Osteoblast lineage cells expressing high levels of Runx2 enhance hematopoietic progenitor cell proliferation and function
Brahmananda R. ChittetiYing‐Hua ChengDrew A. StreicherSonia Rodriguez‐RodriguezNadia CarlessoEdward F. SrourMelissa A. Kacena
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Abstract Although osteoblasts (OB) play a key role in the hematopoietic stem cell (HSC) niche, little is known as to which specific OB lineage cells are critical for the enhancement of stem and progenitor cell function. Unlike hematopoietic cells, OB cell surface phenotypic definitions are not well developed. Therefore, to determine which OB lineage cells are most important for hematopoietic progenitor cell (HPC) function, we characterized OB differentiation by gene expression and OB function, and determined whether associations existed between OB and HPC properties. OB were harvested from murine calvariae, used immediately (fresh OB) or cultured for 1, 2, or 3 weeks prior to their co‐culture with Lin − Sca1 + c‐kit + (LSK) cells for 1 week. OB gene expression, alkaline phosphatase activity, calcium deposition, hematopoietic cell number fold increase, CFU fold increase, and fold increase of Lin − Sca1 + cells were determined. As expected, HPC properties were enhanced when LSK cells were cultured with OB compared to being cultured alone. Initial alkaline phosphatase and calcium deposition levels were significantly and inversely associated with an increase in the number of LSK progeny. Final calcium deposition levels and OB culture duration were inversely associated with all HPC parameters, while Runx2 levels were positively associated with all HPC properties. Since calcium deposition is associated with OB maturation and high levels of Runx2 are associated with less mature OB lineage cells, these results suggest that less mature OB better promote HPC proliferation and function than do more mature OB. J. Cell. Biochem. 111: 284–294, 2010. © 2010 Wiley‐Liss, Inc.Keywords:
RUNX2
Progenitor
Lineage (genetic)
RUNX2
Osteopontin
Periosteum
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Runx2 and Sp7 are essential transcription factors for osteoblast differentiation. However, the molecular mechanisms responsible for the proliferation of osteoblast progenitors remain unclear. The early onset of Runx2 expression caused limb defects through the Fgfr1-3 regulation by Runx2. To investigate the physiological role of Runx2 in the regulation of Fgfr1-3, we compared osteoblast progenitors in Sp7-/- and Runx2-/- mice. Osteoblast progenitors accumulated and actively proliferated in calvariae and mandibles of Sp7-/- but not of Runx2-/- mice, and the number of osteoblast progenitors and their proliferation were dependent on the gene dosage of Runx2 in Sp7-/- background. The expression of Fgfr2 and Fgfr3, which were responsible for the proliferation of osteoblast progenitors, was severely reduced in Runx2-/- but not in Sp7-/- calvariae. Runx2 directly regulated Fgfr2 and Fgfr3, increased the proliferation of osteoblast progenitors, and augmented the FGF2-induced proliferation. The proliferation of Sp7-/- osteoblast progenitors was enhanced and strongly augmented by FGF2, and Runx2 knockdown reduced the FGF2-induced proliferation. Fgfr inhibitor AZD4547 abrogated all of the enhanced proliferation. These results indicate that Runx2 is required for the proliferation of osteoblast progenitors and induces proliferation, at least partly, by regulating Fgfr2 and Fgfr3 expression.
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The LIM-homeodomain transcription factor Lmx1b plays a key role in body pattern formation during development. Although Lmx1b is essential for the normal development of multiple tissues, its regulatory mechanism in bone cells remains unclear. Here, we demonstrated that Lmx1b negatively regulates bone morphogenic protein 2 (BMP2)-induced osteoblast differentiation. Overexpressed Lmx1b in the osteoblast precursor cells inhibited alkaline phosphatase (ALP) activity and nodule formation, as well as the expression of osteoblast maker genes, including runt-related transcription factor 2 (Runx2), alkaline phosphatase (Alpl), bone sialoprotein (Ibsp), and osteocalcin (Bglap). Conversely, the knockdown of Lmx1b in the osteoblast precursors enhanced the osteoblast differentiation and function. Lmx1b physically interacted with and repressed the transcriptional activity of Runx2 by reducing the recruitment of Runx2 to the promoter region of its target genes. In vivo analysis of BMP2-induced ectopic bone formation revealed that the knockdown of Lmx1b promoted osteogenic differentiation and bone regeneration. Our data demonstrate that Lmx1b negatively regulates osteoblast differentiation and function through regulation of Runx2 and provides a molecular basis for therapeutic targets for bone diseases.
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Osteoblasts are specialized mesenchymal cells that are responsible for bone formation. In this study, we examine the role of GATA4 in osteoblast differentiation. GATA4 was abundantly expressed in preosteoblast cells and gradually down-regulated during osteoblast differentiation. Overexpression of GATA4 in osteoblastic cells inhibited alkaline phosphatase activity and nodule formation in osteogenic conditioned cell culture system. In addition, overexpression of GATA4 attenuated expression of osteogenic marker genes, including Runx2, alkaline phosphatase, bone sialoprotein, and osteocalcin, all of which are important for osteoblast differentiation and function. Overexpression of GATA4 attenuated Runx2 promoter activity, whereas silencing of GATA4 increased Runx2 induction. We found that GATA4 interacted with Dlx5 and subsequently decreased Dlx5 binding activity to Runx2 promoter region. Our data suggest that GATA4 acts as a negative regulator in osteoblast differentiation by downregulation of Runx2.
RUNX2
GATA4
Bone sialoprotein
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