Extracellular Fe2+ and Fe3+ modulate osteocytic viability, expression of SOST, RANKL and FGF23, and fluid flow-induced YAP1 nuclear translocation.

Iron overload negatively affects bone mass and strength. However, the impact of iron excess on osteocytes-important bone cells for mechanotransduction and remodeling-is poorly understood. Herein, we examined the effects of iron exposure on osteocytes during their maturation process. We discovered that iron overload caused apoptosis of osteocytes in early and late stages of differentiation. Notably, the expression of key proteins for iron entry was downregulated during differentiation, suggesting that mature osteocytes were less susceptible to iron toxicity due to limited iron uptake. Furthermore, iron overload also enriched a subpopulation of mature osteocytes, as indicated by increased expression of Dmp1, a gene encoding protein for bone mineralization. These iron-exposed osteocytes expressed high levels of Sost, Tnfsf11 and Fgf23 transcripts. Consistently, we demonstrated that exogenous FGF23 stimulated the formation and survival of osteoclasts, suggesting its regulatory role in bone resorption. In addition, iron overload downregulated the expression of Cx43, a gene encoding gap junction protein in the dendritic processes, and impaired YAP1 nuclear translocation in response to fluid flow in differentiated osteocytes. It can be concluded that iron overload induces cellular adaptation in differentiating osteocytes, resulting in insensitivity to mechanical stimulation and potential disruption of the balance in bone remodeling.

Mild-intensity physical activity prevents cardiac and osseous iron deposition without affecting bone mechanical property or porosity in thalassemic mice.

Thalassemia causes anemia, ineffective erythropoiesis, bone loss and iron accumulation in several tissues, e.g., liver, bone and heart, the last of which leads to lethal cardiomyopathy and arrhythmia. Although exercise reportedly improves bone density in thalassemic mice, exercise performance is compromised and might pose risk of cardiovascular accident in thalassemic patients. Therefore, we sought to explore whether mild-intensity physical activity (MPA) with 30-50% of maximal oxygen consumption was sufficient to benefit the heart and bone. Herein, male hemizygous ß-globin knockout (BKO) mice and wild-type littermates were subjected to voluntary wheel running 1 h/day, 5 days/week for 3 months (MPA group) or kept sedentary (SDN; control). As determined by atomic absorption spectroscopy, BKO-MPA mice had less iron accumulation in heart and bone tissues compared with BKO-SDN mice. Meanwhile, the circulating level of fibroblast growth factor-23-a factor known to reduce serum iron and intestinal calcium absorption-was increased early in young BKO-MPA mice. Nevertheless, MPA did not affect duodenal calcium transport or body calcium retention. Although MPA restored the aberrant bone calcium-phosphorus ratio to normal range, it did not change vertebral calcium content or femoral mechanical properties. Microstructural porosity in tibia of BKO-MPA mice remained unaltered as determined by synchrotron radiation X-ray tomographic microscopy. In conclusion, MPA prevents cardiac and bone iron accumulation, which is beneficial to thalassemic patients with limited physical fitness or deteriorated cardiac performance. However, in contrast to moderate-intensity exercise, MPA does not improve bone mechanical properties or reduce bone porosity.

CFTR-mediated anion secretion in parathyroid hormone-treated Caco-2 cells is associated with PKA and PI3K phosphorylation but not intracellular pH changes or Na+/K+-ATPase abundance.

