Transforming growth factor-beta1 (TGF-beta1) induces human osteoclast apoptosis by up-regulating Bim

Abstract

Transforming growth factor-beta1 (TGF-beta1) is the most abundant TGF-beta isoform detected in bone and is an important functional modulator of osteoclasts. TGF-beta1 can induce osteoclast apoptosis; however, the apoptotic pathways involved in this process are not known. We show here that human osteoclasts express both type-I and type-II TGF-beta receptors. In the absence of survival factors, TGF-beta1 (1 ng/ml) induced osteoclast apoptosis. The expression of activated caspase-9, but not that of caspase-8, was increased by TGF-beta1 stimulation, and the rate of TGF-beta1-induced apoptosis was significantly lower in the presence of a caspase-9 inhibitor. To study further the mechanisms involved in TGF-beta1-induced osteoclast apoptosis, we investigated TGF-beta1 signaling, which primarily involves the Smad pathway, but also other pathways that may interfere with intracellular modulators of apoptosis, such as mitogen-activated protein (MAP) kinases and Bcl2 family members. We show here that early events consisted of a trend toward increased expression of extracellular signal-regulated kinase (ERK), and then TGF-beta1 significantly induced the activation of p38 and Smad2 in a time-dependent manner. These signaling cascades may activate the intrinsic apoptosis pathway, which involves Bim, the expression of which was increased in the presence of TGF-beta1. Furthermore, the rate of TGF-beta1-induced osteoclast apoptosis was lower when Bim expression was suppressed, and inhibiting the Smad pathway abolished Bim up-regulation following TGF-beta stimulation. This could correspond to a regulatory mechanism involved in the inhibition of osteoclast activity by TGF-beta1.

FIGURE 1.

TGF-β receptor expression and TGF-β-induced apoptosis in human osteoclasts. A, at the end of the cultures, the expression of the TGF-β-RI and -RII receptors was evaluated by immunocytochemistry using specific antibodies. Polyclonal goat IgGs were used as negative controls. B, the percentage of labeled cells was determined for each TGF-β R type. Results are expressed as the percentage of labeled MNCs over total MNCs (mean ± S.E.), and three independent experiments were conducted. C, at the end of the CBM cultures, after the M-CSF and RANKL had been removed, different concentrations of TGF-β1 were added 24 h before the apoptosis was determined (0.1–10 ng/ml). The same experiments were performed in the presence of RANKL 100 ng/ml. Staurosporine (1 μm for 3 h) was used as a positive control of apoptosis induction. Cells with three or more nuclei were counted, and results of five independent experiments are expressed as the percentage of apoptotic MNCs over the total population of MNCs, mean ± S.E. **, p < 0.01; ***, p < 0.001, treated versus untreated. D, to quantify the number of apoptotic osteoclasts, TACS Blue labeling was used, resulting in blue staining of the nuclei of the apoptotic cells (positive multinucleated cells are delineated by black arrows, whereas non-apoptotic nuclei were stained pink/red (empty arrows) with a Nuclear Fast Red counterstain).

FIGURE 2.

