Pentraxin-3 Is a TSH-Inducible Protein in Human Fibrocytes and Orbital Fibroblasts

Abstract

CD34(+) fibrocytes are bone marrow-derived monocyte progenitor cells that traffic to sites of tissue injury and repair. They putatively infiltrate the orbit in thyroid-associated ophthalmopathy where they appear to transition into CD34(+) orbital fibroblasts (OFs) that interact with residential CD34(-) fibroblasts. A unique phenotypic attribute of fibrocytes and CD34(+) OFs is their expression of the functional thyrotropin receptor (TSHR) and other "thyroid-specific" proteins. When activated through TSHR, fibrocytes express a number of cytokines and other inflammatory genes. Here we sought to determine whether pentraxin-3 (PTX-3), an acute-phase protein involved in inflammation and autoimmunity, might be induced by TSH in fibrocytes and OFs. These cells were collected from patients with Graves disease and healthy individuals. PTX-3 mRNA levels were determined by real-time PCR, protein was determined by ELISA and Western blot, and PTX-3 gene promoter activity was assessed with reporter assays. PTX-3 expression was induced by TSH in both cell types, regardless of the health status of the donor and was a consequence of increased steady-state PTX-3 mRNA levels. M22, a TSHR-activating monoclonal antibody, also induced PTX-3. The induction could be attenuated by dexamethasone and by IGF-I receptor-blocking antibodies, teprotumumab and 1H7. TSH effects were mediated through phosphatidylinositol 3-kinase/AKT, mammalian target of rapamycin/p70(s6k), Janus tyrosine kinase 2 pathways, and enhanced PTX-3 mRNA stability. These findings indicate that PTX-3 is a TSH target gene, the expression of which can be induced in fibrocytes and OFs. They suggest that PTX-3 might represent a previously unidentified nexus between the thyroid axis and the mechanisms involved in tissue remodeling.

Figure 1.

Time course of PTX-3 protein induction in fibrocytes by bTSH. Confluent cultures were incubated without or with bTSH (5 mIU/mL) for the intervals indicated along the abscissas. A, Medium was collected and subjected to a PTX-3–specific ELISA. Data are expressed as means ± SD of triplicates from 3 independent experiments. P < .05 compared with baseline. B, Cellular protein was extracted for Western blot analysis. C, Bands from B were quantified by densitometric analysis.

Figure 2.

Time course of PTX-3 mRNA induction in fibrocytes (A) and GD-OFs (B) by bTSH. Confluent cells from a single healthy fibrocyte donor and from a single GD-OF donor were shifted to medium containing 1% FBS for 12 hours, and then bTSH (5 mIU/mL) was added for the intervals indicated along the abscissas. C, Graded concentrations of bTSH indicated along the abscissa were added to culture medium for 6 hours. Total RNA was extracted, reverse transcribed, and subjected to RT-PCR for PTX-3 mRNA. Data are expressed as means ± SD of triplicates from 3 independent experiments. *, P < .05; **, P < .01; ***, P < .001; compared with baseline.

Figure 3.

A, TSH induction of PTX-3 mRNA in multiple strains of fibrocytes, GD-OFs, and H-OFs. Five strains of fibrocytes from patients with GD, 5 from healthy donors, and 3 strains each of GD-OFs and H-OFs were treated with nothing (control) or bTSH (5 mIU/mL) for 6 hours. Total RNA was extracted and subjected to RT-PCR for PTX-3 mRNA. B, Fibrocytes were treated with nothing (control) or bTSH 5 mIU/mL for 72 hours, monolayers were harvested, and RNA was extracted and subjected to RT-PCR for TSHR mRNA. C, Fibrocytes were treated with nothing, bTSH, or M22 (2 μg/mL) for 6 hours, and then RNA was subjected to RT-PCR for PTX-3 mRNA. D, Fibrocyte cultures were treated with nothing, bTSH, or Dex (10 nM) for 6 hours. RNA was subjected to RT-PCR for PTX-3 mRNA. Data are expressed as means ± SD of triplicates from 3 independent experiments. P values are as indicated.

Figure 4.

A, Anti-IGF-IR blocking antibodies, RV001 and 1H7, attenuate the induction by TSH of PTX-3 in fibrocytes. Confluent cultures were pretreated with nothing, RV001 (50 μg/mL) or 1H7 (10 μg/mL) for 16 hours and then incubated without or with bTSH (5 mIU/mL) for an additional 6 hours. Cellular RNA was subjected to RT-PCR for PTX-3 mRNA. B, Confluent fibrocyte cultures were treated with nothing, bTSH (5 mIU/mL), IGF-I (10 ng/mL), or the combination of bTSH and IGF-I for 6 hours. Cellular RNA was subjected to RT-PCR of PTX-3 mRNA. Data are expressed as mean ± SD of triplicate replicates.

Figure 5.

A, Fibrocytes were pretreated with nothing or AKTi (1 μM), LY294002 (10 μM), AG490 (75 μM), rapamycin (RAPA) (20 nM) for 1 hour before incubation with bTSH (5 mIU/mL) for 6 hours. RNA was subjected to RT-PCR for PTX3 mRNA. Data are expressed as means ± SD of triplicates from 3 independent experiments. *, P < .0001 compared with control; #, P < .0001 compared with bTSH alone. B and C, Cultures were pretreated as in A and then treated with bTSH (5 mIU/mL) for 90 minutes. Cell lysates were subjected to Western blot analysis of AKT, pAKT, JAK2, pJAK2, p70^S6K, and pp70^S6K.

Figure 6.

Effects of bTSH on PTX-3 gene promoter activity and mRNA stability, and its dependence on intermediate protein synthesis in fibrocytes. A, PTX3 gene promoter-reporter constructs were transfected into fibrocytes as described in Materials and Methods. Cultures were treated with nothing or bTSH (5 mIU/mL) for 2 hours, harvested, and luciferase (LUC) activity was determined. B, bTSH (5 mIU/mL) was added to the medium of confluent cell layers for 6 hours and replaced with fresh medium containing DRB (50 μM) without or with bTSH (5 mIU/mL). RNA was harvested at the times indicated along the abscissa and subjected to RT-PCR for PTX-3 mRNA. C, Confluent cultures were pretreated with cycloheximide (CHX) (10 μg/mL) for 1 hour and then treated alone or in combination with bTSH (5 mIU/mL) for 6 hours. RNA was extracted and subjected to RT-PCR for PTX-3 mRNA. Data are expressed as means ± SD of triplicates from 3 independent experiments.