Hoxa5 overexpression correlates with IGFBP1 upregulation and postnatal dwarfism: evidence for an interaction between Hoxa5 and Forkhead box transcription factors
Isabelle Foucher1,
Michel Volovitch1,2,
Monique Frain3,
J. Julie Kim4,
Jean-Claude Souberbielle5,
Lixia Gan6,
Terry G. Unterman6,7,
Alain Prochiantz1,* and
Alain Trembleau1,2
1 CNRS UMR 8542, Ecole normale supérieure, 46 rue d’Ulm, 75230 Paris Cedex 05, France
2 Université Denis Diderot-Paris VII, UFR de Biologie, 2 place Jussieu, 75005 Paris, France
3 INSERM U-368, Ecole normale supérieure, 46 rue d’Ulm, 75230 Paris Cedex 05, France
4 Department of Obstetrics and Gynecology, University of Illinois at Chicago College of Medicine and Chicago Area Veterans Healthcare System (West Side Division), Chicago, IL 60612, USA
5 Laboratoire de Physiologie, Hôpital Necker, 149 rue de Sèvres, 75015 Paris, France
6 Department of Medicine, University of Illinois at Chicago College of Medicine and Chicago Area Veterans Healthcare System (West Side Division), Chicago, IL 60612, USA
7 Department of Physiology and Biophysics, University of Illinois at Chicago College of Medicine and Chicago Area Veterans Healthcare System (West Side Division), Chicago, IL 60612, USA
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Fig. 1. B4B2A5m construct and expression in E9.5 embryos. (A) Schematic of the B4B2A5m construct. HD, homeodomain; SVpA, SV40 polyadenylation signal; CR5, CR6, CR7 and CR8 are the primers used in PCR and RT-PCR assays (for details see Materials and Methods section). (B,C) Hoxa5m transgene expression in E9.5 embryos, visualized by in toto hybridization. (B) Two sibling embryos: one heterozygous transgenic (left) and one control (right). Hoxa5 transgene is observed in r3 and r5, and in the somites. (C) Flat mount of the rhombencephalon of a B4B2A5m mouse, illustrating Hoxa5m transgene expression in r3 and r5 (A, anterior; P, posterior).
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Fig. 2. B4B2A5m mice have an impaired postnatal growth profile. (A,B) Sibling mice of matched sex, one control (left) and one transgenic (right) are shown together at two ages: 2 weeks (A, males) or 5.5 weeks (B, females). (C) Postnatal growth curves comparing the weight of control (n=4) and transgenic (n=7) male mice during the first 7 weeks of postnatal development. The growth rate of transgenic mice is significantly reduced during the second and third postnatal weeks. Growth resumes normally after 3 weeks but the mice remain smaller throughout adulthood.
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Fig. 3. B4B2A5m mice dwarfism is GH- and T3/T4- independent. (A,B) X-Gal detection of lacZ expression in pituitary (200 µm section, A) and whole thyroid gland (B) of adult B4B2lacZ mice. lacZ is strongly expressed in many cells of the pituitary anterior lobe (AL), but less so in scattered cells of the intermediate (IL) and posterior lobe (PL). (C) Northern blot analysis of GH mRNA expression in control (CTRL) and transgenic B4B2A5m male mice. No significant difference in GH mRNA concentration is observed between control (n=4) and transgenic mice (n=4). GH mRNA was normalized using the S26 probe (see Materials and Methods). (D) RIA measurements of circulating GH in control (CTRL males, n=8; CTRL females, n=4) and transgenic B4B2A5m (TG males, n=8; TG females n=5) mice. No significant difference could be seen in GH concentration between transgenic and control mice. (E) RIA measurement of total T3 and T4 thyroid hormones in control and transgenic mice of both sexes (n=6 for all groups). Transgenic mice present no alteration in levels of circulating thyroid hormones. All experiments shown in this figure were performed at 3.5 weeks of age.
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Fig. 4. Decreased liver IGF1 expression and circulating IGF1. (A) Levels of circulating IGF1 in control (CTRL males n=8; CTRL females n=4) and in B4B2A5m mice (TG males n=8; TG females n=5). Circulating IGF1 is significantly decreased in transgenic mice of both sexes (male: P<0.0032; female: P<0.018). (B) Northern blot analysis of liver IGF1 mRNA expression. IGF1 mRNA signal (two transgenic B4B2A5m and two control mice) was normalized with a GAPDH probe. The histogram shows that, compared with control, IGF1 mRNA levels in transgenic animals are halved (P<0.05).
