LGR5 Activates Noncanonical Wnt Signaling and Inhibits Aldosterone Production in the Human Adrenal.
BACKGROUND
Aldosterone synthesis and cellularity in the human adrenal zona glomerulosa (ZG) is sparse and patchy, presumably due to salt excess. The frequency of somatic mutations causing aldosterone-producing adenomas (APAs) may be a consequence of protection from cell loss by constitutive aldosterone production.


OBJECTIVE
The objective of the study was to delineate a process in human ZG, which may regulate both aldosterone production and cell turnover.


METHODS
This study included a comparison of 20 pairs of ZG and zona fasciculata transcriptomes from adrenals adjacent to an APA (n = 13) or a pheochromocytoma (n = 7).


METHODS
Interventions included an overexpression of the top ZG gene (LGR5) or stimulation by its ligand (R-spondin-3).


METHODS
A transcriptome profile of ZG and zona fasciculata and aldosterone production, cell kinetic measurements, and Wnt signaling activity of LGR5 transfected or R-spondin-3-stimulated cells were measured.


RESULTS
LGR5 was the top gene up-regulated in ZG (25-fold). The gene for its cognate ligand R-spondin-3, RSPO3, was 5-fold up-regulated. In total, 18 genes associated with the Wnt pathway were greater than 2-fold up-regulated. ZG selectivity of LGR5, and its absence in most APAs, were confirmed by quantitative PCR and immunohistochemistry. Both R-spondin-3 stimulation and LGR5 transfection of human adrenal cells suppressed aldosterone production. There was reduced proliferation and increased apoptosis of transfected cells, and the noncanonical activator protein-1/Jun pathway was stimulated more than the canonical Wnt pathway (3-fold vs 1.3-fold). ZG of adrenal sections stained positive for apoptosis markers.


CONCLUSIONS
LGR5 is the most selectively expressed gene in human ZG and reduces aldosterone production and cell number. Such conditions may favor cells whose somatic mutation reverses aldosterone inhibition and cell loss.
Clinical Pharmacology Unit (L.H.S., J.Z., A.E.D.T., S.G., J.J.M., E.A.B.A., A.P.D., M.J.B.) and Cardiovascular Division (N.F., H.Y., M.R.B.), Department of Medicine, University of Cambridge, Cambridge National Institute for Health Research (S.G.N.), Biomedical Research Centre, Department of Clinical Biochemistry, Addenbrooke's Hospital, University of Cambridge Metabolic Research Laboratories (G.S.Y.), Institute of Metabolic Science, Addenbrooke's Hospital, and Human Research Tissue Bank (W.Z.), Cambridge University Hospitals Foundation Trust, Addenbrooke's Hospital, Cambridge CB2 0QQ, United Kingdom; Department of Medicine (E.A.B.A.), Faculty of Medicine, The National University of Malaysia Medical Centre, Kuala Lumpur 56000, Malaysia; and Medical Research Council Cancer Unit (G.M.), University of Cambridge, Cambridge CB2 0XZ, United Kingdom.
Lalarukh Haris
LH
Shaikh
Clinical Pharmacology Unit (L.H.S., J.Z., A.E.D.T., S.G., J.J.M., E.A.B.A., A.P.D., M.J.B.) and Cardiovascular Division (N.F., H.Y., M.R.B.), Department of Medicine, University of Cambridge, Cambridge National Institute for Health Research (S.G.N.), Biomedical Research Centre, Department of Clinical Biochemistry, Addenbrooke's Hospital, University of Cambridge Metabolic Research Laboratories (G.S.Y.), Institute of Metabolic Science, Addenbrooke's Hospital, and Human Research Tissue Bank (W.Z.), Cambridge University Hospitals Foundation Trust, Addenbrooke's Hospital, Cambridge CB2 0QQ, United Kingdom; Department of Medicine (E.A.B.A.), Faculty of Medicine, The National University of Malaysia Medical Centre, Kuala Lumpur 56000, Malaysia; and Medical Research Council Cancer Unit (G.M.), University of Cambridge, Cambridge CB2 0XZ, United Kingdom.
Junhua
J
Zhou
Clinical Pharmacology Unit (L.H.S., J.Z., A.E.D.T., S.G., J.J.M., E.A.B.A., A.P.D., M.J.B.) and Cardiovascular Division (N.F., H.Y., M.R.B.), Department of Medicine, University of Cambridge, Cambridge National Institute for Health Research (S.G.N.), Biomedical Research Centre, Department of Clinical Biochemistry, Addenbrooke's Hospital, University of Cambridge Metabolic Research Laboratories (G.S.Y.), Institute of Metabolic Science, Addenbrooke's Hospital, and Human Research Tissue Bank (W.Z.), Cambridge University Hospitals Foundation Trust, Addenbrooke's Hospital, Cambridge CB2 0QQ, United Kingdom; Department of Medicine (E.A.B.A.), Faculty of Medicine, The National University of Malaysia Medical Centre, Kuala Lumpur 56000, Malaysia; and Medical Research Council Cancer Unit (G.M.), University of Cambridge, Cambridge CB2 0XZ, United Kingdom.
