PERK is essential for neonatal skeletal development to regulate osteoblast proliferation and...
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PERK Is Essential for Neonatal Skeletal Development to Regulate Osteoblast Proliferation and Differentiation JIANWEN WEI, XIAOYI SHENG, DAORONG FENG, BARBARA McGRATH AND DOUGLAS R. CAVENER * Department of Biology, Pennsylvania State University, University Park, Pennsylvania Loss of function mutations of Perk (eukaryotic translation initiation factor 2 alpha kinase 3) in humans and mice cause severe neonatal developmental defects, including diabetes, growth retardation and multiple skeletal dysplasias. Comprehensive analyses on bone tissue, at the cellular and molecular level in PERK-deficient mice demonstrated that neonatal Perk/ mice are severely osteopenic, which is caused by a deficiency in the number of mature osteoblasts, impaired osteoblast differentiation, and reduced type I collagen secretion. Impaired differentiation of osteoblasts in Perk KO mice was associated with decreased expression of Runx2 and Osterix, key regulators of osteoblast development. Reduced cell proliferation and reduced expression of key cell cycle factors including cyclin D, cyclin E, cyclin A, Cdc2, and CDK2 occur in parallel with the differentiation defect in mutant osteoblasts. In addition, the trafficking and secretion of type I collagen is compromised as manifested by abnormal retention of procollagen I in the endoplasmic reticulum, and reduced mature collagen production and mineralization. Taken together, these studies identify PERK as a novel regulator of skeletal development and osteoblast biology. J. Cell. Physiol. 217: 693–707, 2008. ß 2008 Wiley-Liss, Inc. Skeletal patterning and bone growth comprise the major developmental processes of skeletal system. Skeletal patterning initiates during embryonic development, when skeletal elements form as cartilage templates. Bone growth extends from the fetal stage through juvenile development as the cartilage skeleton is replaced by bone and the skeleton rapidly expands. The most rapid bone growth occurs during the neonatal period in concert with a dramatic overall increase in somatic growth (Deftos, 1998). Bone growth is largely determined by the expansion of osteoblasts, which secrete the major protein components of bone matrix along with regulatory factors such as insulin-like growth factors and receptor activator of NFkappaB ligand (RANKL) that modulate bone development (Canalis, 1993; Udagawa et al., 1999; Zhao et al., 2000). Osteoblasts are derived from a precursor pool of committed mesenchymal stem cells and preosteoblasts (Blair et al., 2007a). The formation, expansion, and maintenance of the osteoblast precursor pool determine the steady-state osteoblast mass. The runt-related transcription factor 2 (Runx2)/Osterix signaling pathway is essential for osteoblastic lineage commitment from mesenchymal progenitors (Ducy et al., 1997; Nakashima et al., 2002), whereas the activating transcription factor 4 (ATF4) regulates terminal differentiation of mature osteoblasts (Yang et al., 2004). Type I collagen is the major bone matrix protein secreted by osteoblasts, and is essential for proper bone formation and strength. Mutations in type I collagen result in skeletal defects in humans, underscoring the importance of this protein to the health and function of the skeletal system. For example, the low bone mass seen in Osteogenesis Imperfecta (OI) patients stems from an abnormal retention of mutant or improperly modified type I procollagen in the endoplasmic reticulum (ER) that leads to reduced mature collagen content in the bone matrix (Morello et al., 2006; Martin and Shapiro, 2007). The high frequency of bone fracture seen in OI patients during the perinatal and neonatal periods also occurs in Osteogenesis Imperfecta murine (oim) models of the disease and this consistency of phenotype within mammals points to the importance of proper osteoblast function and type I collagen synthesis for normal neonatal skeletal development (Paterson et al., 1984; Chipman et al., 1993; Forlino et al., 1999). Previously, we reported that genetic disruption of the Perk, the eukaryotic translation initiation factor 2 alpha kinase 3 (a.k.a. EIF2AK3 and PEK) in mice resulted in an OI-like phenotype including growth retardation and skeletal deformities (Zhang et al., 2002). PERK, a single-pass type I ER membrane protein kinase contains a luminal regulatory domain and a cytoplasmic eIF2a kinase domain that is conserved among the four-member family of kinases that regulate translation initiation in mammals (Shi et al., 1998; Harding et al., 1999). PERK is negatively regulated by the ER chaperones 78 kDa glucose regulated protein/ Immunoglobulin heavy chain-binding protein (GRP78/BIP) and 94 kDa glucose-regulated protein (GRP94). The high concentration of calcium within the ER lumen promotes binding of PERK to these ER chaperones and inhibits PERK’s dimerization and activation (Ma et al., 2002; Liang et al., 2006). In vitro cell culture studies have demonstrated that PERK is activated during ER stress when the folding capacity of the ER is compromised by Ca 2þ depletion or when the protein load exceeds the capacity of the ER chaperones to regulate proper folding (Harding et al., 1999; Wek and Cavener, 2007). Loss of function mutations of Perk in humans causes Wolcott–Rallison syndrome (WRS), an autosomal recessive disorder that is characterized by the early onset of type I Contract grant sponsor: NIH; Contract grant number: AR49816. *Correspondence to: Douglas R. Cavener, Department of Biology, 110 Life Science Building, Pennsylvania State University, University Park, PA 16802. E-mail: [email protected] Received 25 March 2008; Accepted 18 June 2008 Published online in Wiley InterScience (www.interscience.wiley.com.), 6 August 2008. DOI: 10.1002/jcp.21543 ORIGINAL ARTICLE 693 Journal of Journal of Cellular Physiology Cellular Physiology ß 2008 WILEY-LISS, INC.
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ORIGINAL ARTICLE 693J o u r n a l o fJ o u r n a l o f
CellularPhysiologyCellularPhysiology
PERK Is Essential for NeonatalSkeletal Development toRegulate Osteoblast Proliferationand Differentiation
JIANWEN WEI, XIAOYI SHENG, DAORONG FENG,BARBARA McGRATH AND DOUGLAS R. CAVENER*
Department of Biology, Pennsylvania State University, University Park, Pennsylvania
Loss of function mutations of Perk (eukaryotic translation initiation factor 2 alpha kinase 3) in humans and mice cause severe neonataldevelopmental defects, including diabetes, growth retardation and multiple skeletal dysplasias. Comprehensive analyses on bone tissue, atthe cellular and molecular level in PERK-deficient mice demonstrated that neonatal Perk�/�mice are severely osteopenic, which is causedby a deficiency in the number of mature osteoblasts, impaired osteoblast differentiation, and reduced type I collagen secretion. Impaireddifferentiation of osteoblasts in Perk KO mice was associated with decreased expression of Runx2 and Osterix, key regulators ofosteoblast development. Reduced cell proliferation and reduced expression of key cell cycle factors including cyclin D, cyclin E, cyclin A,Cdc2, and CDK2 occur in parallel with the differentiation defect in mutant osteoblasts. In addition, the trafficking and secretion of type Icollagen is compromised as manifested by abnormal retention of procollagen I in the endoplasmic reticulum, and reduced mature collagenproduction and mineralization. Taken together, these studies identify PERK as a novel regulator of skeletal development andosteoblast biology.
J. Cell. Physiol. 217: 693–707, 2008. � 2008 Wiley-Liss, Inc.
Contract grant sponsor: NIH;Contract grant number: AR49816.
*Correspondence to: Douglas R. Cavener, Department of Biology,110 Life Science Building, Pennsylvania State University, UniversityPark, PA 16802. E-mail: [email protected]
Received 25 March 2008; Accepted 18 June 2008
Published online in Wiley InterScience(www.interscience.wiley.com.), 6 August 2008.DOI: 10.1002/jcp.21543
Skeletal patterning and bone growth comprise the majordevelopmental processes of skeletal system. Skeletal patterninginitiates during embryonic development, when skeletalelements form as cartilage templates. Bone growth extendsfrom the fetal stage through juvenile development as thecartilage skeleton is replaced by bone and the skeleton rapidlyexpands. The most rapid bone growth occurs during theneonatal period in concert with a dramatic overall increase insomatic growth (Deftos, 1998). Bone growth is largelydetermined by the expansion of osteoblasts, which secrete themajor protein components of bone matrix along withregulatory factors such as insulin-like growth factors andreceptor activator of NFkappaB ligand (RANKL) that modulatebone development (Canalis, 1993; Udagawa et al., 1999; Zhaoet al., 2000). Osteoblasts are derived from a precursor pool ofcommitted mesenchymal stem cells and preosteoblasts (Blairet al., 2007a). The formation, expansion, and maintenance of theosteoblast precursor pool determine the steady-stateosteoblast mass. The runt-related transcription factor 2(Runx2)/Osterix signaling pathway is essential for osteoblasticlineage commitment from mesenchymal progenitors (Ducyet al., 1997; Nakashima et al., 2002), whereas the activatingtranscription factor 4 (ATF4) regulates terminal differentiationof mature osteoblasts (Yang et al., 2004).
Type I collagen is the major bone matrix protein secreted byosteoblasts, and is essential for proper bone formation andstrength. Mutations in type I collagen result in skeletal defects inhumans, underscoring the importance of this protein to thehealth and function of the skeletal system. For example, the lowbone mass seen in Osteogenesis Imperfecta (OI) patients stemsfrom an abnormal retention of mutant or improperly modifiedtype I procollagen in the endoplasmic reticulum (ER) that leadsto reduced mature collagen content in the bone matrix(Morello et al., 2006; Martin and Shapiro, 2007). The highfrequency of bone fracture seen in OI patients during theperinatal and neonatal periods also occurs in OsteogenesisImperfecta murine (oim) models of the disease and thisconsistency of phenotype within mammals points to theimportance of proper osteoblast function and type I collagen
� 2 0 0 8 W I L E Y - L I S S , I N C .
synthesis for normal neonatal skeletal development (Patersonet al., 1984; Chipman et al., 1993; Forlino et al., 1999).Previously, we reported that genetic disruption of the Perk, theeukaryotic translation initiation factor 2 alpha kinase 3 (a.k.a.EIF2AK3 and PEK) in mice resulted in an OI-like phenotypeincluding growth retardation and skeletal deformities (Zhanget al., 2002).
