Description and Characterisation of a Splicing-Variant of Insulin-Like Growth Factor-Binding Protein-7 (IGFBP-7) Expressed in Human Granulosa Cells

Zusammenfassung

Fragestellung und Methodik

Insulin-like growth factor (IGF)-binding protein (IGFBP)-related proteins (IGFBP-rPS) sind kürzlich beschriebene zysteinreiche Proteine, die am aminoterminalen Ende ausgeprägte Strukturhomologien zu den bisher beschriebenen IGFBPs haben. IGFBPs sind in einer Vielzahl biologischer Funktionen involviert, einschließlich der Wachstumsregulierung und der Anheftung endothelialer Zellen. Ihre Wirkung können sie sowohl in Abhängigkeit von IGF als auch unabhängig von IGF entfalten. Im Verlauf eines Projektes zur Identifizierung und Charakterisierung von angiogenetisch wirksamen Faktoren haben wir eine Spleißvariante von IGFBP-rP-1/IGFBP-7 identifiziert (IGFBP-7 v) und charakterisiert, die in humanen Primärkulturen von Granulosazellen exprimiert wird, aber nicht in Tumorzelllinien, die von Ovarial-, Brust- und Zervixkarzinomen abgeleitet sind (HeLa, SW756; Caski; Ovcar-3, MDA-MP453).

Ergebnisse

IGFBP-7 v ist um 165 Nukleotide im ersten Exon verkürzt, was einem um 55 Aminosäuren verkürztem Protein entspricht. Diese Deletion umschließt das IGFBP-typische Aminosäure-Motiv, welches für die Bindung von IGF verantwortlich gemacht wird.

Schlussfolgerung

Wir vermuten daher, dass diese natürlich vorkommende Spleißvariante von IGFBP-7 unabhängig von der Bindung von IGF wirken kann.

Abstract

Objective

Insulin-like growth factor (IGF)-binding protein (IGFBP)-related proteins (IGFBP-rPs) are recently described cysteine-rich proteins that share significant amino terminal structural similarity with the conventional IGFBPs and are involved in a variety of biological functions, including growth regulation and endothelial cell attachment and spreading. In an effort to screen for angiogenic factors in human granulosa cells (GC), we have identified and characterised a splice variant of IGFBP-7/IGFBP-rP-1 (IGFBP-7 v).

Methods

The presence of IGFBP-7 and IGFBP-7 v variant was detected by RT-PCR in human granulosa cells derived from women undergoing in vitro fertilisation therapy. The nucleotide sequence was determined after subcloning and sequencing according to standard techniques. Computer analyses were performed at the ExPASy Molecular Biology server using the ClustalW alignment and protein analysis programms (http://www.expasy.ch).

Results and Conclusions

The splice variant of IGFBP-7 (IGFBP-7 v) is 165 nucleotides smaller than conventional IGFBP-7 transcripts. The deletion occurs within exon 1 but does not lead to a frameshift or an aberrant termination of the deduced protein sequence. However, the IGFBP-7 consensus motif is deleted. In contrast to non-tumorigenic granulosa cells, IGFBP-7 v is not expressed in tumor cell lines of ovarian and cervical origins. Computer based analysis of IGFBP-7 v demonstrates that the deduced amino acid sequence lacks the IGFBP typical domain [(G)CGCCXXC], which is supposed to be involved in IGF binding suggesting an IGF independent action of IGFBP-7 v.

