Maf promotes osteoblast differentiation in mice by mediating the age-related switch in mesenchymal cell differentiation

Aging leads to the disruption of the homeostatic balance of multiple biological systems. In bone marrow multipotent mesenchymal cells undergo differentiation into various anchorage-dependent cell types, including osteoblasts and adipocytes. With age as well as with treatment of antidiabetic drugs such as thiazolidinediones, mesenchymal cells favor differentiation into adipocytes, resulting in an increased number of adipocytes and a decreased number of osteoblasts, causing osteoporosis. The mechanism behind this differentiation switch is unknown. Here we show an age-related decrease in the expression of Maf in mouse mesenchymal cells, which regulated mesenchymal cell bifurcation into osteoblasts and adipocytes by cooperating with the osteogenic transcription factor Runx2 and inhibiting the expression of the adipogenic transcription factor Pparg. The crucial role of Maf in both osteogenesis and adipogenesis was underscored by in vivo observations of delayed bone formation in perinatal Maf^–/– mice and an accelerated formation of fatty marrow associated with bone loss in aged Maf^+/– mice. This study identifies a transcriptional mechanism for an age-related switch in cell fate determination and may provide a molecular basis for novel therapeutic strategies against age-related bone diseases.

A progressive and irreversible accumulation of DNA damage, which is triggered by telomere shortening and various stressors such as oxidative stress, contributes to cellular senescence and organismal aging (1, 2), but how aging is related to the disruption of the homeostatic balance of cell differentiation from a common progenitor is not well understood. Bone marrow contains multipotent mesenchymal progenitor cells, which differentiate into various anchorage-dependent cell types, including adipocytes and osteoblasts (3, 4). With age, mesenchymal cells in the bone marrow become inclined to undergo differentiation into adipocytes rather than osteoblasts (5–7), resulting in an increased number of adipocytes and a decreased number of osteoblasts, causing osteoporosis. Adipocytes are also known to directly inhibit functions of other cells in the bone marrow, including hematopoietic stem cells and osteoblasts (8–11). Since an increase in marrow fat along with bone loss is observed in diabetic patients treated with thiazolidinediones (TZDs) (12), understanding the mechanism of this differentiation switch has substantial relevance to both the management of age-related osteoporosis and secondary osteoporosis after such drug treatment. However, the change in mesenchymal cell differentiation cannot be adequately explained by cellular senescence or the cell cycle arrest caused by DNA damage. While estrogen deficiency causes postmenopausal osteoporosis (6, 13), it has been suggested that the downregulation of cytokines and hormones, such as IGF1, TGFB1, IL-11, and growth hormone, is correlated with age-related bone loss (6, 14, 15). However, a cell-intrinsic mechanism that regulates the age-related switch in mesenchymal cell differentiation remains to be elucidated. Here we report an age-related decrease in the expression of Maf in mesenchymal cells and present evidence that Maf regulates mesenchymal cell bifurcation into osteoblasts and adipocytes. This study establishes the crucial role of the Maf-mediated transcriptional program in the physiological and age-related regulation of mesenchymal cell lineage, which may facilitate the development of new therapeutic strategies against bone and metabolic diseases.

