1α,25(OH)2D3-glycosides from Solanum glaucophyllum leaves extract induce myoblasts differentiation through p38 MAPK and AKT activation.

We have previously shown that Solanum glaucophyllum leaf extract (SGE) increases VDR protein levels and promotes myoblast differentiation. Here, we investigated whether p38 MAPK and AKT are involved in SGE actions. Cell-cycle studies showed that SGE prompted a peak of S-phase followed by an arrest in the G0/G1-phase through p38 MAPK. Time course studies showed that p38 MAPK and AKT phosphorylation were statistically increased by SGE (10 nM) or synthetic 1α,25(OH)2D3 (1 nM) treatment. Furthermore, p38 MAPK and AKT inhibitors, SB203580 and LY294002 respectively, suppressed myoblasts fusion induced by SGE or synthetic 1α,25(OH)2D3 We have also studied differentiation genes by qRT-PCR. myoD1 mRNA increased significantly by SGE (24-72 h) or 1α,25(OH)2D3 (24 h) treatment. mRNA expression of myogenin also increased upon SGE or 1α,25(OH)2D3 treatment. Finally, MHC2b mRNA expression, a late differentiation marker, was increased significantly by both compounds at 72 h compared to control. Taken together, these results suggest that SGE, as synthetic 1α,25(OH)2D3, promotes myotube formation through p38 MAPK and AKT activation.

The proapoptotic protein Bim is up regulated by 1α,25-dihydroxyvitamin D3 and its receptor agonist in endothelial cells and transformed by viral GPCR associated to Kaposi sarcoma.

We have previously shown that 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] and its less calcemic analog TX 527 induce apoptosis via caspase-3 activation in endothelial cells (SVEC) and endothelial cells transformed by the viral G protein-coupled receptor associated to Kaposi sarcoma (vGPCR). In this work, we studied whether intrinsic apoptotic pathway could be activated by changing the balance between anti and pro-apoptotic proteins. Time response qRT-PCR analysis demonstrated that the mRNA level of anti-apoptotic gene Bcl-2 decreased after 12h and increased after 48h treatment with 1α,25(OH)2D3 or TX 527 in SVEC and vGPCR cells, whereas its protein level remained unchanged through time. mRNA levels of pro-apoptotic gene Bax significantly increased only in SVEC after 24 and 48h treatment with 1α,25(OH)2D3 and TX 527 although its protein levels remained unchanged in both cell lines. Bim mRNA and protein levels increased in SVEC and vGPCR cells. Bim protein increase by 1α,25(OH)2D3 and TX 527 was abolished when the expression of vitamin D receptor (VDR) was suppressed. On the other hand, Bortezomib (0.25-1nM), an inhibitor of NF-κB pathway highly activated in vGPCR cells, increased Bim protein levels and induced caspase-3 cleavage. Altogether, these results indicate that 1α,25(OH)2D3 and TX 527 trigger apoptosis by Bim protein increase which turns into the activation of caspase-3 in SVEC and vGPCR cells. Moreover, this effect is mediated by VDR and involves NF-κB pathway inhibition in vGPCR.

Age-related changes in the response of intestinal cells to 1α,25(OH)2-vitamin D3.

The hormonally active form of vitamin D(3), 1α,25(OH)(2)-vitamin D(3), acts in intestine, its major target tissue, where its actions are of regulatory and developmental importance: regulation of intracellular calcium through modulation of second messengers and activation of mitogenic cascades leading to cell proliferation. Several causes have been postulated to modify the hormone response in intestinal cells with ageing, among them, alterations of vitamin D receptor (VDR) levels and binding sites, reduced expression of G-proteins and hormone signal transduction changes. The current review summarizes the actual knowledge regarding the molecular and biochemical basis of age-impaired 1α,25(OH)(2)-vitamin D(3) receptor-mediated signaling in intestinal cells. A fundamental understanding why the hormone functions are impaired with age will enhance our knowledge of its importance in intestinal cell physiology.

