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. 2007 Mar;170(3):1121-33.
doi: 10.2353/ajpath.2007.061000.

A role for type 1alpha corticotropin-releasing hormone receptors in mediating local changes in chronically inflamed tissue

Jennifer A Ralph  1 , Davide Zocco, Barry Bresnihan, Oliver Fitzgerald, Alice N McEvoy, Evelyn P Murphy
Affiliations

A role for type 1alpha corticotropin-releasing hormone receptors in mediating local changes in chronically inflamed tissue

Jennifer A Ralph et al. Am J Pathol. 2007 Mar.

Abstract

Peripheral corticotropin-releasing hormone (CRH) is an important regulator of localized inflammatory responses. The aim of this study is to define the pathological signaling pathways in which peripheral CRH receptor-mediated responses reside. We report that PECAM-1-expressing synovial membrane endothelial cells are the principal source of CRH receptor subtype 1alpha in chronically inflamed synovial tissue (ST). Analysis of ST from an early arthritis patient cohort (n = 9) established that expression of CRH-R1alpha significantly (P < 0.03) colocalized with PECAM-1 and E-selectin expression in vivo. Freshly excised ST explants released a mediator(s) that acts to promote CRH-R1alpha mRNA to levels present in inflamed human synovium (n = 8). We tested the ability of conditioned medium and individual inflammatory mediators to modulate CRH-R1alpha expression. Histamine selectively induced the expression of CRH-R1alpha, and these effects were mediated through the histamine receptor type 1. Ectopic expression of CRH-R1alpha in normal human endothelial and synoviocyte cells resulted in the induction of the orphan receptor NR4A2 through the reconstitution of cAMP/protein kinase A/cAMP response element-binding protein signaling and identified a role for CRH in modulating nuclear factor kappaB transcriptional activity. CRH enhanced the expression of nitric-oxide synthase (NOS III) to promote NO production from CRH-R1alpha-expressing cells. These data establish a role for CRH receptor-mediated responses in regulating vascular changes associated with chronic synovitis.

