In vitro effects of 1α,25(OH)₂D₃-glycosides from Solbone A (Solanum glaucophyllum leaves extract; Herbonis AG) compared to synthetic 1α,25(OH)₂D₃ on myogenesis.

The presence of glycoside derivatives of 1α,25(OH)2D3 endows plants to gradual release of the free bioactive form of 1α,25(OH)2D3 from its glycoconjugates by endogenous animal tissue glycosidases. This results in increased half-life of the hormone in blood when purified plant fractions are administered for therapeutic purposes. In this work, we evaluated the role 1α,25(OH)2D3-glycosides enriched natural product (Solbone A) from Solanum glaucophyllum leaf extract compared with synthetic 1α,25(OH)2D3 on myogenic differentiation in C2C12 myoblasts.

Molecular mechanisms associated with PTHrP-induced proliferation of colon cancer cells.

Parathyroid Hormone-related Protein (PTHrP) is normally produced in many tissues and is recognized for its endocrine, paracrine, autocrine and intracrine modes of action. PTHrP is also implicated in different types of cancer and its expression correlates with the severity of colon carcinoma. Using the human colon cell line Caco-2 we recently obtained evidence that PTHrP, through a paracrine pathway, exerts a protective effect under apoptotic conditions.

Involvement of ERK1/2, p38 MAPK, and PI3K/Akt signaling pathways in the regulation of cell cycle progression by PTHrP in colon adenocarcinoma cells.

Parathyroid hormone-related peptide (PTHrP) is distributed in most fetal and adult tissues, and its expression correlates with the severity of colon carcinoma. Recently we obtained evidence that in Caco-2 cells, a cell line from human colorectal adenocarcinoma, exogenous PTHrP increases the number of live cells, via ERK1/2, p38 MAPK, and PI3-kinase and induces the expression of cyclin D1, a cell cycle regulatory protein. In this study, we further investigated the role of PTHrP in the regulation of the cell cycle progression in these intestinal cells.

Cell cycle arrest and apoptosis induced by 1α,25(OH)2D3 and TX 527 in Kaposi sarcoma is VDR dependent.

We have previously shown that 1α,25(OH)2-Vitamin D3 [1α,25(OH)2D3] and its less calcemic analog TX 527 inhibit the proliferation of endothelial cells transformed by the viral G protein-coupled receptor associated to Kaposi sarcoma (vGPCR) and this could be partially explained by the inhibition of the NF-κB pathway. In this work, we further explored the mechanism of action of both vitamin D compounds in Kaposi sarcoma. We investigated whether the cell cycle arrest and subsequent apoptosis of endothelial cells (SVEC) and SVEC transformed by vGPCR (SVEC-vGPCR) elicited by 1α,25(OH)2D3 and TX 527 were mediated by the vitamin D receptor (VDR).

Role of PTHrP in human intestinal Caco-2 cell response to oxidative stress.

We have previously demonstrated that parathyroid hormone (PTH) induces apoptosis in human colon adenocarcinoma Caco-2 cells but the effects of its tumoral analog PTH-related peptide (PTHrP) in this cell line are still unknown. In the present work we investigated whether PTHrP, as PTH, is able to induce Caco-2 cell apoptosis or if it exerts protective effects under apoptotic conditions. Using Caco-2 cells cultured under serum deprivation in the presence or absence of PTHrP we demonstrated that, differently to PTH, its analog employed at the same concentration (10(-8)M) is not a pro-apoptotic hormone.

Parathyroid hormone and the regulation of cell cycle in colon adenocarcinoma cells.

Parathyroid hormone (PTH) functions as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis. In this study, we investigated the role of PTH in the regulation of the cell cycle in human colon adenocarcinoma Caco-2 cells. Flow cytometry analysis revealed that PTH (10(-8)M, 12-24h) treatment increases the number of cells in the G0/G1 phase and diminishes the number in both phases S and G2/M.

PTH inactivates the AKT survival pathway in the colonic cell line Caco-2.

