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Eliminating the Synthesis of Mature Lamin A Reduces Disease Phenotypes in Mice Carrying a Hutchinson-Gilford Progeria Syndrome Allele

2008; Elsevier BV; Volume: 283; Issue: 11 Linguagem: Inglês

10.1074/jbc.m708138200

ISSN

1083-351X

Autores

Shao H. Yang, Xin Qiao, Emily Farber, Sandy Y. Chang, Loren G. Fong, Stephen G. Young,

Tópico(s)

Trace Elements in Health

Resumo

Hutchinson-Gilford progeria syndrome is caused by the synthesis of a mutant form of prelamin A, which is generally called progerin. Progerin is targeted to the nuclear rim, where it interferes with the integrity of the nuclear lamina, causes misshapen cell nuclei, and leads to multiple aging-like disease phenotypes. We created a gene-targeted allele yielding exclusively progerin (LmnaHG) and found that heterozygous mice (LmnaHG/+) exhibit many phenotypes of progeria. In this study, we tested the hypothesis that the phenotypes elicited by the LmnaHG allele might be modulated by compositional changes in the nuclear lamina. To explore this hypothesis, we bred mice harboring one LmnaHG allele and one LmnaLCO allele (a mutant allele that produces lamin C but no lamin A). We then compared the phenotypes of LmnaHG/LCO mice (which produce progerin and lamin C) with littermate LmnaHG/+ mice (which produce lamin A, lamin C, and progerin). LmnaHG/LCO mice exhibited improved body weight curves (p < 0.0001), reduced numbers of spontaneous rib fractures (p < 0.0001), and improved survival (p < 0.0001). In addition, LmnaHG/LCO fibroblasts had fewer misshapen nuclei than LmnaHG/+ fibroblasts (p < 0.0001). A likely explanation for these differences was uncovered; the amount of progerin in LmnaHG/LCO fibroblasts and tissues was lower than in LmnaHG/+ fibroblasts and tissues. These studies suggest that compositional changes in the nuclear lamina can influence both the steady-state levels of progerin and the severity of progeria-like disease phenotypes. Hutchinson-Gilford progeria syndrome is caused by the synthesis of a mutant form of prelamin A, which is generally called progerin. Progerin is targeted to the nuclear rim, where it interferes with the integrity of the nuclear lamina, causes misshapen cell nuclei, and leads to multiple aging-like disease phenotypes. We created a gene-targeted allele yielding exclusively progerin (LmnaHG) and found that heterozygous mice (LmnaHG/+) exhibit many phenotypes of progeria. In this study, we tested the hypothesis that the phenotypes elicited by the LmnaHG allele might be modulated by compositional changes in the nuclear lamina. To explore this hypothesis, we bred mice harboring one LmnaHG allele and one LmnaLCO allele (a mutant allele that produces lamin C but no lamin A). We then compared the phenotypes of LmnaHG/LCO mice (which produce progerin and lamin C) with littermate LmnaHG/+ mice (which produce lamin A, lamin C, and progerin). LmnaHG/LCO mice exhibited improved body weight curves (p < 0.0001), reduced numbers of spontaneous rib fractures (p < 0.0001), and improved survival (p < 0.0001). In addition, LmnaHG/LCO fibroblasts had fewer misshapen nuclei than LmnaHG/+ fibroblasts (p < 0.0001). A likely explanation for these differences was uncovered; the amount of progerin in LmnaHG/LCO fibroblasts and tissues was lower than in LmnaHG/+ fibroblasts and tissues. These studies suggest that compositional changes in the nuclear lamina can influence both the steady-state levels of progerin and the severity of progeria-like disease phenotypes. Mutations in LMNA yield a host of different human diseases, including muscular dystrophy, partial lipodystrophy, and Hutchinson-Gilford progeria syndrome (HGPS) 3The abbreviations used are:HGPSHutchinson-Gilford progeria syndromeHGHutchinson-Gilford alleleLCOlamin C-only alleleRTreverse transcriptionBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolAAaliphatic amino acid. (1Muchir A. Worman H.J. Physiology (Bethesda). 