Estrogen receptor mRNA is expressed in vivo in rat calvarial periosteum

i"~[U T T E R W O R T H

II1E,N EMAN N

Estrogen receptor mRNA is expressed in vivo in rat calvarial periosteum Kim C. Westerlind, Gobinda Sarkar, Mark E. Bolander, and Russell T. Turner Departments of Orthopedic Research and Biochemistr)" and Molecular Biology, Mayo Foundation, Rochester, Minnesota, USA

Estrogen deficiency is well recognized as a cause of bone loss m rats and humans. Likewise, treatment with estrogen results in prevention of this loss. Initially, this effect was thought to be indirectly mediated but, more recently, estrogen receptors (ER) have been reported in osteosarcoma cells and primary cultures originating from surgical waste, suggesting a direct effect of this steroid hormone. Detection of ER in skeletal tissues, however, has remained elusive. The purpose of this investigation was to establish the efficacy of the highly sensitive reverse-transcription polymerase chain reaction (RT-PCR) technique to detect ER in a well defined skeletal tissue (calvarial periosteum) that is responsive to the hormone. Primers were made specific to rat ER sequences. Total RNA was extracted from rat uterus, liver, spleen, and the periosteum using an organic solvent method, cDNA was synthesized from 2 Ixg total RNA. cDNA corresponding to 40 ng total RNA/sample produced intense PCR products for ER. In descending order of intensi~' were uterus, liver, bone, and spleen. Important(v, a similar time-course for estrogen-induced down-regulation of steady-state rnRNA levels for alkaline phosphatase and osteonectin was observed in calvarial periosteum and tissues known to express estrogen receptors. These data provide in vivo evidence of ER mRNA in bone and suggest that at least some of estrogen's action on bone is directly modulated. (Steroids 60:484-487, 1995)

Keywords: estrogen receptor; mRNA; bone; osteoporosis

Introduction Osteoporosis is a disease characterized by a reduction in bone mass leading to a propensity for atraumatic fractures, i Ovarian hormone deficiency, in animals and humans, is known to result in this bone loss, and the use of estrogen replacement therapy exerts a protective effect on the skeleton. 2 Although these phenomena are well documented, the underlying mechanism for estrogen's action is unclear. Specific high affinity receptors for estrogen have been detected in classic estrosen target organs (uterus, liver, breast) but not in bone/'4 Until recently, the prevailing view was that the effect of estrogen on bone was indirectly mediated. In 1988 Eriksen et al. 5 and Komm et al. 6 detected low levels of estrogen receptors (ER) in cultured human bone cells and rat and human osteosarcoma cell lines, respectively, indicating that bone cell activity may be directly modulated by the hormone. Although these findings are persuasive, the effects of long-term cell culture on expresAddress reprint requests to Kim C. Westerlind, Ph.D., AMC Cancer Research Center, 1600 Pierce Street, Denver, CO 80214, USA. Received September 1, 1994; accepted March 28, 1995.

Steroids 60:484---487, 1995 © 1995 by Elsevier Science Inc.

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sion of ER by bone cells is not established. Further, the effects of the hormone on cells in monoculture have been unimpressive, inconsistent, and difficult to reconcile with the known effects of the hormone on the skeletons of laboratory animals and humans.2 To date, detection of ER in vivo in skeletal tissue has been extraordinarily difficult, in all likelihood, due to the inadequate sensitivity of the various techniques utilized. Polymerase chain reaction (PCR) is a tool that has become increasingly useful in molecular biology research. Using microgram amounts of total RNA reverse-transcribed into cDNA, specific sequences or templates can be amplified and viewed by agarose gel electrophoresis. These signals would otherwise be undetectable by conventional techniques (e.g., Northern analysis, dot blot, hybridization etc.). This technique has been used to detect ER mRNA in reproductive tissues 7 and has been applied to bone research over the last few years. 8"9 The purpose of the present investigation was to attempt to detect the presence of ER mRNA in vivo from calvaria as well as uterus, liver, and spleen using the highly sensitive reverse-transcription polymerase chain reaction (RT-PCR) technique. The calvarial periosteum was chosen because the

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Detection o f estrogen receptor mRNA in bone: Westerlind et al. histology of this osteogenic tissue is simple and well characterized, and estrogen has been shown to regulate the differentiation and activity of the osteoblasts. ~o Experimental

