The PiT1 protein, encoded by the SLC20A1 gene, was initially described as a sodium-dependent inorganic phosphate transporter expressed in most tissues and long regarded as fulfilling a housekeeping role in phosphate homeostasis. It is now known to play far more specific roles, including regulation of TNFα-induced apoptosis, erythropoiesis, cell proliferation, osteoblast and chondrocyte differentiation, liver development, and insulin signaling. Complete knockout of the Pit1 gene indeed results in embryonic lethality in mice, despite a compensatory increase in PiT2 messenger RNA, underscoring the lack of functional redundancy between these two transporters. Several observations also pointed to a link between PiT1 and the transcription factor NF-κB, a cornerstone of inflammatory responses: Pit1 expression increases upon activation of the NF-κB pathway, whereas transcription of NF-κB target genes decreases in the absence of PiT1. These clues led the authors to investigate the role of PiT1 in inflammation triggered by lipopolysaccharide (LPS), a constituent of the cell wall of Gram-negative bacteria.
To this end, the team combined in vitro and in vivo models. Bone marrow–derived macrophages from PiT1-deficient mice (Mx1-Cre; Pit1lox/lox model) were stimulated with LPS, and mice of the same genotype received LPS intraperitoneally. Cytokine production, wound healing, generation of reactive oxygen species, and the molecular steps of NF-κB activation were monitored using a range of approaches: immunoassays, flow cytometry, in vitro wound healing assays, chemiluminescence measurement, luciferase assays on the Pit1 promoter, and chromatin immunoprecipitation.
The results outline a reciprocal regulatory loop. In the absence of PiT1, secretion of MCP-1 and IL-6 by stimulated macrophages is reduced, as are the serum concentrations of these cytokines in LPS-treated mice. PiT1 deficiency is also associated with reduced wound healing in vitro and lower production of reactive oxygen species. Mechanistically, PiT1-deficient cells display attenuated IκB degradation and reduced nuclear translocation of the NF-κB p65 subunit. Conversely, LPS stimulation induces PiT1 expression, an effect abolished by an NF-κB inhibitor. The authors further show that p65 activates the Pit1 promoter and binds to it directly in response to LPS.
This work establishes a previously unknown function of PiT1 in the response to LPS and sheds light on the mechanism by which NF-κB regulates its expression: activated by LPS, NF-κB induces PiT1, which in turn promotes NF-κB activation and the transcription of pro-inflammatory genes such as Mcp-1, Tnfα, and Il-6.