Parathyroid hormone–related peptide (PTHrP) is a positive regulator of chondrocyte proliferation during bone development. In embryonic mice lacking PTHrP, chondrocytes stop proliferating prematurely, with accelerated differentiation. Because the bone phenotype of mice lacking the cyclin-dependent kinase inhibitor p57Kip2 is the opposite of the PTHrP-null phenotype, we hypothesized that PTHrP’s proliferative actions in chondrocytes might be mediated by opposing p57. We generated p57/PTHrP-null embryos, which showed partial rescue of the PTHrP-null phenotype. There was reversal of the loss of proliferative chondrocytes in most bones, with reversal of the accelerated differentiation that occurs in the PTHrP-null phenotype. p57 mRNA and protein were upregulated in proliferative chondrocytes in the absence of PTHrP. Metatarsal culture studies confirmed the action of PTHrP to decrease p57 mRNA and protein levels in a model in which parathyroid hormone (PTH), used as an analog of PTHrP, increased chondrocyte proliferation rate and the length of the proliferative domain. PTH treatment of p57-null metatarsals had no effect on proliferation rate in round proliferative chondrocytes but still stimulated proliferation in columnar chondrocytes. These studies suggest that the effects of PTHrP on both the rate and extent of chondrocyte proliferation are mediated, at least in part, through suppression of p57 expression.
Helen E. MacLean, Jun Guo, Melissa C. Knight, Pumin Zhang, David Cobrinik, Henry M. Kronenberg
Based on the fact that aging is associated with a reciprocal decrease of osteogenesis and an increase of adipogenesis in bone marrow and that osteoblasts and adipocytes share a common progenitor, this study investigated the role of PPARγ, a key regulator of adipocyte differentiation, in bone metabolism. Homozygous PPARγ-deficient ES cells failed to differentiate into adipocytes, but spontaneously differentiated into osteoblasts, and these were restored by reintroduction of the PPARγ gene. Heterozygous PPARγ-deficient mice exhibited high bone mass with increased osteoblastogenesis, but normal osteoblast and osteoclast functions, and this effect was not mediated by insulin or leptin. The osteogenic effect of PPARγ haploinsufficiency became prominent with aging but was not changed upon ovariectomy. The PPARγ haploinsufficiency was confirmed to enhance osteoblastogenesis in the bone marrow cell culture but did not affect the cultures of differentiated osteoblasts or osteoclast-lineage cells. This study demonstrates a PPARγ-dependent regulation of bone metabolism in vivo, in that PPARγ insufficiency increases bone mass by stimulating osteoblastogenesis from bone marrow progenitors.
Toru Akune, Shinsuke Ohba, Satoru Kamekura, Masayuki Yamaguchi, Ung-il Chung, Naoto Kubota, Yasuo Terauchi, Yoshifumi Harada, Yoshiaki Azuma, Kozo Nakamura, Takashi Kadowaki, Hiroshi Kawaguchi
The role of TGF-β/bone morphogenetic protein signaling in the chondrogenic differentiation of human synovial fibroblasts (SFs) was examined with the adenovirus vector–mediated gene transduction system. Expression of constitutively active activin receptor–like kinase 3 (ALK3CA) induced chondrocyte-specific gene expression in SFs cultured in pellets or in SF pellets transplanted into nude mice, in which both the Smad and p38 pathways are essential. To analyze downstream cascades of ALK3 signaling, we utilized adenovirus vectors carrying either Smad1 to stimulate Smad pathways or constitutively active MKK6 (MKK6CA) to activate p38 pathways. Smad1 expression had a synergistic effect on ALK3CA, while activation of p38 MAP kinase pathways alone by transduction of MKK6CA accelerated terminal chondrocytic differentiation, leading to type X collagen expression and enhanced mineralization. Overexpression of Smad1 prevented MKK6CA-induced type X collagen expression and maintained type II collagen expression. In a mouse model of osteoarthritis, activated p38 expression as well as type X collagen staining was detected in osteochondrophytes and marginal synovial cells. These results suggest that SFs can be differentiated into chondrocytes via ALK3 activation and that stimulating Smad pathways and controlling p38 activation at the proper level can be a good therapeutic strategy for maintaining the healthy joint homeostasis and treating degenerative joint disorders.
