Orphan nuclear receptor NR4A2 induces transcription of the immunomodulatory peptide hormone prolactin

Abstract

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.

Keywords: Arthritis; Orphan nuclear receptor; Prolactin; Promoter; Synoviocytes; Transcription.

Figure 1

NR4A2 induces expression of genes involved in synovial hyperplasia and inflammation. A. K4IM human synoviocytes were transduced in triplicate with lentiviral particles containing control empty or CMV-NR4A2 expression vectors. RNA was harvested after 48 hours and NR4A2 expression was measured by RT-qPCR using GAPDH as a control. ***p < 0.001. B. Pooled triplicate cDNA from the same experiment was analyzed on TaqMan Human Angiogenesis qPCR arrays. Gene expression data was normalized to 4 housekeeping genes and the genes most highly induced by NR4A2 are displayed.

Figure 2

NR4A2 induces PRL mRNA and protein secretion. A. K4IM human synoviocytes were transduced in triplicate with lentiviral particles containing control empty or CMV-NR4A2 expression vectors. RNA was harvested after 48 hours and PRL expression was measured by RT-qPCR using GAPDH as a control. **p < 0.005. B. Conditioned media from transduced K4IM cells was collected after 48 hours and secreted PRL was measured by ELISA. **p < 0.005. C. HFLS derived from normal synovial tissue were transduced in triplicate with lentiviral particles containing control empty or CMV-NR4A2 expression vectors. RNA was harvested after 48 hours and PRL expression was measured by RT-qPCR using GAPDH as a control. *p < 0.01.

Figure 3

NR4A receptors are required for PGE ₂ induction of PRL. A. Triplicate wells of K4IM synoviocytes were left untreated or stimulated with PGE₂ (1 μM) in serum-free media for the times indicated. NR4A2 expression levels were measured by RT-qPCR. *p < 0.05. B. PRL expression levels were measured by RT-qPCR in the same experiment. *p < 0.05, **p < 0.005. C. Scrambled shRNA or NR4A1-3 shRNA transduced K4IM synoviocytes were left untreated or treated with PGE₂ for one hour. NR4A2 expression levels were measured by RT-qPCR. **p < 0.005. D. Scrambled shRNA or NR4A1-3 shRNA transduced K4IM synoviocytes were left untreated or treated with PGE₂ for 24 hours. PRL expression levels were measured by RT-qPCR. **p < 0.005.

Figure 4

NR4A2 transactivates the distal PRL promoter in synoviocytes. A. RT-PCR was conducted with primers specific for exon 1A and exon 2 of PRL or GAPDH and products were resolved on an agarose gel. PRL primers yielded the expected product size of 275 bp, indicating the presence of exon 1A in PRL transcripts. GAPDH specific primers produced the expected product size of 451 bp. Lanes 1–3 contained the following PCR templates: cDNA from control transduced K4IM cells, cDNA from NR4A2 transduced K4IM cells, and negative control. B. K4IM synoviocytes were transiently transfected in triplicate with a distal PRL promoter (−3000 bp) luciferase reporter and control or NR4A2 expression vectors as indicated. Relative luciferase units (RLUs) were measured in cell lysates 48 hours post-transfection. **p < 0.005.

Figure 5

DNA-binding domain of NR4A2 is required for transactivation of PRL promoter. A. K4IM synoviocytes were transiently transfected in triplicate with distal PRL promoter luciferase reporters (−3000 bp, −270 bp, −32 bp) and control or NR4A2 expression vectors as indicated. Relative luciferase units (RLUs) were measured in cell lysates 48 hours post-transfection. **p < 0.005. B. K4IM synoviocytes were transiently transfected in triplicate with a distal PRL promoter (−270 bp) luciferase reporter and control, NR4A2, or NR4A2 C283G (DNA binding domain mutant) expression vectors as indicated. Relative luciferase units (RLUs) were measured in cell lysates 48 hours post-transfection. *p < 0.05.

Figure 6

PRL regulates expression of genes involved in inflammation, proliferation, and cell survival. A. K4IM synoviocytes were treated in triplicate with TNF-α (10 ng/mL) or PGE₂ (1 μM) for 24 hours. PRL-R was measured by RT-qPCR. ***p < 0.001. B. cDNA from K4IM synoviocytes treated with rhPRL (1 μg/mL, 24 hours) was analyzed on JAK/STAT signaling pathway qPCR arrays. Gene expression data was normalized to 3 housekeeping genes and the genes most highly regulated by rhPRL are displayed.