Claudia Buitrago

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.

1α,25(OH)2D3‐dependent modulation of Akt in proliferating and differentiating C2C12 skeletal muscle cells

We previously reported that 1α,25‐dihydroxy‐vitamin D3 [1α,25(OH)2D3] induces non‐transcriptional rapid responses through activation of Src and MAPKs in the skeletal muscle cell line C2C12. In the present study we investigated the modulation of Akt by the secosteroid hormone in C2C12 cells at proliferative stage (myoblasts) and at early differentiation stage. In proliferating cells, 1α,25(OH)2D3 activates Akt by phosphorylation in Ser473 in a time‐dependent manner (5–60 min). When these cells were pretreated with methyl‐beta‐cyclodextrin to disrupt caveolae microdomains, hormone‐induced activation of Akt was suppressed. Similar results were obtained by siRNA silencing of caveolin‐1 expression, further indicating that hormone effects on cell membrane caveolae are required for downstream signaling. PI3K and p38 MAPK, but not ERK1/2, participate in 1α,25(OH)2D3 activation of Akt in myoblasts. The involvement of p38 MAPK in Akt phosphorylation by the hormone probably occurs through MAPK‐activated protein kinase 2 (MK2), which is activated by the steroid. In addition, the participation of Src in Akt phosphorylation by 1α,25(OH)2D3 was demonstrated using the inhibitor PP2 and antisense oligodeoxynucleotides that suppress Src expression. We also observed that PI3K participates in hormone‐induced proliferation. During the early phase of C2C12 cell differentiation 1α,25(OH)2D3 also increases Akt phosphorylation and activates Src. Of relevance, Src and PI3K are involved in Akt activation and in MHC and myogenin increased expression by 1α,25(OH)2D3. Altogether, these data suggest that 1α,25(OH)2D3 upregulates Akt through Src, PI3K, and p38 MAPK to stimulate myogenesis in C2C12 cells. J. Cell. Biochem. 113: 1170–1181, 2012.

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.

Caveolae and caveolin-1 are implicated in 1α,25(OH)2-vitamin D3-dependent modulation of Src, MAPK cascades and VDR localization in skeletal muscle cells☆

We previously reported that 1alpha,25(OH)2D3 induces non-transcriptional rapid responses through activation of MAPKs in C2C12 skeletal muscle cells. However, there is little information on the molecular mechanism underlying the initiation of 1alpha,25(OH)2D3 signaling through this pathway. Plasma membrane components have been involved in some non-genomic effects. In this work, we investigated the role of caveolae and caveolin-1 (cav-1) in 1alpha,25(OH)2D3-stimulation of c-Src and MAPKs. When proliferating cells were pretreated with methyl beta cyclodextrin (MbetaCD), a caveolae disrupting agent, under conditions in which cell morphology is not affected and no signs of apoptosis are observed, 1alpha,25(OH)2D3-dependent activation of ERK1/2, p38 MAPK and c-Src was suppressed. Similar results were obtained by siRNA technology whereby silencing of cav-1 expression abolished activation of c-Src and MAPKs induced by the hormone. By confocal immunocytochemistry it was observed that cav-1 colocalizes with c-Src in the periplasma membrane zone at basal conditions. Hormone treatment disrupted the colocalization of these proteins and redistributed them into cytoplasm and nucleus. Co-immunoprecipitation assays corroborated these observations. Changes in VDR localization after 1alpha,25(OH)2D3 exposure were also investigated. Confocal microscopy images showed that the hormone induces VDR translocation to the plasma membrane, and this effect is abolished by MbetaCD. Altogether, these data suggest that caveolae is involved upstream in c-Src-MAPKs activation by 1alpha,25(OH)2D3 and that VDR and cav-1 participate in the rapid signaling elicited by the hormone.

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

In chick skeletal muscle cell primary cultures, we previously demonstrated that 1α,25(OH)2‐vitamin D3 [1α,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 1α,25(OH)2D3 within 1 min phosphorylates and increases the activity of p38 MAPK. The immediately upstream mitogen‐activated protein kinase kinases 3/6 (MKK3/MKK6) were also phosphorylated by the hormone suggesting their participation in p38 activation. 1α,25(OH)2D3 was able to dephosphorylate/activate the ubiquitous cytosolic tyrosine kinase c‐Src in C2C12 cells and studies with specific inhibitors imply that Src participates in hormone induced‐p38 activation. Of relevance, 1α,25(OH)2D3 induced in the C2C12 line the stimulation of mitogen‐activated protein kinase activating protein kinase 2 (MAPKAP‐kinase 2) and subsequent phosphorylation of heat shock protein 27 (HSP27) in a p38 kinase activation‐dependent manner. Treatment with the p38 inhibitor, SB203580, blocked p38 phosphorylation caused by the hormone and inhibited the phosphorylation of its downstrean substrates. 1α,25(OH)2D3 also promotes the phosphorylation of c‐jun N‐terminal protein kinases (JNK 1/2), the response is fast (0.5–1 min) and maximal phosphorylation of the enzyme is observed at physiological doses of 1α,25(OH)2D3 (1 nM). The relative contribution of ERK‐1/2, p38, and JNK‐1/2 and their interrelationships in hormonal regulation of muscle cell proliferation and differentiation remain to be established. J. Cell. Biochem. 97: 698–708, 2006.