A genome-wide screening of transcription factors involved in the age-related decrease in bone formation. To identify the transcription factors involved in age-related bone loss, we performed a genome-wide screening of mRNAs expressed in cells derived from mouse calvaria during osteoblastogenesis. Among 1,470 transcription factors, we identified 163 genes related to osteogenic function; the identifying characteristic of these factors was that their expression was increased by more than 4-fold during osteoblastogenesis (Figure 1A and Supplemental Table 1, A and B; supplemental material available online with this article; doi: 10.1172/JCI42528DS1). To identify age-related genes, we also comprehensively analyzed the mRNAs expressed by bone marrow stromal cells (BMSCs) derived from 8- and 32-week-old mice, which resulted in identifications of 179 genes, the expression of which was decreased in the aged mice by more than 2 fold (Supplemental Table 1C). Fifty-two genes met both criteria (Supplemental Table 1D), from which we selected 7 genes preferentially expressed in BMSCs (with an average difference greater than 200). Among these 7 genes, we identified Maf (also known as c-Maf) to be the most highly expressed in the BMSCs. We confirmed that the expression of Maf increased during osteoblastogenesis, using calvarial and BMSCs (Figure 1A, right top, and Supplemental Figure 1A), and decreased with age (Figure 1A, right bottom, and Supplemental Figure 1B). Maf, a basic leucine zipper transcription factor, is known to be involved in the regulation of diverse developmental processes such as lens fiber elongation (16) and Th2 cell differentiation (17, 18). Although it has been documented that transcription factors such as ΔFosb, Taz, Esr1, Msx2, and Cebpβ regulate the bifurcation of osteoblast/adipocyte differentiation (7, 19, 20), the expression of ΔFosb was increased in aged BMSCs (Supplemental Figure 2) and the other 4 factors were not included in the 179 age-related genes (Supplemental Table 1C). These results suggest that Maf is one of the potential candidate genes underlying an age-related decrease in osteoblastogenesis.

Figure 1

Impaired bone formation in Maf^–/– mice. (A) A genome-wide screening of transcription factor mRNAs during in vitro differentiation of osteoblasts (OBs) and a comparison of their expression between 8- and 32-week-old BMSCs. The increase in Maf expression during osteoblastogenesis was confirmed in calvarial osteoblasts (RNA blot analysis, right top). Maf expression was markedly lower in BMSCs derived from the aged mice (real-time RT-PCR analysis, right bottom). Screening results are summarized in the Venn diagram. *P < 0.05; **P < 0.01. (B) Alizarin red/alcian blue staining of E17 embryos (top). Top view of calvaria (bottom). Images in B are composites. (C) Histology (von Kossa staining) and microcomputed tomography analysis of WT and Maf^–/– littermates at P0 (n = 3). Scale bar: 100 μm. (D) ALP and von Kossa staining of osteogenic fronts (OFs) in the calvaria of WT and Maf^–/– littermates. Scale bar: 100 μm. (E) Expression of Bglap1 in the calvaria of WT and Maf^–/– mice (in situ hybridization). Scale bar: 100 μm.

The indispensable role of Maf in osteogenesis. Since Maf^–/– mice usually die immediately after birth (16), we investigated the role of Maf in osteogenesis by analyzing the skeletal development of perinatal Maf^–/– mice. Bone formation was severely impaired in both the long and calvarial bones in the embryos of Maf^–/– mice (Figure 1B), and bone volume was decreased in newborn Maf^–/– mice (Figure 1C, Table 1, and Supplemental Figure 3). The formation of alkaline phosphatase–positive (ALP-positive) cells on an osteogenic front was markedly impaired in the calvaria of newborn Maf^–/– mice (Figure 1D). In situ hybridization analysis revealed the expression of osteoblast genes, such as Bglap1 (encoding osteocalcin), but not of Runx2 was much lower in the embryos of Maf^–/– mice than WT mice (Figure 1E and Supplemental Figure 4, A and B), although the proliferating or apoptotic osteoblast numbers were not different (Supplemental Figure 4C). Since the osteoclast number was decreased in Maf^–/– mice, possibly in a cell-autonomous manner (Supplemental Figure 5), it is unlikely that abnormal osteoclastic bone resorption contributes to the low bone mass phenotype in Maf^–/– mice. When a neomycin-resistance gene cassette was inserted into the Maf locus, chondrocyte development was reported to be affected in the mutant mice, but the mice were not perinatally lethal (18). In the current study, in which the coding sequence of Maf was entirely replaced by the LacZ cassette, the mice were perinatally lethal and exhibited more severe chondrocyte abnormalities (Supplemental Figure 6). It is difficult to rule out the possibility that a defect in chondrocytes contributes to a skeletal phenotype in long bone, but Maf^–/– mice exhibited a defective bone formation in flat bones, like the calvaria (Figure 1B), which are not formed by endochondral bone formation, suggesting a role of Maf in osteoblasts. To analyze a cell-autonomous defect in osteoblasts further, osteoblast differentiation was evaluated in an in vitro culture system of osteoblast precursor cells derived from the calvaria of newborn Maf^–/– mice. ALP activity and bone nodule formation were markedly suppressed in Maf^–/– cells (Figure 2A), but neither the proliferation nor apoptosis was affected (Figure 2B). These results collectively indicate that a complete lack of Maf led to an osteopenic phenotype, due to impaired osteoblast differentiation and bone formation.