Ada E D
AE
Teo
Clinical Pharmacology Unit (L.H.S., J.Z., A.E.D.T., S.G., J.J.M., E.A.B.A., A.P.D., M.J.B.) and Cardiovascular Division (N.F., H.Y., M.R.B.), Department of Medicine, University of Cambridge, Cambridge National Institute for Health Research (S.G.N.), Biomedical Research Centre, Department of Clinical Biochemistry, Addenbrooke's Hospital, University of Cambridge Metabolic Research Laboratories (G.S.Y.), Institute of Metabolic Science, Addenbrooke's Hospital, and Human Research Tissue Bank (W.Z.), Cambridge University Hospitals Foundation Trust, Addenbrooke's Hospital, Cambridge CB2 0QQ, United Kingdom; Department of Medicine (E.A.B.A.), Faculty of Medicine, The National University of Malaysia Medical Centre, Kuala Lumpur 56000, Malaysia; and Medical Research Council Cancer Unit (G.M.), University of Cambridge, Cambridge CB2 0XZ, United Kingdom.
Sumedha
S
Garg
Clinical Pharmacology Unit (L.H.S., J.Z., A.E.D.T., S.G., J.J.M., E.A.B.A., A.P.D., M.J.B.) and Cardiovascular Division (N.F., H.Y., M.R.B.), Department of Medicine, University of Cambridge, Cambridge National Institute for Health Research (S.G.N.), Biomedical Research Centre, Department of Clinical Biochemistry, Addenbrooke's Hospital, University of Cambridge Metabolic Research Laboratories (G.S.Y.), Institute of Metabolic Science, Addenbrooke's Hospital, and Human Research Tissue Bank (W.Z.), Cambridge University Hospitals Foundation Trust, Addenbrooke's Hospital, Cambridge CB2 0QQ, United Kingdom; Department of Medicine (E.A.B.A.), Faculty of Medicine, The National University of Malaysia Medical Centre, Kuala Lumpur 56000, Malaysia; and Medical Research Council Cancer Unit (G.M.), University of Cambridge, Cambridge CB2 0XZ, United Kingdom.
Sudeshna Guha
SG
Neogi
Clinical Pharmacology Unit (L.H.S., J.Z., A.E.D.T., S.G., J.J.M., E.A.B.A., A.P.D., M.J.B.) and Cardiovascular Division (N.F., H.Y., M.R.B.), Department of Medicine, University of Cambridge, Cambridge National Institute for Health Research (S.G.N.), Biomedical Research Centre, Department of Clinical Biochemistry, Addenbrooke's Hospital, University of Cambridge Metabolic Research Laboratories (G.S.Y.), Institute of Metabolic Science, Addenbrooke's Hospital, and Human Research Tissue Bank (W.Z.), Cambridge University Hospitals Foundation Trust, Addenbrooke's Hospital, Cambridge CB2 0QQ, United Kingdom; Department of Medicine (E.A.B.A.), Faculty of Medicine, The National University of Malaysia Medical Centre, Kuala Lumpur 56000, Malaysia; and Medical Research Council Cancer Unit (G.M.), University of Cambridge, Cambridge CB2 0XZ, United Kingdom.
Nichola
N
Figg
Clinical Pharmacology Unit (L.H.S., J.Z., A.E.D.T., S.G., J.J.M., E.A.B.A., A.P.D., M.J.B.) and Cardiovascular Division (N.F., H.Y., M.R.B.), Department of Medicine, University of Cambridge, Cambridge National Institute for Health Research (S.G.N.), Biomedical Research Centre, Department of Clinical Biochemistry, Addenbrooke's Hospital, University of Cambridge Metabolic Research Laboratories (G.S.Y.), Institute of Metabolic Science, Addenbrooke's Hospital, and Human Research Tissue Bank (W.Z.), Cambridge University Hospitals Foundation Trust, Addenbrooke's Hospital, Cambridge CB2 0QQ, United Kingdom; Department of Medicine (E.A.B.A.), Faculty of Medicine, The National University of Malaysia Medical Centre, Kuala Lumpur 56000, Malaysia; and Medical Research Council Cancer Unit (G.M.), University of Cambridge, Cambridge CB2 0XZ, United Kingdom.
