Short communication / Open Access

DOI: 10.31488/bjcr.1000101

Intracellular Transport of Urocortin I in Cancer Cells

Keiichi Ikeda*1, Katsuyoshi Tojo2, Yoshinobu Manome1

  1. Core Research Facilities for Basic Science (Division of Molecular Cell Biology), Research Center for Medical Sciences, The Jikei University School of Medicine, Japan
  2. The Jikei University School of Medicine, Japan

*Corresponding author: Dr. Keiichi Ikeda, Core Research Facilities for Basic Science (Division of Molecular Cell Biology), Research Center for Medical Sciences, The Jikei University School of Medicine, 3-25-8, Nishishinbashi, Minato-ku, Tokyo 105-8461, Japan, Tel: 81-3-3433-1111;

Abstract

Corticotrophin-releasing hormone and its related peptides are identified as stress response substances in the endocrine system and/or peripherally activated stress responsive substances. Urocortins (UCNs) are reported to be abundantly expressed in both normal tissues and cancer cells such as glioblastomas, and gastric adenocarcinomas, and UCN I immunoreactivity has been detected in human tissue and blood, indicating UCN I is secreted from cells. Recently, a previous study reported that UCN I immunoreactivity was detected in the nucleus of clear cell renal cell carcinomas. We therefore tracked the intracellular transport of UCN I and found that it was transported and secreted from cells via a constitutive pathway in the A172 glioblastoma cell line. In the present review, we described the present knowledge concerning the intracellular transport of UCN I in cancer cells.

Keywords: CRH; glioblastoma; protein transport; urocortin

Introduction

Corticotrophin-releasing hormone (CRH/CRF) was identified in 1981 [1] and, thereafter, various related peptides including its receptors (CRF1 and CRF2) [2-5] and urocortins (UCN I, II, and III, or stresscopin and stresscopin-related peptides) [6-9] were identified. CRH is well-known as a key endocrine hormone of the hypothalamus-pituitary-adrenal axis. It exerts its action through CRF receptors, including both CRF1 and CRF2. UCN I is similar to CRH in that UCN I exerts its action via CRF1 and CRF2, but acts as a peripherally-activated stress response substance or neurotransmitter/neuropeptide in the central nervous system rather than as an endocrine component [6, 10-12]. The amino acid sequences of UCN II and III are also similar to CRH and UCN I, but their affinities for CRF receptors are highly selective to CRF2 [7-9].

Expression of CRH and related peptides in normal and cancer cells

In normal tissue, CRF1 is found in the brain (including the anterior lobe of the pituitary), skin, aortic endothelial cells, gastrointestinal tract, hepatocytes, pancreatic β cells, mast cells, and myometrium, while CRF2 is found in the brain, aortic smooth muscle cells, human aortic endothelial cells, human umbilical vein endothelial cells, heart, skeletal muscle, gastrointestinal tract, hepatocytes, pancreatic β-cells, and myometrium [13, 14].

CRH is mainly distributed in the central nervous system and also expressed in the skin. UCN I is ubiquitously distributed not only in the central nervous system (including the pituitary gland) but also in normal peripheral tissues, such as skin, thyroid gland, heart, aortic and venous cells, stomach, liver, gastrointestinal tract, colon, kidney, adrenal gland, lymph organs, gallbladder, prostate, testis, uterus, placenta, and myometrium [13, 14]. UCN I mRNA and immunoreactivity have been detected in cells, culture media, and human clinical samples [15-20], indicating that UCN I may act in an autocrine and/or paracrine fashion and may be secreted from cells. UCN II and III, which are specific ligands of CRF2, are distributed in the central nervous system, heart, endothelial cells, adrenal gland, pancreas, placenta, granulosa-lutein cells, and gastrointestinal tract.

CRH mRNA and/or protein have been detected in glioblastoma cell lines, pituitary cancers, Ewing’s sarcoma, thyroid cancers, small cell lung cancers, pulmonary adenocarcinomas, gastric adenocarcinomas, pancreatic cancers, colorectal cancers, breast cancers, clear cell renal cell carcinomas, NCI-H295R human adrenal carcinoma cells, endometrial cancers, and ovarian cancers [14]. UCN I, II, and III have also been reported to be expressed in various cancers and cancer cell lines, including in human glioblastoma cell lines, pituitary adenomas, malignant melanoma cells, thyroid carcinomas, pheochromocytomas, breast cancers, gastric adenocarcinoma cells, colon cancers, primary and metastatic liver carcinomas, pancreatic ductal adenocarcinomas, clear cell renal carcinomas, adrenal tumors and NCI-H295R human adrenal carcinoma cells, human endometrial carcinomas, and human prostate adenocarcinomas [13, 14]. UCNs exert suppressive or proliferative effects on cancer cells, directly and indirectly, via CRF1 and/or CRF2 [14].

