Laura P. Zanello

Stimulation by 1alpha,25(OH)2-vitamin D3 of whole cell chloride currents in osteoblastic ROS 17/2.8 cells. A structure-function study.

1α,25-Dihydroxyvitamin D3(1α,25(OH)2D3) can generate biological responses via genomic and nongenomic mechanisms. This article reports for the first time the effects of 1α,25(OH)2D3 and structurally related analogs on whole cell chloride currents in osteoblastic cells. 1α,25(OH)2D3 promoted the rapid enhancement of outwardly rectifying Cl− currents in 93% of the osteoblasts in a concentration-dependent manner, with a maximal increase of about 4-fold between 0.5 and 5 nm. This effect of 1α,25(OH)2D3 was blocked by 1 nm stereoisomer 1β,25(OH)2D3when added to the bath before 1α,25(OH)2D3. On the other hand, 1 nm of the 6-s-cis locked analog 1α,25(OH)2-lumisterol3 significantly increased by about 2.2-fold outward Cl− currents in the ROS 17/2.8 cells, whereas the increase promoted by same concentration of the 6-s-trans locked analog 1α,25(OH)2-tachysterol (0.8-fold) was significantly lower, suggesting that the 6-s-cis locked or steroid-like form was preferred over the extended 6-s-trans conformer to promote these rapid effects of the hormone. We conclude that the agonist effects of 1α,25(OH)2D3 in osteoblasts at the cellular membrane level seem to be determined by some structural features of the molecule which may be crucial for its interaction with a putative membrane receptor in the cell surface.

Rapid and genomic biological responses are mediated by different shapes of the agonist steroid hormone, 1α, 25(OH)2vitamin D3

The hormone 1alpha,25(OH)2vitamin D3 (1,25-D) produces biological responses via both genomic and rapid mechanisms. The genomic responses are linked to a nuclear receptor, while the rapid responses are believed to utilize other signal transduction pathways that are likely linked to a putative cell membrane receptor for 1,25-D. The natural seco-steroid, 1,25-D, is capable of facile rotation about its 6,7 single carbon bond to permit generation of a continuum of potential ligand shapes extending from the 6-s-cis (6C) to the 6-s-trans (6T). To identify the shape of the conformer(s) that can serve as agonists for the genomic and rapid responses, we synthesized two families of analogs that were locked in either the 6T or 6C conformation. We found that 6T-locked analogs were inactive or significantly less active than 1,25-D in both rapid responses (transcaltachia or the rapid stimulation of intestinal Ca2+ absorption in perfused chick intestine, stimulation of whole cell chloride currents in osteoblastic ROS 17/2.8 cells, and stimulation of phosphorylation of mitogen-activated protein kinase in promyelocytic NB4 leukemic cells) and in genomic responses (induction of osteocalcin in human MG-63 osteoblastic cells). For genomic responses, the 6C-locked analogs bound poorly to the nuclear receptor and were much less potent than 1,25-D. In contrast, the 6C-locked analogs were potent agonists of the three rapid responses studied and had activities equivalent to 1,25-D. These results demonstrate that the signal transduction pathways that support rapid and genomic responses can discriminate between different shapes of the conformationally flexible 1,25-D.

Biocompatibility of corrosion-resistant zeolite coatings for titanium alloy biomedical implants

Titanium alloy, Ti6Al4V, is widely used in dental and orthopedic implants. Despite its excellent biocompatibility, Ti6Al4V releases toxic Al and V ions into the surrounding tissue after implantation. In addition, the elastic modulus of Ti6Al4V ( approximately 110GPa) is significantly higher than that of bone (10-40GPa), leading to a modulus mismatch and consequently implant loosening and deosteointegration. Zeolite coatings are proposed to prevent the release of the toxic ions into human tissue and enhance osteointegration by matching the mechanical properties of bone. Zeolite MFI coatings are successfully synthesized on commercially pure titanium and Ti6Al4V for the first time. The coating shows excellent adhesion by incorporating titanium from the substrate within the zeolite framework. Higher corrosion resistance than the bare titanium alloy is observed in 0.856M NaCl solution at pHs of 7.0 and 1.0. Zeolite coatings eliminate the release of cytotoxic Al and V ions over a 7 day period. Pluripotent mouse embryonic stem cells show higher adhesion and cell proliferation on the three-dimensional zeolite microstructure surface compared with a two-dimensional glass surface, indicating that the zeolite coatings are highly biocompatible.

