Ana Russo de Boland

Non-genomic signal transduction pathway of vitamin D in muscle

The secosteroid hormone 1,25(OH)2-vitamin D3 rapidly activates voltage-dependent Ca2+ channels of the L-type in skeletal and cardiac muscle cells by a non-genomic mechanism which involves guanine nucleotide binding (G) protein-medicated stimulation of the adenylate cyclase/cAMP/protein kinase A messenger system. Modifications in calmodulin intracellular distribution induced by PKA-dependent membrane protein phosphorylation may participate in the fast regulation of muscle Ca2+ influx by 1,25(OH)2D3. The protein kinase C pathway also plays a role modulating 1,25(OH)2D3 signal transduction in muscle by cross-talk with the PKA system. The hormone sequentially activates phospholipases C and D providing diacylglycerol for PKC activation and inositol triphosphate for intracellular Ca2+ mobilization. In addition, 1,25(OH)2D3 rapidly stimulates phospholipase A2 generating arachidonic acid for the eicosanoid pathway. Specificity of hormone effects suggests that binding to a muscle membrane-bound receptor mediates these events.

1α,25-Dihydroxy-vitamin-D3-induced Store-operated Ca2+ Influx in Skeletal Muscle Cells MODULATION BY PHOSPHOLIPASE C, PROTEIN KINASE C, AND TYROSINE KINASES

In skeletal muscle cells the steroid hormone 1α,25-dihydroxy-vitamin-D3(1,25(OH)2D3) nongenomically promotes Ca2+ release from intracellular stores and cation influx through both l-type and store-operated Ca2+(SOC) channels. In the present work we evaluated the regulation and kinetics of the 1,25(OH)2D3-stimulated SOC influx in chick muscle cells. Stimulation with 10−9 m 1,25(OH)2D3 in Ca2+-free medium resulted in a rapid (40–60 s) but transient [Ca2+] i rise, which correlated with sterol-dependent inositol 1,4,5-trisphosphate production. The SOC influx stimulated by the hormone was insensitive to bothl-type channel antagonists and polyphosphoinositide-specific phospholipase C (PPI-PLC) inhibitors but was fully inhibitable by La3+ and Ni2+. PPI-PLC blockade prior to 1,25(OH)2D3 stimulation suppressed both the [Ca2+] i transient and the SOC influx. 1,25(OH)2D3-induced SOC entry was markedly increased after 3 min of treatment (30% above basal) and then rapidly reached a steady-state level. The sterol-stimulated SOC influx was prevented by protein kinase C and tyrosine kinase inhibitors but unaffected by blockade of the protein kinase A pathway. None of these inhibitors altered the thapsigargin-induced SOC entry, suggesting the operation of a signaling mechanism different from that for sterol-dependent SOC influx. The present results indicate that 1,25(OH)2D3-induced activation of PPI-PLC is upstream to Ca2+ influx through SOC channels and point for a role of both protein kinase C and tyrosine kinases but not protein kinase A in the regulation of the sterol-dependent SOCE pathway.

Activation of Src kinase in skeletal muscle cells by 1,25-(OH)2-vitamin D3 correlates with tyrosine phosphorylation of the vitamin D receptor (VDR) and VDR-Src interaction

The rapid effect of 1α,25(OH)2‐vitamin D3 [1α,25(OH)2D3] on tyrosine kinase Src and its relationship to the vitamin D receptor (VDR) was investigated to further characterize the hormone signaling mechanism in chick muscle cells. Exposure of cultured myotubes to 1α,25(OH)2D3 caused a time‐dependent increase in Src activity, which was evident at 1 min (one‐fold) and reached a maximum at 5 min (15‐fold). Immunoblotting with anti‐phosphotyrosine antibody of immunoprecipitated Src showed that the hormone decreased Src tyrosine phosphorylation state with maximal effects at 5 min. Using a database for protein consensus motifs we found a putative tyrosine phosphorylation site (amino acids 164–170: KTFDTTY) within the primary sequence of the chick VDR. When the myotube VDR was immunoprecipitated it appeared onto SDS‐PAGE gels as a single band of 58 kDa recognized by an anti‐phosphotyrosine antibody. Prior treatment of cells with 1α,25(OH)2D3 significantly increased tyrosine phosphorylation of the VDR (two‐ to three‐fold above basal levels). In agreement with Src being a SH2‐domain containing protein involved in recognition of tyrosine‐phosphorylated targets, immunoprecipitation with anti‐Src antibody under native conditions followed by blotting with anti‐VDR antibody, or using the antibodies in inverse order, showed that the VDR co‐precipitates with Src, thus indicating the existence of a VDR/Src complex. Stimulation with the cognate VDR ligand significantly increased formation of the complex with respect to basal conditions. These results altogether provide the first evidence to date for 1α,25(OH)2D3 activation involving Src association to tyrosine phosphorylated VDR. J. Cell. Biochem. 79:274–281, 2000.

