Daniela Capiati

1,25(OH)2‐vitamin D3 induces translocation of the vitamin D receptor (VDR) to the plasma membrane in skeletal muscle cells

1,25‐dihydroxy‐vitamin D3 (1,25(OH)2D3), the hormonally active form of vitamin D3, acts through two different mechanisms. In addition to regulating gene expression via the specific intracellular vitamin D receptor (VDR), 1,25(OH)2D3 induces rapid, non‐transcriptional responses involving stimulation of transmembrane signal transduction pathways. The activation of second messengers supports the hypothesis that a membrane‐bound steroid receptor similar to those that mediate peptide hormone biology exists. Skeletal muscle is a target tissue for 1,25(OH)2D3. Avian embryonic skeletal muscle cells (myoblasts/myotubes) have been shown to respond both genomically and non‐genomically to the hormone. The present study provides evidence indicating that short‐term treatment (1–10 min) with 1,25(OH)2D3 induces translocation of the VDR from the nuclear to the microsomal fraction in chick myoblasts. This translocation is blocked by colchicine, genistein, or herbimycin, suggesting the involvement of microtubular transport and tyrosine kinase/s in the relocation of the receptor. By isolation of plasma membranes, it was demonstrated that the hormone increases the amounts of VDR specifically in this fraction. These results suggest that the nuclear VDR may be the receptor that mediates the non‐genomic effects of 1,25(OH)2D3 in chick myoblasts. J. Cell. Biochem. 86: 128–135, 2002.

Participation of protein kinase C α in 1,25-dihydroxy-vitamin D3 regulation of chick myoblast proliferation and differentiation

Changes in morphology and DNA synthesis in cultured myoblasts in response to 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3] have previously suggested that the vitamin D hormone may affect muscle cell proliferation and differentiation. However, this interpretation was not substantiated by measurement of specific biochemical markers of myogenesis. To study the effect of 1,25(OH)2D3 on muscle development, chicken embryo myoblasts were cultured for 1-6 days in the presence or absence of 1,25(OH)2D3 (10(-9) M). The hormone increased DNA synthesis and decreased creatine kinase activity, indicating stimulation of cell proliferation and inhibition of myogenesis, in undifferentiated myoblasts (1 day of culture). At longer culture intervals, when myoblasts elongate and fuse to form differentiated myotubes, 1,25(OH)2D3 promoted myogenesis, as indicated by an inhibition of DNA synthesis and an increase in specific muscle differentiation markers as creatine kinase activity and myosin expression. The role of protein kinase C (PKC) in mediating the effects of hormone and the likely PKC isoform involved were also investigated. Increased PKC activity was observed during 1,25(OH)2D3 stimulation of myoblast proliferation whereas inhibition of PKC activity accompanied the effects of the hormone on myoblast differentiation. The specific PKC inhibitor calphostin suppressed hormone potentiation of DNA synthesis in proliferating myoblasts. 1,25(OH)2D3-dependent changes in the expression of PKC isoforms alpha, beta, delta, epsilon and zeta during myogenesis were investigated by Western blot analysis. The early stimulation of myoblast proliferation by the hormone mainly correlated to increased PKC alpha expression whereas decreased PKC alpha levels were observed during the subsequent activation of myoblast differentiation. These results support that 1,25(OH)2D3 has a function in embryonic muscle growth and maturation, and PKC alpha may participate in the signal transduction pathway which mediates the response to the hormone.

Role of protein kinase C in 1,25(OH)2‐vitamin D3 modulation of intracellular calcium during development of skeletal muscle cells in culture