NFκB pathway is down-regulated by 1α,25(OH)(2)-vitamin D(3) in endothelial cells transformed by Kaposi sarcoma-associated herpes virus G protein coupled receptor.

We have previously demonstrated that 1α,25 dihydroxy-vitamin D(3) (1α,25(OH)(2)D(3)) has antiproliferative effects on the growth of endothelial cells transformed by the viral G protein-coupled receptor associated to Kaposi sarcoma (vGPCR). In this work, we have investigated whether 1α,25(OH)(2)D(3) exerts its growth inhibitory effects by inhibiting the Nuclear Factor κ B (NFκB) pathway which is highly activated by vGPCR. Cell proliferation studies demonstrated that 1α,25(OH)(2)D(3), similarly to bortezomib, a proteosome inhibitor that suppresses the activation of NFκB, reduced the proliferation of endothelial cells transformed by vGPCR (SVEC-vGPCR). The activity of NFκB in these cells decreased by 70% upon 1α,25(OH)(2)D(3) treatment. Furthermore, time and dose response studies showed that the hormone significantly decreased NFκB and increased IκBα mRNA and protein levels in SVEC-vGPCR cells, whereas in SVEC only IκBα increased significantly. Moreover, NFκB translocation to the nucleus was inhibited and occurred by a mechanism independent of NFκB association with vitamin D(3) receptor (VDR). 1α,25(OH)(2)D(3)-induced increase in IκBα required de novo protein synthesis, and was independent of MAPK and PI3K/Akt pathways. Altogether, these results suggest that down-regulation of the NFκB pathway is part of the mechanism involved in the antiproliferative effects of 1α,25(OH)(2)D(3) on endothelial cells transformed by vGPCR.

[Vitamin D and cancer: antineoplastic effects of 1α,25(OH)2-vitamin D3]. / Vitamina D y cáncer: acción antineoplásica de la 1α,25(OH)2-vitamina D3.

The hormonal form of vitamin D, 1α,25(OH)2-vitamin D3 (1α,25(OH)2D3), in addition of playing a central role in the control of calcium homeostasis in the body, regulates the growth and differentiation of different cell types, including cancer cells. At present several epidemiologic and clinical studies investigate the effect of the hormone in these cells due to the interest in the therapeutic use of 1α,25(OH)2D3 and analogues with less calcemic activity for prevention or treatment of cancer. This review describes vitamin D endocrine system, its mechanism of action, its antineoplastic activity and provides information about the latest advances in the study of new hormone analogues with less calcemic activity for cancer treatment.

Vitamina D y cáncer: acción antineoplásica de la 1α, 25(OH)2 -vitamina D3 / Vitamin D and cancer: antineoplastic effects of 1α,25(OH)2-vitamin D3

La forma hormonalmente activa de la vitamina D, 1α,25(OH)2-vitamina D3 (1α,25(OH)2D3), además de desempeñar un rol crucial en el mantenimiento de la homeostasis de calcio en el cuerpo, también regula el crecimiento y la diferenciación de diferentes tipos celulares, incluyendo células cancerosas. Actualmente hay numerosos estudios que investigan los efectos de la hormona en estas células, debido al interés en el uso terapéutico del 1α,25(OH)2D3 y de análogos con menor actividad calcémica para el tratamiento o prevención del cáncer. En este trabajo de revisión se describe el sistema endocrino de la vitamina D, su mecanismo de acción, su acción antineoplásica y se provee información sobre los últimos avances en el estudio de nuevos análogos de la hormona con menos actividad calcémica para el tratamiento del cáncer.

The hormonal form of vitamin D, 1α,25(OH)2-vitamin D3 (1α,25(OH)2D3), in addition of playing a central role in the control of calcium homeostasis in the body, regulates the growth and differentiation of different cell types, including cancer cells. At present several epidemiologic and clinical studies investigate the effect of the hormone in these cells due to the interest in the therapeutic use of 1α,25(OH)2D3 and analogues with less calcemic activity for prevention or treatment of cancer. This review describes vitamin D endocrine system, its mechanism of action, its antineoplastic activity and provides information about the latest advances in the study of new hormone analogues with less calcemic activity for cancer treatment.