Figure 2

Regulation of osteoblast differentiation and Bglap1 expression by Maf in cooperation with Runx2. (A) ALP and alizarin red staining of WT and Maf^–/– calvarial cells. ALP activity and bone nodule formation were quantitated. (B) Proliferation and apoptosis of WT and Maf^–/– calvarial cells. (C) mRNA expression of osteoblast-specific genes in WT and Maf^–/– calvarial cells (GeneChip analysis). (D) Bglap1 expression in WT and Maf^–/– calvarial cells cultured with osteogenic medium for 7 days (RNA blot analysis). (E) Schematic of 5 MARE-like sequences (MARE1–MARE5) in the regulatory region of Bglap1, and Bglap1-luc variants harboring point mutation(s) in MARE-like sequences. pDHS and dDHS indicate proximal and distal DNase hypersensitive sites, respectively (23). Arrows indicate the primer set used for ChIP. Numbers within ovals represent corresponding MARE sequences. Ovals with “X”s indicate sequences without that respective MARE sequence. (F) Effect of Maf on the Bglap1-luc variants. (G) Recruitment of Maf to the Bglap1 promoter region containing MARE1–MARE3. Calvarial cells cultured with osteogenic medium for 7 days were analyzed by ChIP. (H) Effect of Runx2 and AP-1 family members on Maf-mediated activation of 1050Oc-luc. *P < 0.05; **P < 0.01.

Table 1

Skeletal development of perinatal Maf^–/– mice

Maf regulates osteoblast differentiation in cooperation with Runx2. The expression of various osteoblast-specific genes, including Bglap1, was severely suppressed in Maf^–/– cells (Figure 2, C and D), but Runx2, a well-known transcriptional regulator of Bglap1 (21, 22), was normally expressed in Maf^–/– mice (Supplemental Figure 4B). It is notable that Maf expression was not decreased in Runx2^–/– calvarial osteoblasts (Supplemental Figure 7). These results prompted us to investigate whether Maf directly regulates the Bglap1 promoter. As expected, 5 Maf recognition element–like (MARE-like) sequences were contained in the 1,050-base Bglap1 promoter region (Figure 2E). A reporter gene assay indicated that Maf activates the Bglap1 promoter mainly through a region containing 3 proximal MARE-like sequences (MARE1–MARE3), which was included in the proximal DNase hypersensitive site (23) and partially overlapped with osteoblast-specific element 1 (OSE1) (22) (Figure 2F). ChIP experiments showed that Maf is recruited to the region containing MARE1–MARE3 in primary osteoblasts (Figure 2G), suggesting that Maf directly regulates the Bglap1 promoter.

To gain insight into the transcriptional partners of Maf, we analyzed the Maf transcriptional network using a systems biology approach, based on protein-protein interaction databases and our own GeneChip analysis. Fos, Jun, Atf4, Nfat, and Runx2 were included among the transcription factors predicted to interact with Maf (Supplemental Figure 8). The function of these candidate partners was examined in the Maf-mediated activation of the Bglap1 promoter, which was found to be exclusively enhanced by the addition of Runx2 (Figure 2H and data not shown). Consistent with this, the transcriptional activity of Maf on the Bglap1 promoter was markedly decreased in Runx2^–/– cells (Supplemental Figure 9). Maf bound to Runx2 in an immunoprecipitation assay (Figure 3 and Supplemental Figure 10), and immunohistochemical analysis showed that Maf was colocalized with Runx2 in calvarial osteoblasts (Supplemental Figure 11). Thus, these results suggest that Maf controls osteoblast differentiation by regulating osteoblastic gene expression mainly in cooperation with Runx2.

