Cbfa1 Gene Expression Research Articles

Integrin‐associated tyrosine kinase FAK affects Cbfa1 expression

Following cell adhesion, focal adhesion kinase (FAK) autophosphorylates on tyrosine and regulates intracellular signaling cascades that regulate cell growth and differentiation. The hypothesis of this study was FAK mediates osteoblast differentiation dependent Cbfa1 expression. Slowly mineralizing UI and rapidly mineralizing UMR-106-01 BSP osteoblasts formed focal adhesions; however, the level of FAK in UI focal adhesions was less than that seen in BSP cells. UI cultures had less FAK expression (p < 0.05) along with elevated levels of FAK phosphotyrosine in comparison to rapidly mineralizing BSP cultures. Mineralization decreased in a dose-dependent manner in response to Herbimycin A, a tyrosine kinase inhibitor. Overexpression of FAK in UI cells led to a fourfold increase in Cbfa1 gene expression (p < 0.02), and an increase in Cbfa1 protein expression. These results suggest that the integrin-associated tyrosine kinase FAK contributes to the regulation of the osteoblast differentiation in part through the regulation of Cbfa1 expression.

Estrogen Receptor Agonists and Immune System in Ovariectomized Mice

Several data implicate the immune system in bone lost after estrogen deficiency, however, some of the effects on the immune system of estrogen deficiency or of estrogen receptor (ER) modulation are not well established. In this study, the effect of ER agonists on the immune system in ovariectomized mice is analyzed. Mice were ovariectomized and were administered 17beta-estradiol (E2), raloxifene (RAL) or genistein (GEN). The effect of a 4-week treatment on bone turnover and on several parameters that reflect the status of the immune system was studied. Results show that ovariectomy provoked both uterine atrophy and thymic hypertrophy. Although RAL corrected thymic hypertrophy, only E2 corrected both. Ovariectomized mice showed increased levels of serum calcium and cathepsin K gene expression and decreased levels of serum alkaline phosphatase (ALP) activity, which suggests that there is a persistent alteration in bone metabolism. Moreover, ovariectomy increased B-cells and CD25+ cells, and decreased the percentages of T-cells and Cbfa1 gene expression in bone marrow (BM). All ER agonists corrected, although to different degrees, changes induced by the ovariectomy. Furthermore, results showed that it is essential to adjust ER agonist doses to avoid immunosuppression, since all ER agonists decreased BM T-cell levels.

Hyperphosphatemia and vascular calcification in end-stage renal disease

Vascular calcification is a common finding in atherosclerosis and a serious problem in uremic patients. Because of the correlation of hyperphosphatemia and vascular calcification, the ability of extracellular inorganic phosphate levels to regulate human aortic smooth muscle cell (HSMC) culture mineralization in vitro was examined. HSMC cultured in media containing normal physiologic levels of inorganic phosphate (1.4 mM) did not mineralize. In contrast, HSMC cultured in media containing phosphate levels comparable with those seen in hyperphosphatemic individuals (>1.4 mM) showed dose-dependent increases in mineral deposition. Mechanistic studies showed that elevated phosphate treatment of HSMC also enhanced the expression of the osteoblastic differentiation markers osteocalcin and osf2/Cbfa-1. The effects of elevated phosphate on HSMC were mediated by a sodium-dependent phosphate cotransporter (NPC) as indicated by the ability of the specific NPC inhibitor phosphonoformic acid to dose-dependently inhibit phosphate-induced calcium deposition as well as osteocalcin and Cbfa-1 gene expression. The NPC in HSMC was identified as Pit-1, a member of the novel type III NPCs. These data suggest that elevated phosphate may directly stimulate HSMC to undergo phenotypic changes that predispose to calcification and offers a novel explanation of the phenomenon of vascular calcification under hyperphosphatemic conditions. Furthermore, we examined the factors affecting peripheral vascular calcification in 332 nondiabetic hemodialysis patients. There were 45 nondiabetic patients with vascular calcification. In multivariate logistic regression, the significant factors affecting vascular calcification were advanced age, longer duration of hemodialysis, increased phosphate concentrations, male gender, and lower predialysis diastolic pressure. Our findings suggest that an elevated phosphate level may directly stimulate HSMC to undergo phenotypic changes that predispose to calcification and offer a novel explanation of the phenomenon of vascular calcification under hyperphosphatemic conditions.

