O. Sharon

Lipoxygenase-derived Metabolites Are Regulators of Peroxisome Proliferator-activated Receptor γ-2 Expression in Human Vascular Smooth Muscle Cells

BACKGROUNDnPeroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of the nuclear receptor family that has been implicated in cell differentiation and proliferation, glucose metabolism, macrophage development, and inflammatory responses. PPAR-gamma can be activated by a range of synthetic substances and also by products of lipid oxidation such as oxidized low-density lipoprotein, 13-hydroxyoctadecadienoic acid (13-HODE) and 15-hydroxyeicosatetraenoic acid (15-HETE). Since 12- and 15-lipoxygenase (12- and 15-LO) are expressed in human vascular smooth muscle cells (VSMCs), we set out to investigate the possible relation between PPAR-gamma and LO system in these cells.nnnMETHODSnIn vitro experiments in human VSMC using standard methods.nnnRESULTSnThe LO products, 12-HETE (10(-7) mol/l), 15-HETE (10(-7) mol/l) and 13-HODE (10(-7) mol//l) increased the expression of PPAR-gamma-2 messenger RNA (mRNA) in VSMC (by 100, 50, and 100%, respectively. Rosiglitazone (1-10 micromol/l) was found to upregulate both the mRNA expression of two LO enzymes, platelet-type 12-lipoxygenase (12-LO; +70%) and 15-lipoxygenase type 2 (15-LO; +60%), and the secretion of their eicosanoid products 12- and 15-HETE. In addition, rosiglitazone-induced a threefold increase in PPAR-gamma-2 mRNA expressions and modest 50% rise in PPAR-gamma-1 mRNA expression. The effect of rosiglitazone on PPAR-gamma-2 could be entirely blocked by the LO inhibitor baicalein and restored by the addition of exogenous 12-HETE.nnnCONCLUSIONSnThese results suggest a novel amplification cycle in which PPAR-gamma activation induces production of 12- and 15-LO-derived metabolites which in turn feed back to upregulate PPAR-gamma-2s own expression. The implications of this link in VSMC pathophysiology remain to be elucidated.

Vitamin D metabolites and analogs induce lipoxygenase mRNA expression and activity as well as reactive oxygen species (ROS) production in human bone cell line

Vitamin D metabolites and its less-calcemic analogs (vitamin D compounds) are beneficial for bone and modulate cell growth and energy metabolism. We now analyze whether 25(OH)D(3) (25D), 1,25(OH)(2)D(3) (1,25D), 24,25(OH)(2)D(3) (24,25D), JKF1624F(2)-2 (JKF) or QW1624F(2)-2 (QW) regulate lipooxygenase (LO) mRNA expression and its products; hydroxyl-eicosatetraenoic acid (12 and 15HETE) formation, as well as reactive oxygen species (ROS) production in human bone cell line (SaOS2) and their interplay with modulation of cell proliferation and energy metabolism. All compounds except 25D increased 12LO mRNA expression and modulated 12 and 15HETE production whereas ROS production was increased by all compounds, and inhibited by NADPH oxidase inhibitors diphenyleneiodonium (DPI) and N-acetylcysteine (NAc). Baicaleine (baic) the inhibitor of 12 and 15LO activity blocked only slightly the stimulation of DNA synthesis by all compounds, whereas DPI inhibited almost completely the stimulation of DNA and CK by all compounds. Treatments of cells with 12 or 15HETE increased DNA synthesis and CK that were only slightly inhibited by DPI. These results indicate that vitamin D compounds increased oxidative stress in osteoblasts in part via induction of LO expression and activity. The increased ROS production mediates partially elevated cell proliferation and energy metabolism, whereas the LO mediation is not essential. This new feature of vitamin D compounds is mediated by intracellular and/or membranal binding sites and its potential hazard could lead to damage due to increased lipid oxidation, although the transient mediation of ROS in cell proliferation is beneficial to bone growth in a yet unknown mechanism.

