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Different inhibitory actions of IGFBP-1, -2 and -4 on IGF-I effects in vascular smooth muscle cells
T Gustafsson1, P Andersson, H J Arnqvist
1Department of Biomedicine and Surgery, Division of Cell Biology, Faculty of Health Sciences, Linköping University, Linköping, Sweden.
Abstract:
IGF-I is involved in the regulation of metabolism, growth and migration of vascular smooth muscle cells (VSMCs). We have studied how IGFBP-1, -2 and -4 modulate IGF-I-induced DNA and protein synthesis in cultured rat VSMCs. DNA and protein synthesis were measured as incorporation of [3H]thymidine and [3H]leucine into DNA and protein respectively. Western immunoblot was used to detect IGFBPs in conditioned medium and solution hybridization was used to measure IGFBP gene expression. IGF-I stimulated DNA synthesis with an EC50 of 44 pM, reaching a maximal effect at 1 nM. An IGF-I concentration of 1 nM was subsequently used in the experiments with IGFBPs. IGFBP-1 and IGFBP-4 acted in an inhibitory manner on IGF-I-induced DNA synthesis with calculated IC50 values of 1.6 nM and 6.2 nM respectively. IGFBP-2 (16 nM) also inhibited the growth response to IGF-I, but this effect could only be obtained if the two peptides were pre-incubated together for 2 h prior to addition to the cells. IGFBP-1, -2 and -4 inhibited IGF-I-induced protein synthesis in a similar way. Immunoblot of the incubation medium showed little degradation of IGFBP-2 and -4 for up to 24 h. mRNA for IGFBP-2 and -4, but not for IGFBP-1 was detected in the VSMCs. Endogenous IGFBP-2 and -4 could be detected by immunoblot in the conditioned medium but only if it was concentrated. In conclusion, IGFBP-1, -2 and -4, of which IGFBP-2 and -4 may be locally derived, act as inhibitors with different potencies on IGF-I effects in VSMCs.
Insights
Insulin-like growth factor-binding proteins (IGFBPs) -1, -2, and -4 inhibit insulin-like growth factor-I (IGF-I)-induced vascular smooth muscle cell growth. IGFBP-2 and -4 may be produced locally by these cells.
Area of Science:
- Cell Biology
- Endocrinology
- Vascular Biology
Background:
- Insulin-like growth factor-I (IGF-I) regulates vascular smooth muscle cell (VSMC) metabolism, growth, and migration.
- Insulin-like growth factor-binding proteins (IGFBPs) modulate IGF-I bioavailability and activity.
Purpose of the Study:
- To investigate the modulatory effects of IGFBP-1, IGFBP-2, and IGFBP-4 on IGF-I-induced DNA and protein synthesis in cultured rat VSMCs.
- To determine the gene expression and detect the presence of IGFBPs in VSMCs.
Main Methods:
- Cell culture of rat VSMCs.
- Measurement of DNA and protein synthesis via [3H]thymidine and [3H]leucine incorporation.
- Western immunoblotting for IGFBP detection.
- Solution hybridization for IGFBP gene expression analysis.
Main Results:
- IGF-I stimulated VSMC DNA synthesis with an EC50 of 44 pM.
- IGFBP-1 and IGFBP-4 inhibited IGF-I-induced DNA synthesis (IC50: 1.6 nM and 6.2 nM, respectively).
- IGFBP-2 also inhibited DNA synthesis when pre-incubated with IGF-I.
- All tested IGFBPs inhibited IGF-I-induced protein synthesis.
- IGFBP-2 and -4 mRNA were detected in VSMCs, suggesting local production.
- IGFBP-2 and -4 were detected in conditioned medium, with minimal degradation over 24 hours.
Conclusions:
- IGFBP-1, -2, and -4 act as inhibitors of IGF-I's effects on VSMC proliferation and protein synthesis.
- IGFBP-2 and -4 may be locally produced within the VSMC environment.
- These findings highlight the complex regulatory role of IGFBPs in VSMC function.