Proteasomal Degradation of Runx2 Shortens Parathyroid Hormone-induced Anti-apoptotic Signaling in Osteoblasts
2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês
10.1074/jbc.m307444200
ISSN1083-351X
AutoresTeresita Bellido, Arshad Ali, Lilian I. Plotkin, Qiang Fu, Igor Gubrij, Paula K. Roberson, Robert S. Weinstein, Charles A. O’Brien, Stavros C. Manolagas, Robert L. Jilka,
Tópico(s)NF-κB Signaling Pathways
ResumoIt is unknown why sustained elevation of parathyroid hormone (PTH) stimulates bone resorption, whereas intermittent administration stimulates bone formation. We show in mice that daily injections of PTH attenuate osteoblast apoptosis, thereby increasing osteoblast number, bone formation rate, and bone mass, but do not affect osteoclast number. In contrast, sustained elevation of PTH, achieved either by infusion or by raising endogenous hormone secretion with a calcium-deficient diet, does not affect osteoblast apoptosis but increases osteoclast number. Attenuation of apoptosis by PTH in cultured osteoblastic cells requires protein kinase A-mediated phosphorylation and inactivation of the pro-apoptotic protein Bad as well as transcription of survival genes, like Bcl-2, mediated by CREB (cAMP response element-binding protein) and Runx2. But, PTH also increases proteasomal proteolysis of Runx2. Moreover, the anti-apoptotic effect of PTH is prolonged by inhibition of proteasomal activity, by overexpressing a dominant negative form of the E3 ligase (ubiquitin-protein isopeptide ligase) that targets Runx2 for degradation (Smurf1), or by overexpressing Runx2 itself. The duration of the anti-apoptotic effect of PTH, thus, depends on the level of Runx2, which in turn is decreased by PTH via Smurf1-mediated proteasomal proteolysis. The self-limiting nature of PTH-induced survival signaling might explain why intermittent administration of the hormone is required for bone anabolism. It is unknown why sustained elevation of parathyroid hormone (PTH) stimulates bone resorption, whereas intermittent administration stimulates bone formation. We show in mice that daily injections of PTH attenuate osteoblast apoptosis, thereby increasing osteoblast number, bone formation rate, and bone mass, but do not affect osteoclast number. In contrast, sustained elevation of PTH, achieved either by infusion or by raising endogenous hormone secretion with a calcium-deficient diet, does not affect osteoblast apoptosis but increases osteoclast number. Attenuation of apoptosis by PTH in cultured osteoblastic cells requires protein kinase A-mediated phosphorylation and inactivation of the pro-apoptotic protein Bad as well as transcription of survival genes, like Bcl-2, mediated by CREB (cAMP response element-binding protein) and Runx2. But, PTH also increases proteasomal proteolysis of Runx2. Moreover, the anti-apoptotic effect of PTH is prolonged by inhibition of proteasomal activity, by overexpressing a dominant negative form of the E3 ligase (ubiquitin-protein isopeptide ligase) that targets Runx2 for degradation (Smurf1), or by overexpressing Runx2 itself. The duration of the anti-apoptotic effect of PTH, thus, depends on the level of Runx2, which in turn is decreased by PTH via Smurf1-mediated proteasomal proteolysis. The self-limiting nature of PTH-induced survival signaling might explain why intermittent administration of the hormone is required for bone anabolism. Cyclic activation of cell surface receptors often leads to a different biologic response than sustained activation. A classic example is the stimulation of luteinizing hormone and follicle stimulating hormone production by pituitary gonadotroph cells in response to pulsatile gonadotropin releasing hormone (GnRH) versus the inhibition of hormone production by continuous GnRH treatment (1.Belchetz P.E. Plant T.M. Nakai Y. Keogh E.J. Knobil E. Science. 