2004; 19: 309-314Crossref PubMed Scopus (51) Google Scholar, 2Burke B. Stewart C.L. Nat. Rev. Mol. Cell Biol. 2002; 3: 575-585Crossref PubMed Scopus (356) Google Scholar, 3Worman H.J. Bonne G. Exp. Cell Res. 2007; 313: 2121-2133Crossref PubMed Scopus (492) Google Scholar). LMNA yields two principal proteins, prelamin A and lamin C (4Lin F. Worman H.J. J. Biol. Chem. 1993; 268: 16321-16326Abstract Full Text PDF PubMed Google Scholar). Prelamin A terminates with a CAAX motif (i.e. CSIM), which triggers farnesylation and methylation of a carboxyl-terminal cysteine (5Beck L.A. Hosick T.J. Sinensky M. J. Cell Biol. 1990; 110: 1489-1499Crossref PubMed Scopus (172) Google Scholar, 6Young S.G. Fong L.G. Michaelis S. J. Lipid Res. 2005; 46: 2531-2558Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Following these modifications, the carboxyl-terminal portion of prelamin A (including the farnesylcysteine methyl ester) is clipped off by ZMPSTE24 and degraded, releasing mature lamin A (6Young S.G. Fong L.G. Michaelis S. J. Lipid Res. 2005; 46: 2531-2558Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 7Corrigan D.P. Kuszczak D. Rusinol A.E. Thewke D.P. Hrycyna C.A. Michaelis S. Sinensky M.S. Biochem. J. 2005; 387: 129-138Crossref PubMed Scopus (146) Google Scholar). Lamin C does not contain a CAAX motif and is not farnesylated. Lamins A and C are important proteins of the nuclear lamina, a filamentous meshwork that lines the inner nuclear membrane (1Muchir A. Worman H.J. Physiology (Bethesda). 2004; 19: 309-314Crossref PubMed Scopus (51) Google Scholar, 2Burke B. Stewart C.L. Nat. Rev. Mol. Cell Biol. 2002; 3: 575-585Crossref PubMed Scopus (356) Google Scholar, 3Worman H.J. Bonne G. Exp. Cell Res. 2007; 313: 2121-2133Crossref PubMed Scopus (492) Google Scholar). In the mouse, a complete absence of lamin A and lamin C leads to muscular dystrophy and death by 6 weeks of age (8Sullivan T. Escalante-Alcalde D. Bhatt H. Anver M. Bhat N. Nagashima K. Stewart C.L. Burke B. J. Cell Biol. 1999; 147: 913-919Crossref PubMed Scopus (971) Google Scholar). However, prelamin A and lamin A appear to be dispensable, as mice that are homozygous for a "lamin C-only" allele (LmnaLCO/LCO) are healthy and robust (9Fong L.G. Ng J.K. Lammerding J. Vickers T.A. Meta M. Cote N. Gavino B. Qiao X. Chang S.Y. Young S.R. Yang S.H. Stewart C.L. Lee R.T. Bennett C.F. Bergo M.O. Young S.G. J. Clin. Investig. 2006; 116: 743-752Crossref PubMed Scopus (195) Google Scholar). Hutchinson-Gilford progeria syndrome Hutchinson-Gilford allele lamin C-only allele reverse transcription 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol aliphatic amino acid. Hutchinson-Gilford progeria syndrome is a rare pediatric progeroid syndrome associated with the production of a mutant form of prelamin A (6Young S.G. Fong L.G. Michaelis S. J. Lipid Res. 2005; 46: 2531-2558Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 10Eriksson M. Brown W.T. Gordon L.B. Glynn M.W. Singer J. Scott L. Erdos M.R. Robbins C.M. Moses T.Y. Berglund P. Dutra A. Pak E. Durkin S. Csoka A.B. Boehnke M. Glover T.W. Collins F.S. Nature. 2003; 423: 293-298Crossref PubMed Scopus (1633) Google Scholar, 11Sandre-Giovannoli de A. Bernard R. Cau P. Navarro C. Amiel J. Boccaccio I. Lyonnet S. Stewart C.L. Munnich A. Merrer Le M. Lévy N. Science. 2003; 300: 2055Crossref PubMed Scopus (1112) Google Scholar). Patients with HGPS appear normal at birth but soon develop multiple disease phenotypes resembling premature aging (12Debusk F.L. Burke B. Stewart C.L. J. Pediatr. 1972; 80: 697-724Abstract Full Text PDF PubMed Scopus (288) Google Scholar). HGPS is almost always caused by a point mutation in LMNA that alters pre-mRNA splicing, resulting in a mutant form of prelamin A, commonly called progerin, with a 50-amino acid internal deletion (10Eriksson M. Brown W.T. Gordon L.B. Glynn M.W. Singer J. Scott L. Erdos M.R. Robbins C.M. Moses T.Y. Berglund P. Dutra A. Pak E. Durkin S. Csoka A.B. Boehnke M. Glover T.W. Collins F.S. Nature. 2003; 423: 293-298Crossref PubMed Scopus (1633) Google Scholar, 11Sandre-Giovannoli de A. Bernard R. Cau P. Navarro C. Amiel J. Boccaccio I. Lyonnet S. Stewart C.L. Munnich A. Merrer Le M. Lévy N. Science. 