In previous works, we found that PTH promotes the apoptosis of human Caco-2 intestinal cells, through the mitochondrial pathway. This study was conducted to investigate the modulation of different players implicated in the AKT survival pathway in PTH-induced intestinal cell apoptosis. We demonstrate, for the first time, that PTH modulates AKT phosphorylation in response to apoptosis via the serine/threonine phosphatase PP2A.

1 Alpha,25-dihydroxyvitamin D3 and its TX527 analog inhibit the growth of endothelial cells transformed by Kaposi sarcoma-associated herpes virus G protein-coupled receptor in vitro and in vivo.

The Kaposi sarcoma-associated herpes virus-G protein-coupled receptor is a key molecule in the pathogenesis of Kaposi sarcoma, playing a central role in promoting vascular endothelial growth factor-driven angiogenesis and spindle cell proliferation. We studied the effects of 1 alpha,25-dihydroxyvitamin D(3) [1 alpha,25(OH)(2)D(3)] and the analog TX527 on the proliferation of endothelial cells (SVECs) and SVECs transformed by the viral G protein-coupled receptor (SVEC-vGPCR). 1 alpha,25(OH)(2)D(3) and TX527 decreased SVEC-vGPCR and SVEC numbers, the response being time dependent and similar in both cell lines.

Extracellular ATP activates MAP kinase cascades through a P2Y purinergic receptor in the human intestinal Caco-2 cell line.

Background: ATP exerts diverse effects on various cell types via specific purinergic P2Y receptors. Intracellular signaling cascades are the main routes of communication between P2Y receptors and regulatory targets in the cell.

Methods And Results: We examined the role of ATP in the modulation of ERK1/2, JNK1/2, and p38 MAP kinases (MAPKs) in human colon cancer Caco-2 cells.

Expression and subcellular distribution of native estrogen receptor beta in murine C2C12 cells and skeletal muscle tissue.

We have recently described the expression and intracellular localization of ER alpha in murine C2C12 cells and skeletal muscle tissue. In separate studies, a protective role of 17beta-estradiol against apoptosis exerted mainly at the mitochondrial level was also shown in the C2C12 muscle cell line. However, this functional evidence was in accordance with the participation of ER beta.

MAP kinases in proliferating human colon cancer Caco-2 cells.

The mitogen-activated protein kinase (MAPK) cascade is one of the most ubiquitous signal transduction systems and is rapidly activated by various stimuli, such as cellular stress and death. The Caco-2 cell line is an in vitro model for colon cancer studies. We investigated the activation status of the ERK1/2, p38, JNK1/2, and ERK5 kinases and their respective upstream intracellular activators in Caco-2 cells induced to proliferate by 10% fetal bovine serum (FBS).

The early phase of programmed cell death in Caco-2 intestinal cells exposed to PTH.

The regulation of apoptosis is critical for ensuring the homeostasis of an organism. As such, the cell has derived various mechanisms to precisely control the balance between survival and apoptotic signaling. Parathyroid hormone (PTH) function as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis.

17beta-estradiol signaling in skeletal muscle cells and its relationship to apoptosis.

17beta-estradiol exerts an antiapoptotic action in skeletal muscle cells through extranuclear ERalpha and beta. This protective action, mainly involves a non-genomic mechanism of ERK1/2 and PI3K/Akt activation and BAD phosphorylation. ERbeta plays a major role in the inhibition of apoptosis by 17beta-estradiol at the level of mitochondria, whereas ERalpha and ERbeta mediate the activation of Akt to the same extent, suggesting differential involvement of ER isoforms depending on the step of the apoptotic/survival pathway involved.

Vitamin D receptor levels and binding are reduced in aged rat intestinal subcellular fractions.

The hormonal form of vitamin D, 1alpha,25(OH)(2)-vitaminD(3) [1alpha,25(OH)(2)D(3)], stimulates signal transduction pathways in intestinal cells. To gain insight into the relative importance of the vitamin D receptor (VDR) in the rapid hormone responses, the amounts and localization of the VDR were evaluated in young (3 months) and aged (24 months) rat intestinal cells. Immune-fluorescence and Western blot studies showed that VDR levels are diminished in aged enterocytes.

