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Rescue of High Glucose Impairment of Cultured Human Osteoblasts Using Cinacalcet and Parathyroid Hormone - PubMed

Rescue of High Glucose Impairment of Cultured Human Osteoblasts Using Cinacalcet and Parathyroid Hormone

V A Shahen et al. Calcif Tissue Int. 2023 Apr.

Abstract

Patients with type 2 diabetes mellitus (T2DM) experience a higher risk of fractures despite paradoxically exhibiting normal to high bone mineral density (BMD). This has drawn into question the applicability to T2DM of conventional fracture reduction treatments that aim to retain BMD. In a primary human osteoblast culture system, high glucose levels (25 mM) impaired cell proliferation and matrix mineralization compared to physiological glucose levels (5 mM). Treatment with parathyroid hormone (PTH, 10 nM), a bone anabolic agent, and cinacalcet (CN, 1 µM), a calcimimetic able to target the Ca2+-sensing receptor (CaSR), were tested for their effects on proliferation and differentiation. Strikingly, CN+PTH co-treatment was shown to promote cell growth and matrix mineralization under both physiological and high glucose conditions. CN+PTH reduced apoptosis by 0.9-fold/0.4-fold as measured by Caspase-3 activity assay, increased alkaline phosphatase (ALP) expression by 1.5-fold/twofold, increased the ratio of nuclear factor κ-B ligand (RANKL) to osteoprotegerin (OPG) by 2.1-fold/1.6-fold, and increased CaSR expression by 1.7-fold/4.6-fold (physiological glucose/high glucose). Collectively, these findings indicate a potential for CN+PTH combination therapy as a method to ameliorate the negative impact of chronic high blood glucose on bone remodeling.

Keywords: Cinacalcet; Diabetes; Osteoblasts; PTH; Parathyroid hormone.

© 2023. The Author(s).

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Conflict of interest statement

All authors have no conflicts of interest.

Figures

Fig. 1
Fig. 1

A Phase contrast microscopy of primary human osteoblasts (HOBs) in monolayer culture at 10X. B Confocal microscopy images of a monolayer culture of primary human osteoblasts taken with × 10 objective (scale bar = 10 μm) stained for CaSR (green) or Homer (red). Nuclei (blue) were stained with DAPI

Fig. 2
Fig. 2

Osteoblastic proliferation and cell number. The phase object confluency (%) of primary human osteoblasts cultured in physiological (5 mM) or high (25 mM) glucose conditions and treated with a vehicle (1 µL DMSO/mL), cinacalcet (CN, 1 µM), parathyroid hormone (PTH, 10 nM), or CN+PTH for 7 days. A The time required for primary human osteoblasts to double their initial confluency. B Osteoblast number after 7 days of treatments. Data are presented as mean fold change from vehicle ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 3
Fig. 3

Caspase-3 and alkaline phosphatase (ALP) activity assays. Osteoblasts were treated with vehicle, CN, PTH, or CN+PTH as previously described under physiological or high glucose conditions. A Caspase-3 activity and B ALP activity in cells after 7 days of treatment. Data are presented as mean fold change from vehicle ± SEM. Note that vehicle is 5 mM (physiological) glucose. *p < 0.05 and ***p < 0.001

Fig. 4
Fig. 4

Matrix mineralization in CN, PTH, and CN+PTH-treated human osteoblasts. Osteoblasts were treated with vehicle, CN, PTH, or CN+PTH as previously described under physiological or high glucose conditions for 7 days. A Alizarin Red S staining at day 7 of treatment B Elution of stain showing quantitative Ca.2+ deposition levels. Data are presented as mean fold change from vehicle ± SEM. *p < 0.05 and ***p < 0.001

Fig. 5
Fig. 5

The expression levels of RUNX2 and OCN. Gene expression from immunoblots was quantified for A RUNX2 and B OCN protein expression under different glucose conditions and treatment with vehicle, CN, PTH, and CN+PTH. In panels, blots are shown above, with quantitation below as mean-fold change from physiological glucose vehicle ± SEM after normalization to the expression of the housekeeping gene, β-tubulin. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6
Fig. 6

The expression levels of RANKL and OPG. Gene expression from immunoblots was quantified for A RANKL and B OPG protein expression under different glucose conditions and treatment with vehicle, CN, PTH, and CN+PTH. In panels, blots are shown above, with quantitation below as mean-fold change from vehicle ± SEM after normalization to the expression of the housekeeping gene, β-tubulin. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 7
Fig. 7

The expression levels of CaSR. Gene expression from immunoblots was quantified for CaSR under different glucose conditions and treatment with vehicle, CN, PTH, and CN+PTH. In panels, blots are shown above, with quantitation below as mean-fold change from vehicle ± SEM after normalization to the expression of the housekeeping gene, β-tubulin. ***p < 0.001

Fig. 8
Fig. 8

The effects of cinacalcet (CN) and parathyroid hormone (PTH) on osteoblasts. CN acts on and increases the sensitivity of the Ca2+ sensing receptor (CaSR) to Ca2+. Ca2+ activates the CaSR and PTH activates the PTH receptor (PTH-R). The activation of the receptors leads to several transcriptional effects. Firstly, receptor activation upregulates runt-related transcription factor 2 (RUNX2). This increases the expression of osteocalcin (OCN) and enhances alkaline phosphatase (ALP) activity, which are hallmarks of osteoblastic activity. The increase in activity then results in enhanced mineralization and hence bone formation. RUNX2 also stimulates differentiation and maturation and inhibits apoptosis by reducing caspase-3 activity. This stimulates osteoblastic proliferation, leading to more cells. Secondly, receptor activation upregulates receptor activator of nuclear factor κ-B ligand (RANKL). This increases the ratio of RANKL to its inhibitor, osteoprotegerin (OPG), which can then stimulate osteoclastic activity and bone resorption. Finally, activating the CaSR and PTH-R also leads to the upregulation of the CaSR, creating a positive feedback loop

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