- Email: [email protected]
6) by decreasing the expression of peroxisome proliferator-activated receptor gamma 2 (PPARγ2)
Experimental Gerontology 39 (2004) 333–338 www.elsevier.com/locate/expgero
1,25(OH)2D3 inhibits bone marrow adipogenesis in senescence accelerated mice (SAM-P/6) by decreasing the expression of peroxisome proliferator-activated receptor gamma 2 (PPARg2) Gustavo Duquea,b,c,*, Michael Macorittoc, Richard Kremerc a
Division of Geriatric Medicine, Department of Medicine, 3755, Cote Sainte Catherine, Montreal, Que., Canada H3T 1E2 b Lady Davis Institute for Medical Research, Jewish general Hospital, Montreal, Que., Canada c Calcium Research Laboratory, McGill University, Montreal, Que., Canada Received 23 July 2003; received in revised form 6 November 2003; accepted 20 November 2003
Abstract Senile osteoporosis is the endpoint of a continuum that starts after the third decade of life when peak bone mass is attained and then is followed by a progressive and irreversible decline in bone mass. One of the mechanisms that could explain this is the increasing levels of adipogenesis in bone marrow seen with increasing age, probably due to alterations in the differentiation of mesenchymal stem cells (MSC). Senescence accelerated mice (SAM-P/6) constitute an accepted model for senile osteoporosis since their loss of bone mineral density is clearly due to high levels of adipogenesis and a deficit in osteoblastogenesis. It is known that MSC expressing a ligand-activated transcription factor known as peroxisome proliferators-activated receptor gamma 2 (PPARg2) are committed to differentiate into adipocytes. The regulation of PPARg2 activation may play a role in the control of adipogenic differentiation of MSC and thus contribute to their differentiation into osteoblasts in order to form new bone. Our previous studies have shown that the active form of vitamin D (1,25(OH)2D3) plays a role as a bone forming agent because it induces osteoblastogenesis and inhibits adipogenesis in bone marrow of SAM-P/6 mice. To elucidate the role of 1,25(OH)2D3 on the expression of PPARg2 we treated 4-month old SAM-P/6 mice with 1,25(OH)2D3 (18 pmol/24 h) or vehicle during 6 weeks. Initially we found that with aging the levels of PPARg2 expression increase in bone marrow of SAM-P/6 ðp , 0:001Þ: We then measured the changes in the expression of PPARg2 by semi-quantitative reverse transcription-polymerase chain reaction and immunofluorescence. We found a significant reduction of PPARg2-expressing cells in 1,25(OH)2D3-treated (32% ^6) as compared to vehicle (76% ^ 5) treated mice ðp , 0:01Þ: In summary, this study shows that the administration of 1,25(OH)2D3 in an in vivo model of senile osteoporosis is associated with reduction in PPARg2 a key transcription factor for the adipose differentiation of MSC. q 2003 Elsevier Inc. All rights reserved. Keywords: Senile osteoporosis; SAM-P/6; Vitamin D; Osteoblastogenesis; Adipogenesis; MAD cells; PPARg2
1. Introduction Senile osteoporosis is the consequence of three major cellular events: declining levels of osteoblastogenesis, increasing number of apoptotic osteoblasts and osteocytes, and increasing levels of bone marrow adipogenesis (Duque and Prestwood, 2003; Chan and Duque, 2002; Justesen et al., 2001; Kirkland et al., 2002). Increasing levels of bone marrow adipogenesis is probably due to the lipid redis* Corresponding author. Address: Division of Geriatric Medicine, Department of Medicine, McGill University, 3755, Cote Sainte Catherine, Montreal, Que., Canada H3T 1E2. Tel.: þ 1-514-340-7501; fax: þ 1-514-340-7547. E-mail address: [email protected] (G. Duque). 0531-5565/$ - see front matter q 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2003.11.008
tribution that happens with aging consisting of a dramatic reduction in the size of fat deposits with concomitant raise of lipid deposits in muscle, bone marrow and other tissues (Kirkland et al., 2002). Although it is known that bone marrow adipogenesis increases with aging (Kirkland et al., 2002; Lecka-Czernik et al., 2002; Moore and Dawson, 1990; Spiteller, 2001) the underlying mechanisms to explain this phenomenon remain unclear. Adipocytes belong to the group of mesenchymal cells and share the same precursors as the osteoblasts, myocytes and chondrocytes. Mesenchymal stem cells (MSC) differentiate into pre-adipocytes and thus into mature adipocytes after exposure to multiple factors including glucocorticoids, insulin growth factor-1, agents
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that increase pre-adipocyte cyclic AMP and other hormonal effectors (Bianco and Gehron Robey, 2000; Kirkland et al., 2002). After exposure to these factors, changes in transcription factors occur with the subsequent genes activation and development of fat cell phenotype (Kirkland and Dobson, 1997). The two main transcription factors involved in the differentiation of MSC into adipocytes are the peroxisome proliferator-activated receptor gamma 2 (PPARg2) and CCAT enhancer binding protein alpha (C/EBPa) (Clarke et al., 1997). The expression of one of these two factors is required for adipocytes differentiation to proceed (Kirkland et al., 2002). Increasing levels of adipogenesis in aging bone is probably due to ‘dysdifferentiation of mesenchymal cells into mesenchymal adipocyte-like default cells (MAD cells)’ (Kirkland et al., 2002). The number of MAD cells increases with aging and are able to induce adjacent cells to convert into MAD cells which extend the process (Kirkland et al., 2002; Kirkland and Dobson, 1997). The increasing number of MSC that become MAD cells with aging within the bone marrow is probably the consequence of increasing levels of endogenous PPARg2 (Duque, 2003) and might in part explain the progressive decline on osteoblastogenesis seen in senile osteoporosis. Although MAD cells remain in a stage of dysdifferentiation they keep their potential to differentiate into the osteoblastic lineage (Kirkland et al., 2002) under appropriate stimulation. Our group has found that the active form of vitamin D, 1,25-dyhydroxyvitamin D (1,25(OH)2D3), inhibits adipogenesis and increases osteoblastogenesis (Duque et al., unpublished data) in senescence accelerated mouse P/6 (SAM-P/6) an accepted model of senile osteoporosis (Kajkenova et al., 1997; Takeda et al., 1997). Although the osteogenic effect of 1,25(OH)2D3 in vitro and in vivo is well known (Hida et al, 1998; Suda et al., 2003) its role on regulating bone marrow adipogenesis in vivo and the transcription factors involved in this process remain unclear. In the present study we attempt to elucidate the mechanism that explains the anti-adipogenic effect of 1,25(OH)2D3 by assessing initially the levels of bone marrow expression of PPARg2 in this model and subsequently by looking at the potential effect of 1,25(OH)2D3 on PPARg2 levels which could explain the replacement of adipocytes by active osteoblasts after treatment.
