Mechanisms of the regulation of EGF receptor gene expression by calcitriol and parathyroid hormone in UMR 106-01 cells.
BACKGROUND
We have previously demonstrated that parathyroid hormone (PTH) and calcitriol increase the expression of epidermal growth factor receptors (EGFR) in UMR 106-01 osteoblast-like cells. The effect of PTH is mediated by cAMP and it involves an increase in the level of EGFR mRNA. The present studies were designed to investigate the mechanisms involved in the regulation of EGFR expression by PTH and calcitriol.


METHODS
To examine the mechanism of the effect of calcitriol on EGFR expression, confluent cultures of UMR 106-01 cells were exposed to calcitriol and levels of EGFR mRNA were determined by reverse transcription-polymerase chain reaction (RT-PCR). In order to study the effect of calcitriol on EGFR gene transcription, a candidate vitamin D-responsive element (VDRE) was identified in the EGFR gene promoter and complimentary 30-mer oligonucleotides spanning this region were tested for binding to recombinant VDR using EMSA. Transcriptional activity in response to calcitriol and PTH was tested in UMR 106-01 cells stably transfected with a luciferase reporter construct containing the full length EGFR gene promoter. The effect of calcitriol on EGFR mRNA stability was examined in transcriptionally arrested cells.


RESULTS
Treatment with calcitriol resulted in a time and dose dependent increase in EGFR mRNA levels in confluent cultures of UMR 106-01 osteoblast-like cells. Using EMSA, we demonstrated that the putative human EGFR VDRE binds to recombinant VDR in a retinoid X receptor (RXR)-dependent manner; however, calcitriol failed to increase transcriptional activity from a luciferase reporter construct containing the full-length EGFR gene promoter in stably transfected UMR 106-01 cells. Therefore, EGFR mRNA degradation was examined in transcriptionally arrested cells and calcitriol was found to prolong the half life of EGFR mRNA. Treatment of the cultures with PTH resulted in a ninefold increase in luciferase activity after four hours of exposure, a finding that was reproduced by treatment with forskolin.


CONCLUSIONS
These studies demonstrate that the calciotropic hormones PTH and calcitriol increase EGF receptor expression by different mechanisms. The former increases EGFR gene transcription whereas the latter increases EGFR mRNA stability.
González
Esther A
EA
Division of Nephrology, Saint Louis University School of Medicine, St. Louis, Missouri 63110, USA. gonzalea@slu.edu
Disthabanchong
Sinee
S
Kowalewski
Rodney
R
Martin
Kevin J
KJ
D002117:Calcitriol / Q000494:pharmacology*; D002120:Calcium Channel Agonists / Q000494:pharmacology*; D002460:Cell Line; D066246:ErbB Receptors / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010446:Peptide Fragments / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
D002120:Calcium Channel Agonists; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D012333:RNA, Messenger; D066246:ErbB Receptors; D002117:Calcitriol