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CBFA1 and Topoisomerase I mRNA Levels Decline During Cellular Aging of Human Trabecular Osteoblasts

^a Danish Centre for Molecular Gerontology, Laboratory of Cellular Ageing, Department of Molecular and Structural Biology
^b University Department of Endocrinology and Metabolism, University of Aarhus, Denmark.

Suresh I. S. Rattan, Laboratory of Cellular Ageing, Department of Molecular and Structural Biology, University of Aarhus, Gustav Wiedsvej 10 C, DK-8000 Aarhus C, Denmark E-mail: rattan{at}imsb.au.dk.

Decision Editor: Jay Roberts, PhD

	Abstract

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In order to understand the reasons for age-related impairment of the function of bone forming osteoblasts, we have examined the steady-state mRNA levels of the transcription factor CBFA1 and topoisomerase I during cellular aging of normal human trabecular osteoblasts, by the use of semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR). There is a progressive and significant reduction of the CBFA1 steady-state mRNA level down to 50% during cellular aging of human osteoblasts. In comparison to the normal cells, human osteosarcoma cell lines SaOS-2 and KHOS/NP, and the SV40-transformed human lung fibroblast cell line MRC5V2 have 20 to 40% higher levels of CBFA1 mRNA. Similar levels of CBFA1 mRNA are detectable in normal human skin fibroblasts, and these cells also exhibit an age-related decline to the same extent. In addition, the expression of topoisomerase I is reduced by 40% in senescent osteoblasts, and the mRNA levels are significantly higher (40–70%) in transformed osteoblasts and fibroblasts. These changes in gene expression may be among the causes of impaired osteoblast functions, resulting in reduced bone formation during aging.

WE have demonstrated that serially passaged cultures of normal human trabecular osteoblasts exhibit limited proliferative capacity and undergo cellular aging in vitro ⁽¹⁾⁽²⁾. This is similar to the well-known Hayflick phenomenon of replicative senescence described for several other normal human diploid cell types including fibroblasts, keratinocytes, and lymphocytes (for a recent review see ref. ⁽³⁾). We have documented a number of changes during serial passaging of human trabecular osteoblasts, which include altered morphology and cytoskeleton; increase in cell size and in senescence-associated ß-galactosidase activity; a reduction in macromolecular synthesis; and reduced production of alkaline phosphatase and collagen type I ⁽²⁾. We have also reported a decrease in mean telomere restriction fragment length ⁽⁴⁾, an upregulation of putative gerontogenes, particularly Apo J and GTP- ⁽⁵⁾, and a reduction in the expression of a number of osteoblast-specific genes during cellular aging of osteoblasts ⁽⁶⁾. Although full implications of these observations are not clear at present, some of these events are thought to be crucial for the observed cessation of proliferation in vitro, others will eventually lead to reduction in osteoblast-specific functions.

Osteoblasts are known to be essential for bone formation in vivo, and decreased bone formation is the primary cause of age related bone loss during bone remodeling, in severe cases leading to osteoporosis ⁽⁷⁾⁽⁸⁾. Recently the CBFA1 transcription factor was identified as being important for osteoblast functions ⁽⁹⁾. CBFA1 is also referred to as PEBP2A (polyoma enhancer–binding protein) and AML3 (acute myelogenous leukemia), and it shares homology with runt, a Drosophila pair-rule gene ^(10)(11). When the gene is disrupted in mice, the result is a complete lack of ossification due to an arrest in osteoblast development in homozygous mice, which die just after birth ^(12)(13). Mutations in the gene are associated with cleidocranial dysplasia, a human autosomal inherited skeletal disorder, indicating its importance during osteoblast differentiation in humans as well ⁽¹⁴⁾. CBFA1 heterodimerises with Cbfß and the heterodimer binds to consensus elements in the promoter region of genes known to be regulated during osteoblast development ^(15)(16)(17). The heterodimer is localized at the nuclear matrix, whereby a link is established between the promoter region and the nuclear matrix ⁽¹⁸⁾. The CBFA1 gene is comprised of nine exons numbered -1 to 7 and eight introns, and alternative splicing events give rise to several isoforms ^(19)(20). A mouse isoform of CBFA1, termed Osf2, in which a novel N-terminus arises due to translation initiation in exon -1, has been reported as the osteoblast-specific product compared to the more generally expressed isoform ⁽⁹⁾. However, in human osteoblasts, this N-terminal sequence is not expressed ⁽²⁰⁾. Because there is no information available on age-related changes in the expression of CBFA1, we have undertaken studies to determine its mRNA levels in human osteoblasts undergoing aging in culture.

