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Effect of Serial Passage on Gene Expression in MC3T3-E1 Preosteoblastic Cells: A Microarray Study
Biochemical and Biophysical Research Communications 281, 1120 –1126 (2001) doi:10.1006/bbrc.2001.4458, available online at http://www.idealibrary.com on
Effect of Serial Passage on Gene Expression in MC3T3E1 Preosteoblastic Cells: A Microarray Study Weibiao Huang,* ,† Brian Carlsen,* ,† George H. Rudkin,* ,† Nathan Shah,* Chi Chung,* ,† Kenji Ishida,‡ Dean T. Yamaguchi,§ and Timothy A. Miller* ,† ,1 *Plastic Surgery Section, §GRECC, and ‡Research Service, VA Greater Los Angeles Healthcare System, Los Angeles, California 90073; and †Department of Surgery, UCLA School of Medicine, Los Angeles, California 90095
Received January 17, 2001
The osteoblastic function of mouse preosteoblastic MC3T3-E1 cells, as measured by alkaline phosphatase activity and osteocalcin secretion, decreases after serial passage. To uncover genes responsible for decreased osteoblastic function in high-passage cells, we have studied passage-dependent change of gene expression in MC3T3-E1 cells. Changes in the expression pattern of 2000 selected genes were examined simultaneously by comparing mRNA levels between MC3T3-E1 cells at passage 20 and passage 60 using the cDNA microarray analysis. Significant changes in the steady-state abundance of 27 mRNAs were observed in response to different passage numbers, including 17 known genes, 4 ESTs with homology to known genes, and 6 genes with no previously described function or homology. Northern blot analysis was used to verify and quantify the expression of selected genes, and revealed a significant higher level of up- and downregulation compared to microarray data. These results indicate the existence of a significant change in gene expression in osteoblastic cells undergoing serial passages. Such changes might be responsible for a reduction in bone regeneration in older osteoblasts. Potential roles of selected genes in bone aging are discussed. © 2001 Academic Press
Replicative senescence is defined by the failure of a culture of primary cells to proliferate after a certain number of passages. Serial cultivation of normal cell strains results in the shortening of telomeres (1), which represents a typical cellular phenotype of an aging organism. Senescent cells are not stimulated to divide by serum, display enlarged cell size, and are associated with an altered pattern of gene expression (2, 3). Studies in senescent fibroblasts have characterized agedependent modifications at both cellular and gene lev1
To whom correspondence should be addressed. Fax: 310-4784538. E-mail: [email protected]. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
els. Comparison of gene expression among different cell types with similar gross senescent phenotypes has shown that gene expression patterns differ greatly in a cell-type-specific manner (4); therefore, separate studies are needed to understand age-dependent modification of gene expression in different cell types. To date, there has been no systematic study to examine changes in gene expression associated with senescence or cell aging in osteoblasts. Osteoblasts are bone-lining cells responsible for the production of bone matrix constituents, including collagen and ground substance. The reduced ability of older osteoblasts to form bone might be responsible for the bone loss that occurs with age (5). Understanding differences in gene expression between young and old osteoblastic cells should provide some insight into ageassociated bone loss. Recent studies indicate that replicative senescence associated with serial passage in cell culture has considerable relevance to aging in vivo (2, 3). Based on these observations, we initiated our aging studies in the well-established mouse MC3T3-E1 cell line. MC3T3 cells are clonal preosteoblastic cells derived from newborn mouse calvaria (6). Their proliferation rate is significantly reduced after a finite period of time. In general, cells above passage 60 became significantly less proliferative than cells at passage 20 or lower. After passage 60, MC3T3-E1 cells demonstrate phenotypes that are typical to cells undergoing replicative senescence. We have demonstrated in a previous study that serial passage alters MC3T3-E1 cell morphology, and significantly diminishes gap junction mediated cell– cell communication, osteoblastic function, TGF-1-mediated cell proliferation, and responsiveness to TGF-1 and BMP-2 (7). However, little is known about alteration of gene expression during the process of serial passage or cell aging. The conventional analysis of the regulation and function of genes has largely been driven by step-by-step studies of individual genes and proteins, which are highly focused and target only specific genes. To gain
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FIG. 1. Image of microarray used to analyze gene expression level in cells at passage 20 and 60. Total RNA was extracted from both cultures and each RNA sample was used as a template for synthesis of cDNA probes. The probe for cells at passage 20 was labeled with Cy-3 (green) while the probe for cells at passage 60 was labeled with Cy-5 (red). The probes were mixed and hybridized to a microarray slide. The slides were scanned in a dual-laser scanning confocal microscope (Genetic MicroSystem) and images were analyzed using a software program called Scanalyze (written by Michael Eisen, Stanford University). Green dots represented genes whose expression is decreased with serial passage while red dots presented genes whose expression is increased. Yellow dots indicate no change in gene expression. The dots identified illustrate genes under each of the described conditions.
