Psoralen stimulates osteoblast differentiation through activation of BMP signaling

Osteoporosis is a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. In order to improve the treatment of osteoporosis, identification of anabolic and orally available agents with minimal side effects is highly desirable. Psoralen is a coumarin-like derivative extracted from Chinese herbs, which have been used to treat bone diseases for thousands of years. However, the role of Psoralen in osteoblast function and the underlying molecular mechanisms remain poorly understood. In this study, we found that Psoralen promoted osteoblast differentiation in primary mouse calvarial osteoblasts in a dose-dependent manner, demonstrated by up-regulation of expressions of osteoblast-specific marker genes including type I collagen, osteocalcin and bone sialoprotein and enhancement of alkaline phosphatase activity. We further demonstrated that Psoralen up-regulated the expression of Bmp2 and Bmp4 genes, increased the protein level of phospho-Smad1/5/8, and activated BMP reporter (12xSBE-OC-Luc) activity in a dose-dependent manner, as well as enhanced the expression of Osx, the direct target gene of BMP signaling. Deletion of the Bmp2 and Bmp4 genes abolished the stimulatory effect of Psoralen on the expression of osteoblast marker genes, such as Col1, Alp, Oc and Bsp. Our results suggest that Psoralen acts through the activation of BMP signaling to promote osteoblast differentiation and demonstrate that Psoralen could be a potential anabolic agent to treat patients with bone loss-associated diseases such as osteoporosis.     

Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene.

Osteoblasts are cells of mesodermal origin that play a pivotal role during bone growth and mineralization. The mechanisms governing osteoblast-specific gene expression are still unknown. To understand these mechanisms, we analyzed the cis-acting elements of mouse osteocalcin gene 2 (mOG2), the best-characterized osteoblast-specific gene, by DNA transfection experiments in osteoblastic and nonosteoblastic cell lines and by DNA-binding assays. 5' deletion analysis of an mOG2 promoter-luciferase chimeric gene showed that a region located between -147 and -34 contained most if not all of the regulatory elements required for osteoblast-specific expression. Three different binding sites, called A, B, and C, for factors present in nuclear extracts of osteoblasts were identified in this short promoter by DNase I footprint assays. In gel retardation assays, the A element, located between bp -64 and -47, bound a factor present only in nuclear extracts of osteoblastic cell lines and nonmineralizing primary osteoblasts. The B element, located between bp -110 and -83, bound a ubiquitously expressed factor. The C element, located between bp -146 and -132, bound a factor present only in nuclear extracts of osteoblastic cell lines and nonmineralizing and mineralizing primary osteoblasts. When cloned upstream of a minimum osteocalcin promoter or a heterologous promoter, multimers of the A element strongly increased the activities of these promoters in osteoblastic cell lines at two different stages of differentiation but in no other cell line; we named this element osteocalcin-specific element 1 (OSE1). Multimers of the C element increased the activities of these promoters predominantly in a differentiated osteoblastic cell line; we named this element OSE2. This study demonstrates that two distinct cis-acting elements are responsible for osteoblast expression of mOG2 and provides for the first time a functional characterization of osteoblast-specific cis-acting elements. We speculate that these two elements may be important at several stages of osteoblast differentiation.     

CCAAT/enhancer-binding protein-beta has a role in osteoblast proliferation and differentiation

CCAAT/enhancer binding protein beta (C/EBPbeta) is known to play an important role in the expression of several genes necessary for bone development and homeostasis including osteocalcin, IGF-1, and IL-6. In this study, we show that C/EBPbeta protein levels and, consequently, DNA-binding activity are temporally regulated, dramatically decreasing upon differentiation of MC3T3-E1 mouse osteoblasts. Corresponding with these results, the constitutive expression of C/EBPbeta LAP in MC3T3-E1 osteoblasts increased proliferation and suppressed osteogenic differentiation. Thus, C/EBPbeta LAP not only appears to participate in the regulation of genes associated with mature bone physiology, but is also a critical regulator of osteoblast growth and differentiation.     

Development and characterization of a mouse floxed <Emphasis Type="Italic">Bmp2</Emphasis> osteoblast cell line that retains osteoblast genotype and phenotype

Bone morphogenetic protein 2 (Bmp2) is essential for osteoblast differentiation and osteogenesis. Generation of floxed Bmp2 osteoblast cell lines is a valuable tool for studying the effects of Bmp2 on osteoblast differentiation and its signaling pathways during skeletal metabolism. Due to relatively limited sources of primary osteoblasts, we have developed cell lines that serve as good surrogate models for the study of osteoblast cell differentiation and bone mineralization. In this study, we established and characterized immortalized mouse floxed Bmp2 osteoblast cell lines. Primary mouse floxed Bmp2 osteoblasts were transfected with pSV3-neo and clonally selected. These transfected cells were verified by PCR and immunohistochemistry. To determine the genotype and phenotype of the immortalized cells, cell morphology, proliferation, differentiation and mineralization were analyzed. Also, expression of osteoblast-related gene markers including Runx2, Osx, ATF4, Dlx3, bone sialoprotein, dentin matrix protein 1, osteonectin, osteocalcin and osteopontin were examined by quantitative RT-PCR and immunohistochemistry. These results showed that immortalized floxed Bmp2 osteoblasts had a higher proliferation rate but preserved their genotypic and phenotypic characteristics similar to the primary cells. Thus, we, for the first time, describe the development of immortalized mouse floxed Bmp2 osteoblast cell lines and present a useful model to study osteoblast biology mediated by BMP2 and its downstream signaling transduction pathways.