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Increasing the length of progerin's isoprenyl anchor does not worsen bone disease or survival in mice with Hutchinson-Gilford progeria syndrome

2008; Elsevier BV; Volume: 50; Issue: 1 Linguagem: Inglês

10.1194/jlr.m800424-jlr200

1539-7262

Brandon S.J. Davies, Shao H. Yang, Emily Farber, Roger Lee, Suzanne B. Buck, Douglas Andres, H. Peter Spielmann, Brian Agnew, Fuyuhiko Tamanoi, Loren G. Fong, Stephen G. Young,

Retinoids in leukemia and cellular processes

Hutchinson-Gilford progeria syndrome (HGPS) is caused by the synthesis of a truncated prelamin A, commonly called progerin, that contains a carboxyl-terminal farnesyl lipid anchor. The farnesyl lipid anchor helps to target progerin to membrane surfaces at the nuclear rim, where it disrupts the integrity of the nuclear lamina and causes misshapen nuclei. Several lines of evidence have suggested that progerin's farnesyl lipid anchor is crucial for the emergence of disease phenotypes. Because a geranylgeranyl lipid is ∼45-fold more potent than a farnesyl lipid in anchoring proteins to lipid membranes, we hypothesized that a geranylgeranylated version of progerin might be more potent in eliciting disease phenotypes. To test this hypothesis, we used gene targeting to create mice expressing geranylgeranylated progerin (LmnaggHG/+). We then compared LmnaggHG/+ mice, side-by-side, with otherwise identical mice expressing farnesylated progerin (LmnaHG/+). Geranylgeranylation of progerin in LmnaggHG/+ cells and farnesylation of progerin in LmnaHG/+ cells was confirmed by metabolic labeling. Contrary to our expectations, LmnaggHG/+ mice survived longer than LmnaHG/+ mice. The LmnaggHG/+ mice also exhibited milder bone disease. The steady-state levels of progerin, relative to lamin C, were lower in LmnaggHG/+ mice than in LmnaHG/+ mice, providing a potential explanation for the milder disease in LmnaggHG/+ mice. Hutchinson-Gilford progeria syndrome (HGPS) is caused by the synthesis of a truncated prelamin A, commonly called progerin, that contains a carboxyl-terminal farnesyl lipid anchor. The farnesyl lipid anchor helps to target progerin to membrane surfaces at the nuclear rim, where it disrupts the integrity of the nuclear lamina and causes misshapen nuclei. Several lines of evidence have suggested that progerin's farnesyl lipid anchor is crucial for the emergence of disease phenotypes. Because a geranylgeranyl lipid is ∼45-fold more potent than a farnesyl lipid in anchoring proteins to lipid membranes, we hypothesized that a geranylgeranylated version of progerin might be more potent in eliciting disease phenotypes. To test this hypothesis, we used gene targeting to create mice expressing geranylgeranylated progerin (LmnaggHG/+). We then compared LmnaggHG/+ mice, side-by-side, with otherwise identical mice expressing farnesylated progerin (LmnaHG/+). Geranylgeranylation of progerin in LmnaggHG/+ cells and farnesylation of progerin in LmnaHG/+ cells was confirmed by metabolic labeling. Contrary to our expectations, LmnaggHG/+ mice survived longer than LmnaHG/+ mice. The LmnaggHG/+ mice also exhibited milder bone disease. The steady-state levels of progerin, relative to lamin C, were lower in LmnaggHG/+ mice than in LmnaHG/+ mice, providing a potential explanation for the milder disease in LmnaggHG/+ mice. Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disease characterized by a number of aging-like disease phenotypes (e.g., osteoporosis, loss of hair, reduced adipose tissue, occlusive vascular disease, and thin skin with a wizened appearance) (1Debusk F.L The Hutchinson-Gilford progeria syndrome..J. Pediatr. 