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Figures

Figure 1
Figure 1
PECAM-1 (CD31)-expressing endothelia are a major source of CRH-R1α in human inflammatory arthritis. A: Representative reverse transcriptase (RT)-PCR products generated using CRH-R1α and GAPDH-specific primers after amplification of mRNA from human myometrium (MM), primary SMECs, and primary HMVECs; MW, 100-bp DNA molecular weight markers. B: Flow cytometric analysis of cultured primary SMECs and HMVECs using antibodies directed against PECAM-1 (CD31) and ICAM-1 (CD54). C: Representative immunostaining for CRH-R1 and PECAM-1 in synovial tissue from a patient with early PsA. Synovial tissue sections were incubated with a Cy3-labeled CRH-R1 antibody. CRH-R1 immunofluorescence is indicated by intense red staining on the vascular endothelium and discrete perivascular cells. Subsequently, the same section was incubated with fluorescein isothiocyanate-labeled immune serum directed against PECAM-1, as indicated by the green fluorescence. Superimposition results in distinct yellow fluorescence, confirming the colocalization of CRH-R1 with PECAM-1-expressing endothelium. BV, blood vessel. Original magnification, ×200.
Figure 2
Figure 2
Coexpression of CRH-R1α with activation and angiogenic molecules on endothelial cells in early inflamed human synovial tissue. Representative serial PsA synovial tissue cryosections were stained with immune serum directed against CRH-R1 (A), von Willebrand factor (factor VIII) (B), PECAM-1 (CD31) (C), E-selectin (D), αvβ3 (E), or isotype-matched nonimmune IgG (F). Positive cells are indicated by dark brown staining. Nuclei are counterstained with hematoxylin. LL, lining layer; SL, sublining layer. Original magnification for each stain, ×100 (A–F).
Figure 3
Figure 3
CRH-R1α mRNA can be induced in normal HMVECs to equivalent transcript levels measured in human inflamed synovial tissue and human myometrium. A: RT-PCR analysis was performed using primers for CRH-R1α and GAPDH with total RNA extracted from HMVECs left untreated (NT) or coincubated for 24 hours with MoCM, PsA synovial tissue (PsA1 and PsA2), and human myometrium (MM1 and MM2). B: HMVECs left untreated (NT) or coincubated for 24 hours with synovial biopsies (RA, PsA, and OA Bx) or MoCM and human myometrium (MM3 and MM4). C and D: Densitometric analysis representing CRH-R1α mRNA levels in HMVECs. Values are the means ± SEM. For HMVECs, each value is representative of three separate experiments. For human synovial tissue, n = 8 and myometrium, n = 8.
Figure 4
Figure 4
Modulation of CRH-R1α mRNA levels in HMVECs by the vasoactive mediator histamine. A: Expression of CRH-R1α, E-selectin, and GAPDH mRNA was analyzed using total RNA isolated from HMVECs left untreated (NT), treated for 24 hours with histamine (10−2 mol/L), PGE2 (10−6 mol/L), TNFα (10 ng/ml), IL-1β (10 ng/ml), or MoCM and human myometrium (MM1 and MM2). B: HMVECs left untreated (NT) or treated for 4 hours with TNFα (10 ng/ml) or IL-1β (10 ng/ml). C: HMVECs left untreated (NT) or treated for 24 hours with increasing concentrations of histamine (10−7 to 10−4 mol/L). D: HMVECs left untreated (NT) or treated for 24 hours with histamine (10−5 mol/L) in the absence or presence of increasing concentrations of mepyramine (0.1, 1.0, and 10.0 μmol/L).
Figure 5
Figure 5
Ectopic expression of CRH-R1α in normal human synoviocytes and HMVECs. A: Synoviocytes nucleofected with 1.0 or 2.0 μg of pc-CRH-R1α for 24 hours were stained with a Cy3-labeled CRH-R1 antibody (C20) or isotype-matched nonimmune IgG. CRH-R1α expression is indicated by intense red fluorescence. Original magnification, ×400. Inset: 4,6-diamidino-2-phenylindole staining, visible as blue fluorescence, distinguishes the nucleus and localizes CRH-R1α to the cytoplasmic membrane. Original magnification, ×600. B and C: RT-PCR analysis of CRH-R1α and GAPDH mRNA expression in synoviocytes and HMVECs after nucleofection with or without the CRH-R1α cDNA.
Figure 6
Figure 6
CRH-induced NR4A2 mRNA expression is dependent on CRH-R1α. HMVECs (A) and synoviocytes (B) were nucleofected with or without 2.0 μg of pc-CRH-R1α for 24 hours, left untreated, or treated with CRH (10−8 mol/L) or PGE2 (10−6 mol/L) for a further 1 to 3 hours. Total RNA was extracted, and RT-PCR was performed using primers specific for CRH-R1α, NR4A2, and GAPDH.
Figure 7
Figure 7
CRH signaling, via CRH-R1α, can modulate CREB and NF-κB transcriptional activity. Synoviocytes were nucleofected with 2.0 μg of pc-LacZ or 2.0 μg of pc-CRH-R1α together with 500 ng of a CRE-tk-LUC reporter plasmid (A) or 500 ng of a κB-tk-LUC reporter plasmid (B). After nucleofection, cells were treated with increasing concentrations of CRH (10−8 to 10−6 mol/L), forskolin (FOR, 25 μmol/L), or TNFα (10 ng/ml) for 24 hours. Values are presented as fold induction after normalization. All transfection experiments were performed in triplicate dishes. The mean ± SEM of four individual experiments is shown.
Figure 8
Figure 8
CRH promotes nitric oxide production from CRH-R1α-expressing cells. A: HMVECs were nucleofected with or without pc-CRH-R1α and seeded into 12-well plates (300,000 cells/well). Cells were left unstimulated or stimulated with increasing concentrations of CRH (10−8 to 10−6 mol/L) or a combination of IL-1β (25 ng/ml), TNFα (25 ng/ml), and interferon-γ (1000 U/ml) for 24 hours. Cell culture medium was harvested, and nitric oxide production was determined by measuring nitrite production using the Griess reaction. Values are the means ± SEM from three separate experiments. B: Synoviocytes transfected with pc-CRH-R1α for 24 hours were treated with CRH (10−8 mol/L) for a further 1 to 3 hours. Expression of NOS III and GAPDH mRNA was analyzed using total RNA. Each gel is representative of three separate experiments.
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