Constitutive and functional expression of runt-related transcription factor-2 by microglial cells

Highlights

• Runx2 is constitutively expressed by microglia.

• Exposure to ATP leads to upregulation of Runx2 in microglia.

• Inhibition of Ca²⁺/calmodulin signals prevents Runx2 upregulation by ATP.

• The upregulation is seen in hematopoietic, but not mesenchymal, lineage cells.

• Runx2 knockdown prolongs retraction of process extension.

Abstract

Runt-related transcription factor-2 (Runx2) is the master regulator of osteoblastogenesis with an ability to promote differentiation of mesenchymal stem cells into the osteoblastic lineage. We have previously shown constitutive and functional expression of Runx2 by astroglial cells. In this study, we investigated the possible expression of Runx2 by both murine microglia and microglial cell line BV-2 cells. Runx2 expression was seen in cultured microglia and BV-2 cells, while sustained exposure to 1 mM ATP led to a significant but transient increase in mRNA and corresponding protein expression of Runx2 within 24 h. The increase in Runx2 expression was invariably prevented by several chemicals with antagonistic properties for P2X7 purinergic receptor, calmodulin and calcineurin in BV-2 cells, with a P2X7 receptor agonist more than quadrupling Runx2 expression. A significant increase in Runx2 expression was seen in osteoclastic cells, but not in osteoblastic or chondrocytic cells, when exposed to a high concentration of ATP. In BV2-cells with control siRNA, a significant decrease was found in the number of cells with at least one process within 3 h after the exposure to 1 mM ATP, followed by an increase up to 24 h. However, Runx2 siRNA significantly deteriorated the property to induce delayed process extension during 6–24 h after exposure to ATP along with drastically decreased Runx2 protein levels. These results suggest that Runx2 is constitutively and functionally expressed by microglial cells with responsiveness to ATP for upregulation in the murine brain.

Introduction

Microglia are derived from parenchymal tissue macrophages at a population over 10% of cells found in the central nervous system (CNS). In the healthy adult brain, microglia are present with at least one branched process as often referred to as “ramified microglia”, which are thought to be quiescent under the normal conditions, with actively surveying their environments using these branched processes (Wake et al., 2009). When they sense unusual signs and/or signals during infection, inflammation, trauma, ischemia and other neurodegenerative abnormalities with those motile processes, in contrast, they immediately transform their features from a surveying type into an active form called “activated microglia”. Activated microglia show several typical profiles functionally different from those in ramified microglia, such as process retraction, migration, proliferation and phagocytosis, in addition to releasing pro-inflammatory cytokines (Hanisch and Kettenmann, 2007, Kreutzberg, 1996, Nakajima et al., 1996). In addition, ramified microglia are also shown to migrate with extremely motile processes to monitor brain parenchyma even in a presumed resting state in vivo (Nimmerjahn et al., 2005).

A large amount of intracellular ATP is leaked from neighboring injured and dead cells to induce diverse functional alterations through activation of a variety of purinergic receptors expressed at the surface in ramified and activated microglia. Purinergic receptors are classified into P1 and P2 receptor subclasses according to the agonist specificity; adenosine for the P1 subclass and ATP for the P2 subclass, respectively (Burnstock and Kennedy, 1985, Koizumi et al., 2013). P1 receptors are all metabotropic receptor isoforms grouped into A1, A2A, A2B and A3 subtypes on the basis of their intracellular signals and molecular nucleotide sequential homology, whereas P2 receptors are further classified into ionotropic P2X and metabotropic P2Y subtypes. Recent studies indicate that purinergic receptor activation plays a central role in determining phenotypes of activated microglia (Koizumi et al., 2013).

Runt-related transcriptional factor-2 (Runx2; also known as Cbfa1, PEBP2α and AML3) is a cell-specific member of the Runt family of transcription factors (Runx1-3) with a critical role in cellular differentiation steps in osteoblasts and chondrocytes (Kagoshima et al., 1993). In osteoblasts, Runx2 forms a heterodimer with the partner protein core binding factor β (Cbfβ) to recognize the particular DNA element osteoblast specific element (OSE2) at the upstream of different target gene promoters, followed by induction of osteoblastic differentiation for subsequent regulation of expression of a variety of genes characteristic to the osteoblast phenotype. By contrast, recent studies including ours have demonstrated that Runx2 is also expressed in non-osseous tissues such as brain (Jeong et al., 2008, Takarada and Yoneda, 2009). We have shown that both Runx2 and Cbfβ are expressed by rat neocortical astrocytes and C6 glioma cells to lead to transactivation of several downstream target genes such as matrix metalloproteinase-13 (MMP13) as seen in osteoblasts (Takarada and Yoneda, 2009). However, little attention has been paid to the expression of Runx2 by other cells residing in the CNS.

In the present study, therefore, we investigated expression of the master regulator of osteoblastic differentiation Runx2 by microglial cells to elucidate the possible novel physiological and/or pathological significance of the Runx2 signaling pathway outside bone in the maintenance of brain functions using primary cultured mouse brain microglia and mouse microglial cell line BV-2 cells.