Modeling growth factor activity during proinflammatory stress: methodological considerations in assessing cytokine modulation of IGF binding proteins released by cultured bovine kidney epithelial cells.
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.
Elsasser
T H
TH
US Department of Agriculture, Agricultural Research Service, Growth Biology Laboratory, Beltsville, MD 20705, United States. elsasser@anri.barc.usda.gov
Caperna
T J
TJ
Ward
P J
PJ
Sartin
J L
JL
Steele
B P
BP
Li
C
C
Kahl
S
S
D000818:Animals; D002417:Cattle; D002460:Cell Line; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000235:genetics; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000235:genetics; D018969:Insulin-Like Growth Factor Binding Proteins / Q000235:genetics / Q000378:metabolism*; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D018971:Insulin-Like Growth Factor Binding Protein 2; D018972:Insulin-Like Growth Factor Binding Protein 3; D018969:Insulin-Like Growth Factor Binding Proteins; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha