and induced apoptosis. The DHEA-induced inhibition of metatarsal growth and growth plate chondrocyte proliferation and hypertrophy/differentiation was nullified by culturing metatarsals with DHEA in the presence of ICI 182,780, an inhibitor of estrogen receptor, but not in the presence of Casodex, an inhibitor of androgen receptor. Lastly, nuclear factor-κB DNA binding activity was inhibited by the addition of DHEA in the medium of cultured chondrocyte. Our findings indicate that DHEA suppressed bone growth by acting directly at growth plate through estrogen receptor. Such growth inhibition is mediated by decreased chondrocyte proliferation and hypertrophy/differentiation and by increased chondrocyte apoptosis.

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IGF-I signalling in bone growth: Inhibitory actions of dexamethasone and IL-1β

Growth Hormone & IGF Research ◽

10.1016/j.ghir.2007.05.002 ◽

2007 ◽

Vol 17 (5) ◽

pp. 435-439 ◽

Cited By ~ 30

Author(s):

Vicky E. MacRae ◽

S. Faisal Ahmed ◽

Talat Mushtaq ◽

Colin Farquharson

Keyword(s):

Bone Growth ◽

Growth Inhibitory ◽

Igf I

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Direct administration of testosterone increases rat tibial epiphyseal growth plate width

Acta Endocrinologica ◽

10.1530/acta.0.1210401 ◽

1989 ◽

Vol 121 (3) ◽

pp. 401-405 ◽

Cited By ~ 36

Author(s):

Song Guang Ren ◽

Saul Malozowski ◽

Prosper Sanchez ◽

Donald E. Sweet ◽

D. Lynn Loriaux ◽

...

Keyword(s):

Growth Plate ◽

Bone Growth ◽

Serum Testosterone ◽

Male Rats ◽

Testosterone Enanthate ◽

Epiphyseal Growth Plate ◽

Igf I ◽

Direct Administration ◽

Tibial Epiphyseal ◽

Plate Width

Abstract. Local injection of hormones into the tibial epiphyseal growth plate offers a possible model to answer whether sex steroids can affect bone growth directly. To answer this question, we injected different doses of testosterone enanthate (4, 40, 120 and 400 μg/100 g of rat weight) once into the tibial epiphyseal growth plate of castrated 35-day-old male rats. The contralateral tibia was injected with sesame oil and served as control. All animals were sacrificed at age 42 days. Tibias were removed for measurement of epiphyseal growth plate width and blood was collected for measurement of serum IGF-I and testosterone. The lower doses of testosterone enanthate (4, 40 and 120 μg/100 g) did not produce any significant change in epiphyseal growth plate width. Testosterone at the largest dose tested (400 μg/100 g) increased epiphyseal growth plate width by about 15% compared to control (p < 0.01). At this dose, serum testosterone was not increased, suggesting that the effect on epiphyseal growth plate width was not due to higher systemic testosterone concentrations. No differences in IGF-I levels were observed among the groups. We conclude that direct administration of testosterone enanthate at a dose of 400 μg/100 g into the rat tibial epiphyseal growth plate can increase epiphyseal growth plate width.

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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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Smad6/Smurf1 overexpression in cartilage delays chondrocyte hypertrophy and causes dwarfism with osteopenia

The Journal of Cell Biology ◽

10.1083/jcb.200311015 ◽

2004 ◽

Vol 165 (3) ◽

pp. 433-445 ◽

Cited By ~ 80

Author(s):

Mitsuru Horiki ◽

Takeshi Imamura ◽

Mina Okamoto ◽

Makoto Hayashi ◽

Junko Murai ◽

...

Keyword(s):

Signal Transduction ◽

Transgenic Mice ◽

Organ Culture ◽

Bone Morphogenetic Proteins ◽

Endochondral Ossification ◽

Bone Growth ◽

Regulatory Factor ◽

Chondrocyte Proliferation ◽

Chondrocyte Hypertrophy

Biochemical experiments have shown that Smad6 and Smad ubiquitin regulatory factor 1 (Smurf1) block the signal transduction of bone morphogenetic proteins (BMPs). However, their in vivo functions are largely unknown. Here, we generated transgenic mice overexpressing Smad6 in chondrocytes. Smad6 transgenic mice showed postnatal dwarfism with osteopenia and inhibition of Smad1/5/8 phosphorylation in chondrocytes. Endochondral ossification during development in these mice was associated with almost normal chondrocyte proliferation, significantly delayed chondrocyte hypertrophy, and thin trabecular bone. The reduced population of hypertrophic chondrocytes after birth seemed to be related to impaired bone growth and formation. Organ culture of cartilage rudiments showed that chondrocyte hypertrophy induced by BMP2 was inhibited in cartilage prepared from Smad6 transgenic mice. We then generated transgenic mice overexpressing Smurf1 in chondrocytes. Abnormalities were undetectable in Smurf1 transgenic mice. Mating Smad6 and Smurf1 transgenic mice produced double-transgenic pups with more delayed endochondral ossification than Smad6 transgenic mice. These results provided evidence that Smurf1 supports Smad6 function in vivo.

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