Defined Carboxy-Terminal Fragments of Insulin-Like Growth Factor (IGF) Binding Protein-2 Exert Similar Mitogenic Activity on Cultured Rat Growth Plate Chondrocytes as IGF-I

The IGF/IGF binding protein (IGFBP) system is an important component in the hormonal regulation of longitudinal growth. Evidence from in vitro studies indicates that IGFBPs may have IGF-independent effects. We analyzed the biological activity of intact IGFBP-2 and defined carboxy-terminal IGFBP-2 fragments isolated from human hemofiltrate in two cell culture systems of the growth plate: rat growth plate chondrocytes in primary culture and the mesenchymal chondrogenic cell line RCJ3.1C5.18. The IGFBP-2 fragments IGFBP-2167–279, IGFBP-2167–289, and IGFBP-2104–289 exerted a strong (2- to 3-fold) mitogenic effect on growth plate chondrocytes, which was comparable with IGF-I in equimolar concentrations (7.8 nm) but was not mediated through the type 1 IGF receptor. In a dose-response experiment, the most effective concentration of IGFBP-2104–289 for the stimulation of cell proliferation was 10 nm. This biological activity of IGFBP-2 fragments was associated with cell membrane binding, demonstrated by Western blot analysis of fractionated cell lysates and immunohistochemistry. Whereas intact IGFBP-2 did not modulate chondrocyte proliferation, partially reduced (by dithiothreitol) full-length IGFBP-2 stimulated cell proliferation to a comparable extent (3.4-fold) as carboxy-terminal IGFBP-2 fragments. The mitogenic activity of these IGFBP-2 fragments and of partially reduced full-length IGFBP-2 was mediated through the use of the MAPK/ERK 1/2. These data imply a novel role of naturally occurring IGFBP-2 fragments for the endocrine and paracrine/autocrine regulation of longitudinal growth.

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Insulin-Like Growth Factor (IGF)-I Stimulates Cell Proliferation and Induces IGF Binding Protein (IGFBP)-3 and IGFBP-5 Gene Expression in Cultured Growth Plate Chondrocytes via Distinct Signaling Pathways

Endocrinology ◽

10.1210/en.2005-0324 ◽

2005 ◽

Vol 146 (7) ◽

pp. 3096-3104 ◽

Cited By ~ 45

Author(s):

Daniela Kiepe ◽

Sonia Ciarmatori ◽

Andreas Hoeflich ◽

Eckhard Wolf ◽

Burkhard Tönshoff

Keyword(s):

Signaling Pathways ◽

Growth Plate ◽

Intracellular Signaling ◽

De Novo ◽

Growth Plate Chondrocytes ◽

Intracellular Signaling Pathways ◽

Igf I ◽

Rat Growth ◽

Igf Binding ◽

Igfbp 3

Abstract The bioactivity of IGF-I in the cellular microenvironment is modulated by both inhibitory and stimulatory IGF binding proteins (IGFBPs), whose production is partially under control of IGF-I. However, little is known on the IGF-mediated regulation of these IGFBPs in the growth plate. We therefore studied the effect of IGF-I on IGFBP synthesis and the involved intracellular signaling pathways in two cell culture models of rat growth plate chondrocytes. In growth plate chondrocytes in primary culture, incubation with IGF-I increased the concentrations of IGFBP-3 and IGFBP-5 in conditioned cell culture medium in a dose- and time-dependent manner. Coincubation of IGF-I with specific inhibitors of the p42/44 MAPK pathway (PD098059 or U0126) completely abolished the stimulatory effect of IGF-I on IGFBP-3 mRNA expression but did not affect increased IGFBP-5 mRNA levels. In contrast, inhibition of the phosphatidylinositol-3 kinase signaling pathway by LY294002 abrogated both IGF-I-stimulated IGFBP-3 and -5 mRNA expression. Comparable results regarding IGFBP-5 were obtained in the mesenchymal chondrogenic cell line RCJ3.1C5.18, which does not express IGFBP-3. The IGF-I-induced IGFBP-5 gene expression required de novo mRNA transcription and de novo protein synthesis. These data suggest that IGF-I modulates its activity in cultured rat growth plate chondrocytes by the synthesis of both inhibitory (IGFBP-3) and stimulatory (IGFBP-5) binding proteins. The finding that IGF-I uses different and only partially overlapping intracellular signaling pathways for the regulation of two IGFBPs with opposing biological functions might be important for the modulation of IGF bioactivity in the cellular microenvironment.