1978; 202: 631-633Crossref PubMed Scopus (1086) Google Scholar). The differential response of the skeleton to intermittent versus continuous elevation of parathyroid hormone (PTH) 1The abbreviations used are: PTHparathyroid hormoneBMDbone mineral densityRunx2runt related transcription factor 2dndominant negativenGFPenhanced green fluorescent protein containing a nuclear localization sequenceDBAdibutyryl-cAMPIGF-1insulin-like growth factor 1wtwild typeCREBcAMP response element-binding proteinANOVAanalysis of varianceRANKLreceptor activator of NFκB ligandPKAprotein kinase ASmurfSmad ubiquitin regulatory factorILinterleukinLIFleukemia inhibitory factorMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseZ-E(Ot-Bu)AL-pNAbenzyloxycarbonyl-Glu(O-t-butyl)-Ala-Leu-p-nitroanilide.1The abbreviations used are: PTHparathyroid hormoneBMDbone mineral densityRunx2runt related transcription factor 2dndominant negativenGFPenhanced green fluorescent protein containing a nuclear localization sequenceDBAdibutyryl-cAMPIGF-1insulin-like growth factor 1wtwild typeCREBcAMP response element-binding proteinANOVAanalysis of varianceRANKLreceptor activator of NFκB ligandPKAprotein kinase ASmurfSmad ubiquitin regulatory factorILinterleukinLIFleukemia inhibitory factorMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseZ-E(Ot-Bu)AL-pNAbenzyloxycarbonyl-Glu(O-t-butyl)-Ala-Leu-p-nitroanilide. is another long known example with important clinical and therapeutic implications. Indeed, chronic elevation of the hormone as in primary or secondary hyperparathyroidism stimulates bone resorption leading to bone loss (2.Guo C.Y. Thomas W.E.G. al-Dehaimi A.W. Assiri A.M.A. Eastell R. J. Clin. Endocrinol. Metab. 1996; 81: 3487-3491Crossref PubMed Scopus (144) Google Scholar, 3.Slatopolsky E. Brown A. Dusso A. Kidney Int. Suppl. 1999; 73: 14-19Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). In contrast, intermittent administration of PTH stimulates new bone formation by increasing osteoblast number (4.Dempster D.W. Cosman F. Parisien M. Shen V. Endocr. Rev. 1993; 14: 690-709Crossref PubMed Scopus (660) Google Scholar). Heretofore, a mechanistic explanation for the dependence of this effect on repeated transient increases in PTH has remained unknown. Albeit, intermittent PTH administration by daily injections was recently approved by the United States Food and Drug Administration as the first form of osteoporosis therapy that increases bone mass de novo, reverses the bone deficit, and dramatically reduces the incidence of fractures (5.Neer R.M. Arnaud C.D. Zanchetta J.R. Prince R. Gaich G.A. Reginster J.Y. Hodsman A.B. Eriksen E.F. Ish-Shalom S. Genant H.K. Wang O. Mitlak B.H. N. Engl. J. Med. 2001; 344: 1434-1441Crossref PubMed Scopus (3816) Google Scholar).We had previously shown that daily injections of PTH to mice for 28 days reduces the prevalence of osteoblast apoptosis and increases the number of osteoblasts (6.Jilka R.L. Weinstein R.S. Bellido T. Roberson P. Parfitt A.M. Manolagas S.C. J. Clin. Invest. 1999; 104: 439-446Crossref PubMed Scopus (885) Google Scholar), providing an explanation for the increase in bone formation rate and bone mineral density (BMD) seen with intermittent PTH administration. In the studies reported herein we show that in contrast to daily injections, sustained elevation of PTH in mice does not reduce the prevalence of osteoblast apoptosis but, as expected, stimulates osteoclastogenesis. Furthermore, we show that the anti-apoptotic effect of PTH requires runt related transcription factor 2 (Runx2)-mediated transcription of survival genes like Bcl-2 and is short-lived because