(Igfbp5) Compromises Survival, Growth, Muscle Development, and Fertility in Mice

Insulin-like growth factor-binding protein 5 (Igfbp5) compromises survival, growth, muscle development, and fertility in mice Dervis A. M. Salih*, Gyanendra Tripathi*, Cathy Holding*, Tadge A. M. Szestak*, M. Ivelisse Gonzalez*, Emma J. Carter*, Laura J. Cobb*, Joan E. Eisemann†, and Jennifer M. Pell*‡ *Signalling Programme, The Babraham Institute, Cambridge CB2 4AT, United Kingdom; and †Department of Animal Science, North Carolina State University, Raleigh, NC 27695 Communicated by Michael J. Berridge, The Babraham Institute, Cambridge, United Kingdom, January 23, 2004 (received for review October 3, 2003) The insulin-like growth factors (IGFs) are essential for development; include a consensus nuclear localization signal (17) and serine͞ bioavailable IGF is tightly regulated by six related IGF-binding pro- threonine phosphorylation sites. These primary motifs are con- teins (IGFBPs). Igfbp5 is the most conserved and is developmentally served, signifying that putative IGF-dependent and -independent up-regulated in key lineages and pathologies; in vitro studies suggest functions have been maintained. that IGFBP-5 functions independently of IGF interaction. Genetic We have therefore pursued the hypothesis that IGFBP-5 has a ablation of individual Igfbps has yielded limited phenotypes because significant role in growth and development. IGFBP function has of substantial compensation by remaining family members. There- been investigated in vivo by using gene ablation by homologous fore, to reveal Igfbp5 actions in vivo, we generated lines of transgenic recombination (e.g., ref. 18); however, to date Igfbp-null mice have mice that ubiquitously overexpressed Igfbp5 from early develop- exhibited a limited phenotype because of compensation and re- ment. Signiﬁcantly increased neonatal mortality, reduced female dundancy from remaining IGFBPs. We therefore decided to take fertility, whole-body growth inhibition, and retarded muscle devel- an alternative approach and generate transgenic mice overexpress- opment were observed in Igfbp5-overexpressing mice. The magni- ing Igfbp5. These mice are significantly growth-retarded and exhibit tude of the response in individual transgenic lines was positively the most severe phenotype of any Igfbp genetic manipulation in vivo correlated with Igfbp5 expression. Circulating IGFBP-5 concentrations to date, providing convincing evidence that IGFBP-5, by either increased a maximum of only 4-fold, total and free IGF-I concentra- IGF-dependent or -independent mechanisms, is important in de- tions increased up to 2-fold, and IGFBP-5 was detected in high Mr termining cell fate. complexes; however, no detectable decrease in the proportion of free IGF-I was observed. Thus, despite only modest changes in IGF and Materials and Methods IGFBP concentrations, the Igfbp5-overexpressing mice displayed a Transgene Construction. The murine Igfbp5-coding region [in phenotype more extreme than that observed for other Igfbp genetic pEMSVscribe ␣2 (5), P. Rotwein, Oregon Health Sciences Uni- models. Although growth retardation was obvious prenatally, max- versity, Portland] was amplified by PCR (primer sequences are imal inhibition occurred postnatally before the onset of growth given in Supporting Text, which is published as supporting infor- hormone-dependent growth, regardless of Igfbp5 expression level, mation on the PNAS web site) with use of PfuTurbo (Stratagene) revealing a period of sensitivity to IGFBP-5 during this important to include the native translation initiation sequence of Igfbp5 and to stage of tissue programming. introduce a C-terminal myc epitope. The EcoRI-restricted, 893-bp Igfbp5-myc amplicon was inserted downstream of the cytomegalo- he insulin-like growth factors (IGF-I and -II) are essential for virus enhancer, chicken ␤-actin promoter, and rabbit ␤-globin Tgrowth and development (1). Six high-affinity IGF-binding splice acceptor, and upstream to the simian virus 40 polyadenyla- proteins (IGFBP-1 to IGFBP-6; refs. 2 and 3) strictly orchestrate tion signal of a pCAGGS expression vector (J. Miyazaki, Osaka IGF action. Despite their considerable sequence homology, each University Medical School, Osaka; ref. 19 and Fig. 1A). The exhibits a discrete expression pattern and possesses an individual CMV-␤A͞Igfbp5-myc transgene was isolated from the plasmid by subset of motifs, signifying that although IGFBPs have common PstI͞SalI double digestion, QIAquick gel purification (Qiagen, actions, they may also have unique properties. Valencia, CA) and serial ethanol precipitations. IGFBP-5 is the most conserved of the IGFBPs (4) and has been highlighted as a focal regulatory factor during the development of Generation of Igfbp5-Overexpressing Mice. Transgenic mice were several key cell