Elsevier

Peptides

Volume 21, Issue 12, December 2000, Pages 1799-1809
Peptides

Urocortin and corticotropin-releasing factor receptor expression in the human colonic mucosa

https://doi.org/10.1016/S0196-9781(00)00335-1Get rights and content

Abstract

Urocortin is a newly identified member of the CRF neuropeptide family. Urocortin has been found to bind with high affinity to CRF receptors. The present study investigated urocortin and CRF receptor expression in human colonic mucosa. Non-pathologic sections of adult colorectal tissues were obtained from patients with colorectal cancer at surgery. Urocortin expression was examined using immunohistochemistry and messenger (m) RNA in situ hybridization. Isolated lamina propria mononuclear cells (LPMC) and epithelial cells were also analyzed by flow cytometry for the characterization of urocortin-positive cells, and by RT-PCR for detection of urocortin, CRF, and CRF receptor mRNA. Urocortin peptide distribution at various stages of human development (n = 35, from 11 weeks of gestation to 6 years of age) was examined by immunohistochemistry using surgical and autopsy specimens. Immunoreactive urocortin and urocortin mRNA were predominantly detected in lamina propria macrophages. Urocortin peptide expression was detected from as early as three months of age, but not before birth or in neonates. Urocortin, CRF receptor type 1 and type 2α mRNA were detected in LPMC. CRF receptor type 2β mRNA, a minor isoform in human tissues, was also detected in LPMC, but at lower levels. Urocortin is locally synthesized in lamina propria macrophages and may act on lamina propria inflammatory cells as an autocrine/paracrine regulator of the mucosal immune system. The appearance of urocortin after birth indicates that the exposure to dietary intake and/or luminal bacteria after birth may contribute to the initiation of urocortin expression in human gastrointestinal tract mucosa.

Introduction

Urocortin (Ucn), a newly identified mammalian member of the corticotropin-releasing factor (CRF) neuropeptide family, has recently been cloned from the rat midbrain [48]. Ucn has been shown to be involved in the modulation of various biologic activities, including prolonged hypotensive effects following peripheral injection [43], [48], mild hypertensive effects after central administration [43], suppression of appetite [43], inhibition of extravasation and subsequent inflammatory processes [46], modulation of body fluid balances [15], [16], suppression of encephalomyelitis [36], and regulation of cardiac contractility [32] in experimental animal models. These actions are all currently considered to be mediated via CRF receptors. Ucn has been found to bind with high affinity to CRF receptor type 1 (CRF-R1) and type 2 (CRF-R2), and has been proposed to be an endogenous ligand for CRF-R2, based upon its anatomic localization within the brain [48].

Human (h) Ucn has 95% homology in amino acid sequence to rat (r) Ucn and 43% to rat/human (r/h) CRF [8]. In humans, the presence of immunoreactive Ucn and Ucn messenger (m) RNA has been demonstrated in the placenta [34], the pituitary gland and its neoplasm [21], the brain, including the cerebral cortex, thalamus, hypothalamus, pons, medulla oblongata, and cerebellum [20], [21], [45], the spinal cord [20], and in lymphocytes [2]. Human CRF-R1 is expressed in the cortex, brainstem, pituitary, and testis [7], [49]. Recently, two isoforms of CRF-R2 have been detected in rat and subsequently termed CRF-R2α and CRF-R2β [28]. The mRNA for rat CRF-R2α is found exclusively in the brain, whereas the mRNA for CRF-R2β appears to be expressed both in the brain and in the periphery, with the highest abundance in the heart and skeletal muscle, and lower levels in the lung and intestine [27]. Human CRF-R2α is considered to be the major CRF-R2 isoform in skeletal muscle, heart, and brain tissues. However, CRF-R2β appears to be a minor CRF-R2 isoform in humans, in contrast to rodents [26], [47].

Several neuropeptides, such as neurotensin, vasoactive intestinal polypeptide (VIP), substance P, and somatostatin, have been demonstrated to be expressed in the human gastrointestinal tract, as well as in the central nervous system. Recently, Kawahito et al. demonstrated the presence of CRF in the human colonic mucosa [22], [23]. Oki et al. reported that relatively high concentrations of immunoreactive Ucn was detected in the rat stomach and colon [31]. Harada et al. recently demonstrated Ucn mRNA expression in the enteric nervous system of the rat [17]. However, the expression of Ucn and CRF receptors has not been examined in the human gastrointestinal tract.

Therefore, in this study, we first examined Ucn expression in the human adult colon using immunohistochemistry and mRNA in situ hybridization. We then characterized Ucn-positive cells using flow cytometric analysis and reverse transcriptase-polymerase chain reaction (PCR) (RT-PCR) analysis of isolated lamina propria mononuclear cells (LPMC) and epithelial cells. We also examined Ucn peptide expression in human colonic mucosa at various stages of human development (n = 35, from 11 weeks of gestation to 6 years of age) using immunohistochemistry to characterize the ontogeny of this neuropeptide in the human gastrointestinal tract. In addition, we examined the presence of CRF, CRF-R1, -R2α, and -R2β mRNA in the human gastrointestinal tract by RT-PCR analysis in LPMC and epithelial cell fractions for further characterization of gastrointestinal Ucn.

Section snippets

Human colorectal tissue specimens

The research protocol for this study was approved by the ethics committee at Tohoku University School of Medicine. Specimens of normal adult gastrointestinal tract were obtained at surgery at Tohoku University Hospital, Sendai, Japan. Normal adult specimens, gross and macroscopically normal areas of colorectal tissues (ascending colon, n = 3; transverse colon, n = 3; descending colon, n = 2; sigmoid colon, n = 3; rectum, n = 3), were obtained from patients undergoing surgery for non-obstructing

Immunoreactive Ucn in adult colorectal specimens

In 10% formalin-fixed and paraffin-embedded specimens, relatively marked cytoplasmic Ucn immunoreactivity was detected in inflammatory cells in the superficial lamina propria using both anti-r/hUcn21–35 and anti-rUcn1–40 antibodies. Inflammatory cells weakly positive for Ucn were sporadically detected in the middle or basal lamina propria. However, inflammatory cells in colonic solitary lymphatic follicles were negative. The morphologic features and distribution of Ucn-positive lamina propria

Discussion

In this study, we demonstrated the expression of Ucn peptide in human colonic mucosa. Marked Ucn immunoreactivity was detected in lamina propria inflammatory cells throughout the whole layer of the intestine. Relatively weak Ucn immunoreactivity was also detected in epithelial cells. Analysis of serial tissue sections revealed that the great majority of these Ucn-positive lamina propria cells corresponded to CD68-positive macrophages. Flow cytometric analysis of isolated LPMC also revealed that

Acknowledgements

We thank Andrew D. Darnel for critical review of the manuscript, and Fumiko Date for technical advice.

This work was supported, in part, by a Grant-in-Aid for Cancer Research 7–1 from the Ministry of Health and Welfare, Japan, a Grant-in-Aid for Scientific Research on Priority Area (A-11137301) from The Ministry of Education, Science and Culture, Japan, a Grant-in-Aid for Scientific Research (B-11470047) from Japan Society for the Promotion of Science, and a grant from The Naitou Foundation, and

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