Figure 3

Physical interaction of Maf with Runx2. Maf and MafΔN, but not MafΔC, bound to Runx2. Runx2 and Runx2ΔC, but not Runx2ΔN, bound to Maf. Maf contains a transactivated domain (acidic domain), a histidine cluster (His rich), glycine stretches (Gly rich), and a basic leucine zipper domain (bZip domain). The asterisk indicates nonspecific bands.

Maf suppresses adipogenesis by the downregulation of Pparg. We further characterized the Maf^–/– calvarial cells using gene set enrichment analysis (GSEA), which revealed adipocyte-related genes to be highly enriched in Maf^–/– cells (Supplemental Figure 12). Indeed, GeneChip data showed that the expression of adipocyte genes was upregulated in Maf^–/– calvarial cells, even under the conditions optimized for osteoblast differentiation (Figure 4A). Maf^–/– cells differentiated into oil red O–positive adipocytes more efficiently than WT cells (Figure 4B). Real-time RT-PCR analysis confirmed the expression of Pparg, the key transcription factor for adipogenesis, as well as that of Fabp4, Slc2a4, Lpl, Acc1, and Cd36, downstream effectors of Pparγ (24–26), to be markedly elevated in Maf^–/– cells (Figure 4C and Supplemental Figure 13). To further investigate the role of Maf in osteoblast and adipocyte differentiation, we ectopically expressed Maf in C3H10T1/2 cells, a stromal cell line with a capacity to differentiate into both osteoblasts and adipocytes, and found that the introduction of Maf resulted in a severe blockade of adipocyte differentiation and an enhancement of osteoblast differentiation (Figure 4D and Supplemental Figure 14). Similar results were obtained using another stromal cell line, ST2, and BMSCs (Supplemental Figure 15, A–C). In addition, short hairpin RNA-mediated knockdown of Maf in ST2 cells resulted in a blockade of osteoblast differentiation and an acceleration of adipocyte differentiation (Supplemental Figure 15, D and E). Similar results were obtained using BMSCs (Supplemental Figure 15F). These results suggest that Maf promotes osteoblast differentiation and inhibits adipocyte differentiation in a cell-autonomous manner.

Figure 4

Maf inhibition of adipocyte differentiation by suppressing Cebpδ/α-mediated induction of Pparg. (A) mRNA expression of adipocyte-specific genes in WT and Maf^–/– calvarial cells cultured with osteogenic medium (GeneChip analysis). (B) Adipocyte formation in WT and Maf^–/– calvarial cells cultured with osteogenic medium (oil red O staining). (C) Expression of Pparg and Fabp4 in WT and Maf^–/– calvarial cells cultured with osteogenic medium (real-time RT-PCR analysis). (D) Effect of Maf overexpression on adipocyte and osteoblast differentiation of C3H10T1/2 cells. Scale bar: 200 μm. (E) Effect of Crebbp overexpression on Maf-mediated inhibition of Cebpδ activation of the Pparg promoter. (F) Inhibition of interaction between Cebpδ and Crebbp by Maf. *P < 0.05; **P < 0.01.