We have also studied the expression level of the topoisomerase I (topo I) gene during cellular aging of osteoblasts. This is because topo I is essential for the survival of cells, as it is involved in removing the topological stress that inadvertently arises during DNA replication, transcription, and possibly DNA repair, thus it is involved in maintaining genomic stability ⁽²¹⁾. Topo I produces transient, single-stranded nicks in the DNA and reduces supercoiling by controlled rotation of the DNA ^(22)(23). The enzyme can be involved in decreasing stability as well, when the covalent link between DNA and topo I gets trapped by drug interaction or by loss of the free DNA strand ⁽²²⁾. Topo I was reported to be active in ribosomal DNA (rDNA) regions ⁽²⁴⁾, and decreased levels of topo I and II have been shown to lead to the excision of extrachromosomal rDNA rings in yeast cells ⁽²⁵⁾; these rings accumulate during yeast aging and can even induce aging ⁽²⁶⁾. However, there has been some controversy about the changes in the expression of topo I during cellular aging of fibroblasts ^(27)(28), and its expression during cellular senescence of osteoblasts has not been measured previously.

Here we report an age-related decline in mRNA levels of the transcription factor CBFA1 and an essential survival gene topo I in human bone cells undergoing aging in vitro.

	Materials and Methods

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Cell Cultures
The following cell types and cell lines have been used in this study: (a) normal human trabecular osteoblast culture, designated M15, established from trabecular bone biopsies from a 15-year-old healthy male following a procedure described by Kassem and colleagues ⁽²⁹⁾; (b) human diploid skin fibroblast culture, designated ASS-2, established from breast biopsies from a 20-year-old healthy female donor ⁽³⁰⁾; (c) spontaneously transformed human osteosarcoma cell line SaOS-2 and Kirsten murine sarcoma virus–transformed osteoblast cell line KHOS/NP, both purchased from the American True Type Culture Collection (Rockville, MD); and (d) the SV40-transformed human embryonic lung fibroblast cell line MRC5V2 ⁽³¹⁾.

Cells were grown in complete medium consisting of Dulbecco's Minimal Essential Medium (DMEM, Bio Whittaker, Belgium) supplemented with 10% fetal calf serum (FCS, In Vitro, Denmark), 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin (Bio Whittaker). Cells were grown at 37°C in humidified, 5% CO₂ and 95% air. Such cell cultures are refered to as heterogeneous/nonsynchronized cell cultures (H). G₁-arrested, synchronized cells (S) were obtained by serum-starvation of cells for 72 hours in DMEM containing 0.2% FCS and tested by the incorporation of 5'-bromo-2'-deoxyuridine, using labeling and detection kit II following a protocol supplied by Boehringer Mannheim (Mannheim, Germany). Restimulation after starvation was done for 18 hours in complete medium (restimulated cells, R).

Serial Passaging
Almost confluent cultures of M15 and ASS-2 were trypsinized and split at a 1:8, 1:4, or 1:2 ratio, depending on the cell density, and medium was changed twice a week. Serially passaged cultures were considered to have become senescent and completed their replicative life span when no increase in cell number had occurred within 1 month. At each subculturing, the number of cells was counted using a Coulter Counter (Coulter Electronics, UK), and the number of population doublings (PD) was calculated as log(N/N₀)/log2, where N is the output cell density at the time of splitting and N₀ is the input cell density. Cumulative population doubling level (CPDL) attained after serial passaging was referred to as 100% life span completed. In this study cells were considered to be young if they had completed <30% life span and cells were considered to be old if they had completed >90% life span.

Cells at different PD levels were checked for senescence- associated ß-galactosidase activity at neutral pH, which is considered as a marker of cellular aging ⁽³²⁾. In brief, cells were fixed in 2% formaldehyde and 0.2% glutaraldehyde in phosphate-buffered saline (PBS) for 5 minutes, washed in PBS, and stained overnight at 37°C in 1 mg/mL X-gal, 40 mM citric acid/sodium phosphate buffer (pH 6), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM sodium chloride, and 2 mM magnesium chloride. For analysis of morphological changes, cells were fixed in -20°C methanol for 30 minutes and thereafter stained with Giemsa stain for 1 hour. After washing in tap water, cells were analyzed under a microscope.

