Phosphate Provides an Extracellular Signal That Drives Nuclear Export of Runx2/Cbfa1 in Bone Cells

During embryogenesis, the expression of mammalian stanniocalcin (STC1) in the appendicular skeleton suggests its involvement in the regulation of longitudinal bone growth. Such a role is further supported by the presence of dwarfism in mice overexpressing STC1. Yet, the STC 1 inhibitory effect on growth may be related to both postnatal metabolic abnormalities and prenatal defective bone formation. In our study, we used an organ culture system to evaluate the effects of STC on growth plate chondrogenesis, which is the primary determinant of longitudinal bone growth. Fetal rat metatarsal bones were cultured in the presence of recombinant human STC (rhSTC). After 3 days, rhSTC suppressed metatarsal growth, growth plate chondrocyte proliferation and hypertrophy/differentiation, and extracellular matrix synthesis. In addition, rhSTC increased the number of apoptotic chondrocytes in the growth plate. In cultured chondrocytes, rhSTC increased phosphate uptake, reduced chondrocyte proliferation and matrix synthesis, and induced apoptosis. All these effects were reversed by culturing chondrocytes with rhSTC and phosphonoformic acid, an inhibitor of phosphate transport. The rhSTC-mediated inhibition of metatarsal growth and growth plate chondrocyte proliferation and hypertrophy/differentiation was abolished by culturing metatarsals with rhSTC and phosphonoformic acid. Taken together, our findings indicate that STC1 inhibits longitudinal bone growth directly at the growth plate. Such growth inhibition, likely mediated by an increased chondrocyte phosphate uptake, results from suppressed chondrocyte proliferation, hypertrophy/differentiation, and matrix synthesis and by increased apoptosis. Last, the expression of both STC1 and its binding site in the growth plate would support an autocrine/paracrine role for this growth factor in the regulation of growth plate chondrogenesis. Stanniocalcin (STC)2 is a glycoprotein first identified as a secretory product of the corpuscles of Stannius, an endocrine gland unique to bony fish (1, 2). The primary function of STC in fish is to prevent hypercalcemia (3) by inhibiting calcium uptake by the gills and gut and stimulating phosphate reabsorption by the kidneys (1, 4).The mammalian homolog of STC (STC1) has been found in humans, rats, and mice (5-7). Human and mouse STC1 proteins are closely related to each other (98% amino acid identity) and share 80% amino acid identity with fish STC (5). Compared with fish STC, STC1 seems to have a preferential effect on phosphate metabolism than on calcium metabolism (8, 9), with evidence suggesting that sodium-dependent P i (NaP i ) transporter(s) may be a target of STC1 activity (10). Although fish STC is primarily expressed in one organ (the corpuscle of Stannius), mammalian STC1 is virtually ubiquitous. The distribution of STC protein and/or mRNA in multiple organs such as kidney, intestine, heart, thyroid, lung, placenta, brain (11-15), and bone (with virtually undetectable serum levels) (11) would suggest a paracrine rather than an endocrine ...

Section: Discussionsupporting

confidence: 56%

Stanniocalcin 1 Acts as a Paracrine Regulator of Growth Plate Chondrogenesis

Yoshiko

Luca

2006

“…In their study the expression of hydroxyapatite nucleator proteins such as BSP was not evaluated. However, the authors observed that P i stimulus lowered the secretion of OC (73).…”

Section: Discussionmentioning

confidence: 90%

“…Indeed, the requirement of inorganic phosphate (P i ) has been shown to be important during osteoblastic differentiation not only as a component of hydroxyapatite minerals but also as a signal event preceding mineralization (72). In their study of the role of P i during mineralization, Fujita et al (73) unexpectedly observed that P i quickly evacuated Runx2 from the nuclei in osteoblastic MC3T3-E1 cells. Accelerated nodule formation and mineralization occurred the following days of culture, suggesting that this ultimate phase of osteoblastic differentiation does not necessitate the presence of nuclear Runx2, which concords with our data.…”

Section: Discussionmentioning

confidence: 99%

Runx2- and Histone Deacetylase 3-mediated Repression Is Relieved in Differentiating Human Osteoblast Cells to Allow High Bone Sialoprotein Expression