Regulation of muscle cell Ca2+ metabolism by 1,25‐dihydroxy‐vitamin D3 [1,25(OH)2D3] is mediated by the classic nuclear mechanism and a fast, nongenomic mode of action that activates signal transduction pathways. The role of individual protein kinase C (PKC) isoforms in the regulation of intracellular Ca2+ levels ([Ca2+]i) by the hormone was investigated in cultured proliferating (myoblasts) and differentiated (myotubes) chick skeletal muscle cells. 1,25(OH)2D3 (10−9 M) induced a rapid (30‐ to 60‐s) and sustained (>5‐min) increase in [Ca2+]i which was markedly higher in myotubes than in myoblasts. The effect was suppressed by the PKC inhibitor calphostin C. In differentiated cells, PKC activity increased in the particulate fraction and decreased in cytosol to a greater extent than in proliferating cells after 5‐min treatment with 1,25(OH)2D3. By Western blot analysis, these changes were correlated to translocation of the PKC α isoform from cytosol to the particulate fraction, which was more pronounced in myotubes than in myoblasts. Specific inhibition of PKC α activity using antibodies against this isoform decreased the 1,25(OH)2D3‐induced [Ca2+]i sustained response associated with Ca2+ influx through voltage‐dependent calcium channels. Neomycin, a phospholipase C (PLC) inhibitor, blocked its effects on [Ca2+]i, PKC activity, and translocation of PKC α. Exposure of myotubes to 1,2‐dioleyl‐rac‐glycerol (1,2‐diolein), also increased [Ca2+]i, PKC activity, and the amount of PKC α associated with the particulate fraction. Changes in [Ca2+]i induced by diolein were inhibited by calphostin C and nifedipine. The results indicate that PKC α activation via PLC‐catalyzed phosphoinositide hydrolysis is part of the mechanism by which 1,25(OH)2D3 regulates muscle intracellular Ca2+ through modulation of the Ca2+ influx pathway of the Ca2+ response to the sterol. J. Cell. Biochem. 77:200–212, 2000.

Non-genomic stimulation of tyrosine phosphorylation cascades by 1,25(OH)2D3 by VDR-dependent and -independent mechanisms in muscle cells

Studies with different cell types have shown that modulation of various of the fast as well as long-term responses to 1,25(OH)(2)D(3) depends on the activation of tyrosine kinase pathways. Recent investigations of our laboratory have demonstrated that 1,25(OH)(2)D(3) rapidly stimulates in muscle cells tyrosine phosphorylation of PLC-gamma and the growth-related proteins MAPK and c-myc. We have now obtained evidence using antisense technology indicating that VDR-dependent activation of Src mediates the fast stimulation of tyrosine phosphorylation of c-myc elicited by the hormone. This non-genomic action of 1,25(OH)(2)D(3) requires tyrosine phosphorylation of the VDR. Immunoprecipitation under native conditions coupled to Western blot analysis revealed 1,25(OH)(2)D(3)-dependent formation of complexes between Src and the VDR and c-myc. However, the activation of MAPK by the hormone was only partially mediated by the VDR and required in addition increased PKC and intracellular Ca(2+). Following its phosphorylation, MAPK translocates into the nucleus where it regulates c-myc transcription. Altogether these results indicate that tyrosine phosphorylation plays a role in the stimulation of muscle cell growth by 1,25(OH)(2)D(3). Data were also obtained involving tyrosine kinases and the VDR in hormone regulation of the Ca(2+) messenger system by mediating the stimulation of store-operated calcium (SOC; TRP) channels. Congruent with this action, 1,25(OH)(2)D(3) induces a rapid translocation of the VDR to the plasma cell membrane which can be blocked by tyrosine kinase inhibitors. Of mechanistic relevance, an association between the VDR and TRP proteins with the participation of the scaffold protein INAD was shown.

Serine/Threonine Protein Phosphatases type 2A and their roles in stress signaling

Serine/Threonine protein phosphatases are ubiquitous enzymes in all eukaryotes but many of their physiological roles in plants remain unknown. The available results have demonstrated critical functions for these enzymes in the regulation of adaptive stress responses, and recent studies have directed attention to the functional roles of Ser/Thr phosphatases type 2A (PP2A) as components of stress signaling pathways. This review is focused primarily on plant PP2As and their participation in the control of biotic and abiotic stress responses.

