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Circulating IGF-binding protein 7 (IGFBP7) levels are elevated in patients with endometriosis or undergoing diabetic hemodialysis.

Kutsukake M, Ishihara R, Momose K, Isaka K, Itokazu O, Higuma C, Matsutani T, Matsuda A, Sasajima K, Hara T, Tamura K - Reprod. Biol. Endocrinol. (2008)

Bottom Line: Decreased IGFBP7 expression has been observed in some cancers and leiomyomata.Using this new ELISA, we demonstrated that cultured uterine cells secrete IGFBP7 into the medium.There were no differences in serum IGFBP7 levels in women at different stages of the menstrual cycle.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Endocrine Pharmacology, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 193-0392, Japan. kutsu@ps.toyaku.ac.jp

ABSTRACT

Background: Insulin-like growth factor-binding protein-7 (IGFBP7) is a secretory protein with a molecular mass of approximately 30 kDa. It is abundantly expressed in the uterine endometrium during the secretory phase of the menstrual cycle. Decreased IGFBP7 expression has been observed in some cancers and leiomyomata.

Methods: To determine whether serum IGFBP7 levels reflect changes in uterine IGFBP7 expression in humans during the menstrual cycle, and to examine whether serum IGFBP7 levels are altered in patients with various disorders, we developed a novel, dual-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Firstly, concentrations of IGFBP7 released into the medium were determined in cultured endometrial stromal and glandular cells. Blood samples were collected from women who had normal menstrual cycles and who had been diagnosed with endometriosis. Serum from hemodialysis patients and gastrointestinal cancers was also used to determine the IGFBP7 levels.

Results: Using this new ELISA, we demonstrated that cultured uterine cells secrete IGFBP7 into the medium. Patients with endometriosis and those with type II diabetes mellitus undergoing hemodialysis had significantly higher serum concentrations of IGFBP7 than the relevant control subjects. There were no differences in serum IGFBP7 levels in women at different stages of the menstrual cycle. Furthermore, serum IGFBP7 levels in patients with colorectal, esophageal, or endometrial cancer were not different than normal healthy subjects.

Conclusion: Our observations suggest that IGFBP7 is associated with the pathophysiology of endometriosis and diabetes mellitus, and that serum IGFBP7 levels do not reflect enhanced uterine expression of IGFBP7 mRNA during the menstrual cycle.

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Determination of IGFBP7 protein levels in conditioned media from human ESCs and GE cells. The conditioned media of subconfluent cells was collected at the indicated times and stored frozen until use. (A) Representative standard curve obtained by ELISA using recombinant human IGFBP7 in PBS containing 0.1% BSA. Data represents the means of four replicates. (B) IGFBP7 levels in conditioned media over time. The conditioned medium of GE cells was measured at the 24 h time point only. Gray column; IGFBP7 levels in the medium of ESCs treated for 24 h with IGFBP7 siRNA (10 pmol). The data represents the means ± SE of three separate cultures, assayed in duplicate.
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Figure 1: Determination of IGFBP7 protein levels in conditioned media from human ESCs and GE cells. The conditioned media of subconfluent cells was collected at the indicated times and stored frozen until use. (A) Representative standard curve obtained by ELISA using recombinant human IGFBP7 in PBS containing 0.1% BSA. Data represents the means of four replicates. (B) IGFBP7 levels in conditioned media over time. The conditioned medium of GE cells was measured at the 24 h time point only. Gray column; IGFBP7 levels in the medium of ESCs treated for 24 h with IGFBP7 siRNA (10 pmol). The data represents the means ± SE of three separate cultures, assayed in duplicate.

Mentions: We developed a novel IGFBP7 ELISA using two anti-IGFBP7 antibodies, as described in Materials and Methods. The standard curve generated with recombinant IGFBP7 was linear between 0.3 and 20 ng/ml (Fig. 1A). We recently demonstrated that IGFBP7 is expressed in the human uterus, and that IGFBP7 mRNA levels are up-regulated in ESCs and glandular epithelial cells during the receptive phase of the menstrual cycle [8]. We first determined whether cultured primary ESCs and EM1 glandular epithelial cells (GE cells) secreted IGFBP7. IGFBP7 gradually accumulated in the culture medium of ESCs and GE cells over a 24 h period of incubation, reaching levels of 55 and 25 ng/ml, respectively, at 24 h (Fig. 1B). When ESCs were treated with IGFBP7 siRNA to knock-down the expression of IGFBP7, the protein levels of IGFBP7 were reduced by 86% (approximately 7.5 ng/mL). These results confirmed that cultured ESCs secrete IGFBP7, and demonstrated that ESCs secrete about 2-fold more IGFBP7 than GE cells.


