Tian-Lan Zhang

Fibronectin enhances in vitro vascular calcification by promoting osteoblastic differentiation of vascular smooth muscle cells via ERK pathway

The process of vascular calcification presents several features similar to osteogenesis in which fibronectin (FN) acts as a regulator of osteoblastic differentiation and the ERK signal pathway is involved. In order to find whether FN promotes the osteoblastic differentiation of vascular smooth muscle cells (VSMCs) through the ERK signal pathway, we investigated the effect of FN on the calcification of VSMCs by using an in vitro cell model. VSMCs cultured in plates with FN (0–20 µg/cm2) coating were induced to calcify by 10 mM sodium β‐glycerophosphate (β‐GP). FN exacerbated VSMC calcification in a dose‐ and time‐dependent manner, as indicated by the number of calcifying nodules per slide and the amount of calcium in the deposition. Data from RT‐PCR and immunoblotting assay revealed that FN also enhanced the expression of several phenotypic markers of osteoblasts, including alkaline phosphatase (ALP) activity, osteocalcin (OC), and Osf2/Cbfa1, a key transcription factor in osteoblastic differentiation. Furthermore, a specific inhibitor for ERK, PD98059 (10 µM), significantly suppressed the effect of FN on calcification and phenotypic marker expression. These findings seem to suggest that FN enhanced vascular calcification by promoting the osteoblastic differentiation of VSMCs via ERK signal pathway. J. Cell. Biochem. 99: 1343–1352, 2006.

Lanthanum enhances in vitro osteoblast differentiation via pertussis toxin‐sensitive gi protein and ERK signaling pathway

Converging lines of evidence suggest that lanthanum tends to deposit in bone. The influence of lanthanum ion (La3+) on osteoblast differentiation and the related mechanism are essential to understanding its effect on bone metabolism. In this study, La3+ treatment enhanced in vitro osteoblast differentiation as evidenced by promoting alkaline phosphatase (ALP) activity, osteocalcin (OC) secretion, and matrix mineralization. The expressions of osteoblast‐specific genes of Cbfa‐1, osteopontin (OPN), and bone sialoprotein (BSP) were all increased in the presence of La3+, but no change was observed in that of type I collagen (COL‐I). Further studies demonstrated that La3+ treatment enhanced phosphorylation of extracellular signal‐regulated kinase (ERK). Inhibition of ERK activation by U0126 suppressed the effects of La3+ on osteoblast activity. Moreover, pretreatment of the cells with pertussis toxin (PTx), a Gi protein inhibitor, suppressed the La3+‐enhanced ERK phosphorylation and osteoblast differentiation. These findings suggest that La3+ exposure enhances in vitro osteoblast differentiation and the effect depends on ERK phosphorylation via PTx‐sensitive Gi protein signaling. J. Cell. Biochem. 105: 1307–1315, 2008.

Effects of Lanthanum on Composition, Crystal Size, and Lattice Structure of Femur Bone Mineral of Wistar Rats

The application of lanthanum (La) in industry, medicine, and agriculture may cause accumulation of the element in human body. This article examines the effects of La on the femur bone mineral of male Wistar rats after administration of La(NO3)3 by gavage at the dose of 2.0 mg La(NO3)3 · kg−1 · day−1 over a 6-month period. Chemical analysis confirmed La accumulation in bone and loss in bone mineral. Thermogravimetric analysis showed a decrease in the mineral-to-matrix ratio and an increase in carbonate content. Fourier-transform infrared spectrometry revealed elevation in the contents of labile carbonate and acidic phosphate. The synchrotron radiation small-angle X-ray scattering study presented a smaller mean thickness of the mineral crystals in the bone of La-treated rats. The synchrotron radiation-extended X-ray absorption fine structure analysis indicated that the La treatment resulted in a lowered disorder in the crystals. The smaller size, more adsorbed labile carbonate, and more acidic phosphate made the bone mineral easier to dissolve, as revealed in the kinetic measurement of bone demineralization. These findings suggest that La retards bone maturation of rats.

