Effect of fluoride on osteocyte-driven osteoclastic differentiation.

Excessive systemic uptake of inorganic fluorides causes disturbances of bone homeostasis. The mechanism of skeletal fluorosis is still uncertain. This study aimed to study the effect of fluoride on osteocyte-driven osteoclastogenesis and probe into the role of PTH in this process. IDG-SW3 cells seeded in collagen-coated constructs were developed into osteocyte-like cells through induction of mineral agents. Then, osteocyte-like cells were exposed to fluoride in the presence or absence of parathyroid hormone (PTH). Cell viability and their capacity to produce receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG) and sclerostin (SOST) were detected by MTT and Western blot assays, respectively. Finally, a transwell coculture system using osteocyte-like cells seeded in the low compartment, and osteoclast precursors added in the inserts was developed to observe the osteocyte-driven osteoclasogenesis response to fluoride with or without PTH, and the expression of molecules involved in this mechanism were measure by real time RT-PCR. Results showed that osteocytes withstood a toxic dose of fluoride, and yet PTH administration significantly reduced osteocytes viability. PTH amplified the effect of fluoride on the expression of osteoclastogenesis-related molecules in osteocyte, but did not enlarged the stimulating effect of fluoride on osteoclastogenesis drove by osteocyte coculture. Gene expression levels of TRAP, RANK, JNK and NFAtc1 significantly increased in fluoride affected osteoclast precursor cocultured with osteocyte-like cells. The impact of fluoride on osteocyte-driven osteoclast differentiation was stronger than that of PTH. In conclusion, osteocyte played a pivotal role on the mechanism underlying fluoride-affected osteoclastogenesis in which RANK-JNK-NFATc1 signaling pathway was involved, and PTH had a significant impact in this process.

Different Effects of Fluoride Exposure on the Three Major Bone Cell Types.

Fluoride accumulates and is toxic to bones. Clinical bone lesions occur in a phased manner, being less severe early in the natural course of skeletal fluorosis. Previous research rarely focused on osteocyte, osteoclast, and osteoblast at the same time, although these three types of cells are involved in the process of fluorosis. In this study, commitment of bone cells was performed according to their respective characteristics. Osteocyte-like cells were verified by protein expression of sclerostin (SOST) in IDG-SW3 cell culture with mineral medium. Positive tartrate-resistant acid phosphatase (TRACP) staining, characteristic of osteoclasts, is observed in RAW264.7 cells after administration of RANKL. We successfully purified a high percentage (94%) of bone mesenchymal stem cells (BMSCs) co-expressing CD34 and CD44. Parallel studies were performed to observe cell viability and apoptosis rates in osteocyte, osteoclast, and osteoblast like cells by using MTT and Annexin V FITC assays. Our results demonstrated that osteocytes have a strong tolerance to high fluoride concentrations, while osteoclasts are more sensitive to changes of fluoride dose. The range of anabolic action of fluoride concentration on osteoblast was narrow. Notably, fluoride exposure aggravated apoptosis of osteocyte and osteoclast induced by administration of PTH and TGF-ß, respectively. In short, three types of bone cells display disparate responses to fluoride exposure and to PTH- and TGF-ß-induced apoptosis.

The antiresoptive effects of recombinant Lingzhi-8 protein against retinoic acid-induced osteopenia.

The antiresorptive agents still are the mainstay of osteoporosis treatment. This study aimed to investigate the efficacy of recombinant Lingzhi-8 (rLZ-8) on osteoclast in vitro and bone resorption in vivo. The rLZ-8 protein was derived from Ganoderma lucidum transformation and produced by a genetic system. Receptor activator of nuclear factor kappa-Β ligand induced RAW 264.7 cells to differentiate into osteoclastic cells in vitro. Cells were exposed to different doses of rLZ-8 for 7 days to measure differences of osteoclastic differentiation, apoptosis rate and gene expression. rLZ-8 was labeled with Alexa Fluor 568 to observe its intracellular distribution under super-resolution light microscopy. In addition, retinoic acid was administered to female rats for 14 days to develop osteopenia changes. Different doses of rLZ-8 were simultaneously administered to rats treated with retinoic acid to observe changes of bone mineral density, biochemical parameters and organ weight ratio. Results indicated that rLZ-8 regulated receptor activator of nuclear factor kappa-Β (RANK) - tumor necrosis factor receptor-associated factor 6 (TRAF6) - c-Jun N-terminal kinase (JNK) signaling pathway, by which rLZ-8 inhibited osteoclastic differentiation and promoted osteoclastic apoptosis. Through 3D-structured illumination microscopy, it was observed that rLZ-8 entered RAW264.7 cells and accumulated gradually into the cytoplasm but little into nucleus. Administration with rLZ-8 reversed loss of bone mass and improved ALP activity in osteoporotic rats. Low-to high-dose rLZ-8 treatments displayed little toxic effects on rat organs and did not seem to impact their overall health. All data suggested that rLZ-8 has possible action against osteoporosis.

Requirement of TGFß Signaling for Effect of Fluoride on Osteoblastic Differentiation.

Research focused on transforming growth factor ß (TGFß) signaling in osteoblast is gradually increasing, whereas literature is rare in terms of fluorosis. This work aimed to investigate how TGFß signaling participated in regulation of the osteoblast by different doses of fluoride treatment. Bone marrow stem cells (BMSCs) were developed into osteoblastic cells and exposed to 1, 4, and 16 mg/L F- with and without 10 ng/mL of TGFß. Cell viability and differentiation state of osteoblast under different settings were measured by means of cell counting kit and analysis of alkaline phosphatase (ALP) activity as well as formation of mineral nodules. Real-time PCR was utilized to test expression of ALP and Runt-related transcription factor 2 (Runx2) at gene level. The gene expression of TGFß signaling effectors was also investigated, such as TGFß receptors (TßRs), smad3, and mitogen-activated protein kinases (MAPK). Results demonstrated that fluoride treatment exhibited action on osteoblast viability and osteogenic differentiation and upregulated expression of TßR2, smad3, and MAPK in this process. Administration of TGFß strengthened ALP activity but attenuated formation of mineral nodules. Co-treatment of TGFß and low-dose fluoride increased ALP activity compared to same dose of single fluoride treatment, whereas it inhibited mineral nodule formation. Administration of TGFß reversed the suppression of high-dose fluoride on osteogenic differentiation of BMSCs. Taken together, studies revealed that TßR2 acted as a target for fluoride and TGFß treatment on BMSCs, and smad3 and MAPK were involved in the mechanism of fluoride regulating osteogenic differentiation. Together, our data indicated that TGFß receptor-mediated signaling through smad3 and MAPK was required for modulation of fluoride on osteoblast viability and differentiation, and activating TßR2-smad3 signaling pathway reversed suppression of osteoblasts differentiation by high dose of fluoride treatment.