2. Materials and methods 2.1. Animals SAM-P/6 mice were kindly provided by Dr Toshio Takeda (Kyoto University, Kyoto, Japan). Male and female were housed in cages in a limited access room restricted to aging mice (light:dark 12 h:12 h). Bedding, food and water were given as previously described
(Duque et al., 2002). Animal husbandry adhered to Canadian Council on Animal Care Standards, and all protocols were approved by the McGill University Health Center Animal Care Utilization Committee. The colony was free of any parasitic, bacterial, or viral pathogens as determined by a sentinel program. At 4 months of age, mice (n ¼ 12 per group) in a 50:50 ratio of males and females were implanted with Alzet osmotic mini-pumps (Alzet, Cupertino, CA, USA) containing 1,25(OH)2D3 (LEO Pharmaceuticals, Ballerup, Denmark) prepared as previously described (El Abdaimi et al., 2000) and delivering a constant infusion of either 18 pmol/ 24 h of 1,25(OH)2D3 or vehicle alone. Osmotic mini-pumps were replaced after 3 weeks for a total of 6 weeks of treatment. Blood calcium levels were monitored at timed intervals by microchemistry (Kodak Ektachrome, Mississauga, ON, Canada) and corrected for albumin concentrations, when levels where below normal, according to the formula: Plasma total calcium þ [40plasma albumin] £ 0.02. Untreated 4- and 10-month old as well as 6 weeks 1,25(OH)2D3 and vehicle-treated animals were sacrificed and femora isolated. One femur was used for histological analysis; the other femur was cut longitudinally and bone marrow cells flushed with Dubelcco’s Modified Medium (DMEM) without serum, separated from red blood cells and suspended in 1 ml of conservation buffer Easy-Kit mini prep (Qiagen, Valencia, CA, USA) for further RNA extraction. 2.2. Oil red O staining of adipose tissue in bone marrow To identify bone marrow adipogenesis, bone sections from all mice were stained using oil red O (SIGMA-Aldrich, St Louis, MI, USA). Sections were washed with 70% ethanol and then stained with 60% oil red in isopropanol. After 20 min, sections were washed, counterstained with hematoxylin and then analyzed under light microscopy, with adipose tissue staining strong red with blue nuclei. 2.3. Detection of PPARg2 by immunofluorescence Bone sections were treated as previously described (Duque et al., 2002). After fixation in 4% paraformaldehyde, sections were washed with PBS and then incubated in 10% normal blocking serum in PBS for 20 min to suppress non-specific binding of IgG. Sections were incubated with PPARg2 monoclonal mouse antibody (sc7196, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1/500 in PBS with 1.5% normal blocking serum overnight at 4 8C and then incubated for 45 min with fluorescent conjugated secondary antibody (FITC-Santa Cruz, Santa Cruz, CA, USA) diluted to 2 mg/ml in PBS with 1.5% normal blocking. Control slides were incubated with rabbit IgG, according to manufacturer instructions, and triplicate tests and control slides were included in immunodetection.
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2.4. Quantification of bone marrow cells expressing PPARg2 Each longitudinal section was analyzed in six zones of 0.8 mm starting from 1 to 28 spongiosa interface and moving in the direction of the diaphysis as previously described (Duque et al., 2002; Tomkinson et al., 1998). The sections were scored for the total number of bone marrow cells expressing PPARg2. In all cases at least 100 cells were assessed per section. Under fluorescent microscopy positive cells for PPARg2 showed intense nuclear green fluorescence, negative controls showed barely detectible nuclear and cytoplasmic fluorescence. 2.5. RNA isolation Total RNA was extracted from 106 bone marrow cells obtained from both 1,25(OH)2D3 and vehicle-treated mice according to a protocol for single-step RNA isolation based on Easy-Kit mini prep (Qiagen, Valencia, CA, USA). Aliquots of total RNA were separated in sterile tubes and quantified. 2.6. Semi-quantitative reverse transcription-polymerase chain reaction RT-PCR Total RNA isolated from bone marrow cells (1 mg) was reverse transcribed with a primer specific to the gene of interest using Qiagen One Step RT-PCR Enzyme Mix (Qiagen, Valencia, CA, USA). The resulting cDNA was amplified by 35 polymerase chain reaction cycles with an annealing temperature of 58 8C. Amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as control under the same conditions. Negative controls (samples without the transcriptase enzyme or with water in place of RNA) were included at every step of sample preparation. A region matching bases 21 –671 calculated from the denoted translational start site of the PPARg2 cDNA (gene bank accession number UO9138) was amplified by PCR. The following primers were used: PPARg2 (50 -GCCCAGGTTTGCTGAATG-30 upstream and 50 -TGAAGACTCATGTCTCTC-30 downstream); and GAPDH (50 -GAAGGTGAAGGTCGGAGTCA-30 upstream and 50 -GAAGATGGTGATGGGATTTC-30 downstream). For PPARg2 the expected size of the amplification product was 650 bp. Amplified products were electrophoresed on a 1.5% agarose gel. The signals were quantified by densitometry and expression ratios were normalized according to glyceraldehydes-3-phosphate dehydrogenase (GAPDH) density. 2.7. Statistical methods Twelve mice per group were used in both experiments. Experiments were pursued in duplicate. Statistical comparisons are based on one-way analysis of variance or Student’s
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T-test. A probability value of p , 0:05 was considered significant. All results are expressed as mean ^ SEM.
