Limited Proteolysis of the IGF Binding Protein-2 (IGFBP-2) by a Specific Serine Protease Activity in Early Breast Milk

Elmlinger, Martin W.; Grund, R; Buck, Michael; Wollmann, H; Feist, Norman; Weber, Matthias M.; Speer, Christian P.; Ranke, Michael B.

doi:10.1203/00006450-199907000-00013

Cited by 30 publications

(33 citation statements)

References 18 publications

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“…Partial hydrolysis of milk proteins by co-secreted proteases would release peptide fragments including di-and tripeptides, and those may be reabsorbed by PEPT2 into epithelial cells. In addition, specific hydrolysis of biologically active proteins, such as binding proteins for insulin-like growth factors in human milk, has been reported (14) that also would release short-chain peptides. These peptides either could be submitted to hydrolysis by surface-bound aminopeptidases (5, 13) to yield free amino acids or may be transported in intact form into the epithelial cells.…”

Section: Discussionmentioning

confidence: 99%

Peptide transport in the mammary gland: expression and distribution of PEPT2 mRNA and protein

Groneberg

Döring

Theis

et al. 2002

American Journal of Physiology-Endocrinology and Metabolism

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The lactating mammary gland utilizes free plasma amino acids as well as those derived by hydrolysis from circulating short-chain peptides for protein synthesis. Apart from the major route of amino acid nitrogen delivery to the gland by the various transporters for free amino acids, it has been suggested that dipeptides may also be taken up in intact form to serve as a source of amino acids. The identification of peptide transporters in the mammary gland may therefore provide new insights into protein metabolism and secretion by the gland. The expression and distribution of the high-affinity type proton-coupled peptide transporter PEPT2 were investigated in rat lactating mammary gland as well as in human epithelial cells derived from breast milk. By use of RT-PCR, PEPT2 mRNA was detected in rat mammary gland extracts and human milk epithelial cells. The expression pattern of PEPT2 mRNA revealed a localization in epithelial cells of ducts and glands by nonisotopic high resolution in situ hybridization. In addition, immunohistochemistry was carried out and showed transporter immunoreactivity in the same epithelial cells of the glands and ducts. In addition, two-electrode voltage clamp recordings using PEPT2-expressing Xenopus laevis oocytes demonstrated positive inward currents induced by selected dipeptides that may play a role in aminonitrogen handling in mammalian mammary gland. Taken together, these data suggest that PEPT2 is expressed in mammary gland epithelia, in which it may contribute to the reuptake of short-chain peptides derived from hydrolysis of milk proteins secreted into the lumen. Whereas PEPT2 also transports a variety of drugs, such as selected β-lactams, angiotensin-converting enzyme inhibitors, and antiviral and anticancer metabolites, their efficient reabsorption via PEPT2 may reduce the burden of xenobiotics in milk.

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Section: Discussionmentioning

confidence: 99%

Peptide transport in the mammary gland: expression and distribution of PEPT2 mRNA and protein

Groneberg

Döring

Theis

et al. 2002

American Journal of Physiology-Endocrinology and Metabolism

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“…To avoid the IGF measurement interfering with the IGFBPs present in breast milk, we used IGF-1 and IGF-2 assays that made use of the principle of excess IGF-2 or IGF-1, respectively. This is a standard approach to make IGFBP assays more reliable (7,8). The sensitivity of all assays was Ͻ0.2 ng/mL.…”

Section: Methodsmentioning

confidence: 99%

“…The RIAs were competitive binding assays for serum utilizing 125 I-labeled IGF-1, IGF-2, IGFBP-2, or IGFBP-3. Using human milk whey as the assay matrix instead of serum, the RIAs had been evaluated previously for linearity, precision, and recovery (7,8, and unpublished data). The precision of measurement of IGF-I, IGF-II, IGFBP-2, and IGFBP-3 were 7.5, 8.9, 7.8, and 9.2% coefficient of variation (CV), respectively.…”

Section: Methodsmentioning

confidence: 99%

Effects of Different CMV-Heat-Inactivation-Methods on Growth Factors in Human Breast Milk