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Effect of growth hormone on steroidogenesis, insulin-like growth factor-I (IGF-I) and IGF-binding protein-1 production and DNA synthesis in cultured human luteinized granulosa cells

Journal of Endocrinology ◽

10.1677/joe.0.1400313 ◽

1994 ◽

Vol 140 (2) ◽

pp. 313-319 ◽

Cited By ~ 19

Author(s):

P Ovesen ◽

H J Ingerslev ◽

H Ørskov ◽

T Ledet

Keyword(s):

Cell Proliferation ◽

Growth Factor ◽

Granulosa Cell ◽

Granulosa Cells ◽

Ovarian Function ◽

Thymidine Incorporation ◽

Factor I ◽

Igf I ◽

Igf Binding ◽

Igf Binding Protein

Abstract Numerous clinical and experimental observations have suggested that GH is important in ovarian function. We have investigated the effect of GH alone and GH in combination with FSH on the secretion of oestradiol, progesterone, insulin-like growth factor-I (IGF-I) and IGF-binding protein-1 (IGFBP-1) and on [3H]thymidine incorporation in cultured human luteinized granulosa cells. Granulosa cells from patients undergoing treatment for in vitro fertilization were isolated and cultured for 2 days in culture medium with 10% serum. After this preincubation, the medium was removed and the cells were incubated with GH (1, 10 and 100 μg/l) with or without FSH in serum-free medium and in the presence of [3H]methylthymidine (2 μCi/ml). GH alone resulted in a significant dose-dependent increase of oestradiol (P<0·05) and in IGFBP-1 (P<0·002) in the medium. The release of IGF-I was undetectable and there was no increase in [3H]thymidine incorporation with GH alone. Neither GH nor FSH alone stimulated granulosa cell proliferation or progesterone release, while the combination induced increases (P<0·001) in both. The stimulatory effect of GH on steroidogenesis, IGFBP-1 production and granulosa cell proliferation supports a putative role for GH in the regulation of ovarian function. Journal of Endocrinology (1994) 140, 313–319

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Production of recombinant porcine IGF-binding protein-5 and its effect on proliferation of porcine embryonic myoblast cultures in the presence and absence of IGF-I and Long-R3-IGF-I

Journal of Endocrinology ◽

10.1677/joe.1.06037 ◽

2005 ◽

Vol 185 (1) ◽

pp. 197-206 ◽

Cited By ~ 14

Author(s):

M S Pampusch ◽

G Xi ◽

E Kamanga-Sollo ◽

K J Loseth ◽

M R Hathaway ◽

...

Keyword(s):

Cell Proliferation ◽

Affinity Chromatography ◽

Binding Protein ◽

Myogenic Cell ◽

Type I ◽

Dependent Manner ◽

Western Blots ◽

Igf I ◽

Igf Binding ◽

Igf Binding Protein

IGF-binding protein-5 (IGFBP-5) is produced by porcine embryonic myogenic cell (PEMC) cultures and is secreted into the medium. IGFBP-5 may play some role in myogenesis and/or in changes in myogenic cell proliferation that accompany differentiation. IGFBP-5 reportedly may either suppress or stimulate proliferation or differentiation of cultured cells depending on cell type and culture conditions. Additionally, IGFBP-5 has been shown to possess both IGF-dependent and IGF-independent actions in some cell types. The goal of this study was to produce recombinant porcine IGFBP-5 (rpIGFBP-5) and assess its IGF-I-dependent and IGF-I-independent actions on the proliferation of PEMCs. To accomplish this, we have expressed porcine IGFBP-5 in the baculovirus system, purified and characterized the expressed rpIGFBP-5 and produced an anti-porcine IGFBP-5 antibody that neutralizes the biological activity of porcine IGFBP-5. rpIGFBP-5, purified to 98% homogeneity using nickel affinity chromatography and IGF-I affinity chromatography, suppressed IGF-I-stimulated proliferation of PEMCs in a concentration-dependent manner (P>0.05). rpIGFBP-5 also suppressed Long-R3-IGF-I-stimulated proliferation of PEMCs (P>0.05), even in the presence of significant molar excess of Long-R3-IGF-I compared with rpIGFBP-5, demonstrating the IGF-independent activity that rpIGFBP-5 possesses in PEMCs, since Long-R3-IGF-I is an IGF analog that has very low affinity for the IGFBPs but retains its ability to bind to the type I IGF receptor and thereby can stimulate proliferation. The anti-rpIGFBP-5 IgY produced against rpIGFBP-5 specifically recognized native porcine IGFBP-5 in PEMC media that also contained porcine IGFBP-2, -3, and -4. This antibody is capable of neutralizing the effects of both rpIGFBP-5 and endogenously produced porcine IGFBP-5 on PEMCs as well as detecting IGFBP-5 in Western blots. The production of rpIGFBP-5 and a neutralizing antibody to porcine IGFBP-5 provides a powerful tool to investigate the role of IGFBP-5 in porcine myogenic cell proliferation and differentiation. The data provided here demonstrated that IGFBP-5 has the potential to affect proliferation of PEMCs during critical periods of in vitro muscle cell development and therefore may impact the capacity for ultimate postnatal muscle mass development in vivo.