Hedgehog signaling enhances core-binding factor a1 and receptor activator of nuclear factor-kappaB ligand (RANKL) gene expression in chondrocytes.

Hedgehog signaling is considered to play a crucial role in chondrogenesis by regulation through a network of cytokine actions, which is not fully understood. We examined the effect of hedgehog signaling on the expression of core-binding factor a1 (Cbfa1), a critical transcription factor for the development of bone and cartilage. Primary chondrocytes prepared from the costal cartilage of newborn mice were treated with N-terminal fragment of recombinant murine sonic hedgehog (rmShh-N). Northern blot analysis indicated that Cbfa1 mRNA expression levels in the chondrocyte cultures were elevated by the treatment with rmShh-N. rmShh-N treatment enhanced 1.8 kb Cbfa1 promoter activity in chondrocytes, suggesting the presence of transcriptional control. As Cbfa1-binding site(s) have been located in the promoter of the receptor activator of nuclear factor-kappaB (RANK) ligand (RANKL) gene, we also examined RANKL expression. rmShh-N treatment upregulated RANKL and RANK mRNA expression levels in chondrocytes. Interestingly, RANKL suppressed the hedgehog enhancement of alkaline phosphatase activity in chondrocytes, suggesting the presence of a link between these signaling molecules. We conclude that hedgehog signaling activates Cbfa1 gene expression through its promoter in chondrocytes, and also activates and interacts with RANKL to maintain cartilage development.

Implant surface roughness affects osteoblast gene expression.

The transcription factor Cbfa1 regulates osteoblast differentiation and expression of genes necessary for the development of a mineralized phenotype. The purpose of this study was to determine if Cbfa1 and BSPII gene expression are influenced by implant surface microtopography. Osteoblasts were cultured on 600-grit (grooved) or sandblasted (roughened) cpTi implant discs. Mineralization was evaluated by Alizarin-Red-S staining. Real Time PCR was used for quantitative analysis of Cbfa1 and BSPII gene expression. Enhanced mineralization was seen in osteoblasts grown on roughened implant surfaces relative to tissue culture plastic. Real Time PCR showed significant (P < 0.05) increases in Cbfa1 gene expression in cells grown on roughened, as compared with grooved, implant surfaces. BSPII gene expression was also increased on rough surfaces in the UMR cells, but was reduced in the rat calvarial osteoblast cultures. These results suggest that osteoblast gene expression and mineralization are affected by roughened implant surface microtopographies during osseointegration of dental implants.

Expression of adipogenesis markers in a murine stromal cell line treated with 15-deoxy Δ12,14-prostaglandin J2, interleukin-11, 9-cis retinoic acid and vitamin K2

Recent studies have demonstrated that bone marrow stromal cells can undergo adipogenesis or osteoblastogenesis in vivo, and in vitro, and that peroxisome proliferator-activated receptor γ (PPAR γ) plays a central role in the control of adipocyte differentiation. In the present study, we treated a murine stromal cell line (TMS-14) with a cocktail of dexamethasone, insulin and glucose (DIG cocktail), which caused the cells to convert to fat-laden cells with adipocyte-like morphology. We also exposed TMS-14 cells to DIG cocktail followed by 15-deoxy Δ12,14-prostaglandin J2 (15d-PGJ2), a ligand of PPAR γ, interleukin- 11 (IL-11), 9-cis retinoic acid (9-cis RA) and vitamin K2. 15d-PGJ2 enhanced DIG cocktail-induced adipogenesis, whereas IL-11, 9-cis RA and vitamin K2 each inhibited adipogenesis induced by DIG cocktail. The gene expressions of four adipogenesis markers, PPAR γ 2, adipocyte P2 (aP2), adipocyte determination and differentiation factor 1 (ADD1), and fatty acid synthase (FAS) were enhanced by DIG cocktail and these expressions were more enhanced by 15d-PGJ2, in contrast they were attenuated by 9-cis RA. IL-11 also attenuated the adipogenesis markers except ADD1. Western blotting showed that 15d-PGJ2 enhanced the levels of PPAR γ, C/EBP α and RXR α proteins, while IL-11 and 9-cis RA decreased the level of PPAR γ protein, but not C/EBP α protein and vitamin K2 decreased the level of C/EBP α protein. We also tested the effect of 15d-PGJ2 on osteoblastogenesis, using TMS-12 cells, another stromal cell clone from the same mouse, which differentiate into osteoblasts spontaneously. 15d-PGJ2 did not affect osteoblastogenesis, as detected by von Kossa staining and Cbfa-1 gene expression. These data indicate that 15d-PGJ2 enhances the expression of both PPAR γ and C/EBP α and as a result it stimulates adipogenesis in murine bone marrow cells.