insight into the global gene expression of an organism in response to various stimuli, it is necessary to develop and implement more sophisticated methods for gene expression analysis and gene discovery. An important milestone of this process has been the development of DNA microarray (8, 9). In this method, DNA probes representing cDNA clones are arrayed onto glass slides and hybridized with fluorescence-labeled cDNA targets. The microarray technology provides a format for the simultaneous measurement of the expression level of thousands of genes in a single hybridization assay. The technology has a wide range of
applications including gene expression profiling, comparative genomics and genotyping (10 –12). This current study was designed to determine the change of gene expression profile in mouse preosteoblastic MC3T3-E1 cells undergoing serial passage. Using cDNA microarrays containing 2000 sequenceverified mouse genes, we compared the gene expression profile between cells at passage number 20 and 60. Our results showed that significant changes in gene expression occur when MC3T3-E1 cells undergo serial passage. The isolated genes represent a wide variety of functional groups, including osteoblastic markers, fac-
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tors involved in reconstruction of extracellular matrix, extra-cellular matrix proteins, transcriptional factors, translational factors as well as an enzyme that can potentially modify telomerase. These findings may significantly improve our understanding of age-related changes in gene expression in osteoblastic cells. MATERIALS AND METHODS Cell culture. MC3T3-E1 cells were cultured in alpha-minimal essential medium (␣-MEM) (Sigma) supplemented with sodium bicarbonate, penicillin-streptomycin suspension, and 10% fetal bovine serum (FBS) (Hyclone). 2.5 ⫻ 10 5 and 1 ⫻ 10 6 cells, at passage 20 and 60, respectively, were seeded onto 10 cm culture plates and allowed to become confluent within 3 days. Total RNA preparation. Confluent cultures from 10-cm culture dishes were harvested by trypsinization followed by the addition of alpha-MEM medium with 10% FBS to deactivate trypsin. Typically, 5 ⫻ 10 6 cells were harvested and collected in a 15-ml conical tube by centrifugation at 1200 rpm in a Beckman bench top centrifuge. Cells were washed by phosphate saline buffer and subjected to RNA preparation using the RNeasy kit (Qiagen) according to the manufacturer’s protocol. cDNA microarrays. Mouse cDNA microarrays containing 2000 sequence-verified genes were obtained from the UCLA Microarray Core Facility. cDNA fragments representing 2000 unique genes were arrayed on glass slides using robotic printing. A housekeeping gene, GAPDH was also printed on the same array to serve as an internal control. Human Cot I DNA and poly(A) were used as negative controls. Fluorescent labeling and hybridization. Total RNA was labeled indirectly using aminoallyl dUTP and appropriate fluorescence. Briefly, 10 g of total RNA was mixed with 1 g of oligo dT primer (Pharmacia) in a total volume of 15.5 l. The mixture was incubated at 70°C for 10 min and chilled on ice for 10 min. Reverse transcription was carried out in a total volume of 30 l containing 1⫻ first strand buffer (Life Technology), 10 mM DTT, 1.9 l of Superscript RT II (Life Technology), 0.5 mM of each of dATP, dGTP and dCTP, 0.3 mM of dTTP and 0.2 mM of 5-(3-aminoallyl)-2⬘-deoxyuridine 5⬘-triphosphate (Sigma). The reaction was incubated at 42°C for 2 hours. 10 l of 1N NaOH and 10 l of 0.5 M EDTA was added and incubated at 65°C for 15 min to hydroxylize RNA. 25 l of 1 M Tris–HCl (pH 7.4) was added to neutralize the reaction. Unincorporated nucleotides and reagents were removed by transferring the solution into a Microcon 30 (Millipore) concentrator pre-filled with 450 l of water and spinning at 12,000 rpm in a microfuge for 8 min. The flowthrough of the Microcon 30 was removed. The clean-up process was repeated twice by refilling the original filter. The cDNA sample was eluted, dried in a speed vacuum and resuspended in 4.5 l of water. The resuspended sample was then mixed with 4.5 l of monofunctional dye NHS-ester Cy3 (for cDNA from cells at passage 20) or Cy5 (for cDNA from cells at passage 60) (Amersham) suspended in 0.1 M sodium bicarbonate (pH 9.0). The mixture was incubated at room temperature for 1 h in the dark. The reaction was quenched by incubating with 4.5 l of 4 M hydroxylamine at room temperature for an additional 15 min in the dark. To remove unincorporated/quenched dyes, the reactions of Cy3 and Cy5 were combined, loaded onto a Qia-quick PCR purification spin column (Qiagen). Purification was performed as per the manufacturer’s protocol. The Qia-quick eluate (probe) was dried in speed vacuum and resuspended with 18 l of water, 3.6 l of 20⫻ SSC, 1.8 l of 10 mg/ml poly(A) DNA (Amersham) and 0.54 l of 10% SDS. The probe was incubated at 100°C for 2 minutes and applied to a prepared microarray (glass slide based). Hybridization was carried out at 65°C overnight in a hybridization chamber (Corning).
Analysis of microarray data. Microarray images from two-color fluorescent hybridizations were scanned using a fluorescent laser scanner (Genetic MicroSystem) at the UCLA Microarray Core Facility. The scan results were saved on a ZIP disk under TIFF format and subsequently analyzed using a program called ScanAlyze (written by Michael Eisen, Stanford University). Images were gridded to locate the spot corresponding to each gene. Following gridding, information on each spot, including fluorescence intensities, background intensities and fluorescence ratios, was extracted using ScanAlyze and presented as a Microsoft Excel file. Fluorescence ratios were ultimately used to determine differentiation of gene expression. Genes with ratio number above 2 or under 0.5, i.e., 2 fold induction or repression, were selected and subjected to further analysis. Northern blot. Plasmids containing cDNAs for genes of interest were obtained from the UCLA microarray Core Facility. Probes were made from amplifying plasmid DNAs using universal primers. The total RNA from cultured MC3T3-E1 cells was isolated using RNeasy kit (Qiagen). The total RNA was quantified by a spectrophotometer at wavelength of 260 nm. Northern blots were performed as described (13). Briefly, total RNA was separated on formaldehyde denaturing 1% agarose gels. The gels were blotted onto nylon membrane using a vacuum transfer device (Pharmacia) according to the manufacturer’s instruction. The membrane was UV cross-linked and hybridized using 32P-labeled probes for 16 h. Hybridized membranes were then washed twice in 2⫻ SSC, 0.1% SDS at room temperature followed by a high-stringency wash in 0.2⫻ SSC, 0.1% SDS at 50°C for 30 min. The relative amounts of mRNA were detected by autoradiography. The GAPDH gene, whose expression does not change as passage number increases, was used as a control.