Circulating IGF-binding protein 7 (IGFBP7) levels are elevated in patients with endometriosis or undergoing diabetic hemodialysis.

Kutsukake M, Ishihara R, Momose K, Isaka K, Itokazu O, Higuma C, Matsutani T, Matsuda A, Sasajima K, Hara T, Tamura K - Reprod. Biol. Endocrinol. (2008)

Determination of IGFBP7 protein levels in conditioned media from human ESCs and GE cells. The conditioned media of subconfluent cells was collected at the indicated times and stored frozen until use. (A) Representative standard curve obtained by ELISA using recombinant human IGFBP7 in PBS containing 0.1% BSA. Data represents the means of four replicates. (B) IGFBP7 levels in conditioned media over time. The conditioned medium of GE cells was measured at the 24 h time point only. Gray column; IGFBP7 levels in the medium of ESCs treated for 24 h with IGFBP7 siRNA (10 pmol). The data represents the means ± SE of three separate cultures, assayed in duplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2600820&req=5

Figure 1: Determination of IGFBP7 protein levels in conditioned media from human ESCs and GE cells. The conditioned media of subconfluent cells was collected at the indicated times and stored frozen until use. (A) Representative standard curve obtained by ELISA using recombinant human IGFBP7 in PBS containing 0.1% BSA. Data represents the means of four replicates. (B) IGFBP7 levels in conditioned media over time. The conditioned medium of GE cells was measured at the 24 h time point only. Gray column; IGFBP7 levels in the medium of ESCs treated for 24 h with IGFBP7 siRNA (10 pmol). The data represents the means ± SE of three separate cultures, assayed in duplicate.
Mentions: We developed a novel IGFBP7 ELISA using two anti-IGFBP7 antibodies, as described in Materials and Methods. The standard curve generated with recombinant IGFBP7 was linear between 0.3 and 20 ng/ml (Fig. 1A). We recently demonstrated that IGFBP7 is expressed in the human uterus, and that IGFBP7 mRNA levels are up-regulated in ESCs and glandular epithelial cells during the receptive phase of the menstrual cycle [8]. We first determined whether cultured primary ESCs and EM1 glandular epithelial cells (GE cells) secreted IGFBP7. IGFBP7 gradually accumulated in the culture medium of ESCs and GE cells over a 24 h period of incubation, reaching levels of 55 and 25 ng/ml, respectively, at 24 h (Fig. 1B). When ESCs were treated with IGFBP7 siRNA to knock-down the expression of IGFBP7, the protein levels of IGFBP7 were reduced by 86% (approximately 7.5 ng/mL). These results confirmed that cultured ESCs secrete IGFBP7, and demonstrated that ESCs secrete about 2-fold more IGFBP7 than GE cells.

Bottom Line: Decreased IGFBP7 expression has been observed in some cancers and leiomyomata.Using this new ELISA, we demonstrated that cultured uterine cells secrete IGFBP7 into the medium.There were no differences in serum IGFBP7 levels in women at different stages of the menstrual cycle.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Endocrine Pharmacology, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 193-0392, Japan. kutsu@ps.toyaku.ac.jp

ABSTRACT

Background: Insulin-like growth factor-binding protein-7 (IGFBP7) is a secretory protein with a molecular mass of approximately 30 kDa. It is abundantly expressed in the uterine endometrium during the secretory phase of the menstrual cycle. Decreased IGFBP7 expression has been observed in some cancers and leiomyomata.

Methods: To determine whether serum IGFBP7 levels reflect changes in uterine IGFBP7 expression in humans during the menstrual cycle, and to examine whether serum IGFBP7 levels are altered in patients with various disorders, we developed a novel, dual-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Firstly, concentrations of IGFBP7 released into the medium were determined in cultured endometrial stromal and glandular cells. Blood samples were collected from women who had normal menstrual cycles and who had been diagnosed with endometriosis. Serum from hemodialysis patients and gastrointestinal cancers was also used to determine the IGFBP7 levels.

Results: Using this new ELISA, we demonstrated that cultured uterine cells secrete IGFBP7 into the medium. Patients with endometriosis and those with type II diabetes mellitus undergoing hemodialysis had significantly higher serum concentrations of IGFBP7 than the relevant control subjects. There were no differences in serum IGFBP7 levels in women at different stages of the menstrual cycle. Furthermore, serum IGFBP7 levels in patients with colorectal, esophageal, or endometrial cancer were not different than normal healthy subjects.

Conclusion: Our observations suggest that IGFBP7 is associated with the pathophysiology of endometriosis and diabetes mellitus, and that serum IGFBP7 levels do not reflect enhanced uterine expression of IGFBP7 mRNA during the menstrual cycle.

Show MeSH
Related in: MedlinePlus