PERK, a single-pass type I ER membrane protein kinasecontains a luminal regulatory domain and a cytoplasmic eIF2akinase domain that is conserved among the four-member familyof kinases that regulate translation initiation in mammals (Shiet al., 1998; Harding et al., 1999). PERK is negatively regulated bythe ER chaperones 78 kDa glucose regulated protein/Immunoglobulin heavy chain-binding protein (GRP78/BIP) and94 kDa glucose-regulated protein (GRP94). The highconcentration of calcium within the ER lumen promotes bindingof PERK to these ER chaperones and inhibits PERK’sdimerization and activation (Ma et al., 2002; Liang et al., 2006). Invitro cell culture studies have demonstrated that PERK isactivated during ER stress when the folding capacity of the ER iscompromised by Ca2þ depletion or when the protein loadexceeds the capacity of the ER chaperones to regulate properfolding (Harding et al., 1999; Wek and Cavener, 2007).
Loss of function mutations of Perk in humans causesWolcott–Rallison syndrome (WRS), an autosomal recessivedisorder that is characterized by the early onset of type I
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diabetes, growth retardation and multiple skeletal dysphasia,including epiphyseal dysplasia, bone fractures and laterosteoporosis (Delepine et al., 2000; Biason-Lauber et al., 2002;Brickwood et al., 2003). Perk knockout mice duplicate all of themajor defects seen in WRS patients, and are therefore avaluable animal model for the study of PERK’s functions inmammalian development and physiology (Harding et al., 2001;Zhang et al., 2002). Previously we have shown that PERK hasdistinct functions in the pancreatic b-cells and the exocrineacinar cells (Zhang et al., 2006; Iida et al., 2007). PERK isintrinsically required during fetal and neonatal development forproper proliferation and differentiation of the insulin-secretingb-cells of the endocrine pancreas (Zhang et al., 2006). Incontrast, it is dispensable for exocrine pancreas developmentbut is required postnatally to maintain acinar cell viability (Iidaet al., 2007). Surprisingly, neither of these very differentpancreatic defects was associated with an ER stress response,which the cell culture models predicted would occur if PERKwere ablated. These observations suggest that PERK has tissue-specific and stage-specific functions that regulate development.
The cellular and developmental basis of the severe skeletaldefects in humans and mice deficient for PERK is currentlyunknown. In the present study, we show that deficiency ofPERK in mice leads to severe neonatal osteopenia, which isassociated with defects in osteoblast proliferation anddifferentiation and the trafficking of type I procollagen.
Materials and MethodsMouse strains
Generation of Perk global knockout (Perk�/�) and floxed Perkallele strains has been previously described (Zhang et al., 2002). Togenerate osteoblast specific conditional Perk mutant mice(ObPerk�/�), heterozygous floxed Perk mice that contain onefloxed Perk allele and one Perk null allele, were crossed with atransgenic strain carrying the Cre recombinase under the controlof the Col2.3 promoter to driver Cre expression at the matureosteoblast stage (Liu et al., 2004). To isolate homogenouspopulations of osteoblasts for molecular and cellular analysis, globalPerk�/� mice were crossed with pOBCol3.6GFPtpz orpOBCol2.3GFPemd transgenic strains, which specificallyexpresses a yellow variant of green fluorescent protein GFP topaz(GFPtpz, Ex/Em¼ 514/527 nm) in preosteoblasts or emerald greenfluorescent protein (GFPemd, Ex/Em¼ 487/509 nm) in matureosteoblasts, respectively (Kalajzic et al., 2002a). The InstitutionalAnimal Care and Use Committee at the Pennsylvania StateUniversity approved all mouse handling procedures.
MicroCT analysis
Mouse tibia and vertebrae were isolated from P1.5 and P10 miceand preserved in 70% ethanol at room temperature. The proximaltibia and L1 lumbar vertebra were scanned in a mCT 40 MicroCTsystem at 6mm resolution (Scanco Medical AG, Southeastern, PA).Three-dimensional quantitative histomorphometry analyses andimaging of bone structure were done on the trabecular structuresof L1 vertebra using the mCT Evaluation Program v4.4A (ScancoMedical AG). For vertebral trabecular bone analysis, the middleregion of the L1 vertebrae was selected. The parameters measuredwere based on the internationally accepted standard for thepresentation of histomorphometric data (Parfitt et al., 1987).Trabecular bone volume (BV/TV) is the volume of bone tissueexpressed as a percentage of the total tissue volume of the bone.Trabecular number (TbN) reflects the number of trabeculae that aline through the bone would hit per millimeter of its length.Trabecular thickness (Tb.Th) is the thickness of trabeculae.Trabecular separation (Tb.Sp.) is the distance between trabeculae.Bone surface density (BS/BV) is the surface area of the trabeculae(BS) in relation to the total volume (BV) of bone. Structural model
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index (SMI) reflects the curvature of the structure, or how muchthe trabecular elements bulge out. SMI normally ranges from 0 to 3,with 0 being a totally plate-like structure and 3 indicating a rod-likestructure (Hildebrand et al., 1999).
Histomorphometry, histological andimmunocytochemistry analysis
For measurement of mineral apposition rate (MAR), mice weresequentially injected intraperitoneally (IP) with calcein (25 mg/kgbody weight) at postnatal day 3 (P3) and P8 and then sacrificed atP10. Femurs were isolated, fixed in 4% formaldehyde, embedded inmethyl methacrylate resin and then cut into 10 mm thin sections.Cortical MAR was determined by measuring the distance betweenthe two fluorochrome labels in the cortical bone region, usingfluorescence microscopy (Parfitt et al., 1987). The mineralapposition rate (MAR) reflects the rate at which new bone wasdeposited in a radial direction.
For histological analysis, 5 mm-femur bone cryo-sections fromP4-P12 animals were prepared as described previously (Jiang et al.,2005). Immunocytochemistry of primary osteoblasts was carriedout according to a standard protocol using rabbit anti-type ICollagen (Rockland Immunochemicals Inc., Gilbertsville, PA),mouse anti-PDI (Assay Designs, Inc., Ann Arbor, MI), goatanti-type I procollagen and rabbit anti-HSP47 (Santa CruzBiotechnology, Inc., Santa Cruz, CA) and appropriate secondaryantibodies conjugated with either Alexa Fluor 488 or 555 dyes(Invitrogen Corporation, Carlsbad, CA). Nuclei were stained withDAPI (Molecular Probes). Images from Fluorescence and Vonkossa (5% silver nitrate) stained femur sections were sequentiallycaptured using a Nikon E1000 microcope. Bone parameters,including cortical mineral apposition rate, number of GFP positivemature osteoblasts, GFP intensity per osteoblast, bone surface inprimary spongiosa and number of osteocytes per cortical bonearea, were measured using the Image Pro Plus software (Phase 3Imaging Systems). The background brightness and contrast werekept equivalent for all images that were directly compared so as notto distort any apparent differences. For all histomorphometricanalyses, the results from five mice of each genotype are shown.
Primary osteoblast culture
Mouse calvarial osteoblast cells were isolated from P5 mice bysequential collagenase digestions as described previously (Kalajzicet al., 2002a). Calvarial cells from a single mouse were cultured withDMEM 10% FCS in T25 flask for a week. For differentiation analysis,calvarial cells were replated at 1� 105 cells/well in 12-well platesand allowed to grow for an additional week. Differentiation wasinduced by adding aMEM medium supplemented with 10% FCS,50 mg/ml ascorbic acid and 4 mM b-glycerophosphate.
For the in vivo proliferation assay, P10 mice were intraperitoneallyinjected with 60 mg BrdU (Sigma, St. Louis, MO)/kg body weight.Mice were sacrificed 3 h after injection and primary calvarial cellswere isolated as described above. Calvarial cells from fraction 2 to5 werepooled and fixed in 4% phosphate-bufferedparaformaldehydefor 10 min at room temperature. The fixed cells were then evenlyspread onto the slides and air-dried overnight at roomtemperature. BrdU incorporation was detected using acombination of rabbit anti-GFP-FTIC (1:1,000, Abcam, Cambridge,MA) and mouse anti-BrdU (1:25; DAKO, Carpinteria, CA)according to a standard protocol.
For in vitro proliferation, calvarial cells isolated from P5 micewere replated at 5� 104/chamber in a 4-well slide chamber. Cellswere incubated with 10 mM BrdU for 3 h and then BrdUincorporation was examined as described above.
To examine the cell cycle functions, osteoblasts were firstsynchronized by serum-starvation for 72 h followed by re-feedingwith DMEM supplemented with 30% FCS. Cells were thencollected at 0, 8, and 24-h time points and fixed with 70% ethanol.The DNA content per cell was determined by standard propidium
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iodide (PI) staining. Flow cytometry was run on an XL-MCL flowcytometer (Beckman-Coulter, Miami Lakes, FL). The cell cycleprofile was analyzed with Mcycle (Phoenix flow, San Diego, CA).
Cell viability was assayed using, TdT-mediated dUTP Nick-EndLabeling (TUNEL, In Situ Cell Death Detection Kit, TMR red(Roche Applied Science, Indianapolis, IN)) following themanufacturer’s protocol.
For FACS sorting, single cell suspensions from day 7 and 14 daysosteoblast cultures were prepared as described previously(Kalajzic et al., 2005). FACS was done on an influx V-GS CytometryWorkbench (Cytopeia Inc., Seattle, WA) using Spigot software.Fluorescence was excited at 488 nm and emission was collectedusing 525 nm band-pass filter.
Measurement of intracellular GFP and alkaline phosphatase(ALP) content in cultured osteoblasts
Total osteoblast cellular protein was extracted with RIPA buffer(1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS, 1� PBS,pH 8.0) containing 1� protease and phosphatase inhibitor cocktails(Sigma) and the protein concentration was determined using theBio-Rad protein assay reagent. GFP intensity was measured(Ex¼ 485, Em¼ 530) in 100 ml of protein extract using microplatefluorescence reader (FLx 800, Bio-Tek Instruments Inc., Winooski,VT). ALP activity was determined using standard p-nitrophenolateprotocol (Dimai et al., 1998).
