Antiproliferative Effects of Insulin-like Growth Factor-binding Protein-3 in Mesenchymal Chondrogenic Cell Line RCJ3.1C5.18

Spagnoli, Anna; Hwa, Vivian; Horton, William A.; Lunstrum, Gregory P.; Roberts, Charles T.; Chiarelli, Francesco; Torello, Monica; Rosenfeld, Ron G.

doi:10.1074/jbc.m005088200

Cited by 64 publications

(79 citation statements)

References 46 publications

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“…We then differentiated C5.18 cells for an extended time course toward terminally differentiated hypertrophic-like chondrocytes. 18 Differentiation of these cells was confirmed by increased gene expression of chondrocyte markers Acan, Col2a1, Col10a1, and Alpl as well as nodule formation and glycosaminoglycan production ( Fig. S1A-H).…”

Section: Pharmacological Inhibitors Of Lysosomal Activity Stimulated mentioning

confidence: 77%

Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

et al. 2015

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These authors contributed equally to this publication.Keywords: autophagy, bafilomycin, bone, chondrocyte, lysosome, MTOR, MTORC1Abbreviations: 3-MA, 3-methyladenine; ACAN, aggrecan; ACP5/TRAP, acid phosphatase 5, tartrate-resistant; ACTB, actin, b; ALPL, alkaline phosphatase, liver/bone/kindey; ATG, autophagy related; ATG5cKO, ATG5 conditional knockout in chondrocytes; BC, avidin-biotin complex; Baf, bafilomycin A 1 ; bGP, b-glycerophosphate; BrdU, bromodeoxyuridine; C5.18 cells, RCJ 3.1C5.18 rat mesenchymal cell line; COL2A1, collagen, type II, a 1; COL10A1, collagen, type X, a 1; CQ, chloroquine; DAPI, 4 0 , 6-diamidino-2-phenylindole, dihydrochloride; Dox, doxycycline; EIF4EBP1, eukaryotic translation initiation factor 4E binding protein 1; FBS, fetal bovine serum; GAG, glycosaminoglycan; IGF1, insulin-like growth factor 1 (somatomedin C); LAMP2, lysosomalassociated membrane protein 2; MEFs, mouse embryonic fibroblasts; MAP1LC3A, microtubule-associated protein 1 light chain 3 a;MTOR, mechanistic target of rapamycin (serine/threonine kinase); MTORC, MTOR complex; p-, phosphorylated-; PFA, paraformaldehyde; RPS6, ribosomal protein S6; RPS6KB1/p70(S6K)-a/PS6K/S6K1, ribosomal protein S6 kinase, 70kDa, polypeptide 1; RPTOR, regulatory associated protein of MTOR, complex 1; SGK1, serum/glucocorticoid regulated kinase 1; TSC, tuberous sclerosis complex; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; v-ATPase, vacuolar-type H C -ATPase.Mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) is a protein-signaling complex at the fulcrum of anabolic and catabolic processes, which acts depending on wide-ranging environmental cues. It is generally accepted that lysosomes facilitate MTORC1 activation by generating an internal pool of amino acids. Amino acids activate MTORC1 by stimulating its translocation to the lysosomal membrane where it forms a super-complex involving the lysosomal-membrane-bound vacuolar-type H C -ATPase (v-ATPase) proton pump. This translocation and MTORC1 activation require functional lysosomes. Here we found that, in contrast to this well-accepted concept, in epiphyseal chondrocytes inhibition of lysosomal activity by v-ATPase inhibitors bafilomycin A 1 or concanamycin A potently activated MTORC1 signaling. The activity of MTORC1 was visualized by phosphorylated forms of RPS6 (ribosomal protein S6) and EIF4EBP1, 2 well-known downstream targets of MTORC1. Maximal RPS6 phosphorylation was observed at 48-h treatment and reached as high as a 12-fold increase (p < 0.018). This activation of MTORC1 was further confirmed in bone organ culture and promoted potent stimulation of longitudinal growth (p < 0.001). Importantly, the same effect was observed in ATG5 (autophagy-related 5)-deficient bones suggesting a macroautophagy-independent mechanism of MTORC1 inhibition by lysosomes. Thus, our data show that in epiphyseal chondrocytes lysosomes inhibit MTORC1 in a macroautophagy-independent manner and this inhibition likely depends on v-ATPase activity.

