DGKζ Downregulation Enhances Osteoclast Differentiation and Bone Resorption Activity Under Inflammatory Conditions.

Bone homeostasis is maintained by a balance between resorption of the bone matrix and its replacement by new bone. Osteoclasts play a crucially important role in bone metabolism. They are responsible for bone resorption under pathophysiological conditions. Differentiation of these cells, which are derived from bone marrow cells, depends on receptor activator of NF-κB ligand (RANKL). RANKL-induced osteoclastogenesis is regulated by the phosphoinositide (PI) signaling pathway, in which diacylglycerol (DG) serves as a second messenger in signal transduction. In this study, we examined the functional implications of DG kinase (DGK), an enzyme family responsible for DG metabolism, for osteoclast differentiation and activity. Of DGKs, DGKζ is most abundantly expressed in osteoclast precursors such as bone marrow-derived monocytes/macrophages. During osteoclast differentiation from precursor cells, DGKζ is downregulated at the protein level. In this regard, we found that DGKζ deletion enhances osteoclast differentiation and bone resorption activity under inflammatory conditions in an animal model of osteolysis. Furthermore, DGKζ deficiency upregulates RANKL expression in response to TNFα stimulation. Collectively, results suggest that DGKζ is silent under normal conditions, but it serves as a negative regulator in osteoclast function under inflammatory conditions. Downregulation of DGKζ might be one factor predisposing a person to osteolytic bone destruction in pathological conditions. J. Cell. Physiol. 232: 617-624, 2017. © 2016 Wiley Periodicals, Inc.

Reciprocal regulation of p53 and NF-κB by diacylglycerol kinase ζ.

Diacylglycerol kinase (DGK) participates in lipid mediated-signal transduction. It phosphorylates diacylglycerol (DG) to phosphatidic acid (PA), thereby regulating the balanced control of these second messenger actions. Previous reports have described that one DGK family, DGKζ, is closely involved in stress responses under various conditions. Cellular stress response, a physiological process enabling cells to cope with an altered environment, is finely tuned through various signaling cascades and their molecular crosstalk. The major components of stress response are p53 and NF-κB. p53 generally serves as a proapoptotic transcriptional factor, whereas NF-κB promotes resistance to programmed cell death under most circumstances. Recent studies have suggested that DGKζ facilitates p53 degradation in cytoplasm through ubiquitin proteasome system and that DGKζ deletion upregulates p53 protein levels under basal and DNA-damage conditions. Counter-intuitively, however, DGKζ deletion suppresses p53 transcriptional activity despite increased p53 levels. In contrast, DGKζ knockdown engenders enhancement of NF-κB pathway in response to cytokines such as TNF-α and IL-1ß. In response to these cytokines, DGKζ downregulation accelerates phosphorylation of the p65 subunit and its nuclear translocation, thereby enhancing NF-κB transcriptional activity. Furthermore, DGKζ deficiency is shown to promote increased association of p65 subunit with the transcriptional cofactor CBP. It is particularly interesting that this association is observed even under basal conditions in the absence of stimulation. These findings suggest that DGKζ plays a role in sequestration of the limiting pool of CBP/p300 between the NF-κB p65 subunit and p53, and that DGKζ downregulation shifts CBP/p300 toward the NF-κB subunit to regulate reciprocally antagonistic phenotypes of these transcription factors.

Downregulation of diacylglycerol kinase ζ enhances activation of cytokine-induced NF-κB signaling pathway.

The transcription factor NF-κB family serves as a key component of many pathophysiological events such as innate and adaptive immune response, inflammation, apoptosis, and oncogenesis. Various cell signals trigger activation of the regulatory mechanisms of NF-κB, resulting in its nuclear translocation and transcriptional initiation. The diacylglycerol kinase (DGK) family, a lipid second messenger-metabolizing enzyme in phosphoinositide signaling, is shown to regulate widely various cellular processes. Results of recent studies suggest that one family member, DGKζ, is closely involved in immune and inflammatory responses. Nevertheless, little is known about the regulatory mechanism of DGKζ on NF-κB pathway in cytokine-induced inflammatory signaling. This study shows that siRNA-mediated DGKζ knockdown in HeLa cells facilitates degradation of IκB, followed by nuclear translocation of NF-κB p65 subunit. In addition, DGKζ-deficient MEFs show upregulation of p65 subunit phosphorylation at Serine 468 and 536 and its interaction with CBP transcriptional coactivator upon TNF-α stimulation. These modifications of p65 subunit might engender enhanced NF-κB transcriptional reporter assay of DGKζ knockdown cells. These findings provide further insight into the regulatory mechanisms of cytokine-induced NF-κB activation.