Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Stat1 functions as a cytoplasmic attenuator of Runx2 in the transcriptional program of osteoblast differentiation Sunhwa Kim,1,5 Takako Koga,1,2,5 Miho Isobe,1,2 Britt E. Kern,3 Taeko Yokochi,1 Y. Eugene Chin,4 Gerard Karsenty,3 Tadatsugu Taniguchi,1,6 and Hiroshi Takayanagi1,2 1Department of Immunology, Graduate School of Medicine and Faculty of Medicine, University of Tokyo, Tokyo 113-0033, Japan; 2PRESTO, Japan Science and Technology Corporation (JST), Kawaguchi, Saitama 332-0012, Japan; 3Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA; 4Department of Pathology and Laboratory Medicine, Brown University School of Medicine, Providence, Rhode Island 02912, USA Bone remodeling is central to maintaining the integrity of the skeletal system, wherein the developed bone is constantly renewed by the balanced action of osteoblastic bone formation and osteoclastic bone resorption. In the present study, we demonstrate a novel function of the Stat1 transcription factor in the regulation of bone remodeling. In the bone of the Stat1-deficient mice, excessive osteoclastogenesis is observed, presumably caused by a loss of negative regulation of osteoclast differentiation by interferon (IFN)-␤. However, the bone mass is unexpectedly increased in these mice. This increase is caused by excessive osteoblast differentiation, wherein Stat1 function is independent of IFN signaling. Actually, Stat1 interacts with Runx2 in its latent form in the cytoplasm, thereby inhibiting the nuclear localization of Runx2, an essential transcription factor for osteoblast differentiation. The new function of Stat1 does not require the Tyr 701 that is phosphorylated when Stat1 becomes a transcriptional activator. Our study provides a unique example in which a latent transcription factor attenuates the activity of another transcription factor in the cytoplasm, and reveals a new regulatory mechanism in bone remodeling. [Keywords: Stat1; Runx2; osteoblast; bone remodeling] Supplemental material is available at http://www.genesdev.org. Received June 3, 2003; revised version accepted June 24, 2003. The integrity of the vertebrate skeletal system is main- and bone tumors, whereas excessive bone formation or tained by two distinct regulatory processes: the embry- defective bone resorption lead to osteosclerosis or osteo- onic developmental and postnatal regulatory processes petrosis (Rodan and Martin 2000; Takayanagi et al. (Olsen et al. 2000; Karsenty and Wagner 2002). In the 2000a; Teitelbaum 2000). Thus, the investigation of the former process, the development of the skeletal system regulatory mechanism of differentiation of these two cell depends on the differentiation of chondrocytes, osteo- types is important in the understanding of the physiol- blasts, and osteoclasts, all of which coordinate endo- ogy and pathology of the skeletal system. chondral and membranous bone formation. On the other Previously, we have shown that both type I and type II hand, in the latter process, referred to as bone remodel- interferon systems (i.e., IFN-␣/␤ and IFN-␥ systems) are ing, the bone matrix is constantly degraded by osteo- critical for the regulation of the skeletal system by sup- clasts and deposited by osteoblasts (Manolagas 2000). pressing osteoclastogenesis (Takayanagi et al. 2000b, The balanced action of these two cell types is critical for 2002b). These two IFNs exert their inhibitory functions the normal homeostasis of the skeletal system in adults. by distinct mechanisms, as briefly described below Therefore, tipping this balance in favor of either cell type (Stark et al. 1998; Taniguchi et al. 2001; Takayanagi et al. sometimes leads to pathological conditions. Excessive 2002c). During osteoclast differentiation, induced by bone resorption is seen in autoimmune arthritis, peri- RANKL (receptor activator of NF-␬B ligand), the IFN-␤ odontitis, postmenopausal osteoporosis, Paget’s disease, gene is induced in osteoclast precursor cells and IFN-␤ inhibits the differentiation by interfering with the RANKL-induced expression of c-Fos, an essential tran- 5These authors contributed equally to this work. scription factor for osteoclastogenesis. This inhibition is 6Corresponding author. dependent on the IFN-activated transcription factor, in- E-MAIL [email protected]; FAX 81-3-5841-3450. terferon stimulated gene factor 3 (ISGF3), which consists Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ gad.1119303. of activated Stat1, Stat2, and a member of the family of GENES & DEVELOPMENT 17:1979–1991 © 2003 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/03 $5.00; www.genesdev.org 1979 Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Kimetal. interferon regulatory factors (IRFs), IRF-9. On the other notype in the skeletal system of the mice deficient in the hand, IFN-␥ is critical in the T-cell-mediated regulation Stat1 gene (Stat1−/− mice). The bone of the mutant mice of osteoclastogenesis. RANKL expression is induced by exhibits excessive osteoclastogenesis, which is expected activated T cells, but RANKL signaling is negatively from our previous report demonstrating the critical role regulated by IFN-␥, which is also induced by the acti- of the IFN-activated ISGF3 in negative regulation of os- vated T-cells, thereby balancing RANKL signaling (Kong teoclastogenesis. Surprisingly, however, the bone mass et al. 1999; Takayanagi et al. 2000b). Stat1 (signal trans- is increased in these mice, suggesting a possibility that ducer and activator of transcription 1) is required for this Stat1 plays a critical role(s) in inhibiting the bone-form- IFN-␥ signal-mediated inhibition, in which the phos- ing process and that this inhibitory function dominates phorylated Stat1 forms a homodimer, termed IFN-␥-ac- over the inhibitory function of bone-resorbing process in tivated factor (GAF). Despite the distinct mechanisms of the context of IFN signaling. regulating osteoclast differentiation by IFN-␤ and IFN-␥, To date, the negative regulatory mechanism of bone Stat1 is involved in both mechanisms, suggesting the formation has been poorly understood. We show here integral role of Stat1 in the regulation of bone metabo- that Stat1 is involved in attenuating the transcriptional lism. activity of Runx2 in a unique manner, that is, by inter- In this regard, it is interesting that an activating mu- action with Runx2 in the cytoplasm. This new function tation in FGFR3 results in human achondroplasia, one of of Stat1 is independent of IFN signaling. We further the most common congenital diseases characterized by show that the in vitro differentiation of osteoblasts de- dwarfism, and that Stat1 is involved in this pathogenesis rived from the Stat1−/− mice is increased, which is ac- (Rousseau et al. 1994; Deng et al. 1996). The skeletal companied by the up-regulation of the DNA-binding ac- malformation in achondroplasia is caused by the sup- tivity of Runx2. Our results thus reveal a novel physi- pression of chondrocyte proliferation by excessive FGF ological function of Stat1 in the regulation of osteoblast signaling, leading to impaired endochondral ossification. differentiation in the postnatal stage, and offer an inter- FGF induces the phosphorylation of Stat1, and the sup- esting example of an otherwise latent transcription fac- pressive effect of FGF on chondrocyte proliferation is not tor in the cytoplasm that has an active and physiologi- observed in the skeletal tissue derived from mice lacking cally critical role. Stat1, suggesting that activation of Stat1 is involved in the suppressive effect of FGF on chondrocyte prolifera- tion in endochondral bone formation during embryogen- Results esis (Su et al. 1997; Sahni et al. 1999, 2001). Notwith- Increased bone mass in the Stat1−/− mice standing, it is unknown at present if and how Stat1 is involved in the control of transcriptional program(s) of To investigate the physiological function of Stat1 in the the skeletal system at the postnatal stage. skeletal system, we examined the bone phenotype of The transcription factor Runx2, also called Cbfa1, is a Stat1−/− mice (Meraz et al. 1996) at the age of 12 wk. We runt family transcription factor, and it plays a central reported previously that both Stat1 and IRF-9 are re- role in the determination of osteoblast differentiation quired for IFN-␤-mediated inhibition of osteoclastogen- (Ducy et al. 1997, 2000). Targeted disruption of Runx2 esis, suggesting that Stat1 participates in this inhibition results in the complete lack of bone formation by osteo- in the context of ISGF3 (Takayanagi et al. 2002b). Con- blasts, revealing that Runx2 is essential for both endo- sistently, we observed an increased osteoclast number chondral and membranous bone formation (Komori et al. and enhanced osteoclastic bone resorption in the tibia 1997). The haploinsufficiency of the Runx2 gene leads to of the Stat1−/− mice (Fig. 1A). Tartrate-resistant acid cleidocranial dysplasia in humans, and Runx2+/− mice phosphatase (TRAP) staining of the epiphyseal area of show a similar phenotype characterized by abnormal adult mice shows that the Stat1−/− mice have signifi- membranous ossification, suggesting that the expression cantly more osteoclasts in vivo than the wild-type (WT) level of Runx2 is closely related to the promotion of littermates (Fig. 1B), an observation similar to the mice