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Interaction of IGF-I and 1α,25(OH)2D3 on receptor expression and growth stimulation in rat growth plate chondrocytes

Kidney International ◽

10.1046/j.1523-1755.1998.00884.x ◽

1998 ◽

Vol 53 (5) ◽

pp. 1152-1161 ◽

Cited By ~ 26

Author(s):

Günter Klaus ◽

Lutz Weber ◽

Julian Rodríguez ◽

Porfirio Fernández ◽

Thomas Klein ◽

...

Keyword(s):

Growth Plate ◽

Growth Stimulation ◽

Receptor Expression ◽

Growth Plate Chondrocytes ◽

Igf I ◽

Rat Growth

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Proepithelin Stimulates Growth Plate Chondrogenesis via Nuclear Factor-κB-p65-dependent Mechanisms

Journal of Biological Chemistry ◽

10.1074/jbc.m110.201368 ◽

2011 ◽

Vol 286 (27) ◽

pp. 24057-24067 ◽

Cited By ~ 8

Author(s):

Shufang Wu ◽

Weijin Zang ◽

Xu Li ◽

Hongzhi Sun

Keyword(s):

Cell Proliferation ◽

Growth Plate ◽

Bone Growth ◽

Nuclear Translocation ◽

Nuclear Factor Κb ◽

Pyrrolidine Dithiocarbamate ◽

Growth Plate Chondrocytes ◽

Cell Proliferation And Differentiation ◽

Rat Growth ◽

And Function

Proepithelin, a previously unrecognized growth factor in cartilage, has recently emerged as an important regulator for cartilage formation and function. In the present study, we provide several lines of evidences in proepithelin-mediated induction of cell proliferation, differentiation, and apoptosis in the metatarsal growth plate. Proepithelin-mediated stimulation of metatarsal growth and growth plate chondrogenesis was neutralized by pyrrolidine dithiocarbamate, a known NF-κB inhibitor. In rat growth plate chondrocytes, proepithelin induced NF-κB-p65 nuclear translocation, and nuclear NF-κB-p65 initiated its target gene cyclin D1 to regulate chondrocyte functions. The inhibition of NF-κB-p65 expression and activity (by p65 short interfering RNA (siRNA) and pyrrolidine dithiocarbamate, respectively) in chondrocytes reversed the proepithelin-mediated induction of cell proliferation and differentiation and the proepithelin-mediated prevention of cell apoptosis. Moreover, the inhibition of the phosphatidylinositol 3-kinase and Akt abolished the effects of proepithelin on NF-κB activation. Finally, using siRNA and antisense strategies, we demonstrated that endogenously produced proepithelin by chondrocytes is important for chondrocyte growth in serum-deprived conditions. These results support the hypothesis that the induction of NF-κB activity of in growth plate chondrocytes is critical in proepithelin-mediated growth plate chondrogenesis and longitudinal bone growth.

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Effect of recombinant porcine IGF-binding protein-3 on proliferation of embryonic porcine myogenic cell cultures in the presence and absence of IGF-I

Journal of Endocrinology ◽

10.1677/joe.0.1760227 ◽

2003 ◽

Vol 176 (2) ◽

pp. 227-235 ◽

Cited By ~ 37

Author(s):

MS Pampusch ◽

E Kamanga-Sollo ◽

ME White ◽

MR Hathaway ◽

WR Dayton

Keyword(s):