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Section: Pharmacological Inhibitors Of Lysosomal Activity Stimulated mentioning

confidence: 77%

Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

et al. 2015

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“…RCJ3·1C5·18 cells are derived from rat calvaria mesenchymal cells and over 2 weeks of culture, without requiring biochemical and oncogenic transformation, they sequentially differentiate from MCCs to terminally differentiated chondrocytes in a highly predictable and reproducible manner (Grigoriadis et al 1996, Lunstrum et al 1999, Spagnoli et al 2001. Furthermore, RCJ3·1C5·18 cells do not express IGFs or IGFBP-3, so the action of these peptides can be studied without interference from endogenous proteins (Spagnoli et al 2001).…”

Section: Introductionmentioning

confidence: 99%

“…Using RCJ3·1C5·18 chondrogenic cells as an established model to study MCC fate during the chondrogenesis process in vitro, we have previously reported that: IGFBP-3 has IGF-independent antiproliferative and apoptotic effects in MCCs; the IGFBP-3 apoptotic effect in MCCs requires STAT-1 expression and activation; and IGFBP-3 modulates the chondrocytic differentiation rate by regulating MCC growth (Spagnoli et al 2001, Longobardi et al 2003. RCJ3·1C5·18 cells are derived from rat calvaria mesenchymal cells and over 2 weeks of culture, without requiring biochemical and oncogenic transformation, they sequentially differentiate from MCCs to terminally differentiated chondrocytes in a highly predictable and reproducible manner (Grigoriadis et al 1996, Lunstrum et al 1999, Spagnoli et al 2001. Furthermore, RCJ3·1C5·18 cells do not express IGFs or IGFBP-3, so the action of these peptides can be studied without interference from endogenous proteins (Spagnoli et al 2001).…”

Section: Introductionmentioning

confidence: 99%

“…Well characterized as an IGF carrier, IGFBP-3 has been extensively reported to exert pleiotropic effects on diverse cell types and to have a broad range of functions that are independent from its binding to IGF. Through this IGF-independent action, IGFBP-3 has been shown to control cell proliferation and to induce or enhance apoptosis (Cohen et al 1993, Oh et al 1995b, Valentinis et al 1995, Lalou et al 1996, Spagnoli et al 2001, Longobardi et al 2003. Using RCJ3·1C5·18 chondrogenic cells as an established model to study MCC fate during the chondrogenesis process in vitro, we have previously reported that: IGFBP-3 has IGF-independent antiproliferative and apoptotic effects in MCCs; the IGFBP-3 apoptotic effect in MCCs requires STAT-1 expression and activation; and IGFBP-3 modulates the chondrocytic differentiation rate by regulating MCC growth (Spagnoli et al 2001, Longobardi et al 2003.…”

Section: Introductionmentioning

confidence: 99%

“…Through this IGF-independent action, IGFBP-3 has been shown to control cell proliferation and to induce or enhance apoptosis (Cohen et al 1993, Oh et al 1995b, Valentinis et al 1995, Lalou et al 1996, Spagnoli et al 2001, Longobardi et al 2003. Using RCJ3·1C5·18 chondrogenic cells as an established model to study MCC fate during the chondrogenesis process in vitro, we have previously reported that: IGFBP-3 has IGF-independent antiproliferative and apoptotic effects in MCCs; the IGFBP-3 apoptotic effect in MCCs requires STAT-1 expression and activation; and IGFBP-3 modulates the chondrocytic differentiation rate by regulating MCC growth (Spagnoli et al 2001, Longobardi et al 2003. RCJ3·1C5·18 cells are derived from rat calvaria mesenchymal cells and over 2 weeks of culture, without requiring biochemical and oncogenic transformation, they sequentially differentiate from MCCs to terminally differentiated chondrocytes in a highly predictable and reproducible manner (Grigoriadis et al 1996, Lunstrum et al 1999, Spagnoli et al 2001.…”

Section: Introductionmentioning

confidence: 99%

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Signaling cross-talk between IGF-binding protein-3 and transforming growth factor-β in mesenchymal chondroprogenitor cell growth