3. Results 3.1. Bone marrow expression of PPARg2 increases with aging in SAM-P/6 mice Analysis of PPARg2 mRNA showed a markedly lower level of content in 4-month old as compared 10-month old SAM-P/6 mice (Fig. 1, upper panel). Densitometric quantification of the signals produced by the hybridization in each treatment group, normalized according with GAPDH RNA expression indicated an increase in PPARg2 RNA content in the older group (Fig. 1, lower panel) (* p , 0:001). 3.2. 1,25(OH)2D3 decreases the number of PPARg2expressing cells within the bone marrow of SAM-P/6 mice A marked reduction in bone marrow adiposity was seen in 1,25(OH)2D3-treated as compared with non-treated mice (Fig. 2A and B). When PPARg2-expressing cells were quantified by immunofluorescence we found a significant reduction of PPARg2-expressing cells in 1,25(OH)2D3 (32% ^ 6) treated as compared to vehicle-treated (76% ^ 5) mice (Fig. 2C –E) ðp , 0:01Þ:
Fig. 1. Bone marrow expression of PPARg2 increases with aging in SAMP/6 mice. Total RNA was extracted from bone marrow cells from femora of 4- and 10-month old SAM-P/6 mice as described in Section 2. Expression levels of PPARg2 were determined by semi-quantitative RT-PCR and compared to GADPH. Representative data from two separate experiments is shown (* p , 0:001).
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Fig. 2. Effect of 1,25(OH)2D3 on bone marrow adipogenesis. Longitudinal sections of proximal tibiae from 1,25(OH)2D3 were stained with oil red O for adipose tissue (panels A and B). Hematoxylin was used as counterstaining. Adipocytes (AD) stained strong red with blue nuclei while all other bone marrow cells were stained light blue by hematoxylin. A marked decrease in adipocyte staining (AD) and grease content in the bone marrow (BM) is seen in 1,25(OH)2D3-treated (panel B) as compared to vehicle-treated mice (panel A). (Magnification 100 £ ). Detection of PPARg2 by immunofluorescence. After decalcification, bone samples were embedded in low-melting paraffin, coronary sections were made for the epiphyseal parts and the shaft, respectively. Sections were treated as described in Section 2. In both cases sections were incubated with PPARg2 antibody. Each longitudinal section was analyzed as previously described (Duque et al., 2002; Tomkinson et al., 1998). The percentage of nuclear PPARg2-expressing cells was determined by counting groups of 100 cells per zone (C and D). The bars in panel E show the percentage of PPARg2-expressing cells in each one of the groups. There is a significantly lower number of PPARg2-expressing osteoblasts in 1,25(OH)2D3 (panel D) treated vs. vehicle-treated mice (panel C). Determinations were done in triplicate (* p , 0:001).
3.3. 1,25(OH)2D3 inhibits PPARg2 mRNA expression in bone marrow cells of SAM-P/6 mice Semi-quantitative RT-PCR analysis of PPARg2 in bone marrow cells of 1,25(OH)2D3-treated vs. untreated
SAM-P/6 mice demonstrated a significant reduction in the expression of PPARg2 in the 1,25(OH)2D3-treated group as measured by densitometry and normalized according to GAPDH RNA expression (Fig. 3) ðp ,0:001Þ:
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Fig. 3. 1,25(OH)2D3 inhibits PPARg2 mRNA expression in bone marrow cells of SAM-P/6 mice. Total RNA was extracted from bone marrow cells from femora of both 1,25(OH)2D3-treated and vehicle-treated SAM-P/6 mice as described in Section 2. Expression levels of PPARg2 were determined by semi-quantitative RT-PCR and compared to GADPH. The figure shows a significant reduction in PPARg2 expression in 1,25(OH)2D3-treated as compared to vehicle-treated mice. Representative data from three separate experiments is shown (* p , 0:001).
4. Discussion The process of bone marrow adipogenesis that occurs with aging is due to either alterations in cell differentiation (Chan and Duque, 2002; Kirkland et al., 2002) or to increasing levels of lipid oxidation (Lecka-Czernik et al., 2002). These two processes induce an increase in the number of MAD cells, which although partially differentiated into adipocytes, maintain their potential to re-differentiate into osteoblasts after appropriate stimulation. The re-differentiation of MAD cells into osteoblasts might be induced by stimulating osteogenic or repressing adipogenic transcription factors (Duque, 2003; Garcia-Palacios et al., 2001). In addition to its known effect as inducer of osteoblastic differentiation and inhibitor of osteoblasts apoptosis (Duque et al., 2002), 1,25(OH)2D3 has been demonstrated to inhibit adipogenesis induced by thiazolidenione, a potent stimulator of PPARg2 (Hida et al., 1998). Although we have previously found that the administration of 1,25(OH)2D3 in SAM-P/6 mice increase bone mass and osteoblastogenesis while inhibiting bone marrow adipogenesis (Duque et al., unpublished data) the mechanism(s) to explain this effect remains unknown. In this study, our initial aim was to determine if the higher levels of adipogenesis seen in aging SAM-P/6 mice (Kajkenova et al., 1997) correlate with higher levels of PPARg2 within the bone marrow. Previously, we have
