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Modeling growth factor activity during proinflammatory stress: methodological considerations in assessing cytokine
T H Elsasser1, T J Caperna, P J Ward
1US Department of Agriculture, Agricultural Research Service, Growth Biology Laboratory, Beltsville, MD 20705, United States. elsasser@anri.barc.usda.gov
Insights
Tumor necrosis factor-alpha (TNF-α) influences insulin-like growth factor-binding proteins (IGFBPs) by altering gene expression and promoting degradation. Culture conditions significantly impact TNF-α
Area of Science:
- Cell Biology
- Molecular Biology
- Endocrinology
Background:
- Insulin-like growth factor-binding proteins (IGFBPs) modulate insulin-like growth factor (IGF) bioavailability.
- Tumor necrosis factor-alpha (TNF-α) is a key proinflammatory cytokine with diverse cellular effects.
- The interplay between TNF-α and IGFBPs in cellular responses is not fully understood.
Purpose of the Study:
- To investigate the mechanisms by which TNF-α affects IGFBPs in epithelial cells.
- To model the impact of proinflammatory stimuli on IGFBP expression and stability.
- To elucidate the role of culture conditions in mediating TNF-α's effects on IGFBPs.
Main Methods:
- Madin-Darby bovine kidney (MDBK) epithelial cells were utilized as a model system.
- Cells were treated with forskolin or recombinant TNF-α under varying culture conditions, including protease inhibitors (PIs) and bovine serum albumin (BSA) concentrations.
- IGFBP-3 gene expression, media protein content, and protease activity were analyzed.
Main Results:
- Forskolin increased IGFBP-3 gene expression and protein levels.
- TNF-α enhanced basal and forskolin-induced IGFBP-3 gene expression.
- TNF-α's effect on media IGFBP levels was dependent on culture conditions; protease activity degraded IGFBPs in low BSA/no PI conditions, while high BSA/PI preserved IGFBP levels.
Conclusions:
- TNF-α influences IGFBP levels through a dual mechanism of modulating gene expression and enhancing proteolytic degradation.
- Culture conditions, specifically the presence of protease inhibitors and sufficient albumin, are critical in determining the net effect of TNF-α on IGFBPs.
- The MDBK cell model demonstrates that proinflammatory responses can alter IGFBP homeostasis via protease release.
Abstract:
The present research was conducted to model potential mechanisms through which IGFBPs might be affected by a key proinflammatory response initiating cytokine tumor necrosis factor (TNF-)-alpha. Madin-Darby bovine kidney epithelial (MDBK) cells, known to release IGFBPs in response to several stimuli, were grown under several conditions and challenged with forskolin (F) or recombinant TNF-alpha for 24h. Forskolin increased IGFBP-3 gene expression and media content of BP-3 protein. TNF-alpha increased basal and augmented F-mediated IGFBP-3 gene expression. However, TNF-alpha effects on the measurable media content of IGFBPs were influenced by culture conditions; in the absence of added protease inhibitors (PIs) or sufficient media albumin concentration (high BSA, 1mg/ml), the effect of TNF-alpha was to decrease (P<0.02) measurable IGFBPs. In the presence of PI and high BSA, media IGFBP-3 levels were shown to be increased by TNF-alpha consistent with the gene expression data. Changes in media IGFBP-3 protease activity were examined further to explain the observed effects of TNF-alpha on production and destruction of IGFBPs in media. When recombinant human IGFBP-3 (500 ng/ml) was added to PI-free, low BSA 100 microg/ml) media from TNF-treated MDBK cells, less than 10% of the BP-3 was recognizable by Western blot in 30 min; conversely, inclusion of High BSA and PI in media resulted in attenuation of the protease effect on the IGFBPs. The data suggest that the MDBK model of cellular response to proinflammatory stimulus is affected by culture conditions and that TNF-alpha affects media content of IGFBPs through effects on IGFBP gene expression coupled with degradation of IGFBPs via enhanced proteolytic enzyme release.
