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ER Gene Expression in Human Primary Osteoblasts: Evidence for the Expression of Two Receptor Proteins

Stefanie Denger, George Reid, Martin Ko, Gilles Flouriot, Dominik Parsch, Heike Brand, Kenneth S. Korach, Vera Sonntag-Buck and Frank Gannon

European Molecular Biology Laboratory (S.D., G.R., M.K., H.B., V.S.-B., F.G.), 69117 Heidelberg, Germany; Endocrinologie Moléculaire de la Reproduction (G.F.), UPRES-A CNRS 6026, Campus de Beaulieu, 35042 Rennes cedex, France; Department of Orthopedics (D.P.), University of Heidelberg, 69118 Heidelberg, Germany; and National Institute of Environmental Health Sciences (K.S.K.), Research Triangle Park, North Carolina 27709

Address all correspondence and requests for reprints to: Stefanie Denger, EMBL, Meyerhofstrasse 1, 69012 Heidelberg, Germany. E-mail: denger{at}embl-heidelberg.de

The beneficial influence of E2 in the maintenance of healthy bone is well recognized. However, the way in which the actions of this hormone are mediated is less clearly understood. Western blot analysis of ER in osteoblasts clearly demonstrated that the well characterized 66-kDa ER was only one of the ER isoforms present. Here we describe a 46-kDa isoform of ER, expressed at a level similar to the 66-kDa isoform, that is also present in human primary osteoblasts. This shorter isoform is generated by alternative splicing of an ER gene product, which results in exon 1 being skipped with a start codon in exon 2 used to initiate translation of the protein. Consequently, the transactivation domain AF-1 of this ER isoform is absent. Functional analysis revealed that human (h)ER46 is able to heterodimerize with the full-length ER and also with ERß. Further, a DNA-binding complex that corresponds to hER46 is detectable in human osteoblasts. We have shown that hER46 is a strong inhibitor of hER66 when they are coexpressed in the human osteosarcoma cell line SaOs. As a functional consequence, proliferation of the transfected cells is inhibited when increasing amounts of hER46 are cotransfected with hER66. In addition to human bone, the expression of the alternatively spliced ER mRNA variant is also detectable in bone of ER knockout mice.

These data suggest that, in osteoblasts, E2 can act in part through an ER isoform that is markedly different from the 66-kDa receptor. The expression of two ER protein isoforms may account, in part, for the differential action that estrogens and estrogen analogs have in different tissues. In particular, the current models of the action of estrogens should be reevaluated to take account of the presence of at least two ER protein isoforms in bone and perhaps in other tissues.

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