Vitamin D Receptor–Dependent 1α,25(OH)2 Vitamin D3–Induced Anti‐Apoptotic PI3K/AKT Signaling in Osteoblasts

Osteoblast apoptosis plays a crucial role in bone remodeling. Physiological doses of 1α,25(OH)2‐vitamin D3 (1,25D) protect osteoblasts against apoptosis by means of mechanisms only partially understood. We studied activation of an Akt survival cascade downstream of 1,25D nongenomic stimulation of phosphatidylinositide‐3′‐kinase (PI3K) in osteoblastic cells. We measured a dose‐ and time‐dependent 1,25D induction of Akt phosphorylation (p‐Akt) in cultured osteoblastic cells. Maximal response was achieved with 10 nM 1,25D after 5 min. We found that staurosporine (STSP)‐induced apoptosis was significantly reduced in 1,25D‐pretreated osteoblasts. 1,25D prosurvival effects were abolished when cells were preincubated with inhibitors of PI3K activation. By means of siRNA silencing, we proved that 1,25D induction of p‐Akt requires a classic vitamin D receptor (VDR) in osteoblasts. Furthermore, non‐osteoblastic CV‐1 cells transfected with an enhanced green fluorescent protein (EGFP)‐VDR construct responded to 1,25D treatment with a rapid p‐Akt response associated with increased cell survival not detected in native, nontransfected cells. We measured increased levels of p‐Akt substrates p‐Bad and p‐FKHR and significantly reduced activity of caspases 8 and 3/7 after 1,25D treatment. In addition, 1,25D‐induced protection against apoptosis was abolished when osteoblasts were preincubated with pertussis toxin. We conclude that anti‐apoptotic effects of 1,25D in osteoblasts occur through nongenomic activation of a VDR/PI3K/Akt survival pathway that includes phosphorylation of multiple p‐Akt substrates and reduction of caspase activities.

1α,25(OH)2 Vitamin D3 actions on ion channels in osteoblasts

Membrane-initiated cellular responses to steroids include modulation of ion channel activities via signal transduction pathways. However, the molecular mechanisms involved in nongenomic actions remain only partially understood. Our research has focused on the rapid effects of 1alpha,25(OH)(2) Vitamin D(3) [1,25D] on L-type Ca(2+) [L-Ca] and DIDS-sensitive Cl(-) channels in osteoblasts. Physiological nanomolar concentrations of hormonally active 1,25D promote rapid (1-5 min) potentiation of outward Cl(-) currents in osteosarcoma ROS 17/2.8 cells and mouse primary osteoblasts. In addition, 1,25D increases inward barium currents through L-Ca channels at low depolarizing potentials within seconds in a fashion similar to the 1,4-dihydropyridine [DHP] agonist Bay K8644. We found that second messenger cAMP is involved in 1,25D potentiation of Cl(-) and Ca(2+) channels. Nongenomic 1,25D effects on ion channel activities in osteoblasts appear to involve different mechanisms that include a possible direct interaction with the L-Ca channel molecule, on one hand, and signaling through the cAMP pathway, on the other. Rapid 1,25D actions on Cl(-) and Ca(2+) currents seem to couple to secretory activities in osteoblasts, thus contributing to bone mass formation.

1α,25(OH)2-Vitamin D3 stimulation of secretion via chloride channel activation in Sertoli cells