PTH itself also stimulates the proteasomal degradation of Runx2 (also known as Cbfa1, Osf2, Aml3, and PEBP2αA). The self-limiting nature of PTH-induced survival signaling suggests a mechanistic explanation for the necessity of intermittent administration to elicit the bone anabolic properties of this hormone.EXPERIMENTAL PROCEDURESAnimal Studies—Female Swiss-Webster mice (4–6 months old, Harlan, Indianapolis, IN) were maintained and used in accordance with National Institutes of Health guidelines. The Institutional Animal Care and Use Committees of the University of Arkansas School for Medical Sciences and the Central Arkansas Veterans Healthcare System approved the animal protocols. Mice were fed standard rodent diet (Agway RMH 3000; Arlington Heights, IL) containing 22% protein, 0.97% calcium, and 0.85% phosphorus. The age of the animals used in each experiment varied by 2 weeks or less, and mice with a body weight 10% higher or lower than the mean of the group were excluded.Mice were given daily subcutaneous injections of human PTH-(1–34), human PTH-(1–84) (Bachem California, Inc., Torrance, CA) or vehicle (0.9% saline, 0.01 mm β-mercaptoethanol, 0.1% acetic acid) as described (6.Jilka R.L. Weinstein R.S. Bellido T. Roberson P. Parfitt A.M. Manolagas S.C. J. Clin. Invest. 1999; 104: 439-446Crossref PubMed Scopus (885) Google Scholar). Alternatively, mice were infused with PTH-(1–84) using 3- or 7-day micro-osmotic pumps delivering hormone at 1 or 0.5 μl/h, respectively (Durect Corp., Cupertino, CA). Pumps were implanted into an inter-scapular subcutaneous pocket under Metaphane anesthesia as previously described (7.Grey A. Mitnick M.A. Masiukiewicz U. Sun B.H. Rudikoff S. Jilka R.L. Manolagas S.C. Insogna K. Endocrinology. 1999; 140: 4683-4690Crossref PubMed Scopus (130) Google Scholar). There was no effect of PTH administration whether by injection or infusion on body weight (not shown). In the experiments shown in Fig. 1c, untreated mice were used as controls in view of evidence that injected vehicle had no effect on the skeleton (Fig. 1b), and our previous studies showing that infused vehicle had no effect on the level of serum pyridinoline cross-links (7.Grey A. Mitnick M.A. Masiukiewicz U. Sun B.H. Rudikoff S. Jilka R.L. Manolagas S.C. Insogna K. Endocrinology. 1999; 140: 4683-4690Crossref PubMed Scopus (130) Google Scholar). To elevate endogenous PTH, 5-month-old C57Bl/6 mice (Jackson Laboratories, Bar Harbor, ME) were fed a diet containing 0.01% calcium and 0.45% phosphorous (#960177, ICN Biomedicals, Aurora, OH). Preliminary studies showed that the circulating level of PTH-(1–84) was elevated from a base-line value of 12 ± 7 to 44 ± 20 pg/ml (n = 6, p < 0.01 by paired t test) 1 day after placing the mice on the calcium-deficient diet. Tetracycline HCl (30 mg/kg of body weight, intraperitoneally) was given 6 and 2 days before sacrifice in some experiments to permit measurement of bone formation rate.BMD was measured by dual-energy x-ray absorption using a QDR 2000 Plus densitometer (Hologic Inc., Bedford, MA) adapted for measurement of murine skeletal subregions with the aid of customized software developed by Hologic for use with mice. Resolution was enhanced by increasing the line spacing and using a smaller collimator. Sensitivity was increased by reducing the scan speed (8.Jilka R.L. Weinstein R.S. Takahashi K. Parfitt A.M. Manolagas S.C. J. Clin. Invest. 1996; 97: 1732-1740Crossref PubMed Scopus (296) Google Scholar, 9.Weinstein R.S. Jilka R.L. Parfitt A.M. Manolagas S.C. J. Clin. Invest. 