Follicle stimulating hormone receptor in mesenchymal stem cells integrates effects of glycoprotein reproductive hormones

Abstract

Previously we reported that follicle stimulating hormone (FSH) affects bone degradation in human cells and in follicle stimulating hormone receptor (FSH-R) null mice. Here we describe a FSH-R knockout bone-formation phenotype. We used mesenchymal stem cells (MSCs), osteoblast precursors that express FSH-R, to determine whether FSH regulates bone formation. FSH stimulates MSC cell adhesion 1-3 h and proliferation at 24 h after addition. On the basis of phylogenetic and clinical precedents, we also examined effects of pregnant levels of human chorionic gonadotropin (hCG) on MSCs. We found effects similar to those of FSH, and RNAi knockdown of FSH-R abrogated both FSH and hCG effects on MSCs. In contrast to effects on MSCs, neither FSH nor hCG had significant effects on osteoblast maturation. Also in MSCs, short-term treatment by FSH and hCG altered signaling pathways for proliferation, including Erk1/2 phosphorylation. Our results show augmentation of MSC proliferation by either FSH at menopausal levels or hCG at normal pregnant levels. We conclude that FSH-R participates in regulation of MSC precursor pools in response to either FSH or hCG, integrating the effects of these two glycoprotein hormones.

Keywords: Erk1/2; FSH; hCG; osteoblast; osteoporosis.

Figure 1. FSH-R–mediated effects on bone formation and bone degradation

(A). Whole vertebral cross section of the Fshr^−/− mouse calcein labeled 5 and 1 d before sacrifice. In these 3 mm fields, the wild-type mouse (left) has typical short regions of labeling, while the knockout (right) shows a unique pattern with long regions of active bone formation. (B). Hematoxylin-stained cross sections at 20× (fields are 600 µm across) show typical variability in the wild-type (left), but uniform and smooth swiss cheese–like trabecular bone in the Fshr^−/−. (C). A much greater proportion of bone was double labeled in the FSH-R^−/− animal (p < 0.01). (D). Total calcein label was unchanged. (E). Linear cross-sectional length of calcein labeling was increased in the FSH-R^−/− animal (p < 0.01), reflecting the size of bone forming units. (F). Trabecular thickness was increased in the Fshr^−/− consistent with previous work, but had marginal statistical difference (p = 0.06) in this case.

Figure 2. Effects of FSH (25 ng/ml) and hCG (1000 ng/ml) on human MSCs adhesion and proliferation

(A). Adhesion of MSCs in MTT assays at 30 min - 3h. At each time point, mean ± SD is shown, n = 4; ** indicates p < 0.01. (B). Effects on proliferation. After plating MSCs for 4, 12, or 24 h cell proliferation was measured using MTT. Mean ± SD, n = 4; ** indicates p < 0.01. (C). Live images of control and treated MSCs at 24 h time point. (D). Mitotic events in MSCs treated with FSH and hCG. Events were counted from phase images by observers blinded to conditions studied. Mean ±SD, n = 12; * p < 0.05 relative to the control.

Figure 3. Effects of MSCs transfection with FSH-R RNAi on FSH-R mRNA expression, protein expression, and on cell proliferation

(A). FSH-R mRNA in MSCs after 24 h of transfection. The band at 320 bp is the type 1 isoform; the band at 140 bp is the type 2 isoform. (B). FSH-R protein in MSCs. Immunoblotting of lysates after 48 h of transfection. (C). Proliferative response of MSCs to FSH or hCG after transfection with FSH-R RNAi. MSCs were transfected with FSH-R RNAi or scrambled control RNAi for 48 h followed by 12 h treatment with FSH or hCG. Proliferation was measured by MTT assay (** p < 0.01 relative to scrambled control RNAi; n = 4, mean ± SD).

Figure 4. Osteoblast differentiation with and without FSH or hCG

(A–B). Alkaline phosphatase activity in MSCs cultured in bone differentiation medium, B, relative to growth medium, A. (C). Mineral deposition (black) demonstrated by optical density in transmitted light. One of two duplicate cultures with similar results is shown. Fields are 0.5 cm across. (D–H). QPCR analysis for osteoblast specific gene expression in MSCs treated with FSH or hCG. All assays were normalized to GAPDH. One of three experiments with similar findings is shown.

Figure 5. Erk1/2 phosphorylation with and without FSH or hCG treatment in growing MSCs

(A). Immunoblotting of lysates of MSCs treated with FSH or hCG for 10 min. (B). c-Myc and p27 protein expression in MSCs after addition of FSH or hCG for 24 h. One of three blots with similar findings is shown. (C–D). Effects of MSCs transfection with FSH-R RNAi on Erk1/2 phosphorylation. MSCs were transfected with FSH-R RNAi or control scrambled RNAi for 48 h followed by 10 min treatment with FSH or hCG. One of two blots with similar findings is shown.