2003; 300: 2055Crossref PubMed Scopus (1112) Google Scholar). Progerin retains the carboxyl-terminal CAAX motif and is farnesylated and methylated (13Dechat T. Shimi T. Adam S.A. Rusinol A.E. Andres D.A. Spielmann H.P. Sinensky M.S. Goldman R.D. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 4955-4960Crossref PubMed Scopus (214) Google Scholar). However, the 50-amino acid deletion eliminates the site for the ZMPSTE24-mediated cleavage reaction, abrogating further processing to mature lamin A. Progerin accumulates at the nuclear rim and impairs the integrity of the nuclear lamina, resulting in misshapen nuclei and multiple disease phenotypes (10Eriksson M. Brown W.T. Gordon L.B. Glynn M.W. Singer J. Scott L. Erdos M.R. Robbins C.M. Moses T.Y. Berglund P. Dutra A. Pak E. Durkin S. Csoka A.B. Boehnke M. Glover T.W. Collins F.S. Nature. 2003; 423: 293-298Crossref PubMed Scopus (1633) Google Scholar, 11Sandre-Giovannoli de A. Bernard R. Cau P. Navarro C. Amiel J. Boccaccio I. Lyonnet S. Stewart C.L. Munnich A. Merrer Le M. Lévy N. Science. 2003; 300: 2055Crossref PubMed Scopus (1112) Google Scholar). Importantly, rare LMNA mutations leading to particularly high levels of progerin synthesis are associated with particularly severe forms of progeria (14Moulson C.L. Fong L.G. Gardner J.M. Farber E.A. Go G. Passariello A. Grange D.K. Young S.G. Miner J.H. Hum. Mutat. 2007; 28: 882-889Crossref PubMed Scopus (89) Google Scholar). Yang et al. (15Yang S.H. Bergo M.O. Toth J.I. Qiao X. Hu Y. Sandoval S. Meta M. Bendale P. Gelb M.H. Young S.G. Fong L.G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10291-10296Crossref PubMed Scopus (237) Google Scholar) created a gene-targeted Hutchinson-Gilford allele, LmnaHG, which yields exclusively progerin. Mouse fibroblasts heterozygous for the mutant allele (LmnaHG/+) express large amounts of progerin and have an increased frequency of misshapen nuclei (15Yang S.H. Bergo M.O. Toth J.I. Qiao X. Hu Y. Sandoval S. Meta M. Bendale P. Gelb M.H. Young S.G. Fong L.G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10291-10296Crossref PubMed Scopus (237) Google Scholar). LmnaHG/+ mice appear normal at birth, but by 6-8 weeks of age they manifest hallmarks of progeria (slow growth, weight loss, loss of adipose tissue, and osteolytic lesions in ribs and other bones) (16Yang S.H. Meta M. Qiao X. Frost D. Bauch J. Coffinier C. Majumdar S. Bergo M.O. Young S.G. Fong L.G. J. Clin. Investig. 2006; 116: 2115-2121Crossref PubMed Scopus (231) Google Scholar). Osteolytic lesions in the ribs result in numerous spontaneous fractures. Most LmnaHG/+ mice die by ∼28 weeks of age. Mice with a double dose of progerin (LmnaHG/HG) are severely affected and die by 3 weeks of age (16Yang S.H. Meta M. Qiao X. Frost D. Bauch J. Coffinier C. Majumdar S. Bergo M.O. Young S.G. Fong L.G. J. Clin. Investig. 2006; 116: 2115-2121Crossref PubMed Scopus (231) Google Scholar). Genetic studies in both humans and mice have shown that higher levels of progerin are associated with more severe disease phenotypes (14Moulson C.L. Fong L.G. Gardner J.M. Farber E.A. Go G. Passariello A. Grange D.K. Young S.G. Miner J.H. Hum. Mutat. 2007; 28: 882-889Crossref PubMed Scopus (89) Google Scholar, 16Yang S.H. Meta M. Qiao X. Frost D. Bauch J. Coffinier C. Majumdar S. Bergo M.O. Young S.G. Fong L.G. J. Clin. Investig. 2006; 116: 2115-2121Crossref PubMed Scopus (231) Google Scholar). Thus far, however, no one has investigated whether alterations in the composition of the nuclear lamina could affect the severity of disease. Progerin is targeted to the nuclear rim and directly interacts with other nuclear lamins (17Delbarre E. Tramier M. Coppey-Moisan M. Gaillard C. Courvalin J.C. Buendia B. Hum. Mol. Genet. 