Isolation of RNA Total cellular RNA was isolated from the uterus, liver, spleen, and the periosteurn of the calvaria from 3-month-old intact female rats using a modified organic solvent method.ll The periostea was removed as previously described, to

Reverse transcription Two p.g of total RNA extracted from bone, liver, uterus, and spleen were reverse-transcribed into corresponding complimentary DNA's (cDNA) using: 0.25 U Random primers (BoehringerMannheim, Indianapolis, IN, USA), 0.3 ng Oligo dT-15, and 50 U of AMV reverse transcriptase (Boehringer-Mannheim, Indianapolis, IN, USA). Samples were incubated for 2 h at 42°C in a 20 p.L solution containing: 50 mM Tris-HC1 (pH 8.3), 8 mM MgCI2, 30 mM KC1, 1 mM dithiothreitol, 40 U of RNAsin (Promega, Madison, WI, USA), and 2 mM each dATP, dCTP, dGTP, dTTP.

PCR Amplification of ER message was initially evaluated in rat uterine mRNA with several pairs of primers made against the published sequence of the rat ER (data not shown). 12 Primers were generated with an Applied Biosystems DNA Synthesizer (Foster City, CA, USA). The specific primers which demonstrated the greatest efficiency and least background framed a 517 base pair length nucleotide sequence. The primers were 1) 5' GAGTCCTCAGATGGTGTT 3', starting at nucleotide position 2018 and 2) 5' ACATGTTGCT~ACG 3', starting at nucleotide position 1501. The primers were non-overlapping and 3' to the DNA binding domain. They overlapped in part the putative estrogen binding domain. Following determination of the optimal oligonucleotide primers to utilize, 1 I~L of each cDNA (corresponding to 40 ng total RNA) was denatured at 94°C for 5 min then amplified for 40 cycles of PCR which included denaturation at 94°C for 1 rain, annealing at 50°(2 for 2 min, and extension at 72°C for 2 min in a DNA Thermal Cycler (Perkin-Elmer Cetus, Norwalk, CT, USA). The PCR was carded out in a 20 p,L reaction in the presence of: 2 p,L 10 x PCR buffer [10 x PCR buffer: 500 mM KCI, 100 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2] , 2 I~L 10 x dNTP [10 x dNTP = 2 mM each dATP, dCTP, dGTP, dTFP], 0.5 U Amplitaq (Perkin-Elmer, Norwalk, CT, USA), and 2-10 pmol of specific primer sets. After PCR, the amplified products were electropboresed in a 2.5% agarose gel along with DNA size standard 0X174 digested with HaeIII and stained with ethidium bromide for viewing under UV light.

Northern analysis Membranes with RNA isolated from liver, uteri, and calvaria periosteum of ovariectomized animals were probed for osteonectin [ON 13] and alkaline phosphatase [APt4]. Both genes have sequences homologous to the putative estrogen response element and are expressed in liver (AP), uterus (AP,ON), and bone (AP,ON). Animals (4 rats per time point) were injected with the estrogen diethystilbesterol (DES; 5 p,g in 0.1 mL 0.05% ethanol) or vehicle and sacrificed 1, 2, 3, or 12 h post-administration. One group received DES or vehicle via an implanted controlled release pellet (2.5 mg/3 wk). These animals were sacrificed 2 weeks after implantation. Electrophoresis, transfer, and hybridization techniques are described elsewhere.l°

Results A single band, of expected size - 5 0 0 base pairs, was visualized in each PCR amplified product (Figure 1). Qualitatively, in order of decreasing intensity, the uterus appeared to have the highest concentration of ER m R N A followed by liver, bone, and spleen. This order of intensity was replicated in 3 independent experiments using R N A isolated from different animals. Restriction digestion with Rsal confirmed the presence of 2 bands ( - 4 0 0 and 100 bp) (Figure 2). Estrogen treatment resulted in rapid decreases in steadystate m R N A levels for alkaline phosphatase in bone, liver, and uterus (Figure 3) and osteonectin in bone and uterus (Figure 4). The kinetics of the responses were similar for all tissues.

Discussion Polymerase chain-reaction amplification has been used in other tissues to detect the presence of m R N A species that were undetectable by Northern analysis. The data from the present study indicate the presence of estrogen receptor mRNA in the calvarial periosteum of 3-month-old ovaryintact female rats. The calvarial periosteum was studied because of the prevalence of differentiated bone cells I ° and because the

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Restriction digestion To confirm that the amplified PCR product was the ER sequence, aliquots from PCR products were digested with Rsal (1 cutting site in the PCR amplified template) to cut the segment into a 418 and 99 bp sequence. Digestion was performed according to manufacturers instructions. Intact and digested samples were analyzed by electrophoresis in 3% agarose gel stained by ethidium bromide.

