Inhibition of Prolyl Hydroxylase Domain-Containing Protein Suppressed Lipopolysaccharide-Induced TNF-α Expression

Abstract

Objective-Prolyl hydroxylase domain-containing proteins (PHDs) play pivotal roles in oxygen-sensing system through the regulation of ␣-subunit of hypoxia-inducible factor (HIF), a key transcription factor governing a large set of gene expression to adapt hypoxia. Although tissue hypoxia plays an essential role in maintaining inflammation, the role of PHDs in the inflammatory responses has not been clearly determined. Here, we investigated the role of PHDs in lipopolysaccharide (LPS)-induced tumor necrosis factor ␣ (TNF-␣) induction in macrophages. Methods and Results-Northern blot analysis and ELISA revealed that LPS-induced TNF-␣ upregulation was strongly suppressed by PHD inhibitors, dimethyloxallyl glycine (DMOG), and TM6008 in RAW264.7 macrophages. DMOG suppressed LPS-induced TNF-␣ upregulation in HIF-1␣-depleted cells and HIF-1␣ overexpression failed to suppress the induction of TNF-␣. DMOG rather suppressed LPS-induced NF-B transcriptional activity. Downregulation of Phd1 or Phd2 mRNA by RNA interference partially attenuated LPS-induced TNF-␣ induction. DMOG also inhibited LPS-induced TNF-␣ production in peritoneal macrophages as well as human macrophages. Conclusions-PHD inhibition by DMOG or RNA interference inhibited LPS-induced TNF-␣ upregulation in macrophages possibly through NF-B inhibition, which is independent of HIF-1␣ accumulation. This study suggests that PHDs are positive regulators of LPS-induced inflammatory process, and therefore inhibition of PHD may be a novel strategy for the treatment of inflammatory diseases. Key Words: tumor necrosis factor -alpha Ⅲ prolyl hydroxylase domain-containing protein Ⅲ hypoxia-inducible factor Ⅲ inflammation Ⅲ hypoxia I nflammation is a fundamental process for the protection of our body against outside pathogen. Tissues with inflammation are characterized by several features including the accumulation of inflammatory cells such as macrophages, lymphocytes, and neutrophils, limited blood supply attributable to impaired local microcirculation, and abnormal angiogenesis. 1 Inflammatory cells are metabolically active and consume a large amount of oxygen and nutrient. These cells are, therefore, eventually exposed to hypoxic and nutrient-deprived condition. 2 Thus, the inflammatory cells need to adapt these hypoxic conditions to perpetuate inflammatory reaction. 3 The reduced oxygen concentration is directly sensed by an innate oxygen-sensing system. 4 -6 The hypoxia-inducible factor (HIF) is a key transcription factor that mediates cellular adaptive responses to hypoxia. 7 HIF is a heterodimer consisting of an oxygen-labile ␣-subunit and a stable ␤-subunit. The stability of the ␣-subunit of HIF-1 and HIF-2 (HIF-1␣ and HIF-2␣) is regulated through the hydroxylation at the 4-position of specific proline residues in HIF-1␣ and HIF-2␣ by prolyl hydroxylase domain-containing proteins (PHDs). 8,9 Because PHD activity depends on the availability of molecular oxygen, PHDs are able to serve as a sensor for oxygen concentration. Under normal oxygen concentration, HIF-␣ i...

The Prolyl Hydroxylase PHD3 Identifies Proinflammatory Macrophages and Its Expression Is Regulated by Activin A

“…However, based on the role of activin A in inflammatory responses (43,44) and its ability to support proinflammatory macrophage polarization and limit the acquisition of anti-inflammatory markers like IL-10 (29), it is tempting to speculate that activin A contributes to PHD3 expression in macrophages within inflamed gut and that macrophage EGLN3 gene expression identifies macrophages exhibiting an M1-skewed proinflammatory polarization. If so, and despite the difficulties in extrapolating polarization marker expression between the human and murine systems (40), our results would be in agreement with the proposed role of PHDs as positive regulators of the LPS-induced inflammatory process (45) and the link between HIF1a induction and M1 polarization (6). The correlation between PHD3 and macrophage M1 polarization is further supported by the restriction of PHD3 expression to a TAM subset that exhibits a low level of M2 polarization-associated markers and for which location differed from that of M2-polarized macrophages (highly positive for CD163 and FRb).…”

Regulation of IL-1β–induced NF-κB by hydroxylases links key hypoxic and inflammatory signaling pathways

“…Furthermore, the noncanonical NF-κB-signaling pathway, which is also TRAF6 independent, is unaffected by hydroxylase inhibition (36). However, LPS-induced NF-κB activity, which is TRAF6 dependent, is down-regulated (15,37). We found that similar to LPS, IL-1β-induced NF-κB activity is down-regulated by hydroxylase inhibition.…”

Inhibition of Prolyl Hydroxylase Domain-Containing Protein Downregulates Vascular Angiotensin II Type 1 Receptor

“…Recent studies showed that inhibition of PHD, using different PHD inhibitors, lowered the levels of proinflammatory cytokines, such as tumor necrosis factor-␣, in different models. [22][23][24] Tumor necrosis factor-␣ and interleukin 1␤ have been reported to transcriptionally enhance the AT 1 R gene expression in cardiac fibroblasts. [25][26][27] Therefore, reduction of Ang II-induced cytokine production may also be responsible for CoCl 2 -induced AT 1 R downregulation in vivo.…”