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found that both adiposity and PPARg2 expression increase in C57BL/6J mice (Duque, 2003). Due to recent reports of C57BL/6J as a useful model to study some aspects of agerelated bone loss (Halloran et al., 2002) we quantified bone marrow adiposity and PPARg2 expression in 4- and 24month old C57BL/6J. We found that, comparable to the present study, both adiposity and PPARg2 expression increases in bone marrow cells of old C57BL/6J mice (Duque, 2003). Initially, we assessed the levels of PPARg2 by using semi-quantitative RT-PCR. We found a significant increase in the expression of PPARg2 RNA in old (10 months) vs. young (4 months) SAM-P/6 mice which might partially explain the predominance of adipogenesis within the bone marrow of aging SAM-P/6 mice. Furthermore, after treating 4-month old SAM-P/6 mice with 1,25(OH)2D3 we found a marked decrease in adiposity within the bone marrow of 1,25(OH)2D3-treated animals, as compared to vehicle-treated animals (Fig. 2A and B). In addition, to determine if 1,25(OH)2D3 has an effect on PPARg2 expression we performed immunofluorescence and semi-quantitative RT-PCR quantification of PPARg2 in bone marrow obtained from 1,25(OH)2D3-treated and vehicle-treated animals. Overall, we found a very significant inhibitory effect of 1,25(OH)2D3 on PPARg2 mRNA expression in 1,25(OH)2D3-treated animals as compared with vehicle treated after both fluorescence quantification of PPARg2-expressing cells (Fig. 2C –E) and mRNA levels of PPARg2 within the bone marrow (Fig. 3). Given that the main goal in the treatment of senile osteoporosis is to increase the number of active osteoblasts and subsequently increase bone formation and turnover (Duque, 2001) we ought to replace adipose cells with new mature osteoblasts. The active form of vitamin D (1,25(OH)2D3) has been shown to be effective as an inducer of osteoblastogenesis (Suda et al., 2003) and more recently as an inhibitor of osteoblast apoptosis (Duque et al., 2002) as well as adipogenesis with the subsequent gain in bone mass (Duque et al., unpublished data). Although previous studies have demonstrated that 1,25(OH)2D3 inhibits PPARg2 in vitro (Kelly and Gimble, 1998) its inhibitory effect on PPARg2 in vivo was still unclear. In this study we have found that the anti-adipogenic effect of 1,25(OH)2D3 is associated with the inhibition of PPARg2 expression within the bone marrow of a mouse model for senile osteoporosis. From the mechanistic point of view, although PPARg2 and vitamin D receptors (VDR) belong to the same family of nuclear receptors, their possible interactions have not been elucidated. PPARg2 is a ligand-binding nuclear transcription factor in the same super-family of retinoid, thyroid, and steroid receptors whose natural ligand remains unknown (Duque, 2003). Although there is evidence that VDR and PPAR share their affinity for nuclear co-activators (Ren et al., 2000) there is no evidence that
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supports the possibility of either a competitive inhibition of binding to RXR or by a more direct ligand competition. Further studies will be required to elucidate these possibilities. In summary, in this study we suggest that the inhibitory effect of 1,25(OH)2D3 on adipogenesis in an in vivo model of senile osteoporosis is obtained through the inhibition of PPARg2, a key transcription factor for adipocytes differentiation. The intrinsic mechanisms to explain this interaction are still unknown, but the demonstration of this antagonistic effect of two steroid compounds in bone may open an important area of research for new therapeutic interactions in the future.
Acknowledgements Dr Duque holds a bursary from the Fonds de la Recherche du Sante´ du Que´bec. We thank Dr Lise Binderup (LEO Pharmaceuticals, Ballerup, Denmark) for kindly providing us with 1,25(OH)2D3 and Dr Susan Gold for her critical review of the manuscript.
References Bianco, P., Gehron Robey, P., 2000. Marrow stromal stem cells. J. Clin. Invest. 105, 1663– 1668. Chan, G.K., Duque, G., 2002. Age-related bone loss: old bone, new facts. Gerontology 48, 62 –71. Clarke, S.L., Robinson, C.E., Gimble, J.M., 1997. CAAT/enhancer binding proteins directly modulate transcription from the peroxisome proliferator-activated receptor gamma 2 promoter. Biochem. Biophys. Res. Commun. 240, 99–103. Duque, G., 2001. Anabolic agents to treat osteoporosis in older people: is there still place for fluoride? Fluoride for treating postmenopausal osteoporosis. J. Am. Geriatr. Soc. 49, 1387–1389. Duque, G., 2003. Will reducing adipogenesis in bone increase bone mass?: PPAR gamma 2 as a key target in the treatment of age related bone loss. Drug News Perspectives 16, 1 –6. Duque, G., Prestwood, K.M., 2003. Osteoporosis. In: Hazzard, W.R., Blass, J.R., Halter, J.B., Ouslander, J.G., Tinetti, M.E., (Eds.), Principles of Geriatric Medicine and Gerontology, McGraw-Hill, New York, pp. 973 – 986. Duque, G., El-Abdaimi, K.E., Macoritto, M., Miller, M.M., Kremer, R., 2002. Estrogens (E2) regulate expression and response of 1,25dihydroxyvitamin D3 receptors in bone cells: changes with aging and hormone deprivation. Biochem. Biophys. Res. Commun. 299, 446– 454. El Abdaimi, K., Dion, N., Papavasiliou, V., Cardinal, P.E., Binderup, L., Goltzman, D., Ste-Marie, L.G., Kremer, R., 2000. The vitamin D
analogue EB 1089 prevents skeletal metastasis and prolongs survival time in nude mice transplanted with human breast cancer cells. Cancer Res. 60, 4412–4418. Garcia-Palacios, V., Morita, I., Murota, S., 2001. Expression of adipogenesis markers in a murine stromal cell line treated with 15-deoxy Delta(12,14)-prostaglandin J2, interleukin-11, 9-cis retinoic acid and vitamin K2. Prostaglandins Leukot. Essent. Fatty Acids 65, 215–221. Halloran, B.P., Ferguson, V.L., Simske, S.J., Burghardt, A., Venton, L.L., Majumdar, S., 2002. Changes in bone structure and mass with advancing age in the male C57BL/6J mouse. J. Bone Miner. Res. 17, 1044–1050. Hida, Y., Kawada, T., Kayahashi, S., Ishihara, T., Fushiki, T., 1998. Counteraction of retinoic acid and 1,25-dihydroxyvitamin D3 on upregulation of adipocyte differentiation with PPARgamma ligand, an antidiabetic thiazolidinedione, in 3T3-L1 cells. Life Sci. 62, PL205–PL211. Justesen, J., Stenderup, K., Ebbesen, E.N., Mosekilde, L., Steiniche, T., Kassem, M., 2001. Adipocyte tissue volume in bone marrow is increased with aging and in patients with osteoporosis. Biogerontology 2, 165–171. Kajkenova, O., Lecka-Czernik, B., Gubrij, I., Hauser, S.P., Takahashi, K., Parfitt, A.M., Jilka, R.L., Manolagas, S.C., Lipschitz, D.A., 1997. Increased adipogenesis and myelopoiesis in the bone marrow of SAMP6, a murine model of defective osteoblastogenesis and low turnover osteopenia. J. Bone Miner. Res. 12, 1772–1779. Kelly, K.A., Gimble, J.M., 1998. 