Hiroaki Seto, Satoshi Kamekura, Toshiki Miura, Aiichiro Yamamoto, Hirotaka Chikuda, Toru Ogata, Hisatada Hiraoka, Hiromi Oda, Kozo Nakamura, Hisashi Kurosawa, Ung-il Chug, Hiroshi Kawaguchi, Sakae Tanaka
The gp130-dependent cytokines, which signal through at least two intracellular pathways, regulate osteoclast and osteoblast formation. To define their roles in regulating bone mass, we analyzed mice in which gp130 signaling via either the signal transducer and activator of transcription (STAT) 1/3 (gp130ΔSTAT/ΔSTAT) or SHP2/ras/MAPK (gp130Y757F/Y757F) pathway was attenuated. In gp130ΔSTAT/ΔSTAT mice, trabecular bone volume (BV/TV) and turnover were normal, but bone length was reduced by premature growth plate closure, indicating an essential role for gp130-STAT1/3 signaling in chondrocyte differentiation. In contrast, while bone size was normal in gp130Y757F/Y757F mice, BV/TV was reduced due to high bone turnover, indicated by high osteoclast surface/bone surface (OcS/BS) and osteoblast surface/bone surface (ObS/BS). Furthermore, generation of functional osteoclasts from bone marrow of gp130Y757F/Y757F mice was elevated, revealing that while gp130 family cytokines stimulate osteoclastogenesis through the osteoblast lineage, gp130, via SHP2/Ras/MAPK, inhibits osteoclastogenesis in a cell lineage–autonomous manner. Genetic ablation of IL-6 in gp130Y757F/Y757F mice exacerbated this osteopenia by reducing ObS/BS without affecting OcS/BS. Thus, while IL-6 is critical for high bone formation in gp130Y757F/Y757F mice, it is not involved in the increased osteoclastogenesis. In conclusion, gp130 is essential for normal bone growth and trabecular bone mass, with balanced regulation depending on selective activation of STAT1/3 and SHP2/ras/MAPK, respectively. Furthermore, the latter pathway can directly inhibit osteoclastogenesis in vivo.
Natalie A. Sims, Brendan J. Jenkins, Julian M.W. Quinn, Akira Nakamura, Markus Glatt, Matthew T. Gillespie, Matthias Ernst, T. John Martin
VEGF is crucial for metaphyseal bone vascularization. In contrast, the angiogenic factors required for vascularization of epiphyseal cartilage are unknown, although this represents a developmentally and clinically important aspect of bone growth. The
Christa Maes, Ingrid Stockmans, Karen Moermans, Riet Van Looveren, Nico Smets, Peter Carmeliet, Roger Bouillon, Geert Carmeliet
We describe the effects of the overexpression of noggin, a bone morphogenetic protein (BMP) inhibitor, on osteoblast differentiation and bone formation. Cells of the osteoblast and chondrocyte lineages, as well as bone marrow macrophages, showed intense β-gal histo- or cytostaining in adult noggin+/– mice that had a LacZ transgene inserted at the site of noggin deletion. Despite identical BMP levels, however, osteoblasts of 20-month-old C57BL/6J and 4-month-old senescence-accelerated mice (SAM-P6 mice) had noggin expression levels that were approximately fourfold higher than those of 4-month-old C57BL/6J and SAM-R1 (control) mice, respectively. U-33 preosteoblastic cells overexpressing the noggin gene showed defective maturation and, in parallel, a decreased expression of Runx-2, bone sialoprotein, osteocalcin, and RANK-L. Noggin did not inhibit the ligandless signaling and pro-differentiation action of the constitutively activated BMP receptor type 1A, ca-ALK-3. Transgenic mice overexpressing noggin in mature osteocalcin-positive osteoblasts showed dramatic decreases in bone mineral density and bone formation rates with histological evidence of decreased trabecular bone and CFU-osteoblast colonies at 4 and 8 months. Together, the results provide compelling evidence that noggin, expressed in mature osteoblasts, inhibits osteoblast differentiation and bone formation. Thus, the overproduction of noggin during biological aging may result in impaired osteoblast formation and function and hence, net bone loss.
Xue-Bin Wu, Yanan Li, Adina Schneider, Wanqin Yu, Gopalan Rajendren, Jameel Iqbal, Matsuo Yamamoto, Mohammad Alam, Lisa J. Brunet, Harry C. Blair, Mone Zaidi, Etsuko Abe
The mechanisms through which estrogen prevents bone loss are uncertain. Elsewhere, estrogen exerts beneficial actions by suppression of reactive oxygen species (ROS). ROS stimulate osteoclasts, the cells that resorb bone. Thus, estrogen might prevent bone loss by enhancing oxidant defenses in bone. We found that glutathione and thioredoxin, the major thiol antioxidants, and glutathione and thioredoxin reductases, the enzymes responsible for maintaining them in a reduced state, fell substantially in rodent bone marrow after ovariectomy and were rapidly normalized by exogenous 17-β estradiol. Moreover, administration of N-acetyl cysteine (NAC) or ascorbate, antioxidants that increase tissue glutathione levels, abolished ovariectomy-induced bone loss, while L-buthionine-(S,R)-sulphoximine (BSO), a specific inhibitor of glutathione synthesis, caused substantial bone loss. The 17-β estradiol increased glutathione and glutathione and thioredoxin reductases in osteoclast-like cells in vitro. Furthermore, in vitro NAC prevented osteoclast formation and NF-κB activation. BSO and hydrogen peroxide did the opposite. Expression of TNF-α, a target for NF-κB and a cytokine strongly implicated in estrogen-deficiency bone loss, was suppressed in osteoclasts by 17-β estradiol and NAC. These observations strongly suggest that estrogen deficiency causes bone loss by lowering thiol antioxidants in osteoclasts. This directly sensitizes osteoclasts to osteoclastogenic signals and entrains ROS-enhanced expression of cytokines that promote osteoclastic bone resorption.