25 hydroxy-vitamin D3-1α hydroxylase expression and activity in cultured human osteoblasts and their modulation by parathyroid hormone, estrogenic compounds and dihydrotestosterone

Human osteoblasts (hOB) produce and respond to 1,25(OH)(2)D(3) (1,25D), suggesting an autocrine/paracrine system. We therefore examined hormonal modulation of the expression and activity of 25 hydroxy-vitamin D(3)-1alpha hydroxylase (1-Ohase) in hOB. Cells from pre- and post-menopausal women or men, were treated with estrogenic compounds and 1-OHase expression and activity were measured. 1-OHase mRNA expression was highest in pre-menopausal women hOB and was increased by all hormones tested. In post-menopausal hOB all hormones except biochainin A (BA) and genistein (G) increased 1-OHase mRNA expressions to less extent. In male-derived hOB only dihydrotestosterone (DHT) and carboxy BA (cBA) increased 1-OHase mRNA expression. 1,25D production from 25(OH)D(3) had a K(m) of approximately 769-400 ng/ml (1.92-1.07 microM) and V(max) of 31.3-17.4 ng/ml (0.078-0.044 microM/60 min/5 x 10(6)cells) respectively, and was increased by all hormones except raloxifene (Ral) with higher stimulation in pre- than in post-menopausal cells. Only BA was almost five times more potent in pre- rather than post-menopausal hOBs. In male hOB only DHT and cBA increased 1,25D production whereas estradiol-17beta (E(2)) had no effect and BA decreased it. These results provide evidence for the expression of 1-OHase mRNA and production of 1,25D in hOBs, which are age and sex dependent and are hormonally modulated. The role of this local autocrine/paracrine 1,25D system in bone physiology deserves further investigation.

Membranal effects of phytoestrogens and carboxy derivatives of phytoestrogens on human vascular and bone cells: New insights based on studies with carboxy-biochanin A☆

Estradiol-17beta (E2) and some phytoestrogens induce a biphasic effect on DNA synthesis in cultured human vascular smooth muscle cells (VSMC), i.e., stimulation at low concentrations and inhibition at high concentrations. These compounds also increase the specific activity of creatine kinase (CK) as well as intracellular Ca2+ concentration in both VSMC and human female-derived cultured bone cells (OBs), and stimulate ERK1/2 phosphorylation in VSMC. At least some of these effects are exerted via membranal binding sites (mER), as would appear from observations that protein-bound, membrane impermeant estrogenic complexes can mimic the effect of E2 on DNA synthesis, intracellular Ca2+ concentration and MAPK, but not on CK activity. We now extend these studies by examining the effects of a novel carboxy-derivative of biochanin A, 6-carboxy-biochanin A (cBA) in VSMC and human osteoblasts in culture. cBA increased DNA synthesis in VSMC in a dose-dependent manner and was able to maintain this effect when linked to a cell membrane impermeable protein. In VSMC both cBA and estradiol, in their free or protein-bound forms induced a steep and immediate rise in intracellular calcium. Both the free and protein-bound conjugates of cBA and estradiol increased net MAPK-kinase activity. Neither the stimulatory effect of cBA nor the inhibitory effect of estradiol on DNA synthesis in VSMC could be shown in the presence of the MAPK-kinase inhibitor UO126. The presence of membrane binding sites for both estradiol and cBA was supported by direct visualization, using fluorescence labeling of their respective protein conjugates, E2-BSA and cBA-ovalbumin. Furthermore, these presumed membrane ER for estradiol and cBA were co-localized. In cultured human osteoblasts, cBA stimulated CK activity in a dose related fashion, which paralleled the increase in CK induced by estradiol per se, confirming the estrogenic properties of cBA in human bone cells. Both the free and protein-bound forms of cBA elicited immediate and substantial increments in intracellular Ca2+, similar to, but usually larger than the responses elicited by estradiol per se. cBA also increased ERalpha and suppressed ERbeta mRNA expression in human osteoblasts. Cultured human osteoblasts also harbor membrane binding sites for protein-bound form of cG, which are co-localized with the binding sites for protein-bound estradiol. The extent to which these properties of the novel synthetic phytoestrogen derivatives may be utilized to avert human vascular and/or bone disease requires further study.