Realgar-induced differentiation is associated with MAPK pathways in HL-60 cells

The clinical efficacy and safety of realgar (arsenic sulfide, As4S4) in the treatment of acute promyelocytic leukemia in China have given rise to an upsurge in research on the underlying mechanism. We prepared realgar nanoparticles (RNPs) to examine their effect on the differentiation of HL‐60 cells. Treatment with RNPs at 6 μM for 72 h induced cell differentiation that was assessed by morphological change, NBT reductive ability, and elevation of CD11b expression at both mRNA and protein levels. The RNP‐induced differentiation was synergized, enhanced and suppressed by the inhibition of p38 MAPK, JNK and ERK pathways, respectively. Our findings demonstrate that MAPK signaling pathways are closely related to the RNP‐induced differentiation in HL‐60 cells.

Cholestane-3β,5α,6β-triol inhibits osteoblastic differentiation and promotes apoptosis of rat bone marrow stromal cells

Converging lines of evidence suggest that oxidized lipids, long recognized as a risk factor in atherogenesis, also contribute to osteoporosis, but the underlying mechanism is not understood in detail. The effect of atherogenesis related factors including oxysterols on the differentiation and survival of marrow stromal cells (MSCs) would be very important in understanding the link between atherosclerosis and osteoporosis. In the present study, the effect of oxysterol cholestane‐3β,5α,6β‐triol (Triol) on osteoblastic differentiation and apoptosis of primary rat bone MSCs as well as the related mechanisms were studied. Triol inhibited MSCs osteoblastic differentiation as demonstrated by inhibition of alkaline phosphatase activity, osteocalcin secretion, and matrix mineralization. In the other aspect, Triol promoted MSCs apoptosis, as characterized by condensed or fragmented nuclei as well as active externalization of phosphatidyl serine to the cell surface. In addition, Triol was found to induce increases of intracellular Ca2+ and Ca2+‐dependent reactive oxygen species generation in MSCs. These effects were involved in the action of Triol on apoptosis, but not on osteoblastic differentiation of MSCs. These results suggested that Triol might contribute to the decreased bone formation by inhibition of osteoblastic differentiation and promotion of apoptosis of MSCs, providing insights about common factors underlying the pathogenesis of atherosclerosis and osteoporosis.

Lanthanum suppresses osteoblastic differentiation via pertussis toxin-sensitive G protein signaling in rat vascular smooth muscle cells.

A major cellular event in vascular calcification is the phenotypic transformation of vascular smooth muscle cells (VSMCs) into osteoblast‐like cells. After demonstrating that lanthanum chloride (LaCl3) suppresses hydrogen peroxide‐enhanced calcification in rat calcifying vascular cells (CVCs), here we report its effect on the osteoblastic differentiation of rat VSMCs, a process leading to the formation of CVCs. Cells were isolated from aortic media of male SD rats, and passages between three and eight were cultured in Dulbeccols Modified Eagles Medium (DMEM) containing 10% fetal bovine serum (FBS) and 10 mM β‐glycerophosphate (β‐GP) in the presence or absence of LaCl3. Exposure of cells to LaCl3 suppressed the β‐GP‐induced elevations in calcium deposition, alkaline phosphatase (ALP) activity, and Cbfa1/Runx2 expression, as well as the concomitant loss of SM α‐actin. Furthermore, LaCl3 activated the phosphorylation of extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK), and the blockage of either pathway with a specific inhibitor abolished the effects of LaCl3. In addition, pretreatment of the cells with pertussis toxin (PTx), an inhibitor of G protein‐mediated signaling pathway, repealed all the changes induced by LaCl3. These findings demonstrate that LaCl3 suppresses the β‐GP‐induced osteoblastic differentiation and calcification in rat VSMCs, and its effect is mediated by the activation of both ERK and JNK MAPK pathways via PTx‐sensitive G proteins. J. Cell. Biochem. 108: 1184–1191, 2009.

Gadolinium-promoted precipitation of calcium phosphate is associated with profibrotic activation of RAW 264.7 macrophages.