1998; 102: 274-282Crossref PubMed Scopus (1379) Google Scholar). Serum was obtained by retroorbital bleeding for measurement of osteocalcin by radioimmunoassay (Biomedical Technologies Inc., Stoughton, MA), total calcium by colorimetric determination (Sigma), pyridinoline cross-links by enzyme immunoassay (Quidel Corp., San Diego, CA), or murine PTH-(1–84) or human PTH-(1–84) by enzyme-linked immunosorbent assay (Immunotopics, Inc., San Clemente, CA). Lumbar vertebrae (L1-L4) and femurs were embedded in methyl methacrylate. Histomorphometric analysis of undecalcified bone sections and detection of apoptotic osteoblasts by in situ nick-end labeling was performed as previously described (9.Weinstein R.S. Jilka R.L. Parfitt A.M. Manolagas S.C. J. Clin. Invest. 1998; 102: 274-282Crossref PubMed Scopus (1379) Google Scholar, 10.Weinstein R.S. Chen J.R. Powers C.C. Stewart S.A. Landes R.D. Bellido T. Jilka R.L. Parfitt A.M. Manolagas S.C. J. Clin. Invest. 2002; 109: 1041-1048Crossref PubMed Scopus (323) Google Scholar). Osteoclasts were identified by staining for tartrate-resistant acid phosphatase and osteoblasts by their juxtaposition to osteoid, which can be distinguished from bone by its light blue color following staining with toluidine blue (11.Baron R. Vignery A. Neff L. Silverglate A. Santa Maria A. Recker R.R. Bone Histomorphometry: Techniques and Interpretation. CRC Press, Inc., Boca Raton, FL1983: 13-35Google Scholar).Cell Cultures and in Vitro Quantification of Apoptosis—HeLa cells, osteoblastic OB-6 cells, and osteoblastic cells from neonatal murine calvaria were prepared and used as described (12.Kousteni S. Bellido T. Plotkin L.I. O'Brien C.A. Bodenner D.L. Han L. Han K. DiGregorio G.B. Katzenellenbogen J.A. Katzenellenbogen B.S. Roberson P.K. Weinstein R.S. Jilka R.L. Manolagas S.C. Cell. 2001; 104: 719-730Abstract Full Text Full Text PDF PubMed Google Scholar, 13.Lecka-Czernik B. Gubrij I. Moerman E.J. Kajkenova O. Lipschitz D.A. Manolagas S.C. Jilka R.L. J. Cell. Biochem. 1999; 74: 357-371Crossref PubMed Scopus (442) Google Scholar, 14.Jilka R.L. Weinstein R.S. Bellido T. Parfitt A.M. Manolagas S.C. J. Bone Miner. Res. 1998; 13: 793-802Crossref PubMed Scopus (466) Google Scholar). Calvaria cells from fos-deficient mice (15.Wang Z.-Q. Ovitt C. Grigoriadis A.E. Möhle-Steinlein U. Rüther U. Wagner E.F. Nature. 1992; 360: 741-745Crossref PubMed Scopus (797) Google Scholar) were provided by L. McCauley, University of Michigan, Ann Arbor, MI. Calvaria cells from Bcl-2-deficient mice and wild-type siblings were prepared from neonatal mice from our breeding colony of B6;129S2-Bcl2tm1Sjk heterozygotes (The Jackson Laboratory, Bar Harbor, ME). The genotype of each mouse was established by the presence of neo and Bcl2 sequences by PCR of tail genomic DNA and confirmed by Western blotting of calvaria cell lysates. OB-6 cells harboring the rTA(TET-OFF) protein were infected with a tetracycline-regulated retroviral construct to conditionally express dominant negative (dn) Runx2 (16.Fu Q. Jilka R.L. Manolagas S.C. O'Brien C.A. J. Biol. Chem. 2002; 277: 48868-48875Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar).Cells were seeded at 1.5 × 104/cm2 and maintained in the presence of α-minimal essential medium supplemented with 10% fetal bovine serum (calvaria) or 2% fetal bovine serum (OB-6 cells) for 18–24 h before the addition of inhibitors, PTH, or pro-apoptotic stimuli. Apoptotic cells were quantified by trypan blue staining (14.Jilka R.L. Weinstein R.S. Bellido T. Parfitt A.M. Manolagas S.C. J. Bone Miner. Res. 1998; 13: 793-802Crossref PubMed Scopus (466) Google Scholar) or nuclear morphology (12.Kousteni S. Bellido T. Plotkin L.I. O'Brien C.A. Bodenner D.L. Han L. Han K. DiGregorio G.B. Katzenellenbogen J.A. Katzenellenbogen B.S. Roberson P.K. Weinstein R.S. Jilka R.L. Manolagas S.C. Cell. 2001; 104: 719-730Abstract Full Text Full Text PDF PubMed Google Scholar, 17.Plotkin L.I. Weinstein R.S. Parfitt A.M. Roberson P.K. Manolagas S.C. Bellido T. J. Clin. Invest. 