1,25-Dihydroxy vitamin D3 inhibits adipocyte differentiation and gene expression in murine bone marrow stromal cell clones and primary cultures. Endocrinology 139, 2622–2628. Kirkland, J.L., Dobson, D.E., 1997. Preadipocyte function and aging: links between age-related changes in cell dynamics and altered fat tissue function. J. Am. Geriatr. Soc. 45, 959–967. Kirkland, J.L., Tchkonia, T., Pirtskhalava, T., Han, J., Karagiannides, I., 2002. Adipogenesis and aging: does aging make fat go MAD? Exp. Gerontol. 37, 757 –767. Lecka-Czernik, B., Moerman, E.J., Grant, D.F., Lehmann, J.M., Manolagas, S.C., Jilka, R.L., 2002. Divergent effects of selective peroxisome proliferator-activated receptor-gamma 2 ligands on adipocyte versus osteoblast differentiation. Endocrinology 143, 2376–2384. Moore, S.G., Dawson, K.L., 1990. Red and yellow marrow in the femur: age related changes in appearance at MR imaging. Radiology 175, 219 –223. Ren, Y., Behre, E., Ren, Z., Zhang, J., Wang, Q., Fondell, J.D., 2000. Specific structural motifs determine TRAP220 interactions with nuclear hormone receptors. Mol. Cell Biol. 20, 5433–5446. Spiteller, G., 2001. Lipid peroxidation in aging and age-dependent diseases. Exp. Gerontol. 36, 1425–1457. Suda, T., Ueno, Y., Fujii, K., Shinki, T., 2003. Vitamin D and bone. J. Cell. Biochem. 88, 259– 266. Takeda, T., Hosokawa, M., Higuchi, K., 1997. Senescence-accelerated mouse (SAM): a novel murine model of senescence. Exp. Gerontol. 32, 105 –109. Tomkinson, A., Gevers, E.F., Wit, J.M., Reeve, J., Noble, B.S., 1998. The role of estrogen in the control of rat osteocyte apoptosis. J. Bone Miner. Res. 13, 1243–1250.
1,25(OH)2D3 inhibits bone marrow adipogenesis in senescence accelerated mice (SAM-P/6) by decreasing the expression of peroxisome proliferator-activated receptor gamma 2 (PPARg2) Gustavo Duquea,b,c,*, Michael Macorittoc, Richard Kremerc a
Division of Geriatric Medicine, Department of Medicine, 3755, Cote Sainte Catherine, Montreal, Que., Canada H3T 1E2 b Lady Davis Institute for Medical Research, Jewish general Hospital, Montreal, Que., Canada c Calcium Research Laboratory, McGill University, Montreal, Que., Canada Received 23 July 2003; received in revised form 6 November 2003; accepted 20 November 2003
Abstract Senile osteoporosis is the endpoint of a continuum that starts after the third decade of life when peak bone mass is attained and then is followed by a progressive and irreversible decline in bone mass. One of the mechanisms that could explain this is the increasing levels of adipogenesis in bone marrow seen with increasing age, probably due to alterations in the differentiation of mesenchymal stem cells (MSC). Senescence accelerated mice (SAM-P/6) constitute an accepted model for senile osteoporosis since their loss of bone mineral density is clearly due to high levels of adipogenesis and a deficit in osteoblastogenesis. It is known that MSC expressing a ligand-activated transcription factor known as peroxisome proliferators-activated receptor gamma 2 (PPARg2) are committed to differentiate into adipocytes. The regulation of PPARg2 activation may play a role in the control of adipogenic differentiation of MSC and thus contribute to their differentiation into osteoblasts in order to form new bone. Our previous studies have shown that the active form of vitamin D (1,25(OH)2D3) plays a role as a bone forming agent because it induces osteoblastogenesis and inhibits adipogenesis in bone marrow of SAM-P/6 mice. To elucidate the role of 1,25(OH)2D3 on the expression of PPARg2 we treated 4-month old SAM-P/6 mice with 1,25(OH)2D3 (18 pmol/24 h) or vehicle during 6 weeks. Initially we found that with aging the levels of PPARg2 expression increase in bone marrow of SAM-P/6 ðp , 0:001Þ: We then measured the changes in the expression of PPARg2 by semi-quantitative reverse transcription-polymerase chain reaction and immunofluorescence. We found a significant reduction of PPARg2-expressing cells in 1,25(OH)2D3-treated (32% ^6) as compared to vehicle (76% ^ 5) treated mice ðp , 0:01Þ: In summary, this study shows that the administration of 1,25(OH)2D3 in an in vivo model of senile osteoporosis is associated with reduction in PPARg2 a key transcription factor for the adipose differentiation of MSC. q 2003 Elsevier Inc. All rights reserved. Keywords: Senile osteoporosis; SAM-P/6; Vitamin D; Osteoblastogenesis; Adipogenesis; MAD cells; PPARg2
1. Introduction Senile osteoporosis is the consequence of three major cellular events: declining levels of osteoblastogenesis, increasing number of apoptotic osteoblasts and osteocytes, and increasing levels of bone marrow adipogenesis (Duque and Prestwood, 2003; Chan and Duque, 2002; Justesen et al., 2001; Kirkland et al., 2002). Increasing levels of bone marrow adipogenesis is probably due to the lipid redis* Corresponding author. Address: Division of Geriatric Medicine, Department of Medicine, McGill University, 3755, Cote Sainte Catherine, Montreal, Que., Canada H3T 1E2. Tel.: þ 1-514-340-7501; fax: þ 1-514-340-7547. E-mail address: [email protected] (G. Duque). 0531-5565/$ - see front matter q 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2003.11.008
tribution that happens with aging consisting of a dramatic reduction in the size of fat deposits with concomitant raise of lipid deposits in muscle, bone marrow and other tissues (Kirkland et al., 2002). Although it is known that bone marrow adipogenesis increases with aging (Kirkland et al., 2002; Lecka-Czernik et al., 2002; Moore and Dawson, 1990; Spiteller, 2001) the underlying mechanisms to explain this phenomenon remain unclear. Adipocytes belong to the group of mesenchymal cells and share the same precursors as the osteoblasts, myocytes and chondrocytes. Mesenchymal stem cells (MSC) differentiate into pre-adipocytes and thus into mature adipocytes after exposure to multiple factors including glucocorticoids, insulin growth factor-1, agents