1972; 80: 697-724Abstract Full Text PDF PubMed Scopus (288) Google Scholar, 2Merideth M.A Gordon L.B. Clauss S. Sachdev V. Smith A.C. Perry M.B. Brewer C.C. Zalewski C. Kim H.J. Solomon B. et al.Phenotype and course of Hutchinson-Gilford progeria syndrome..N. Engl. J. Med. 2008; 358: 592-604Crossref PubMed Scopus (484) Google Scholar). HGPS is caused by point mutations in LMNA, the gene encoding prelamin A and lamin C (3Eriksson 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. et al.Recurrent de novo point mutations in lamin A cause Hutchinson–Gilford progeria syndrome..Nature. 2003; 423: 293-298Crossref PubMed Scopus (1632) Google Scholar). These point mutations alter splicing of the prelamin A pre-mRNA and lead to the production of a mutant prelamin A protein, commonly called progerin, containing an internal 50-amino-acid deletion (3Eriksson 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. et al.Recurrent de novo point mutations in lamin A cause Hutchinson–Gilford progeria syndrome..Nature. 2003; 423: 293-298Crossref PubMed Scopus (1632) Google Scholar). Progerin is targeted to the cell nucleus by a nuclear localization signal (4Lin F. Worman H.J. Structural organization of the human gene encoding nuclear lamin A and nuclear lamin C..J. Biol. Chem. 1993; 268: 16321-16326Abstract Full Text PDF PubMed Google Scholar), and a significant amount of progerin is located at the rim of the nucleus adjacent to the inner nuclear membrane (5Yang S.H Bergo M.O. Toth J.I. Qiao X. Hu Y. Sandoval S. Meta M. Bendale P. Gelb M.H. Young S.G. et al.Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts with a targeted Hutchinson-Gilford progeria syndrome mutation..Proc. Natl. Acad. Sci. USA. 2005; 102: 10291-10296Crossref PubMed Scopus (237) Google Scholar, 6Goldman R.D Shumaker D.K. Erdos M.R. Eriksson M. Goldman A.E. Gordon L.B. Gruenbaum Y. Khuon S. Mendez M. Varga R. et al.Accumulation of mutant lamin A causes progressive changes in nuclear architecture in Hutchinson-Gilford progeria syndrome..Proc. Natl. Acad. Sci. USA. 2004; 101: 8963-8968Crossref PubMed Scopus (832) Google Scholar). Progerin leads to dramatically misshapen cell nuclei (3Eriksson 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. et al.Recurrent de novo point mutations in lamin A cause Hutchinson–Gilford progeria syndrome..Nature. 2003; 423: 293-298Crossref PubMed Scopus (1632) Google Scholar, 5Yang S.H Bergo M.O. Toth J.I. Qiao X. Hu Y. Sandoval S. Meta M. Bendale P. Gelb M.H. Young S.G. et al.Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts with a targeted Hutchinson-Gilford progeria syndrome mutation..Proc. Natl. Acad. Sci. USA. 2005; 102: 10291-10296Crossref PubMed Scopus (237) Google Scholar) and is responsible for all of the aging-like disease phenotypes in HGPS (3Eriksson 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. et al.Recurrent de novo point mutations in lamin A cause Hutchinson–Gilford progeria syndrome..Nature. 2003; 423: 293-298Crossref PubMed Scopus (1632) Google Scholar, 7Yang S.H Meta M. Qiao X. Frost D. Bauch J. Coffinier C. Majumdar S. Bergo M.O. Young S.G. Fong L.G. Treatment with a protein farnesyltransferase inhibitor improves disease phenotypes in mice with a targeted Hutchinson-Gilford progeria syndrome mutation..J. Clin. Invest. 2006; 116: 2115-2121Crossref PubMed Scopus (231) Google Scholar). Like wild-type prelamin A, progerin contains a carboxyl-terminal CaaX motif, which triggers farnesylation of the carboxyl-terminal cysteine (the “C” of the CaaX motif) (3Eriksson 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. et al.Recurrent de novo point mutations in lamin A cause Hutchinson–Gilford progeria syndrome..Nature. 