1999; 104: 1363-1374Crossref PubMed Scopus (775) Google Scholar). The latter was used in experiments involving expression constructs wherein cells were co-transfected with enhanced green fluorescent protein containing a nuclear localization sequence (nGFP) to permit assessment of nuclear morphology in fluorescent cells. Apoptotic cells were defined as those exhibiting nuclear fragmentation and/or chromatin condensation, a sine qua non feature of programmed cell death. Because apoptosis is only determined in the transfected cells, this approach has the advantage of being independent of differences in the efficiency of transfection among experiments and eliminates the contribution of untransfected cells that would otherwise mask the effect of the construct being studied. At least 225 transfected cells were evaluated, and the identity of the samples was unknown to the person scoring the live and dead cells. Experiments were repeated at least once and assessed by a different blinded reader. Critical findings were confirmed by in situ nick-end labeling, measurement of caspase-3 activity, or immunodetection of active caspase-3 as described in Supplemental Methods and Supplemental Fig. 1. H89, dibutyryl-cAMP (DBA), and RpcAMP were purchased from Biomol (Plymouth Meeting, PA), human insulin-like growth factor 1 (IGF-1) and neutralizing antibodies to interleukin (IL)-6, IL-11, or leukemia inhibitory factor (LIF) from R&D Systems (Minneapolis, MN), α-cyano-(3-methoxy,4-hydroxy,5-iodo)cinnamoyl-(3′,4′-dihydroxyphenyl) ketone tyrphostin (AG538), lactacystin, and Z-E(Ot-Bu)AL-pNA from Calbiochem-Novabiochem, actinomycin and etoposide from Sigma, and PD98059 from New England Biolabs Inc. (Beverly, MA). IGF binding protein 4 was a gift from S. Mohan (Loma Linda Veterans Affairs Medical Center).Data are presented as % dead cells or as % of stimulus-induced apoptosis in the absence of PTH or DBA. The latter was calculated using the formula (% DCA+P – % DCP)/(% DCA – % DCV) × 100, where DC = dead cells, A = death-inducing agent, P = PTH or DBA, and V = vehicle. The value of %DCA – % DCV was designated as 100%. The S.D. of the % of stimulus-induced apoptosis value was calculated by combining the S.D. values of the components of the equation as previously described (18.Kendall M. Kendalls' Advanced Theory of Statistics. Oxford University Press, New York1987: 324-325Google Scholar). None of the pharmacologic agents or expression constructs affected basal cell viability, except as noted in the legend of Fig. 7.Fig. 7The duration of the anti-apoptotic effect of PTH is limited by PTH-induced proteasomal degradation of Runx2.a, OB-6 cells were treated with vehicle, lactacystin (10 μm), or the proteasome activator Z-E(Ot-Bu)AL-pNA (100 μm) for 30 min followed by the addition of 50 nm PTH. After 1 h, etoposide was added, and apoptosis was evaluated by trypan blue staining 6 or 24 h later. b and d–g, OB-6 cells transfected with the indicated constructs (along with nGFP) were treated with PTH followed by the addition of etoposide. Nuclear morphology of fluorescent cells was determined 6 or 24 h later. Basal apoptosis was increased to ∼20% in cells transfected with dn Bad and treated with lactacystin and in cells cotransfected with dn Bad and dn Smurf1, as compared with 5% in cells transfected with vector or the other constructs. An additional 15% of the cells were killed by etoposide in all conditions. Data shown represent the mean (±S.D.) of triplicate determinations. *, p < 0.05 versus cells maintained in the absence of PTH by ANOVA. c, upper panel, OB-6 cell lysates were analyzed by Western blotting using