Protein expression of urotensin II, urotensin-related peptide and their receptor in the lungs of patients with lymphangioleiomyomatosis

Abstract

Urotensin II (UII) and urotensin-related peptide (URP) are vasoactive neuropeptides with wide ranges of action in the normal mammalian lung, including the control of smooth muscle cell proliferation. UII and URP exert their actions by binding to the G-protein coupled receptor-14 known as UT. Lymphangioleiomyomatosis (LAM) is a disease of progressive lung destruction resulting from the excessive growth of abnormal smooth muscle-like cells that exhibit markers of neural crest origin. LAM cells also exhibit inactivation of the tumor suppressor tuberin (TSC2), excessive activity of ‘mammalian target of rapamycin (mTOR), and dysregulated cell growth and proliferation. In the present study we examined the expression and distribution of UII and UT in the lungs of patients with LAM. There was abundant expression of UII, URP and UT proteins in the interstitial nodular lesions of patients with LAM. By immunohistochemistry, UII, URP and UT were co-localized with HMB45, a diagnostic marker of LAM. Immunoreactivity for UII, URP and UT was also evident over the pulmonary epithelium, pulmonary vasculature and inflammatory cells. Western blotting revealed the presence of greater UT expression in the lungs of patients with LAM compared to normal human lungs. UT expression correlated with mTOR activity, as indicated by increased phosphorylation of S6 in LAM samples. These findings demonstrate for the first time the presence of UII, URP and their receptor in the lesions of patients with LAM, and suggest a possible role in the pathogenesis of the disease.

Introduction

Urotensin II (UII) is a small ancient peptide (11–14 amino acids) that was originally isolated from the urophysis, a neurosecretory organ, of the goby fish [3], [37]. The peptide is conserved from invertebrates to humans, shares similar structure to somatostatin, and is expressed in the nervous system and cardiopulmonary system of various species [1]. UII has a wide range of physiologic actions mediated through binding to the G-protein coupled receptor-14 named UT [1], [28], [34], [36]. Pharmacologic studies have shown UII to be the most potent vasconstricor peptide identified to date, though its vasoactive properties vary depending on the species and type of vessels investigated [10].

The bioactivity of UII is highly variable, and has been attributed in part to the levels of UT in any given tissue [11]. For example, UII constricted human coronary, radial and pulmonary arteries, which are tissues that exhibit abundant expression of UT [1], [12], [13], [30]. On the other hand, UII can also exert endothelium-dependant vasorelaxation through release of nitric oxide and prostaglandin I₂ in the aorta, as well as coronary and mesenteric arteries [4], [17], [18], [26], [27], [44]. Others reported no significant effect for UII on pulmonary arteries of humans and animals [44]. Apart from its vascular effects, UII contracted airway smooth muscle cells of human, cat and rat bronchi [2], [19]. UII also stimulates proliferation of the smooth muscle cells of pulmonary and systemic arteries [9], [40], [49], epithelial cells [33], cardiac [20] and adventitial fibroblast [8], as well as umbilical endothelial cells [42], [43].

UII is expressed mainly in the aorta, heart, kidneys, central nervous system and spinal cord [1]. The UII receptor, UT, is expressed in the heart, brain, lungs, brain, skeletal muscles, bladder and pancreas [1], [23], [28], [34], [36]. UT is also expressed in vascular and airway smooth muscle cells, as well as pulmonary vasculature [31], [32]. Recent studies have demonstrated the presence of another ligand for UT named urotensin-related peptide URP [7], [45]; the detailed expression of URP in the human lungs remains to be elucidated. The proliferative effects of UII, a neuropeptide, suggested a potential role in neoplastic diseases of neuroendocrine origin. In agreement, increased UT expression, as well as proliferation in response to UII, was demonstrated in adrenal tumors [47], [50]. We hypothesized that UII and its receptor might play a role in lymphangioleiomyomatosis (LAM), a pulmonary and lymphatic disease caused by abnormal smooth muscle-like cells that also express neural crest markers.

Lymphangioleiomyomatosis (LAM) is a rare disease of young women characterized by abnormal proliferation of smooth muscle-like cells (LAM cells) in the pulmonary interstitium. The nodular proliferation of LAM cells leads to cystic destruction of the lungs and respiratory failure. Recent evidence describing the recurrence of LAM after lung transplantation and the presence of LAM cells with the same mutation in the lung, lymph nodes and kidney suggests that LAM cells are clonal, and can metastasize [24]. Although the origin of LAM cells is unknown, they express markers of smooth muscle differentiation (e.g., α-actin), as well as proteins associated with neural crest lineage (e.g., GP100). Recognition of GP100, by the HMB45 antibody is diagnostic of LAM. Given that urotensin signaling might play an important role in the pathophysiology of neoplastic processes of neural lineage, we sought to determine the expression of UII, URP and UT in the lungs of patients with LAM and compare that to those of normal individuals.