Gonadotropin-releasing hormone stimulates biliary proliferation by paracrine/autocrine mechanisms

Abstract

During cholestatic liver disease, there is dysregulation in the balance between biliary growth and loss in bile duct-ligated (BDL) rats modulated by neuroendocrine peptides via autocrine/paracrine pathways. Gonadotropin-releasing hormone (GnRH) is a trophic peptide hormone that modulates reproductive function and proliferation in many cell types. We evaluated the autocrine role of GnRH in the regulation of cholangiocyte proliferation. The expression of GnRH receptors was assessed in a normal mouse cholangiocyte cell line (NMC), sham, and BDL rats. The effect of GnRH administration was evaluated in normal rats and in NMC. GnRH-induced biliary proliferation was evaluated by changes in intrahepatic bile duct mass and the expression of proliferation and function markers. The expression and secretion of GnRH in NMC and isolated cholangiocytes was assessed. GnRH receptor subtypes GnRHR1 and GnRHR2 were expressed in cholangiocytes. Treatment with GnRH increased intrahepatic bile duct mass as well as proliferation and function markers in cholangiocytes. Transient knockdown and pharmacologic inhibition of GnRHR1 in NMC decreased proliferation. BDL cholangiocytes had increased expression of GnRH compared with normal rats, accompanied by increased GnRH secretion. In vivo and in vitro knockdown of GnRH decreased intrahepatic bile duct mass/cholangiocyte proliferation and fibrosis. GnRH secreted by cholangiocytes promotes biliary proliferation via an autocrine pathway. Disruption of GnRH/GnRHR signaling may be important for the management of cholestatic liver diseases.

Figure 1

A: Representative immunofluorescence for GnRHR₁ (red) and GnRHR₂ (green) in liver sections co-stained with cytokeratin-19 (CK-19) show their expression on bile ducts. Receptor and CK-19 localization in bile ducts are indicated by white arrows. B: Immunohistochemistry showing the expression of GnRHR₁ and GnRHR₂ in bile ducts, the expression increases after bile duct ligation (BDL) (Table 1). Positive staining for GnRHR₁ and GnRHR₂ in bile ducts is shown by black arrows. Scale bar = 25 μm (A). Original magnification: ×40 (B).

Figure 2

A: Administration of gonadotropin-releasing hormone (GnRH) to normal rats enhances intrahepatic bile duct mass (IBDM) compared with saline-treated rats. Data are means ± SEM of 10 cumulative values obtained from two slides for each group. For semiquantitative analysis, see Table 1. Cytokeratin-19–positive bile ducts are indicated by red arrows. B: PCNA expression increases in cholangiocytes from rats treated with GnRH compared with controls. Data are means ± SEM of five replicates. C–F: cAMP levels and expression of SR, CFTR, and AE2 increases in cholangiocytes from GnRH-treated rats compared with control rats. Data are means ± SEM of three evaluations. ^∗P < 0.05 versus cholangiocytes from control rats. Original magnification: ×40 (A).

Figure 3

A: Gonadotropin-releasing hormone (GnRH) expression decreases in the hypothalamus of normal rats treated with GnRH and in bile duct-ligated (BDL) rats compared with normal rats, and increases in the hypothalamus of BDL rats treated with GnRH Vivo-Morpholino compared with BDL control rats. Data are means ± SEM of three experiments. B: Representative immunohistochemistry for GnRH in liver sections from normal and BDL rats. The immunoreactivity of GnRH increases in bile ducts from BDL compared with normal rat (Table 1). Red arrows indicate bile ducts showing positivity for GnRH. C and D: GnRH expression and levels increase in cholangiocytes (C) and cholangiocyte supernatant (D) from BDL compared with normal rats, respectively. Data are means ± SEM of six evaluations. ^∗P < 0.05 versus normal rats; ^†P < 0.05 versus BDL rats. Original magnification: ×40 (B).

Figure 4

A and B: The administration of gonadotropin-releasing hormone (GnRH) Vivo-Morpholino to bile duct-ligated (BDL) rats decreases the immunoreactivity of GnRH in liver sections (red arrows) (Table 1) and GnRH secretion in cholangiocyte supernatant. C–E: The administration of GnRH Vivo-Morpholino to BDL rats decreases intrahepatic bile duct mass (IBDM) in liver sections (green arrows; C). Proliferating cell nuclear antigen (PCNA) expression, and cAMP levels compared with BDL control rats. n = 3 (D); n = 6 (E). ^∗P < 0.05 versus BDL rats treated with mismatch-Morpholino. Original magnification: ×20 (A and C). CK-19, cytokeratin-19.

Figure 5

A: The administration of gonadotropin-releasing hormone (GnRH) Vivo-Morpholino to bile duct-ligated (BDL) rats decreases the immunoreactivity of interstitial collagen in liver sections evaluated by Sirius red staining compared with BDL rats treated with mismatch-Morpholino. Collagen deposition around bile ducts is indicated by black arrows. B and C: The administration of GnRH Vivo-Morpholino to BDL rats decreases the expression of α-SMA, collagen type I α(Col1A1), and fibronectin-1 (Fn-1) in purified cholangiocytes (B) and total liver tissue (C) compared with BDL control rats. Data are means ± SEM of 12 real-time PCR reactions. ^∗P < 0.05 versus BDL rats treated with mismatch-Morpholino. Original magnification: ×20 (A).

Figure 6

A: By immunofluorescence, normal mouse cholangiocyte cell line (NMC) expresses both GnRHR₁ and GnRHR₂. Specific immunoreactivity of representative fields is shown in red and nuclei were stained with DAPI (blue). B: GnRH (10 to 100 nmol/L) induces the proliferation of NMC in a dose-dependent manner. C: Gonadotropin-releasing hormone (GnRH) increases PCNA expression in NMC compared with basal levels. Data are means ± SEM of five replicates. D: GnRH (50 nmol/L)-induced increase in NMC proliferation is blocked when NMC are treated with the specific GnRHR₁ antagonist. E: GnRH-mediated increase in NMC proliferation is preferentially blocked when GnRHR₁ is silenced. Basal proliferative activity decreases in si-GnRHR₁ NMC compared with control NMC. Data are means ± SEM of five replicates. F: cAMP levels increase in NMC treated with GnRH. NMC treated with secretin was used as the positive control. Data are means ± SEM of six replicates. ^∗P < 0.05 versus basal (C and E) or versus control NMC (F); ^†P < 0.05 versus NMC treated with GnRH alone (B and D) or versus cells treated with control vector and GnRH (E). Scale bar = 25 μm (A).

Figure 7

A and B:In vitro, normal mouse cholangiocyte cell line (NMC) also express gonadotropin-releasing hormone (GnRH) and secrete GnRH at both the apical and basolateral domains. C: Incubation of NMC with the media of short-term cultures of bile duct-ligated (BDL) cholangiocytes increases the proliferation of these cells. GnRH-stimulation of NMC proliferation is blocked by preincubation with a GnRH-neutralizing antibody. Data are means ± SEM of four replicates. D and E: The shGnRH NMC (approximately 90% knockdown) shows lower proliferative activity compared with Neg-transfected NMC. Data are means ± SEM of five replicates. F: The supernatant of shGnRH NMC increases NMC proliferation at a lower extent compared with the supernatant of Neg-transfected NMC, which display a higher proliferative capacity because they secrete more GnRH. Data are means ± SEM of three experiments. ^∗P < 0.05 versus basal; ^†P < 0.05 versus Neg-transfected NMC. Scale bar = 10 μm.