Vascular calcification and inorganic phosphate

Vascular calcification is highly correlated with elevated serum phosphate levels in uremic patients. To shed light on this process, we examined the ability of extracellular inorganic phosphate (Pi) levels to regulate human aortic smooth muscle cell (HSMC) culture mineralization in vitro. When cultured in media containing normal physiological levels of Pi (1.4 mmol/L Pi), HSMC grew in monolayers and did not mineralize. In contrast, HSMC cultured in media containing Pi levels comparable to those seen in hyperphosphatemic individuals (> 1.4 mmol/L), showed dose-dependent increases in cell culture calcium deposition. Mechanistic studies showed that elevated Pi treatment of HSMC also enhanced the expression of the osteogenic markers, osteocalcin and Cbfa-1. The effects of elevated Pi on HSMC were mediated by a sodium-dependent phosphate cotransporter (NPC), as indicated by the ability of the specific NPC inhibitor, phosphonoformic acid (PFA), to dose-dependently inhibit Pi-induced calcium deposition as well as osteocalcin and Cbfa-1 gene expression. Using polymerase chain reaction and Northern blot analyses, the NPC in HSMC was identified as Pit-1 (Glvr-1), a member of the type III NPCs. Interestingly, platelet-derived growth factor-BB (PDGF-BB), a potent atherogenic stimulus, increased the maximum velocity (Vmax) but not the affinity (Km) of phosphate uptake, enhanced the expression of Pit-1 mRNA, and induced HSMC culture calcification in a time- and dose-dependent manner. Importantly, in the presence of PDGF, HSMC culture calcification occurred under normophosphatemic conditions. These data suggest that elevated Pi may directly stimulate HSMC to undergo phenotypic changes that predispose to calcification and may help explain both the phenomena of human metastatic calcification under hyperphosphatemic conditions as well as increased calcification in PDGF-rich atherosclerotic lesions. © 2001 by the National Kidney Foundation, Inc.

Regulation of human cbfa1 gene transcription in osteoblasts by selective estrogen receptor modulators (SERMs)

Cbfa1, a transcription factor critical for bone formation, plays a key role in osteoblast recruitment and differentiation. We have cloned and characterized a 3.0 KB 5′-flanking region of the human cbfa1 gene isolated from a P1 human genomic library. DNA sequencing revealed several known canonical nuclear transcription factor binding sites, including two AP1 and six OSE2 binding sites in the proximal promoter region. Although no estrogen (E2)-response element (ERE) binding sites were identified, E2 has been shown to regulate gene activity via AP1 promoter sites. We examined the effect of selective estrogen receptor modulators (SERMs) on human cbfa1 gene promoter activity using cell-based luciferase reporter transcriptional assays. Three characterized SERMs, tamoxifen, raloxifene, and ICI 178,180, all upregulated cbfa1–luciferase (cbfa1Luc) gene activity 5- to 10-fold in a dose-dependent manner. This effect was mediated by both ERα and ERβ. Mutational analysis demonstrated that the minimal promoter region for basal of SERM-activated transcription was mapped to adjacent AP1-like and OSE2 binding sites within −93 and +7 of the transcription start condon. Further, electrophoretic mobility shift assays (EMSAs) demonstrate that ICI 178,180 increased binding of AP1 and OSE2 site by ERα and cbfa1, respectively. These studies suggest that SERMs can modulate bone-specific cbfa1 gene expression in a human osteosarcoma cell line.

Phosphate regulation of vascular smooth muscle cell calcification.