2003; 423: 293-298Crossref PubMed Scopus (1632) Google Scholar). After endoproteolytic release of the last three amino acids of the protein (i.e., the aaX of the CaaX motif), the carboxyl-terminal farnesylcysteine is methylated (8Young S.G Fong L.G. Michaelis S. Prelamin A, Zmpste24, misshapen cell nuclei, and progeria—New evidence suggesting that protein farnesylation could be important for disease pathogenesis..J. Lipid Res. 2005; 46: 2531-2558Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 9Dechat T. Shimi T. Adam S.A. Rusinol A.E. Andres D.A. Spielmann H.P. Sinensky M.S. Goldman R.D. Alterations in mitosis and cell cycle progression caused by a mutant lamin A known to accelerate human aging..Proc. Natl. Acad. Sci. USA. 2007; 104: 4955-4960Crossref PubMed Scopus (214) Google Scholar). Progerin lacks the sequences required for the ZMPSTE24-mediated processing step that would ordinarily clip off the farnesylated and methylated cysteine and release mature lamin A (8Young S.G Fong L.G. Michaelis S. Prelamin A, Zmpste24, misshapen cell nuclei, and progeria—New evidence suggesting that protein farnesylation could be important for disease pathogenesis..J. Lipid Res. 2005; 46: 2531-2558Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Thus, progerin terminates with a farnesylcysteine α-methyl ester (9Dechat T. Shimi T. Adam S.A. Rusinol A.E. Andres D.A. Spielmann H.P. Sinensky M.S. Goldman R.D. Alterations in mitosis and cell cycle progression caused by a mutant lamin A known to accelerate human aging..Proc. Natl. Acad. Sci. USA. 2007; 104: 4955-4960Crossref PubMed Scopus (214) Google Scholar). Progerin's carboxyl-terminal lipid anchor is thought to be important for targeting the protein to the inner nuclear membrane (5Yang S.H Bergo M.O. Toth J.I. Qiao X. Hu Y. Sandoval S. Meta M. Bendale P. Gelb M.H. Young S.G. et al.Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts with a targeted Hutchinson-Gilford progeria syndrome mutation..Proc. Natl. Acad. Sci. USA. 2005; 102: 10291-10296Crossref PubMed Scopus (237) Google Scholar). When the farnesylation of progerin is blocked with a protein farnesyltransferase inhibitor (FTI), progerin is mislocalized away from the nuclear rim and the frequency of misshapen nuclei is reduced (5Yang S.H Bergo M.O. Toth J.I. Qiao X. Hu Y. Sandoval S. Meta M. Bendale P. Gelb M.H. Young S.G. et al.Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts with a targeted Hutchinson-Gilford progeria syndrome mutation..Proc. Natl. Acad. Sci. USA. 2005; 102: 10291-10296Crossref PubMed Scopus (237) Google Scholar). An FTI also ameliorates disease phenotypes in mouse models of progeria (7Yang S.H Meta M. Qiao X. Frost D. Bauch J. Coffinier C. Majumdar S. Bergo M.O. Young S.G. Fong L.G. Treatment with a protein farnesyltransferase inhibitor improves disease phenotypes in mice with a targeted Hutchinson-Gilford progeria syndrome mutation..J. Clin. Invest. 2006; 116: 2115-2121Crossref PubMed Scopus (231) Google Scholar, 10Fong L. Frost D. Meta M. Qiao X. Yang S. Coffinier C. Young S. A protein farnesyltransferase inhibitor ameliorates disease in a mouse model of progeria..Science. 2006; 311: 1621-1623Crossref PubMed Scopus (257) Google Scholar, 11Yang S.H Qiao X. Fong L.G. Young S.G. Treatment with a farnesyltransferase inhibitor improves survival in mice with a Hutchinson-Gilford progeria syndrome mutation..Biochim. Biophys. Acta. 2008; 1781: 36-39Crossref PubMed Scopus (65) Google Scholar). The fact that a farnesyl group would help to anchor progerin to the nuclear membrane is not surprising. In in vitro lipid bilayer studies, farnesylation of synthetic peptides increases their partitioning to the surface of phospholipids vesicles, proportionately reducing their concentration in the aqueous phase (12Silvius J.R l'Heureux F. Fluorimetric evaluation of the affinities of isoprenylated peptides for lipid bilayers..Biochemistry. 