Parathyroid hormone (PTH) has previously been shown to enhance the transepithelial secretion of Cl- and HCO3 - across the intestinal epithelia including Caco-2 monolayer, but the underlying cellular mechanisms are not completely understood. Herein, we identified the major signaling pathways that possibly mediated the PTH action to its known target anion channel, i.e., cystic fibrosis transmembrane conductance regulator anion channel (CFTR). Specifically, PTH was able to induce phosphorylation of protein kinase A and phosphoinositide 3-kinase. Since the apical HCO3 - efflux through CFTR often required the intracellular H+/HCO3 - production and/or the Na+-dependent basolateral HCO3 - uptake, the intracellular pH (pHi) balance might be disturbed, especially as a consequence of increased endogenous H+ and HCO3 - production. However, measurement of pHi by a pH-sensitive dye suggested that the PTH-exposed Caco-2 cells were able to maintain normal pH despite robust HCO3 - transport. In addition, although the plasma membrane Na+/K+-ATPase (NKA) is normally essential for basolateral HCO3 - uptake and other transporters (e.g., NHE1), PTH did not induce insertion of new NKA molecules into the basolateral membrane as determined by membrane protein biotinylation technique. Thus, together with our previous data, we concluded that the PTH action on Caco-2 cells is dependent on PKA and PI3K with no detectable change in pHi or NKA abundance on cell membrane.

Autophagic stress; a new cellular response to nanoparticles. Could it be a new strategy for inhibition of liver cancer cell invasion and metastasis?

We herein report a new biological consequence from a unique interaction between nanoparticles of ferric-tannic complexes (Fe-TA NPs) and liver cancer cells (HepG2.2.15). The Fe-TA NPs were found to accumulate into the cells via specific cellular uptake mechanisms and thereafter disturbed cellular autophagy and cellular pH homeostasis, which led the cells to undergo autophagic stress and eventual death. According to biophysical analysis, the cells undergoing autophagic stress were found to lose their capability of attachment, migration, and movement. Similarly, KEGG analysis demonstrated the down-regulation of TGF-beta indicating that the autophagic stress is capable of reducing cancer cell invasion. Therefore, the Fe-TA NPs could be considered beneficial as a new pharmaceutical nanoplatform for liver cancer treatment via induction of autophagic stress.

Tannic acid (TA): A molecular tool for chelating and imaging labile iron.

This report presents the potential utilization of tannic acid (TA) as a natural iron chelator. TA is capable of binding with small ferric complexes without competitive binding with endogenous iron-containing molecules such as ferritin and transferrin. It was observed that the extracellular iron binding of TA resulted in the formation of self-assembled Fe3+-TA complexes, which were then taken up by HepG2 cells via phagocytosis pathway with autophagy-inducing properties. Obviously, TA was found to inhibit iron-induced HepG2 cell growth. However, cellular interactions and biological responses to the treatment were found to depend on availability of iron. Based on the results of the iron efflux experiment, it can be stated that TA has the capability to mobilize iron from cells in the form of assembled Fe3+-TA complexes. Interestingly, TA-mediated cellular iron influx and efflux were successfully monitored via MRI. The results of this study suggest that TA can be used as a molecular tool for chelating and imaging labile iron. This might be a promising approach for prevention and treatment of iron-associated cancer or other iron overload disorders.

Characterization and cellular studies of molecular nanoparticle of iron (III)-tannic complexes; toward a low cost magnetic resonance imaging agent.

Herein, a new magnetic resonance imaging (MRI) agent based on molecular nanoparticles of iron(III)-tannic complexes (Fe-TA NPs) is reported. The paramagnetic and molecularlike Fe-TA NPs were successfully synthesized at room temperature within a few minutes without the use of any toxic agents or expensive equipment. The coordination states of the Fe-TA NPs were pH-dependent. The r1 relaxivity values of the bis-dominated and tris-dominated structures of the Fe-TA NPs were determined to be 6.31 and 5.24 mM-1 s-1, respectively, by using a Philips Achieva 1.5T MRI scanner. The Fe-TA NPs were 177 ± 12 nm in diameter (hydrodynamic size) with a zeta potential value of -28 ± 0.9 mV, dispersing very well in aqueous solution and were highly stable in phosphate buffered saline buffer (pH 7.4) containing competitive ligands and metals. From in vitro studies, it was evident that the Fe-TA NPs exhibited good biocompatibility, with high cellular uptake in HepG2 cells. Clearly, the Fe-TA NPs were found to induce signal enhancement in the T1-weighted image of the HepG2 cells. As a result, it can be stated that the Fe-TA NPs may have the potential for being developed as low-cost and clinically translatable magnetic resonance imaging agents in the near future.