antibodies to Runx2 or β actin. Values shown represent the mean (±S.D.) fold change in 3 replicate experiments (*, p < 0.05 versus untreated cells at the same time point). c, lower panel, cell lysates were immunoprecipitated (IP) with anti-Runx2 antibody and analyzed by Western blotting (WB) with anti-ubiquitin antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Receptor Activator of NFκB Ligand (RANKL) mRNA Detection— Total RNA was isolated from primary osteoblastic cells or mouse tibiae and analyzed by Northern blotting as previously described (16.Fu Q. Jilka R.L. Manolagas S.C. O'Brien C.A. J. Biol. Chem. 2002; 277: 48868-48875Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar) or by RNase protection analysis using a commercial kit (Pharmingen). Bcl-2 mRNA was quantified by TaqMan by real-time reverse transcriptase polymerase chain reaction using the ABI PRISM 7700 sequence detector (Applied Biosystems, Foster City, CA). Murine Bcl-2 mRNA was amplified with 5′-GCGACTTCGCCGAGATGT-3′ and 5′-CACCACCGTGGCAAAGC-3′ and detected with labeled 5′-ACCTGACGCCCTTCA-3′. Murine glyceraldehyde-3-phosphate dehydrogenase was amplified with 5′-ATGTCGTGGAGTCTACTGGTGTCTT-3′ and 5′-TTGGCTCCACCCTTCAAGTG-3′ and detected with labeled 5′-CACCACCATGGAGAAG-3′. The expression level of Bcl-2 mRNA was calculated relative to that of glyceraldehyde-3-phosphate dehydrogenase as the value 2 to the power of the negative value of the difference between the threshold cycles for Bcl-2 and glyceraldehyde-3-phosphate dehydrogenase amplification. The efficiency of target amplification for both mRNA species was equal.DNA Constructs and Transient Transfections—The following expression constructs were used: wild type (wt) Bad and Bad mutants from X-M Zhou (Apoptosis Technology, Inc. Cambridge, MA) (19.Zhou X.M. Liu Y. Payne G. Lutz R.J. Chittenden T. J. Biol. Chem. 2000; 275: 25046-25051Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar); dn cAMP response element-binding protein (CREB), dn Fos, and dn CCAAT/enhancer binding protein β from C. Vinson (NCI, National Institutes of Health, Bethesda, MD) (20.Ahn S. Olive M. Aggarwal S. Krylov D. Ginty D.D. Vinson C. Mol. Cell. Biol. 1998; 18: 967-977Crossref PubMed Scopus (445) Google Scholar); murine wt Runx2 (21.Ducy P. Zhang R. Goeffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3603) Google Scholar) and dn Runx2 (22.Ducy P. Starbuck M. Priemel M. Shen J. Pinero G. Geoffroy V. Amling M. Karsenty G. Genes Dev. 1999; 13: 1025-1036Crossref PubMed Scopus (703) Google Scholar) from P. Ducy and G. Karsenty (Baylor College of Medicine, Houston, TX); Runx2-(1–443) from D. Chen and G. Mundy (University of Texas Health Science Center at San Antonio); wt Smad ubiquitin regulatory factor (Smurf) 1 and dn Smurf1 from G. Thomsen (Stony Brook University, Stony Brook, NY) (23.Zhu H. Kavsak P. Abdollah S. Wrana J.L. Thomsen G.H. Nature. 1999; 400: 687-693Crossref PubMed Scopus (674) Google Scholar); PTH receptor from E. Schipani (24.Schipani E. Karga H. Karaplis A.C. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Abou-Samra A.-B. Jüppner H. Endocrinology. 1993; 132: 2157-2165Crossref PubMed Scopus (153) Google Scholar); dn Elk-1 from S. Safe (Texas A&M University, College Station, TX) (25.Duan R. Xie W. Burghardt R.C. Safe S. J. Biol. Chem. 2001; 276: 11590-11598Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar); dn activator protein-1 (TAM67) from T. Chambers (University of Arkansas for Medical Sciences, Little Rock, AR) (26.Brown P.H. Chen T.K. Birrer M.J. Oncogene. 