Sertoli cell secretory activities are highly dependent on ion channel functions and critical to spermatogenesis. The steroid hormone 1alpha,25(OH)2-vitamin D3 (1,25(OH)2-D3) stimulates exocytosis in different cell systems by activating a nongenotropic vitamin D receptor (VDR). Here, we described 1,25(OH)2-D3 stimulation of secretion via Cl(-) channel activation in the mouse immature Sertoli cell line TM4. 1,25(OH)2-D3 potentiation of chloride currents was dependent on hormone concentration, and correlated with a significant increase in whole-cell capacitance within 20-40 min. In addition, Cl(-) currents were potentiated by the nongenomic VDR agonist 1alpha,25(OH)2 lumisterol D3 (JN), while 1,25(OH)2-D3 potentiation of channels was suppressed by nongenomic VDR antagonist 1beta,25(OH)2-vitamin D3 (HL). Treatment of TM4 cells with PKC and PKA activators PMA and forskolin respectively, increased Cl(-) currents significantly, while PKC and PKA inhibitors Go6983 and H-89, respectively, abolished 1,25(OH)2-D3 stimulation of Cl(-) currents, suggesting phosphorylation pathways in 1,25(OH))2-D3 mediated channel responses. RT-PCR demonstrated the expression of outwardly rectifying ClC-3 channels in TM4 cells. Taken together, our results demonstrate a PKA/PKC-dependent 1,25(OH)2-D3/VDR nongenotropic pathway leading to Cl(-) channel and exocytosis activation in Sertoli cells. We conclude that 1,25(OH)2-D3 appears to be a modulator of male reproductive functions at least in part by stimulating Sertoli cell secretory functions.

Multiple molecular mechanisms of 1α,25(oh)2-vitamin D3 rapid modulation of three ion channel activities in osteoblasts

Rapid nongenomic responses to steroids include modulation of ion channel activities on the cell membrane of target cells, but little is known about the molecular mechanisms involved. In this paper we investigate the mechanisms underlying the combined action of the secosteroid hormone 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)(2)D3] on three different ion channel types in rat osteoblasts, which include a voltage-gated L-type Ca(2+) channel, a mechanosensitive Cl(-) channel, and a stretch-activated cation (SA-Cat) channel. We found that physiological nanomolar concentrations of 1alpha,25(OH)(2)D3 rapidly modify the overall electrical activity of the membrane in ROS 17/2.8 cells. 1alpha,25(OH)(2)D3 increases the osteoblast L-type Ca(2+) channel activity at low depolarizing voltages in a fashion similar to the 1,4-dihydropyridine (DHP) agonist Bay K8644. At highly depolarizing potentials 1alpha,25(OH)(2)D3 potentiates volume-sensitive Cl(-) currents through mechanisms that may involve a putative membrane receptor. We show for the first time that 1alpha,25(OH)(2)D3 also increases inward currents through SA-Cat channels at positive membrane voltages in a dose-dependent manner. Contrary to our expectations, the stereoisomer 1beta,25(OH)(2)D3, which suppresses 1alpha,25(OH)(2)D3 activation of osteoblast Cl(-) currents, mimicked 1alpha,25(OH)(2)D3 agonist effects on Ca(2+) and SA-Cat channel activities. Cyclic AMP is involved in 1alpha,25(OH)(2)D3 effects on both Ca(2+) and SA-Cat channels, but not in Cl(-) channels. We conclude that 1alpha,25(OH)(2)D3 rapid effects on ion channel activities in ROS 17/2.8 cells occur through multiple mechanisms that, on the one hand, involve a possible direct interaction with the L-type Ca(2+) channel molecule and, on the other hand, molecular pathways that may include a putative membrane receptor.

1α,25(OH)2-vitamin D3 membrane-initiated calcium signaling modulates exocytosis and cell survival

1α,25(OH)2-vitamin D3 (1,25D) is considered a bone anabolic hormone. 1,25D actions leading to bone formation involve gene transactivation, on one hand, and modulation of cytoplasmic signaling, on the other. In both cases, a functional vitamin D receptor (VDR) appears to be required. Here we study 1,25D-stimulated calcium signaling that initiates at the cell membrane and leads to exocytosis of bone materials and increased osteoblast survival. We found that rapid 1,25D-induction of exocytosis couples to cytoplasmic calcium increase in osteoblastic ROS 17/2.8 cells. In addition, we found that elevation of cytoplasmic calcium concentration is involved in 1,25D anti-apoptotic effects via Akt activation in ROS 17/2.8 cells and non-osteoblastic CV-1 cells. In both cases, 1,25D-stimulated elevation of intracellular calcium is due in part to activation of L-type Ca2+ channels. We conclude that 1,25D bone anabolic effects that involve increased intracellular Ca2+ concentration in osteoblasts can be explained at two levels. At the single-cell level, 1,25D promotes Ca2+-dependent exocytotic activities. At the tissue level, 1,25D protects osteoblasts from apoptosis via a Ca2+-dependent Akt pathway. Our studies contribute to the understanding of the molecular basis of bone diseases characterized by decreased bone formation and mineralization.