Vascular calcification is a common finding in atherosclerosis and a serious problem in diabetic and uremic patients. Because of the correlation of hyperphosphatemia and vascular calcification, the ability of extracellular inorganic phosphate levels to regulate human aortic smooth muscle cell (HSMC) culture mineralization in vitro was examined. HSMCs cultured in media containing normal physiological levels of inorganic phosphate (1.4 mmol/L) did not mineralize. In contrast, HSMCs cultured in media containing phosphate levels comparable to those seen in hyperphosphatemic individuals (>1.4 mmol/L) showed dose-dependent increases in mineral deposition. Mechanistic studies revealed that elevated phosphate treatment of HSMCs also enhanced the expression of the osteoblastic differentiation markers osteocalcin and Cbfa-1. The effects of elevated phosphate on HSMCs were mediated by a sodium-dependent phosphate cotransporter (NPC), as indicated by the ability of the specific NPC inhibitor phosphonoformic acid, to dose dependently inhibit phosphate-induced calcium deposition as well as osteocalcin and Cbfa-1 gene expression. With the use of polymerase chain reaction and Northern blot analyses, the NPC in HSMCs was identified as Pit-1 (Glvr-1), a member of the novel type III NPCs. These data suggest that elevated phosphate may directly stimulate HSMCs to undergo phenotypic changes that predispose to calcification and offer a novel explanation of the phenomenon of vascular calcification under hyperphosphatemic conditions. The full text of this article is available at http://www.circresaha.org.

Transcriptional autoregulation of the bone related CBFA1/RUNX2 gene

The runt related transcription factor CBFA1 (AML3/PEBP2αA/RUNX2) regulates expression of several bone- and cartilage-related genes and is required for bone formation in vivo. The gene regulatory mechanisms that control activation and repression of CBFA1 gene transcription during osteoblast differentiation and skeletal development are essential for proper execution of the osteogenic program. We have therefore defined functional contributions of 5′ regulatory sequences conserved in rat, mouse and human CBFA1 genes to transcription. Deletion analysis reveals that 0.6 kB of the bone-related rat or mouse CBFA1 promoter (P1, MASNS protein isoform) is sufficient to confer transcriptional activation, and that there are multiple promoter domains which positively and negatively regulate transcription. Progressive deletion of promoter segments between nt −351 and −92 causes a striking 30- to 100-fold combined decrease in promoter activity. Additionally, 5′ UTR sequences repress reporter gene transcription 2- to 3-fold. Our data demonstrate that CBFA1 is a principal DNA binding protein interacting with the 5′ region of the CBFA1 gene in osseous cells, that there are at least three CBFA1 recognition motifs in the rat CBFA1 promoter, and that there are three tandemly repeated CBFA1 sites within the 5′ UTR. We find that forced expression of CBFA1 protein downregulates CBFA1 promoter activity and that a single CBFA1 site is sufficient for transcriptional autosuppression. Thus, our data indicate that the CBFA1 gene is autoregulated in part by negative feedback on its own promoter to stringently control CBFA1 gene expression and function during bone formation. J. Cell. Physiol. 184:341–350, 2000. © 2000 Wiley-Liss, Inc.

Transcriptional autoregulation of the bone related CBFA1/RUNX2 gene.

The runt related transcription factor CBFA1 (AML3/PEBP2alphaA/RUNX2) regulates expression of several bone- and cartilage-related genes and is required for bone formation in vivo. The gene regulatory mechanisms that control activation and repression of CBFA1 gene transcription during osteoblast differentiation and skeletal development are essential for proper execution of the osteogenic program. We have therefore defined functional contributions of 5' regulatory sequences conserved in rat, mouse and human CBFA1 genes to transcription. Deletion analysis reveals that 0.6 kB of the bone-related rat or mouse CBFA1 promoter (P1, MASNS protein isoform) is sufficient to confer transcriptional activation, and that there are multiple promoter domains which positively and negatively regulate transcription. Progressive deletion of promoter segments between nt -351 and -92 causes a striking 30- to 100-fold combined decrease in promoter activity. Additionally, 5' UTR sequences repress reporter gene transcription 2- to 3-fold. Our data demonstrate that CBFA1 is a principal DNA binding protein interacting with the 5' region of the CBFA1 gene in osseous cells, that there are at least three CBFA1 recognition motifs in the rat CBFA1 promoter, and that there are three tandemly repeated CBFA1 sites within the 5' UTR. We find that forced expression of CBFA1 protein downregulates CBFA1 promoter activity and that a single CBFA1 site is sufficient for transcriptional autosuppression. Thus, our data indicate that the CBFA1 gene is autoregulated in part by negative feedback on its own promoter to stringently control CBFA1 gene expression and function during bone formation.