Giles S
GS
Yeo
Clinical Pharmacology Unit (L.H.S., J.Z., A.E.D.T., S.G., J.J.M., E.A.B.A., A.P.D., M.J.B.) and Cardiovascular Division (N.F., H.Y., M.R.B.), Department of Medicine, University of Cambridge, Cambridge National Institute for Health Research (S.G.N.), Biomedical Research Centre, Department of Clinical Biochemistry, Addenbrooke's Hospital, University of Cambridge Metabolic Research Laboratories (G.S.Y.), Institute of Metabolic Science, Addenbrooke's Hospital, and Human Research Tissue Bank (W.Z.), Cambridge University Hospitals Foundation Trust, Addenbrooke's Hospital, Cambridge CB2 0QQ, United Kingdom; Department of Medicine (E.A.B.A.), Faculty of Medicine, The National University of Malaysia Medical Centre, Kuala Lumpur 56000, Malaysia; and Medical Research Council Cancer Unit (G.M.), University of Cambridge, Cambridge CB2 0XZ, United Kingdom.
Haixiang
H
Yu
Clinical Pharmacology Unit (L.H.S., J.Z., A.E.D.T., S.G., J.J.M., E.A.B.A., A.P.D., M.J.B.) and Cardiovascular Division (N.F., H.Y., M.R.B.), Department of Medicine, University of Cambridge, Cambridge National Institute for Health Research (S.G.N.), Biomedical Research Centre, Department of Clinical Biochemistry, Addenbrooke's Hospital, University of Cambridge Metabolic Research Laboratories (G.S.Y.), Institute of Metabolic Science, Addenbrooke's Hospital, and Human Research Tissue Bank (W.Z.), Cambridge University Hospitals Foundation Trust, Addenbrooke's Hospital, Cambridge CB2 0QQ, United Kingdom; Department of Medicine (E.A.B.A.), Faculty of Medicine, The National University of Malaysia Medical Centre, Kuala Lumpur 56000, Malaysia; and Medical Research Council Cancer Unit (G.M.), University of Cambridge, Cambridge CB2 0XZ, United Kingdom.
Janet J
JJ
Maguire
Clinical Pharmacology Unit (L.H.S., J.Z., A.E.D.T., S.G., J.J.M., E.A.B.A., A.P.D., M.J.B.) and Cardiovascular Division (N.F., H.Y., M.R.B.), Department of Medicine, University of Cambridge, Cambridge National Institute for Health Research (S.G.N.), Biomedical Research Centre, Department of Clinical Biochemistry, Addenbrooke's Hospital, University of Cambridge Metabolic Research Laboratories (G.S.Y.), Institute of Metabolic Science, Addenbrooke's Hospital, and Human Research Tissue Bank (W.Z.), Cambridge University Hospitals Foundation Trust, Addenbrooke's Hospital, Cambridge CB2 0QQ, United Kingdom; Department of Medicine (E.A.B.A.), Faculty of Medicine, The National University of Malaysia Medical Centre, Kuala Lumpur 56000, Malaysia; and Medical Research Council Cancer Unit (G.M.), University of Cambridge, Cambridge CB2 0XZ, United Kingdom.
Wanfeng
W
Zhao
Clinical Pharmacology Unit (L.H.S., J.Z., A.E.D.T., S.G., J.J.M., E.A.B.A., A.P.D., M.J.B.) and Cardiovascular Division (N.F., H.Y., M.R.B.), Department of Medicine, University of Cambridge, Cambridge National Institute for Health Research (S.G.N.), Biomedical Research Centre, Department of Clinical Biochemistry, Addenbrooke's Hospital, University of Cambridge Metabolic Research Laboratories (G.S.Y.), Institute of Metabolic Science, Addenbrooke's Hospital, and Human Research Tissue Bank (W.Z.), Cambridge University Hospitals Foundation Trust, Addenbrooke's Hospital, Cambridge CB2 0QQ, United Kingdom; Department of Medicine (E.A.B.A.), Faculty of Medicine, The National University of Malaysia Medical Centre, Kuala Lumpur 56000, Malaysia; and Medical Research Council Cancer Unit (G.M.), University of Cambridge, Cambridge CB2 0XZ, United Kingdom.