2006; 15: 1113-1122Crossref PubMed Scopus (102) Google Scholar); hence, it seemed possible that changes in lamin composition might influence disease phenotypes elicited by the mutant allele. In this study, we hypothesized that compositional changes in the nuclear lamina might modulate disease phenotypes in mice harboring the LmnaHG allele. To explore this hypothesis, we took advantage of the existence of a lamin C-only allele, LmnaLCO, which produces lamin C but no lamin A. In our studies, we compared the severity of disease phenotypes in LmnaHG/LCO mice (which produce progerin and lamin C) and littermate LmnaHG/+ mice (which produce mature lamin A, lamin C, and progerin). Our data indicate that the severity of disease phenotypes elicited by the LmnaHG allele is diminished in the setting of the LmnaLCO allele (i.e. in the setting of a nuclear lamina devoid of mature lamin A). Genetically Modified Mice—Male chimeric mice, generated by injecting C57BL/6 blastocysts with LmnaHG/+ embryonic stem cells (9Fong L.G. Ng J.K. Lammerding J. Vickers T.A. Meta M. Cote N. Gavino B. Qiao X. Chang S.Y. Young S.R. Yang S.H. Stewart C.L. Lee R.T. Bennett C.F. Bergo M.O. Young S.G. J. Clin. Investig. 2006; 116: 743-752Crossref PubMed Scopus (195) Google Scholar, 15Yang S.H. Bergo M.O. Toth J.I. Qiao X. Hu Y. Sandoval S. Meta M. Bendale P. Gelb M.H. Young S.G. Fong L.G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10291-10296Crossref PubMed Scopus (237) Google Scholar), were bred with LmnaLCO/+ female mice (9Fong L.G. Ng J.K. Lammerding J. Vickers T.A. Meta M. Cote N. Gavino B. Qiao X. Chang S.Y. Young S.R. Yang S.H. Stewart C.L. Lee R.T. Bennett C.F. Bergo M.O. Young S.G. J. Clin. Investig. 2006; 116: 743-752Crossref PubMed Scopus (195) Google Scholar, 15Yang S.H. Bergo M.O. Toth J.I. Qiao X. Hu Y. Sandoval S. Meta M. Bendale P. Gelb M.H. Young S.G. Fong L.G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10291-10296Crossref PubMed Scopus (237) Google Scholar), and Lmna+/+, LmnaHG/+ (n = 16), and LmnaHG/LCO (n = 26) offspring were analyzed. Genotyping was performed by PCR (9Fong L.G. Ng J.K. Lammerding J. Vickers T.A. Meta M. Cote N. Gavino B. Qiao X. Chang S.Y. Young S.R. Yang S.H. Stewart C.L. Lee R.T. Bennett C.F. Bergo M.O. Young S.G. J. Clin. Investig. 2006; 116: 743-752Crossref PubMed Scopus (195) Google Scholar, 15Yang S.H. Bergo M.O. Toth J.I. Qiao X. Hu Y. Sandoval S. Meta M. Bendale P. Gelb M.H. Young S.G. Fong L.G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10291-10296Crossref PubMed Scopus (237) Google Scholar) and by Western blotting with an antibody against lamin A/C (sc-6215, Santa Cruz Biotechnology) (15Yang S.H. Bergo M.O. Toth J.I. Qiao X. Hu Y. Sandoval S. Meta M. Bendale P. Gelb M.H. Young S.G. Fong L.G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10291-10296Crossref PubMed Scopus (237) Google Scholar). Cell Culture, Protein Extraction, and Western Blotting—Primary mouse embryonic fibroblasts were prepared from embryonic day 13.5 mouse embryos (18Bergo M.O. Gavino B. Ross J. Schmidt W.K. Hong C. Kendall L.V. Mohr A. Meta M. Genant H. Jiang Y. Wisner E.R. Bruggen van N. Carano R.A.D. Michaelis S. Griffey S.M. Young S.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13049-13054Crossref PubMed Scopus (352) Google Scholar). To generate cell extracts for Western blots, early passage fibroblasts were washed with phosphate-buffered saline, and urea-soluble extracts were prepared as described (19Dalton M. Sinensky M. Methods Enzymol. 1995; 250: 134-148Crossref PubMed Scopus (17) Google Scholar). For tissue extraction, 150 mg of fresh mouse tissues were frozen with liquid nitrogen and ground into a powder with a mortar and pestle. The powder was then resuspended in 0.5 ml of urea solubilization buffer (19Dalton M. Sinensky M. Methods Enzymol. 