IGF-I differentially regulates IGF-binding protein expression in primary mammary fibroblasts and epithelial cells
- Department of Animal Sciences, Rutgers The State University of New Jersey, 108 Foran Hall, 59 Dudley Road, New Brunswick, New Jersey 08901-8520, USA
- 1Department of Animal Science, College of Agriculture and Life Sciences, University of Vermont, 570 Main Street, Burlington, Vermont 05405, USA
- (Requests for offprints should be addressed to J M Fleming; Email: jfleming{at}AESOP.Rutgers.edu)
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Figure 1IGF-I differentially regulates IGFBP in primary BMFs and BME cells. Confluent cells were rinsed twice in serum-free DMEM followed by 20 h serum starvation, then treated with serum-free media ± IGF-I (100 ng/ml) for 48 h. Conditioned media containing equal amounts of protein (BME, 6 μg; MAC-T, 5 μg; BMF, 2 μg) for each pair of samples were separated by SDS-PAGE. Following transfer, the membranes were ligand blotted with 125I-labeled IGF-I (A) or 125I-labeled IGF-II (B). Results are representative of a minimum of three experiments.
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Figure 2Identification of IGFBP secreted by primary BMFs and BME cells. Confluent cells were rinsed twice in serum-free DMEM followed by 20 h serum starvation, then treated with serum-free media ± IGF-I (100 ng/ml) for 48 h. Representative immunoblots of media conditioned by IGF-I-stimulated primary epithelial cells and fibroblasts for 48 h are shown. Media were concentrated and separated by SDS-PAGE. After transfer, the membranes were immunoblotted with specific IGFBP antisera. Results are representative of a minimum of three experiments.
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Figure 3IGF-I increases IGFBP-3 mRNA levels in a dose-responsive manner in primary BME cells. Confluent cells were rinsed twice in serum-free DMEM followed by 20 h serum starvation, then exposed to serum-free media ± increasing doses of IGF-I for 8 h. Total RNA was isolated and analyzed by Northern blotting via hybridization with 32P-labeled bovine IGFBP-2, -3, -6 and 18S cDNA. (A) Representative Northern blot. (B) Relative intensity by PhosphorImager analysis (corrected for 18S): data represent means ± s.e.m. of at least three separate experiments. IGF treatment was determined as significant, P<0.001, (ANOVA) with a post-hoc REGWQ multiple-range test indicating groups b and c are significant compared with group a, and group c is significant compared with group b.
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Figure 4Temporal induction of IGFBP mRNA levels by IGF-I in primary BME cells. Confluent cells were rinsed twice in serum-free DMEM followed by 20 h serum starvation, then exposed to serum-free media ± IGF-I (100 ng/ml) for indicated times. Total RNA was isolated and analyzed by Northern blotting by hybridizing with 32P-labeled bovine IGFBP-2, -3, -6 or 18S cDNA. (A) Representative Northern blot. (B and C) Relative intensity by PhosphorImager analysis for IGFBP-3 and -2 respectively: data represent means ± s.e.m. of at least three separate experiments. *Significance of P<0.05 for overall protection (ANOVA).
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Figure 5IGF-I regulates IGFBP-4 mRNA levels in primary BME cells. Confluent cells were rinsed twice in serum-free DMEM followed by 20 h serum starvation, then exposed to serum-free media ± IGF-I (100 ng/ml) for indicated times. Total RNA was isolated, DNase treated and analyzed by qRT-PCR as described in the Materials and Methods. Data represent means ± s.e.m. of two separate experiments.
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Figure 6IGF-I increases IGFBP-5, -4 and -3 mRNA levels in a dose-responsive manner in primary BMFs. Confluent cells were rinsed twice in serum-free DMEM followed by 20 h serum starvation, then exposed to serum-free media ± increasing doses of IGF-I for 8 h. Total RNA was isolated and analyzed by Northern blotting via hybridization with 32P-labeled bovine IGFBP-2, -3, -6 or human IGFBP-4 and -5 cDNA. (A) Representative Northern blot. (B and C) Relative intensity by PhosphorImager analysis of IGFBP-5 and -4 respectively: data represent means ± s.e.m. of at least three separate experiments. IGF-treatment was determined significant at P<0.02 (ANOVA) with a post-hoc REGWQ multiple-range test indicating that group b is significantly different compared with group a.
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Figure 7Temporal induction of IGFBP mRNA levels by IGF-I in primary BMFs. Confluent cells were rinsed twice in serum-free DMEM followed by 20 h serum starvation, then exposed to serum-free media ± IGF-I (100 ng/ml) for indicated times. Total RNA was isolated and analyzed by Northern blotting via hybridization with 32P-labeled bovine IGFBP-2, -3, -6 or human IGFBP-4 and -5 cDNA. (A) Representative Northern blot. (B, C and D) Relative intensity by PhosphorImager analysis of IGBP-5, -4 and -3 respectively: data represent means ± s.e.m. of at least three separate experiments. *Significance of P<0.05 for overall protection (ANOVA); Dunn–Sidak correction for individual test, P<0.01.
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Figure 8IGF-I differentially regulates IGFBP mRNA levels in primary BME cells compared with BMFs. Confluent cells were rinsed twice in serum-free DMEM followed by 20 h serum starvation, then exposed to serum-free media ± IGF-I (100 ng/ml) for indicated times. Total RNA was isolated, DNase treated and analyzed by qRT-PCR as described in the Materials and Methods. The graph represents fold increase over serum-free controls (shown in parentheses), data represent means ± s.e.m. of at least two separate experiments.
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Figure 9Primary BMFs secrete PAPP-A, an IGF-dependent protease of IGFBP-4. Confluent cells were rinsed, serum-starved for 20 h, then exposed to serum-free media for 48 h. (A) Lane 1, media (15 ml) were concentrated, separated by SDS-PAGE and immunoblotted with an antibody to the PAPP-A/proMBP complex; lane 2, 100 ng of purified human PAPP-A/proMBP protein positive control. (B and C) Representative autoradiogram of ligand blots from an immuno-neutralization study of IGFBP-4 protease activity: 50 μl of media were incubated ± IGF-I (100 ng/ml) and ± an antibody to PAPP-A/proMBP complex at 37 °C for 20 h, then separated by SDS-PAGE. After transfer, the membrane was ligand blotted with 125I-labeled IGF-II (B) and 125I-labeled IGF-I (C). Results are representative of a minimum of three experiments.
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Figure 10IGF-I and -II mRNA is present in primary BMFs but not BME cells. Confluent cells were rinsed twice in serum-free DMEM followed by 20 h serum starvation and exposed to serum-free media for 8 h. Total RNA was isolated, DNAse treated then analyzed by RT-PCR with primers designed against bovine IGF-I, -II and the IGF-IR. Figure shows representative ethidium bromide stain of PCR products separated by agarose gel electrophoresis. Results are representative of three experiments.
- © 2005 Society for Endocrinology