Real-time quantitative RT-PCR analysis
Long bones (femurs and tibias) from P10 mice were freed of softtissue and bone marrow, and then homogenized in Qiazol lysisreagent (QIAGEN Inc., Valencia, CA). RNA samples from longbones, cultured primary osteoblasts and sorted osteoblasts wereprepared using RNeasy Mini kit (QIAGEN), following themanufacturer’s instructions. Random primed reverse transcription(RT) was carried out using M-MLV reverse transcriptase (Promega,Madison, WI) according to the manufacturer’s protocol. Real-timequantitative RT-PCR reactions were run on an ABI Prism 7000Sequence Detection System using the qPCR Core Kit for SYBRGreen I (Eurogentec, San Diego, CA) and analyzed using ABI Prism7000 SDS Software (Applied Biosystems, Foster City, CA). RelativemRNA levels of all genes were first normalized to the levels of actinand/or a-tubulin and then normalized to the average of WT levels.The primer sequences for the transcripts quantified by this methodare shown in Table 1.
TABLE 1. PCR Primers Used for Real-Time Quantitative RT-PCR
Gene Forward primer
Actin 5(-GCCCTGAGGCTCTTTTCC-3(Alp 5(-CATGTGATGGCGTATGCCTC-3(Asns 5(-GCTCAGAGTGCCTGCAGTCC-3(a-Tubulin 5(-CCTGGAACCCACGGTCATC-3(Bip 5(-ACCCTTACTCGGGCCAAATT-3(Bsp 5(-GACCAGTGTTGGCACCCAG-3(Chop 5(-CCAACAGAGGTCACACGCAC-3(Col1a1 5(-CACCCTCAAGAGCCTGAGTC-3(Col2a1 5(-TTCCACTTCAGCTATGGCGAT-3(Dmp1 5(-TTCCAGGGTGTCTCCCACTC-3(Glyt1 5(-GGCACTGAACGCAAGAGTCTG-3Ocn 5(-GCCTTCATGTCCAAGCAGGA-3(Opn 5(-CACCTCTCACATGAAGAGCGG-3Osterix 5(-CCAGCCTCTGGCTATGCAAA-3(Phex 5(-TTGTCTAGTCAAAGGCGAGCC-3Rankl 5(-CCTGATGAAAGGAGGGAGCA-3(Runx2 5(-GGAGCTCGGCGGAGTAGTTC-3(Sars1 5(-GGGACAGAGAGGCCGAGAAC-3(Slc3a2 5(-CCTGCTGTTGACCAGCTCCT-3(Slc7a5 5(-GAGTGTGGCATTGGCTTCG-3(Sox9 5(-TCCAGCAAGAACAAGCCACA-3(Wars 5(-ATATCCAGTGCCTCATCCCG-3(Xbp1-S 5(-CTGAGTCCGAATCAGGTGCAG-3Xbp1-T 5(-TGGCCGGGTCTGCTGAGTCCG-3
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Immunoblot analysis of bone tissues and primary osteoblasts
The procedures for total protein extraction and immunoblotanalysis for long bones and primary osteoblasts has been describedpreviously (Zhang et al., 2002). Rabbit polyclonal antibodies (Cdk2,Cdc2, cyclin A, cyclin D1, cyclin E, Runx2, ATF4), mousemonoclonal antibodies (cyclin D1 and actin) were purchased fromSanta Cruz Biotechnology, Inc. The other primary antibodiesincluded rabbit anti-collagen I (Rockland), rabbit abit-phospo-Ser251ATF4 (Gift from Dr. Gerard Karsenty), mouse anti-a-tubulin(Sigma), mouse anti BM28 (BD Transduction Laboratory,Lexington, KY). Horseradish peroxidase-conjugated anti-mouse,anti-rabbit and anti-goat secondary antibodies were purchasedfrom Sigma. Immunoreactive signals were detected using the ECLPlus kit (GE Healthcare, Piscataway, NJ) on a Storm 860Phosphorimager (GE Healthcare). Signal quantification wasmeasured using ImageQuant 5.1 software (GE Healthcare).
Statistics
Statistical analysis was performed using Student’s t-test; P< 0.05was accepted as significant. Error bars represent the standarderror of mean (SEM).
ResultsPERK is required for normal neonatal bone development
Perk�/� mice exhibit reduced mineralization in flat bones(skull), long bones (ulna and radius) and vertebrae suggestingthat both intramembraneous and endochondral ossification aredefective (Zhang et al., 2002). To characterize the structuraland architectural abnormalities of the skeleton of Perk�/�micethe first lumbar vertebra (L1) and the proximal end of the tibiaof neonatal mice were analyzed by micro-computedtomography (mCT). Compared to wild type (WT) littermates,Perk�/� mice showed a remarkable reduction in bone densityin the cortical and trabecular region of bones (Fig. 1A–D,G,H),and delayed formation of secondary ossification centers in theproximal tibia (Fig. 1E,F).
Three dimensional histomorphometric analysis of L1vertebrae trabeculae at day 1.5 and day 10 revealed a significantreduction of bone volume (55–74%), trabecular number(31–33%) and trabecular thickness (16–36%) in Perk�/� mice(Fig. 1I–K), whereas trabecular separation and bone surface
Reverse primer
5(-TGCCACAGGATTCCATACCC-3(5(-GGCACAGTGGTCAAGGTTGG-3(5(-GCACTCAGACACTGCACGGA-3(5(-AAGAGCTGGCGGTAGGTGC-3(5(-AGAGCGGAACAGGTCCATGT-3(5(-TGTCTGCTGAAACCCGTTCAG-3(5(-TGACTGGAATCTGGAGAGCGA-3(5(-GTTCGGGCTGATGTACCAGT-3(5(-GACGTTAGCGGTGTTGGGAG-3(5(-TGCACGTGATCCCTGTGTTC-3(
( 5(-CATCCCTTTGGCACCTTTTC-3(5(-GCGCCGGAGTCTGTTCACTA-3(
( 5(-CAGGGATGACATCGAGGGAC-3(5(-AGGAAATGAGTGAGGGAAGGGT-3(
( 5(-CCCCTCGACACCCTTTCTC-3(5(-TGGAATTCAGAATTGCCCGA-3(5(-CTGTGGTTACCGTCATGGCC-3(5(-AACACCTGCTCTGTCCACGG-3(5(-GCCAGTGGCATTCAAAAACC-3(5(-GCAGAATCCACTTGGGCTTG-3(5(-TGCAGATGCGGGTACTGGT-3(5(-GGTTTAGGATGGCCGATCCT-3(
( 5(-GTCCATGGGAAGATGTTCTGG-3(( 5(-GTCCATGGGAAGATGTTCTGG-3(
Fig. 1. MicroCTanalysisshowsthatPERKKOmiceexhibitneonatalosteopenia.Micro-CTimagesof2dayold(A–D,G,H)and18dayold(E,F)mousebones. A,C,E,G show WT bones and B,D,F,H are Perk�/� bones. A,B: Longitudinal cross section of proximal tibias shows a remarkable reduction intrabecular bone mineralization and cortical bone thickness in Perk�/� mouse. E,F: The epiphysis and metaphysis of proximal tibias reveal delayedformationofsecondaryossificationcenter(thestructureindicatedbyarrow)inPerk�/�mice.C,D:L1vertebralbodiesandtheirtrabecularstructure(G,H)showseverereduction intrabecularboneinPerk�/�mice. I–Nare3Dquantitativehistomorphometricdataofvertebral trabecularbonefromday 1.5 (n U 7 of each genotype) and day 10mice (n U 3 of each genotype): (I) Trabecular volume (BV/TV; %), (J) Trabecular number (TbN;/mm), (KTrabecular thickness (Tb.Th; mm), (L) Trabecular Separation (Tb.Sp; mm), (M) Bone Surface Density (BS/BV; 1/mm), (N) Structural Model Index(SMI). The definitions of bone parameters are described in Materials and Methods Section. Black bars U WT mice and white bars U Perk�/� miceScale bar: 1 mm. Error bars represent W SEM. Asterisks indicate that Perk�/� mice are significantly different from WT mice at P < 0.05.
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)
.
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density were both significantly increased (Fig. 1L,M). Thestructure model index increased approximately twofold inPerk�/� (Fig. 1N) suggesting the trabeculae in Perk�/� micewas rod-like, which is different from the normal plate-likestructure seen in WT trabeculae. These defects were seen asearly as P1.5 when bone density reaches a minimal threshold foranalysis bymCT. The severity and early appearance of low bonedensity at birth suggests that the skeletal abnormalities inPERK-deficient mice are due to developmental defects intrinsicto PERK-dependent regulation and not due to secondarypostnatal metabolic defects.
To identify and quantify osteoblasts in bone tissue, anosteoblast-specific marker transgene (Col2.3GFPemd) that
Fig. 2. HistologicalanalysisofPerk�/� femurrevealsreducedosteoblastaimages of day 4 Col2.3GFPemd Perk�/� and PerkR/R litter mate control femature osteoblasts exhibit green fluorescence. Primary spongiosa regionarrows.B,C:Quantifiedparametersofprimaryspongiosa fromday4Perk�per bone perimeter (n U 4 of each genotype). C: Expression of Col2.3GFPcorticalbonemineralizationappositionrate(MAR)ofday10mice(n U 4ofefrom day 10 mice (n U 4 of each genotype). Results from at least three micindicatewherePerk�/�micearesignificantlydifferentfromWTmiceatP <www.interscience.wiley.com.]