Caspase activation in response to TGF-β stimulation. At the end of the CBM cultures, osteoclasts were cultured in a serum-reduced medium and deprived of RANKL and M-CSF for 24 h. They were then either treated or not with TGF-β1 (1 ng/ml) for 3, 6, 8, or 24 h or with staurosporine (1 μm) for 3 h as a positive control for apoptosis. A, Western blot analysis revealed the presence of the full-length caspase-8 (57 kDa) and of two fragments (43/45 kDa), corresponding to the cleaved caspase-8. The full-length caspase-9 appears at 47 kDa, and the two cleaved fragments appear at 35/37 kDa. B, immunocytochemistry was performed using antibodies specifically directed against the cleaved caspase-8 or the cleaved caspase-9 or normal serum as a negative control. Positive cells are indicated by black arrows, and negative cells are indicated by white arrows. NT, untreated. C, the expression of cleaved caspase-8 and caspase-9 was evaluated in these cultures in the presence of TGF-β1 (1 ng/ml) or staurosporine (1 μm). Results are expressed as the percentage of labeled MNCs from five different experiments (mean ± S.E.). D, caspase inhibitors (caspase-8 inhibitor Z-IETD, 10 μm, and caspase-9 inhibitor Z-LEHD 10 μm) were added 1 h before TGF-β1 stimulation, and osteoclast apoptosis was evaluated after 24 h using a terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL)-derived method. Staurosporine (1 μm) was used as the apoptotic inducer control. Results are expressed as the percentage of apoptotic MNCs over total MNCs (mean ± S.E.). Eight independent experiments were conducted. *, p < 0.05; **, p < 0.01, versus untreated. DMSO, dimethyl sulfoxide.

FIGURE 3.

TGF-β1 activates signaling pathway components. At the end of the cultures, mature osteoclasts were treated with 1 ng/ml TGF-β1 for the times indicated. Then cells were rinsed on ice with cold phosphate-buffered saline, extracts were harvested, and 50 μg of protein were analyzed by Western blotting for the indicated phosphorylated (p) or total protein. We analyzed the band intensities using NIH ImageJ software, and the results are presented on each graph on the right of Western blot. Western blots of JNK (A), ERK (B), p38 (C), and Smad2 (D) are shown. The phosphorylation results are normalized for the total protein intensity and presented as relative phosphorylation versus the untreated cells (mean ± S.E.) for five different experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.005, treated versus untreated.

FIGURE 4.

Expression of Bcl-2 homologs after TGF-β stimulation. At the end of the CBM cultures, mature osteoclasts were treated with TGF-β1 at 1 ng/ml for the time indicated. A, expression of four Bcl-2 homologs (Bcl-2, Bid, Bim, and Bax) was analyzed by Western blot. B, the intensity of the bands was analyzed using NIH ImageJ Software for four different experiments, protein expressions are normalized relative to the respective actin intensity, and the results are expressed as the expression relative to that of the untreated cells (0 h) (mean ± S.E.) (*, p < 0.05; ***, p < 0.005, treated versus untreated).

FIGURE 5.

Effect of blocking Bim expression on TGF-β-induced caspase-9 activation and apoptosis in human osteoclasts. At the end of the culture, the cells were transfected with Bim siRNA 48 h before stimulation. 24 h before stimulation, the cells were placed in medium-reduced serum and deprived of RANKL and M-CSF. Cells were then stimulated with TGF-β1 at a concentration of 1 ng/ml for the times indicated. A, using Western blot (WB), the transfection of Bim siRNA was controlled by measuring Bim expression in wild type (WT) in unsilencing control (CTL) or Bim siRNA-transfected cells. B, the expression of cleaved caspase-9 was determined by immunocytochemistry in wild type and Bim siRNA-transfected cells after exposure to different TGF-β1 stimulation times (1 ng/ml). The results are expressed as the percentage of positive cleaved caspase-9 MNCs under each experimental condition for four different experiments. C, TGF-β1-induced apoptosis was evaluated in cells that had or had not been transfected with Bim siRNA and were then either treated or not with TGF-β1 (1 ng/ml) for 24 h or with staurosporine (1 μm) for 3 h. Results are expressed as the percentage of apoptotic MNCs over total MNCs (mean ± S.E.). Six independent experiments were conducted. **, p < 0.01; ***, p < 0.001; treated versus untreated (NT). D, expression of Bim was studied by Western blot in cells transfected by unsilencing control siRNA, Smad2, siRNA, or Bim siRNA in control experiments 48 h before stimulation with TGF-β1 (1 ng/ml) for 6 h as described above. Results are expressed as the expression relative to that of the untreated cells (three experiments). *, p < 0.05; treated versus untreated, °°, p < 0.01 versus NT wild type.