Demonstration of estrogen receptor mRNA in bone using in situ reverse-transcriptase polymerase chain reaction.
Falling estrogen levels affect the female skeleton profoundly. Following menopause, estrogen lack is a major cause of osteoporosis. The site of estrogen action in human bone, however, is unclear, but responsive cells must express the estrogen receptor (ER). One obstacle to localizing these cells is that mRNA for ER is expressed in low copy number. Hence, conventional molecular techniques are either too insensitive to detect receptor transcripts (in situ hybridization) or necessitate amplification of RNA extracted from tissue [Northern analysis and polymerase chain reaction (PCR)], thus failing to identify the specific target cells within the mixed-cell population of bone. In situ PCR (IS-PCR) is a technique that combines the sensitivity of PCR with the localization of conventional in situ hybridization. The technique has previously been used primarily to detect single-copy genes and viral DNA within cells. More recently, incorporation of a reverse-transcriptase reaction (IS-RT-PCR) has allowed the technique to be used to identify rare mRNAs within tissues. We have therefore applied the technique of IS-RT-PCR to localize ER mRNA first in human breast tumors, a known positive tissue, and then in bone. Using conventional riboprobe in situ hybridization, ER transcripts were not detectable in any bone cells within sections taken from normal bone and several actively remodeling bone tissues, namely, Paget's disease, renal hyperparathyroidism, and healing fracture callus. The technique of IS-RT-PCR, however, allowed amplification of transcripts to a detectable level. Following two cycles of amplification, hybridization signal was observed in osteoblasts and to a lower level in osteoclasts and occasional osteocytes. This positive signal was more obvious after five cycles, particularly in osteoclasts and osteocytes. After ten cycles, although signal was increased in osteoclasts and osteocytes, it appeared to be decreased in osteoblasts, suggesting that overamplification leads to loss of target complex from these cells. We conclude that several cell types in human bone express ER mRNA in vivo.
Hoyland
J A
JA
Department of Osteoarticular Pathology, University of Manchester, UK. jhoyland@fs2.scg.man.ac.uk
Mee
A P
AP
Baird
P
P
Braidman
I P
IP
Mawer
E B
EB
Freemont
A J
AJ
D001853:Bone Marrow / Q000737:chemistry; D001842:Bone and Bones / Q000737:chemistry*; D001943:Breast Neoplasms / Q000737:chemistry; D006801:Humans; D017403:In Situ Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D011960:Receptors, Estrogen / Q000235:genetics*; D012016:Reference Values; D014158:Transcription, Genetic
D012333:RNA, Messenger; D011960:Receptors, Estrogen