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expresses a color variant of GFP (emerald; emd) in matureosteoblasts was genetically introduced into the background ofPerk�/� and wild type mice. We found that the number ofGFP-positive osteoblasts in the primary spongiosa of the femurof neonatal Perk�/� mice (P4) was reduced by 39% (Fig. 2A,B).Moreover, the GFPemd signal intensity within each osteoblastwas significantly reduced in neonatal Perk�/� mice (Fig. 2C),suggesting that Perk�/� osteoblasts are less mature than WT.The observed difference in GFPemd signal is relatively small(5%) which is likely to be underestimated as we found thatCol1a1 mRNA was reduced by 30% (Fig. 3B). To evaluateosteoblast function in vivo, the mineralization apposition rate(MAR) in cortical bone was measured in tibia using a sequential
ndosteocytenumberandimpairedosteoblastactivity.A:Fluorescencemur sections and their primary spongiosa region. Col2.3GFP positive(PS) and cortical bone area (CB) are indicated by white dashed
/�andWTmice: (B)NumberofCol2.3GFPpositivematureosteoblastper mature osteoblasts (n U 4 of each genotype). D: Quantification ofachgenotype).E:Numberofosteocytespermm2 ofcorticalboneareae of each genotype are shown. Error bars represent W SEM. Asterisks0.05.[Colorfigurecanbeviewedintheonline issue,which isavailableat
Fig. 3. Reduced mRNA expression level of bone cell markers in neonatal Perk�/�mice lone bones. Real-time quantitative RT-PCR of total longbone RNA from day 10 mice (n U 6 of each genotype). The results include osteoblast markers: (A) alkaline phosphatase (Alp), (B) type I collagen(Col1a1), (C) receptor activator of NFkappaB ligand (Rankl), (D) osteocalcin (Ocn), (E) bone sialoprotein (Bsp), (F) osteopontin (Opn), andosteoblast specific transcriptional factors (G) Osterix and (H) Runx2; osteoclast markers: (I) tartrate-resistant acidic phosphatase (Trap)and (J) cathepsin K (Ctsk); and osteocyte markers: (K) dentin matrix protein 1 (Dmp1) and (L) phosphate-regulating gene with homology toendopeptidasesontheXchromosome(Phex).Errorbars represent W SEM.Asterisks indicatewherePerk�/�micearesignificantlydifferent fromWT mice at P < 0.05.
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calcein injection protocol. The MAR of 10-day-oldPerk�/� mice was reduced to 50% of WT controls (Fig. 2D).Collectively, these data show that Perk�/� mice manifestsevere osteopenia during the early neonatal stage, which isassociated with reduced osteoblast number and boneformation.
Deficiency of PERK impairs osteoblast activity in vivo
To investigate the cellular basis of osteopenia in Perk�/� mice,gene expression of key markers of osteoblast development wasquantified in total long bones of P10 Perk�/� mice and WTcontrols. Expression of mature osteoblast markers including
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alkaline phosphatase (Alp, Fig. 3A), type I Collagen (Col1a1,Fig. 3B), Rankl (Fig. 3C), osteocalcin (Ocn, Fig. 3D) and bonesialoprotein (Bsp, Fig. 3E) were significantly down-regulated to60–70% of control levels in Perk�/� mutants while expressionof osteopontin (Opn, Fig. 3F), an early osteoblast marker,equaled that of wildtype mice. Expression levels of Osterix andRunx2 were reduced by about 15% on average in Perk�/� micebut were not significantly different than wild-type (Fig. 3G,H).Expression of tartrate-resistant acid phosphatase (Trap, Fig. 3I)and cathepsin K (Ctsk, Fig. 3J), two osteoclast specific markers,were both diminished in Perk�/� mice to about 55% of wildtype mice. The decrease of TRAP and CTSK expression wascorrelated with reduced TRAP enzyme activity in serum and
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bone extracts and with a reduced CTSK protein level in totalbone protein (data not shown), indicating osteoclast activitywas also compromised in Perk�/� mice. The impairedosteoclast activities in the absence of PERK were at leastpartially correlated with the reduced Rankl expression in bonetissues (Fig. 3C).
PERK also impacts osteocyte gene expression, as revealed byreduced expression of osteocyte marker gene dentin matrixprotein 1(Dmp1) and phosphate-regulating gene withhomology to endopeptidases on the X chromosome (Phex)(Fig. 3K,L). Moreover, histological analysis showed that thenumber of osteocytes in cortical bone was significantly reducedin Perk�/�mice (Fig. 2E). Since osteocytes derive directly frommature osteoblasts during bone development this observedreduction in osteocyte number is likely a consequence ofimpaired differentiation or reduction in the number of matureosteoblasts in Perk�/� mice. Taken together, these bone cellmarker expression profile results suggest that Perk�/� miceexhibit low bone turnover osteopenia that is mainly due todefects in osteoblasts.
To identify cell autonomous functions of PERK inosteoblasts, Perk expression was specifically ablated inosteoblasts (ObPerk�/�) by crossing mice carrying a floxedallele of Perk with the Col12.3-Cre deletor strain that expressesthe Cre recombinase in mature osteoblasts. The efficiency ofCre-mediated deletion in ObPerk�/� bone tissue at postnatalday 10 was estimated by PCR to be 70%. Incomplete deletion islikely due to a combination of non-osteoblasts in the bone tissueanalyzed and by failure of Cre to be expressed or successfullydelete Perk in some of the osteoblasts. Real-time quantitativeRT-PCR revealed that ObPerk�/� mice have a significantreduction of alkaline phosphatase, type I collagen andosteocalcin in long bone at day 10 (Fig. 4A). These genes were
Fig. 4. Molecular phenotypes of osteoblast specific ObPerk�/�micelong bones. A: Real-time quantitative RT-PCR of total long bone RNAfrom day 10 mice (n U 7 of each genotype). The results show relativeexpression of osteoblast markers: alkaline phosphatase (Alp), type Icollagen (Col1a1) and osteocalcin (Ocn). Error bars represent W SEM.Asterisks indicate that Perk�/� mice are significantly different withWT mice at P < 0.05. B: Immunoblot analysis of procollagen of totallong bone protein from day 10 mice. Left part shows representiveresults from global Perk�/� mice. Right part shows representiveresults from osteoblast specific ObPerk�/�mice. Results represent atleast three independent experiments.
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are also reduced in bone tissue of global Perk�/� mice but to agreater extent than what was seen in the osteoblast specificknockout, which is likely due to the incomplete deletion of Perkin the osteoblasts of the ObPerk�/� mice. Furthermore,immunoblotting revealed that the level of procollagen in thelong bones of ObPerk�/� mice was two- to threefold greaterthan in WT, while the level of mature type I collagen wasdecreased (Fig. 4B, right part). This increase in immuno-reactiveprocollagen at the apparent expense of the mature form waspreviously seen in global Perk�/� mice (Fig. 4B, left part andZhang et al., 2002). Collectively, these findings indicate thatPERK plays a cell autonomous role in regulation of osteoblastdifferentiation and collagen processing.
PERK is required for normal osteoblast differentiationand maturation
To study the role of PERK in regulation of osteoblastdifferentiation, primary calvarial osteoblasts from transgenicCol2.3-GFPemd Perkþ/þ and Perk�/� mice were cultured inosteoblast differentiation medium and the differentiation ofthese cells was monitored by observing the acquisition of GFPfluorescence in culture and by assessing the cellular content ofGFP and ALP in total protein extracts every 4 days over a 20-dayperiod. In culture, mature Perk�/� osteoblasts, as identified bythe expression of the Col2.3GFP, appeared later and fewer innumber than the WT controls (Fig. 5A). Expression levels ofboth the transgenic mature osteoblast marker Col2.3GFP(Fig. 5B) and the endogenous osteoblast differentiation markerALP (Fig. 5C) were dramatically lower in Perk�/� cells,suggesting impaired or delayed osteoblast differentiation.Consistent with these observations, the mRNA expression ofmature osteoblast markers, including type I collagen,osteocalcin and bone sialoprotein were substantially reduced atall time points in the Perk�/� primary calvarial osteoblasts overthe 20-day time course (Fig. 5D–F). They formed smaller andmuch less mineralized bone nodules after 20 days of culture asdetected by Von Kossa staining which is consistent with theobserved reduction in MAR seen in vivo (Fig. 5A, far right part).
The in vivo and in vitro data suggest that reduced geneexpression of osteoblast markers in Perk mutant long bonesmay be due to a reduction in osteoblast number and/or delayedmaturation (Fig. 2B,C). To determine if the reduction inosteoblasts markers in bone tissues of Perk KO mice is only dueto fewer osteoblasts rather than changes in gene expression,the expression of specific mRNAs was measured in purifiedpreosteoblasts and osteoblasts. To isolate osteoblasts andpreosteoblasts we utilized the Col3.6 GFPtpz andCol2.3GFPemd transgenes that are expressed in matureosteoblasts and preosteoblasts, respectively (Kalajzic et al.,2002a). Based upon fluorescence differences of these twotransgenes, populations of presumptive preosteoblasts (day 7 inculture) and mature osteoblasts (day 14 in culture) wereobtained by FACS, achieving a purity that was greater than 95%.The expression profile of osteoblast markers in the purifiedosteoblast fractions was determined by real-time quantitativeRT-PCR. As expected, loss of Perk resulted in a 33–50%decrease in the expression of osteoblast markers (Alp, Col1a1,Ocn and Bsp) in preosteoblasts (Fig. 5I) and an 18–25%reduction in these transcripts was seen in mature osteoblasts(Fig. 5J). Opn was down regulated in preosteoblasts, but not inmature osteoblasts. These data confirm that bothdifferentiation and maturation of osteoblasts are impaired inthe absence of PERK.