1994; 33: 3014-3022Crossref PubMed Scopus (240) Google Scholar). On the other hand, it is important to emphasize that a farnesyl lipid is not a particularly strong membrane anchor. An n-alkyl chain with only 11 carbons is as effective as the 15-carbon branch-chain farnesyl group in promoting membrane binding (12Silvius J.R l'Heureux F. Fluorimetric evaluation of the affinities of isoprenylated peptides for lipid bilayers..Biochemistry. 1994; 33: 3014-3022Crossref PubMed Scopus (240) Google Scholar). Also, a farnesyl lipid is only ∼2% as potent as a 20-carbon geranylgeranyl lipid in anchoring peptides to lipid membranes (12Silvius J.R l'Heureux F. Fluorimetric evaluation of the affinities of isoprenylated peptides for lipid bilayers..Biochemistry. 1994; 33: 3014-3022Crossref PubMed Scopus (240) Google Scholar). The latter differences persist when the isoprenylcysteine is methylated (12Silvius J.R l'Heureux F. Fluorimetric evaluation of the affinities of isoprenylated peptides for lipid bilayers..Biochemistry. 1994; 33: 3014-3022Crossref PubMed Scopus (240) Google Scholar). Because the targeting of progerin to the nuclear rim is thought to be important in eliciting disease phenotypes in vivo and because a geranylgeranyl lipid is considerably stronger than a farnesyl group in promoting membrane binding, we hypothesized that a geranylgeranylated version of progerin would elicit more severe disease phenotypes than a farnesylated progerin. There is a straightforward path to examine this hypothesis. Gene-targeting approaches can be used to create mice expressing progerin (5Yang S.H Bergo M.O. Toth J.I. Qiao X. Hu Y. Sandoval S. Meta M. Bendale P. Gelb M.H. Young S.G. et al.Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts with a targeted Hutchinson-Gilford progeria syndrome mutation..Proc. Natl. Acad. Sci. USA. 2005; 102: 10291-10296Crossref PubMed Scopus (237) Google Scholar, 7Yang S.H Meta M. Qiao X. Frost D. Bauch J. Coffinier C. Majumdar S. Bergo M.O. Young S.G. Fong L.G. Treatment with a protein farnesyltransferase inhibitor improves disease phenotypes in mice with a targeted Hutchinson-Gilford progeria syndrome mutation..J. Clin. Invest. 2006; 116: 2115-2121Crossref PubMed Scopus (231) Google Scholar), and it is possible to trigger protein geranylgeranylation by altering the last amino acid of progerin's CaaX motif (13Zhang F.L Casey P.J. Protein prenylation: molecular mechanisms and functional consequences..Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1743) Google Scholar). In this study, we produced knock-in mice that expressed either a farnesylated or a geranylgeranylated version of progerin. Except for the length of the isoprenyl lipid anchor on the carboxyl-terminal cysteine of progerin, the two groups of mice were identical. We characterized both groups of mice, side-by-side, for severity of disease phenotypes. To create a mutant Lmna allele yielding a geranylgeranylated progerin (LmnaggHG), we used a gene-targeting vector identical to one used previously to create a mutant allele yielding farnesylated progerin (LmnaHG) (5Yang S.H Bergo M.O. Toth J.I. Qiao X. Hu Y. Sandoval S. Meta M. Bendale P. Gelb M.H. Young S.G. et al.Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts with a targeted Hutchinson-Gilford progeria syndrome mutation..Proc. Natl. Acad. Sci. USA. 2005; 102: 10291-10296Crossref PubMed Scopus (237) Google Scholar), except for a point mutation in exon 12 (in the 5′ arm of the vector) that changes the methionine of progerin's CaaX motif to a leucine (i.e., CSIM to CSIL). The mutation was introduced into the 5′-arm of the vector by site-directed mutagenesis with the QuikChange kit (Stratagene, La Jolla, CA) and primer 5′-CTCCCAGAACTGCAGCATTCTGTAATCTGGGACCTGCCAGG -3′ (and the complementary reverse primer). The integrity of the gene-targeting vector was verified by restriction endonuclease digestion and DNA sequencing (5Yang S.H Bergo M.O. Toth J.I. Qiao X. Hu Y. Sandoval S. Meta M. Bendale P. Gelb M.H. Young S.G. et al.Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts with a targeted Hutchinson-Gilford progeria syndrome mutation..Proc. Natl. Acad. Sci. USA. 2005; 102: 10291-10296Crossref PubMed Scopus (237) Google Scholar). After electroporating 129/OlaHsd embryonic stem cells with the gene-targeting vector, targeted clones were identified by Southern blotting with EcoRI-cleaved genomic DNA and a 348-bp 5′-flanking probe (5Yang S.H Bergo M.O. Toth J.I. Qiao X. Hu Y. Sandoval S. Meta M. Bendale P. Gelb M.H. Young S.G. et al.Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts with a targeted Hutchinson-Gilford progeria syndrome mutation..Proc. Natl. Acad. Sci. USA. 2005; 102: 10291-10296Crossref PubMed Scopus (237) Google Scholar). Targeted ES cells were microinjected into C57BL/6 blastocysts, and the resulting chimeras were bred with C57BL/6 females to generate LmnaggHG/+ mice. In parallel, LmnaHG/+ ES cells (5Yang S.H Bergo M.O. Toth J.I. Qiao X. Hu Y. Sandoval S. Meta M. Bendale P. Gelb M.H. Young S.G. et al.Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts with a targeted Hutchinson-Gilford progeria syndrome mutation..Proc. Natl. Acad. Sci. USA. 2005; 102: 10291-10296Crossref PubMed Scopus (237) Google Scholar, 7Yang S.H Meta M. Qiao X. Frost D. Bauch J. Coffinier C. Majumdar S. Bergo M.O. Young S.G. Fong L.G. Treatment with a protein farnesyltransferase inhibitor improves disease phenotypes in mice with a targeted Hutchinson-Gilford progeria syndrome mutation..J. Clin. Invest. 2006; 116: 2115-2121Crossref PubMed Scopus (231) Google Scholar) were used to generate chimeras, which were bred to generate LmnaHG/+ mice. Because LmnaggHG/+ and LmnaHG/+ mice were generated from chimeras, they were identical (with one C57BL/6 chromosome and one 129/OlaHsd chromosome), except for the single-nucleotide difference in exon 12 of Lmna. Genotyping of mice was performed by PCR with genomic DNA from tail biopsies (5Yang S.H Bergo M.O. Toth J.I. Qiao X. Hu Y. Sandoval S. Meta M. Bendale P. Gelb M.H. Young S.G. et al.Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts with a targeted Hutchinson-Gilford progeria syndrome mutation..Proc. Natl. Acad. Sci. USA. 2005; 102: 10291-10296Crossref PubMed Scopus (237) Google Scholar). The mice were fed a chow diet and housed in a virus-free barrier facility with a 12-h light-dark cycle. Body weights were measured weekly. All mouse experiments were approved by UCLA's Animal Research Committee. Primary mouse embryonic fibroblasts (MEFs) were prepared from E13.5 embryos (14Bergo M.O Gavino B. Ross J. Schmidt W.K. Hong C. Kendall L.V. Mohr A. Meta M. Genant H. Jiang Y. et al.Zmpste24 deficiency in mice causes spontaneous bone fractures, muscle weakness, and a prelamin A processing defect..Proc. Natl. Acad. Sci. USA. 2002; 99: 13049-13054Crossref PubMed Scopus (352) Google Scholar), plated in 6-well plates, and grown to 75% confluency. To assess nuclear shape abnormalities, early-passage MEFs were grown on coverslips and then fixed and permeabilized as described previously (15Fong L.G Ng J.K. Meta M. Cote N. Yang S.H. Stewart C.L. Sullivan T. Burghardt A. Majumdar S. Reue K. et al.Heterozygosity for Lmna deficiency eliminates the progeria-like phenotypes in Zmpste24-deficient mice..Proc. Natl. Acad. Sci. USA. 2004; 101: 