Double inhibition of cAMP and mTOR signalling may potentiate the reduction of cell growth in ADPKD cells.
BACKGROUND
ADPKD is a renal pathology caused by mutations of PKD1 and PKD2 genes, which encode for polycystin-1 (PC1) and polycystin-2 (PC2), respectively. PC1 plays an important role regulating several signal transducers, including cAMP and mTOR, which are involved in abnormal cell proliferation of ADPKD cells leading to the development and expansion of kidney cysts that are a typical hallmark of this disease. Therefore, the inhibition of both pathways could potentiate the reduction of cell proliferation enhancing benefits for ADPKD patients.


METHODS
The inhibition of cAMP- and mTOR-related signalling was performed by Cl-IB-MECA, an agonist of A3 receptors, and rapamycin, respectively. Protein kinase activity was evaluated by immunoblot and cell growth was analyzed by direct cell counting.


RESULTS
The activation of A3AR by the specific agonist Cl-IB-MECA causes a marked reduction of CREB, mTOR, and ERK phosphorylation in kidney tissues of Pkd1flox/-: Ksp-Cre polycystic mice and reduces cell growth in ADPKD cell lines, but not affects the kidney weight. The combined sequential treatment with rapamycin and Cl-IB-MECA in ADPKD cells potentiates the reduction of cell proliferation compared with the individual compound by the inhibition of CREB, mTOR, and ERK kinase activity. Conversely, the simultaneous application of these drugs counteracts their effect on cell growth, because the inhibition of ERK kinase activity is lost.


CONCLUSIONS
The double treatment with rapamycin and Cl-IB-MECA may have synergistic effects on the inhibition of cell proliferation in ADPKD cells suggesting that combined therapies could improve renal function in ADPKD patients.
Section of Biochemistry, Molecular Biology and Medical Genetics, Department of Biomedical and Surgical Specialty Sciences, University of Ferrara, via Fossato di Mortara, 74, 44121, Ferrara, Italy.
Lucia
L
de Stephanis
Section of Biochemistry, Molecular Biology and Medical Genetics, Department of Biomedical and Surgical Specialty Sciences, University of Ferrara, via Fossato di Mortara, 74, 44121, Ferrara, Italy.
Anna
A
Bonon
Section of Pharmacology, Department of Medical Sciences, University of Ferrara, via Fossato di Mortara, 17-19, 44121, Ferrara, Italy.
Katia
K
Varani
Section of Pathological Anatomy and Molecular Diagnostic, Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, via Aldo Moro 8, 44124, Ferrara, Italy.
Giovanni
G
Lanza
Section of Pathological Anatomy and Molecular Diagnostic, Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, via Aldo Moro 8, 44124, Ferrara, Italy.
Roberta
R
Gafà
Section of Pathology, Oncology and Experimental Biology, Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, via Fossato di Mortara 64/b, 44121, Ferrara, Italy.
Paolo
P
Pinton
Dibit 1 San Raffaele, via Olgettina 60, 20132, Milan, Italy.
Monika
M
Pema
Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, 06520, USA.
Stefan
S
Somlo
Dibit 1 San Raffaele, via Olgettina 60, 20132, Milan, Italy.
Alessandra
A
Boletta
Section of Biochemistry, Molecular Biology and Medical Genetics, Department of Biomedical and Surgical Specialty Sciences, University of Ferrara, via Fossato di Mortara, 74, 44121, Ferrara, Italy. dsn@unife.it.
Gianluca
G
Aguiari
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D058909:Adenosine A3 Receptor Agonists / Q000494:pharmacology*; D000818:Animals; D050882:CREB-Binding Protein / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects*; D000242:Cyclic AMP / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004195:Disease Models, Animal; D004357:Drug Synergism; D004359:Drug Therapy, Combination; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D020022:Genetic Predisposition to Disease; D006801:Humans; D007668:Kidney / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010641:Phenotype; D010766:Phosphorylation; D016891:Polycystic Kidney, Autosomal Dominant / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*; D020123:Sirolimus / Q000494:pharmacology*; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D050396:TRPP Cation Channels / Q000172:deficiency / Q000235:genetics; D013997:Time Factors
D000241:Adenosine; D058909:Adenosine A3 Receptor Agonists; D050882:CREB-Binding Protein; D000242:Cyclic AMP; D048049:Extracellular Signal-Regulated MAP Kinases; D047428:Protein Kinase Inhibitors; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; D050396:TRPP Cation Channels; C494363:CREBBP protein, human; C494364:Crebbp protein, mouse; C546842:MTOR protein, human; C546843:mTOR protein, mouse; C090034:2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide
ADPKD; CREB; Cell growth; ERK signalling; Sequential double treatment; mTOR