RESULTS Gene Expression Pattern of MC3T3-E1 Cells during Serial Passage We have demonstrated in a previous study that MC3T3-E1 cells exhibit significant changes in growth and differentiation after reaching passage 60 (7). During the process of serial passage, MC3T3-E1 cells demonstrate reduced growth, reduced response to BMP-2 and TGF-, as well as decreased osteoblastic function. Serial passage also causes a decrease in the number of S-phase positive cells and a decrease in the rates of DNA synthesis in MC3T3-E1 cells (Peterson and Yamaguchi, unpublished results). All these phenotypic changes are indicative of cell aging in osteoblasts (14). To further explore the molecular mechanisms responsible for these aging-associated changes, we compared the gene expression pattern between cells at passage 20 and 60 using a cDNA microarray assay. In each experiment, total RNA was used as template for synthesis of Cy-3 or Cy-5 labeled cDNA probes and hybridized to a microarray containing 2000 mouse cDNA fragments. The fluorescence intensity of each cDNA spot was measured as described under Materials and Methods. To ensure reproducibility of microarray results, we repeated the assay twice using the same total RNA samples. The genes which displayed at least 2-fold difference in the level of transcripts in all three independent experiments were selected as genes that are differentially expressed during serial passage of MC3T3-E1 cells. Figure 1 represents a typical hybrid-
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List of Differentially Expressed Genes during Serial Passage Clone ID
Description
Fold
Genes up-regulated during serial passage 481134 571759 620546 483753 902923 536464 876166 903370 524540 482943 948567 904738 652644 532186 481322 620221 920268 922371 922965 777549
ESTs Secreted phosphoprotein 1 Protein phosphatase 3, catalytic subunit, alpha isoform ESTs Tissue inhibitor of metalloproteinase 2 Phospholipase A2 group VII (platelet-activating factor acetylhydrolase) ESTs, highly similar to INTERFERON-INDUCIBLE PROTEIN [Rat] ESTs, highly similar to NADH-CYTOCHROME B5 REDUCTASE [Rat] odz (odd Oz/ten-m) homolog (Drosophila) related 1 Synaptonemal complex protein 3 Mus musculus receptor activity modifying protein 2 mRNA, Speckle-type POZ protein Diaphorase 4 (NADH/NADPH) CUG triplet repeat, RNA-binding protein 2 High mobility group protein I ESTs, weakly similar to P9513.2 gene product [S. cerevisiae] Ring finger protein 11 Eukaryotic translation initiation factor 4, gamma 2
5.4 4.1 3.9 3.4 2.8 2.6 2.6 2.5 2.3 2.2 2.2 2.1 2.1 2.1 2.1 2.1 2.1 2.0 2.0 2.0
Genes down-regulated during serial passage 808000 658260 888553 480620 619970 820167 482170
Immediate early response 5 ESTs ESTs, Highly similar to protocadherin [R. norvegicus] Procollagen, type VI, alpha 3 ESTs Cysteine rich protein 61 Four jointed box 1 (Drosophila)
2.6 2.3 2.1 2.0 2.0 2.0 2.0
Note. Clone ID and description of genes are adopted from the information provided by the UCLA Microarray Core Facility. Listed on the top are the up-regulated genes while on the bottom are the down-regulated genes. Fold of change for each individual gene is the average of three independent microarray experiments, and is presented under the column “Fold.”
ization result in which the cDNA probe derived from cells at passage 20 is labeled with Cy-3 fluorochrome (green) and the cDNA probe from cells at passage 60 is labeled with Cy-5 fluorochrome (red). Green (e.g., clone ID 619970) and red (e.g., protein phosphatase 3) fluorescent signals indicate greater expression in passage 20 and passage 60, respectively, while yellow (e.g., zipcode binding protein 1) fluorescent signals equivalent level of expression. Using a cutoff value of 2-fold, which is conventionally used by similar studies, we isolated 20 genes which displayed a ⬎2-fold increase in the levels of transcript between cells at passage 20 and 60 in multiple independent experiments. Only 7 genes were found to be down-regulated more than 2-fold during the process of serial passage. A list of identified genes is shown in Table 1. Among the up-regulated genes during cell aging, osteopontin (secreted phosphoprotein 1) is a prominent osteoblastic marker which can function as an inhibitor of mineralization. Factors involved in a wide variety of biological
processes were also up-regulated, including transcription factors (such as high mobility group protein 1 (HMG1)) and translation factors (translation initiation factor 4, gamma 2). We also found significantly elevated expression of tissue inhibitor of metalloproteinase 2 (TIMP-2), a gene product responsible for the regulation of the activity of metalloproteinase-2 which is a major member of a family of proteolytic enzymes involved in remodeling of extra-cellular matrix and normal morphogenesis (15). Interestingly, expression of a gene encoding for the alpha isoform of protein phosphatase 3 was also highly up-regulated. This increase might have significant implications during the process of cellular aging because another family member of protein phosphatase, namely protein phosphatase 2A, has been found to be involved in deactivating telomerase, an enzyme whose activity diminishes with aging (16). Included in the down-regulated genes were those encoding extracellular matrix proteins such as procollagen (type VI, alpha3) and a gene similar to protocadherin. Other down-regulated genes included
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FIG. 2. Northern blot analysis of gene expression during serial passage. To verify microarray results on selected genes, we subjected the same total RNA samples used in microarray assays to Northern blot analysis. Names for selected genes (see text for abbreviations) were labeled at the right. Band intensities were measured by densitometry and used to quantify the fold of change, which is given at the left. (A) GAPDH control, (B) down-regulated genes, (C) up-regulated genes.
genes encoding immediate early response 5, cysteinerich protein 61 and four jointed box 1, as well as two unknown genes. Verification of Expression of Selected Genes To verify the microarray results, we performed Northern blots using the same total RNA samples prepared for microarray studies. Probes for each tested gene were PCR products amplified from plasmid DNAs containing appropriate cDNAs. Among up-regulated genes, we selected osteopontin, protein phosphatase 3, TIMP-2, HMG1 and two highly up-regulated ESTs for Northern blot verification (Fig. 2C). From the list of down-regulated genes, we selected genes encoding procollagen VI and immediate early response 5 to be verified (Fig. 2B). A housekeeping GAPDH gene, whose expression was unchanged during senescence, was chosen to serve as a control (Fig. 2A). Our Northern blot results, shown in Fig. 2, demonstrated identical expression pattern for all selected genes as revealed by microarray analysis. The results indicated that our
microarray data, derived from three independent experiments, were highly reproducible and reliable. Quantification of Northern blot bands using densitometry showed change of expression level for all of the genes are was more significant than the original quantification given by microarray hybridization. For example, our microarray data showed a 4.1-fold increase of osteopontin gene expression (Table 1) while the Northern blot results showed a 15.1-fold increase (Fig. 2C). Likewise, a 2.6-fold decrease in expression of immediate early response 5 gene (Table) revealed by microarray assay was revised to a 11.9-fold decrease by Northern blot analysis (Fig. 2B). Comparison of these results illustrated that significant changes have occurred in gene expression during serial passage or senescence of osteoblastic cells. Although we observed identical patterns of expression using two independent measurements, the quantitative discrepancy revealed by comparing these two measurements indicates that there are likely some target genes not identified by microarray assay alone.