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that increase pre-adipocyte cyclic AMP and other hormonal effectors (Bianco and Gehron Robey, 2000; Kirkland et al., 2002). After exposure to these factors, changes in transcription factors occur with the subsequent genes activation and development of fat cell phenotype (Kirkland and Dobson, 1997). The two main transcription factors involved in the differentiation of MSC into adipocytes are the peroxisome proliferator-activated receptor gamma 2 (PPARg2) and CCAT enhancer binding protein alpha (C/EBPa) (Clarke et al., 1997). The expression of one of these two factors is required for adipocytes differentiation to proceed (Kirkland et al., 2002). Increasing levels of adipogenesis in aging bone is probably due to ‘dysdifferentiation of mesenchymal cells into mesenchymal adipocyte-like default cells (MAD cells)’ (Kirkland et al., 2002). The number of MAD cells increases with aging and are able to induce adjacent cells to convert into MAD cells which extend the process (Kirkland et al., 2002; Kirkland and Dobson, 1997). The increasing number of MSC that become MAD cells with aging within the bone marrow is probably the consequence of increasing levels of endogenous PPARg2 (Duque, 2003) and might in part explain the progressive decline on osteoblastogenesis seen in senile osteoporosis. Although MAD cells remain in a stage of dysdifferentiation they keep their potential to differentiate into the osteoblastic lineage (Kirkland et al., 2002) under appropriate stimulation. Our group has found that the active form of vitamin D, 1,25-dyhydroxyvitamin D (1,25(OH)2D3), inhibits adipogenesis and increases osteoblastogenesis (Duque et al., unpublished data) in senescence accelerated mouse P/6 (SAM-P/6) an accepted model of senile osteoporosis (Kajkenova et al., 1997; Takeda et al., 1997). Although the osteogenic effect of 1,25(OH)2D3 in vitro and in vivo is well known (Hida et al, 1998; Suda et al., 2003) its role on regulating bone marrow adipogenesis in vivo and the transcription factors involved in this process remain unclear. In the present study we attempt to elucidate the mechanism that explains the anti-adipogenic effect of 1,25(OH)2D3 by assessing initially the levels of bone marrow expression of PPARg2 in this model and subsequently by looking at the potential effect of 1,25(OH)2D3 on PPARg2 levels which could explain the replacement of adipocytes by active osteoblasts after treatment.
2. Materials and methods 2.1. Animals SAM-P/6 mice were kindly provided by Dr Toshio Takeda (Kyoto University, Kyoto, Japan). Male and female were housed in cages in a limited access room restricted to aging mice (light:dark 12 h:12 h). Bedding, food and water were given as previously described
(Duque et al., 2002). Animal husbandry adhered to Canadian Council on Animal Care Standards, and all protocols were approved by the McGill University Health Center Animal Care Utilization Committee. The colony was free of any parasitic, bacterial, or viral pathogens as determined by a sentinel program. At 4 months of age, mice (n ¼ 12 per group) in a 50:50 ratio of males and females were implanted with Alzet osmotic mini-pumps (Alzet, Cupertino, CA, USA) containing 1,25(OH)2D3 (LEO Pharmaceuticals, Ballerup, Denmark) prepared as previously described (El Abdaimi et al., 2000) and delivering a constant infusion of either 18 pmol/ 24 h of 1,25(OH)2D3 or vehicle alone. Osmotic mini-pumps were replaced after 3 weeks for a total of 6 weeks of treatment. Blood calcium levels were monitored at timed intervals by microchemistry (Kodak Ektachrome, Mississauga, ON, Canada) and corrected for albumin concentrations, when levels where below normal, according to the formula: Plasma total calcium þ [40plasma albumin] £ 0.02. Untreated 4- and 10-month old as well as 6 weeks 1,25(OH)2D3 and vehicle-treated animals were sacrificed and femora isolated. One femur was used for histological analysis; the other femur was cut longitudinally and bone marrow cells flushed with Dubelcco’s Modified Medium (DMEM) without serum, separated from red blood cells and suspended in 1 ml of conservation buffer Easy-Kit mini prep (Qiagen, Valencia, CA, USA) for further RNA extraction. 2.2. Oil red O staining of adipose tissue in bone marrow To identify bone marrow adipogenesis, bone sections from all mice were stained using oil red O (SIGMA-Aldrich, St Louis, MI, USA). Sections were washed with 70% ethanol and then stained with 60% oil red in isopropanol. After 20 min, sections were washed, counterstained with hematoxylin and then analyzed under light microscopy, with adipose tissue staining strong red with blue nuclei. 2.3. Detection of PPARg2 by immunofluorescence Bone sections were treated as previously described (Duque et al., 2002). After fixation in 4% paraformaldehyde, sections were washed with PBS and then incubated in 10% normal blocking serum in PBS for 20 min to suppress non-specific binding of IgG. Sections were incubated with PPARg2 monoclonal mouse antibody (sc7196, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1/500 in PBS with 1.5% normal blocking serum overnight at 4 8C and then incubated for 45 min with fluorescent conjugated secondary antibody (FITC-Santa Cruz, Santa Cruz, CA, USA) diluted to 2 mg/ml in PBS with 1.5% normal blocking. Control slides were incubated with rabbit IgG, according to manufacturer instructions, and triplicate tests and control slides were included in immunodetection.