Runx2 and Osterix are suppressed in Perk�/� osteoblasts
To determine if the delayed osteoblast differentiation in Perk�/�mice is caused by misregulation of key bone differentiationfactors, the mRNA levels of Runx2 and Osterix were quantified
Fig. 5. Perk�/� calvarial osteoblasts showed impaired differentiation and maturation. Primary calvarial osteoblasts isolated fromP4 PerkR/R and Perk�/�mice were grown in osteoblast differentiation medium for 20 days. A: fluorescence images of Col2.3GFP positive matureosteoblasts(greencells) fromday8today20calvarialcellcultures(magnificationof40T).ThefarrightpartsareimagesofVonKossastainingofday28 calvarial cell cultures. Mineralization nodules (MN) stain as dark spots and are indicated by red dashed arrows. B: Quantification ofCol2.3GFPemdsignalduring 20 days differentiation period (Ex U 480, Em U 530). C: Quantificationof cellular alkaline phosphatase activity duringthe 20 day differentiation period. D–H: Time serial expression profile of total RNA from day 0 to day 20 osteoblast cultures. The bars indicaterelative expression as normalized to WT level at at day 0. Osteoblast markers analyzed include: Col1a1(D), Ocn (E) and Bsp; and chondrocytemarkers: sexdeterminingregionYbox9(Sox9) (I)andtype II collagen(Col2a1) (H). Results representat least three independent experiments. I,J:Real-time quantitative RT-PCR of total RNA of purifed osteoblast from day 7 and day 14 osteoblast cultures. The results show osteoblast markers:Alp, Col1a1, Ocn, Bsp and Opn. I: Expression profile of FACS sorted Col3.6GFPtpz positive preosteoblasts from day 7 cultures (n U 3 of eachgenotypes). J: Expression profile of FACS sorted Col2.3GFPemd positive osteoblasts from day 14 cultures (n U 2 of each genotypes). The relativemRNAlevel isnormalized totheaverage expression level ofWT samples.Error bars represent W SEM. Asterisks indicate where Perk�/�micearesignificantly different from WT mice at P < 0.05. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
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during the differentiation of primary calvarial osteoblasts and inFACS purified preosteoblasts and mature osteoblasts. Theexpression of Runx2 in Perk�/� cultures from day 0 to day 8was reduced by 20–35% compared to WT and the differencebecame more evident at days 12–20 (Fig. 6A). A similarreduction was seen in Osterix mRNA in Perk�/� cultures(Fig. 6B). Down-regulation of RUNX2 was further confirmed atthe protein level in day 0 culture samples, which displayed a 44%reduction in Perk�/� cells (Fig. 6D,E). Furthermore, in purifiedpreosteoblasts, mRNA levels of Runx2 and Osterix were52% and 46% of WT, whereas in mature osteoblasts, their levelswere the same as WT (Fig. 6C), suggesting that PERK is notrequired to maintain normal levels of Runx2 and Osterix inmature osteoblasts.
Runx2/Osterix signaling plays dual roles in the regulation ofosteogenesis. On one hand it upregulates expression of genes
Fig. 6. Expressionofosteoblast specifictranscriptional factors inPerk�/�from day 0 to day 20 osteoblast cultures. C: mRNA expression of Runx2 andosteoblasts (n U 2of eachgenotype). D: ImmunoblotsofRunx2 in primary ctheimmunoblotsrevealsreductionofRunx2proteinexpression(n U 6ofeacells from day 0 cultures of both genotypes (top part) and phospo-Ser251genotypes. Results represent two independent experiments. G: mRNA expfrom day 10 mice of WT (n U 8), Atf4�/� (n U 3) and Perk�/� (n U 5). The1 (Glyt1), seryl-aminoacyl-tRNA synthetase (Sars), solute carrier family 3tryptophanyl-tRNA synthetase (Wars). H: Immunoblots of type I procollagbars represent W SEM. Asterisks indicate where Perk�/� mice or Atf4�/�
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that specify osteoblast lineage; on the other hand, it preventsmultipotent or pluripotent mesenchymal progenitors fromdifferentiating into other types of bone cells such aschondrocytes, myoblasts and adipocytes (Kobayashi et al.,2000; Morriss-Kay, 2001). To test if the suppression of Runx2/Osterix signaling in Perk�/� calvarial cells alters thedifferentiation potential of mesenchymal progenitors, mRNAlevels of chondrocyte, myoblast and adipocyte marker geneswere analyzed by realtime quantitative RT-PCR. In wild-typecalvarial osteoblasts, the mRNA level of sex determining regionY box 9 (Sox9), an essential transcription factor forchondrogenic lineage commitment, rapidly declined by 80%from day 0 to day 4, whereas the decline of Sox9 inPerk�/� calvarial osteoblasts was significantly reduced anddelayed (Fig. 5G). The expression of type II collagen (Col2a1), achondrocyte specific marker, was higher in Perk�/� cultures
osteoblast.A,B:TimecourseofmRNAexpressionofRunx2andOsterixOsterix in purified presoteoblast (n U 3 of each genotype) and mature
alvarial cells from day 0culturesof both genotypes. E: Quantificationofchgenotype).F: ImmunoblotsoftotalATF4protein inprimarycalvariaATF4 protein in total long bone extracts from day 10 mice of bothression profile of ATF4 transcriptional targets in total long bones RNAtested genes include asparagine synthase (Asns), glycine transporter
member 2 (Slc3a2), solute carrier family 7 member 5 (Slc7a5) anden in total long bone extracts from day 10 WT and Atf4�/�mice. Errorare significantly different from WT mice at P < 0.05.
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than WT at all time points after day 4 (Fig. 5H), while theexpression of the myoblast marker, myogenic differentiation1 (MyoD) and the adipocyte marker fatty acid binding protein 4(Fabp4) remained at WT levels (data not shown). The enhancedexpression of chondrocyte markers could represent eitherincreased chondrogenesis or an immature stage such as theosteochondral progenitors that express both osteoblast andchondrocyte markers. Perk�/� mice do not exhibit increasedchondrogenesis and therefore it is more likely that PERK plays arole in the maturation of the osteochondral progenitor cellsrather than determining their fate.
ATF4 is not a major downstream target of PERK inregulation of osteoblast biology
ATF4 is a known translational target of PERK activation incultured fibroblasts. To determine whether or not ATF4expression is impacted by the loss of PERK in vivo we quantifiedATF4 at the mRNA and protein levels in bone tissue and infreshly isolated osteoblasts using real-time quantitative RT-PCR and immunoblot analysis. In the absence of Perk, proteinlevels of both total ATF4 and its osteoblastic active form(phospo-Ser251 ATF4) were comparable to WT controls(Fig. 6F). Recent studies have shown that ATF4 regulatesosteoblast differentiation and function by controlling a set ofgenes that regulate amino acid metabolism (Harding et al., 2003;Elefteriou et al., 2006). The mRNA levels of ATF4 downstreamtargets asparagine synthase (Asns), glycine transporter 1 (Glyt1),seryl-aminoacyl-tRNA synthetase (Sars), solute carrier family3 member 2 (Slc3a2), solute carrier family 7 member 5 (Slc7a5)and tryptophanyl-tRNA synthetase (Wars), were significantlydecreased in Atf4�/�mice bone tissue to about 40–60% of theWT controls (Fig. 6G) and these results are consistent with theprevious findings. In contrast, the expression of these geneswas either unchanged (Sars, Slc3a2 and Wars), or significantlyincreased (Asns, Glyt1 and Slc7a5) in Perk mutant bone tissue ascompared to controls (Fig. 6G). Unlike PERK deficiency(Fig. 4B), loss of ATF4 resulted in reduced levels of type Iprocollagen (Fig. 6H). Thus, we conclude that the osteoblastdefects seen in Perk�/� mice are not due to reducedexpression or activity of ATF4.
ER retention of type I procollagen in Perk�/� osteoblastsdoes not affect their viability
The appearance of abnormally high intracellular levels of type Iprocollagen (Fig. 4B) and distended ER in Perk�/� osteoblasts(Zhang et al., 2002), raised the possibility that osteoblasts inPerk�/�mice may be prone to ER stress-induced cell death. Topinpoint the intracellular localization of type I procollagen inPerk�/� osteoblasts, double immunostaining of type I collagenand protein disulfide isomerase (PDI), an ER luminal chaperone,was performed on freshly isolated calvarial osteoblasts from10-day-old mice. A fraction of mutant osteoblasts with afrequency of 1.57� 0.11% (n¼ 3 of Perk�/� mice) displayedintensive collagen staining that was co-localized with PDI (Fig. 7,
Fig. 7. Abnormal attenuation of type I procollagen in the ER of Perk�/� otype I collagen (Col1, green) and the ER chaperone Protein Disulphide Iso(A1–3) and WT littermates (A4–6). Nuclei are stained with DAPI (blue). Cocolor. A Perk�/�osteoblast with abnormal collagen staining is indicated in Aby immunoflurescent staining in Perk�/� (B7–12) and WT (B1–6) primar(B1–3andB7–9)ofascorbicacid.Co-localizationoftypeIProcollagen(red)color. In the absence of ascorbic acid (NT/non-treated), type I procollageof ascorbic acid treament (AC/ascorbic acid treated), only faint procollagenare still detected in a fraction of Perk�/� cells (B10–12: indicated by whitefrom day 10 Perk�/� mice and WT littermates. (n U 5 of each genotype).spliced form (Xbp1-S), total form (Xbp1-T) and ratio of spliced form to toC/EBPhomologousprotein(Chop).D:TUNELassayinprimarycalvariacellbars represent W SEM. Asterisks indicate where Perk�/� mice are significsignificant difference is found between Perk�/� mice and WT mice.
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A1–A3 part, indicated by arrows). This abnormal collagenstaining osteoblasts were never seen in WT mice (Fig. 7, A4–A6part). We speculate that the very high level of procollagen-Iexpression in these relatively rare cells represent the extrememanifestation of the pronounced ER retention defect observedby western blot analysis of a large population of cells.
To determine if the abnormal ER retention of procollagen inPerk�/� induced an ER stress response, mRNA expressionprofiles of ER stress markers were analyzed in total long bone.Unexpectedly, the expression level of the classic ER stressmarkers was either unchanged, as seen for the X-box bindingprotein 1 (Xbp1) spliced form (Xbp1-S), ratio of Xbp1-S to totalform of Xbp1 (Xbp1-T) and Chop, or was significantly decreased,as seen for Bip and Xbp1-T mRNA (Fig. 7C). These ER stressmarkers were substantially induced by treatment of osteoblastswith 10 mM DTT confirming that these cells have the capacityto mount an ER stress response if suitably stimulated to do so(data not shown). Moreover, cell death as assessed by TUNEL,Caspase 3 and lactate dehydrogenase assays, showed nosignificant difference between Perk�/� and WT osteoblastsunder either in vivo or in vitro conditions (Fig. 7D and data notshown).