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DISCUSSION The significance of studies in bone aging is apparent due to wide occurrence of age-related bone diseases such as osteoporosis. In an attempt to isolate genes involved in the aging of bone cells, we have used MC3T3-E1 cells undergoing serial passage as a model of in vitro aging. In this study, we have identified 27 senescence-associated genes using a cDNA microarray containing 2000 sequence-verified mouse genes. We selected 8 genes for Northern blot verification. All of them were confirmed to be differentially expressed, indicating that our microarray data are consistent with the results of traditional gene expression analysis. During the aging process, the ability of osteoblasts to form new bone is greatly diminished (5). Our results showed that osteopontin gene expression is highly elevated in cells at passage 60. It has been shown that osteopontin accumulated at the late stage of mineralization can function as an inhibitor of mineralization (17, 18). Thus, this significantly increased osteopontin expression might be accountable for the decreased activity of mineralization and bone formation in aged cells. It is also noteworthy that osteopontin is a multifunctional factor and has been shown to be up-regulated in older macrophages compared to younger cells according to a separate study (19). Another interesting gene identified by this study is protein phosphatase 3. It has been shown that telomerase activity can be regulated by protein phosphorylation. For example, incubation of cell nuclear telomerase extracts with protein phosphatase 2A (PP2A) abolished the telomerase activity (16). Since decreased telomerase activity is closely related to cell aging, it is likely that elevated expression of protein phosphatase 3 could result in decreased telomerase activity, which could further result in the arrest of proliferation. Differentiation of osteoblasts involves the biosynthesis and organization of bone extracellular matrix, which can lead to the formation of mineralized bone. Studying the gene expression of matrix proteins and components involved in matrix reconstruction is crucial in understanding how bone formation is regulated. Our study has identified several genes that are closely related to extracellular matrix formation, namely TIMP-2, procollagen VI and a gene highly similar to protocadherin. Among these genes, TIMP-2 is involved in the reconstruction of extracellular matrix by binding to matrix metalloproteinases, and also possesses mitogenic activities on a number of cell types (15). It has been shown to inhibit basic fibroblast growth factorinduced human endothelial cell growth (20). The inhibitory effect of growth by TIMP-2 is consistent with its overexpression in senescent cells. Procollagen VI has been shown to play important roles for interleukin-4 induced mineralization, type I collagen accumulation, and hydroxyapatite accumulation in human periosteal
osteoblast-like cells (21). Reduction of procollagen VI production is therefore expected in aged osteoblasts where bone formation is significantly decreased. We believe that the expression patterns of some known target genes are consistent with senescent or aging phenotypes and have great implications in the aging studies of osteoblasts. We are currently characterizing some of the isolated genes to determine their regulatory roles in bone formation. Comparing our gene expression data in osteoblasts with those in fibroblasts, we have found similar expression patterns among some of the genes. For example, genes encoding tissue inhibitor of metalloproteinase 2 and enzymes involved in NADH metabolism are elevated in both types of cells during senescence (22, 23), indicating that there might be a certain set of senescence-associated genes responsible for common phenotypes in different cell types during cellular aging. In conclusion, we have demonstrated that gene expression is significantly altered in MC3T3-E1 cells undergoing serial passage. Dissection of the identified genes suggests that a wide range of biological processes are involved during cell aging. Confirmation by Northern blot analyses showed a more drastic change of gene expression than that revealed by microarray studies. Verification of the data demonstrated that our microarray results are reproducible and consistent with results from Northern blot analysis. ACKNOWLEDGMENTS We thank the UCLA Microarray Core Facility for providing cDNA microarrays and cDNA probes used in the above study. We thank Dr. William Peterson for critical reading of the manuscript and sharing information on his studies with MC3T3-E1 cells. This study was supported by a Merit Review Grant of the Department of Veterans Affairs and the Thomas and Arlene Bannon Foundation Research Fund to T.A.M.
REFERENCES
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1. Harley, C. B., Futcher, A. B., and Greider, C. W. (1990) Nature 345, 458 – 460. 2. Cristofalo, V. J., Volker, C., Francis, M. K., and Tresini, M. (1998) Crit. Rev. Eukaryot. Gene Expression 8, 43– 80. 3. Faragher, R. G., and Kipling, D. (1998) BioEssays 20, 985–991. 4. Shelton, D. N., Chang, E., Whittier, P. S., Choi, D., and Funk, W. D. (1999) Curr. Biol. 9, 939 –945. 5. Liang, C. T., Barnes, J., Seedor, J. G., Quartuccio, H. A., Bolander, M., Jeffrey, J. J., and Rodan, G. A. (1992) Bone 13, 435– 441. 6. Sudo, H., Kodama, H. A., Amagai, Y., Yamamoto, S., and Kasai, S. (1983) J. Cell Biol. 96, 191–198. 7. Chung, C. Y., Iida-Klein, A., Wyatt, L. E., Rudkin, G. H., Ishida, K., Yamaguchi, D. T., and Miller, T. A. (1999) Biochem. Biophys. Res. Commun. 265, 246 –251. 8. Schena, M., Shalon, D., Davis, R. W., and Brown, P. O. (1995) Science 270, 467– 470. 9. Schena, M., Shalon, D., Heller, R., Chai, A., Brown, P. O., and Davis, R. W. (1996) Proc. Natl. Acad. Sci. USA 93, 10614 –10619.
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10. Lander, E. S. (1999) Nat. Genet. 21, 3– 4. 11. Ferea, T. L., and Brown, P. O. (1999) Curr. Opin. Genet. Dev. 9, 715–722. 12. Brown, P. O., and Botstein, D. (1999) Nat. Genet. 21, 33–37. 13. Sambrook, J., Maniatis, T., and Fritsch, E. F. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 14. Kassem, M., Ankersen, L., Eriksen, E. F., Clark, B. F., and Rattan, S. I. (1997) Osteoporos. Int. 7, 514 –524. 15. Nagase, H., and Woessner, J. F., Jr. (1999) J. Biol. Chem. 274, 21491–21494. 16. Li, H., Zhao, L. L., Funder, J. W., and Liu, J. P. (1997) J. Biol. Chem. 272, 16729 –16732.
17. MacNeil, R. L., Berry, J., D’Errico, J., Strayhorn, C., Piotrowski, B., and Somerman, M. J. (1995) Connect. Tissue Res. 33, 1–7. 18. Gerstenfeld, L. C. (1999) J. Bone Miner. Res. 14, 850 – 855. 19. Rollo, E. E., and Denhardt, D. T. (1996) Immunology 88, 642– 647. 20. Murphy, A. N., Unsworth, E. J., and Stetler-Stevenson, W. G. (1993) J. Cell Physiol. 157, 351–358. 21. Ishibashi, H., Harumiya, S., and Koshihara, Y. (1999) Biochim. Biophys. Acta 1472, 153–164. 22. Zeng, G., and Millis, A. J. (1994) Exp. Cell Res. 213, 148 –155. 23. Kodama, S., Yamada, H., Annab, L., and Barrett, J. C. (1995) Exp. Cell Res. 219, 82– 86.
