CXCR4 is a response gene for parathyroid hormone which affects osteoblast and osteoclast function in vitro

Abstract

Aims: To investigate the role of CXCR4 in response to teriparatide (TPTD) treatment in osteoblasts and osteoclasts.

Methods: Primary murine and human osteoblasts and osteoclasts, MC3T3 cell lines, and hMSC-TERT4 cell lines were treated with TPTD and/or AMD3100, a pharmacological inhibitor of CXCR4. Changes in gene expression, osteoblast viability, mobility, mineralization capacity, and alkaline phosphatase activity were investigated. Osteoclastogenesis and cell fusion were also assessed in response to both treatments.

Results: TPTD increased messenger RNA levels of CXCR4 in all stages of both murine and human osteoblast differentiation. Mineralization analysis showed that CXCR4 was involved in bone matrix formation in response to TPTD. Alkaline phosphatase activity was also impaired by CXCR4 inhibition at early stages of osteoblast differentiation, while it was promoted at late stages, suggesting that CXCR4 could produce a delay in osteoblast maturation. Moreover, we also found a direct activation of osteoclastogenesis after TPTD treatment in murine and human osteoclasts. This process seems to involve CXCR4 activity, since AMD3100-induced CXCR4 inhibition led to a reduction in both murine and human osteoclastogenesis. This process, however, could not be prevented by TPTD treatment.

Conclusion: Our results suggest that CXCR4 is a responsive gene to TPTD treatment, involved in the regulation of osteoblast and osteoclast generation and function. Further in vivo studies are required to confirm this role, and to determine whether pharmacological strategies targeting CXCR4 could potentially improve the treatment outcome for osteoporotic patients.

Fig. 1

CXCR4 expression at RNA level in response to teriparatide (TPTD) treatment in: a) MC3T3 undifferentiated pre-osteoblasts (biological replicates = 4; Kruskal-Wallis test); b) seven days of osteoblast differentiation (n = 3 to 6; Kruskal-Wallis test); and c) three weeks of osteoblast differentiation (biological replicates = 3). Levels are relative to control CXCR4 expression at each timepoint. d) CXCR4 expression at RNA level in response to TPTD treatment in human mesenchymal stem cells during the differentiation process. Housekeeping gene mix refers to the average mean of Ct values of ubiquitin C (UBC), TATA-box binding protein (TBP), and hypoxanthine phosphoribosyltransferase 1 (HPRT1) genes. Values are shown as median (IQR). Biological replicates = 6. One-sample Wilcoxon signed-rank test was used to compare each timepoint to the control levels. Dotted line shows the control fold change. mRNA, messenger RNA.

Fig. 2

Median (IQR) changes in cell viability during murine MC3T3-E1 osteoblast differentiation for two weeks in response to AMD3100 and teriparatide (TPTD) (biological replicates = 3). Kruskal-Wallis non-parametric test was used to calculate the p-value. CNT, control.

Fig. 3

Migration analysis of MC3T3-E1 differentiated osteoblasts in response to teriparatide (TPTD) or AMD3100. a) Visual representation of the migration (remaining wound area represented by yellow, mobility area represented by purple). b) Quantification of the mobility in the different treatments (median % migration (IQR), n = 3, each biological replicate representing the mean of four technical replicates). Statistical test: Kruskal-Wallis with Dunn’s multiple comparisons test (TPTD vs TPTD + AM3100). CNT, control.

Fig. 4

Mineralization rate of MC3T3-E1 osteoblasts at different stages of differentiation in response to teriparatide (TPTD) and AMD3100. a) Alizarin red staining; quantification of mineralizing nodules after treatment b) one week, c) two weeks, and d) three weeks of osteoblast differentiation (median (IQR), biological replicates = 3). P-values calculated with Kruskal-Wallis with Dunn’s multiple comparisons test (TPTD vs TPTD + AM3100). CNT, control.

Fig. 5

Alkaline phosphatase (ALP) activity during osteoblast differentiation in response to teriparatide (TPTD) and AMD3100. a) Representative pictures. b) Quantification of ALP activity after three weeks of osteoblast differentiation (median (IQR), biological replicates = 3). The p-value was calculated with Kruskal-Wallis non-parametric test. CNT, control.

Fig. 6

Effect of teriparatide (TPTD) and AMD3100 on alkaline phosphatase (ALP) activity in primary human osteoblasts from two donors (mean (SD), biological replicates = 6). a) Donor 1 results. b) Donor 2 results. CNT, control.

Fig. 7

Murine osteoclastogenesis in response to teriparatide (TPTD) and AMD3100. a) Tartrate-resistant acid phosphatase (TRAcP) staining for osteoclasts (OC) from young mice (scale bar: 100 µm). Median numbers of osteoclasts from: b) young mice at day 2 of differentiation; c) young mice at day 4 of differentiation; d) aged mice at day 3 of differentiation; and e) aged mice at day 5 of differentiation (error bars represent IQRs, biological replicates = 3; each biological replicate represents the mean of eight technical replicates). P-values were calculated using Kruskal-Wallis with Dunn’s multiple comparisons test (TPTD vs TPTD + AM3100). CNT, control.

Fig. 8

Number of osteoclasts (OCs) in five human subjects after the different treatments at a) three days and b) seven days of differentiation. Each point represents the mean of eight technical replicates. All p-values calculated with paired t-test. Ctrl, control; RANKL, receptor activator of nuclear factor-κB ligand; TPTD, teriparatide.

Fig. 9

Number of nuclei per osteoclast (OC) in five human subjects after the different treatments at a) three days and b) seven days of differentiation. Each point represents the mean of eight technical replicates. All p-values calculated with paired t-test. Ctrl, control; RANKL, receptor activator of nuclear factor-κB ligand; TPTD, teriparatide.