Vitamin D analogs induce lipoxygenase mRNA expression and activity as well as reactive oxygen species (ROS) production in human bone cells

Vitamin D metabolites or its less-calcemic analogs (JKF or QW) are beneficial for bone biology. We analyzed whether or not 25(OH)D3 (25), 1,25(OH)2D3 (1,25), JKF or QW regulate lipooxygenase (LO) enzymes expression and their products hydroxyeicosatetraenoic acid (12 and 15 HETE) formation as well as reactive oxygen species (ROS) production in human bone cell lines (SaOS2 and hFOB) and primary cultured human bone cells (Obs) from males or females. All compounds except 25 increased LOs mRNA expression and HETE production in female or male Obs. ROS formation was induced by JKF and QW in both cell lines, and was inhibited by different inhibitors. Baicalein (baic) an inhibitor of 12 and 15 LO activity, inhibited partially ROS formation by JKF or QW in SaSO2 and hFOB. JKF-stimulated DNA synthesis in female Obs was inhibited by baic but unchanged by addition of HETE or HETE with baic. These results indicate that vitamin D increased oxidative stress in bone cells is in part via induction of LO expression and activity. This new feature of vitamin D is probably mediated by intracellular and/or membranal receptors and its potential hazard could lead to potential damage due to increased lipid oxidation.

Calciotrophic hormones and hyperglycemia modulate vitamin D receptor and 25 hydroxyy vitamin D 1-α hydroxylase mRNA expression in human vascular smooth muscle cells

Estrogen receptors (ERα and ERβ), the vitamin D receptor (VDR) and 25 hydroxyy vitamin D 1-α hydroxylase (1OHase) mRNA are expressed in vascular smooth muscle cells (VSMC). In these cells estrogenic hormones modulate cell proliferation as measured by DNA synthesis (DNA). In the present study we determined whether or not the calciotrophic hormones PTH 1-34 (PTH) and less- calcemic vitamin D analog QW as well as hyperglycemia can regulate DNA synthesis and CK. E2 had a bimodal effect on VSMC DNA synthesis, such that proliferation was inhibited at 30nM but stimulated at 0.3nM. PTH at 50nM increased, whereas QW at 10nM inhibited DNA synthesis. Hyperglycemia inhibited the effects on high E2, QW and PTH on DNA only. Both QW and PTH increased ERα mRNA expression, but only PTH increased ERβ expression. Likewise, both PTH and QW stimulated VDR and 1OHase expression and activity. ERβ, VDR and 1OHase expression and activity were inhibited by hyperglycemia, but ERα expression was unaffected by hyperglycemia. In conclusion, calcitrophic hormones modify VSMC growth and concomitantly affect ER expression in these cells as well as the endogenous VSMC vitamin D system elements, including VDR and 1OHase. Some of the later changes may likely participate in growth effects. Of importance in the observation is that several regulatory effects are deranged in the presence of hyperglycemia, particularly the PTH- and vitamin D-dependent up regulation of VDR and 1OHase in these cells. The implications of these effects require further studies. This article is part of a Special Issue entitled 17th Vitamin D Workshop.

Vitamin D less-calcemic analog modulates the expression of estrogen receptors, vitamin D receptor and 1α-hydroxylase 25-hydroxy vitamin D in human thyroid cancer cell lines

Estrogen receptors (ERs) are expressed in various non-reproductive cancer cell types. Some cancer types express 1α-hydroxylase 25-hydroxy vitamin D (1OHase) whose product, 1,25(OH)2D3 can retard cancer cell proliferation. Thyroid carcinoma cell growth is apparently promoted by estrogens, but whether or not this interaction is modified by vitamin D metabolites/analogs is presently unknown. Here we assessed the effect of a less calcemic vitamin D analog [JK 1624 F2-2 (JKF)] in three human thyroid cancer cell lines: ARO (anaplastic carcinoma), NPA (papillary carcinoma) and MRO (follicular carcinoma). (1) All cell lines expressed both ERα and ERβ, vitamin D receptor (VDR) and 1OHase mRNA quantified by Real Time PCR. There was a general abundance of ERβ over ERα expression, such that the ratio of ERβ to ERα mRNA was >1000:1, 228:1 and 7.7:1 in ARO, MRO and NPA cells, respectively. (2) JKF up regulated ERβ expression in ARO (by 110±15%) and MRO (by 280±10%) but down regulated ERβ in NPA cells (by 40±15%). The expression of VDR was up regulated by JKF in NPA (21±6%), down regulated in ARO (-24±7%) and not affected in MRO. (3) All three human thyroid cancer cell lines were found to express 1OHase, which was up regulated by JKF in MRO (350±25%) and NPA (35±8%) but down regulated in ARO (-20±5%). This is the first report to describe direct regulation of VDR and 1OHase expression by a vitamin D analog in human thyroid cancer cells. A functional role for the vitamin D system in human thyroid cancer is suggested by the finding that the vitamin D analog can affect ERs expression which is in turn involved in estrogen-induced cell growth in an ER-type specific manner in these cells.