To understand the mechanism underlying the Maf-mediated inhibition of Pparg expression and adipogenesis, we examined the effect of Maf on the transcriptional activity of Cebp, the family members of which are involved in the regulation of Pparg (27, 28). Overexpression of Maf clearly suppressed the activation of the Pparg promoter by Cebpα and Cebpδ but not that by Cebpβ (Figure 4E and data not shown). Since the introduction of mutation(s) into MAREs in the Pparg promoter did not affect the inhibitory effect of Maf on Cebpδ activity, it is unlikely that Maf inhibited the Pparg promoter activity by directly binding to MAREs (Supplemental Figure 16, A and B). EMSA revealed that Maf did not affect the DNA binding activity of Cebpδ, further supporting Maf-mediated inhibition of the Pparg promoter being independent of Maf binding to DNA (Supplemental Figure 16C). In addition, AP-1 family members and Runx2 did not affect Maf-mediated inhibition of the Pparg promoter (Supplemental Figure 16D). As it has been documented that Maf and Cebpδ commonly use CREB-binding protein gene (Crebbp) as their crucial transcriptional coactivator (29) (Supplemental Figure 8), we inferred that competition for the limited amount of Crebbp accounts for the inhibitory effect of Maf on the Cebpδ activity. As expected, the interaction of Cebpδ with Crebbp was suppressed by the overexpression of Maf (Figure 4F). Overexpression of Crebbp recovers the Maf-mediated inhibition of the effect of Cebpδ on the Pparg promoter (Figure 4E), lending support to the notion that Maf inhibits Cebpδ activity due to Crebbp squelching, although this may not be the sole mechanism underlying Maf inhibition of adipogenesis.

Decreased Maf expression accelerates age-related osteoporosis and fatty bone marrow. Although the perinatal lethality of Maf^–/– mice renders it difficult to evaluate the development of fatty marrow with aging, the expression of Pparg in the bone marrow is much higher in perinatal Maf^–/– mice than WT mice (Figure 5A), suggesting that the adipogenesis is enhanced in vivo in the case of Maf deficiency. Haploinsufficiency of Maf did not affect bone formation in embryos or neonatal mice (Figure 1B and Supplemental Figure 17). At the age of 22 weeks, however, histological analysis revealed that the bone marrow was filled with adipocytes characterized by fat vacuoles, and the bone volume was reduced in the Maf^+/– mice (Figure 5, B and C, and Table 2). In contrast, no abnormalities in cartilage were found in the adult or neonatal Maf^+/– mice (Supplemental Figures 6 and 18). The accelerated fatty marrow formation was accompanied by a decrease in osteoblast number and bone formation (Figure 5D), while osteoclastic bone resorption was not affected in the Maf^+/– mice, with the serum calcium and phosphate levels being normally maintained (Supplemental Figures 19–21). Thus, haploinsufficiency of Maf results in enhanced adipogenesis and decreased osteogenesis in vivo, which was obvious at an advanced age, suggesting that the decreasing level of Maf with age contributes to the age-related switch in mesenchymal cell differentiation into adipocytes rather than osteoblasts.

Figure 5

Increased adipogenesis in the Maf deficiency. (A) Expression of Pparg in the tibia of WT and Maf^–/– littermates (in situ hybridization). Scale bar: 100 μm. (B) Histological analysis of the bone marrow of 22-week-old WT and Maf^+/– female mice (femur, toluidine blue staining). Yellow boxed regions in the top panels are shown at higher magnification in the bottom panels. Note that Maf^+/– bone marrow is filled with adipocytes. The number, but not the size, of adipocytes was significantly increased (n = 6). Scale bar: 500 μm (top row); 50 μm (bottom row). (C) Three-dimensional microcomputed tomography images of the femurs of 22-week-old WT and Maf^+/– mice. Scale bar: 200 μm. (D) Parameters of osteoblastic bone formation in the bone morphometric analysis (n = 6; 22–26 weeks old). *P < 0.05; **P < 0.01.

Table 2

Skeletal development of 22-week-old Maf^+/– mice

To determine whether forced expression of Maf in mesenchymal cells would rescue an aging phenotype of Maf^+/– mice, we overexpressed Maf in calvarial cells by retroviral transfer and transplanted them into the femurs of aged mice (Supplemental Figure 22). Maf^+/– mice transplanted with Maf-transduced calvarial cells had a higher trabecular bone mass (but not cortical bone mass) and a decreased number of intramedullary adipocytes compared with those transplanted with mock-infected calvarial cells (Figure 6A and Table 3). We observed similar results when we transplanted Maf-transduced calvarial cells into aged WT mice (Supplemental Figure 22D). These results indicate that overexpression of Maf resulted in effective restoration of both an accelerated aging phenotype in Maf^+/– mice and age-related changes in WT mice.