lineages, e.g., myoblasts (5) and neural cells (6, 7). generated by pronuclear injection of the linear transgene into In mice, Igfbp5 is expressed in the embryo from early development, fertilized zygotes from C57BL͞6J ϫ CBA͞CA superovulated dams principally in the myotomal component of the somites and devel- mated to males of the same genotype. Founders were identified by oping central nervous system (8). Postnatally, serum IGFBP-5, in Southern blot analysis and PCR. common with IGFBP-3, forms a ternary complex with IGF-I or IGF-II and the acid-labile subunit (9). Igfbp5 is up-regulated in the Growth and Development of Igfbp5 Mice. Mice were housed in a aggressive pediatric cancer, rhabdomyosarcoma (10), in the pro- controlled-barrier unit (12 h light͞12 h dark at 21 Ϯ 2°C) and fed gression of prostate cancers to androgen independence (11), and in standard laboratory diet ad libitum. WT (Ϫ/Ϫ), hemizygous (Ϫ/ϩ), smooth muscle-derived uterine leiomyoma (12), indicating a func- and homozygous (ϩ/ϩ) animals were weighed weekly from birth to tion in neoplasia. 8 wk and killed by decapitation (postneonatal mice were anesthe- IGFBP-5 initially binds IGFs with high affinity, principally by an tized first). Selected tissues were dissected; muscle was represented N-terminal motif (13), and inhibits IGF activity by preventing IGF by the gastrocnemius, plantaris, and soleus muscle bundle. interaction with the type 1 receptor. It is further subject to regulated posttranslational modifications (3) to induce conformational changes that decrease its affinity for IGFs and allow their effective Abbreviations: IGF, insulin-like growth factor; IGFBP, insulin-like growth factor-binding delivery to the type 1 receptor. However, IGFBPs are also multi- protein; Tg, transgenic; en, embryonic day n. functional proteins that can regulate cell function independent of ‡To whom correspondence should be addressed. E-mail: jenny.pell@bbsrc.ac.uk. IGF signaling (14–16); key motifs that may mediate these functions © 2004 by The National Academy of Sciences of the USA 4314–4319 ͉ PNAS ͉ March 23, 2004 ͉ vol. 101 ͉ no. 12 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400230101 Downloaded by guest on September 27, 2021 DMSO, followed by nonreducing 7.5% SDS͞PAGE (22) and immunoblotting with anti-myc Ab. Real-Time PCR. Total RNA (2 ␮g) was reverse-transcribed with SuperScript II RT (Invitrogen), and resultant cDNA was subjected to real-time PCR (Applied Biosystems). The mRNA levels of Igf1, Igfbp3, Igfbp5, and ␤-actin were assessed (primer sequences given in Supporting Text). The fluorescence of SYBR Green bound to double-stranded DNA was measured after each PCR cycle as the reaction proceeded to completion. A ␤-actin dilution series during the linear phase was used to normalize RNA levels between samples. Histology. Embryos were embedded in optimum cutting tempera- ture compound and frozen in dry-ice-cooled isopentane. Sagittal sections (10 ␮m) were stained with Gills III hematoxylin͞eosin (BDH). Statistics. Mice were grouped according to line, sex, and genotype. Differences between genotypes and lines at selected times were determined with Student’s t test (confirmed by using the Mann– Whitney test) or by two-way ANOVA. Values are expressed as the mean Ϯ SEM in all figures. Results Fig. 1. Igfbp5-myc expression is widespread but at different levels in the three Derivation of Igfbp5-Overexpressing Lines: Low Copy Number. Zy- lines. (A) Schematic of the Igfbp5-myc construct. CMV-IE, immediate early cyto- gotes were microinjected with Igfbp5-myc transgene to generate megalovirus. (B) Hybridization of Igfbp5 cDNA probe to mRNA isolated from three Igfbp5-overexpressing lines. Southern and slot blot analyses hemizygous skeletal muscle at 8 wk (mice from WT females ϫ Tg males). (C) (Fig. 7, which is published as supporting information on the Endogenous and Tg mRNA levels from key tissues of line 3 mice at 8 wk. PNAS web site) revealed differential sites of integration and a single copy of Igfbp5-myc per haploid genome for all founders. Other transgenes driven by this promoter have produced lines Southern and Northern Blot Analyses. Tail genomic DNA was containing up to 20 copies of transgene (23), and mouse models digested with EcoRI for Southern blot analysis. Slot-blot analysis of overexpressing other Igfbps have invariably displayed multiple genomic DNA, in parallel with WT genomic DNA that had been copy numbers (e.g., ref. 24). spiked using a serial dilution of transgene, confirmed copy number, PHYSIOLOGY and distinguished homo- and hemizygotes. Blots were hybridized Ubiquitous Expression of Igfbp5-myc. Northern blot analyses re- with a random-primed, labeled (High Prime protocol, Roche vealed relative transgene expression levels between lines (transcript Diagnostics),

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