The stimulation of MAP kinase by 1,25(OH)2-vitamin D3 in skeletal muscle cells is mediated by protein kinase C and calcium

In previous work we have demonstrated that the steroid hormone 1,25(OH)(2)-vitamin D(3) [1,25(OH)(2)D(3)] stimulates in skeletal muscle cells the phosphorylation and activity of the extracellular signal-regulated mitogen-activated protein (MAP) kinase isoforms ERK1 and ERK2. In the present study we evaluated the involvement of Ca(2+) and protein kinase C (PKC) on 1,25(OH)(2)D(3)-induced activation of MAP kinase. The hormone response was found to depend on PKC stimulation since it was attenuated by the PKC inhibitors calphostin C (100 nM) and bisindolylmaleimide I (30 nM) and PKC downregulation by prolonged treatment with the phorbol ester TPA (1 microM). Removal of external Ca(2+), chelation of intracellular Ca(2+) with BAPTA (5 microM), inhibition of phosphoinositide-phospholipase C (PLC) by neomycin, the calmodulin antagonist fluphenazine (50 microM) and the specific inhibitor of calmodulin kinase II, KN-62 (10 microM), significantly decreased 1,25(OH)(2)D(3)-activation of MAP kinase. In addition, the Ca(2+)-channel blocker verapamil (5 microM) suppressed hormone-induced MAP kinase activity in these cells. Furthermore, the Ca(2+)-mobilizing agent thapsigargin and the Ca(2+)-inophore A23187 paralleled the phosphorylation of MAP kinase observed with 1,25(OH)(2)D(3). Taken together, these results indicate that PKC and Ca(2+) are two upstream activators mediating the effects of 1,25(OH)(2)D(3) on MAP kinase in skeletal muscle cells.

Rapid 1,25(OH)2-vitamin D3 stimulation of calcium uptake by rat intestinal cells involves a dihydropyridine-sensitive cAMP-dependent pathway

The acute effects of 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3) on Ca2+ influx in isolated rat enterocytes were studied. The hormone significantly increased 45Ca2+ uptake by the cells within 1-10 min in a specific dose-dependent manner (10(-11)-10(-9) M) since 25(OH)D3 and 24,25(OH)2D3 were devoid of activity. The effects of 1,25(OH)2D3 were mimicked by the Ca2+ channel agonist BAY K8644 and completely abolished by nifedipine (1 microM) and verapamil (10 microM). Incubation of duodenal cells with 1,25(OH)2D3 rapidly (1-5 min) increased cAMP levels. Forskolin caused a rapid increase in Ca2+ uptake by enterocytes which was similar to the action of the hormone. Moreover, pretreatment of cells with the specific cAMP inhibitor Rp-cAMPS suppressed the changes in 45Ca influx induced by 1,25(OH)2D3. These results provide the first evidence involving Ca2+ channel activation through the cAMP pathway by 1,25(OH)2D3 in mammalian intestinal cells.