Martin R
MR
Bennett
Clinical Pharmacology Unit (L.H.S., J.Z., A.E.D.T., S.G., J.J.M., E.A.B.A., A.P.D., M.J.B.) and Cardiovascular Division (N.F., H.Y., M.R.B.), Department of Medicine, University of Cambridge, Cambridge National Institute for Health Research (S.G.N.), Biomedical Research Centre, Department of Clinical Biochemistry, Addenbrooke's Hospital, University of Cambridge Metabolic Research Laboratories (G.S.Y.), Institute of Metabolic Science, Addenbrooke's Hospital, and Human Research Tissue Bank (W.Z.), Cambridge University Hospitals Foundation Trust, Addenbrooke's Hospital, Cambridge CB2 0QQ, United Kingdom; Department of Medicine (E.A.B.A.), Faculty of Medicine, The National University of Malaysia Medical Centre, Kuala Lumpur 56000, Malaysia; and Medical Research Council Cancer Unit (G.M.), University of Cambridge, Cambridge CB2 0XZ, United Kingdom.
Elena A B
EA
Azizan
Clinical Pharmacology Unit (L.H.S., J.Z., A.E.D.T., S.G., J.J.M., E.A.B.A., A.P.D., M.J.B.) and Cardiovascular Division (N.F., H.Y., M.R.B.), Department of Medicine, University of Cambridge, Cambridge National Institute for Health Research (S.G.N.), Biomedical Research Centre, Department of Clinical Biochemistry, Addenbrooke's Hospital, University of Cambridge Metabolic Research Laboratories (G.S.Y.), Institute of Metabolic Science, Addenbrooke's Hospital, and Human Research Tissue Bank (W.Z.), Cambridge University Hospitals Foundation Trust, Addenbrooke's Hospital, Cambridge CB2 0QQ, United Kingdom; Department of Medicine (E.A.B.A.), Faculty of Medicine, The National University of Malaysia Medical Centre, Kuala Lumpur 56000, Malaysia; and Medical Research Council Cancer Unit (G.M.), University of Cambridge, Cambridge CB2 0XZ, United Kingdom.
Anthony P
AP
Davenport
Clinical Pharmacology Unit (L.H.S., J.Z., A.E.D.T., S.G., J.J.M., E.A.B.A., A.P.D., M.J.B.) and Cardiovascular Division (N.F., H.Y., M.R.B.), Department of Medicine, University of Cambridge, Cambridge National Institute for Health Research (S.G.N.), Biomedical Research Centre, Department of Clinical Biochemistry, Addenbrooke's Hospital, University of Cambridge Metabolic Research Laboratories (G.S.Y.), Institute of Metabolic Science, Addenbrooke's Hospital, and Human Research Tissue Bank (W.Z.), Cambridge University Hospitals Foundation Trust, Addenbrooke's Hospital, Cambridge CB2 0QQ, United Kingdom; Department of Medicine (E.A.B.A.), Faculty of Medicine, The National University of Malaysia Medical Centre, Kuala Lumpur 56000, Malaysia; and Medical Research Council Cancer Unit (G.M.), University of Cambridge, Cambridge CB2 0XZ, United Kingdom.
Grahame
G
McKenzie
Clinical Pharmacology Unit (L.H.S., J.Z., A.E.D.T., S.G., J.J.M., E.A.B.A., A.P.D., M.J.B.) and Cardiovascular Division (N.F., H.Y., M.R.B.), Department of Medicine, University of Cambridge, Cambridge National Institute for Health Research (S.G.N.), Biomedical Research Centre, Department of Clinical Biochemistry, Addenbrooke's Hospital, University of Cambridge Metabolic Research Laboratories (G.S.Y.), Institute of Metabolic Science, Addenbrooke's Hospital, and Human Research Tissue Bank (W.Z.), Cambridge University Hospitals Foundation Trust, Addenbrooke's Hospital, Cambridge CB2 0QQ, United Kingdom; Department of Medicine (E.A.B.A.), Faculty of Medicine, The National University of Malaysia Medical Centre, Kuala Lumpur 56000, Malaysia; and Medical Research Council Cancer Unit (G.M.), University of Cambridge, Cambridge CB2 0XZ, United Kingdom.
Morris J
MJ
Brown
D000311:Adrenal Glands / Q000166:cytology / Q000378:metabolism*; D000450:Aldosterone / Q000096:biosynthesis*; D002452:Cell Count; D015536:Down-Regulation / Q000235:genetics; D020869:Gene Expression Profiling; D006801:Humans; D007150:Immunohistochemistry; D046228:Microarray Analysis; D043562:Receptors, G-Protein-Coupled / Q000502:physiology*; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000235:genetics; D060449:Wnt Signaling Pathway / Q000235:genetics*; D015383:Zona Fasciculata / Q000378:metabolism; D015384:Zona Glomerulosa / Q000166:cytology / Q000378:metabolism
C116884:LGR5 protein, human; D043562:Receptors, G-Protein-Coupled; D000450:Aldosterone