Onset Of Osteoblast Differentiation Research Articles

Phosphoproteomic profiling reveals a defined genetic program for osteoblastic lineage commitment of human bone marrow-derived stromal stem cells.

Bone marrow–derived mesenchymal stem cells (MSCs) differentiate into osteoblasts upon stimulation by signals present in their niche. Because the global signaling cascades involved in the early phases of MSCs osteoblast (OB) differentiation are not well-defined, we used quantitative mass spectrometry to delineate changes in human MSCs proteome and phosphoproteome during the first 24 h of their OB lineage commitment. The temporal profiles of 6252 proteins and 15,059 phosphorylation sites suggested at least two distinct signaling waves: one peaking within 30 to 60 min after stimulation and a second upsurge after 24 h. In addition to providing a comprehensive view of the proteome and phosphoproteome dynamics during early MSCs differentiation, our analyses identified a key role of serine/threonine protein kinase D1 (PRKD1) in OB commitment. At the onset of OB differentiation, PRKD1 initiates activation of the pro-osteogenic transcription factor RUNX2 by triggering phosphorylation and nuclear exclusion of the histone deacetylase HDAC7.

Abstract 1094: Transcriptional activation by pRB/p107 and coordination with SWI/SNF

Abstract The retinoblastoma susceptibility gene, Rb1, is closely linked with osteosarcoma as well as retinoblastoma. The basis of such ties between individual susceptibility genes and tumors of specific tissue origin is a central question in cancer biology. Functional analysis of pRB and the closely related family members, p107 and p130, has tended to focus on repression of proliferation, but genetic evidence from our lab and others indicates an active requirement for pRB in osteoblast differentiation that might relate more directly with osteosarcoma susceptibility. Our previous studies linked pRB with activation of the early osteoblast marker, alkaline phosphatase (the product of the Akp2 gene). Here we have investigated the functional relationships between pRB and the SWI/SNF complex in Akp2 activation. Chromatin immunoprecipitation (ChIP) assays reveal the Akp2 promoter is targeted directly by all three pRB family members. Only p130 occupies the promoter prior to induction, while pRB and p107 target Akp2 directly during activation, and concurrent with association of the global co-activator p300. Akp2 is also a direct target of SWI/SNF chromatin remodeling complexes. These ATPase-powered complexes help orchestrate the programmatic shifts in gene expression necessary for progression from a self-renewing precursor state to a state of terminal differentiation. In osteoblasts, SWI/SNF complexes that repress osteogenic gene expression in order to maintain the precursor state are characterized by inclusion of the BRM ATPase. The alternative ATPase, BRG1, characterizes complexes that activate osteogenic genes during terminal differentiation. Thus, the onset of osteoblast differentiation is marked by a switch from BRM to BRG1 complexes on the Akp2 promoter. Our analysis shows that the BRM to BRG1 switch is accomplished in a precise two-step mechanism, with dissociation of BRM-containing SWI/SNF dependent on p300, and association of BRG1-containing SWI/SNF dependent on pRB/p107. Binding of RNA polymerase-II is dependent in turn on BRG1. Thus, in contrast to their widely studied role in cell cycle repression, pRB/p107 play a directly activating role in osteogenic gene expression, specifically in recruitment of the activating SWI/SNF complex. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1094.

EBF2 Regulates Osteoblast-Dependent Differentiation of Osteoclasts

Communication between bone-depositing osteoblasts and bone-resorbing osteoclasts is required for bone development and homeostasis. Here, we identify EBF2, a member of the early B cell factor (EBF) family of transcription factors that is expressed in osteoblast progenitors, as a regulator of osteoclast differentiation. We find that mice homozygous for a targeted inactivation of Ebf2 show reduced bone mass and an increase in the number of osteoclasts. These defects are accompanied by a marked downregulation of the osteoprotegerin (Opg) gene, encoding a RANK decoy receptor. EBF2 binds to sequences in the Opg promoter and transactivates the Opg promoter in synergy with the Wnt-responsive LEF1/TCF:beta-catenin pathway. Taken together, these data identify EBF2 as a regulator of RANK-RANKL signaling and osteoblast-dependent differentiation of osteoclasts.