Gadolinium-based contrast agents are now being linked to nephrogenic systemic fibrosis (NSF). The exact mechanism by which gadolinium species act in the pathogenesis of NSF is not fully understood. In this study, we evaluated the effect of gadolinium chloride (GdCl(3)) on the precipitation of calcium phosphate, and examined the role of the gadolinium-containing precipitates in the profibrotic activation of macrophages. In a free-drift system, the induction time was markedly reduced with increasing concentration of GdCl(3), accompanied by alterations of morphology and composition of the precipitates. In complete cell culture medium, the addition of GdCl(3) resulted in formation of particles around 200-300 nm. In an in vitro cellular model with RAW 264.7 macrophages, GdCl(3) increased the production of TGF-beta1 and IL-6 via the activation of PKC and ERK signaling pathway. Our findings demonstrate that GdCl(3) promotes calcium phosphate precipitation and induces profibrotic activation of macrophages.

Lanthanum chloride bidirectionally influences calcification in bovine vascular smooth muscle cells

Vascular calcification (VC) is frequent prevalence in patients with chronic kidney disease (CKD) and atherosclerosis. Lanthanum carbonate is used as an orally administered phosphate‐binding agent to reduce the gastrointestinal absorption of phosphate and ameliorate VC in advanced CKD. In this study, we used bovine vascular smooth muscle cells as a model VC in vitro and studied the effects of lanthanum chloride on calcium deposition. Exposure of cells to LaCl3 at the concentration of 0.1 µM suppressed the β‐glycerophosphate‐induced alkaline phosphatase activity and calcium deposition. Furthermore, LaCl3 upregulated the β‐glycerophosphate‐suppressed expression of calcium‐sensing receptor. In contrast to the inhibitory effect of LaCl3 on calcium deposition, higher level lanthanum (50 µM) was found to promote immediately precipitation of calcium phosphate in cell culture medium. At this concentration, LaCl3 was found to induce cell apoptosis which involves caspases‐9 and ‐3. These data indicate that the promotory effect of LaCl3 on calcium deposition is likely mediated by induction of apoptosis. Our in vitro findings do suggest that, in the context of raised lanthanum, greater attention should be paid to potential toxic effects associated to the use of lanthanide‐based drugs. J. Cell. Biochem. 113: 1776–1786, 2012.

Association of oxidative stress with realgar-induced differentiation in human leukemia HL-60 cells.

Background: Realgar (arsenic sulfide, As4S4) has been shown to have clinical efficacy in patients with newly diagnosed and relapsed acute promyelocytic leukemia. Mechanistic studies have demonstrated that realgar is able to induce cell differentiation. Methods: The oxidative stress in the realgar-induced differentiation was examined with human leukemia HL-60 cells. Cell differentiation was evaluated by the expression of cell surface antigen CD11b and nitroblue tetrazolium assay. The activities of catalase and superoxide dismutase were measured spectrophotometrically. Flow cytometry was used to assess cell cycle distribution and apoptosis, the cellular level of reactive oxygen species (ROS) and glutathione, as well as mitochondrial transmembrane potential (MTP). Results: The realgar-induced differentiation was enhanced by hydrogen peroxide, and preceded with drastic changes in ROS and catalase, as well as small changes in superoxide dismutase and the reduced form of glutathione. MTP values at 24 h were in linear proportion to the CD11b expression at 48 h when no apoptosis was observed. Conclusion: Oxidative stress and stress-related MTP decrease are associated with realgar-induced differentiation in HL-60 cells.

Nature of cerium(III)- and lanthanum(III)-induced aggregation of human erythrocyte membrane proteins

To clarify the nature of the aggregation of membrane proteins (MP) induced by lanthanide cations (Lns), the interaction of cerium(III) (Ce3+) and lanthanum(III)(La3+) with erythrocyte membrane proteins was studied by means of SDS-PAGE, light scattering measurement, fluorescence, CD and FTIR spectra. The results showed that Ce3+ and La3+ induce protein aggregation not only by Lns non-covalent binding and cross-linking, but also by oxidative cross-linking through disulfide bond formation. As demonstrated by intrinsic fluorescence, CD and FTIR spectra studies, the aggregation was accompanied by the conformation changes with tryptophane residues exposing to more hydrophobic environment and the decreasing alpha-helix and beta-sheet contents. By stopped-flow studies, protein aggregation was shown to be a slow change, which is initiated by rapid Lns binding and then followed by subsequent conformational changes.