RNA Isolation
RNA was extracted by the method of Davis and colleagues ⁽³³⁾. Briefly, cells were washed twice in Hank's buffer and lysed in 4 M guanidine isothiocyanate (GITC), 25 mM sodium acetate (NaAc) pH 6, and 0.83% ß-mercaptoethanol. The lysate was layered on top of 5.7 M CsCl, final concentration 0.5 M, and centrifuged at 32,000 rpm for 20 hours in a SW40 rotor. The supernatant was discarded, RNA was redissolved in diethylpyrocarbamate treated H₂O (DEPC-H₂O), and NaAc pH 6 was added to a final concentration of 0.3 M. RNA was precipitated using 2.5 volumes 100% ethanol and by incubating for 30 minutes at -80°C. Finally, RNA was pelleted by centrifugation, redissolved in DEPC-H₂O and stored at -80°C.

RT-PCR
Noncompetitive quantitative RT-PCR was performed by reverse transcription (RT) of a known amount of RNA and subsequently running triplicate polymerase chain reactions (PCR) with specific primers ^(34)(35). For each primerset the optimal hybridization temperature and primer concentration were determined, and the number of cycles was set within the linear range of the amplification. The PCR products were quantified by either using -³²P-dATP (Amersham, Buckinghamshire, UK) incorporation during PCR, followed by scintillation-counting of correctly sized bands excised from agarose gels after electrophoresis, or by measuring intensity of bands visualized by UV-irradiation of ethidium bromide (EtBr)-stained gels using a Gel-Doc and Molecular Analyst software (BioRad, CA). In the latter case, intensity of the 653-basepair (bp) molecular-weight marker band was used to correct for differences in gel thickness and EtBr differences between gels. Initially, semiquantitative RT-PCR was performed on RNA isolated from the osteosarcoma cell line SaOS-2 to characterize the two different methods for quantifying the PCR products. Both methods gave reproducible results, and were comparable in sensitivity and detection range (data not shown). Therefore, for further studies the rapid and relatively inexpensive Gel-Doc method was used, and the use of radioactivity was avoided.

For RT, 4 µg RNA in 3 µL DEPC-H₂O, 1 µL oligo d(T)₁₆ (50 pmol/µL), and 2 µL random hexamer d(NTP)₆ (100 pmol/µL) was heated at 65°C for 10 minutes, followed by incubation on ice for 5 minutes. RT was then performed by adding 3 µL AMV RTase (24 U, Promega, Madison, WI), 2 µL dNTP mix (dATP, dGTP, dCTP, and dTTP each at 20 mM), 4 µL 5x AMV buffer (50 mM MgCl₂, 250 mM KCl, 250 mM Tris pH 8.3, 50 mM dithiotreitol [DTT], 2.5 mM spermidine), and 1 µL RNase inhibitor (20 U, Promega), and by incubating at 42°C for >4 hours. One twentieth of the RT product was used for a PCR and a control containing H₂O was included each time a PCR was performed. Reactions were performed by initially incubating 0.1 µL (5 U/µL) Taq Polymerase (Promega, Madison, Wisconsin) with 0.1 µL Antitaq (Clontech, Palo Alto, CA) in 0.4 µL anti-Taq buffer for 5 minutes at room temperature. This was then added to 2 µL dNTP mix (2 mM), 1 µL MgCl₂ (25 mM), 1.7 µL 10x Taq buffer (500 mM KCl, 100 mM Tris pH 9, 1% Triton X-100), 1 µL of each primer (3–10 µM), 3 µL RT product, and, in the case of radioactive-labeling, 1 µCi -³²P-dATP. The Taq polymerase amplification was then performed by initially denaturing at 94°C (2 minutes), followed by 20 to 44 cycles at 94°C (1 minute), 55°C (2 minutes), and 72°C (2 minutes). Finally the samples were incubated at 72°C (8 minutes).

Primers
Primers were chosen to avoid the possible amplification of genomic DNA, that would give rise to a larger sized PCR product, which, however, was never seen. Identity of the PCR products was verified by sequencing (data not shown). For each primer set the optimal conditions were found using RNA isolated from young heterogeneous cultures of osteoblasts and PCR products were only compared when obtained during the exponential phase of amplification. Primer concentration and number of cycles differed markedly (Table 1 ), thus indicating that CBFA1 and topo I mRNAs were less abundant than glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA. It must be noted though, that primer and product characteristics influence the efficiency of amplification as well.