1995; 250: 134-148Crossref PubMed Scopus (17) Google Scholar) and ground in a glass tissue grinder for an additional 2 min. Samples were then sonicated and centrifuged at 14,000 × g for 10 min. The supernatant fluid was collected and analyzed by Western blotting. The protein extracts were electrophoresed on 4-12% gradient polyacrylamide BisTris gels (Invitrogen); the size-fractionated proteins were then electrophoretically transferred to a sheet of nitrocellulose membrane for Western blotting. The antibody dilutions were 1:400 for anti-lamin A/C goat IgG (sc-6215, Santa Cruz Biotechnology), 1:1000 for anti-actin goat IgG, and 1:6000 for horseradish peroxidase-labeled anti-goat IgG (sc-2020, Santa Cruz Biotechnology). Antibody binding was detected with the ECL Plus chemiluminescence system (GE Healthcare) and exposure to x-ray film. Western blots were also analyzed with the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE). In those experiments, the nitrocellulose membrane was rinsed in phosphate-buffered saline for 5 min before incubation in Odyssey blocking buffer. The membrane was incubated with the primary antibodies in blocking solution for 1 h at room temperature. The antibody dilutions were 1:400 for anti-lamin A/C rabbit IgG (sc-20681, Santa Cruz Biotechnology, Santa Cruz, CA) and 1:1000 for anti-actin goat IgG (sc-1616, Santa Cruz Biotechnology). After washing the membrane three times for 10 min in phosphate-buffered saline containing 0.1% Tween 20, it was incubated with 1:5000 IRDye 700 anti-goat IgG antibody and 1:5000 IRDye 800 anti-rabbit IgG antibody (both from Rockland Immunochemicals) in Odyssey blocking buffer. After washing, the membranes were scanned on an Odyssey infrared imaging system, and images were acquired and analyzed according to the manufacturer's instructions. RNA Extraction, cDNA Synthesis, and RT-PCR—Total RNA was extracted from primary fibroblasts and mouse tissues with the RNeasy mini kit (Qiagen, Valencia, CA) and quantified with a NanoDrop Spectrophotometer (NanoDrop Technologies, Wilmington, DE). The integrity of the RNA was verified with an ethidium bromide-stained 1.5% agarose gel. Reverse transcriptase reactions were carried out with 1 μg of RNA that had been treated with DNase (DNA-Free, Ambion, Foster City, CA), Superscript III (Invitrogen), and a mixture of oligo(dT) (Invitrogen) and random primers (Invitrogen). The cDNA samples were incubated with RNase (RNase H, Ambion, Foster City, CA) and then diluted to 10 ng/μl for PCRs. The progerin and prelamin A cDNAs were amplified with primers corresponding to exon 10 and exon 12 sequences (forward, 5′-CTGACCATGGTTGAGGACAA-3′; reverse, 5′-TTAGGAGAGCAGGCCTGAAG-3′). Arbp, used as the internal control, was amplified with primers 5′-ACTGAGATTCGGGATATGCTGT-3′ and 5′-TCCTAGACCAGTGTTCTGAGCTG-3′. All primers were designed with Primer 3 software. The RT-PCR was performed with AmpliTaq (ABI Foster City, CA) and 50 ng of cDNA. The thermal cycling conditions were as follows: 95 °C for 3 min, 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 15 s, for a total of 26 cycles. The RT-PCR fragment spanning exon 10-12 was 456 bp in the wild-type Lmna allele and 306 bp in the LmnaHG allele. The RT-PCR fragment for Arbp was 108 bp. 10 μl of Arbp PCR products were mixed with 25 μl of lamin A and progerin PCR products and loaded onto a 1.3% agarose gel containing SYTO 60 nucleic acid stain (Invitrogen) (1:16,000 dilution). Images of the gels were acquired with the Odyssey infrared imaging system, and band intensity was quantified according to the manufacturer's instructions. Immunofluorescence Microscopy—Early passage fibroblasts (matched for passage number) were grown on coverslips, fixed with 3% paraformaldehyde, permeabilized with 0.2% Triton X-100, and blocked with bovine serum albumin (20Fong L.G. Ng J.K. Meta M. Cote N. Yang S.H. Stewart C.L. Sullivan T. Burghardt A. Majumdar S. Reue K. Bergo M.O. Young S.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 18111-18116Crossref PubMed Scopus (164) Google Scholar). Cells were incubated for 60 min with antibody against lamin A (1:200, sc-20680, Santa Cruz Biotechnology). After washing, cells were stained with anti-rabbit Cy3-conjugated secondary antibody (Jackson ImmunoResearch) and 4′,6-diamidino-2-phenylindole (to visualize DNA). Images were obtained on an Axiovert 200 MOT microscope (Carl Zeiss, Thornwood, NY) with a 63×/1.25 oil immersion objective and processed with Axo-Vision 4.2 software (Zeiss). The number of misshapen nuclei (exhibiting blebs or folds) was scored by three independent observers blinded to genotype. Differences in the