Blocking FSH action attenuates osteoclastogenesis

Abstract

A direct effect of FSH on bone turnover via stimulation of osteoclast formation has been reported. Here we show that monoclonal or polyclonal antibodies to FSH inhibit osteoclast formation induced by FSH to an extent similar to that noted in FSH receptor (FSHR) knockout cells. Furthermore, we document the amplification of FSHR cDNA from well-characterized human CD14+ osteoclast precursors and osteoclasts, and the direct sequencing of the PCR products to definitively establish the expression of FSHRs. At these sites, the FSHR was expressed predominantly as an isoform that omits exon 9, a linker between the FSH-binding region and a long, invariant signaling domain of the receptor. These data provide compelling evidence for expression of a FSH receptor isoform in osteoclasts and their precursors.

Figure 1. FSH increases osteoclastic differentiation from mouse marrow cells, but the effect is abrogated by neutralizing antibodies

A. Blocking antibodies for FSH reverse the effect of FSH on osteoclast formation. Bone marrow cells from 6 month old mice were isolated and osteoclast differentiation was induced with murine RANKL and murine CSF-1 [1]. This increased osteoclast formation by ~20%; duplicate experiments are shown. In the first experiment monoclonal anti-FSHβ was added in excess and this eliminated eliminating the effect of FSH (p = 0.001). In the second experiment polyclonal anti-FSHβ also returned osteoclast formation to background (p = 0.003). B. The effect of FSHR deletion on osteoclast formation is shown. Multinucleated TRAP-expressing osteoclasts 5-days after RANK-L treatment were attenuated in FSHR^−/− cultures compared with FSHR^+/− or wild type (p < 0.001 FSHR^−/− relative to WT littermates). The size of the effect was similar to that seen when FSH is added during osteoclast formation in wild type cells (A). C. Photomicrographs of multinucleated TRAP-expressing cells in wild type, FSHR^+/−, and FSHR^−/− marrow cell cultures with 30 ng/ml of FSH during differentiation in RANKL and CSF-1. The knockout cells produce fewer multinucleated cells.

Figure 2. An alternatively spliced FSH receptor transcript is expressed in human osteoclasts, CD14+ monocytes, and CD14-depleted monocytic cells

(A) FSHR isoform 1 is expressed in the ovary, but a truncated form is the main form found in monocytes and osteoclasts. Primer set 1 (Methods), amplifying across exon 9, shows the full length FSHR fragment from distal exon 8 to early exon 10, and the smaller isoform missing exon 9. The smaller, type 2 isoform, 140 bp, is barely visible in ovary, but is the major product in osteoclasts made from CD14 cells with 14 day incubation in RANKL and CSF-1. Exon 9 is a short extracellular exon just distal to the FSH-binding sequence and proximal to the invariant transmembrane signaling region, exon 10. Results from three reactions in a temperature gradient PCR are shown. Further reactions were annealed at 54°C. (B) FSHR isoform 2 with exon 9 omitted. Primer set 2, the forward primer of which extends across the exon 8–10 boundary, was used, and the products of 30 cycles of amplification were then re-amplified with an internal nested primer set (131 bp) for a further 20 cycles. Transcript of this isoform was seen in fractions of peripheral blood mononuclear cells (unselected, CD14-selected, CD14-depleted, and osteoclasts from CD14 cells), and ovarian control (COV) cells. Presence of the smaller isoform, at low levels, in ovarian cells was previously described (see text).

Figure 3. Sequence confirmation of an alternatively spliced FSH receptor transcript

(A) (i) Full length FSHR transcript noted in osteoclasts. Bands at 320 bp from agarose gels (A) were excised, re-amplified 10 cycles, and (ii) direct sequencing of the PCR products was performed. Sequence of the FSHR from osteoclasts using the forward primer, extending across the exon 9–10 boundary, is shown in color below the reference genomic sequence. The ovarian product also matched the reference sequence (not shown). The osteoclast sequence had several unresolved bases (pink) and two mismatches (underlined), which are probably misreads due to low level signal, but might also be amplification errors. Bases in yellow are primer pair 1. (B) The FSHR isoform 2, missing exon 9, is expressed in osteoclasts and CD14 cells. Bands at 140 bp from agarose gels (i) were excised, re-amplified using primer set 2 for 10 cycles, and direct sequencing of the products was performed (ii). Sequence from the reverse primer, extending across the exon 8–10 boundary, is shown in color below the reference genomic sequence, for both osteoclasts (top color sequence) and CD14 cell (lower color sequence) amplicons. Bases in yellow are primer pair 2. The reference sequence was also obtained from control ovarian cells, and in the PCR product amplified from CD14 depleted cells (neither shown). As in (A), unresolved bases are shown in pink and misreads are underlined.