Lamour

Detry

Sanchez

et al. 2007

Bone sialoprotein (BSP) is a bone matrix glycoprotein whose expression coincides with terminal osteoblastic differentiation and the onset of mineralization. In this study we show that BSP expression is considerably increased in confluent Saos-2 human osteosarcoma cells and in differentiating normal human osteoblasts, concomitantly with the decrease of Runx2, a key transcription factor controlling bone formation. Therefore, we investigated the role of Runx2 in the regulation of BSP expression in Saos-2 cells. Using a mobility shift assay, we demonstrated that Runx2 binds to the BSP promoter only in preconfluent cells. Histone deacetylase 3 (HDAC3) has been recently shown to act as a Runx2 co-repressor. Chromatin immunoprecipitation assays demonstrated that both Runx2 and HDAC3 are detectable at the BSP promoter in preconfluent Saos-2 cells but not when they are confluent and overexpress BSP. Consistently, nuclear Runx2 protein level is down-regulated, whereas Saos-2 cells became increasingly confluent. Finally, the suppression of HDAC3, Runx2, or both by RNA interference induced the expression of BSP at both mRNA and protein levels in Saos-2 cells. Our data demonstrate that Runx2 and HDAC3 repress BSP gene expression and that this repression is suspended upon osteoblastic cell differentiation. Both the nuclear disappearance of Runx2 and the non-recruitment of HDAC3 represent new means to relieve Runx2-mediated suppression of BSP expression, thus allowing the acquisition of a fully differentiated and mineralization-competent phenotype by osteoblast cells.

“…Prior in situ analysis of Runx2 isoform expression demonstrated that Runx2-I is expressed predominantly in undifferentiated mesenchymal cells, preosteoblasts, and chondrocytes, as well as during later stages of development within these lineages, whereas Runx2-II expression is limited to later stages of osteoblastic differentiation and terminal hypertrophic chondrocytes (36 -37, 40, 48). In addition, Runx2-II is differentially regulated by bone morphogenetic proteins 2 and 7 (BMP-2 and BMP-7), transforming growth factor ␤1 (TGF-␤1), and 1,25(OH) 2 D 3 in osteoblasts (26,29,35,(52)(53)(54)(55)(56)(57), and tumor necrosis factor ␣ (TNF␣) selectively suppresses Runx2-I (58). Such differences in temporal and spatial expression patterns and regulation imply specialized functions of the isoforms.…”

Section: Differential Actions Of Runx2 Isoformsmentioning

confidence: 99%

Selective Deficiency of the “Bone-related” Runx2-II Unexpectedly Preserves Osteoblast-mediated Skeletogenesis

Xiao

Hjelmeland

Quarles

2004

128

Runx2 (runt-related transcription factor 2) is a master regulator of skeletogenesis. Distinct promoters in the Runx2 gene transcribe the "bone-related" Runx2-II and non-osseous Runx2-I isoforms that differ only in their respective N termini. Existing mutant mouse models with both isoforms deleted exhibit an arrest of osteoblast and chondrocyte maturation and the complete absence of mineralized bone, but they do not distinguish the separate functions of the two N-terminal isoforms. To elucidate the function of the bone-related isoform, we generated selective Runx2-II-deficient mice by the targeted deletion of the distal promoter and exon 1. Homozygous Runx2-II-deficient (Runx2-II ؊/؊ ) mice unexpectedly formed axial, appendicular, and craniofacial bones derived from either intramembranous ossification or mesenchymal cells of the bone collar, but they failed to form the posterior cranium and other bones derived from endochondral ossification. Heterozygous Runx2-II-deficient mice had grossly normal skeletons, but were osteopenic. The commitment of mesenchymal cells ex vivo to the osteoblast lineage occurred in Runx2-II ؊/؊ mice, but osteoblastic gene expression was impaired. Chondrocyte maturation appeared normal, but the zone of hypertrophic chondrocytes was not transformed into metaphyseal bone, leading to widened growth plates in Runx2-II ؊/؊ mice. Compensatory increments in Runx2-I expression occurred in Runx2-II ؊/؊ mice but were not sufficient to normalize osteoblastic maturation or transcriptional activity. Our findings support distinct functions of Runx2-II and -I in the control of skeletogenesis. Runx2-I is sufficient for early osteoblastogenesis and intramembranous bone formation, whereas Runx2-II is necessary for complete osteoblastic maturation and endochondral bone formation.The Runx 1 family of transcription factors consists of three highly conserved mammalian genes, designated Runx1, Runx2, and Runx3 (1-5), that regulate, respectively, hematopoiesis (6), skeletogenesis (7-10), and neuronal development (11)(12). RUNX2 is the master transcription factor essential for skeletogenesis. Targeted disruption of the Runt domain of Runx2 results in the complete lack of both intramembranous and endochondral bone formation due to an arrest in osteoblast and chondrocyte maturation (8,10,(13)(14)(15)(16)(17)(18)(19). Haploinsufficiency of RUNX2 causes cleidocranial dysplasia in humans, and heterozygous Runx2 deficient mice show a similar phenotype, suggesting that the expression level of Runx2 influences the skeletal phenotype (10).Separate promoters control the expression of Runx2 isoforms with different 5Ј-UTRs and N-terminal sequences that bind to the same consensus DNA cis-acting elements, interact with similar co-factors, and demonstrate overlapping transactivation potential in vitro (17-24). The Runx2-II isoform (also called PEBP2␣A, major til-1 and Cbfa1.iso, Osf2) is derived from the distal "bone-related" promoter (P1) and encodes a 528-amino acid protein that begins with the 19 amino acids MASNSLFSAVTPCQQ...