Activation of MAPKs by 1alpha,25(OH)2-Vitamin D3 and 17beta-estradiol in skeletal muscle cells leads to phosphorylation of Elk-1 and CREB transcription factors.

The mitogen activated protein kinases (MAPKs) have been classified into at least six subfamilies, among which ERK1/2, JNK1/2 and p38 MAPK are the most extensively studied. The steroid hormones 1alpha,25-dihydroxy-Vitamin D(3) and 17beta-estradiol promote biological responses through activation of MAPK cascades in various cell types. We previously reported that 1alpha,25(OH)(2)D(3) rapidly (within 1 min) activates p38 MAPK in C2C12 skeletal muscle cells.

PTH regulation of c-Jun terminal kinase and p38 MAPK cascades in intestinal cells from young and aged rats.

In the present study, we examined the role of Parathyroid hormone (PTH) on the c-Jun N-terminal kinase (JNK) 1/2 and p38 mitogen-activated protein kinase (MAPK) members of the MAPK family as it relates to ageing by measuring hormone-induced changes in their activity in enterocytes isolated from young (3 month old) and aged (24 month old) rats. Our results show that PTH induces a transient activation of JNK 1/2, peaking at 1 min (+threefold). The hormone also stimulates JNK 1/2 tyrosine phosphorylation, in a dose-dependent fashion, this effect being maximal at 10 nM.

Age-related alteration of 1alpha,25(OH)2-vitamin D3-dependent activation of p38 MAPK in rat intestinal cells.

In intestinal cells, 1alpha,25(OH)(2)-vitamin D(3) (1alpha,25(OH)(2)D(3)) regulates gene expression via the specific intracellular vitamin D receptor and induces fast non-transcriptional responses involving stimulation of transmembrane signal transduction pathways. In the present study, we analyzed, for the first time, alterations in p38 MAPK response to 1alpha,25(OH)(2)D(3) in rat enterocytes with ageing. In enterocytes from young rats, the hormone increased, in a time- and dose-dependent fashion, the phosphorylation of p38 MAPK, peaking at 3 min (+2-fold).

1alpha,25(OH)(2)-Vitamin D(3) stimulates intestinal cell p38 MAPK activity and increases c-Fos expression.

In intestinal cells, as in other target cells, the steroid hormone 1alpha,25(OH)(2)-Vitamin D(3) (1alpha,25(OH)(2)D(3)) regulates gene expression via the specific intracellular Vitamin D receptor and induces fast non-transcriptional responses involving stimulation of transmembrane signal transduction pathways. We have previously shown that the hormone activates the extracellular signal-regulated mitogen-activated protein (MAP) kinase isoforms ERK1 and ERK2 in rat intestinal cells. In the present study, we have demonstrated that 1alpha,25(OH)(2)D(3) also induces the phosphorylation and activation of p38 MAPK in these cells.

MAP kinases p38 and JNK are activated by the steroid hormone 1alpha,25(OH)2-vitamin D3 in the C2C12 muscle cell line.

In chick skeletal muscle cell primary cultures, we previously demonstrated that 1alpha,25(OH)2-vitamin D3 [1alpha,25(OH)2D3], the hormonally active form of vitamin D, increases the phosphorylation and activity of the extracellular signal-regulated mitogen-activated protein (MAP) kinase isoforms ERK1 and ERK2, their subsequent translocation to the nucleus and involvement in DNA synthesis stimulation. In this study, we show that other members of the MAP kinase superfamily are also activated by the hormone. Using the muscle cell line C2C12 we found that 1alpha,25(OH)2D3 within 1 min phosphorylates and increases the activity of p38 MAPK.

Modulation of tyrosine phosphorylation signalling pathways by 1alpha,25(OH)2-vitamin D3.