Vitamina D y cáncer: acción antineoplásica de la 1α, 25(OH)2 -vitamina D3 / Vitamin D and cancer: antineoplastic effects of 1α,25(OH)2-vitamin D3

La forma hormonalmente activa de la vitamina D, 1α,25(OH)2-vitamina D3 (1α,25(OH)2D3), además de desempeñar un rol crucial en el mantenimiento de la homeostasis de calcio en el cuerpo, también regula el crecimiento y la diferenciación de diferentes tipos celulares, incluyendo células cancerosas. Actualmente hay numerosos estudios que investigan los efectos de la hormona en estas células, debido al interés en el uso terapéutico del 1α,25(OH)2D3 y de análogos con menor actividad calcémica para el tratamiento o prevención del cáncer. En este trabajo de revisión se describe el sistema endocrino de la vitamina D, su mecanismo de acción, su acción antineoplásica y se provee información sobre los últimos avances en el estudio de nuevos análogos de la hormona con menos actividad calcémica para el tratamiento del cáncer.(AU)

The hormonal form of vitamin D, 1α,25(OH)2-vitamin D3 (1α,25(OH)2D3), in addition of playing a central role in the control of calcium homeostasis in the body, regulates the growth and differentiation of different cell types, including cancer cells. At present several epidemiologic and clinical studies investigate the effect of the hormone in these cells due to the interest in the therapeutic use of 1α,25(OH)2D3 and analogues with less calcemic activity for prevention or treatment of cancer. This review describes vitamin D endocrine system, its mechanism of action, its antineoplastic activity and provides information about the latest advances in the study of new hormone analogues with less calcemic activity for cancer treatment.(AU)

Vitamina D y cáncer: acción antineoplásica de la 1α, 25(OH)2 -vitamina D3 / Vitamin D and cancer: antineoplastic effects of 1α,25(OH)2-vitamin D3

La forma hormonalmente activa de la vitamina D, 1α,25(OH)2-vitamina D3 (1α,25(OH)2D3), además de desempeñar un rol crucial en el mantenimiento de la homeostasis de calcio en el cuerpo, también regula el crecimiento y la diferenciación de diferentes tipos celulares, incluyendo células cancerosas. Actualmente hay numerosos estudios que investigan los efectos de la hormona en estas células, debido al interés en el uso terapéutico del 1α,25(OH)2D3 y de análogos con menor actividad calcémica para el tratamiento o prevención del cáncer. En este trabajo de revisión se describe el sistema endocrino de la vitamina D, su mecanismo de acción, su acción antineoplásica y se provee información sobre los últimos avances en el estudio de nuevos análogos de la hormona con menos actividad calcémica para el tratamiento del cáncer.(AU)

The hormonal form of vitamin D, 1α,25(OH)2-vitamin D3 (1α,25(OH)2D3), in addition of playing a central role in the control of calcium homeostasis in the body, regulates the growth and differentiation of different cell types, including cancer cells. At present several epidemiologic and clinical studies investigate the effect of the hormone in these cells due to the interest in the therapeutic use of 1α,25(OH)2D3 and analogues with less calcemic activity for prevention or treatment of cancer. This review describes vitamin D endocrine system, its mechanism of action, its antineoplastic activity and provides information about the latest advances in the study of new hormone analogues with less calcemic activity for cancer treatment.(AU)

Signal transduction pathways associated with ATP-induced proliferation of colon adenocarcinoma cells.