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Effect of Serial Passage on Gene Expression in MC3T3E1 Preosteoblastic Cells: A Microarray Study Weibiao Huang,* ,† Brian Carlsen,* ,† George H. Rudkin,* ,† Nathan Shah,* Chi Chung,* ,† Kenji Ishida,‡ Dean T. Yamaguchi,§ and Timothy A. Miller* ,† ,1 *Plastic Surgery Section, §GRECC, and ‡Research Service, VA Greater Los Angeles Healthcare System, Los Angeles, California 90073; and †Department of Surgery, UCLA School of Medicine, Los Angeles, California 90095
Received January 17, 2001
The osteoblastic function of mouse preosteoblastic MC3T3-E1 cells, as measured by alkaline phosphatase activity and osteocalcin secretion, decreases after serial passage. To uncover genes responsible for decreased osteoblastic function in high-passage cells, we have studied passage-dependent change of gene expression in MC3T3-E1 cells. Changes in the expression pattern of 2000 selected genes were examined simultaneously by comparing mRNA levels between MC3T3-E1 cells at passage 20 and passage 60 using the cDNA microarray analysis. Significant changes in the steady-state abundance of 27 mRNAs were observed in response to different passage numbers, including 17 known genes, 4 ESTs with homology to known genes, and 6 genes with no previously described function or homology. Northern blot analysis was used to verify and quantify the expression of selected genes, and revealed a significant higher level of up- and downregulation compared to microarray data. These results indicate the existence of a significant change in gene expression in osteoblastic cells undergoing serial passages. Such changes might be responsible for a reduction in bone regeneration in older osteoblasts. Potential roles of selected genes in bone aging are discussed. © 2001 Academic Press
Replicative senescence is defined by the failure of a culture of primary cells to proliferate after a certain number of passages. Serial cultivation of normal cell strains results in the shortening of telomeres (1), which represents a typical cellular phenotype of an aging organism. Senescent cells are not stimulated to divide by serum, display enlarged cell size, and are associated with an altered pattern of gene expression (2, 3). Studies in senescent fibroblasts have characterized agedependent modifications at both cellular and gene lev1
To whom correspondence should be addressed. Fax: 310-4784538. E-mail: [email protected]. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
els. Comparison of gene expression among different cell types with similar gross senescent phenotypes has shown that gene expression patterns differ greatly in a cell-type-specific manner (4); therefore, separate studies are needed to understand age-dependent modification of gene expression in different cell types. To date, there has been no systematic study to examine changes in gene expression associated with senescence or cell aging in osteoblasts. Osteoblasts are bone-lining cells responsible for the production of bone matrix constituents, including collagen and ground substance. The reduced ability of older osteoblasts to form bone might be responsible for the bone loss that occurs with age (5). Understanding differences in gene expression between young and old osteoblastic cells should provide some insight into ageassociated bone loss. Recent studies indicate that replicative senescence associated with serial passage in cell culture has considerable relevance to aging in vivo (2, 3). Based on these observations, we initiated our aging studies in the well-established mouse MC3T3-E1 cell line. MC3T3 cells are clonal preosteoblastic cells derived from newborn mouse calvaria (6). Their proliferation rate is significantly reduced after a finite period of time. In general, cells above passage 60 became significantly less proliferative than cells at passage 20 or lower. After passage 60, MC3T3-E1 cells demonstrate phenotypes that are typical to cells undergoing replicative senescence. We have demonstrated in a previous study that serial passage alters MC3T3-E1 cell morphology, and significantly diminishes gap junction mediated cell– cell communication, osteoblastic function, TGF-1-mediated cell proliferation, and responsiveness to TGF-1 and BMP-2 (7). However, little is known about alteration of gene expression during the process of serial passage or cell aging. The conventional analysis of the regulation and function of genes has largely been driven by step-by-step studies of individual genes and proteins, which are highly focused and target only specific genes. To gain
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FIG. 1. Image of microarray used to analyze gene expression level in cells at passage 20 and 60. Total RNA was extracted from both cultures and each RNA sample was used as a template for synthesis of cDNA probes. The probe for cells at passage 20 was labeled with Cy-3 (green) while the probe for cells at passage 60 was labeled with Cy-5 (red). The probes were mixed and hybridized to a microarray slide. The slides were scanned in a dual-laser scanning confocal microscope (Genetic MicroSystem) and images were analyzed using a software program called Scanalyze (written by Michael Eisen, Stanford University). Green dots represented genes whose expression is decreased with serial passage while red dots presented genes whose expression is increased. Yellow dots indicate no change in gene expression. The dots identified illustrate genes under each of the described conditions.