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Fig. 5. Expression of Hoxa5 in HepG2 cells and in liver of control and transgenic mice. (A) Total RNA from HepG2 cells was reverse-transcribed and PCR-amplified using two different couples of primers CR3-CR4 (378 bp) and CR7-CR8 (180 bp) specific for Hoxa5 and recognizing both human and mouse transcripts. (B) PolyA+ RNAs from liver of control (CTR) or transgenic (TG) mice (3.5 weeks), and from E9.5 transgenic embryos (positive control for expression of Hoxa5m) were reverse-transcribed and PCR-amplified using three different couples of primers. CR7-CR8 primers amplify a 180 bp fragment, demonstrating the presence of endogenous Hoxa5 mRNA in control liver and of endogenous Hoxa5 plus transgene-encoded Hoxa5m mRNAs in transgenic livers and embryos. The Hoxa5m transgene-specific CR5-CR6 primers amplify a 286 bp fragment only in transgenic livers and embryo. No amplification is obtained in absence of reverse transcriptase (RT–). First lane, molecular mass markers; last lane, PCR performed in the same conditions on H2O instead of cDNA samples. (C) Liver polyA+ RNAs from control and transgenic mice (1-month-old) were analyzed by quantitative RT-PCR. Equivalence of signal intensity between liver Hoxa5 mRNAs (lower band) and the added standard synthetic RNA (upper band) is obtained at 40 pg for control mice (CTR, arrowhead), and 80 pg for transgenic mice (TG, arrowhead), demonstrating a twofold increase in Hoxa5 mRNA content in transgenic animals.
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Fig. 6. Macroarray analysis and real time PCR of liver cDNAs from B4B2A5m and control mice. (A) Radioactive spots obtained for IGF1, ESP1, IGFBP1 and glucose-6-phosphate (G6P) isomerase from membranes hybridized with labeled cDNAs obtained from control (CTRL) and B4B2A5m livers. Results are normalized using global standardization with AtlasImage 1.5 software. (B) Graphs representing normalized fluorescence intensity (Rn) as a function of cycle number and showing threshold cycles (CT) for IGFBP1 and GAPDH transcripts amplicons.
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Fig. 7. Regulatory actions of Hoxa5 on the IGFBP1 promoter expression in HepG2 and HuF cells. Cells were transfected with the IGFBP1 promoter linked to luciferase, with/without FKHR and/or Hoxa5. (A) in the HepG2 hepatoma cell line, Hoxa5 alone has no effect, but it inhibits the FKHR-dependent activation of the promoter. (B) In the HuF cells, Hoxa5 activates the IGFBP1 promoter, and it cooperates with FKHR to enhance its activity dramatically. Graphs illustrate the result of one representative experiment; each bar represents the mean and s.e.m. from triplicates.
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Fig. 8. Hoxa5 directly interacts with FKHR and HNF3ß. (A) Binding of 35S-labeled FKHR and HNF3ß to GST or GST-Hoxa5 fusion protein. GST or GST-Hoxa5 produced in bacteria were immobilized on glutathione-sepharose beads, which were subsequently loaded with either radioactive luciferase (L), FKHR (F) or HNF3ß (H). The last two lanes on each gel (input) show that the same amounts of radioactive luciferase and FKHR or HNF3ß were added to the beads. The first four lanes demonstrate that FKHR and HNF3ß bind specifically to Hoxa5 (absence of binding on GST alone, and absence of luciferase binding on GST-Hoxa5). (B) Binding of Hoxa5m to GST-FKHR extracted from transfected COS cells. GST or GST-FKHR constructs were co-transfected with Hoxa5m in COS cells, cell extracts were obtained and incubated with Glutathione Sepharose beads. Bound proteins were analyzed by SDS-PAGE and western blot. Whereas Hoxa5m protein is expressed as well in the GST transfected cells as in the GST-FKHR transfected cells, it is pulled down only from cells expressing GST-FKHR, but not from cells expressing GST.
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© The Company of Biologists Ltd 2002
