Identification of IGF binding proteins in bovine milk and the demonstration of IGFBP-3 synthesis and release by bovine mammary epithelial cells.

Abstract

Insulin-like growth factor system components are synthesized and secreted by mammary epithelial cells and multiple IGF binding proteins (IGFBP) are found in milk of various species. This study was conducted to identify the IGFBP in bovine milk, to compare them with those found in blood, and to identify the cell(s) responsible for mammary IGFBP synthesis. Bovine blood, milk, and cell culture-conditioned media were analyzed and characterized with Western ligand blot procedures for specific IGFBP. Electrophoresis and [125I]IGF-II ligand blot analyses of the samples indicated that, unlike serum and mammary primary cell culture-conditioned media, milk required removal of casein in order to accurately disclose all IGFBP. Immunoprecipitation studies identified IGFBP-2, -3, -4, and -5 in blood, milk, and primary cell culture conditioned media. The IGFBP were present at higher concentrations in serum than in milk, and milk concentrations were greater than that shown in conditioned media from primary cultures of bovine mammary cells. Northern analysis detected IGFBP-3 messenger RNA in extracts from fresh tissue and cells in culture, and in situ hybridization studies with fresh tissue utilizing probes for IGFBP-3 and alphaS1-casein showed that the mRNA for IGFBP-3 is predominant in the secretory epithelial cells, when compared to other tissue cell types.

Comparative expression profiling of insulin-like growth factor binding protein-5 in milk of Bos indicus and Bubalus bubalis during lactation

“…A similar observation was reported in bovines by Gibson et al (1999). It has also been suggested that, although IGFBP-5 appear in milk, leakage or biotransport of IGFBP-5 from blood to milk may have happened during the pre-colostrum period leading to an increase in its concentration (Gibson et al, 1999). Vega et al (1991) have reported that high concentrations of IGFBP-5 during the colostrum period may be due to the accumulation of IGF and IGFBP in the pre-colostral milk, 2 to 4 weeks prepartum.…”

“…A comparatively higher concentration of IGFBP-5 during the colostrum period in present study may be due to the accumulation of IGFBP-5 in the mammary gland as a result of pre-parturient accumulation of milk ensuing parturition. A similar observation was reported in bovines by Gibson et al (1999). It has also been suggested that, although IGFBP-5 appear in milk, leakage or biotransport of IGFBP-5 from blood to milk may have happened during the pre-colostrum period leading to an increase in its concentration (Gibson et al, 1999).…”

“…To make sure that IGFBP-5 synthesis took place in the mammary gland, we clearly observed very high level expression of IGFBP-5 transcripts in milk somatic cells of cows during the involution period (data not shown). However, perfusion of IGFBP-5 from blood into mammary secretion during involution cannot be ruled out, as has been reported elsewhere (Gibson et al, 1999). IGFBP-5 has an independent role in inducing extracellular matrix remodeling through plasminogen activator inhibitor-I (Tonner et al, 2000), apart from suppressing the survival action of IGFs.…”

“…After centrifugation, the milk fat formed a uniform layer that was removed by a spatula. The defatted milk was centrifuged at 50 000 × g in a highspeed refrigerated centrifuge (Sorvall RC6 + ) for 2 h (Gibson et al, 1999). After centrifugation, casein was separated as a loose pellet at the bottom of tube.…”

“…Primary bovine mammary epithelial cells (MECs) secrete IGFBP-2, -3, -4, -5 in culture, which are identical to those present in bovine milk (Gibson et al, 1999). IGFBP-5 is also overexpressed during mammary gland involution.…”