Osteopontin (OPN) is a multifunctional protein found in all vertebrates. OPN is expressed in many different cell types, and is consequently found in most tissues and physiological secretions. OPN is involved in a multitude of biological processes, such as activation and regulation of the immune system; biomineralization; tissue-transformative processes, including growth and development of the gut and brain; interaction with bacteria; and many more. OPN is found in the highest concentrations in milk, where it is believed to initiate and regulate developmental, immunological and physiological processes in infants who consume milk. Processes for the isolation of bovine OPN for use in infant formula have been developed, and in recent years, many studies have investigated the effects of the intake of milk OPN. The purpose of this article is to review and compare existing knowledge about the structure and function of milk OPN, with a particular focus on the effects of milk OPN on human health and disease.

Osteopontin (OPN) is a multifunctional protein highly expressed in milk, where it is hypothesized to be involved in immunological signaling via the conserved Arg-Gly-Asp (RGD) integrin-binding sequence. Intervention studies have indicated beneficial effects of orally administered OPN in animal and human infants, but the mechanisms underlying these effects are not well described. To induce physiological effects, OPN must resist gastrointestinal transit in a bioactive form. In this study, we subjected bovine milk OPN to in vitro gastrointestinal transit, and characterized the generated fragments using monoclonal antibody and mass spectrometric analyses. We found that the fragment Trp²⁷-Phe¹⁵¹ containing the integrin-binding RGD sequence resisted in vitro gastric digestion. This resistance was dependent on glycosylation of threonine residues near the integrin-binding sequence in both human and bovine milk OPN. Furthermore, the fragment Trp²⁷-Phe¹⁵¹ retained the ability to interact with integrins in an RGD-dependent process. These results suggest a mechanism for how ingested milk OPN can induce physiological effects via integrin signaling in the intestine.

Quantifying the ability of a compound to modulate cell migration rate is a crucial part of many studies including those on chemotaxis, wound healing and cancer metastasis. Existing migration assays all have their strengths and weaknesses. The "scratch" assay is the most widely used because it seems appealingly simple and inexpensive. However, the scratch assay has some important limitations, as the tool introducing the "wound" might injure/stress the boundary cells and/or harm underlying matrix coatings, which in both cases will affect cell migration. This described method is a Cell Exclusion Zone Assay, in which cell-free areas are created by growing cells around removable silicone stoppers. Upon appropriate staining with fluorescent dyes and microscopically visualizing the monolayers, the migration rate is then quantified by counting the cells (nuclei) intruding the void area left by the silicone insert. In the current study human small intestine epithelial cells were seeded on a physiological substrate matrix to produce collectively migrating monolayers. Different substrates were tested to determine the optimal surface for enterocyte adherence and migration and morphological changes monitored. Recombinant human epidermal growth factor and osteopontin purified from urine were tested to see if the established migration assay produces accurate and reliable migration data with human small intestine cells. The obtained data accurately confirmed that the two bioactive proteins modulate cellular migration in a dose-dependent manner. The presented assay can likely be converted for use with other adherent cell lines or substrate matrices and allows for high throughput, while cost is kept low and versatility high. Co-staining can be applied in order to assay for cell death, different cell types, cell stress and others allowing intricate analysis of migration rate of mixed populations and correction for cell viability.

The effect of food components on brain growth and development has attracted increasing attention. Milk has been shown to contain peptides that deliver important signals to the brains of neonates and infants. In order to reach the brain, milk peptides have to resist proteolytic degradation in the gastrointestinal tract, cross the gastrointestinal barrier and later cross the highly selective blood–brain barrier (BBB). To investigate this, we purified and characterized endogenous peptides from bovine milk and investigated their apical to basal transport by using human intestinal Caco-2 cells and primary porcine brain endothelial cell monolayer models. Among 192 characterized milk peptides, only the α_S1-casein peptide ¹⁸⁵PIGSENSEKTTMPLW¹⁹⁹, and especially fragments of this peptide processed during the transport, could cross both the intestinal barrier and the BBB cell monolayer models. This peptide was also shown to resist simulated gastrointestinal digestion. This study demonstrates that a milk derived peptide can cross the major biological barriers in vitro and potentially reach the brain, where it may deliver physiological signals.

Osteopontin (OPN) is a multifunctional protein abundantly present in human milk, whereas the concentration is significantly lower in bovine milk. Human and bovine milk OPN are structurally similar and both proteins resist gastric digestion and reach the intestines in a bioactive form. Intervention studies have indicated the beneficial effects of supplementing infant formula with bovine milk OPN and several in vivo and in vitro studies have shown that bovine milk OPN positively influences intestinal development. To investigate the functional relationship, we compared the effect of simulated gastrointestinal digested human and bovine milk OPN on gene expression in Caco-2 cells. After incubation, total RNA was extracted and sequenced and transcripts were mapped to the human genome. Human and bovine milk OPN regulated the expression of 239 and 322 genes, respectively. A total of 131 genes were similarly regulated by the OPNs. As a control, a whey protein fraction with a high content of alpha-lactalbumin had a very limited transcriptional impact on the cells. Enrichment data analysis showed that biological processes related to the ubiquitin system, DNA binding, and genes associated with transcription and transcription control pathways were affected by the OPNs. Collectively, this study shows that human and bovine milk OPN have a significant and highly comparable effect on the intestinal transcriptome.

Osteopontin (OPN) is a bioactive integrin-binding protein found in high concentrations in milk, where it is present both as a full-length protein and as several N-terminally derived fragments. OPN resists gastric digestion, and via interaction with receptors in the gut or by crossing the intestinal barrier into circulation, ingested milk OPN may influence physiological processes. The aim of this study was to investigate OPN interaction with intestinal cells and its transport across models of the intestinal barrier. Immunodetection of OPN incubated with Caco-2 cells at 4 °C and 37 °C showed that OPN binds to the intestinal cells, but it is not internalised. Transepithelial transport was studied using mono- and co-cultures of Caco-2 cells and mucus-producing HT29-MTX cells in transwell membranes. OPN was shown to cross the barrier models in a time-, temperature-, and energy-dependent process inhibited by wortmannin, indicating that the transport takes place via the transcytosis pathway. Analyses of the naturally occurring milk mixture of full-length and N-terminal fragments showed that the N-terminal fragments of OPN bound intestinal cells most effectively and that the fragments were transported across the intestinal membrane models. This suggests that proteolytic processing of OPN increases its biological activity after ingestion.