Figure 6

Aging and Maf-mediated regulation of osteoblastogenesis and adipogenesis. (A) Effect of Maf overexpression in mesenchymal cells on an aging phenotype of Maf^+/– mice. Three-dimensional microcomputed tomography images and histological analysis of the bone marrow (toluidine blue staining) of Maf^+/– mice transplanted with Maf-transduced or mock-infected calvarial cells. Scale bar: 200 μm (top row); 100 μm (bottom row). (B) Effect of hydrogen peroxide on Maf expression in WT and Trp53^–/– calvarial osteoblasts (real-time RT-PCR analysis). (C) Effect of N-acetylcys­teine (NAC) administration on an aging phenotype of Maf^+/– mice. Three-dimensional microcomputed tomography images (top row) and histology of the bone marrow (toluidine blue staining, middle and bottom rows) of mice (n = 4). Images in the middle row are shown at higher magnification in the bottom row. Scale bar: 500 μm (top and middle rows); 50 μm (bottom row). (D) Microcomputed tomography and histological analysis of WT and Maf^+/– mice. (E) A model of Maf-mediated reciprocal regulation of osteoblast and adipocyte differentiation. *P < 0.05; **P < 0.01.

Table 3

Skeletal development and adipogenesis in Maf^+/– mice transplanted with Maf-transduced and mock-infected calvarial cells

ROS regulation of Maf expression through Trp53. How is Maf expression regulated during aging? Since it has been reported that age-related bone loss is related to an increased expression of the Wnt inhibitor secreted frizzled-related protein 4 (30) or a decreased production of soluble factors, such as IGF1, TGFB1, IL-11, and bone morphogenetic protein 2 (6, 14, 15), we evaluated the effect of these factors as well as the effect of ROS on Maf expression. Although none of the soluble factors increased Maf expression in osteoblast precursor cells (Supplemental Figure 23), treatment with the hydrogen peroxide led to a marked decrease in Maf expression, which was restored by the addition of the antioxidant N-acetylcysteine (Figure 6B). These results prompted us to investigate whether administration of N-acetylcysteine rescue the bone phenotype of Maf^+/– mice. As expected, administration of N-acetylcysteine led to an increased bone mass and decreased intramedullary fat in Maf^+/– mice (Figure 6, C and D).

Furthermore, to gain mechanistic insight into the downregulation of Maf by ROS, we focused on the regulation of Maf by Trp53, which is reported to be induced by ROS and involved in aging (1, 2, 31, 32). Indeed, the Maf promoter contains multiple Trp53 binding sites and overexpression of Trp53 markedly inhibited the activity of the Maf promoter (Supplemental Figure 24). In addition, the downregulation of Maf by ROS was markedly attenuated in Trp53^–/– osteoblasts (Figure 6B). Although NF-κB is involved in aging (33) and activated by ROS, Maf expression was not restored by an NF-κB inhibitor (Supplemental Figure 25). These results suggest that the oxidative stress that accumulates with aging mediates, at least in part, the age-related decrease in Maf expression through Trp53.

The molecular basis for age-related changes in higher organisms is poorly understood, particularly in the skeletal system. Although many factors have been suggested to regulate the bifurcation of osteoblasts and adipocytes, the function of a few factors has been demonstrated by genetic loss-of-function studies (7), and how the expression level of these factors changes in aging is not well known. A combination of a genome-wide screening and mouse genetic studies led us to identify the expression level of Maf to be both under the influence of aging and a determinant of mesenchymal cell differentiation into osteoblasts and adipocytes (Figure 6E).