To track the movement of intracellular procollagen withinthe secretory pathway in osteoblasts we cultured the cells inthe presence or absence of ascorbic acid and then observedchanges in procollagen localization with respect to HSP47, aprocollagen-specific ER chaperone usingimmunohistochemistry. Culturing osteoblasts in the absence ofascorbate, a necessary co-factor for the prolyl-4-hydroxylaseenzyme, impairs proper procollagen modification and foldingresulting in ER retention that can be reversed when normalascorbate supplementation is restored. In ascorbate-freemedium both WT and mutant cells, procollagen accumulated inthe ER that co-localized with HSP47 (Fig. 7, B1–3 and B7–9part). Two hours after addition of ascorbic acid, the procollagensignal was reduced dramatically in the ER of WT cells (Fig. 7, B4–B6 part). In contrast, Perk�/�cultures exhibited a high level ofprocollagen that remained co-localized with the ER marker in asubstantial number of cells (Fig. 7, B10–B12 part indicated byarrows), suggesting that trafficking of procollagen beyond theER was slowed in Perk�/� osteoblasts. We therefore concludethat while the inability of procollagen to exit the ERundoubtedly contributes to its intracellular accumulation andaberrant localization, it does not induce the IRE1 and ATF6pathways of the unfolded protein response (UPR) or cause celldeath in osteoblasts.
PERK positively regulates osteoblast proliferation
To determine if a defect in cell proliferation might underliereduced osteoblast number in Perk deficient mice,Col3.6GFPtpz transgenic Perk�/� and Perkþ/þ mice wereinjected with BrdU 3 h prior to being sacrificed, and thenprimary calvarial osteoblasts were isolated by sequentialcollagenase digestion and prepared for immunocytochemistry.
steoblast. A: Immunofluorescence images showing the distribution ofmerase (PDI, red) in calvarial osteoblasts from day 10 Perk�/� mice-localization of type I collagen (green) and PDI (red) produces a yellow1–3 by an arrow. B: Subcellular localization of procollagen as revealed
y calvarial cell culture in the presence (B4–6 and B10–12) or absenceandthecollagenspecificERchaperoneHSP47(green)produceayellown is co-localized with HSP47 in both genotypes (B3, B9). After 2 hstaining is seen in the ER of WT cells, but intensive procollagen signals
arrows). C: mRNA expression of ER stress markers in total bone RNAThe ER stress markers tested include X-box binding protein 1 (Xbp1)tal form (Xbp1-S/T), ER chaperone Bip/Grp78 and transcription factorsday0cultureofbothgenotype(n U 2ofeachgenotype,P U 0.48).Errorantly different from WT mice at P < 0.05. NS. means no statistically
Fig. 7.
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BrdU positive cells, which represent the cells in S phase, werecategorized into three cell populations: all cells,osteoprogenitors (Col3.6GFPtpz negative cells), andpreosteoblasts/osteoblasts (Col3.6GFPtpz positive cells).
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Perk�/�mice had significantly fewer proliferating cells in all thethree populations compared to control littermates (Fig. 8A). Toexclude the possibility that decreased osteoblast proliferationin Perk�/� mice was due to secondary metabolic problems
Fig. 8. PERK controls osteoblast proliferation. A: In vivo BrdU incorporation in calvarial cells from day 10 Col3.6GFPtpz Perk�/�mice and WTlittermates (n U 3 of each genotype). The numbers of BrdU-positive osteoblasts were recorded in three populations: total cells, osteoprogenitors(Col3.6GFPtpz negative cells) and preosteoblasts/osteoblasts (Col3.6GFPtpz positive cells). P values of these three popoluations were 5.5E-10,0.024and0.0005,respectively.B: InvitroBrdUincorporationinprimarycalvarialosteoblastsbefore(day0)andafter7-daydifferentiationinculture(n U 3 of each genotype). The numbers of BrdU-positive osteoblasts were counted in total cell culture. Error bars represent W SEM. C: Cell cycleprofile of synchronized serum-induced osteoblasts at 0, 8, and 24 h time points (n U 3 of each genotype). Student’s t-test P values of 8 and24hsamplesofG1,SandG2phasespopulationswere0.012,0.004,0.036,0.000,0.040,and0.002,respectively.Asterisks indicate that Perk�/�miceare significantly different from WT mice at P < 0.05. D: Immunoblot analysis of cell cycle markers in synchronized serum-induced osteoblasts at 0,12, 24, 36, and 48 h time points. Levels of cyclin D1, CDK4, cyclin E, CDK 2, BM28, cyclin A and cdc2 were examined relative to actin. Resultsrepresent at least three independent experiments.
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such as low serum IGF1 and insulin levels (Zhang et al., 2002,2006; Li et al., 2003), Perk�/� osteoblast proliferation wasexamined in primary calvarial osteoblasts. The number of BrdUpositive cells in cultured Perk�/� calvarial osteoblasts wasreduced to 57% and 67% of WT in pre-confluence (day 0) andpost-confluence cultures (day 7), respectively (Fig. 8B).Furthermore, cell cycle analysis of synchronized serum-inducedosteoblasts revealed that there were significantly fewer cells inS and G2/M phases at both the 8 and 24 h time points inPerk�/� cultures, whereas the number of Perk�/� cells in G1phase was significantly elevated compared to WT (Fig. 8C).Collectively, these results indicate that cell cycle progression inosteoblasts is impaired in the absence of PERK.
To explore the molecular basis underlying cell cycleattenuation in Perk�/� osteoblasts, protein levels of cell cycleregulatory factors were examined in protein extracts ofsynchronized serum-induced osteoblasts at 0-, 12-, 24-, 36-,and 48-h time points. Perk�/� osteoblasts showed reducedamounts of cyclin D1(G1/S), cyclin E (G1/S), Cyclin-dependentkinase 2 (CDK2, G1/S), minichromosome maintenancedeficient 2 (BM28, S phase), cyclin A (G1/S and G2/M), and celldivision cycle 2 (CDC2, G1/S and G2/M), whereas the levels ofCyclin-dependent kinase 4 (CDK4, G1/S), cell cycle inhibitorsp16 and p21 were comparable to WT (Fig. 8D, and data not
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shown). These observations coincided with a decreased in thenumber of cells in the S and G2/M phases.
DiscussionPERK regulates neonatal skeletal development
Neonatal Perk�/� mice exhibit severe osteopenia that ismanifested as decreased cortical bone thickness and mineralapposition rate and is correlated with changes in the spongiosaincluding reduced trabecular bone volume and thickness.Perk�/� mice exhibit several other defects including growthretardation, diabetes, and exocrine pancreas atrophy (Zhanget al., 2002; Li et al., 2003) that could indirectly cause theskeletal defects. However, we found that the osteopenicphenotype in Perk�/� mice is evident at birth, prior to theonset of diabetes, exocrine pancreatic atrophy, and growthretardation suggesting that intrinsic defects in the developmentof the skeletal systems underlie the gross skeletal dysplasias.
Within the skeletal system osteoblasts have the mostprofound influence over bone development, and consequentlywe investigated whether abnormalities in osteoblastdevelopment and function might underlie the severeosteopenia in Perk�/� mice. Osteoblast differentiation andproliferation were delayed or reduced both in vivo and in vitro
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culture. Furthermore, a significant reduction of matureosteoblast markers and abnormal accumulation of type Iprocollagen were also detected in bone tissues of theosteoblast specific Perk�/� mice, supporting the hypothesisthat PERK functions cell-autonomously in osteoblasts.Compared to global Perk�/� mice, the bone defects in ObPerk�/� were milder. This could be due to incomplete ordelayed deletion of the Perk gene as dictated by the expressionof the Cre recombinase under the heterologous control of thecollagen-I promoter. We speculate that the Cre-mediateddeletion of the Perk gene does not occur efficiently enoughduring fetal and neonatal development to fully recapitulate themore severe skeletal phenotype of global Perk�/� mice.Similarly in the insulin-secreting b-cells, we have shown thatPerk must be efficiently ablated prior to embryonic day 13.5 inorder to disrupt their normal development and proliferationresulting in neonatal diabetes (Zhang et al., 2006).
PERK is a crucial regulator in osteoblast biology
PERK plays an important role in the differentiation ofosteoblasts as revealed by analysis of gene expression patternsand changes in the development of extracellular matrix.Specifically, the expression of mature osteoblast markers Alp,type I collagen, Ocn, Bsp and Rankl was reduced inPerk�/� osteoblasts in vivo and in vitro, whereas Opn, an earlyosteoblast marker, was normally expressed thus implyingdefects in osteoblast differentiation and/or maturation. Runx2and its transcriptional target Osterix are essential transcriptionfactors for osteoblast lineage commitment (Ducy et al., 1997;Nakashima et al., 2002), and their reduced expression in Perk�/� mice could hinder the ability of mesenchymal stem cells tocommit to the osteoblast lineage. RUNX2 has two cooperativefunctions in osteogenesis: to induce osteoblast specific genesexpression and to repress differentiation of other cell types,including chondrocytes, myoblasts and adipocytes via inhibitionof their specific commitment factors Sox9, MyoD andperoxisome proliferator-activated receptors g (PPARg),respectively (Komori, 2006). Relatively high levels of thechondrocyte lineage markers Sox9 and Col2a1 were seen inPerk�/� osteoblast culture, and no changes of myoblast andadipocyte specific markers were observed suggesting that thedifferentiation defect is first seen in the osteochondralprogenitor lineage. A similar abnormal expression ofchondrocyte marker genes was also observed in Osterixknockout mice (Nakashima et al., 2002) andSATB2�/� osteoblasts, where Runx2 transcription activity isimpaired (Dobreva et al., 2006). Hence, we speculate PERK maybe a key regulator of RUNX2 and OSTERIX. The decreasedexpression of Runx2 in the absence of PERK results in reductionof Osterix and consequently affects the segregation ofosteoblasts from osteochondral progenitors.