1α,25(OH)2‐Vitamin D3 signaling in chick enterocytes: Enhancement of tyrosine phosphorylation and rapid stimulation of mitogen‐activated protein (MAP) kinase

The steroid hormone 1α,25(OH)2–vitamin D3 (1α,25(OH)2D3) generates biological responses in intestinal and other cells via both genomic and rapid, nongenomic signal transduction pathways. We examined the hypothesis that 1α,25(OH)2D3 action in chick enterocytes may be linked to pathways involving tyrosine phosphorylation. Brief exposure of isolated chick enterocytes to 1α,25(OH)2D3 demonstrated increased tyrosine phosphorylation of several cellular proteins (antiphosphotyrosine immunoblots of whole cell lysates) with prominent bands at 42–44, 55–60, and 105–120 Kda. The 42–44 Kda bands comigrated with mitogen‐activated protein (MAP) kinase (immunoblotting with anti‐MAP kinase antibody) The response occurred within 30 s, peaked at 1 min, and was dose‐dependent (0.01–10 nM), with maximal stimulation at 1 nM (three‐ to fivefold). This effect was specific for 1α,25(OH)2D3 since its metabolic precursors 25(OH)D3and vitamin D3 did not increase MAP kinase tyrosine phosphorylation. The tyrosine kinase inhibitor, genistein, blocked 1α,25(OH)2D3‐induced tyrosine phosphorylation of MAP kinase, while staurosporine, a PKC inhibitor, attenuated the hormones effects by 30%. We have evaluated the ability of 1α,25(OH)2D3 analogs, which have complete flexibility around the 6,7 carbon‐carbon bond (6F) or which are locked in either the 6‐s‐cis (6C) or the 6‐s‐trans(6T) shape(s), to activate MAP kinase. Thus, two 6F and one 6C analog stimulated while one 6T analog did not stimulate MAP kinase tyrosine phosphorylation. In addition, 1β,25(OH)2D3, a known antagonist of 1α,25(OH)2D3‐mediated rapid responses, blocked the hormone effects on MAP kinase. We conclude that 1α,25(OH)2D3 and analogs which can achieve the 6‐s‐cis shape (6F and 6C) can increase tyrosine phosphorylation and activation of MAP kinase in chick enterocytes. J. Cell. Biochem. 69:470–482, 1998.

Modulation by 1,25(OH)2-vitamin D3 of the adenylyl cyclase/cyclic AMP pathway in rat and chick myoblasts

We have previously reported that the calciotropic hormone 1,25(OH)2-vitamin D3 stimulates influx of Ca2+ into cultured rat and embryonic chick myoblasts via voltage sensitive Ca(2+)-channels. In the present study, we show that this effect of 1,25(OH)2D3 requires the mediation of the adenylylcyclase signalling system since the hormone-dependent Ca2+ influx is abolished by specific inhibitors of adenylylcyclase and protein kinase A and mimicked by forskolin and dibutyryl cAMP. 1,25(OH)2D3-stimulated elevations in cellular cAMP paralleled increases in Ca2+ uptake, further suggesting a coupling of adenylylcyclase activation and calcium influx. Fluoride and GTP gamma S mimicked 1,25(OH)2D3-stimulation of calcium influx while GDP beta S suppressed the effect of the hormone. Cholera toxin and Bordetella pertussis toxin both increased 45Ca2+ uptake in rat and chick myoblasts. The hormone further increased cholera toxin actions, but was unable to modify pertussis toxin-induced 45Ca2+ uptake, suggesting a similar target of action for pertussis toxin and 1,25(OH)2D3. Incubation of microsomal membranes with the sterol (10 nM, 2 min) markedly displaces (-32%) [35S]GTP gamma S binding to the membranes. ADP-ribosylation of the pertussis toxin-sensitive 41 kDa substrate was significantly increased (+40%) in 1,25(OH)2D3-pretreated cells. These results suggest that 1,25(OH)2D3-stimulated influx of Ca2+ into rat and embryonic chick cultured myoblasts sequentially requires inhibition of a pertussis toxin-sensitive G protein, accumulation of cAMP and activation of dihydropyridine-sensitive Ca(2+)-channels through PKA-mediated phosphorylation events.

The tyrosine kinase c-Src is required for 1,25(OH)2-vitamin D3 signalling to the nucleus in muscle cells.