insight into the global gene expression of an organism in response to various stimuli, it is necessary to develop and implement more sophisticated methods for gene expression analysis and gene discovery. An important milestone of this process has been the development of DNA microarray (8, 9). In this method, DNA probes representing cDNA clones are arrayed onto glass slides and hybridized with fluorescence-labeled cDNA targets. The microarray technology provides a format for the simultaneous measurement of the expression level of thousands of genes in a single hybridization assay. The technology has a wide range of
applications including gene expression profiling, comparative genomics and genotyping (10 –12). This current study was designed to determine the change of gene expression profile in mouse preosteoblastic MC3T3-E1 cells undergoing serial passage. Using cDNA microarrays containing 2000 sequenceverified mouse genes, we compared the gene expression profile between cells at passage number 20 and 60. Our results showed that significant changes in gene expression occur when MC3T3-E1 cells undergo serial passage. The isolated genes represent a wide variety of functional groups, including osteoblastic markers, fac-
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tors involved in reconstruction of extracellular matrix, extra-cellular matrix proteins, transcriptional factors, translational factors as well as an enzyme that can potentially modify telomerase. These findings may significantly improve our understanding of age-related changes in gene expression in osteoblastic cells. MATERIALS AND METHODS Cell culture. MC3T3-E1 cells were cultured in alpha-minimal essential medium (␣-MEM) (Sigma) supplemented with sodium bicarbonate, penicillin-streptomycin suspension, and 10% fetal bovine serum (FBS) (Hyclone). 2.5 ⫻ 10 5 and 1 ⫻ 10 6 cells, at passage 20 and 60, respectively, were seeded onto 10 cm culture plates and allowed to become confluent within 3 days. Total RNA preparation. Confluent cultures from 10-cm culture dishes were harvested by trypsinization followed by the addition of alpha-MEM medium with 10% FBS to deactivate trypsin. Typically, 5 ⫻ 10 6 cells were harvested and collected in a 15-ml conical tube by centrifugation at 1200 rpm in a Beckman bench top centrifuge. Cells were washed by phosphate saline buffer and subjected to RNA preparation using the RNeasy kit (Qiagen) according to the manufacturer’s protocol. cDNA microarrays. Mouse cDNA microarrays containing 2000 sequence-verified genes were obtained from the UCLA Microarray Core Facility. cDNA fragments representing 2000 unique genes were arrayed on glass slides using robotic printing. A housekeeping gene, GAPDH was also printed on the same array to serve as an internal control. Human Cot I DNA and poly(A) were used as negative controls. Fluorescent labeling and hybridization. Total RNA was labeled indirectly using aminoallyl dUTP and appropriate fluorescence. Briefly, 10 g of total RNA was mixed with 1 g of oligo dT primer (Pharmacia) in a total volume of 15.5 l. The mixture was incubated at 70°C for 10 min and chilled on ice for 10 min. Reverse transcription was carried out in a total volume of 30 l containing 1⫻ first strand buffer (Life Technology), 10 mM DTT, 1.9 l of Superscript RT II (Life Technology), 0.5 mM of each of dATP, dGTP and dCTP, 0.3 mM of dTTP and 0.2 mM of 5-(3-aminoallyl)-2⬘-deoxyuridine 5⬘-triphosphate (Sigma). The reaction was incubated at 42°C for 2 hours. 10 l of 1N NaOH and 10 l of 0.5 M EDTA was added and incubated at 65°C for 15 min to hydroxylize RNA. 25 l of 1 M Tris–HCl (pH 7.4) was added to neutralize the reaction. Unincorporated nucleotides and reagents were removed by transferring the solution into a Microcon 30 (Millipore) concentrator pre-filled with 450 l of water and spinning at 12,000 rpm in a microfuge for 8 min. The flowthrough of the Microcon 30 was removed. The clean-up process was repeated twice by refilling the original filter. The cDNA sample was eluted, dried in a speed vacuum and resuspended in 4.5 l of water. The resuspended sample was then mixed with 4.5 l of monofunctional dye NHS-ester Cy3 (for cDNA from cells at passage 20) or Cy5 (for cDNA from cells at passage 60) (Amersham) suspended in 0.1 M sodium bicarbonate (pH 9.0). The mixture was incubated at room temperature for 1 h in the dark. The reaction was quenched by incubating with 4.5 l of 4 M hydroxylamine at room temperature for an additional 15 min in the dark. To remove unincorporated/quenched dyes, the reactions of Cy3 and Cy5 were combined, loaded onto a Qia-quick PCR purification spin column (Qiagen). Purification was performed as per the manufacturer’s protocol. The Qia-quick eluate (probe) was dried in speed vacuum and resuspended with 18 l of water, 3.6 l of 20⫻ SSC, 1.8 l of 10 mg/ml poly(A) DNA (Amersham) and 0.54 l of 10% SDS. The probe was incubated at 100°C for 2 minutes and applied to a prepared microarray (glass slide based). Hybridization was carried out at 65°C overnight in a hybridization chamber (Corning).
Analysis of microarray data. Microarray images from two-color fluorescent hybridizations were scanned using a fluorescent laser scanner (Genetic MicroSystem) at the UCLA Microarray Core Facility. The scan results were saved on a ZIP disk under TIFF format and subsequently analyzed using a program called ScanAlyze (written by Michael Eisen, Stanford University). Images were gridded to locate the spot corresponding to each gene. Following gridding, information on each spot, including fluorescence intensities, background intensities and fluorescence ratios, was extracted using ScanAlyze and presented as a Microsoft Excel file. Fluorescence ratios were ultimately used to determine differentiation of gene expression. Genes with ratio number above 2 or under 0.5, i.e., 2 fold induction or repression, were selected and subjected to further analysis. Northern blot. Plasmids containing cDNAs for genes of interest were obtained from the UCLA microarray Core Facility. Probes were made from amplifying plasmid DNAs using universal primers. The total RNA from cultured MC3T3-E1 cells was isolated using RNeasy kit (Qiagen). The total RNA was quantified by a spectrophotometer at wavelength of 260 nm. Northern blots were performed as described (13). Briefly, total RNA was separated on formaldehyde denaturing 1% agarose gels. The gels were blotted onto nylon membrane using a vacuum transfer device (Pharmacia) according to the manufacturer’s instruction. The membrane was UV cross-linked and hybridized using 32P-labeled probes for 16 h. Hybridized membranes were then washed twice in 2⫻ SSC, 0.1% SDS at room temperature followed by a high-stringency wash in 0.2⫻ SSC, 0.1% SDS at 50°C for 30 min. The relative amounts of mRNA were detected by autoradiography. The GAPDH gene, whose expression does not change as passage number increases, was used as a control.
