FSH-receptor isoforms and FSH-dependent gene transcription in human monocytes and osteoclasts

Abstract

Cells of the monocyte series respond to follicle stimulating hormone (FSH) by poorly characterized mechanisms. We studied FSH-receptors (FSH-R) and FSH response in nontransformed human monocytes and in osteoclasts differentiated from these cells. Western blot and PCR confirmed FSH-R expression on monocytes or osteoclasts, although at low levels relative to ovarian controls. Monocyte and osteoclast FSH-Rs differed from FSH-R from ovarian cells, reflecting variable splicing in exons 8–10. Monocytes produced no cAMP, the major signal in ovarian cells, in response to FSH. However, monocytes and osteoclasts transcribed TNFα in response to the FSH. No relation of expression of osteoclast FSH-R to the sex of cell donors or to exposure to sex hormones was apparent. Controls for FSH purity and endotoxin contamination were negative. Unamplified cRNA screening in adherent CD14 cells after 2 h in 25 ng/ml FSH showed increased transcription of RANKL signalling proteins. Transcription of key proteins that stimulate bone turnover, TNFα and TSG-6, increased 2- to 3-fold after FSH treatment. Smaller but significant changes occurred in transcripts of selected signalling, adhesion, and cytoskeletal proteins. We conclude that monocyte and osteoclast FSH response diverges from that of ovarian cells, reflecting, at least in part, varying FSH-R isoforms.

Introduction

Receptors for pituitary glycoprotein hormones that mediate endocrine response were once believed to be limited to endocrine glands. This is altered by discoveries including thyrotropin receptors (TSH-R) in mesenchymal stem cells that modulate bone turnover [1], [2]. Our studies showed a direct effect of FSH on bone turnover [3]. Studies in murine marrow suggest that this is related to TNFα production by immune cells [4], but the response in human osteoclasts and their precursors to FSH has not been evaluated, beyond that the FSH increases the differentiation of osteoclasts in vitro[3]. Indeed, whether the osteoclast or its precursor are the responsive cells remains uncertain. Precedents for FSH response in macrophages include that FSH-dependent lactate and cAMP production in rat macrophages [5]. Skatchard analysis in these cells was consistent with FSH-R binding [6]. However, the investigators reporting these results were unable to amplify FSH-R by PCR [7].

The FSH receptor is a rhodopsin-like G protein-coupled receptor of ∼60 kD. Glycoprotein hormone receptors act through Gαs (cAMP/cAMP-dependent protein kinase) or Gαi/o pathways [8] both of which occur in osteoclasts [9], [10]. FSH-R has a conserved seven-transmembrane domain structure and an ∼40 kDa extracellular hormone binding domain. There are many precedents for splice variants of the FSH-R [11], [12], [13], which in the ovary has 10 exons, nine encoding the extracellular domain and a large 10th exon with the conserved seven-transmembrane domain. FSH-R variants might signal by alternative mechanisms, including FSH-R omitting the last extracellular domain (type 2) that may represent a developmental form of the FSH-R [13], and forms with a single transmembrane domain or missing a portion of the C-terminal [11], [12], although physiological functions of these alternative transcripts are not clear.

We used affinity isolated CD14 human monocytes and osteoclasts produced in vitro from monocytes to evaluate the presence and activity of FSH-R under conditions with high sensitivity and specificity. Response of cells to FSH at short time points was studied by unamplified cRNA screening to avoid subjective judgements or observer bias. We find that human monocytes and osteoclasts express the FSH receptor at low levels. Receptor activity caused no detectable cAMP production. Several proteins, including TNFα and TSG-6, were transcribed after FSH stimulus. Small but significant changes were consistent with increased osteoclast differentiation and activity in response to FSH.