FSH-receptor isoforms and FSH-dependent gene transcription in human monocytes and osteoclasts
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.
Section snippets
Human cells and media
Procedures were approved by the Institutional Review Board. Human peripheral blood monocytes were separated by centrifugation on a density gradient to isolate cells with specific gravity <1.077. Subjects were between the ages of 18 and 40. For selected assays where indicated affinity purified CD14+ cells were isolated by anti-CD14 immuno-magnetic selection with verification of purity by flow cytometry [14]. Osteoclast differentiation in vitro used recombinant human CSF-1 (M-CSF) and RANKL [15].
Western blots for FSH-R in human monocytes and osteoclasts
Western blots for FSH-R were performed using antibodies raised against an internal peptide of the extracellular domain (Fig. 1A) or an N-terminal FSH-R epitope (Fig. 1B). Ovarian controls showed the receptor clearly; much smaller amounts of protein were detected in osteoclasts or monocytic cells. The FSH-R had a lower apparent Mr than receptor in ovarian cells on 7% polyacrylamide. The difference was confirmed with improved resolution of the monocyte receptor using larger amounts of protein and
Discussion
While low abundance receptors are sometimes of minimal importance, the G-protein-coupled receptors are an exception. One receptor may trigger a cellular response [21]. However, analysis of low-abundance targets requires meticulous technique. The conditions indicated, particularly for PCR analysis represent as accurately as possible the buffers, substrate, cDNA production, and settings on the instruments we used. However, it is recommended that, for replication of our experiments, the annealing
Acknowledgments
Supported by National Institutes of Health GrantsAR053976, AR055208, AR053566 and by the Department of Veteran’s Affairs (USA). M.Z. and L.S. also acknowledge National Institutes of Health GrantsAG23176, DK70526, and DK70526. MZ is a named inventor of a pending patent application related to osteoclastic bone resorption filed by the Mount Sinai School of Medicine (MSSM). In the event the pending or issued patent is licensed, he would be entitled to a share of any proceeds MSSM receives from the
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