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11–14 APRIL, 2018, HELSINKI, FINLAND
13:39–13:42
S1-4 (PP)
RAPAMYCIN REGULATION OF CORE CLOCK GENES
IS ASSOCIATED WITH ALTERED DE-OBSTRUCTION
SIGNATURES COORDINATE WITH PHYSIOLOGY
Karen AITKEN
1
, Annette SCHRODER
2
, Jia-Xin JIANG
3
, Aliza SIEBENALLER
4
,
Thenuka THANABALASINGHAM
5
, Martin SIDLER
6
and Darius BAGLI
7
1) PGCRL room 159420TUV, DSCB, Sickkids Research Institute, Toronto, CANADA - 2) Hospital for Sick Children,
Urology Division3, Department of Surgery; Developmental and Stem Cell Biology, Research Institute, Toronto,
CANADA - 3) Hospital for Sick Children, University of Toronto, Developmental and Stem Cell Biology, Research Institute;
Department of Physiology, Faculty of Arts a, Toronto, CANADA - 4) Hospital for Sick Children, Developmental and Stem
Cell Biology, Research Institute, Toronto, CANADA - 5) H, Developmental and Stem Cell Biology, Research Institute,
Toronto, CANADA - 6) Hospital for Sick Children, University of Toronto, Developmental and Stem Cell Biology, Research
Institute; Institute of Medical Sciences, Faculty of M, Toronto, CANADA - 7) Hospital for Sick Children, University
of Toronto, Developmental and Stem Cell Biology, Research Institute; Department of Surgery, Faculty of Medicine,
Toronto, CANADA
PURPOSE
While rapamycin improves smooth muscle phenotype and gene expression patterns during stretch,
altered matrix, hypoxia and obstruction(Aitken, 2010, Am.J.Path., Schroder, JUrol, 2013, Jiang,
PLOSONE, 2015), the effect of rapamycin on gene expression during de-obstruction(dOB) is
unknown. We hypothesized that rapamycin-induced changes in gene expression during dOB point
to new pathways with functional relevance.
MATERIAL AND METHODS
Sprague-Dawley female rats underwent PBO by tying a silk suture around the proximal urethra and
a 0.9 mm steel rod, leaving only the suture in place. PBO animals were randomized to 6 week PBO
or 6 week PBO plus de-obstruction (dOB). dOB were randomized to vehicle (saline) or rapamycin
for 6 weeks. Shams were performed over 6 and 12 weeks. High-throughput QPCR was performed
to identify genes dysregulated during dOB normalized with rapamycin treatment and correlated with
function. Effects of gene products were examined in human SMC by immunofluorescent staining
of SMC markers. We used transcription factor binding site programs (DAVID, DIRE). Clock gene
dependency of target genes and SMC markers was queried by treating SMC with a reverba agonist
(SR9009) and by performing QPCR and immunofluorescence.
RESULTS
IGFBP7, BMP2, and SOD3 correlated with functional improvements in rapamycin treated animals.
We identified the E-boxes bound by circadian regulators CLOCK/BMAL or NPAS2/BMAL as a po-
tential sites of transcriptional regulation amongst the three genes. We found that obstruction and
dOB upregulated mRNA expression of CLOCK and NPAS2. Rapamycin downregulated mRNA of
CLOCK, NPAS2, alongside the 3 physiologic genes, p<0.01. SR9009 downregulated several core
clock genes alongside IGFBP7, p<0.01. Exogenous IGFBP7 increased SMC hypertrophy in vitro.
CONCLUSIONS
Core clock genes direct expression of the de-obstruction-induced rapamycin-responsive gene
IGFBP7, concordant with bladder smooth muscle cell hypertrophy.