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2.4. Quantification of bone marrow cells expressing PPARg2 Each longitudinal section was analyzed in six zones of 0.8 mm starting from 1 to 28 spongiosa interface and moving in the direction of the diaphysis as previously described (Duque et al., 2002; Tomkinson et al., 1998). The sections were scored for the total number of bone marrow cells expressing PPARg2. In all cases at least 100 cells were assessed per section. Under fluorescent microscopy positive cells for PPARg2 showed intense nuclear green fluorescence, negative controls showed barely detectible nuclear and cytoplasmic fluorescence. 2.5. RNA isolation Total RNA was extracted from 106 bone marrow cells obtained from both 1,25(OH)2D3 and vehicle-treated mice according to a protocol for single-step RNA isolation based on Easy-Kit mini prep (Qiagen, Valencia, CA, USA). Aliquots of total RNA were separated in sterile tubes and quantified. 2.6. Semi-quantitative reverse transcription-polymerase chain reaction RT-PCR Total RNA isolated from bone marrow cells (1 mg) was reverse transcribed with a primer specific to the gene of interest using Qiagen One Step RT-PCR Enzyme Mix (Qiagen, Valencia, CA, USA). The resulting cDNA was amplified by 35 polymerase chain reaction cycles with an annealing temperature of 58 8C. Amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as control under the same conditions. Negative controls (samples without the transcriptase enzyme or with water in place of RNA) were included at every step of sample preparation. A region matching bases 21 –671 calculated from the denoted translational start site of the PPARg2 cDNA (gene bank accession number UO9138) was amplified by PCR. The following primers were used: PPARg2 (50 -GCCCAGGTTTGCTGAATG-30 upstream and 50 -TGAAGACTCATGTCTCTC-30 downstream); and GAPDH (50 -GAAGGTGAAGGTCGGAGTCA-30 upstream and 50 -GAAGATGGTGATGGGATTTC-30 downstream). For PPARg2 the expected size of the amplification product was 650 bp. Amplified products were electrophoresed on a 1.5% agarose gel. The signals were quantified by densitometry and expression ratios were normalized according to glyceraldehydes-3-phosphate dehydrogenase (GAPDH) density. 2.7. Statistical methods Twelve mice per group were used in both experiments. Experiments were pursued in duplicate. Statistical comparisons are based on one-way analysis of variance or Student’s
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T-test. A probability value of p , 0:05 was considered significant. All results are expressed as mean ^ SEM.
3. Results 3.1. Bone marrow expression of PPARg2 increases with aging in SAM-P/6 mice Analysis of PPARg2 mRNA showed a markedly lower level of content in 4-month old as compared 10-month old SAM-P/6 mice (Fig. 1, upper panel). Densitometric quantification of the signals produced by the hybridization in each treatment group, normalized according with GAPDH RNA expression indicated an increase in PPARg2 RNA content in the older group (Fig. 1, lower panel) (* p , 0:001). 3.2. 1,25(OH)2D3 decreases the number of PPARg2expressing cells within the bone marrow of SAM-P/6 mice A marked reduction in bone marrow adiposity was seen in 1,25(OH)2D3-treated as compared with non-treated mice (Fig. 2A and B). When PPARg2-expressing cells were quantified by immunofluorescence we found a significant reduction of PPARg2-expressing cells in 1,25(OH)2D3 (32% ^ 6) treated as compared to vehicle-treated (76% ^ 5) mice (Fig. 2C –E) ðp , 0:01Þ:
Fig. 1. Bone marrow expression of PPARg2 increases with aging in SAMP/6 mice. Total RNA was extracted from bone marrow cells from femora of 4- and 10-month old SAM-P/6 mice as described in Section 2. Expression levels of PPARg2 were determined by semi-quantitative RT-PCR and compared to GADPH. Representative data from two separate experiments is shown (* p , 0:001).
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Fig. 2. Effect of 1,25(OH)2D3 on bone marrow adipogenesis. Longitudinal sections of proximal tibiae from 1,25(OH)2D3 were stained with oil red O for adipose tissue (panels A and B). Hematoxylin was used as counterstaining. Adipocytes (AD) stained strong red with blue nuclei while all other bone marrow cells were stained light blue by hematoxylin. A marked decrease in adipocyte staining (AD) and grease content in the bone marrow (BM) is seen in 1,25(OH)2D3-treated (panel B) as compared to vehicle-treated mice (panel A). (Magnification 100 £ ). Detection of PPARg2 by immunofluorescence. After decalcification, bone samples were embedded in low-melting paraffin, coronary sections were made for the epiphyseal parts and the shaft, respectively. Sections were treated as described in Section 2. In both cases sections were incubated with PPARg2 antibody. Each longitudinal section was analyzed as previously described (Duque et al., 2002; Tomkinson et al., 1998). The percentage of nuclear PPARg2-expressing cells was determined by counting groups of 100 cells per zone (C and D). The bars in panel E show the percentage of PPARg2-expressing cells in each one of the groups. There is a significantly lower number of PPARg2-expressing osteoblasts in 1,25(OH)2D3 (panel D) treated vs. vehicle-treated mice (panel C). Determinations were done in triplicate (* p , 0:001).