18111-18116Crossref PubMed Scopus (164) Google Scholar). Cells were incubated with antibodies against lamin A (1:200, sc-20680, Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h. After washing, cells were incubated with Alexa Fluor 568-conjugated anti-rabbit antibody (1:800, Jackson ImmunoResearch Laboratories, West Grove, PA) and DAPI to visualize DNA. Images were obtained on an Axiovert 200 MOT microscope (Carl Zeiss, Thornwood, NY), and nuclear shape (550–1,500 cells per genotype) was assessed in a blinded fashion (5, 16–18). To assess protein farnesylation, MEFs were incubated for 36 h with an analog of farnesol, 8-anilinogeraniol (AG) (30 μM in dimethyl sulfoxide) (19Troutman J.M Roberts M.J. Andres D.A. Spielmann H.P. Tools to analyze protein farnesylation in cells..Bioconjug. Chem. 2005; 16: 1209-1217Crossref PubMed Scopus (43) Google Scholar). AG is incorporated into anilinogeranyl diphosphate and used by protein farnesyltransferase as a substrate for the modification of CaaX proteins. The incorporation of AG into proteins can be detected by Western blotting with an AG-specific mouse monoclonal antibody (1:5,000) (19Troutman J.M Roberts M.J. Andres D.A. Spielmann H.P. Tools to analyze protein farnesylation in cells..Bioconjug. Chem. 2005; 16: 1209-1217Crossref PubMed Scopus (43) Google Scholar). To prepare samples for Western blots, the A-type lamins were immunoprecipitated with a goat anti-lamin A/C antibody (15 μl) (Santa Cruz Biotechnology) (20Coffinier C. Hudon S.E. Lee R. Farber E.A. Nobumori C. Miner J.H. Andres D.A. Spielmann H.P. Hrycyna C.A. Fong L.G. et al.A potent HIV protease inhibitor, darunavir, does not inhibit ZMPSTE24 or lead to an accumulation of farnesyl-prelamin A in cells..J. Biol. Chem. 2008; 283: 9797-9804Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Proteins were size-fractionated on 4–12% gradient polyacrylamide Bis-Tris gels (Invitrogen, Carlsbad, CA), and the separated proteins were transferred to nitrocellulose membranes for Western blotting. Signals were detected with an Odyssey infrared imaging scanner. The antibody dilutions were 1:200 for anti-lamin A/C goat IgG (sc-6215, Santa Cruz Biotechnology), 1:5,000 for anti-AG mouse IgG, and 1:1,000 for anti-actin goat IgG (sc-1616, Santa Cruz Biotechnology). After washing the membranes, the membranes were incubated with 1:5,000 IRDye 800 anti-goat IgG and 1:5,000 IRDye 680 anti-mouse IgG (Li-Cor, Lincoln, NE). Protein geranylgeranylation was assessed by azido geranylgeranyl alcohol labeling. Cells were incubated with 50 μM azido geranylgeranyl alcohol for 48 h and harvested in 1% SDS and 50 mM Tris, pH 8. Protein extracts were prepared by sonication, chloroform-methanol precipitation, and resuspension in 1% SDS and 50 mM Tris, pH 8. Proteins that had incorporated azido geranylgeranyl alcohol were labeled with tetramethylrhodamine (TAMRA) with the Click-iT Tetramethylrhodamine (TAMRA) Protein Analysis Detection Kit (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. Proteins were size-fractionated on 4–12% gradient polyacrylamide Bis-Tris gels (Invitrogen), and labeled proteins were visualized with the Typhoon 9410 Variable Mode Imager (GE Healthcare, Piscataway, NJ). Following imaging, proteins were transferred to nitrocellulose for Western blotting with anti-lamin A/C goat IgG (sc-6215, Santa Cruz Biotechnology). The interior of the thorax of Lmna+/+, LmnaggHG/+, and LmnaHG/+ mice was photographed, and the number of rib fractures was counted (7Yang S.H Meta M. Qiao X. Frost D. Bauch J. Coffinier C. Majumdar S. Bergo M.O. Young S.G. Fong L.G. Treatment with a protein farnesyltransferase inhibitor improves disease phenotypes in mice with a targeted Hutchinson-Gilford progeria syndrome mutation..J. Clin. Invest. 