Hormonally active vitamin D(3), 1alpha,25(OH)(2)D(3), interacts with the classic vitamin D nuclear receptor that regulates gene transcription and with a putative cell membrane receptor that mediates rapid biological responses. 1alpha,25(OH)(2)D(3) actions on target tissues regulate: mineral metabolism and intracellular Ca(2+); protein kinase cascades leading to cell proliferation, differentiation and apoptosis; muscle growth and contractility; and the immune system. There is evidence for underlying 1alpha,25(OH)(2)D(3)-mediated protein tyrosine phosphorylation signalling in bone, intestine, muscle, epidermal and cancer cells.

PTH and phospholipase A2 in the aging process of intestinal cells.

In this study we analyzed, for the first time, alterations in phospholipase A2 (PLA2) activity and response to parathyroid hormone (PTH) in rat enterocytes with aging. We found that PTH, rapidly stimulate arachidonic acid (AA) release in rat duodenal cells (+1- to 2-fold), an effect that is greatly potentiated by aging (+4-fold). We also found that hormone-induced AA release in young animals is Ca2+-dependent via cPLA2, while AA released by PTH in cells from aged rats is due to the activation of cPLA2 and the Ca2+-independent PLA2 (iPLA2).

1alpha,25(OH)2D3 and parathyroid hormone (PTH) signaling in rat intestinal cells: activation of cytosolic PLA2.

In the current study, we have probed the role of cytosolic phospholipase A2 (cPLA2) activity in the cellular response to the calciotropic hormones, 1alpha,25,dihydroxy-vitamin D(3) [1alpha,25(OH)(2)D(3)] and PTH. Stimulation of rat enterocytes with either hormone, increased release of arachidonic acid (AA) 3H-AA] one-two fold in a concentration and time-dependent manner. The effect of either hormone on enterocytes was totally reduced by preincubation with the intracellular Ca(2+) chelator BAPTA-AM (5 microM), suggesting that the release of AA following cell exposure to the calciotropic hormones occurs mainly through a Ca(2+)-dependent mechanism involving activation of Ca(2+)-dependent cPLA2.

Tyrosine phosphorylation signalling dependent on 1alpha,25(OH)2-vitamin D3 in rat intestinal cells: effect of ageing.

In intestinal cells, as in other target cells, 1alpha,25(OH)(2)D(3) elicits long-term and short-term responses which involve genomic and non-genomic mode of actions, respectively. There is evidence indicating that activation of tyrosine phosphorylation pathways may participate in the responses induced by 1alpha,25(OH)(2)D(3) through its non-genomic mechanism. In this study we have evaluated the involvement of 1alpha,25(OH)(2)D(3) in the tyrosine phosphorylation of PLCgamma and MAPK (ERK1/2) in enterocytes from young (3 months) and aged (24 months) rats.

Characterization of PTH/PTHrP receptor in rat duodenum: effects of ageing.

In rat enterocytes, signaling through the parathyroid hormone (PTH)/PTH-related peptide receptor type 1(PTHR1) includes stimulation of adenylyl cyclase, increases of intracellular calcium, activation of phospholipase C, and the MAP kinase pathway, mechanisms that suffer alterations with ageing. The purpose of this study was to evaluate whether an alteration at the level of the PTH receptor (PTHR1) is the basis for impaired PTH signaling in aged rat enterocytes. Western Blot analysis with a specific monoclonal anti-PTHR1 antibody revealed that a 85 kDa PTH binding component, the size expected for the mature PTH/PTHrP receptor, localizes in the basolateral (BLM) and brush border (BBM) membranes of the enterocyte, being the protein expression about 7-fold higher in the BLM.

Effect of ageing in the early biochemical signals elicited by PTH in intestinal cells.

In previous work, we have demonstrated that rPTH(1-34) increases cytoplasmic calcium concentration ([Ca(2+)](i)) in isolated rat enterocytes. In the present study, we have identified the sources of PTH-mediated increase in [Ca(2+)](I) and the implication of Ca(2+) on hormone early signals in enterocytes isolated from young (3-month-old) and aged (24-month-old) rats. In young enterocytes, PTH raised [Ca(2+)](i) in a dose-dependent manner (1 pM-100 nM).