percentages of misshapen nuclei between cells with different genotypes were assessed with a χ2 test. Mouse Phenotypes—Body weights of Lmna+/+, LmnaHG/+, and LmnaHG/LCO mice were monitored weekly. Body weight curves for different groups of mice were compared with repeated measures analysis of variance and the log-rank test. For survival analysis, Kaplan-Meier analyses were performed, and the log-rank test was used to compare the survival curves. At the time that each mouse died, the heart and lungs were removed, and the thorax was photographed with a digital camera, and the number of rib fractures was counted. The number of rib fractures in different groups of mice was compared with a two-tailed Student's t test. To determine whether the absence of lamin A synthesis would affect the disease phenotypes elicited by the LmnaHG allele, we compared the phenotypes of LmnaHG/LCO and littermate LmnaHG/+ mice. As expected, the LmnaHG/LCO mice synthesized lamin C and progerin, whereas LmnaHG/+ mice produced lamin A, lamin C, and progerin (Fig. 1). LmnaHG/LCO and LmnaHG/+ mice appeared similar at weaning. By 10-15 weeks of age, however, the body weight curves in LmnaHG/LCO mice were significantly better than those of LmnaHG/+ mice (p < 0.0001) (Fig. 2A). These differences were apparent in both males and females (p < 0.0001 for both groups). Kaplan-Meier survival curves revealed that LmnaHG/LCO mice lived longer than LmnaHG/+ mice (p < 0.0001) (Fig. 2B). Again, this difference was significant in both males and females (p < 0.0001). Also, the number of rib fractures was lower in LmnaHG/LCO mice than in LmnaHG/+ mice (p < 0.0001) (Fig. 2C), despite the fact that the LmnaHG/LCO mice were older at the time of death (27 ± 1.65 weeks versus 23 ± 0.70 weeks in LmnaHG/+ mice). A hallmark of LmnaHG/+ fibroblasts is misshapen cell nuclei. Given the less severe disease phenotypes in LmnaHG/LCO mice, we hypothesized that the frequency of misshapen nuclei in LmnaHG/LCO fibroblasts might be lower than in LmnaHG/+ fibroblasts. To test this possibility, we scored nuclear shape abnormalities in primary embryonic fibroblasts from LmnaHG/LCO (n = 5), LmnaHG/+ (n = 3), LmnaLCO/+ (n = 5), and Lmna+/+ (n = 2) mice (generated from two litters). The frequency of misshapen nuclei (i.e. nuclei with folds or blebs) was lower in LmnaHG/LCO fibroblasts than in LmnaHG/+ fibroblasts (p < 0.0001) (Fig. 3, A and B). The number of misshapen nuclei in LmnaLCO/+ mice was somewhat greater than in Lmna+/+ cells (p < 0.001), consistent with results of earlier studies (9Fong L.G. Ng J.K. Lammerding J. Vickers T.A. Meta M. Cote N. Gavino B. Qiao X. Chang S.Y. Young S.R. Yang S.H. Stewart C.L. Lee R.T. Bennett C.F. Bergo M.O. Young S.G. J. Clin. Investig. 2006; 116: 743-752Crossref PubMed Scopus (195) Google Scholar, 21Lammerding J. Fong L.G. Ji J.Y. Reue K. Stewart C.L. Young S.G. Lee R.T. J. Biol. Chem. 2006; 281: 25768-25780Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). To understand the mechanism for the less severe phenotypes in LmnaHG/LCO mice and LmnaHG/LCO fibroblasts, we performed Western blot analyses on mouse fibroblasts and tissues. We reasoned that the less severe phenotypes of LmnaHG/LCO mice and fibroblasts would make sense if the altered composition of the nuclear lamina in LmnaHG/LCO cells (i.e. complete absence of mature lamin A) resulted in a reduced steady-state level of progerin. Indeed, this proved to be the case. Western blots quantified with an Odyssey infrared imaging system revealed that the expression of progerin, relative to actin, was lower in LmnaHG/LCO fibroblasts (n = 8) than in LmnaHG/+ fibroblasts (n = 8) (p < 0.0001) (Fig. 4, A and B). The expression of lamin C, relative to actin, tended to be lower in LmnaHG/+ cells than in LmnaHG/LCO cells (p = 0.057). Similarly, the levels of progerin in the heart and skin of LmnaHG/LCO mice, relative to actin, were lower in LmnaHG/LCO mice than in LmnaHG/+ mice (Fig. 4, C-F). The differences in progerin expression levels could not be ascribed to different mRNA levels. Progerin mRNA levels, as judged by quantitative RT-PCR, were not different in LmnaHG/LCO (n = 8) and LmnaHG/+ fibroblasts (n = 8) (Fig. 5, A and C). Also, progerin mRNA levels in the heart and skin of LmnaHG/LCO and LmnaHG/+ mice were virtually identical (Fig. 5, B and C). Previous studies have established, quite unequivocally, that progerin is the culprit molecule in HGPS (22Glynn M.W. Glover T.W. Hum. Mol. Genet. 