A Twist to Bone Development

The Runx2 transcription factor controls bone development during early mammalian embryogenesis. However, a delay between Runx2 expression and osteoblast differentiation has suggested that this key transcription factor is held in check by another regulatory protein. Bialek et al. have identified two transcription factors of the Twist family, Twist-1 and -2, that inhibit osteoblast differentiation by blocking Runx2 function, which indicates that initiation of skeletogenesis occurs when an inhibition is relieved. Mouse osteoblasts were found to express Twist proteins before they expressed Runx2. After a decrease in Twist expression, expression of Runx2 gene targets commenced. Premature osteoblast differentiation was observed in mice heterozygous for Twist-1. Runx2 expression was not affected in the heterozygous embryos nor in an osteosarcoma cell line overexpressing Twist proteins. Expression of Twist proteins also decreased Runx2 activity in a transactivation-reporter gene assay. Purified recombinant Twist and Runx proteins interacted directly in vitro, and the authors further mapped the sites of interaction in both proteins. The study also points to premature activation of Runx2 as the molecular defect in Saethre-Chotzen syndrome, a skeletal disorder caused by a haplo-insufficiency in the TWIST locus. P. Bialek, B. Kern, X. Yang, M. Schrock, D. Sosic, N. Hong, H. Wu, K. Yu, D. M. Ornitz, E. N. Olson, M. J. Justice, G. Karsenty, A Twist code determines the onset of osteoblast differentiation. Dev. Cell 6 , 423-435 (2004). [Online Journal]

Stretch-mediated Activation of Selective MAPK Subtypes and Potentiation of AP-1 Binding in Human Osteoblastic Cells

It is widely accepted that mechanical loading is necessary to construct the architecture of bone and to maintain bone mass. However, the molecular mechanisms whereby bone cells respond to mechanical stimuli remain elusive. The mitogen-activated protein kinase (MAPK) signaling cascades are known to play a crucial role in the immediate osteoblast response to a variety of bone-active agents. An important component of this response occurs at the transcriptional level and is executed by downstream phosphorylation substrates, most notably, a number of signal-responsive transcription factors. To identify whether the MAPKs are involved in the mechanotransduction process and to determine the effect on down-stream transcription factors, we stimulated human periodontal ligament (hPDL) osteoblast-like cells by mechanical stretching by employing an established in vitro model of continuous stretch application. Whole-cell extracts were prepared from cultivated hPDL cells that were exposed to short-term, continuous mechanical stretch. In-gel kinase assays were used to assess their kinase activity towards the immediate-early gene products c-Jun and c-Fos [constituents of the activator protein-1 (AP-1) transcription factor]. Electrophoretic mobility-shift and southwestern experiments utilizing a DNA sequence that contained a previously undefined atypical AP-1-binding site in the promoter of the human liver/bone/kidney alkaline phosphatase (L/B/K ALP) gene (an early marker for osteoblastic differentiation) were employed to evaluate their specific binding capacity. Selective members of the MAPK family were rapidly induced by stretching, as manifested by their ability to enhance phosphorylation of their cognate substrates c-Jun and, to a lesser extent, c-Fos in the in-gel kinase assay. This induction was accompanied by markedly increased, phospho-c-Jun-containing AP-1-binding activity, as determined by the binding analyses performed with the relevant sequence from the L/B/K ALP promoter. In as much as AP-1 is instrumental in regulating genes activated at the onset of osteoblast differentiation, such as the ALP gene, we pose that an interplay of distinct MAPKs targeting AP-1 components may dictate the osteogenic response of hPDL cells to mechanical stimulation.

The Onset and Progression of Osteoblast Differentiation is Functionally Related to Cellular Proliferation

The relationship of proliferation to the developmental sequence associated with bone cell differentiation was examined in primary osteoblast cultures derived from fetal rat and embryonic chick calvaria. A reciprocal and functional relationship exists between the decline in proliferative activity which occurs during the initial stages of the developmental sequence and the induction of genes encoding osteoblast phenotype proteins associated with matrix maturation and mineralization. This relationship is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phosphatase (AP) and osteopontin (OP) genes immediately following the proliferative period and expression of osteocalcin with the onset of mineralization, and 2) increases in AP and OP when DNA synthesis is inhibited. By determining cellular mRNA levels and rates of mRNA synthesis in isolated nuclei, we found that the down-regulation of cell growth-related genes is modified at both the levels of transcription and mRNA stability. For a histone gene where down-regulation is transcriptionally mediated, we have observed that the shutdown of osteoblast proliferation is associated with the selective loss of the interaction of a promoter binding factor (HiNF-D) with a proximal regulatory element (Site II). A relationship between Site II occupancy by HiNF-D and the onset of osteoblast differentiation is supported by the persistence of Site II-HiNF-D interactions when proliferating rat osteoblasts are growth arrested under conditions that do not induce differentiation; and additionally, by the loss of Site II-HiNF-D interactions during the shut-down of proliferation when HL60 promyelocytic leukemia cells are induced to differentiate into monocytes. Our results are consistent with a requirement of proliferation for expression of genes involved with production, deposition and possibly organization of the osteoblast extracellular matrix. It is also reasonable to postulate that properties of the mineralizing matrix are related to the shut-down of proliferation.