Maf promotes osteoblast differentiation by regulating osteoblast genes, including Bglap1, in cooperation with Runx2. We propose that Maf functions as a modulator of bone formation by regulating the activity of crucial determinants like Runx2. Even in aged mice, bone marrow cells expressed a normal level of Runx2 (based on the screening shown in Figure 1A and Supplemental Table 1), but a low level of Maf expression resulted in a decrease in Runx2-mediated transcriptional activity. Therefore, Maf is a potential candidate to help explain the gradual and moderate decrease in bone formation observed in age-related osteoporosis. We also demonstrated that Maf inhibited adipocyte differentiation through the downregulation of Pparg expression, thus indicating that Maf regulates the bifurcation of the mesenchymal cell lineage into osteoblasts and adipocytes. It is interesting to note that the MAF locus was recently identified as one of the risk loci for obesity (34).

Using genetically modified mice, AP-1 superfamily members Fra1, Junb, and ΔFosb were shown to regulate bone formation (35–37), but ΔFosb was the only member that also regulates adipogenesis (19). However, since the adipogenic function of ΔFosb depends on a non–cell-autonomous mechanism (38), Maf is the only AP-1 superfamily member that has a cell-autonomous role in the regulation of both osteoblast and adipocyte differentiation. Interestingly, Maf also regulated osteoclastogenesis in a cell-autonomous manner, possibly by modulating NFAT activity (Supplemental Figure 5, C and E), suggesting that Maf plays a distinct role in each skeletal cell type.

Since Pparγ inhibits the expression of Maf (39), a small reduction in Maf expression would form a vicious cycle: an increasing expression of Pparg further inhibits Maf expression, leading to a severe reduction in Maf. Antidiabetic drugs — such as TZDs, which activate Pparγ — are known to reduce bone mass and increase fracture risk (12). Interestingly, treatment with rosiglitazone led to decreased Maf expression (data not shown). This is possibly because Pparγ agonists accelerate this vicious cycle through suppression of Maf expression.

Mathematical modeling of gene expression is helpful for understanding how the gradual reduction of Maf expression leads to a dramatic change in cell differentiation. Based on this simulation, if the expression of Maf decreased by more than 60% of the maximum level, the adipogenic genes became preferentially expressed (Supplemental Figure 26 and Supplemental Methods). Reduction of Maf gene expression in neonatal Maf^+/– mice was not sufficient for activation of this switch, as we observed no obvious bone phenotype in neonatal Maf^+/– mice (Figure 1B and Supplemental Figure 17). As the level of Maf gradually decreased with age, they exhibited an osteopenic phenotype with fatty marrow.

Currently, there are few drugs available in the clinic that effectively increase bone formation. The modulation of Maf expression appears to hold considerable promise as what we believe to be a novel antiaging therapeutic target in the skeletal system.

View Supplemental data

We thank H.R. Ueda, M. Asagiri, T. Ando, Y. Kunisawa, T. Honda, Y. Suzuki, T. Kudo, A. Izumi, A. Suematsu, and A. Hirota for discussion and assistance. This work was supported in part by a grant for ERATO from the Takayanagi Osteonetwork Project from the Japan Science and Technology Agency; Grant-in-Aids for Creative Scientific Research and Young Scientist (A and Start-up) from the Japan Society for the Promotion of Science (JSPS); Grant-in-Aid for Challenging Exploratory Research JSPS; grants for the Genome Network Project and Global Center of Excellence Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan; and grants from Tokyo Biochemical Research Foundation, Life Science Foundation of Japan, Yokoyama Foundation for Clinical Pharmacology, and Takeda Science Foundation.

Address correspondence to: Hiroshi Takayanagi, Department of Cell Signaling, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8549, Japan. Phone: 81.3.5803.5471; Fax: 81.3.5803.0192; E-mail: taka.csi@tmd.ac.jp.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J Clin Invest. 2010;120(10):3455–3465. doi:10.1172/JCI42528.

See the related article at c-Maf and you won’t see fat.

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