Expression and localization of estrogen receptor-beta in annulus cells of the human intervertebral disc and the mitogenic effect of 17-beta-estradiol in vitro

Abstract

Background: Recent evidence suggests that estrogens exert effects in different tissues throughout the body, and that the estrogen receptor beta (ERbeta) may be important for the action of estrogen (17-beta-estradiol) on the skeleton. The cellular localization of ERbeta in the human intervertebral disc, however, has not yet been explored.

Methods: Human disc tissue and cultured human disc cells were used for immunocytochemical localization of ERbeta. mRNA was isolated from cultured human disc cells, and RT-PCR amplification of ERbeta was employed to document molecular expression of this receptor. Cultured human disc cells were tested to determine if 17-beta-estradiol stimulated cell proliferation.

Results: In this report data are presented which provide evidence for ERbeta gene expression in human intervertebral disc cells in vivo and in vitro. Culture of annulus cells in the presence of 10-7 M 17-beta-estradiol significantly increased cell proliferation.

Conclusions: These data provide new insight into the biology of cells in the annulus of the intervertebral disc.

Figure 1

Fig. 1A Photomicrograph of disc tissue showing immunolocalization of ERβ in disc cells in the annulus of a cervical disc specimen from a 49 year old female patient. Localization is cytoplasmic. Fig. 1B, negative control. (original magnification × 640).

Figure 2

Fig. 2A: Photomicrograph of immunolocalization of ERβ in cultured disc cells in monolayer culture. Localization is cytoplasmic. Fig. 2B, negative control. (original magnification × 295).

Figure 3

Fig. 3A presents a positive control photomicrograph of a specimen of benign hyperplasia of the prostate; localization is cytoplasmic. Fig. 3B, negative control performed with omission of primary antibody.

Figure 4

RT-PCR amplification of ERβ mRNA in cultured human disc cells from the annulus. Panel A is a photograph of an ethidium bromide-stained agarose gel electrophoresis showing gene expression of ERβ as a single band at the predicted 323 bp size. First lane, base pair controls; mRNA from lumbar disc cells from a 30 year old male (lane 1), mRNA from lumbar disc cells from a 28 year old female (lane 2), mRNA from lumbar disc cells from a 37 year old female (lane 3), mRNA from cells from a thoracic disc from a 35 year old male donor (lane 4). Panel B shows expression of the control HPRT gene in the lanes as described for Panel A (size markers as indicated in Panel A).