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.

Involvement of Tyrosine Kinase Activity in 1α,25(OH)2-vitamin D3 Signal Transduction in Skeletal Muscle Cells

In cultured chick skeletal muscle cells loaded with Fura-2, the tyrosine kinase inhibitors herbimycin A and genistein abolished both the fast inositol 1,4,5-trisphosphatedependent Ca2+ release from internal stores and extracellular Ca2+ influx induced by 1α,25(OH)2-vitamin D3 (1α,25(OH)2D3). Daidzein, an inactive analog of genistein, was without effects. Tyrosine phosphatase inhibition by orthovanadate increased cytosolic Ca2+. Anti-phosphotyrosine immunoblot analysis revealed that 1α,25(OH)2D3 rapidly (0.5–10 min) stimulates in a concentrationdependent fashion (0.1–10 nm) tyrosine phosphorylation of several myoblast proteins, among which the major targets of the hormone could be immunochemically identified as phospholipase Cγ (127 kDa), which mediates intracellular store Ca2+ mobilization and external Ca2+ influx, and the growth-related proteins mitogen-activated protein (MAP) kinase (42/44 kDa) and c-myc (65 kDa). Genistein suppressed the increase in phosphorylation and concomitant elevation of MAPK activity elicited by the sterol. Both genistein and the MAPK kinase (MEK) inhibitor PD98059 abolished stimulation of DNA synthesis by 1α,25(OH)2D3. The sterol-induced increase in tyrosine phosphorylation of c-myc, a finding not reported before for cell growth regulators, was totally suppressed by the specific Src inhibitor PP1. These results demonstrate that tyrosine phosphorylation is a previously unrecognized mechanism involved in 1α,25(OH)2D3 regulation of Ca2+homeostasis in hormone target cells. In addition, the data involve tyrosine kinase cascades in the mitogenic effects of 1α,25(OH)2D3 on skeletal muscle cells.

Activation of MAPKs by 1α,25(OH)2-Vitamin D3 and 17β-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. In this work, using the same muscle cell line, we demonstrate that 1alpha,25(OH)(2)D(3) or 17beta-estradiol phosphorylate and activate ERK1/2 and p38 MAPK after longer treatment intervals, maximal effects seen at 90 and 30 min (ERK1/2) and at 60 and 15 min (p38 MAPK) for these hormones, respectively. Hormone-dependent ERK and p38 activation was abolished by MAPK specific inhibitors U0126 and SB203580. 1alpha,25(OH)(2)D(3) and 17beta-estradiol also induced the phosphorylation of CREB and Elk-1 transcription factors in an ERK1/2-dependent manner. Simultaneous addition of both hormones potentiated CREB phosphorylation. 1alpha,25(OH)(2)D(3)- and 17beta-estradiol-induced c-fos expression, which was mediated by p38 phosphorylation. The action of 17beta-estradiol on c-fos levels was also dependent on ERK1/2. These results suggest that MAPK signalling pathways play an important role in regulating early gene expression through CREB and Elk-1 activation in skeletal muscle cells.

Role of estrogen receptors, PKC and Src in ERK2 and p38 MAPK signaling triggered by 17β-estradiol in skeletal muscle cells.

We have previously reported in C2C12 murine skeletal muscle cells that 10(-8)M 17β-estradiol promotes MAPKs stimulation which in turn mediates the activation of CREB and Elk-1 transcription factors. In this work, we demonstrated that the hormone induces ERK2 phosphorylation (without affecting ERK1 activation) and also stimulates p38 MAPK, both in a dose-dependent manner. Moreover, estrogen receptors involvement in MAPKs activation by the estrogen was studied. The use of ICI182780 (1 μM), an antagonist of ERs, and specific siRNAs to block ERα and ERβ expression, demonstrated that ERα mediates ERK2 activation but not p38 MAPK phosphorylation by 17β-estradiol, and that ERβ isoform is not implicated in MAPKs activation by the hormone. Furthermore, Src and PKC contribution in estrogen stimulation of the MAPKs was investigated. Compounds PP2 and Ro318220, Src and PKC family inhibitors, respectively abrogated ERK2 and p38 MAPK phosphorylation by 17β-estradiol. Of interest, the hormone was able to induce Src and PKCδ activation. In addition, Ro318220 decreased estrogen-dependent Src modulation implicating PKC in hormone upregulation of Src. Accordingly, PP2 and Ro318220 suppressed CREB and Elk-1 phosphorylation as well as c-Fos and c-Jun oncoprotein levels induced by 17β-estradiol. Altogether, these data indicate that 17β-estradiol activates ERK2 through ERα and p38 MAPK in an ERα/β-independent manner and that PKC and Src proteins are key upstream components on MAPKs activation in C2C12 skeletal muscle cells.