	Results

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Serially passaged normal human trabecular osteoblast cell strain M15 exhibited the typical phenomenon of cellular aging and a limited proliferative capacity in culture. A representative longevity curve (Fig. 1A) shows that this particular strain of osteoblasts attained 50 CPDL (=100% life span completed) within a period of 495 days. In order to monitor the senescence status of the osteoblast culture, cells were stained for senescence-associated ß-galactosidase (SA-ß-gal) activity, and with Giemsa stain to characterize the morphological changes these cells undergo during their life span. The osteoblast cultures exhibited the expected age-related increase in SA-ß-gal activity, such that less than 1% of the cells stained positive in young cultures (<30% life span completed), whereas more than 90% of the cells stained positive in old cultures (>95% life span completed, pictures not shown). Fig. 1B shows the longevity curve of ASS-2 skin fibroblasts, which were used for comparative analysis. In these series of experiments the ASS-2 cell strain underwent 35 CPDL (=100% life span completed) in a period of 206 days and was comparable to that reported previously ⁽³⁰⁾.

View larger version (11K): [in this window] [in a new window]   Figure 1. Longevity curves of (A) normal human trabecular osteoblasts and (B) normal human diploid skin fibroblasts serially passaged in vitro.

View larger version (27K): [in this window] [in a new window]   Figure 2. RT-PCR performed on RNA isolated from heterogeneous (H, shaded bars), synchronized (S, white bars), and restimulated (R, black bars) cultures of normal human trabecular osteoblasts M15, and from heterogeneous cultures of immortal osteoblast cell- lines KHOS/NP and SaOS-2 and of immortal lung fibroblast cell-line MRC5V2. (A) Representative EtBr-stained gels. (B) CBFA1 and topo I intensities are expressed as the mean ± SD of the intensity of triplicate reactions relative to the intensity of the 653-bp band, relative to the corresponding GAPDH intensity and finally normalized against young (19% life span completed) heterogeneously growing M15 osteoblasts. Intensities of GAPDH bands, expressed as the mean ± SD of three PCR reactions, relative to the 653-bp band of the marker and normalized to young heterogeneously growing M15 osteoblasts. *p < .01; **p < .001 represent statistically significant deviations from the level in young heterogeneous M15 cell cultures.

View larger version (23K): [in this window] [in a new window]   Figure 3. Amplification of GAPDH and CBFA1 mRNA in heterogeneously growing normal human diploid skin fibroblasts ASS-2. (A) EtBr-stained agarose gels, and (B) mean ± SD intensities of bands expressed relative to the 653-bp band of the marker. CBFA1 is expressed relative to GAPDH intensities and both are normalized against young (34% life span completed) ASS-2 cells. **p < .001 represent statistically significant deviations from the level in young heterogeneous ASS-2 cell cultures.

As regards topo I gene expression, its mRNA levels remained fairly constant in human osteoblasts until about 70% life span was completed (Fig. 2). However, the level was significantly reduced to approximately 60% (p < .01) in nonsynchronized senescent cultures. Whereas the topo I mRNA levels remained constant in heterogeneous, synchronized, and serum-restimulated early passage cultures, late passage osteoblasts exhibited significantly elevated levels of topo I both during starvation and after restimulation (statistical analysis not shown). Furthermore, there was a significantly ( p < .01) higher expression in the immortal cell lines, as compared to the levels in young heterogeneous osteoblasts cell cultures. We have also tried to determine the protein levels of topo I in young and old osteoblasts and fibroblasts, and in the transformed KHOS/NP cell line by Western blotting using a C-terminal–specific antibody. However, at the protein level, the enzyme was only detectable in the KHOS/NP cells and no comparison between young and old cells could be made (results not shown).

	Discussion

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In this study we have characterized age-related changes in the steady-state mRNA levels of CBFA1 and topo I genes, thus facilitating a further understanding of reduced osteoblast functions during cellular aging. Quantification of mRNA relies on the use of an internal control, and in the present study the constitutively expressed housekeeping gene GAPDH was used for this purpose. Although it has been reported that the GAPDH mRNA levels vary to some extent during the cell cycle ^(36)(37), we have observed quite stable levels of the mRNA during the growth phase and G₁-arrest at different passage levels in several cell types. In our hands the mRNA level of this gene therefore qualifies as a useful internal standard.