BACKGROUND: In previous work, we have demonstrated that extracellular adenosine 5'-triphosphate (ATP) acts on intestinal Caco-2 cell P2Y receptors promoting a rapid increase in the phosphorylation of ERK1/2, p46 JNK and p38 MAP kinases (MAPKs). METHODS AND RESULTS: In this study, we investigated whether the extracellular ATP-P2Y receptor signalling pathways were required for the proliferation of Caco-2 cells. Confocal microscopy and immunobloting studies showed that ERK1/2 and JNK translocate into the nucleus of the cells stimulated by ATP, where they participate, together with p38 MAPK, in the phosphorylation of JunD, ATF-1 and ATF-2 transcription factors. In addition, ATP through the activation of MAPKs induces the expression of the immediate early genes products of the Jun family, c-Fos and MAP kinase phosphatase-1 (MKP-1). Moreover, ERK1/2 and p38 MAPK are involved in the phosphorylation of MKP-1 in Caco-2 cells. Of physiological significance, in agreement with the mitogenic role of the MAPK cascade, ATP increased Caco-2 cell proliferation, and this effect was blocked by UO126, SB203580 and SP600125, the specific inhibitors of ERK1/2, p38 MAPK and JNK1/2, respectively. CONCLUSION: Extracellular ATP induces proliferation of Caco-2 human colonic cancer cells by activating MAPK cascades and modulation of transcription factors. GENERAL SIGNIFICANCE: These findings and identification of the specific P2Y subtype receptors involved in the mitogenic effect of ATP on Caco-2 cells might be relevant for understanding tumor cell development, resistance to treatment regimens and the design of new therapeutic strategies.

Mecanismo de acción de PTH en células de adenocarcinoma de colon humano / Mechanism of action of pth on cells of human colonic adenocarcinoma

El concepto sobre el rol de la hormona paratiroidea (PTH) actuando sobre los tejidos blanco clásicos (hueso y riñón), se ha expandido a tejidos no clásicos como el intestino donde ejerce importantes funciones regulatorias. En células intestinales, PTH luego de unirse a su receptor (PTHR1) en la membrana plasmática, activa las vías de señalización de AMPC/PKA, DAG/IP3/PKC, las cascadas de las MAP quinasas y regula la concentración de Ca2+ intracelular. En la línea celular intestinal Caco-2, derivada de adenocarcinoma de colon humano, el tratamiento con PTH en ausencia de suero disminuye el número de células viables e induce cambios morfológicos consistentes con la apoptosis: alteración de los filamentos de actina y consecuentemente de la forma celular, pérdida de las uniones intercelulares, externalización de la fosfatidilserina de membrana, distribución perinuclear de las mitocondrias, condensación nuclear y fragmentación del ADN. Además la hormona induce la desfosforilación de la proteína pro-apoptótica Bad, su disociación de la proteína 14-3-3 y su translocación a las mitocondrias con la consecuente liberación de citocromo c y Smac/Diablo al citosol, lo que resulta en la activación de caspasa-3 y el clivaje de su sustrato PARP. En estas células, PTH además de activar la vía mitocondrial de la apoptosis, inhibe la vía de supervivencia de AKT mediante la acción concertada de la serina-treonina fosfatasa PP2A y la vía del AMPc. El conocimiento de los mecanismos moleculares involucrados en la apoptosis de células intestinales podría ser utilizado para generar fármacos pro-apoptóticos en el tratamiento del cáncer de colon humano.

Pro-apoptotic effects of parathyroid hormone in intestinal cells.

Apoptosis, a form of programmed cell death, is a process fundamental to normal growth and development, immune response, tissue remodeling after injury or insult, and homeostasis of the intestinal epithelium. Recently, it has become apparent that apoptosis is a crucial process in skeletal development and homeostasis, and that signaling by the parathyroid hormone (PTH) receptor can either promote or suppress apoptosis depending on the cellular context. In this study, we evaluated the role of PTH in intestinal apoptosis using human colonic Caco-2 cells. To that end, Caco-2 cells were exposed to PTH (10-8 mol/L) for 1-5 days. Evaluation of cell survival by use of resazurin staining, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) staining, and crystal violet staining revealed that PTH treatment diminishes the number of viable cells. Assessment of cells after PTH treatment by use of propidium iodide showed that the hormone increased the number of red-stained (dead) cells (178%, 5 days). Moreover, we found that the hormone induced disruption of actin filaments with changes to cellular shape, alteration of cell-to-cell junctions, externalization of membrane phosphatidylserine, chromatin condensation, and DNA fragmentation of the nucleus, which are morphological features consistent with apoptosis. In addition, PTH treatment revealed a cytosolic staining pattern of 14-3-3. However, the significance of such differential localization for 14-3-3 function remains unknown. Taken together, the present study suggests that PTH promotes apoptosis in Caco-2 intestinal cells.