O'Rear

Longobardi

Torello

et al. 2005

Journal of Molecular Endocrinology

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Cartilage formation is driven by mesenchymal chondroprogenitor cells (MCCs) that proliferate and differentiate into chondrocytes. The molecular mechanisms by which growth factors regulate MCC fate are not well defined. Insulin-like growth factor binding protein-3 (IGFBP-3) has intrinsic bioactivity that is independent of IGF binding. We previously reported that IGFBP-3 has IGF-independent antiproliferative and apoptotic effects in MCCs, and requires STAT-1 activation to mediate its apoptotic effect. Transforming growth factor-(TGF-) is a key chondroinductive growth factor. The objective of the study is to define the interactions between IGFBP-3 and TGF-in MCC growth and their intracellular signaling pathways. We used the RCJ3·1C5·18 mesenchymal chondrogenic cells that without biochemical or oncogenic transformation progress in culture from MCCs to differentiated chondrocytes. Cell proliferation was assessed in MCCs treated with IGFBP-3 or transfected with IGFBP-3, in the presence or absence of TGF-. To demonstrate that IGFBP-3 effects were IGF-independent an IGFBP-3 analog that lacks IGF binding was used (GGG-IGFBP-3). To determine the functional roles of the TGF--mediated signaling and the STAT-1 pathway, cells were either stably transfected with a dominant negative TGF-type II receptor (MCC-DNT RII) or treated with a STAT-1 morpholino antisense oligonucleotide. We found that in MCCs, TGF-antagonized the antiproliferative effect of IGFBP-3. IGFBP-3 increased the cyclin-dependent kinase inhibitor p21 expression and this effect was abolished by TGF-. Furthermore, TGF-inhibited STAT-1 phosphorylation induced by IGFBP-3. Similarly to TGF-, STAT-1 antisense oligonucleotide inhibited the IGFBP-3 antiproliferative action. Although TGF-in MCC-DNT RII lacked Smad-mediated signaling, it persistently antagonized the IGFBP-3 antiproliferative action. However, TGF-even in MCC-DNT RII cells induced ERK1/2 phosphorylation, and treatment with MEK inhibitor, UO126, inhibited the antagonistic effects of TGF-on IGFBP-3. Furthermore, UO126 blocked the TGF-inhibition of STAT-1 phosphorylation induced by IGFBP-3. Collectively, these results demonstrate cross-talk between the IGFBP-3-dependent STAT-1 signaling and the TGF--dependent ERK pathway that regulates MCC proliferation.

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Biology of insulin‐like growth factors in development

Dupont¹,

Holzenberger²

2003

Birth Defects Research Pt C

179

140

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Insulin-like growth factors (IGFs) provide essential signals for the control of embryonic and postnatal development in vertebrate species. In mammals, IGFs act through and are regulated by a system of receptors, binding proteins, and related proteases. In each of the many tissues dependent on this family of growth factors, this system generates a complex interaction specific to the tissue concerned. Studies carried out over the last decade, mostly with transgenic and gene knockout mouse models, have demonstrated considerable variety in the cell type-specific and developmental stage-specific functions of IGF signals. Brain, muscle, bone, cartilage, pancreas, ovary, skin, and fat tissue have been identified as major in vivo targets for IGFs. Concentrating on several of these organ systems, we review here phenotypic analyses of mice with genetically modified IGF systems. Much progress has also been made in understanding the specific intracellular signaling cascades initiated by the binding of circulating IGFs to their cognate receptor. We also summarize the most relevant aspects of this research. Considerable efforts are currently focused on deciphering the functional specificities of intracellular pathways, particularly the molecular mechanisms by which cells distinguish growth-stimulating insulin-like signals from metabolic insulin signals. Finally, there is a growing body of evidence implicating IGF signaling in lifespan control, and it has recently been shown that this function has been conserved throughout evolution. Very rapid progress in this domain seems to indicate that longevity may be subject to IGF-dependent neuroendocrine regulation and that certain periods of the life cycle may be particularly important in the determination of individual lifespan.

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Antiproliferative Effects of Insulin-like Growth Factor-binding Protein-3 in Mesenchymal Chondrogenic Cell Line RCJ3.1C5.18

Cited by 64 publications

References 46 publications

Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

Signaling cross-talk between IGF-binding protein-3 and transforming growth factor-β in mesenchymal chondroprogenitor cell growth

Biology of insulin‐like growth factors in development

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