Figure 1 Electrophoretic pattern of PeR amplified products. Ethidium bromide staining of PCR products separated in a 2.5% agarose gel. Lane identification: M = DNA size marker; 0 X174 digested with Haelll; S = spleen; B = bone; U = uterus; L = liver; N = no DNA.

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osteogenic layer can be isolated free from non-bone cell contaminants (e.g., muscle and cartilage). ~5 In this tissue, estrogen has been shown to inhibit bone formation and differentiation of preosteoblasts to osteoblasts, decrease osteoblast number and size, and reduce mRNA levels for bone matrix proteins. 10 Bone resorption and endochondral ossification do not occur at this site. The presence of ER mRNA does not prove that ER protein is present and binding assays lack sufficient sensitivity to detect receptors in bone. 3A6 However, the timing of the skeletal response to estrogen administration supports the receptor hypothesis. We have previously reported that estrogen treatment rapidly regulated ( - 3 h) expression of mRNA levels for skeletalsignaling molecules (e.g., IGF-I). 1° This time-frame is similar to that in which growth factor gene expression is regulated in classic estrogen-responsive tissues (e.g., uterus and liver) which are known to have estrogen receptor protein.ST'~8 In addition, estrogen rapidly alters expression of ~oo

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Figure 3 The effect of estrogen administration on m R N A levels for alkaline phosphatase in calvaria periosteum, liver, and uterus. Five micrograms of diethylstilbestrol or vehicle were administered i.p. or via controlled-release pellets to 3-month-old ovariectomized female rats and groups of 4 were sacrificed at 1, 2, and 12 h or 2 weeks post-administration. Total RNA was isolated from specified tissues and data are expressed as a percentage of vehicle-treated controls. O = uterus, A = liver, • = bone.

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Figure 4 The effect of estrogen administration on m R N A levels for osteonectin in calvaria periosteum and uterus. Five micrograms of diethylstilbestrol or vehicle were administered to 3-month-old ovariectomized female rats and groups of 4 were sacrificed 1, 2, 3, and 12 h post-administration, m R N A data are expressed as percentage of vehicle-treated controls. O = uterus, • = bone.

osteonectin and alkaline phosphatase mRNA in bone, liver, and uterus in a time-frame generally believed to only be possible due to a direct receptor-mediated effect. Thus, the present findings are consistent with but are not sufficient to prove that estrogen has direct ER-mediated effects on bone cells. The mRNA for ER was detected in calvarial periosteum in three independent experiments. The strongest signal for ER mRNA was consistently observed in the amplified PCR product from the uterus. The liver was intermediate in strength and the weakest signal was found in the spleen. These findings are consistent with the relative distribution of ER protein in these tissues as assayed by ligand binding. ~9The mRNA signals for ER in the calvarial periosteum were weaker than those of the liver but much stronger than those from the spleen. We have been able to consistently detect ER mRNA in liver and uterus but not bone and spleen following Northern analysis of total cellular RNA. We have, however, detected ER mRNA from bone by Northern analysis after poly A + selection (data not shown). Although this supports the thesis that ER mRNA is expressed in low levels in skeletal tissues, the required pooling of RNA from approximately 50 rats prevents the practical use of Northern analysis of poly A ÷ selected RNA as a method to study regulation of ER expression in skeletal tissues. We have also detected the displaceable binding of [3H]estradiol to bone cells in birds by autoradiography. 16 However, the method is arduous and non-quantitative. The finding of estrogen receptor mRNA in periosteum supports the prevailing view that bone cell activity may be directly modulated by estrogen. With the RT-PCR technique however, comes the prerequisite need for quantitation. Developing quantitative methods using 13-actin as an internal control, standard curves, and competitive PCR using tissue-generated hybrid templates is currently underway in our laboratory. Refinement of these techniques will allow us and others to examine the regulation and influence of estrogen receptors with age, ovarian status, different disease states, and various treatment regimens.

Detection of estrogen receptor mRNA in bone: Westerlind et al.

Acknowledgments

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This work was supported by NIH grant AR41418 and the Mayo Foundation. KC Westerlind was supported by the NIH Endocrine Training Grant DK07352.

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