2005; 14: 2959-2969Crossref PubMed Scopus (196) Google Scholar, 23Goldman R.D. Shumaker D.K. Erdos M.R. Eriksson M. Goldman A.E. Gordon L.B. Gruenbaum Y. Khuon S. Mendez M. Varga R. Collins F.S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8963-8968Crossref PubMed Scopus (832) Google Scholar, 24Scaffidi P. Misteli T. Nat. Med. 2005; 11: 440-445Crossref PubMed Scopus (463) Google Scholar). Higher levels of progerin produce more severe nuclear shape abnormalities than lower levels of progerin (22Glynn M.W. Glover T.W. Hum. Mol. Genet. 2005; 14: 2959-2969Crossref PubMed Scopus (196) Google Scholar, 23Goldman R.D. Shumaker D.K. Erdos M.R. Eriksson M. Goldman A.E. Gordon L.B. Gruenbaum Y. Khuon S. Mendez M. Varga R. Collins F.S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8963-8968Crossref PubMed Scopus (832) Google Scholar). Also, LMNA mutations that yield particularly high levels of progerin result in particularly severe forms of progeria (14Moulson C.L. Fong L.G. Gardner J.M. Farber E.A. Go G. Passariello A. Grange D.K. Young S.G. Miner J.H. Hum. Mutat. 2007; 28: 882-889Crossref PubMed Scopus (89) Google Scholar). In this study, we studied an entirely new issue, whether changes in the composition of the nuclear lamina might modulate the phenotypes associated with the synthesis of a fixed amount of progerin. To explore this issue, we compared disease phenotypes in LmnaHG/+ and LmnaHG/LCO mice. The wild-type allele in LmnaHG/+ mice produces both lamin A and lamin C, whereas the LmnaLCO allele in LmnaHG/LCO mice produces exclusively lamin C (9Fong L.G. Ng J.K. Lammerding J. Vickers T.A. Meta M. Cote N. Gavino B. Qiao X. Chang S.Y. Young S.R. Yang S.H. Stewart C.L. Lee R.T. Bennett C.F. Bergo M.O. Young S.G. J. Clin. Investig. 2006; 116: 743-752Crossref PubMed Scopus (195) Google Scholar). The disease phenotypes in LmnaHG/LCO mice were milder than those in LmnaHG/+ mice; the LmnaHG/LCO mice exhibited significantly improved body weight curves, improved survival, and fewer rib fractures. In addition, LmnaHG/LCO fibroblasts had fewer misshapen nuclei. The differences between LmnaHG/LCO and LmnaHG/+ mice were reproducible and highly significant, but the mechanism was not immediately obvious. However, we reasoned that the most parsimonious explanation for these phenotypic differences would be altered steady-state levels of progerin. Indeed, this was the case; progerin levels in LmnaHG/LCO fibroblasts and tissues were significantly lower than those in LmnaHG/+ mice. Thus, changing the protein composition of the nuclear lamina, by replacing a wild-type Lmna allele with a lamin C-only allele, lowered steady-state levels of progerin and reduced disease phenotypes elicited by the LmnaHG allele. We gained insight into the probable mechanisms for the reduced steady-state levels of progerin in LmnaHG/LCO mice. The quantitative RT-PCR experiments revealed equal amounts of progerin transcripts in LmnaHG/+ and LmnaHG/LCO cells and tissues, so the reduced levels of progerin protein in LmnaHG/LCO cells and tissues cannot be ascribed to changes in progerin transcript levels. It is a formal possibility that the lower progerin levels in LmnaHG/LCO cells and tissues are because of reduced translation of the LmnaHG transcript. However, we believe that it is more likely that the altered composition of the nuclear lamina in LmnaHG/LCO cells and tissues (i.e. a complete absence of mature lamin A) is responsible for the alterations in progerin levels. We suspect that the cellular machinery responsible for the turnover of progerin could be more efficient when that protein is surrounded by lamin C than when it is surrounded by a mixture of lamin A and lamin C. Part of this may relate to the abilities of lamin A, lamin C, and progerin to interact with each other. More than 15 years after a molecular understanding of lamin A and lamin C biogenesis (4Lin F. Worman H.J. J. Biol. Chem. 1993; 268: 16321-16326Abstract Full Text PDF PubMed Google Scholar), no one has conclusively determined whether lamins A and C form heterodimers in the cell or exist solely in homodimers. Recently, however, Delbarre et al. (17Delbarre E. Tramier M. Coppey-Moisan M. Gaillard C. Courvalin J.C. Buendia B. Hum. Mol. Genet. 