Orphan Nuclear Receptor NR4A2 Induces Transcription of the Immunomodulatory Peptide Hormone Prolactin

Background: Nuclear receptor 4A2 (NR4A2) is an orphan nuclear receptor and constitutively active transcription factor expressed at elevated levels in inflamed joint tissues from patients with arthritis. Inflammatory mediators rapidly and potently induce NR4A2 expression in resident joint cells and infiltrating immune cells. This receptor promotes synovial hyperplasia by increasing proliferation of synoviocytes and inducing transcription of matrix degrading enzymes and pro-inflammatory mediators. In order to further elucidate the molecular mechanisms of NR4A2, we conducted a gene expression screen to identify novel transcriptional targets of NR4A2 that may contribute to arthritis progression.

Methods: NR4A2 was over-expressed in human synoviocytes by lentiviral transduction and gene expression changes were measured using qPCR arrays specific for inflammation, proliferation, adhesion, and migration pathways. Subsequent analysis focused on the most potently induced gene prolactin (PRL). Messenger RNA levels of PRL and PRL receptor (PRL-R) were measured by RT-qPCR and protein levels were measured by ELISA. PRL promoter studies were conducted in synoviocytes transiently transfected with NR4A2 and PRL reporter constructs. Molecular responses to PRL in synoviocytes were addressed using qPCR arrays specific for JAK/STAT signaling pathways.

Results: PRL was the most potently induced gene on the qPCR arrays, exhibiting a 68-fold increase in response to ectopic NR4A2. This gene encodes an immunomodulatory peptide hormone with roles in autoimmune diseases and inflammation. Induction of PRL mRNA and secreted protein by NR4A2 was confirmed in subsequent experiments, with increases of 300-fold and 18-fold respectively. Depletion of endogenous NR4A receptors with shRNA reduced basal and PGE2-induced PRL levels by 95%. At the transcriptional level, NR4A2 requires a functional DNA binding domain to transactivate the distal PRL promoter. Deletional analysis indicates that NR4A2 targets a region of the distal PRL promoter spanning -270 to -32 bp. In synoviocytes, recombinant PRL regulates several genes involved in inflammation, proliferation, and cell survival, suggesting that NR4A2 induced PRL may also impact these pathways and contribute to arthritis progression.

Conclusions: These results provide the first evidence for transcriptional regulation of the immunomodulatory peptide hormone PRL by NR4A2 in synoviocytes, and highlight a novel molecular pathway in inflammatory arthritis.