Evidence on the participation of protein kinase C α in the proliferation of cultured myoblasts

There is evidence involving protein kinase C (PKC) in the signal transduction pathways that regulate the differentiation of myoblasts into mature multinucleated muscle cells (myotubes). In order to obtain information on the possible role of individual PKC isozymes in myogenesis, in the present work we investigated the differential expression of PKC isoforms α, β, δ, ϵ, and ζ during muscle cell development in vitro. Chick embryo myoblasts cultured from 1 to 6 days were used as experimental model. Morphological characterization and measurement of specific biochemical parameters in cultures, e.g., DNA synthesis, creatine kinase activity, and myosin levels, revealed a typical muscle cell developmental pattern consisting of an initial proliferation of myoblasts followed by their differentiation into myotubes. PKC activity was high at the proliferative stage, decreased as myoblasts elongated and fused, and increased again in differentiated myotubes. In proliferating myoblasts, the PKC inhibitors calphostin C and bisindolylmaleimide I decreased DNA synthesis whereas in myoblasts undergoing differentiation they exerted the opposite effect, suggesting that PKC plays a role at both stages of myogenesis. Western blot analysis of changes in the expression of PKC isoforms during muscle cell development showed high levels of PKC α in the proliferating phase which markedly decreased as myoblasts differentiated. Treatment with TPA of proliferative myoblasts inhibited DNA synthesis and selectively down‐regulated PKC α, suggesting that this isozyme may have an important role in maintaining myoblast proliferation. On the other hand, an increase in the expression of PKC β, δ, and ϵ was detected during myogenesis, suggesting that one or more of these isoforms may participate in the differentiation process of myoblasts. J. Cell. Biochem. 74:292–300, 1999.

Inhibition of serum-stimulated mitogen activated protein kinase by 1α, 25(OH)2-Vitamin D3 in MCF-7 breast cancer cells

1α,25‐Dihydroxyvitamin D3 [1α,25(OH)2D3], the hormonally active form of vitamin D3, has been shown to be a potent negative growth regulator of breast cancer cells both in vitro and in vivo. 1α,25(OH)2D3 acts through two different mechanisms. In addition to regulating gene transcription via its specific intracellular receptor (vitamin D receptor, VDR), 1α,25(OH)2D3 induces rapid, non‐transcriptional responses involving activation of transmembrane signal transduction pathways, like growth factors and peptide hormones. The mechanisms that mediate the antiproliferative effects of 1α,25(OH)2D3 in breast cancer cells are not fully understood. Particularly, there is no information about the early non‐genomic signal transduction effectors modulated by the hormone. The present study shows that 1α,25(OH)2D3 rapidly inhibits serum induced activation of ERK‐1 and ERK‐2 MAP kinases. The tyrosine kinase Src is involved in the pathway leading to activation of ERK 1/2 by serum. Furthermore, 1α,25(OH)2D3 increases the tyrosine‐phosphorylated state of Src and inhibits its kinase activity, while induces the association of the VDR with Src, either in the presence or absence of serum. In parallel, the hormone rapidly increases the amounts of VDR associated to plasma membranes (PM). Pretreatment with the tyrosine phosphatase inhibitors orthovanadate or bpV (phen) prevented mitogen‐activated protein kinase (MAPK) inhibition by 1α,25(OH)2D3. These data altogether suggest that 1α,25(OH)2D3 inhibits the MAPK cascade by inactivating Src tyrosine kinase through a mechanism mediated by the VDR and tyrosine phosphatases.

MAPK inhibition by 1α,25(OH)2–Vitamin D3 in breast cancer cells.: Evidence on the participation of the VDR and Src☆

1alpha,25-Dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], the hormonally active form of Vitamin D(3), has been shown to be a potent negative growth regulator of breast cancer cells both in vitro and in vivo. 1alpha,25(OH)(2)D(3) acts through two different mechanisms. In addition to regulating gene transcription via its specific intracellular receptor (Vitamin D receptor, VDR), 1alpha,25(OH)(2)D(3) induces, rapid, non-transcriptional responses involving activation of transmembrane signal transduction pathways. The mechanisms that mediate the antiproliferative effects of 1alpha,25(OH)(2)D(3) in breast cancer cells are not fully understood. Particularly, there is no information about the early non-genomic signal transduction effectors modulated by the hormone. The present study shows that 1alpha,25(OH)(2)D(3) rapidly inhibits serum induced activation of ERK-1 and ERK-2 MAP kinases. The non-receptor tyrosine kinase Src is involved in the pathway leading to activation of ERK 1/2 by serum. Furthermore, 1alpha,25(OH)(2)D(3) increases the tyrosine-phosphorylated state of Src as well as it inhibits its kinase activity and induces the association of the VDR with Src. These data suggest that 1alpha,25(OH)(2)D(3) inhibits MAPK by inactivating Src tyrosine kinase through a so far unknown mechanism that seems to be mediated by the VDR.