We have recently shown that the hormonal form of vitamin D3, 1,25(OH)2-vitamin D3 (1,25(OH)2D3), stimulates the enzymatic activity of the non-receptor protein tyrosine kinase c-Src in skeletal muscle cells. In this study we show that intracellular and extracellular Ca2+ chelation with BAPTA and EGTA, respectively, blocked hormone stimulation of c-Src activity/dephosphorylation, indicating that the calcium messenger system is an upstream activator of c-Src. Tyrosine phosphorylation and stimulation of the growth-related mitogen-activated protein kinase (MAPK) by 1,25(OH)2D3 was shown to be dependent on activation of c-Src, since pretreatment with the c-Src specific inhibitor PP1 or muscle cell transfection with an antisense oligodeoxynucleotide directed against c-Src mRNA markedly reduced hormone stimulation of MAPK phosphorylation. Evidence was obtained indicating that MAPK is then translocated to the cell nucleus in active phosphorylated form and induces the expression of c-myc oncoprotein, as the MAPK kinase (MEK) inhibitor PD98059 abolished stimulation of c-myc synthesis by 1,25(OH)2D3. In addition, the hormone rapidly stimulated tyrosine phosphorylation of c-myc. In cells pretreated with PP1 (4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-D3,4-pyrimidine), the 1,25(OH)2D3-induced increase in tyrosine phosphorylation of c-myc was suppressed. Taken together, these results demonstrate that 1,25(OH)2D3 stimulates proliferation-associated signalling pathways in skeletal muscle cells and implicate c-Src kinase as mediator of this response.

Involvement of protein kinase C in the modulation of 1α,25-dihydroxy-vitamin D3-induced 45Ca2+ uptake in rat and chick cultured myoblasts

The calciotropic hormone 1alpha,25-dihydroxy-vitamin D3 (1,25(OH)2D3) has been shown to stimulate both rat and chick myoblast 45Ca2+ uptake via modulation of dihydropyridine-sensitive L-type calcium channels through phosphorylation by the cAMP/protein kinase A pathway. We further investigated the involvement of protein kinases in 1,25(OH)2D3-signal transduction on cultured myoblasts. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) was found to rapidly stimulate myoblast 45Ca2+ uptake, mimicking 1,25(OH)2D3. The effects of PMA were time- (1-5 min) and dose (50-100 nM)-dependent, were mimicked by 1,2-dioctanoylglycerol (DOG) and were specific, since the inactive analogue 4alpha-phorbol was without effect. Analogously to the hormone, PMA-enhanced 45Ca2+ uptake was suppressed by the Ca2+-channel blocker nifedipine (5 microM). 1-(5-isoquinolynsulfonyl)-2-methylpiperazine (H-7), a PKC inhibitor, and down-regulation of PKC by prolonged exposure to PMA (1 microM, 24 h), abolished both PMA and hormone effects on rat and chick cells. As in chick myoblasts, 1,25(OH)2D3 activated PKC in rat myoblasts, with translocation of activity from the cytosol to the cell membrane. Treatment of myoblasts with PMA (100 nM) plus 1,25(OH)2D3 (1 nM) greatly potentiated 45Ca2+ uptake than either agent alone. PMA also increased myoblast cAMP content. These results suggest the involvement of PKC in the mechanism by which 1,25(OH)2D3 rapidly stimulates calcium uptake in both mammalian and avian myoblasts.

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

The classical model of 17β‐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 ERα in the C2C12 skeletal muscle cell line. We first established [3H]17βestradiol binding characteristics in whole cells in culture. Specific and saturable [3H]17βestradiol binding sites of high affinity were then detected in mitochondrial fractions (Kd = 0.43 nM; Bmax = 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 17βestradiol‐BSA conjugates. The immunoreactivity did not translocate into the nucleus by 17β‐estradiol treatment. Western and Ligand blot approaches corroborated the non‐classical localization. Expression and subcellular distribution of ERα proteins were confirmed in C2C12 cells transfected with ERα siRNA and by RT‐PCR employing specific primers. The non‐classical distribution of native pools of ERα in skeletal muscle cells suggests an alternative mode of ER localization/function. J. Cell. Biochem. 104: 1254–1273, 2008.