2006; 116: 2115-2121Crossref PubMed Scopus (231) Google Scholar, 10Fong L. Frost D. Meta M. Qiao X. Yang S. Coffinier C. Young S. A protein farnesyltransferase inhibitor ameliorates disease in a mouse model of progeria..Science. 2006; 311: 1621-1623Crossref PubMed Scopus (257) Google Scholar, 11Yang S.H Qiao X. Fong L.G. Young S.G. Treatment with a farnesyltransferase inhibitor improves survival in mice with a Hutchinson-Gilford progeria syndrome mutation..Biochim. Biophys. Acta. 2008; 1781: 36-39Crossref PubMed Scopus (65) Google Scholar, 15Fong L.G Ng J.K. Meta M. Cote N. Yang S.H. Stewart C.L. Sullivan T. Burghardt A. Majumdar S. Reue K. et al.Heterozygosity for Lmna deficiency eliminates the progeria-like phenotypes in Zmpste24-deficient mice..Proc. Natl. Acad. Sci. USA. 2004; 101: 18111-18116Crossref PubMed Scopus (164) Google Scholar, 18Yang S.H Qiao X. Farber E. Chang S.Y. Fong L.G. Young S.G. Eliminating the synthesis of mature lamin A reduces disease phenotypes in mice carrying a Hutchinson-Gilford progeria syndrome allele..J. Biol. Chem. 2008; 283: 7094-7099Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The thoracic cages of Lmna+/+, LmnaggHG/+, and LmnaHG/+ mice were imaged by compact cone-beam tomography (μCT 40 scanner, Scanco Medical, Bassersdorf, Switzerland) (15Fong L.G Ng J.K. Meta M. Cote N. Yang S.H. Stewart C.L. Sullivan T. Burghardt A. Majumdar S. Reue K. et al.Heterozygosity for Lmna deficiency eliminates the progeria-like phenotypes in Zmpste24-deficient mice..Proc. Natl. Acad. Sci. USA. 2004; 101: 18111-18116Crossref PubMed Scopus (164) Google Scholar, 17Fong L.G Ng J.K. Lammerding J. Vickers T.A. Meta M. Cote N. Gavino B. Qiao X. Chang S.Y. Young S.R. et al.Prelamin A and lamin A appear to be dispensable in the nuclear lamina..J. Clin. Invest. 2006; 116: 743-752Crossref PubMed Scopus (195) Google Scholar). Urea-soluble extracts were prepared from early-passage MEFs and mouse tissues (21Dalton M. Sinensky M. Expression systems for nuclear lamin proteins: farnesylation in assembly of nuclear lamina..in: Patrick J.C Janice E.B Methods Enzymol. Academic Press, 1995: 134-148Crossref Scopus (17) Google Scholar). Extracts were size-fractionated on 4–12% gradient polyacrylamide Bis-Tris gels (Invitrogen), and the separated proteins transferred to nitrocellulose membranes for Western blotting. Antibody dilutions were 1:200 for anti-lamin A/C goat IgG (sc-6215, Santa Cruz Biotechnology), 1:1,000 for anti-actin goat IgG (sc-1616, Santa Cruz Biotechnology), and 1:5,000 for IRDye 800 anti-goat IgG (Rockland Immunochemicals, Gilbertsville, PA). The IR-coupled antibodies were detected with an Odyssey infrared imaging scanner and quantified according to the manufacturer's instructions. The isolation of RNA, cDNA synthesis, and RT-PCR were performed as described (18Yang S.H Qiao X. Farber E. Chang S.Y. Fong L.G. Young S.G. Eliminating the synthesis of mature lamin A reduces disease phenotypes in mice carrying a Hutchinson-Gilford progeria syndrome allele..J. Biol. Chem. 2008; 283: 7094-7099Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Band intensities of the fragments amplified from LmnaggHG, LmnaHG, and Lmna+ transcripts were quantified with the Odyssey infrared imaging system and normalized to 36B4. The number of surviving male and female mice was recorded weekly. Differences were assessed with log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests. Body weight curves were compared with repeated-measures ANOVA. Differences in the number of rib fractures were assessed with a two-tailed Student's t-test. Differences in the percentages of misshapen nuclei were assessed with a χ2 test. We used gene targeting to create a mutant Lmna allele, LmnaggHG, that yields geranylgeranylated progerin (Fig. 1A). The LmnaggHG allele is identical to the LmnaHG allele generated previously (5Yang S.H Bergo M.O. Toth J.I. Qiao X. Hu Y. Sandoval S. Meta M. Bendale P. Gelb M.H. Young S.G. et al.Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts with a targeted Hutchinson-Gilford progeria syndrome mutation..Proc. Natl. Acad. Sci. USA. 2005; 102: 10291-10296Crossref PubMed Scopus (237) Google Scholar), except that the methionine in the CaaX motif was replaced with a leucine (a substitution that triggers protein geranylgeranylation). Multiple targeted ES cell clones were identified by Southern blotting (Fig. 1B), and two were used to create male chimeric mice, which were bred with C57BL/6 females to produce heterozygous knock-in mice (LmnaggHG/+). Offspring were genotyped by PCR (Fig. 1C). As expected, fibroblasts from LmnaggHG/+ embryos produced substantial amounts of progerin protein (Fig. 1D). The carboxyl-terminal leucine in the progerin of LmnaggHG/+ mice was predicted to trigger protein geranylgeranylation. Indeed, metabolic labeling studies revealed that the progerin in LmnaggHG/+ MEFs, but not LmnaHG/+ MEFs, was geranylgeranylated (Fig. 2A). As expected, the progerin in LmnaHG/+ MEFs was farnesylated, as judged by metabolic labeling with a farnesol analog (Fig. 2B). We could not detect farnesylation of progerin in LmnaggHG/+ MEFs (Fig. 2B). Progerin's lipid anchor is important both for the misshapen nuclei phenotype in cultured fibroblasts and disease phenotypes in mice (5Yang S.H Bergo M.O. Toth J.I. Qiao X. Hu Y. Sandoval S. Meta M. Bendale P. Gelb M.H. Young S.G. et al.Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts with a targeted Hutchinson-Gilford progeria syndrome mutation..Proc. Natl. Acad. Sci. USA. 2005; 102: 10291-10296Crossref PubMed Scopus (237) Google Scholar, 7Yang S.H Meta M. Qiao X. Frost D. Bauch J. Coffinier C. Majumdar S. Bergo M.O. Young S.G. Fong L.G. Treatment with a protein farnesyltransferase inhibitor improves disease phenotypes in mice with a targeted Hutchinson-Gilford progeria syndrome mutation..J. Clin. Invest. 2006; 116: 2115-2121Crossref PubMed Scopus (231) Google Scholar). Because a geranylgeranyl anchor binds to membranes more avidly than a farnesyl anchor, we suspected that LmnaggHG/+ mice would exhibit more severe phenotypes than LmnaHG/+ mice. As expected, LmnaggHG/+ MEFs (n = 3 independent cell lines) exhibited a high frequency of misshapen nuclei (Fig. 3A), but to our surprise, the frequency of the misshapen nuclei was not greater than in LmnaHG/+ MEFs (Fig. 3B). One of the most easily quantifiable disease phenotypes in mouse models of progeria is bone fractures at the sites of osteolytic lesions in the ribs (which occur adjacent to the costovertebral joints). The number of rib fractures increases with age (7Yang S.H Meta M. Qiao X. Frost D. Bauch J. Coffinier C. Majumdar S. Bergo M.O. Young S.G. Fong L.G. Treatment with a protein farnesyltransferase inhibitor improves disease phenotypes in mice with a targeted Hutchinson-Gilford progeria syndrome mutation..J. Clin. Invest. 2006; 116: 2115-2121Crossref PubMed Scopus (231) Google Scholar). The rib factures heal poorly, so each fracture can be easily detected by exuberant fracture callus (7Yang S.H Meta M. Qiao X. Frost D. Bauch J. Coffinier C. Majumdar S. Bergo M.O. Young S.G. Fong L.G. Treatment with a protein farnesyltransferase inhibitor improves disease phenotypes in mice with a targeted Hutchinson-Gilford progeria syndrome mutation..J. Clin. Invest. 2006; 116: 2115-2121Crossref PubMed Scopus (231) Google Scholar, 11Yang S.H Qiao X. Fong L.G. Young S.G. Treatmen