The transcription factor CBFA1 is known to be very important for the differentiation of both mouse and human osteoblasts in vivo ^(9)(13)(14). We have found that the levels of CBFA1 mRNA decreased significantly and progressively during cellular aging of human trabecular osteoblasts. As CBFA1 protein is known to bind osteoblast-specific cis-acting elements (OSE2) in genes expressed during osteoblast differentiation ⁽⁹⁾, a reduction in its levels could place it as a possible upstream factor controlling age-related reductions in other genes. The osteocalcin gene is an example of an osteoblast specific gene, which harbors an OSE2, and the mRNA levels of this gene has been shown by us to be downregulated during cellular aging of human osteoblasts ⁽⁶⁾. Therefore, it is likely that the age-related decrease in osteocalcin mRNA levels could be the consequence of a decreased CBFA1 expression, which eventually results in decreased osteoblast functions, such as decreased formation of extracellular matrix and reduced mineralization. Although it is not known whether the reduced mRNA level of CBFA1 mRNA during cellular aging is accompanied by a similar decrease in its protein levels, it was recently demonstrated that cells which have a high level of CBFA1 mRNA after transfection with the CBFA1 gene have increased osteocalcin, osteopontin, and collagen type I mRNA levels ⁽³⁸⁾. It would be of great value to determine changes of CBFA1 mRNA and protein during in vivo aging, and its role in the origin of osteoporosis.

Previously, the expression of CBFA1 has been reported by the use of Northern blotting to be confined to osteoblasts ^(19)(20), though it was initially found to be expressed in T cells ⁽³⁹⁾. However, we detected in addition to high mRNA levels in the two immortal osteoblast cell lines used, a high level of CBFA1 mRNA in SV40-transformed lung fibroblasts as well. This encouraged us to investigate its levels in normal human diploid fibroblasts undergoing aging in vitro. These cells displayed a comparable level of the CBFA1 mRNA, and a significant age-related decrease to about 50% of the level in young cells was also observed. A possible explanation for the detection of CBFA1 mRNA in fibroblasts is that the primers employed in this study do not distinguish between the different isoforms, because exons 0 and 6 are known to be differentially spliced, possibly in a tissue-specific manner ^(19)(20)(40). Therefore, although our studies suggest that there is a general age-related decrease in the global expression of CBFA1, it will be interesting to determine changes in the expression pattern of the different isoforms of CBFA1 with primers encompassing the differentially spliced exons.

Additionally, we have investigated the mRNA levels of the type I class of topoisomerases during cellular aging. Two previous reports ^(27)(28) looking at in vitro aging of fibroblasts have shown opposite results, and we were therefore prompted to investigate the levels in a new cell type and with a more sensitive method. Our results show a 40% decrease in the topo I mRNA level at senescence (>90% osteoblast life span completed), supporting a previous observation in fibroblasts ⁽²⁸⁾. However, it is important to note that as the decrease in topo I mRNA level is not significant until cells are very near senescence, it might just be a consequence of cellular aging in vitro. Although we were unable to detect the topo I protein in normal osteoblasts, due to the low amounts present, it is known that topo I mRNA levels reflect topo I activity very well ⁽²⁸⁾. Therefore, reduced topo I content in aging cells can be one of the factors involved in age-related decrease in genomic stability ⁽⁴¹⁾. It was recently reported that topoisomerase inhibitors actually induce premature cellular senescence ⁽⁴²⁾.

Furthermore, we have observed that the steady-state level of topo I mRNA is increased during serum-starvation in late passage osteoblasts but not in early passage and middle-aged cells. Previously too, a downregulation of topo I mRNA was demonstrated by treatment with the growth inhibitor retinoic acid in young cells, whereas the same treatment resulted in its upregulation in old cells ⁽²⁸⁾. Because topo I can also cause strand breaks when the covalent link between DNA and topo I gets trapped, it is plausible that serum-starvation of senescent cells induces more damage to DNA. Further studies are, however, needed to determine the exact role of increased topo I expression in serum-starved and stressed senescent cells. In comparison, the high expression level of topo I in the immortal cell is consistent with the previously reported higher expression in tumor cells as compared to normal cells ^(43)(44), and may be a reflection of altered growth characteristics in immortal cells.

Our studies thus show that the impairment of functions of human osteoblasts during aging is accompanied by alterations in the expression in various genes which may be the target for developing therapeutic strategies for treatment and prevention of age-related loss in bone formation.

	Acknowledgments

 
We thank Dr. Ole Westergaard and Mikael Lisby (Aarhus, Denmark) for advice concerning topoisomerase I. These studies were performed partially within the framework of EU Biomed-2 shared cost grant GENAGE.

Received February 16, 1999

Accepted September 14, 1999

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