| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 17, 2003 as doi:10.1096/fj.02-0993fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,2
The University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, UK;
* Nuffield Department of Obstetrics and Gynaecology,
Molecular Angiogenesis Laboratory, Cancer Research UK, Weatherall Institute of Molecular Medicine; and
AstraZeneca, CVGI, Alderley Park, Macclesfield, Cheshire, SK10 4TG, UK
2Correspondence: NDOG, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, UK. E-mail: leonid.nikitenko{at}obs-gyn.ox.ac.uk
SPECIFIC AIMS
The peptide adrenomedullin shows a remarkable range of effects on the vasculature that include inter alia, vasodilatation, regulation of permeability, inhibition of endothelial apoptosis, and promotion of angiogenesis. It is becoming clear that either activation or disruption of adrenomedullin signaling might contribute to many pathologies including cardiovascular disease, pulmonary hypertension, neoplastic growth, inflammatory disease, and disorders of the reproductive tract. The aim of the present study was to identify factors that regulate expression of calcitonin-receptor like receptor (CRLR), the mediator of adrenomedullin signaling, in microvascular endothelial cells and therefore potentiate the effects of the peptide on vasculature.
PRINCIPAL FINDINGS
1. Microvascular endothelial cells are the major source of CRLR mRNA in vitro and in vivo
Transcriptional profiling of 7 transmembrane (7TM) G-protein-coupled receptor (GPCR) CRLR in various cell lines demonstrated the predominant expression of the transcript in all primary microvascular endothelial cells lines studied and low expression in a few of the 16 nonendothelial cell lines analyzed. This is in accordance with previous findings on CRLR mRNA localization in blood vessels and further supports the view of endothelial cells as one of the main targets for the peptide in vivo. In the present study CRLR mRNA was found in primary microvascular endothelial cells obtained from lung, skin, endometrium, and myometrium, indicating that the mechanisms investigated here could be relevant to the microvascular bed of various tissues. This finding also supports the view that CRLR is potentially a major mediator of effects of adrenomedullin on the vasculature.
2. Identification of the transcriptional start of the human CRLR gene
The transcriptional start of human CRLR cDNA was identified by 5'-RACE and the subsequent sequencing of the amplified fragment. GenBank Nucleotide Sequence Database (http://www.ncbi.nlm.nih.gov) was searched by BLASTN analysis of the 5'UTR sequence determined by 5'-RACE to identify the 5'-flanking region of the gene by sequence overlap. The 2318 bp proximal 5'-flanking region of human CRLR was isolated by PCR using the corresponding BAC clone as a template, cloned, and subsequently sequenced.
3. Sequence, analysis, and promoter activity of 5'-flanking region of human CRLR
The isolated genomic fragment contains the basal gene promoter of human CRLR, including potential TATA boxes and several GC boxes. When cloned into pGL3 luciferase reporter gene vectors, the constructs of various lengths showed significant promoter activity. Regulatory elements binding known transcription factors such as Sp-1, Pit-1, glucocorticoid receptor, and hypoxia-inducible factor 
(HIF-1) were identified within the reported sequence.
4. Mutation of the hypoxia-response element in the human CRLR promoter results in the reduced promoter activity under hypoxic conditions
The candidate binding site for hypoxia-inducible factor-1 (HIF-1), 5'-GGCGTGTG-3', was identified on the antisense strand between -343 and -347, which contained the core sequence 5'-RCGTG-3' recognized by this transcription factor in the promoter of VEGF, erythropoietin, aldolase, enolase, lactate dehydrogenase, and phospho-glycerophosphate kinase genes. To determine whether this sequence mediates the hypoxia response of the human CRLR gene, a 3 bp substitution within this sequence was introduced into wild-type construct pGL3-CRLR-516 (Fig. 1
A). Basal promoter activity of the resulting construct pGL3-CRLR-516 mut was similar to that of pGL3-CRLR-516 at 20% O2, but no induction under hypoxia was observed (Fig. 1B
).