Goelz¹,

Hihn²,

Hamprecht

et al. 2009

Pediatr Res

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Preterm infants can inoculate virulent cytomegalovirus (CMV) through their mothers' raw breast milk. Complete virus inactivation is achieved only by heat treatment, but the effect on growth factors has never been assessed systematically. Insulin-likegrowth-factor-1-, IGF-2-, insulin-like-growth-factor-binding-protein-2-, and IGFBP-3-concentrations were measured, before and after heating, in 51 breast-milk-samples from 28 mothers, and epidermalgrowth-factor-concentrations in a subgroup of 35 samples from 22 mothers. Two heating methods were applied: Short-term (5 s) pasteurisation at 62, 65, and 72°C, and long-term Holder-Pasteurisation (30 min) at 63°C. IGF-1, IGF-2, IGFBP-2, and IGFBP-3 were measured by RIA, and EGF by ELISA. Heating for 30 min decreased significantly IGF-1 by 39.4%, IGF-2 by 9.9%, IGFBP-2 by 19.1%, and IGFBP-3 by 7.0%. In contrast, IGF-1, IGF-2, IGFBP-2, and IGFBP-3 were not altered significantly when using a short heating duration of 5 s, irrespective of the level of temperature, except for IGF-2 at 62°C for 5 s (p ϭ 0.041) and IGFBP-2 at 72°C for 5 s (p ϭ 0.025). Neither long-nor short-time heating methods changed the concentration of EGF. Only short heating methods (5 s, 62-72°C) can preserve, almost completely, the concentrations of IGFs in human milk, whereas Holder-Pasteurization does not. (Pediatr Res 65: 458-461, 2009) P reterm infants are at risk of acquiring Cytomegalovirus (CMV) infection in their first weeks of life through their mothers' raw breast milk, because CMV-IgG-positive mothers reactivate the virus during lactation and excrete it into their breast milk (1,2). For total inactivation of CMV in breast milk, heating procedures are necessary. Freeze-thawing may reduce the virus count but does not eliminate virulent CMV completely (3-5). There is growing evidence that, in addition to conventional Holder pasteurization (63°C, 30 min), short-term heat inactivation methods are effective (1,3; Müller, D ͓Establishing of a new procedure for the short-time heat pasteurization for inactivating of human Cytomegalievirus in mother's milk͔. Doctoral thesis 2006 at The Medical Faculty of the Eberhard-Karls-University of Tuebingen). However, the effect of the duration of heating and the temperature level achieved on bioactive substances like insulin-like growth factors (IGF), insulin-like growth factor binding proteins (IGFBP), and epidermal growth factor (EGF) in human milk has not yet been assessed systematically. We, therefore, evaluated the concentrations of growth hormones in human breast milk before and after processing with different heat inactivation parameters. METHODSMilk samples. IGF-1, IGF-2, IGFBP-2, and IGFBP-3 were analyzed in 51 freshly expressed breast milk specimens from 28 mothers; 21 of preterm and 7 of term infants. The expressed milk specimens, originating from lactation day 5-46, were stored at Ϫ20°C soon after collection until use. For processing, they were thawed, aliquoted, and labeled as either control or treated. The latter were heated according to fou...

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“…IGFBP-2, the second most frequent IGFBP in the human circulation, is involved in many physiological and pathological conditions and processes: IGFBP-2 is highly expressed during the perinatal period (Blum et al 1993, Lindenbergh-Kortleve et al 1997, while in adults the highest concentrations of IGFBP-2 are found in cerebrospinal fluid (Binoux et al 1991, Arnold et al 1999), mother's milk (Elmlinger et al 1999) and seminal plasma (Rosenfeld et al 1990). IGFBP-2 is also often highly expressed in malignant tumor cells but decreases upon remission (Muller et al 1994, Elmlinger et al 1996, Hoeflich et al 2000, Elmlinger et al 2001, Moore et al 2003, Ranke et al 2003.…”

Section: Introductionmentioning

confidence: 99%

IGF-independent effects of IGFBP-2 on the human breast cancer cell line Hs578T

Frommer¹,

Reichenmiller²,

Schütt³

et al. 2006

Journal of Molecular Endocrinology

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There is evidence that insulin-like growth factor-binding protein (IGFBP-2), a modulator of the actions of IGFs, also has IGF-independent effects in human tumor cell lines. These involve specific binding of IGFBP-2 to a5b1-integrin, followed by alterations in the phosphorylation status of downstream signaling molecules. Previously, IGFBP-2 has also been shown to be associated with cell proliferation, adhesion and migration. Here, we investigated direct effects of IGFBP-2 on apoptosis and alterations in the expression of related proteins. The breast cancer cell line Hs578T, which shows no IGFBP-2 production of its own and is independent of the IGF-I receptor, was treated with human recombinant IGFBP-2 in order to study the changes in gene expression induced by IGFBP-2. The methods employed for this purpose were oligonucleotide microarrays, real-time RT-PCR, western blotting, and immunoassays. Out of the 440 genes covered by the Oligo GEArray Human Cancer Microarray OHS-802, the expression of 77 genes was directly influenced by IGFBP-2. By the use of real-time quantitative RT-PCR, the gene expression of Nuclear Factor (NF)kB, p53, transforming growth factor b (TGF b-1), LAMB1 (Laminin, Beta 1), Bcl-2, and IIp45 was found to be significantly upregulated (by 1$2-to 3$05-fold; all P!0$001). Accordingly, NFkB, p53, and TGF b-1 proteins, as measured by Western blotting and immunoassay, were upregulated O1$5-fold. By using an ELISA-based and a flow cytometry-based apoptosis assay, IGFBP-2 was found to have a pro-apoptotic effect on Hs578T cells. Our results suggest that IGFBP-2-induced gene expressions are of functional significance for proliferation, cell adhesion, cell migration and apoptosis, and showed that IGFBP-2 can promote apoptosis in tumor cells independent of IGF.

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Limited Proteolysis of the IGF Binding Protein-2 (IGFBP-2) by a Specific Serine Protease Activity in Early Breast Milk

Cited by 30 publications

References 18 publications

Peptide transport in the mammary gland: expression and distribution of PEPT2 mRNA and protein

Peptide transport in the mammary gland: expression and distribution of PEPT2 mRNA and protein

Effects of Different CMV-Heat-Inactivation-Methods on Growth Factors in Human Breast Milk

IGF-independent effects of IGFBP-2 on the human breast cancer cell line Hs578T

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