Expression and localization of estrogen receptor alpha in the C2C12 murine skeletal muscle cell line.

The classical model of 17beta-estradiol action has been traditionally described to be mediated by the estrogen receptor (ER) localized exclusively in the nucleus. However, there is increasing functional evidence for extra nuclear localization of ER. We present biochemical, immunological and molecular data supporting mitochondrial-microsomal localization of ER alpha in the C2C12 skeletal muscle cell line. We first established [(3)H]17beta estradiol binding characteristics in whole cells in culture. Specific and saturable [(3)H]17beta estradiol binding sites of high affinity were then detected in mitochondrial fractions (K(d) = 0.43 nM; B(max) = 572 fmol/mg protein). Immunocytological studies revealed that estrogen receptors mainly localize at the mitochondrial and perinuclear level. These results were also confirmed using fluorescent 17beta estradiol-BSA conjugates. The immunoreactivity did not translocate into the nucleus by 17beta-estradiol treatment. Western and Ligand blot approaches corroborated the non-classical localization. Expression and subcellular distribution of ER alpha proteins were confirmed in C2C12 cells transfected with ER alpha siRNA and by RT-PCR employing specific primers. The non-classical distribution of native pools of ER alpha in skeletal muscle cells suggests an alternative mode of ER localization/function.

ATP modulation of mitogen activated protein kinases and intracellular Ca2+ in breast cancer (MCF-7) cells.

In the breast tumor cell line MCF-7, extracellular nucleotides induce transient elevations in intracellular calcium concentration ([Ca(2+)](i)). In this study we show that stimulation with ATP or UTP sensitizes MCF-7 cells to mechanical stress leading to an additional transient Ca(2+) influx. ATP> or =ATPgamma-S> or =UTP>>>ADP=ADPbeta-S elevate [Ca(2+)](i), proving the presence of P2Y(2)/P2Y(4) purinergic receptor subtypes. In addition, cell stimulation with ATP, ATPgamma-S or UTP but not ADPbeta-S induced the phosphorylation of ERK1/2, p38 and JNK1/2 mitogen activated protein kinases (MAPKs). The use of Gd(3+), La(3+) or a Ca(2+)-free medium, inhibited ATP-dependent stress activated Ca(2+) (SAC) influx, but had no effect on MAPK phosphorylation. ATP-induced activation of MAPKs was diminished by two PI-PLC inhibitors and an IP(3) receptor antagonist. These results evidence an ATP-sensitive SAC influx in MCF-7 cells and indicate that phosphorylation of MAPKs by ATP is dependent on PI-PLC/IP(3)/Ca(2+)(i) release but independent of SAC influx in these cells, differently to other cell types.

Implication of Gbetagamma proteins and c-SRC tyrosine kinase in parathyroid hormone-induced signal transduction in rat enterocytes.

