Expression and purification of biologically active IGF-binding proteins using the LCR/Mel expression system

Bagnall, Wendy; Sharpe, Paul T.; Newham, Peter; Tart, Johnathan; Mott, Richard A; Torr, Vanessa R; Forder, Robert A.; Needham, Maurice

doi:10.1016/s1046-5928(02)00549-1

Cited by 5 publications

(4 citation statements)

References 32 publications

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“…Our finding in MCCs differs from reports from previous studies that have shown that glycosylation status of IGFBP-3, although non-essential for IGF binding, influences cell surface binding and antiproliferative responses (Firth & Baxter 1999, Bagnall et al 2003. Different cell surface binding sites and cell types may be responsible for these dissimilarities.…”

Section: Figure 11contrasting

confidence: 98%

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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Section: Figure 11contrasting

confidence: 98%

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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“…Experiments were repeated at least three-times, and representative data are shown. IGFBP-3 induced by treatment with either 1,25(OH) 2 D 3 or TGFb shows multiple bands around 44 kDa, due to post-translational modifications including glycosylation as reported previously [23][24][25].…”

Section: Western Blotting For Igfbp-3supporting

confidence: 79%

1α,25‐dihydroxyvitamin D₃ induced growth inhibition of PC‐3 prostate cancer cells requires an active transforming growth factor beta signaling pathway

Murthy

Weigel

2003

The Prostate

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We find that 1,25(OH)2D3 action in PC-3 cells is mediated through at least two distinct pathways, the TGFbeta pathway and the IGFBP-3 pathway. We show that 1,25(OH)2D3 treatment elevates TGFbeta production and signaling, as well as receptor levels, in PC-3 cells. Further, using a blocking antibody against TGFbeta substantially reduces 1,25(OH)2D3 mediated growth inhibition without affecting IGFBP-3 induction, suggesting that IGFBP-3, alone, is insufficient to inhibit the growth of PC-3 cells.

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“…However, the pattern of response involving pro-and antiapoptotic factors, as well as signals involved in cell proliferation, suggests a complex mechanism of IGFBP-3 action. As most reports on IGFBP-3induced apoptosis describe the use of ng-IGFBP-3, 34,35 we compared the effects of gly-and ng-IGFBP-3 on ML-1 cells in the presence of TNFa or TRAIL. As shown in Figure 4c, both isoforms of IGFBP-3 inhibited apoptosis in response to proapoptotic signals.…”

Section: Igfbp-3 Interferes With Apoptosis Induced By Tnfa and Trailmentioning

confidence: 99%

High IGFBP-3 levels in marrow plasma in early-stage MDS: effects on apoptosis and hemopoiesis

et al. 2005

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The pathophysiology of the myelodysplastic syndromes (MDS) is incompletely understood. Tumor necrosis factor (TNF)alpha levels are elevated, particularly in early-stage MDS, and apoptosis in marrow cells is upregulated. Observations in other models have shown a role for insulin-like growth factor binding protein 3 (IGFBP-3) in TNFalpha-mediated apoptosis. We observed increased levels of IGFBP-3 in the marrow plasma of patients with MDS (P = 0.005) and hypothesized that altered IGFBP-3 levels contribute to the dysregulation of hemopoiesis in MDS by affecting proliferation and apoptosis. Western analysis of marrow plasma from MDS patients revealed an increase in the ratio of intact vs fragmented IGFBP-3 in early-stage MDS (relative to controls) that decreased with MDS disease progression, suggesting increased proteolysis with more advanced disease. Thus, these results provide evidence for dysregulation of IGFBP-3 in patients with MDS. While the data are complex, they are consistent with a modulatory effect of IGFBP-3 on hemopoiesis in MDS. Conceivably, understanding these mechanisms may allow for the development of novel therapeutic strategies.

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Expression and purification of biologically active IGF-binding proteins using the LCR/Mel expression system

Cited by 5 publications

References 32 publications

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

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

1α,25‐dihydroxyvitamin D₃ induced growth inhibition of PC‐3 prostate cancer cells requires an active transforming growth factor beta signaling pathway

High IGFBP-3 levels in marrow plasma in early-stage MDS: effects on apoptosis and hemopoiesis

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Expression and purification of biologically active IGF-binding proteins using the LCR/Mel expression system

Cited by 5 publications

References 32 publications

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

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

1α,25‐dihydroxyvitamin D3 induced growth inhibition of PC‐3 prostate cancer cells requires an active transforming growth factor beta signaling pathway

High IGFBP-3 levels in marrow plasma in early-stage MDS: effects on apoptosis and hemopoiesis

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1α,25‐dihydroxyvitamin D₃ induced growth inhibition of PC‐3 prostate cancer cells requires an active transforming growth factor beta signaling pathway