1994; 9: 791-799PubMed Google Scholar); wt signal transducers and activator of transcription 3 (Stat3) and dn Stat3 from M. Saunders (GlaxoSmithKline) (27.Kaptein A. Paillard V. Saunders M. J. Biol. Chem. 1996; 271: 5961-5964Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar); wt IGF-1 receptor from S. Melmed (Cedars-Sinai Medical Center, Los Angeles, CA) (28.Prager D. Yamasaki H. Weber M.M. Gebremedhin S. Melmed S. J. Clin. Invest. 1992; 90: 2117-2122Crossref PubMed Scopus (33) Google Scholar); dn kinase-deficient IGF-1 receptor from T. Gustafson (Signal Transduction, Metabolex, Inc., Hayward, CA) (29.Cheng Z.Q. Adi S. Wu N.Y. Hsiao D. Woo E.J. Filvaroff E.H. Gustafson T.A. Rosenthal S.M. J. Endocrinol. 2000; 167: 175-182Crossref PubMed Scopus (22) Google Scholar); dn MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase) from N. G. Ahn (Howard Hughes Medical Institute, University of Colorado, Boulder, CO) (30.Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande W.G. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1254) Google Scholar). Cells were transiently co-transfected with the gene of interest and nGFP using LipofectAMINE Plus (Invitrogen) (12.Kousteni S. Bellido T. Plotkin L.I. O'Brien C.A. Bodenner D.L. Han L. Han K. DiGregorio G.B. Katzenellenbogen J.A. Katzenellenbogen B.S. Roberson P.K. Weinstein R.S. Jilka R.L. Manolagas S.C. Cell. 2001; 104: 719-730Abstract Full Text Full Text PDF PubMed Google Scholar). The efficiency of transfection was 40–60% as determined by the number of cells labeled with nGFP.Western Blot Analysis—Rabbit anti-phospho-155Ser-Bad antibody was from Cell Signaling Technology (Beverly, MA), and rabbit anti-Runx2 antibody from Oncogene Research Products (Cambridge, MA). All other antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Cell lysates were obtained as described previously (17.Plotkin L.I. Weinstein R.S. Parfitt A.M. Roberson P.K. Manolagas S.C. Bellido T. J. Clin. Invest. 1999; 104: 1363-1374Crossref PubMed Scopus (775) Google Scholar) and separated in an 8–10% SDS gel followed by immunoblotting with the relevant antibody and development by enhanced chemiluminescence. The intensity of the bands in the autoradiograms was quantified by densitometry. To detect ubiquitinated Runx2, cells were lysed in standard buffer (17.Plotkin L.I. Weinstein R.S. Parfitt A.M. Roberson P.K. Manolagas S.C. Bellido T. J. Clin. Invest. 1999; 104: 1363-1374Crossref PubMed Scopus (775) Google Scholar) containing 5 mmN-ethylmaleimide. After pre-clearing with rabbit IgG, anti-Runx2 antibody was added, and the precipitated protein was subjected to SDS gel electrophoresis and Western blotting with anti-ubiquitin antibody conjugated with horseradish peroxidase.Statistics—Values are reported as the mean ± S.D. Statistical analyses were performed using SigmaStat version 2.0 (SPSS Science, Chicago, IL). Data were analyzed by t test or one-way analysis of variance (ANOVA) after establishing normal distribution of data and homogeneity of variances. Significance values were adjusted with Bonferroni's correction. Pearson Product moment analysis was used to detect relationships between osteoblast apoptosis and indices of bone formation. For longitudinal analysis of the effect of PTH on BMD, a mixed-effects longitudinal ANOVA model was used with SAS software (SAS Institute Inc., Cary, NC) to allow specification of the covariance structure.RESULTSThe Prevalence of Osteoblast Apoptosis Is Inversely Related to Bone Formation in Mice Receiving Intermittent PTH—To firmly link suppression of osteoblast apoptosis and the stimulation of bone formation during intermittent PTH administration, we sought evidence for the correlation of the two phenomena in dose-response- and time-course studies in mice. In these studies we used 3–300 ng PTH-(1–34)/g to determine the minimum effective dose requirement for the anabolic effect of PTH in mice and also to eliminate the possibility that the anti-apoptotic effect of PTH was a phenomenon only associated with the 400 ng/g dose we had used in our earlier work (6.Jilka R.L. Weinstein R.S. Bellido T. Roberson P. Parfitt A.M. Manolagas S.C. J. Clin. Invest. 