Cell Proliferation ◽

Muscle Development ◽

Binding Protein ◽

Myogenic Cell ◽

Complete Suppression ◽

Dependent Manner ◽

Igf I ◽

Igf Binding ◽

Igf Binding Protein ◽

Igfbp 3

IGF-binding protein (IGFBP)-3 is produced by cultured porcine embryonic myogenic cell (PEMC) cultures and is secreted into the medium. Levels of secreted IGFBP-3 and IGFBP-3 mRNA are significantly reduced during differentiation and increase after differentiation is complete, suggesting that IGFBP-3 may play some role in myogenesis and/or in changes in myogenic cell proliferation that accompany differentiation. IGFBP-3 reportedly may either suppress or stimulate proliferation of cultured cells depending on cell type. Additionally, IGFBP-3 has been shown to affect proliferation via both IGF-dependent and IGF-independent mechanisms in some cell types but not all. Currently, the effect, if any, of IGFBP-3 on myogenic cell proliferation is not known. Consequently, the goal of this study was to assess the IGF-I-dependent and IGF-I-independent actions of recombinant porcine IGFBP-3 on proliferation of cultured porcine myogenic cells. To facilitate these investigations, we have expressed porcine IGFBP-3 in the baculovirus system, purified and characterized the expressed recombinant porcine IGFBP-3 (rpIGFBP-3), and produced and characterized an anti-porcine IGFBP-3 antibody that neutralizes the biological activity of porcine IGFBP-3. rpIGFBP-3 suppressed IGF-I-stimulated proliferation of PEMCs in a concentration-dependent manner with equimolar concentrations of IGF-I and rpIGFBP-3, resulting in complete suppression of IGF-I-stimulated proliferation. rpIGFBP-3 also suppressed Long-R3-IGF-I-stimulated proliferation of PEMC, indicating that rpIGFBP-3 possesses IGF-independent activity in this cell system. These data have established that IGFBP-3 has the potential to affect proliferation of PEMCs during critical periods of muscle development that may impact ultimate muscle mass achievable postnatally.

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Protease-Resistant Insulin-Like Growth Factor (IGF)-Binding Protein-4 Inhibits IGF-I Actions and Neointimal Expansion in a Porcine Model of Neointimal Hyperplasia

Endocrinology ◽

10.1210/en.2007-0571 ◽

2007 ◽

Vol 148 (10) ◽

pp. 5002-5010 ◽

Cited By ~ 19

Author(s):

T. C. Nichols ◽

W. H. Busby ◽

E. Merricks ◽

J. Sipos ◽

M. Rowland ◽

...

Keyword(s):

Cell Proliferation ◽

Binding Protein ◽

Neointimal Hyperplasia ◽

Nuclear Antigen ◽

Wild Type ◽

Lesion Development ◽

Resistant Form ◽

Igf I ◽

Igf Binding ◽

Igf Binding Protein

IGF-I has been shown to play a role in the progression of atherosclerosis in experimental animal models. IGF-binding protein-4 (IGFBP-4) binds to IGF-I and prevents its association with receptors. Overexpression of a protease-resistant form of IGFBP-4 has been shown to inhibit the ability of IGF-I to stimulate normal smooth muscle cell growth in mice. Based on these observations, we prepared a protease-resistant form of IGFBP-4 and infused it into hypercholesterolemic pigs. Infusion of the protease-resistant mutant inhibited lesion development by 53.3 ± 6.1% (n = 6; P < 0.01). Control vessels that received an equimolar concentration of IGF-I and the protease-resistant IGFBP-4 showed no reduction in lesion size compared with control lesions that were infused with vehicle. Infusion of a nonmutated form of IGFBP-4 did not significantly inhibit lesion development. Proliferating cell nuclear antigen analysis showed that the mutant IGFBP-4 appeared to inhibit cell proliferation. The area occupied by extracellular matrix was also reduced proportionally compared with total lesion area. Immunoblotting revealed that the mutant IGFBP-4 remained intact, whereas the wild-type IGFBP-4 that was infused was proteolytically cleaved. Further analysis of the lesions revealed that a marker protein, IGFBP-5, whose synthesis is stimulated by IGF-I, was decreased in the lesions that received the protease-resistant, IGFBP-4 mutant, whereas there was no change in lesions that received wild-type IGFBP-4 or the mutant protein plus IGF-I. These findings clearly illustrate that infusion of protease-resistant IGFBP-4 into the perilesion environment results in inhibition of cell proliferation and attenuation of the development of neointima. The findings support the hypothesis that inhibiting IGFBP-4 proteolysis in the lesion microenvironment could be an effective means for regulating neointimal expansion.

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