RESULTS Gene Expression Pattern of MC3T3-E1 Cells during Serial Passage We have demonstrated in a previous study that MC3T3-E1 cells exhibit significant changes in growth and differentiation after reaching passage 60 (7). During the process of serial passage, MC3T3-E1 cells demonstrate reduced growth, reduced response to BMP-2 and TGF-, as well as decreased osteoblastic function. Serial passage also causes a decrease in the number of S-phase positive cells and a decrease in the rates of DNA synthesis in MC3T3-E1 cells (Peterson and Yamaguchi, unpublished results). All these phenotypic changes are indicative of cell aging in osteoblasts (14). To further explore the molecular mechanisms responsible for these aging-associated changes, we compared the gene expression pattern between cells at passage 20 and 60 using a cDNA microarray assay. In each experiment, total RNA was used as template for synthesis of Cy-3 or Cy-5 labeled cDNA probes and hybridized to a microarray containing 2000 mouse cDNA fragments. The fluorescence intensity of each cDNA spot was measured as described under Materials and Methods. To ensure reproducibility of microarray results, we repeated the assay twice using the same total RNA samples. The genes which displayed at least 2-fold difference in the level of transcripts in all three independent experiments were selected as genes that are differentially expressed during serial passage of MC3T3-E1 cells. Figure 1 represents a typical hybrid-
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List of Differentially Expressed Genes during Serial Passage Clone ID
Description
Fold
Genes up-regulated during serial passage 481134 571759 620546 483753 902923 536464 876166 903370 524540 482943 948567 904738 652644 532186 481322 620221 920268 922371 922965 777549
ESTs Secreted phosphoprotein 1 Protein phosphatase 3, catalytic subunit, alpha isoform ESTs Tissue inhibitor of metalloproteinase 2 Phospholipase A2 group VII (platelet-activating factor acetylhydrolase) ESTs, highly similar to INTERFERON-INDUCIBLE PROTEIN [Rat] ESTs, highly similar to NADH-CYTOCHROME B5 REDUCTASE [Rat] odz (odd Oz/ten-m) homolog (Drosophila) related 1 Synaptonemal complex protein 3 Mus musculus receptor activity modifying protein 2 mRNA, Speckle-type POZ protein Diaphorase 4 (NADH/NADPH) CUG triplet repeat, RNA-binding protein 2 High mobility group protein I ESTs, weakly similar to P9513.2 gene product [S. cerevisiae] Ring finger protein 11 Eukaryotic translation initiation factor 4, gamma 2
5.4 4.1 3.9 3.4 2.8 2.6 2.6 2.5 2.3 2.2 2.2 2.1 2.1 2.1 2.1 2.1 2.1 2.0 2.0 2.0
Genes down-regulated during serial passage 808000 658260 888553 480620 619970 820167 482170
Immediate early response 5 ESTs ESTs, Highly similar to protocadherin [R. norvegicus] Procollagen, type VI, alpha 3 ESTs Cysteine rich protein 61 Four jointed box 1 (Drosophila)
2.6 2.3 2.1 2.0 2.0 2.0 2.0
Note. Clone ID and description of genes are adopted from the information provided by the UCLA Microarray Core Facility. Listed on the top are the up-regulated genes while on the bottom are the down-regulated genes. Fold of change for each individual gene is the average of three independent microarray experiments, and is presented under the column “Fold.”
ization result in which the cDNA probe derived from cells at passage 20 is labeled with Cy-3 fluorochrome (green) and the cDNA probe from cells at passage 60 is labeled with Cy-5 fluorochrome (red). Green (e.g., clone ID 619970) and red (e.g., protein phosphatase 3) fluorescent signals indicate greater expression in passage 20 and passage 60, respectively, while yellow (e.g., zipcode binding protein 1) fluorescent signals equivalent level of expression. Using a cutoff value of 2-fold, which is conventionally used by similar studies, we isolated 20 genes which displayed a ⬎2-fold increase in the levels of transcript between cells at passage 20 and 60 in multiple independent experiments. Only 7 genes were found to be down-regulated more than 2-fold during the process of serial passage. A list of identified genes is shown in Table 1. Among the up-regulated genes during cell aging, osteopontin (secreted phosphoprotein 1) is a prominent osteoblastic marker which can function as an inhibitor of mineralization. Factors involved in a wide variety of biological
processes were also up-regulated, including transcription factors (such as high mobility group protein 1 (HMG1)) and translation factors (translation initiation factor 4, gamma 2). We also found significantly elevated expression of tissue inhibitor of metalloproteinase 2 (TIMP-2), a gene product responsible for the regulation of the activity of metalloproteinase-2 which is a major member of a family of proteolytic enzymes involved in remodeling of extra-cellular matrix and normal morphogenesis (15). Interestingly, expression of a gene encoding for the alpha isoform of protein phosphatase 3 was also highly up-regulated. This increase might have significant implications during the process of cellular aging because another family member of protein phosphatase, namely protein phosphatase 2A, has been found to be involved in deactivating telomerase, an enzyme whose activity diminishes with aging (16). Included in the down-regulated genes were those encoding extracellular matrix proteins such as procollagen (type VI, alpha3) and a gene similar to protocadherin. Other down-regulated genes included
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FIG. 2. Northern blot analysis of gene expression during serial passage. To verify microarray results on selected genes, we subjected the same total RNA samples used in microarray assays to Northern blot analysis. Names for selected genes (see text for abbreviations) were labeled at the right. Band intensities were measured by densitometry and used to quantify the fold of change, which is given at the left. (A) GAPDH control, (B) down-regulated genes, (C) up-regulated genes.
genes encoding immediate early response 5, cysteinerich protein 61 and four jointed box 1, as well as two unknown genes. Verification of Expression of Selected Genes To verify the microarray results, we performed Northern blots using the same total RNA samples prepared for microarray studies. Probes for each tested gene were PCR products amplified from plasmid DNAs containing appropriate cDNAs. Among up-regulated genes, we selected osteopontin, protein phosphatase 3, TIMP-2, HMG1 and two highly up-regulated ESTs for Northern blot verification (Fig. 2C). From the list of down-regulated genes, we selected genes encoding procollagen VI and immediate early response 5 to be verified (Fig. 2B). A housekeeping GAPDH gene, whose expression was unchanged during senescence, was chosen to serve as a control (Fig. 2A). Our Northern blot results, shown in Fig. 2, demonstrated identical expression pattern for all selected genes as revealed by microarray analysis. The results indicated that our
microarray data, derived from three independent experiments, were highly reproducible and reliable. Quantification of Northern blot bands using densitometry showed change of expression level for all of the genes are was more significant than the original quantification given by microarray hybridization. For example, our microarray data showed a 4.1-fold increase of osteopontin gene expression (Table 1) while the Northern blot results showed a 15.1-fold increase (Fig. 2C). Likewise, a 2.6-fold decrease in expression of immediate early response 5 gene (Table) revealed by microarray assay was revised to a 11.9-fold decrease by Northern blot analysis (Fig. 2B). Comparison of these results illustrated that significant changes have occurred in gene expression during serial passage or senescence of osteoblastic cells. Although we observed identical patterns of expression using two independent measurements, the quantitative discrepancy revealed by comparing these two measurements indicates that there are likely some target genes not identified by microarray assay alone.
