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

doi:10.1016/j.jsbmb.2010.03.002

The Journal of Steroid Biochemistry and Molecular Biology

Volume 121, Issues 1–2, July 2010, Pages 169-175

https://doi.org/10.1016/j.jsbmb.2010.03.002 Get rights and content

Abstract

We previously reported that 1α,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 1α,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 1α,25(OH)₂D₃-stimulation of c-Src and MAPKs. When proliferating cells were pretreated with methyl beta cyclodextrin (MβCD), a caveolae disrupting agent, under conditions in which cell morphology is not affected and no signs of apoptosis are observed, 1α,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 1α,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 MβCD. Altogether, these data suggest that caveolae is involved upstream in c-Src–MAPKs activation by 1α,25(OH)₂D₃ and that VDR and cav-1 participate in the rapid signaling elicited by the hormone.

Introduction

The steroid hormone 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] acts in target cells by a genomic mode of action where it binds to its receptor (VDR) regulating gene expression [1]. Also, the operation of a non-genomic mechanism associated with rapid hormone regulation of signal transduction pathways has been demonstrated [2]. In many cases, these events appear to be initiated at the plasma membrane level. Particularly, there is growing evidence that VDR localized at the immediate zone of the cell membrane mediates some of these signaling events [3], [4], [5]. We previously demonstrated translocation of the VDR to the cellular membrane and its co-immunoprecipitation with c-Src after 1α,25(OH)₂D₃ exposure of chicken skeletal muscle cells [6], [7]. Moreover, VDR and c-Src participation in hormone-dependent activation of MAPKs in proliferative skeletal muscle cells by the hormone was also well established [8], [9]. But, up to now, membrane components involved in 1α,25(OH)₂D₃ rapid signaling in muscle have not been established.

Caveolae are invaginations of plasma membrane enriched in sphingolipids and cholesterol. These microdomains are specialized lipid rafts that serve as signaling pathway platforms [10]. Robust information indicates that caveolae concentrate specific signal transducer proteins (e.g. tyrosine kinases of Src family and G proteins) as well as caveolins [11], [12]. Caveolins are main structural components of caveolae and also are members of scaffolding cytosolic proteins [13]. In absence of caveolins, no morphologically identifiable caveolae exist [14]. Molecular cloning has identified three caveolins isoforms, caveolin-1, -2 and -3 (cav-1, cav-2 and cav-3) [15]. Although it is known that cav-1 and -2 are present in fibroblasts, endothelial cells and adipocytes whereas cav-3 protein is muscle specific, cav-1 expression was confirmed in undifferentiated skeletal muscle cells in vivo and in vitro [16]. Caveolins play a significant role in different disease phenotypes, specifically in muscular dystrophy. Mice knock-down for cav-1 gene (cav-1 −/−) present abnormalities in skeletal muscle due to cav-1 expression conditioned later appearance of cav-2, so cav-1 absence promotes the pathogenesis called tubular aggregate formation [17]. Modulation of cav-1 levels also controls satellite cell activation during muscle repair [16]. In parallel, cav-1 plays a role in the physiological intracellular signaling network. Cav-1 can bind c-Src via its caveolin-scaffolding domain and thereby “clamp” Src in its inactive configuration [18]. So, the disruption of cav-1–c-Src complex conduces to c-Src activation and this event could be one of the beginning steps of signaling pathway cascades [14].

With respect to the VDR, Norman and collaborators reported that it is present in caveolae-enriched plasma membranes and binds 1α,25(OH)₂D₃ in vivo and in vitro [19]. Plasma membrane requirements for hormone-dependent PKC signaling were observed in chondrocytes and osteoblasts. Specifically, the regulation of growth plate chondrocytes by 1α,25(OH)₂D₃ requires caveolae and cav-1 [4]. Recently it was reported the key role of cav-1 in the compartmentalization of estrogen receptor β (ERβ) to the plasma membrane, thus allowing estradiol to control VDR transcription and expression [20]. In view of the data described, our aim was to investigate the role of caveolae and cav-1 in 1α,25(OH)₂D₃-dependent modulation of kinase cascades and VDR localization in skeletal muscle cells.

Section snippets

Chemicals

1α,25(OH)₂D₃ was kindly provided by Dr. Jan-Paul van de Velde from Solvay Pharmaceuticals (Weesp, The Netherlands). Dulbecco's modified Eagle's medium (DMEM) low glucose, with l-glutamine and HEPES, without phenol red, was from US Biological (Swampscott, MA, USA). Fetal bovine serum (FBS), protein A sepharose and the compound MβCD (methyl beta cyclodextrin) were from Sigma–Aldrich Co. (St. Louis, MO, USA). Primary antibodies: anti-phospho p38, anti-caveolin-1, anti-VDR and secondary antibodies

Effects of cholesterol depletion by MβCD on proliferating skeletal muscle C2C12 cells

MβCD (methyl beta cyclodextrin), a cyclic oligosaccharide employed to remove cholesterol of membrane lipid rafts [25] disrupts caveolae [26]. It is known that cholesterol has a key role on myoblast fusion [27] but there are not reports about the effects of cholesterol depletion by MβCD on proliferative C2C12 cell physiology. We studied then cellular morphology and size and nucleus integrity in cells preincubated for 30 min with this agent. We used the nuclear marker DAPI and the cellular stain

Discussion

Most processes of signal transduction involve activation of sequential cascades of kinases. Among them are the ubiquitous MAPKs that regulate a plethora of responses. Different molecules are required to interact in an orderly manner and activate MAPK pathways. Several reports have shown that some of these components are concentrated within caveolae membranes [28], [29], [30]. Particularly, Src kinases are early key intermediates of ERK1/2 and p38 MAPK pathways that can be compartmentalized into

Acknowledgements

This research was supported by grants from the Agencia Nacional de Promoción Científica yTecnológica, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), and Universidad Nacional del Sur, Argentina.

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^☆: Special issue selected article from the 14th Vitamin D Workshop held at Brugge, Belgium on October 4–8, 2009.

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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☆

Abstract

Introduction

Section snippets

Chemicals

Effects of cholesterol depletion by MβCD on proliferating skeletal muscle C2C12 cells

Discussion

Acknowledgements

Steroids

Biochim. Biophys. Acta

J. Biol. Chem.

Cell

Exp. Cell Res.

J. Biol. Chem.

Am. J. Pathol.

J. Biol. Chem.

Mol. Cell. Endocrinol.

Anal. Biochem.

J. Biol. Chem.

Am. J. Hum. Genet.

J. Biol. Chem.

1,25(OH)2-vitamin D3 receptors: gene regulation and genetic circuitry

FASEB J.

Rapid actions of vitamin D compounds

J. Cell. Biochem.

Minireview: vitamin D receptor: new assignments for an already busy receptor

Endocrinology

The vitamin D sterol–vitamin D receptor ensemble model offers unique insights into both genomic and rapid-response signalling

Sci. Signal.

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

J. Cell. Biochem.

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☆

1,25(OH)₂-vitamin D₃ receptors: gene regulation and genetic circuitry

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