Correlated with reduced differentiation, Perk�/�osteoblasts exhibited reduced proliferation both in mice and inprimary calvarial osteoblast cultures. Reduced osteoblastproliferation in the absence of PERK was associated with a delayof entry into the S phase of the cell cycle and reduced expression of cell cycle promoting factors, including cyclin D1,cyclin E and CDk2 (G1/S), BM28 (S), Cyclin A and CDC2 (G1/SandG2/M). The fact that decreased proliferation rate has alsobeen found in vitro cultured osteoblasts from oim mice and OIpatients (Fedarko et al., 1995), suggests that the proliferationdefect Perk�/� mice could be an adaptive response to theabnormal retention of procollagen in the endoplasmicreticulum.
Besides differentiation and proliferation, PERK also impactsosteoblast function as revealed by reduced mineral appositionrate in Perk�/� mice and impaired mineral nodule formation incultured primary Perk�/� osteoblasts. The reduction in bone
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formation was correlated with reduced mature collagen and islikely caused by aberrant trafficking and processing ofprocollagen through the secretory pathway, which abnormallyaccumulated in the endoplasmic reticulum. These defects couldbe caused by the delay in maturation of Perk�/� osteoblast asrevealed by decreased expression of osteoblast markers oralternatively, the abnormal trafficking of procollagen in earlystages of osteoblast development may be the cause of thedelayed differentiation.
ATF4, an osteoblast specific transcription factor, had beenshown to be essential for osteoblast maturation and functionthrough regulation of collagen synthesis and amino acid import(Yang et al., 2004; Elefteriou et al., 2006). Under conditions ofacute ER stress in vitro, ATF4 is translationally up-regulated viaPERK dependent phosphorylation of eIF2a and is claimed to bemajor downstream target of PERK regulation (Harding et al.,2000). However, in bone tissue of Perk�/� mice, both totalATF4 protein and its osteoblastic active form were normallyexpressed as well as its downstream amino acid metabolismgenes. Moreover, the amount of intracellular procollagen wasreduced in Atf4�/� osteoblasts in contrast to Perk�/�osteoblasts. Therefore, other factors besides ATF4 are likely tobe downstream of PERK-dependent regulation of osteoblastdevelopment and function.
Reduced mature collagen production in Perk�/�osteoblastsis associated with abnormal ER retention of procollagen,resembling one of the molecular features of osteogenesisimperfecta. Based upon cell culture studies of mouse embryonicfibroblasts, over accumulation of ER client proteins shouldresult in an UPR with activation of the three arms of thispathway: PERK, inositol requiring-1 (IRE1), and activatingtranscription factor 6 (ATF6) (Tirasophon et al., 1998; Yoshidaet al., 1998; Harding et al., 1999). In the absence of PERK, IRE1and ATF6 are still activated in cultured fibroblasts treated withpharmacological reagents that induce ER stress (Wu et al.,2007). However, we found that the major downstream targetsof IRE1 and ATF6, such as Bip and Xbp1, were not induced inPerk�/� osteoblasts despite the highly abnormal accumulationof procollagen in the ER. Similarly in Perk�/� insulin-secretingb-cells, proinsulin over accumulates in the ER yet does notresult in the persistent activation of the IRE1 and ATF6 arms ofthe UPR (Zhang et al., 2006). We speculate that either the UPRis highly transient, and therefore cannot be observed in achronic state of ER stress, or that accumulation of ER clientproteins in osteoblasts and b-cells do not elicit a classic UPR.Mutations in the type I collagen gene (osteogenesis imperfecta)also result in abnormal accumulation of type I procollagen in theER and severe osteopenia (Kojima et al., 1998). Activation ofUPR had been found in osteoblasts from some OI patients andoim mice (Lamande et al., 1995; Forlino et al., 2007). Othermolecular and cellular consequences of the osteogenesisimperfecta are distinct from what we have shown inPerk�/� mice. For example the non-lethal oim exhibits normaltype I collagen mRNA levels, normal MAR, increased osteoblastnumber and elevated bone resorption (Kalajzic et al., 2002b),unlike Perk�/�mice. This suggests that a major perturbation ofER functions do not necessarily impact bone development in thesame manner.
PERK plays critical roles in the growth and development ofthe skeletal system during perinatal and neonatal developmentby two independent functions. As shown herein PERK regulatesbone development through regulating differentiation andproliferation of osteoblasts and the secretion of type I collagen.We have previously shown that PERK regulates bone growth,as well as growth of the entire body, through its regulation ofliver-secreted insulin-like growth factor 1(Li et al., 2003). Inaddition, during the same critical developmental period PERKregulates the differentiation, proliferation and function of theinsulin-secreting b-cells. Given that the activity of PERK is
706 W E I E T A L .
regulated by dynamic changes in the endoplasmic reticulumwhere its regulatory domain resides, why does PERK have theseessential roles in fetal and neonatal development? We speculatethat PERK acts as a developmental physiological sensor tomodulate the ER function and capacity of specific secretorytissues whose secretory activities are essential for earlydevelopment. Modulation of secretory capacity of a developingtissue may occur through inducing differentiation, increasingproliferation, and maturation of the secretory apparatus withincells. ER sensing functions of PERK not be limited to assessingthe client load of the ER and the folding status of ER proteins,but also changes in ER calcium levels (Liang et al., 2006), whichare impacted by a wide range of intracellular, intercellular, andextracellular signaling events. In particular, intracellular andextracellular calcium levels are known to be importantdeterminants of bone development and growth as mediated byosteoblast functions (Jorgensen, 2005; Zayzafoon, 2006; Blairet al., 2007b).
Acknowledgments
We thank Dr. David W. Rowe (University of ConnecticutHealth Center) for providing the pOBCol3.6GFPtpz andpOBCol2.3GFPemd strain, Dr. Barbara E. Kream (University ofConnecticut Health Center) for providing the Col2.3-Crestrain, Dr. Gerard Karsenty (Columbia University MedicalCenter) for providing P-Ser251 ATF4 antibody and Dr. TimTownes (University of Alabama) for providing the Atf4knockout strain. We also thanks for Elaine Kunze and SusanMagargee for assisting the FACS (Cytometry Facility, HuckInstitutes of the Life Sciences, Penn State University). This workwas supported by NIH grants AR49816 (D.R.C.).
Literature Cited
Biason-Lauber A, Lang-Muritano M, Vaccaro T, Schoenle EJ. 2002. Loss of kinase activity in apatient with Wolcott-Rallison syndrome caused by a novel mutation in the EIF2AK3 gene.Diabetes 51:2301–2305.
Blair HC, Sun L, Kohanski RA. 2007a. Balanced regulation of proliferation, growth,differentiation, and degradation in skeletal cells. Ann NY Acad Sci 1116:165–173.
Blair HC, Schlesinger PH, Huang CL, Zaidi M. 2007b. Calcium signalling and calciumtransport in bone disease. Subcell Biochem 45:539–562.
Brickwood S, Bonthron DT, Al-Gazali LI, Piper K, Hearn T, Wilson DI, Hanley NA. 2003.Wolcott-Rallison syndrome: Pathogenic insights into neonatal diabetes from new mutationand expression studies of EIF2AK3. J Med Genet 40:685–689.
Canalis E. 1993. Systemic and local factors and the maintenance of bone quality. Calcif TissueInt 53:S90–S92; discussion S92–S93.
Chipman SD, Sweet HO, McBride DJ, Jr., Davisson MT, Marks SC, Jr., Shuldiner AR,Wenstrup RJ, Rowe DW, Shapiro JR. 1993. Defective pro alpha 2(I) collagen synthesis in arecessive mutation in mice: A model of human osteogenesis imperfecta. Proc Natl Acad SciUSA 90:1701–1705.
Deftos L. 1998. Clinical essentials of calcium and skeletal metabolism. 1st edition,Professional Communication Inc. pp 1–208 (Published on-line at Medscape.com).
Delepine M, Nicolino M, Barrett T, Golamaully M, Lathrop GM, Julier C. 2000. EIF2AK3,encoding translation initiation factor 2-alpha kinase 3, is mutated in patients with Wolcott-Rallison syndrome. Nat Genet 25:406–409.
Dimai HP, Linkhart TA, Linkhart SG, Donahue LR, Beamer WG, Rosen CJ, Farley JR,Baylink DJ. 1998. Alkaline phosphatase levels and osteoprogenitor cell numbers suggestbone formation may contribute to peak bone density differences between two inbredstrains of mice. Bone 22:211–216.
Dobreva G, Chahrour M, Dautzenberg M, Chirivella L, Kanzler B, Farinas I, Karsenty G,Grosschedl R. 2006. SATB2 is a multifunctional determinant of craniofacial patterning andosteoblast differentiation. Cell 125:971–986.
Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G. 1997. Osf2/Cbfa1: A transcriptionalactivator of osteoblast differentiation. Cell 89:747–754.
Elefteriou F, Benson MD, Sowa H, Starbuck M, Liu X, Ron D, Parada LF, Karsenty G. 2006.ATF4 mediation of NF1 functions in osteoblast reveals a nutritional basis for congenitalskeletal dysplasiae. Cell Metab 4:441–451.
Fedarko NS, D’Avis P, Frazier CR, Burrill MJ, Fergusson V, Tayback M, Sponseller PD,Shapiro JR. 1995. Cell proliferation of human fibroblasts and osteoblasts in osteogenesisimperfecta: Influence of age. J Bone Miner Res 10:1705–1712.
Forlino A, Porter FD, Lee EJ, Westphal H, Marini JC. 1999. Use of the Cre/loxrecombination system to develop a non-lethal knock-in murine model for osteogenesisimperfecta with an alpha1(I) G349C substitution. Variability in phenotype in BrtlIV mice.J Biol Chem 274:37923–37931.