Insulin - Wachstumsfaktor - Spleißen - Deletion - Tumorzelle

IGF - IGFBP - granulosa cell - splicing - deletion - tumor cell

References

• 1 Hwa V, Oh Y, Rosenfeld R G. The insulin-like growth factor-binding protein (IGFBP) superfamily.  Endocr Rev. 1999;  20 761-787
• 2 Ragosch V, Hundertmark S, Hopp H, Opri F, Weitzel H K. [Insulin-like-growth-factor-binding-protein 1 (IGFBP-1) and fetal fibronectin in diagnosis of premature rupture of fetal membranes].  Geburtsh Frauenheilk. 1996;  56 291-296
• 3 Hwa V, Tomasini-Sprenger C, Bermejo A L, Rosenfeld R G, Plymate S R. Characterization of insulin-like growth factor-binding protein-related protein-1 in prostate cells.  J Clin Endocrinol Metab. 1998;  83 4355-4362
• 4 Damon S E, Haugk K L, Swisshelm K, Quinn L S. Developmental regulation of Mac25/insulin-like growth factor-binding protein-7 expression in skeletal myogenesis.  Exp Cell Res. 1997;  237 192-195
• 5 Zhou J, Adesanya O O, Vatzias G, Hammond J M, Bondy C A. Selective expression of insulin-like growth factor system components during porcine ovary follicular selection.  Endocrinology. 1996;  137 4893-4901
• 6 Swisshelm K, Ryan K, Tsuchiya K, Sager R. Enhanced expression of an insulin growth factor-like binding protein (mac25) in senescent human mammary epithelial cells and induced expression with retinoic acid.  Proc Natl Acad Sci USA. 1995;  92 4472-4476
• 7 Keck C, Neulen J, Breckwoldt M. [Ovarian hyperstimulation syndrome].  Geburtsh Frauenheilk. 1994;  54 315-320
• 8 Ahn W S, Huh S W, Bae S M, Lee I P, Lee J M, Namkoong S E. et al . A major constituent of green tea, EGCG, inhibits the growth of a human cervical cancer cell line, CaSki cells, through apoptosis, G(1) arrest, and regulation of gene expression.  DNA Cell Biol. 2003;  22 217-224
• 9 Cristoforoni P M, Lotzova E, Cook K R, Chuang L T, Morris M, Grossi C E. et al . Oncolytic activity of NK cells against SW-756 squamous cervical carcinoma cell line: role of interferons alpha and gamma and CD54 adhesion molecule in oncolysis.  Gynecol Oncol. 1994;  52 365-372
• 10 Fife R S, Sledge Jr G W. Effects of doxycycline on in vitro growth, migration, and gelatinase activity of breast carcinoma cells.  J Lab Clin Med. 1995;  125 407-411
• 11 Pearson J W, Hedrick E, Fogler W E, Bull R L, Ferris D K, Riggs C W. et al . Enhanced therapeutic efficacy against an ovarian tumor xenograft of immunotoxins used in conjunction with recombinant alpha-interferon.  Cancer Res. 1990;  50 6379-6388
• 12 Nishimura S, Murasugi T, Kubo T, Kaneko I, Meguro M, Marumoto S. et al . RS-4252 inhibits amyloid beta-induced cytotoxicity in HeLa cells.  Pharmacol Toxicol. 2003;  93 29-32
• 13 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.  Anal Biochem. 1987;  162 156-159
• 14 Joliot V, Khelifi C, Wyers M, Dambrine G, Lasserre F, Lemercier P. et al . The noncoding and surface envelope coding sequences of myeloblastosis-associated virus are respectively responsible for nephroblastoma development and renal hyperplasia.  J Virol. 1996;  70 2576-2580
• 15 Akaogi K, Okabe Y, Funahashi K, Yoshitake Y, Nishikawa K, Yasumitsu H. et al . Cell adhesion activity of a 30-kDa major secreted protein from human bladder carcinoma cells.  Biochem Biophys Res Commun. 1994;  198 1046-1053
• 16 Samaras S E, Guthrie H D, Barber J A, Hammond J M. Expression of the mRNAs for the insulin-like growth factors and their binding proteins during development of porcine ovarian follicles.  Endocrinology. 1993;  133 2395-2398
• 17 Ahr A, Holtrich U, Solbach C, Scharl A, Strebhardt K, Karn T. et al . Molecular classification of breast cancer patients by gene expression profiling.  J Pathol. 2001;  195 312-320
• 18 Romisch M, Meier W, Kimmig R, Hepp H. [13-cis retinoic acid and interferon-alfa-2 a as palliative therapy in pretreated, recurrent squamous epithelial carcinoma of the cervix uteri and vulva].  Geburtsh Frauenheilk. 1996;  56 520-524