2006; 15: 1113-1122Crossref PubMed Scopus (102) Google Scholar) examined the polymerization of green fluorescent protein-tagged and red fluorescent protein-tagged wild-type and mutated lamins in the nuclear envelope by measuring fluorescence resonance energy transfer. They concluded that both wild-type lamin A and lamin B1 form homopolymers that interact further in the nuclear lamina. In contrast, their studies suggested that progerin co-assembles with both lamin B1 and lamin A to form a heteropolymer, so that the usual segregation of A-type and B-type lamins is lost (17Delbarre E. Tramier M. Coppey-Moisan M. Gaillard C. Courvalin J.C. Buendia B. Hum. Mol. Genet. 2006; 15: 1113-1122Crossref PubMed Scopus (102) Google Scholar). Assuming that these fluorescence resonance energy transfer studies with green fluorescent protein-tagged and red fluorescent protein-tagged lamins accurately portray the ability of native untagged progerin to interact with other lamin proteins, it is not at all farfetched to imagine that changes in the composition of the lamina could influence the steady-state levels of progerin in the cell. The phenotypic differences between LmnaHG/+ and LmnaHG/LCO mice were unequivocal and highly significant, but we emphasize that the reduction in disease phenotypes in LmnaHG/LCO mice was only partial. The LmnaHG/LCO mice still had markedly abnormal body weight curves, still developed spontaneous bone fractures, and still died prematurely. A similar situation was observed in LmnaHG/+ mice that had been treated with a farnesyltransferase inhibitor. Treatment of LmnaHG/+ mice with a farnesyltransferase inhibitor significantly improved disease phenotypes but that treatment fell far short of a complete cure (15Yang S.H. Bergo M.O. Toth J.I. Qiao X. Hu Y. Sandoval S. Meta M. Bendale P. Gelb M.H. Young S.G. Fong L.G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10291-10296Crossref PubMed Scopus (237) Google Scholar, 25Fong L. Frost D. Meta M. Qiao X. Yang S. Coffinier C. Young S. Science. 2006; 311: 1621-1623Crossref PubMed Scopus (257) Google Scholar, 26Meta M. Yang S.H. Bergo M.O. Fong L.G. Young S.G. Trends Mol. Med. 2006; 12: 480-487Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). This study shows that the absence of mature lamin A affects steady-state progerin levels and progerin-induced disease phenotypes. These studies lay the foundation for a new concept that compositional changes in the nuclear lamina have secondary effects on progerin levels and progeria disease phenotypes. This concept can now be explored in more detail. For example, it would be intriguing to determine whether transgenic overexpression of lamins A and C (27Varga R. Eriksson M. Erdos M.R. Olive M. Harten I. Kolodgie F. Capell B.C. Cheng J. Faddah D. Perkins S. Avallone H. San H. Qu X. Ganesh S. Gordon L.B. Virmani R. Wight T.N. Nabel E.G. Collins F.S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3250-3255Crossref PubMed Scopus (221) Google Scholar) would affect the severity of disease. Similarly, it would be interesting to determine whether a lamin B1 knock-out allele (28Vergnes L. Peterfy M. Bergo M.O. Young S.G. Reue K. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10428-10433Crossref PubMed Scopus (306) Google Scholar) would influence the severity of progeria. Ultimately, these genetic studies could yield clues regarding the functional relevance of different lamin proteins in the pathogenesis of progeria.

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