Jenny M. Lean, Julie T. Davies, Karen Fuller, Christopher J. Jagger, Barrie Kirstein, Geoffrey A. Partington, Zoë L. Urry, Timothy J. Chambers
FGF-23, a novel member of the FGF family, is the product of the gene mutated in autosomal dominant hypophosphatemic rickets (ADHR). FGF-23 has been proposed as a circulating factor causing renal phosphate wasting not only in ADHR (as a result of inadequate degradation), but also in tumor-induced osteomalacia (as a result of excess synthesis by tumor cells). Renal phosphate wasting occurs in approximately 50% of patients with McCune-Albright syndrome (MAS) and fibrous dysplasia of bone (FD), which result from postzygotic mutations of the GNAS1 gene. We found that FGF-23 is produced by normal and FD osteoprogenitors and bone-forming cells in vivo and in vitro. In situ hybridization analysis of FGF-23 mRNA expression identified “fibrous” cells, osteogenic cells, and cells associated with microvascular walls as specific cellular sources of FGF-23 in FD. Serum levels of FGF-23 were increased in FD/MAS patients compared with normal age-matched controls and significantly higher in FD/MAS patients with renal phosphate wasting compared with those without, and correlated with disease burden bone turnover markers commonly used to assess disease activity. Production of FGF-23 by FD tissue may play an important role in the renal phosphate–wasting syndrome associated with FD/MAS.
Mara Riminucci, Michael T. Collins, Neal S. Fedarko, Natasha Cherman, Alessandro Corsi, Kenneth E. White, Steven Waguespack, Anurag Gupta, Tamara Hannon, Michael J. Econs, Paolo Bianco, Pamela Gehron Robey
We have found that the ubiquitin-proteasome pathway exerts exquisite control of osteoblast differentiation and bone formation in vitro and in vivo in rodents. Structurally different inhibitors that bind to specific catalytic β subunits of the 20S proteasome stimulated bone formation in bone organ cultures in concentrations as low as 10 nM. When administered systemically to mice, the proteasome inhibitors epoxomicin and proteasome inhibitor–1 increased bone volume and bone formation rates over 70% after only 5 days of treatment. Since the ubiquitin-proteasome pathway has been shown to modulate expression of the Drosophila homologue of the bone morphogenetic protein-2 and -4 (BMP-2 and BMP-4) genes, we examined the effects of noggin, an endogenous inhibitor of BMP-2 and BMP-4 on bone formation stimulated by these compounds and found that it was abrogated. These compounds increased BMP-2 but not BMP-4 or BMP-6 mRNA expression in osteoblastic cells, suggesting that BMP-2 was responsible for the observed bone formation that was inhibited by noggin. We show proteasome inhibitors regulate BMP-2 gene expression at least in part through inhibiting the proteolytic processing of Gli3 protein. Our results suggest that the ubiquitin-proteasome machinery regulates osteoblast differentiation and bone formation and that inhibition of specific components of this system may be useful therapeutically in common diseases of bone loss.
I.R. Garrett, D. Chen, G. Gutierrez, M. Zhao, A. Escobedo, G. Rossini, S.E. Harris, W. Gallwitz, K.B. Kim, S. Hu, C.M. Crews, G.R. Mundy
Although the role of estradiol in maintaining bone mass is well established, the relative contributions of the estradiol receptors ERα and ERβ and of the androgen receptor (AR) remain controversial. To determine the role of ERα-mediated, ERβ-mediated, and non–ER-mediated mechanisms in maintaining bone mass, gonadectomy and estradiol treatment were studied in ER-knockout mice. Estradiol treatment of ovariectomized ERαβ–/– mice failed to prevent bone loss, precluding significant effects of estradiol on bone through non–ER-signaling pathways. In contrast, estradiol prevented ovariectomy-induced bone loss in ERβ–/– mice, as in WT males and females, indicating that ERα is the major mediator of estradiol effects in bone. No response of bone to estradiol was detected in orchidectomized ERα–/– mice, suggesting estradiol cannot protect bone mass via the AR in vivo. In contrast to female ERαβ–/– and male ERα–/– mice, female ERα–/– mice were partially protected against ovariectomy-induced bone loss by estradiol, confirming that ERβ mediates estradiol effects in bone, but only in females and with a lower efficacy than ERα. We conclude that ERα is the main effector of estradiol’s protective function in bone in both male and female mice, and that, in its absence, AR is not sufficient to mediate this response.
Natalie A. Sims, Philippe Clément-Lacroix, Dominique Minet, Caroline Fraslon-Vanhulle, Martine Gaillard-Kelly, Michèle Resche-Rigon, Roland Baron