3.3. 1,25(OH)2D3 inhibits PPARg2 mRNA expression in bone marrow cells of SAM-P/6 mice Semi-quantitative RT-PCR analysis of PPARg2 in bone marrow cells of 1,25(OH)2D3-treated vs. untreated
SAM-P/6 mice demonstrated a significant reduction in the expression of PPARg2 in the 1,25(OH)2D3-treated group as measured by densitometry and normalized according to GAPDH RNA expression (Fig. 3) ðp ,0:001Þ:
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Fig. 3. 1,25(OH)2D3 inhibits PPARg2 mRNA expression in bone marrow cells of SAM-P/6 mice. Total RNA was extracted from bone marrow cells from femora of both 1,25(OH)2D3-treated and vehicle-treated SAM-P/6 mice as described in Section 2. Expression levels of PPARg2 were determined by semi-quantitative RT-PCR and compared to GADPH. The figure shows a significant reduction in PPARg2 expression in 1,25(OH)2D3-treated as compared to vehicle-treated mice. Representative data from three separate experiments is shown (* p , 0:001).
4. Discussion The process of bone marrow adipogenesis that occurs with aging is due to either alterations in cell differentiation (Chan and Duque, 2002; Kirkland et al., 2002) or to increasing levels of lipid oxidation (Lecka-Czernik et al., 2002). These two processes induce an increase in the number of MAD cells, which although partially differentiated into adipocytes, maintain their potential to re-differentiate into osteoblasts after appropriate stimulation. The re-differentiation of MAD cells into osteoblasts might be induced by stimulating osteogenic or repressing adipogenic transcription factors (Duque, 2003; Garcia-Palacios et al., 2001). In addition to its known effect as inducer of osteoblastic differentiation and inhibitor of osteoblasts apoptosis (Duque et al., 2002), 1,25(OH)2D3 has been demonstrated to inhibit adipogenesis induced by thiazolidenione, a potent stimulator of PPARg2 (Hida et al., 1998). Although we have previously found that the administration of 1,25(OH)2D3 in SAM-P/6 mice increase bone mass and osteoblastogenesis while inhibiting bone marrow adipogenesis (Duque et al., unpublished data) the mechanism(s) to explain this effect remains unknown. In this study, our initial aim was to determine if the higher levels of adipogenesis seen in aging SAM-P/6 mice (Kajkenova et al., 1997) correlate with higher levels of PPARg2 within the bone marrow. Previously, we have
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found that both adiposity and PPARg2 expression increase in C57BL/6J mice (Duque, 2003). Due to recent reports of C57BL/6J as a useful model to study some aspects of agerelated bone loss (Halloran et al., 2002) we quantified bone marrow adiposity and PPARg2 expression in 4- and 24month old C57BL/6J. We found that, comparable to the present study, both adiposity and PPARg2 expression increases in bone marrow cells of old C57BL/6J mice (Duque, 2003). Initially, we assessed the levels of PPARg2 by using semi-quantitative RT-PCR. We found a significant increase in the expression of PPARg2 RNA in old (10 months) vs. young (4 months) SAM-P/6 mice which might partially explain the predominance of adipogenesis within the bone marrow of aging SAM-P/6 mice. Furthermore, after treating 4-month old SAM-P/6 mice with 1,25(OH)2D3 we found a marked decrease in adiposity within the bone marrow of 1,25(OH)2D3-treated animals, as compared to vehicle-treated animals (Fig. 2A and B). In addition, to determine if 1,25(OH)2D3 has an effect on PPARg2 expression we performed immunofluorescence and semi-quantitative RT-PCR quantification of PPARg2 in bone marrow obtained from 1,25(OH)2D3-treated and vehicle-treated animals. Overall, we found a very significant inhibitory effect of 1,25(OH)2D3 on PPARg2 mRNA expression in 1,25(OH)2D3-treated animals as compared with vehicle treated after both fluorescence quantification of PPARg2-expressing cells (Fig. 2C –E) and mRNA levels of PPARg2 within the bone marrow (Fig. 3). Given that the main goal in the treatment of senile osteoporosis is to increase the number of active osteoblasts and subsequently increase bone formation and turnover (Duque, 2001) we ought to replace adipose cells with new mature osteoblasts. The active form of vitamin D (1,25(OH)2D3) has been shown to be effective as an inducer of osteoblastogenesis (Suda et al., 2003) and more recently as an inhibitor of osteoblast apoptosis (Duque et al., 2002) as well as adipogenesis with the subsequent gain in bone mass (Duque et al., unpublished data). Although previous studies have demonstrated that 1,25(OH)2D3 inhibits PPARg2 in vitro (Kelly and Gimble, 1998) its inhibitory effect on PPARg2 in vivo was still unclear. In this study we have found that the anti-adipogenic effect of 1,25(OH)2D3 is associated with the inhibition of PPARg2 expression within the bone marrow of a mouse model for senile osteoporosis. From the mechanistic point of view, although PPARg2 and vitamin D receptors (VDR) belong to the same family of nuclear receptors, their possible interactions have not been elucidated. PPARg2 is a ligand-binding nuclear transcription factor in the same super-family of retinoid, thyroid, and steroid receptors whose natural ligand remains unknown (Duque, 2003). Although there is evidence that VDR and PPAR share their affinity for nuclear co-activators (Ren et al., 2000) there is no evidence that
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supports the possibility of either a competitive inhibition of binding to RXR or by a more direct ligand competition. Further studies will be required to elucidate these possibilities. In summary, in this study we suggest that the inhibitory effect of 1,25(OH)2D3 on adipogenesis in an in vivo model of senile osteoporosis is obtained through the inhibition of PPARg2, a key transcription factor for adipocytes differentiation. The intrinsic mechanisms to explain this interaction are still unknown, but the demonstration of this antagonistic effect of two steroid compounds in bone may open an important area of research for new therapeutic interactions in the future.
Acknowledgements Dr Duque holds a bursary from the Fonds de la Recherche du Sante´ du Que´bec. We thank Dr Lise Binderup (LEO Pharmaceuticals, Ballerup, Denmark) for kindly providing us with 1,25(OH)2D3 and Dr Susan Gold for her critical review of the manuscript.
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