Parathyroid hormone (PTH) interacts in target tissues with a G protein-coupled receptor (GPCR) localized in the plasma membrane. Although activation of GPCR can elicit rapid stimulation of cellular protein tyrosine phosphorylation, the mechanism by which G proteins activate protein-tyrosine kinases is not completely understood. In the present work, we demonstrate that PTH rapidly increases the activity of non-receptor tyrosine kinase c-Src in rat intestinal cells (enterocytes). The response is biphasic, the early phase is fast and transient, peaking at 30 s (+120%), while the second phase progressively increases up to 5 min (+220%). The hormone activates c-Src in intestinal cells through fast changes in tyrosine phosphorylation of the enzyme. The first event in the activation of c-Src is the dephosphorylation of Tyr527 (which happens after a few seconds of PTH treatment), followed by a second event of activation with phosphorylation at Tyr416 (+twofold, 5 min). Removal of external Ca(2+) (EGTA, 0.5 mM) and chelation of intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetracetic acid acetoxymethyl ester (BAPTA) (5 microM) suppressed Tyr527 dephosphorylation and Tyr416 phosphorylation, indicating that Ca(2+) is an upstream activator of c-Src in enterocytes stimulated with PTH. The G protein subunits, Galphas and Gbeta, are associated with c-Src in basal conditions and this association increases two- to threefold in cells treated with PTH. Blocking of Gbeta subunits by preincubation of cells with a Gbeta antibody abolished hormone-dependent c-Src Tyr416 phosphorylation and ERK1/ERK2 activation. The results of this work indicate that PTH activates c-Src in intestinal cells through conformational changes via G proteins and calcium-dependent modulation of tyrosine phosphorylation of the enzyme, and that PTH receptor activation leads via Gbetagamma-c-Src to the phosphorylation of the MAP kinases, ERK1 and ERK2.

Age-related changes in the response of intestinal cells to parathyroid hormone.

The concept of the role(s) of parathyroid hormone (PTH), has expanded from that on acting on the classical target tissues, bone and kidney, to the intestine where its actions are of regulatory and developmental importance: regulation of intracellular calcium through modulation of second messengers and, activation of mitogenic cascades leading to cell proliferation. Several causes have been postulated to modify the hormone response in intestinal cells with ageing, among them, alterations of PTH receptor (PTHR1) binding sites, reduced expression of G proteins and hormone signal transduction changes. The current review summarizes the actual knowledge regarding the molecular and biochemical basis of age-impaired PTH receptor-mediated signaling in intestinal cells. A fundamental understanding of why PTH functions are impaired with age will enhance our understanding of its importance in intestinal cell physiology.

Involvement of PI3-kinase and its association with c-Src in PTH-stimulated rat enterocytes.

Phosphoinositide-3-kinase (PI3K) is a lipid kinase, which phosphorylates the D3 position of phosphoinositides, and is known to be activated by a host of protein tyrosine kinases. PI3K plays an important role in mitogenesis in several cell systems. However, whether parathyroid hormone (PTH) affects the activity and functional roles of PI3K in intestinal cells remain to be determined. The objective of this study was to identify and characterize the PI3K pathway, and its relation to other non-receptor tyrosine kinases in mediating PTH signal transduction in rat enterocytes. PTH dose- and time-dependently increased PI3K activity with a peak occurring at 2 min. The tyrosine kinase inhibitor genistein, c-Src inhibitor PP1 and two structurally different inhibitors of PI3K, LY294002 and wortmannin, suppressed PI3K activity dependent on PTH. Co-immunoprecipitation analysis showed a constitutive association between c-Src and PI3K, which was enhanced by PTH treatment, suggesting that the cytosolic tyrosine kinase forms an immunocomplex with PI3K probably via the N-SH2 domain of the p85alpha regulatory subunit. In response to PTH, tyrosine phosphorylation of p85alpha was enhanced, effect that was abolished by PP1, the inhibitor of c-Src kinase. PTH causes a rapid (0.5-5 min) phosphorylation of Akt/PKB, effect that was abrogated by PI3K inhibitors, indicating that in rat enterocytes, PI3K is an upstream mediator of Akt/PKB activation by PTH. We report here that PI3K is also required for PTH activation of the mitogen-activated protein kinases ERK1 and ERK2. Taken together, the present study demonstrate, for the first time, that PTH rapidly and transiently stimulates PI3K activity and its down effector Akt/PKB in rat enterocytes playing c-Src kinase a central role in PTH-dependent PI3K activation and that PI3K signaling pathway contributes to PTH-mediated MAPK activation.