Electrical responses to 1α,25(OH)2-Vitamin D3 and their physiological significance in osteoblasts

Osteoblasts are a main target for the steroid 1alpha,25(OH)2-Vitamin D3 (1,25D3), where a major outcome is the modulation of the bone remodeling process. 1,25D3 deficiency leads to clinical disorders such as osteomalacia and osteoporosis, characterized by a state of insufficiently calcified tissue and bone loss, respectively. In the osteoblast nucleus, 1,25D3 modulates gene transcription for the synthesis of bone matrix proteins via the Vitamin D receptor (VDR). At the plasma membrane level, 1,25D3 potentiates ion channel functions, activates signal transduction pathways, and increases cytoplasmic calcium concentrations. So far, no clear physiological significance has been attributed to membrane-initiated 1,25D3 actions in single cells. To investigate if (a) 1,25D3 is a modulatory agent of secretion in osteoblasts and (b) the classical VDR is involved in rapid electrical events in the cell membrane, we studied hormone effects on ion channel activities in relation to exocytosis in osteoblasts isolated from VDR knockout (KO) and wild-type (WT) mice. This paper is a retrospect of the electrophysiological studies done in our laboratory to date. We found that 1,25D3-promoted ion channel responses are coupled to secretion in calvarial osteoblasts, and develop only in the presence of a functional nuclear steroid VDR. This 1,25D3-regulated exocytosis in osteoblasts, which takes place within minutes of hormone application, seems to be the natural complement of genomic actions that evolve at a longer time scale. The absence of both 1,25D3 membrane and nuclear effects in VDR KO osteoblasts may explain bone abnormalities typically found in VDR KO mice.

1α,25(OH)2 Vitamin D3 Induction of ATP Secretion in Osteoblasts

In the absence of mechanical stimulation, brief exposure of osteoblasts to 1α,25(OH)2vitamin D3 (1,25D) triggers plasma membrane electrical responses that couple to exocytosis. Here we describe for the first time 1,25D induction of exocytotic ATP release in static ROS 17/2.8 and SAOS‐2 cells and primary calvarial osteoblasts expressing a vitamin D receptor (VDR). We found that 10 nM 1,25D optimally induced 45 ± 1% and 40 ± 1% of partial and complete exocytotic events, respectively, from a 1,25D‐sensitive pool of ATP‐containing secretory vesicles within 60 s. We measured a dose‐dependent 1,25D induction of ATP secretion, with maximal response of ∼6.2‐fold (16.93 ± 1.82 nM for SAOS‐2) and 3.1‐fold (18.89 ± 1.39 nM for ROS 17/2.8) obtained with 10 nM 1,25D compared with basal ATP levels (2.75 ± 0.39 nM, SAOS‐2; 6.09 ± 0.58 nM, ROS 17/2.8 cells). The natural metabolite 25(OH)vitamin D3 (25D, 10 nM) induced a significant 3.6‐fold increase of ATP release in ROS 17/2.8 cells, but there was no induction with the antagonist 1β,25(OH)2vitamin D3 (1β,25D, 10 nM) or the steroid 17β‐estradiol (10 nM). 1,25D‐induced ATP secretion was abolished when cells were preincubated with inhibitors of vesicular exocytosis. siRNA VDR silencing prevented 1,25D stimulation of ATP exocytosis in ROS 17/2.8 and SAOS‐2 cells. Similarly, 1,25D failed to stimulate ATP exocytosis in primary osteoblasts from a VDR knockout mouse. ATP secretion coupled to 1,25D induction of cytosolic calcium and chloride channel potentiation. Rapid 1,25D stimulation of ATP secretion involving nontranscriptional VDR functions in osteoblasts may help explain 1,25D bone anabolic properties.