1999; 104: 439-446Crossref PubMed Scopus (885) Google Scholar). Daily injection of as little as 30 ng of PTH-(1–34)/g for 28 days increased hindlimb and spine BMD. These changes were associated with a reduction in osteoblast apoptosis at both sites as well as an increase in the level of serum osteocalcin, a biochemical index of osteoblast number (Fig. 1a). At the same dose that PTH inhibited osteoblast apoptosis, it also increased osteoblast number, bone formation rate, and the amount of cancellous bone in the distal femur, but the number of osteoclasts was not affected at any dose examined (not shown). The prevalence of osteoblast apoptosis exhibited a strong inverse correlation with three independent measures of bone formation, serum osteocalcin, bone formation rate, and osteoblast number (Fig. 1a). Temporal correlation between osteoblast apoptosis and bone formation was demonstrated in a separate experiment in which mice were given daily injections of 200 ng of PTH-(1–34)/g for 7, 14, or 28 days (Fig. 1b). Spine and hindlimb (not shown) BMD increased time-dependently in the PTH-treated mice, and this increase became statistically significant at days 14 and 28. A decrease in osteoblast apoptosis and an increase in osteoblast number in vertebral bone along with an increase in serum osteocalcin were evident as early as 7 days in mice receiving PTH, as compared with the vehicle controls. These changes persisted throughout the course of the experiment.Sustained PTH Elevation Does Not Affect Osteoblast Apoptosis—We next compared the effects of sustained versus intermittent PTH elevation on the prevalence of osteoblast apoptosis and on the number of osteoblasts and osteoclasts at 2, 4, and 6 days (Fig. 1c). In this experiment we used human PTH-(1–84), the full-length PTH molecule that has identical biological effects as the 1–34 peptide but, unlike 1–34, can be quantified using a commercially available immunoassay. Infusion of 140 ng of PTH-(1–84)/h raised the serum level of the hormone from 13 ± 7 pg/ml to 160 ± 54, 164 ± 56, and 201 ± 66 pg/ml as determined at 2, 4, or 6 days after initiation of the infusion. Sustained elevation of PTH caused an increase in the number of osteoclasts at day 2 and was followed by an increase in osteoblast number at day 4, consistent with the expected increase in bone remodeling by continuous PTH elevation (Fig. 1c and Supplemental Fig. 2). The level of serum pyridinoline cross-links, a biochemical marker of bone resorption, was also elevated by day 6 (Fig. 1c). Consistent with previous findings in the rat (31.Dobnig H. Turner R.T. Endocrinology. 1997; 138: 4607-4612Crossref PubMed Scopus (237) Google Scholar), 6 days of PTH infusion at this dose did not affect vertebral cancellous bone area (not shown). Strikingly, sustained elevation of PTH had no effect on the prevalence of osteoblast apoptosis at any time during the experiment (Fig. 1c), whereas daily injections of 230 ng PTH-(1–84)/g reduced osteoblast apoptosis and increased osteoblast number beginning on day 2. Osteoclast number and pyridinoline cross-links were unchanged by intermittent PTH. Importantly, the increase in osteoblast number seen with daily injections was significantly greater and occurred earlier than that caused by sustained infusion of PTH. Consistent with these differences and the association of increased osteoblast number with reduced apoptosis only in the case of the daily injections, 6 days of intermittent PTH i
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