12S-Lipoxygenase is necessary for human vascular smooth muscle cell survival

Considerable evidence has been published demonstrating the importance of lipoxygenase enzymes for vascular smooth muscle cell (VSMC) growth. The current study sets out to determine whether or not 12-lipoxygenase (12LO) is also important for human placental VSMC survival. Both a pharmacological and two 12LO antisense knockdown approaches were applied. The 12LO inhibitor baicalien induced a 2-2.5-fold increase in cell death, which appeared to result from apoptosis, as indicated by DNA fragmentation, activation of procaspase 3 to caspase 3 and cytochrome C release from the mitochondria to the cytosol. This apoptosis could be prevented by treatment with the 12LO product, 12 hydroxyeicosatetraenoic acid (12HETE). Human platelet-type 12LO-antisense knockdown, by either plasmid transfection or adeno-associated virus (AAV) infection also induced substantial VSMC death over controls, which could also be prevented by treatment with 12HETE, but not 5HETE. Hence, biochemical 12LO inhibition or 12LO-antisense knockdown in VSMC can induce programmed cell death. These observations suggest a previously unrecognized association between human VSMC survivability and 12LO.

The effects of Femarelle and Vitamin D Analog on Lipoxygenase Products and Reactive Oxygen Species (ROS) formation in the Female and Male Human-Derived Cultured Osteoblastic Cells

1. AbstractWe have previously reported that human primary cultured bone cells (hObs) respond to estradiol-17β (E2) and different phytoestrogens and vitamin D analogs by modulating cell proliferation. In the present study hObs derived from biopsies of pre- or post- menopausal females or males were cultured and treated with different estrogens and with the non-calcemic vitamin D JK-1624F2-2 (JKF) and assayed for the expression of 12LO and 15LO mRNA as well as for the synthesis of 12 and 15HETEs and the formation of ROS. All cells express 12LO and 15LO and produce 12 and 15 HETEs. Treatment with all estrogens: E2, daidzein (D) and femarelle (F) as well as JKF modulated the expression of both 12 and 15LO mRNA in pre- or post-menopausal female hObs and to some extent in male hObs. 12 and 15HETEs production was also modulated by these compounds in these cells. ROS formation was increased by all hormones in pre- and post- but not in male hObs. ROS formation was also increased by 12 and 15HETEs in pre- and post- ...

Mutual modulation of femarelle and vitamin D analog activities in human derived female cultured osteoblasts

Abstract Introduction: Pre-, post-menopausal, and male human osteoblasts (hObs) express estradiol-17β (E2) receptors α and β (ERα, ERβ), vitamin D receptor (VDR), and 25-hydroxy vitamin D3 1-α hydroxylase (1OHase) and produce 1,25 (OH)2D3 (1,25D). Pre-treatment with JKF (JKF 1624F2–2) up-regulated estrogenic responsiveness, via modulation of ERs expression and E2 binding. These estrogens induce VDR and 1OHase expression and 1,25D production. Purpose: Compared the effects of femarelle (F) to daidzein (D) and E2 by themselves and their modulation by JKF. Methods: hObs derived from human bones at different ages and sex were cultured, treated with different hormones and analyzed for the different parameters. Results: (1) F, D and E2 stimulated 3[H] thymidine incorporation (DNA) and creatine kinase specific activity (CK) in female but not in male hObs. The responses to E2 and to D unlike to F were up-regulated by JKF. (2) All hormones increased ERα and decreased ERβ mRNA in all hObs. (3) JKF modulated in different ways the expressions of ERα and ERβ mRNA in all hObs. (4) JKF did not significantly modulate the expressions of ERα and ERβ mRNA in all hObs. (5) JKF increased intracellular competitive binding of E2 by all hormones only in female hObs. (6) Pre-treatment with all hormones increased VDR and 1OHase expression and 1,25D formation in pre- and post-, but only JKF modulated it in male hObs. Conclusions: F, D and E2 increase different parameters in hObs. However, pre-treatment with JKF modulates the effect of E2 and D but not of F. F reciprocally modulates mRNA expression and activity of 1OHase which in turn up-regulate ERs expression and activity. These finding may contribute to F’s beneficial role in treatment of post-menopausal bone loss even in vitamin D deficiency.