Secretion of CRH and intracellular transport of UCN I

Recently, Romanov et al. [21, 22] reported that CRH and/or γ-aminobutyric acid co-existed with secretagogin (scgn), a Ca2+ sensor critical for the stimulus-driven assembly of molecular machinery involved in the fast regulated exocytosis of CRH in parvocellular neurons. In addition, scgn-expressing neurons typically contain CRH and glucocorticoid receptor mRNA transcripts, and scgn is co-expressed with CRH in both neuronal soma and axon terminal-like specializations in the paraventricular nucleus. Furthermore, knocking down scgn resulted in accumulation of CRH in parvocellular neuronal soma in the paraventricular nucleus in RNAi-exposed hypothalami, indicating that the vesicular traffic and exocytosis of CRH may be controlled by scgn.

As for UCN I, although the detailed mechanism of UCN I has not been reported, Tezval et al. [23] described the intracellular distribution of UCN I. Briefly, UCN I-like immunoreactivity was detected in the nucleus of clear cell renal cell carcinomas, although no UCN I immunoreactivity in the cytoplasm was detected in adult human kidney cells. Three-dimensional images of clear cell renal cell carcinomas showed that UCN I-like immunoreactivity was distributed in the entire nucleus of clear cell renal cell carcinomas. It is not known whether UCN I acts as a transcription factor, and it should be clarified how UCN I is transported into the nucleus of cancer cell(s).

To confirm the results of Tezval et al. [23], tracked the intracellular transport of UCN I [24]. As described above, we used the UCN I-expressing cancer cell line, A172 human glioblastoma cells, as a platform for UCN I tracking. For the first time, we confirmed the expression of UCN I mRNA expression and UCN I-like immunoreactivity in the human glioblastoma cell lines, A172 and U-138MG. We confirmed the expression of UCN I and UCN I-like immunoreactivity in these cell lines. Using light field microscopy, the results of the UCN I immunohistochemical analyses showed that UCN I-like immunoreactivity was distributed in the cytosolic areas of A172 and U-138MG human glioblastoma cells without nuclear UCN I-like immunoreactivity, indicating the intracellular transport of UCN I was not always into the nucleus. In addition, transmission electron microscopy revealed that UCN I may be transported in secretory vesicles and released by exocytosis. Finally, we planned to track intracellular UCN I transport by live cell imaging because secretory proteins were transported via constitutive or regulatory pathways in the cells. After constructing a plasmid-expressing hybrid protein (UCN I signal peptide, which lacks pharmacologically active sequence, and the fluorescent protein, mCherry), we transfected it into A172 glioblastoma cells. Live cell imaging was performed and images were constructed as time-lapse images with or without modification of intracellular transport by cycloheximide or brefeldin A. Plain images without transport modification resulted in direct transport of fluorescent vesicles, which was not inhibited by cycloheximide. Furthermore, addition of brefeldin A resulted in retrograde transport of the fluorescent vesicles. Based on these results, we concluded that UCN I may be transported via a constitutive pathway.

It is very important to determine whether the peptides are transport across the blood brain barrier (BBB) when the UCN I and/or its analogs are administered exogenously. Unfortunately, Hsuchou et al. [25] reported that UCN I may be transported across the BBB by receptor-mediated endocytosis in neonatal mice but not in adults. These findings implied that UCN I itself or hydrophilic forms of UCN I analogs may be difficult to transport to the brain across the BBB. It is therefore necessary to use hydrophobic forms of UCN I analogs or stimulant the expression of CRH or UCNs in brain tissue for CRF receptor stimulation to be effective in cancer treatment.

Conclusions

Recent studies reported several effects of CRH and its related peptides, especially, UCNs. The roles of these peptides still remained to be elucidated, but some of their beneficial effects have been reported. To utilize such effects, two options, exogenous administration and increasing of endogenous agonist(s), may be used. But in the brain, because exogenous UCN I may not be transported across the BBB, stimulation of UCN I expression or administration of hydrophobic forms of UCN I analogs may be another option if CRF receptor stimulation is effective in cancer treatment in the brain.

Abbreviations

CRH/CRF: Corticotrophin-Releasing Hormone; CRF1: CRF Type 1 Receptor; CRF2: CRF Type 2 Receptor; UCN: Urocortin; scgn: Secretagogin

Conflict of Interest

The authors declare no conflict of interest.

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Received: April 20, 2018;
Accepted: April 27, 2018;
Published: April 29, 2018.

To cite this article : Ikeda K, Tojo K, Manome Y. Intracellular Transport of Urocortin I in Cancer Cells. British Journal of Cancer Research. 2018: 1:3.

© Ikeda K, et al. 2018.