1,25(OH)2-VITAMIN D3 AFFECTS THE SUBCELLULAR DISTRIBUTION OF PROTEIN KINASE C ISOENZYMES IN MUSCLE CELLS

Previous studies have shown the involvement of protein kinase C (PKC) in 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3] regulation of DNA synthesis (long-term effect) and Ca2+ channel activity (short-term effect) in cultured myoblasts. Both events mediate stimulation of myoblast cell proliferation and growth by 1,25(OH)2D3. To characterise further the role of PKC in the hormone mode of action in muscle cells, the presence of PKC isoenzymes in chicken embryo myoblasts and changes in their total cell and subcellular levels after treatment (72 h and 5 min) with 1,25(OH)2D3 (1 nM), 12-O-tetradecanoyl phorbol 13-acetate (TPA; 100 nM) and 1,2-dioctanoyl-rac-glycerol (DOG; 50 microM) were investigated. Western blot analysis provided evidence on the expression of PKC alpha, beta and delta isoforms in avian myoblasts. Two immunoreactive bands of 80 kDa (intact molecule) and 50 kDa (catalytic fragment) were detected for each isoenzyme. 1,25(OH)2D3 and DOG, which increased myoblast PKC activity parallel with the stimulation of DNA synthesis and culture growth and the phorbol ester TPA which induced the opposite changes, exerted differential effects on PKC isoenzymes. Long-term (72 h) treatment with 1,25(OH)2D3 and DOG did not change total PKC isoform levels but decreased the 80 kDa species and increased the release of the catalytic fragment of PKC delta and beta, whereas TPA augmented the total amounts of the three PKC isoforms, increasing the band of 80 kDa of PKC beta and delta and the 50 kDa species for PKC alpha. Subcellular distribution studies showed that the 80 kDa molecule is only present in the cytosolic fraction whereas in the particulate fractions the 50 kDa fragments are detected. Increased amounts of the catalytic fragments of PKC beta and delta both in the nucleus and membranes were observed after 72 h treatment with DOG while 1,25(OH)2D3 increases PKC beta in the nucleus and PKC delta in membranes. TPA induced the appearance of the 50 kDa species of PKC alpha in the nuclear and membrane fractions. The phorbol ester also decreased the catalytic fragments of PKC beta and delta in membranes. Increased levels of PKC beta, and to a lesser extent of PKC delta, in membranes and cytosol could be detected after short exposure (5 min) of myoblasts to 1,25(OH)2D3, DOG and TPA. In conclusion, the data indicate the operation in myoblasts of PKC signal transduction pathways mediated by the Ca(2+)-dependent PKCs alpha and beta and the Ca(2+)-independent PKC delta. Moreover, the results suggest that the beta and delta isoforms of PKC could play a role in the regulation of muscle cell metabolism by 1,25(OH)2D3.

Nuclear Vitamin D (VDR) and Estrogen (ER) Receptors in the Membrane of Muscle and Breast Cancer Cells

1α,25-dihydroxy-vitamin D3 (lα,25(OH)2D3; calcitriol) and estrogen (17β-estradiol) act, as other steroid hormones, through two different mechanisms. In addition to regulating expression of target genes via their specific nuclear receptors (VDR and ER, respectively), both hormones induce fast, non transcriptional responses involving stimulation of transmembrane signal transduction pathways. The rapid nature and specificity by which 1α,25(OH)2D3 and 17β-estradiol trigger the activation of second messengers has led to the concept that interaction with a plasma membrane receptor is responsible for the initiation of their effects. However, there is controversy over its molecular characteristics. Among several models for non-genomic steroid receptor identity, has been proposed the existence of membrane-associated forms of either the classical receptors or alternatively of novel 1α,25(OH)2D3 and 17β- estradiol binding proteins. In this chapter we report the presence of the nuclear VDR and ER in the plasma membrane of avian muscle and mammalian breast cells and furnish data suggesting that they may be involved in non-genomic signalling by their cognate ligands.