Forlino A, Tani C, Rossi A, Lupi A, Campari E, Gualeni B, Bianchi L, Armini A, Cetta G, BiniL, Marini JC. 2007. Differential expression of both extracellular and intracellular proteins is
JOURNAL OF CELLULAR PHYSIOLOGY
involved in the lethal or nonlethal phenotypic variation of BrtlIV, a murine model forosteogenesis imperfecta. Proteomics 7:1877–1891.
Harding HP, Zhang Y, Ron D. 1999. Protein translation and folding are coupled by anendoplasmic-reticulum-resident kinase. Nature 397:271–274.
Harding HP, Novoa I, Zhang Y, Zeng H, Wek R, Schapira M, Ron D. 2000. Regulatedtranslation initiation controls stress-induced gene expression in mammalian cells. Mol Cell6:1099–1108.
Harding HP, Zeng H, Zhang Y, Jungries R, Chung P, Plesken H, Sabatini DD, Ron D. 2001.Diabetes mellitus and exocrine pancreatic dysfunction in perk�/� mice reveals a role fortranslational control in secretory cell survival. Mol Cell 7:1153–1163.
Harding HP, Zhang Y, Zeng H, Novoa I, Lu PD, Calfon M, Sadri N, Yun C, Popko B, PaulesR, Stojdl DF, Bell JC, Hettmann T, Leiden JM, Ron D. 2003. An integrated stress responseregulates amino acid metabolism and resistance to oxidative stress. Mol Cell11:619–633.
Hildebrand T, Laib A, Muller R, Dequeker J, Ruegsegger P. 1999. Direct three-dimensionalmorphometric analysis of human cancellous bone: Microstructural data from spine, femur,iliac crest, and calcaneus. J Bone Miner Res 14:1167–1174.
Iida K, Li Y, McGrath BC, Frank A, Cavener DR. 2007. PERK eIF2 alpha kinase is required toregulate the viability of the exocrine pancreas in mice. BMC Cell Biol 8:38.
Jiang X, Kalajzic Z, Maye P, Braut A, Bellizzi J, Mina M, Rowe DW. 2005. Histological analysisof GFP expression in murine bone. J Histochem Cytochem 53:593–602.
Jorgensen NR. 2005. Short-range intercellular calcium signaling in bone. APMIS Suppl (118):5–36.
Kalajzic I, Kalajzic Z, Kaliterna M, Gronowicz G, Clark SH, Lichtler AC, Rowe D. 2002a.Use of type I collagen green fluorescent protein transgenes to identify subpopulations ofcells at different stages of the osteoblast lineage. J Bone Miner Res 17:15–25.
Kalajzic I, Terzic J, Rumboldt Z, Mack K, Naprta A, Ledgard F, Gronowicz G, Clark SH,Rowe DW. 2002b. Osteoblastic response to the defective matrix in the osteogenesisimperfecta murine (oim) mouse. Endocrinology 143:1594–1601.
Kalajzic I, Staal A, Yang WP, Wu Y, Johnson SE, Feyen JH, Krueger W, Maye P, Yu F, ZhaoY, Kuo L, Gupta RR, Achenie LE, Wang HW, Shin DG, Rowe DW. 2005. Expressionprofile of osteoblast lineage at defined stages of differentiation. J Biol Chem280:24618–24626.
Kobayashi H, Gao Y, Ueta C, Yamaguchi A, Komori T. 2000. Multilineage differentiation ofCbfa1-deficient calvarial cells in vitro. Biochem Biophys Res Commun273:630–636.
Kojima T, Miyaishi O, Saga S, Ishiguro N, Tsutsui Y, Iwata H. 1998. The retention ofabnormal type I procollagen and correlated expression of HSP 47 in fibroblasts from apatient with lethal osteogenesis imperfecta. J Pathol 184:212–218.
Komori T. 2006. Regulation of osteoblast differentiation by transcription factors. J CellBiochem 99:1233–1239.
Lamande SR, Chessler SD, Golub SB, Byers PH, Chan D, Cole WG, Sillence DO, BatemanJF. 1995. Endoplasmic reticulum-mediated quality control of type I collagen production bycells from osteogenesis imperfecta patients with mutations in the pro alpha 1 (I) chaincarboxyl-terminal propeptide which impair subunit assembly. J Biol Chem270:8642–8649.
Li Y, Iida K, O’Neil J, Zhang P, Li S, Frank A, Gabai A, Zambito F, Liang SH, Rosen CJ,Cavener DR. 2003. PERK eIF2alpha kinase regulates neonatal growth by controlling theexpression of circulating insulin-like growth factor-I derived from the liver. Endocrinology144:3505–3513.
Liang SH, Zhang W, McGrath BC, Zhang P, Cavener DR. 2006. PERK (eIF2alpha kinase) isrequired to activate the stress-activated MAPKs and induce the expression of immediate-early genes upon disruption of ER calcium homoeostasis. Biochem J 393:201–209.
Liu F, Woitge HW, Braut A, Kronenberg MS, Lichtler AC, Mina M, Kream BE. 2004.Expression and activity of osteoblast-targeted Cre recombinase transgenes in murineskeletal tissues. Int J Dev Biol 48:645–653.
Ma K, Vattem KM, Wek RC. 2002. Dimerization and release of molecular chaperoneinhibition facilitate activation of eukaryotic initiation factor-2 kinase in response toendoplasmic reticulum stress. J Biol Chem 277:18728–18735.
Martin E, Shapiro JR. 2007. Osteogenesis imperfecta:epidemiology and pathophysiology.Curr Osteoporos Rep 5:91–97.
Morello R, Bertin TK, Chen Y, Hicks J, Tonachini L, Monticone M, Castagnola P, Rauch F,Glorieux FH, Vranka J, Bachinger HP, Pace JM, Schwarze U, Byers PH, Weis M,Fernandes RJ, Eyre DR, Yao Z, Boyce BF, Lee B. 2006. CRTAP is required for prolyl3- hydroxylation and mutations cause recessive osteogenesis imperfecta. Cell127:291–304.
Morriss-Kay GM. 2001. Derivation of the mammalian skull vault. J Anat 199:143–151.Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, de Crombrugghe B.
2002. The novel zinc finger-containing transcription factor osterix is required forosteoblast differentiation and bone formation. Cell 108:17–29.
Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, Ott SM, ReckerRR. 1987. Bone histomorphometry: Standardization of nomenclature, symbols, and units.Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res2:595–610.
Paterson CR, McAllion S, Stellman JL. 1984. Osteogenesis imperfecta after the menopause.N Engl J Med 310:1694–1696.
Shi Y, Vattem KM, Sood R, An J, Liang J, Stramm L, Wek RC. 1998. Identification andcharacterization of pancreatic eukaryotic initiation factor 2 alpha-subunit kinase, PEK,involved in translational control. Mol Cell Biol 18:7499–7509.
Tirasophon W, Welihinda AA, Kaufman RJ. 1998. A stress response pathway from theendoplasmic reticulum to the nucleus requires a novel bifunctional protein kinase/endoribonuclease (Ire1p) in mammalian cells. Genes Dev 12:1812–1824.
Udagawa N, Takahashi N, Jimi E, Matsuzaki K, Tsurukai T, Itoh K, Nakagawa N, Yasuda H,Goto M, Tsuda E, Higashio K, Gillespie MT, Martin TJ, Suda T. 1999. Osteoblasts/stromalcells stimulate osteoclast activation through expression of osteoclast differentiationfactor/RANKL but not macrophage colony-stimulating factor: Receptor activator of NF-kappa B ligand. Bone 25:517–523.
Wek RC, Cavener DR. 2007. Translational control and the unfolded protein response.Antioxid Redox Signal 9:2357–2371.
Wu J, Rutkowski DT, Dubois M, Swathirajan J, Saunders T, Wang J, Song B, Yau GD,Kaufman RJ. 2007. ATF6alpha optimizes long-term endoplasmic reticulum function toprotect cells from chronic stress. Dev Cell 13:351–364.
Yang X, Matsuda K, Bialek P, Jacquot S, Masuoka HC, Schinke T, Li L, Brancorsini S,Sassone-Corsi P, Townes TM, Hanauer A, Karsenty G. 2004. ATF4 is a substrate of RSK2and an essential regulator of osteoblast biology; implication for Coffin-Lowry Syndrome.Cell 117:387–398.
Yoshida H, Haze K, Yanagi H, Yura T, Mori K. 1998. Identification of the cis-actingendoplasmic reticulum stress response element responsible for transcriptional induction
P E R K I S R E Q U I R E D F O R O S T E O B L A S T D E V E L O P M E N T 707
of mammalian glucose-regulated proteins. Involvement of basic leucine zippertranscription factors. J Biol Chem 273:33741–33749.
Zayzafoon M. 2006. Calcium/calmodulin signaling controls osteoblast growth anddifferentiation. J Cell Biochem 97:56–70.
Zhang P, McGrath B, Li S, Frank A, Zambito F, Reinert J, Gannon M, Ma K, McNaughton K,Cavener DR. 2002. The PERK eukaryotic initiation factor 2 alpha kinase is required for thedevelopment of the skeletal system, postnatal growth, and the function and viability of thepancreas. Mol Cell Biol 22:3864–3874.
JOURNAL OF CELLULAR PHYSIOLOGY
Zhang W, Feng D, Li Y, Iida K, McGrath B, Cavener DR. 2006. PERK EIF2AK3 control ofpancreatic beta cell differentiation and proliferation is required for postnatal glucosehomeostasis. Cell Metab 4:491–497.
Zhao G, Monier-Faugere MC, Langub MC, Geng Z, Nakayama T, Pike JW, Chernausek SD,Rosen CJ, Donahue LR, Malluche HH, Fagin JA, Clemens TL. 2000. Targetedoverexpression of insulin-like growth factor I to osteoblasts of transgenic mice: Increasedtrabecular bone volume without increased osteoblast proliferation. Endocrinology141:2674–2682.