|
5. Induction of CRLR, but not RAMPs, mRNA expression in hypoxic microvascular endothelial cells
Hypoxia and cytokine production in septic shock or chronic heart failure, shear stress in hypertension, mechanical stress in heart failure, and hyperglycemia in diabetes induce adrenomedullin secretion by vascular cells. This might contribute to the increased plasma concentrations of the peptide in these conditions and hence constitute a protective action by which cardiovascular homeostasis is maintained. To identify whether expression of any of the components of adrenomedullin receptor system, i.e., CRLR and RAMPs, is influenced by low oxygen tension in microvascular endothelial cells, we performed semiquantitative RT-PCR. This showed up-regulation of both adrenomedullin and CRLR transcripts in microvascular cells in response to hypoxia (Fig. 2
). However, the level of RAMP2 mRNA remained virtually unchanged whereas RAMP1 and 3 mRNAs were below detection limit in microvascular endothelial cells (Fig. 2)
.
|
6. Pathophysiological considerations
Adrenomedullin signaling is of particular significance in endothelial cell biology since the peptide protects cells from apoptosis, promotes angiogenesis, and affects vascular tone and permeability. Therefore, identification of mechanisms of transcriptional regulation of the G-protein-coupled receptor CRLR, as well as other integral parts of the adrenomedullin/CGRP signaling system, in microvascular endothelial cells would contribute to understanding how these cells function in many pathologies including cardiovascular disease, pulmonary hypertension, atherosclerosis, inflammatory disease, disorders of the reproductive tract, and neoplastic growth.
The results from the present study demonstrate the presence of critical cis-acting elements in the human CRLR gene promoter. Of significance is the demonstration for the first time that the human CRLR promoter region has a functional HRE that can be activated by hypoxia in microvascular endothelial cells. Since previous studies have showed induction of adrenomedullin gene by hypoxia, we investigated whether CRLR and RAMPs mRNA expression was also affected by low oxygen tension. We show here for the first time that adrenomedullin and CRLR are both up-regulated under hypoxic conditions in microvascular endothelial cells. This observation indicates that the response of microvascular endothelial cells to elevated levels of exogenous adrenomedullin and calcitonin-gene related peptide (CGRP) in hypoxia could be further enhanced by increased expression of CRLR. In favor of this hypothesis, autocrine/paracrine effectors and their receptors are often regulated in coordinated fashion. For instance, hypoxia up-regulates expression of the components of VEGF signaling system (VEGF and its receptor flt1). The simultaneous transcriptional up-regulation of CRLR and its ligand adrenomedullin in endothelial cells might play a significant role in the vascular responses to hypoxia and ischemia by creating a potent survival loop. In cardiovascular disease, septic shock, and hypertension, where adrenomedullin expression is increased, any factor that alters CRLR expression would directly affect the compensatory mechanisms that induce vasodilatation and decrease permeability in order to reduce the tissue damage found in these pathologies.
In the present study expression of RAMPs was not activated by hypoxia in microvascular cells. However, the presence of basal levels of RAMP2 mRNA indicates that all components of the adrenomedullin signaling system are present in microvascular endothelial cells in both normoxia and hypoxia. The low abundance of RAMPs transcripts in endothelial cells in culture in our study further supports the view that other factors play a more significant role in their expression. Such factors will affect the formation of CRLR/RAMP heterodimers and therefore the transport of the CRLR to the plasma membrane by regulating RAMP expression. In pathological conditions, these factors when acting in combination with hypoxia could further contribute to the alteration of the expression of functional receptors, resulting in modulation of growth/apoptosis of vascular cells and magnitude of the effects of adrenomedullin and CGRP on blood vessel permeability and vasodilatation (Fig. 3
).
|
Of particular interest is the presence of the numerous glucocorticoid receptor binding sites in CRLR promoter. This agrees with the previous finding that levels of CRLR mRNA are elevated after exposure to dexamethasone. Therefore, elevated levels of CRLR mRNA in glucocorticoid-exposed endothelial cells might potentially contribute to the responses of the vasculature to glucocorticoid therapy. Whether excess or prolonged treatment with glucocorticoids contributes to hypertension via regulation of CRLR gene transcription and therefore modulation of effects of adrenomedullin on vasculature needs further investigation.
CONCLUSIONS
In summary, the present study reveals the genomic organization, regulatory components, and the organ- and cell-specific expression pattern of a 7TM GPCR human CRLR. Our findings demonstrate that simultaneous transcriptional up-regulation of CRLR and its ligand adrenomedullin in microvascular endothelial cells could lead to a potent survival loop and therefore might play a significant role in vascular responses to hypoxia and ischemia and in tumor biology (Fig. 3)
.