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DISCUSSION The significance of studies in bone aging is apparent due to wide occurrence of age-related bone diseases such as osteoporosis. In an attempt to isolate genes involved in the aging of bone cells, we have used MC3T3-E1 cells undergoing serial passage as a model of in vitro aging. In this study, we have identified 27 senescence-associated genes using a cDNA microarray containing 2000 sequence-verified mouse genes. We selected 8 genes for Northern blot verification. All of them were confirmed to be differentially expressed, indicating that our microarray data are consistent with the results of traditional gene expression analysis. During the aging process, the ability of osteoblasts to form new bone is greatly diminished (5). Our results showed that osteopontin gene expression is highly elevated in cells at passage 60. It has been shown that osteopontin accumulated at the late stage of mineralization can function as an inhibitor of mineralization (17, 18). Thus, this significantly increased osteopontin expression might be accountable for the decreased activity of mineralization and bone formation in aged cells. It is also noteworthy that osteopontin is a multifunctional factor and has been shown to be up-regulated in older macrophages compared to younger cells according to a separate study (19). Another interesting gene identified by this study is protein phosphatase 3. It has been shown that telomerase activity can be regulated by protein phosphorylation. For example, incubation of cell nuclear telomerase extracts with protein phosphatase 2A (PP2A) abolished the telomerase activity (16). Since decreased telomerase activity is closely related to cell aging, it is likely that elevated expression of protein phosphatase 3 could result in decreased telomerase activity, which could further result in the arrest of proliferation. Differentiation of osteoblasts involves the biosynthesis and organization of bone extracellular matrix, which can lead to the formation of mineralized bone. Studying the gene expression of matrix proteins and components involved in matrix reconstruction is crucial in understanding how bone formation is regulated. Our study has identified several genes that are closely related to extracellular matrix formation, namely TIMP-2, procollagen VI and a gene highly similar to protocadherin. Among these genes, TIMP-2 is involved in the reconstruction of extracellular matrix by binding to matrix metalloproteinases, and also possesses mitogenic activities on a number of cell types (15). It has been shown to inhibit basic fibroblast growth factorinduced human endothelial cell growth (20). The inhibitory effect of growth by TIMP-2 is consistent with its overexpression in senescent cells. Procollagen VI has been shown to play important roles for interleukin-4 induced mineralization, type I collagen accumulation, and hydroxyapatite accumulation in human periosteal
osteoblast-like cells (21). Reduction of procollagen VI production is therefore expected in aged osteoblasts where bone formation is significantly decreased. We believe that the expression patterns of some known target genes are consistent with senescent or aging phenotypes and have great implications in the aging studies of osteoblasts. We are currently characterizing some of the isolated genes to determine their regulatory roles in bone formation. Comparing our gene expression data in osteoblasts with those in fibroblasts, we have found similar expression patterns among some of the genes. For example, genes encoding tissue inhibitor of metalloproteinase 2 and enzymes involved in NADH metabolism are elevated in both types of cells during senescence (22, 23), indicating that there might be a certain set of senescence-associated genes responsible for common phenotypes in different cell types during cellular aging. In conclusion, we have demonstrated that gene expression is significantly altered in MC3T3-E1 cells undergoing serial passage. Dissection of the identified genes suggests that a wide range of biological processes are involved during cell aging. Confirmation by Northern blot analyses showed a more drastic change of gene expression than that revealed by microarray studies. Verification of the data demonstrated that our microarray results are reproducible and consistent with results from Northern blot analysis. ACKNOWLEDGMENTS We thank the UCLA Microarray Core Facility for providing cDNA microarrays and cDNA probes used in the above study. We thank Dr. William Peterson for critical reading of the manuscript and sharing information on his studies with MC3T3-E1 cells. This study was supported by a Merit Review Grant of the Department of Veterans Affairs and the Thomas and Arlene Bannon Foundation Research Fund to T.A.M.
REFERENCES
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1. Harley, C. B., Futcher, A. B., and Greider, C. W. (1990) Nature 345, 458 – 460. 2. Cristofalo, V. J., Volker, C., Francis, M. K., and Tresini, M. (1998) Crit. Rev. Eukaryot. Gene Expression 8, 43– 80. 3. Faragher, R. G., and Kipling, D. (1998) BioEssays 20, 985–991. 4. Shelton, D. N., Chang, E., Whittier, P. S., Choi, D., and Funk, W. D. (1999) Curr. Biol. 9, 939 –945. 5. Liang, C. T., Barnes, J., Seedor, J. G., Quartuccio, H. A., Bolander, M., Jeffrey, J. J., and Rodan, G. A. (1992) Bone 13, 435– 441. 6. Sudo, H., Kodama, H. A., Amagai, Y., Yamamoto, S., and Kasai, S. (1983) J. Cell Biol. 96, 191–198. 7. Chung, C. Y., Iida-Klein, A., Wyatt, L. E., Rudkin, G. H., Ishida, K., Yamaguchi, D. T., and Miller, T. A. (1999) Biochem. Biophys. Res. Commun. 265, 246 –251. 8. Schena, M., Shalon, D., Davis, R. W., and Brown, P. O. (1995) Science 270, 467– 470. 9. Schena, M., Shalon, D., Heller, R., Chai, A., Brown, P. O., and Davis, R. W. (1996) Proc. Natl. Acad. Sci. USA 93, 10614 –10619.
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10. Lander, E. S. (1999) Nat. Genet. 21, 3– 4. 11. Ferea, T. L., and Brown, P. O. (1999) Curr. Opin. Genet. Dev. 9, 715–722. 12. Brown, P. O., and Botstein, D. (1999) Nat. Genet. 21, 33–37. 13. Sambrook, J., Maniatis, T., and Fritsch, E. F. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 14. Kassem, M., Ankersen, L., Eriksen, E. F., Clark, B. F., and Rattan, S. I. (1997) Osteoporos. Int. 7, 514 –524. 15. Nagase, H., and Woessner, J. F., Jr. (1999) J. Biol. Chem. 274, 21491–21494. 16. Li, H., Zhao, L. L., Funder, J. W., and Liu, J. P. (1997) J. Biol. Chem. 272, 16729 –16732.
17. MacNeil, R. L., Berry, J., D’Errico, J., Strayhorn, C., Piotrowski, B., and Somerman, M. J. (1995) Connect. Tissue Res. 33, 1–7. 18. Gerstenfeld, L. C. (1999) J. Bone Miner. Res. 14, 850 – 855. 19. Rollo, E. E., and Denhardt, D. T. (1996) Immunology 88, 642– 647. 20. Murphy, A. N., Unsworth, E. J., and Stetler-Stevenson, W. G. (1993) J. Cell Physiol. 157, 351–358. 21. Ishibashi, H., Harumiya, S., and Koshihara, Y. (1999) Biochim. Biophys. Acta 1472, 153–164. 22. Zeng, G., and Millis, A. J. (1994) Exp. Cell Res. 213, 148 –155. 23. Kodama, S., Yamada, H., Annab, L., and Barrett, J. C. (1995) Exp. Cell Res. 219, 82– 86.
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