The activity of the CRLR promoter under hypoxic conditions is regulated at least in part through hypoxia-responsive regulatory element binding transcription factor HIF-1. Other stimuli (e.g., presence of glucocorticoids) might also have a significant role in the transcriptional regulation of the CRLR and RAMPs expression in pathophysiological conditions and alter the protective role of adrenomedullin on the cardiovascular system. Therefore, the mechanisms of transcriptional regulation of G-protein-coupled receptor CRLR as an integral part of the adrenomedullin signaling system might have a significant role in dysfunction of the microvasculature observed in many pathologies including cardiovascular disease, pulmonary hypertension, atherosclerosis, neoplastic growth, inflammatory disease, and disorders of the reproductive tract.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0993fje; doi: 10.1096/fj.02-0993fje 
This article has been cited by other articles:
|
|
 |
|
  A. Silaghi, V. Achard, O. Paulmyer-Lacroix, T. Scridon, V. Tassistro, I. Duncea, K. Clement, A. Dutour, and M. Grino Expression of adrenomedullin in human epicardial adipose tissue: role of coronary status Am J Physiol Endocrinol Metab, November 1, 2007; 293(5): E1443 - E1450. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Gibbons, R. Dackor, W. Dunworth, K. Fritz-Six, and K. M. Caron Receptor Activity-Modifying Proteins: RAMPing up Adrenomedullin Signaling Mol. Endocrinol., April 1, 2007; 21(4): 783 - 796. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Ramachandran, T. Arumugam, R. F. Hwang, J. K. Greenson, D. M. Simeone, and C. D. Logsdon Adrenomedullin Is Expressed in Pancreatic Cancer and Stimulates Cell Proliferation and Invasion in an Autocrine Manner via the Adrenomedullin Receptor, ADMR Cancer Res., March 15, 2007; 67(6): 2666 - 2675. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S Hagner, U Stahl, T Grimm, M Sturzl, and R E Lang Expression of calcitonin receptor-like receptor in human vascular tumours. J. Clin. Pathol., October 1, 2006; 59(10): 1104 - 1107. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. L. Nikitenko, T. Cross, L. Campo, H. Turley, R. Leek, S. Manek, R. Bicknell, and M. C.P. Rees Expression of Terminally Glycosylated Calcitonin Receptor-Like Receptor in Uterine Leiomyoma: Endothelial Phenotype and Association with Microvascular Density. Clin. Cancer Res., October 1, 2006; 12(19): 5648 - 5658. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. C. Bosco, M. Puppo, C. Santangelo, L. Anfosso, U. Pfeffer, P. Fardin, F. Battaglia, and L. Varesio Hypoxia Modifies the Transcriptome of Primary Human Monocytes: Modulation of Novel Immune-Related Genes and Identification Of CC-Chemokine Ligand 20 as a New Hypoxia-Inducible Gene J. Immunol., August 1, 2006; 177(3): 1941 - 1955. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Uzan, H.-K. Ea, J.-M. Launay, J.-M. Garel, R. Champy, M. Cressent, and F. Liote A Critical Role for Adrenomedullin-Calcitonin Receptor-Like Receptor in Regulating Rheumatoid Fibroblast-Like Synoviocyte Apoptosis J. Immunol., May 1, 2006; 176(9): 5548 - 5558. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. L. Nikitenko, N. Blucher, S. B. Fox, R. Bicknell, D. M. Smith, and M. C. P. Rees Adrenomedullin and CGRP interact with endogenous calcitonin-receptor-like receptor in endothelial cells and induce its desensitisation by different mechanisms. J. Cell Sci., March 1, 2006; 119(Pt 5): 910 - 922. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Nagaya, H. Mori, S. Murakami, K. Kangawa, and S. Kitamura Adrenomedullin: angiogenesis and gene therapy Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2005; 288(6): R1432 - R1437. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G Eisenhofer, T-T Huynh, K Pacak, F M Brouwers, M M Walther, W M Linehan, P J Munson, M Mannelli, D S Goldstein, and A G Elkahloun Distinct gene expression profiles in norepinephrine- and epinephrine-producing hereditary and sporadic pheochromocytomas: activation of hypoxia-driven angiogenic pathways in von Hippel-Lindau syndrome Endocr. Relat. Cancer, December 1, 2004; 11(4): 897 - 911. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Salim, R. P. Nacamuli, E. F. Morgan, A. J. Giaccia, and M. T. Longaker Transient Changes in Oxygen Tension Inhibit Osteogenic Differentiation and Runx2 Expression in Osteoblasts J. Biol. Chem., September 17, 2004; 279(38): 40007 - 40016. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
