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Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.37 no.4 Santiago  2004

http://dx.doi.org/10.4067/S0716-97602004000400021 

 

Biol Res 37: 647-651, 2004

ARTICLE

1a,25(OH)2D3 induces capacitative calcium entry involving a trpc3 protein in skeletal muscle and osteoblastic cells

SANTILLÁN G., KATZ S., BUITRAGO C. and BOLAND R.

Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, San Juan 670 (B8000CN), Bahía Blanca, Argentina

Dirección para Correspondencia


ABSTRACT

This work describes the involvement of TRPC proteins in capacitative calcium entry (CCE) induced by 1a,25-dihydroxy-vitamin-D3 [1a,25(OH)2D3] in chick skeletal muscle and in rat osteoblast-like cells (ROS 17/2.8) and the role of the vitamin D receptor (VDR) in this non-genomic rapid response mediated by the hormone. We propose that an endogenous TRPC3 protein mediates 1a,25(OH)2D3 modulation of CCE in these cells, which seems to implicate VDR-TRPC3 association and the participation of an INAD-like scaffold protein.

Key words: CCE; TRPC; muscle cells; osteoblasts; 1a,25(OH)2D3; VDR; INAD.


INTRODUCTION

1a,25(OH)2D3 induces a fast and sustained increment in [Ca2+]i in skeletal muscle and osteoblastic cells through a non-genomic mechanism of action which depends on both IP3-mediated mobilization of Ca2+ from the endoplasmic reticulum and cation influx, mainly through voltage-dependent Ca2+ channels (3,5). Moreover, recent studies evidence the existence in these cells of a CCE pathway activated by the hormone (1,9).

The TRP (Transient Receptor Potential) proteins, designated as the TRP-canonical (TRPC) subfamily of the larger TRP superfamily gene products in Drosophila melanogaster, function as Ca2+ permeable channels mainly regulated by store depletion when expressed in heterologous systems. There are at least seven homologues of the TRPC invertebrate counterparts described in mammals (TRPC1-TRPC7) (2). In different cell lines, the overexpression of these genes results in CCE sharing heterogeneous patterns of activation and/or modulation (8).

The existence of TRPC proteins and their involvement in the regulation of 1a,25(OH)2D3-activated CCE in chick skeletal muscle and ROS 17/2.8 osteoblastic cells were investigated in this work.

RESULTS AND DISCUSSION

Previously, capacitative calcium entry in response to 1a,25(OH)2D3 or mere store depletion with thapsigargin, a sarco/endoplasmic reticulum Ca2+-ATPase inhibitor, was observed in muscle (chick embryo myoblasts) and ROS 17/2.8 cells. This CCE is a non-selective cation entry route permeable to Mn2+, Sr2+ or Ca2+ (1,9).

There is evidence demonstrating that expression of certain mammalian TRP proteins induces an increment of non-selective cation channels, making them good candidates for mediating capacitative calcium entry (2). Other lines of evidence suggest that members of the TRPC3/6/7 subfamily would form CCE channels. Generally, the overexpression of these genes into different cell types show an increased capacitative Ca2+ entry sharing heterogenous patterns of activation and/or modulation (8). However, there is no available evidence to date that demonstrates the direct involvement of TRP proteins as mediators of endogenous CCE.

By RT-PCR two fragments of 443 bp and 190 bp in myoblasts and of 390 bp and 201 bp in osteoblastic cells were obtained and sequenced at the University of Chicago Cancer Research Center DNA Sequence Facility (Chicago, IL, USA). The sequences have been submitted to the GenBankTM (accession numbers AY130980 and AY130981 for muscle and AF313482 and AF313481 for osteoblastic cell sequences, respectively). These fragments were found to encode continuous reading frames of 116, 63, 131 and 67 amino acids. The 116 and 63 amino acid fragments showed 71-95% of identity with human, mouse and rat TRPC3, the 131 and 67 amino acids fragments exhibited 80-100% sequence identity with TRPC3 from the same species. Since the primers used showed highly conserved regions in TRPC3/6/7 mammalian sequences and low homology with other avian or mammalian proteins non-related to the TRPC family, we can assume that the results obtained strongly suggest the existence of mRNA coding for TRPC proteins in chick skeletal muscle cells and in rat ROS 17/2.8 cells. In addition, by Northern blot analysis using as probe a 446 bp nucleotide sequence from HEK293 cells, a major band in myoblasts similar to that found in the latter cells corresponding to a size of ~ 4.4 Kb was observed. Transcripts of about 3.5 kb and 4.3 kb were found in osteoblastic cells and rat brain (positive control), respectively, employing as hybridization probe the 201 bp PCR fragment (not shown). As observed in other cellular systems, the significantly smaller size of the ROS 17/2.8 cell transcript compared to that expressed in rat brain suggests that a splicing variant of TRPC3 might be expressed in these cells (7). However, because the full sequence of the osteoblastic TRPC3 is not known, we cannot totally exclude the presence of other TRPs in both cell types. A single band of about 140 kDa (muscle cells) and 110 kDa (osteoblastic cells) similar to the control (rat brain) was detected by western blot analysis using polyclonal anti-TRPC3 antibodies (not shown). Altogether, the data are consistent with the endogenous expression of the TRPC3 isoform in avian skeletal muscle and rat osteoblastic cells.

Additionally, the role of a TRPC3-like protein in 1a,25(OH)2D3 modulation of CCE in muscle and osteoblastic cells was examined by incorporation of antisense ODNs directed against specific regions of TRPC3 mRNA coupled to fluorimetric measurements of CCE induced by either 1a,25(OH)2D3 or thapsigargin. Microinjection of an antisense-ODN directed against EWKFAR, a highly conserved region observed in almost all TRPC proteins, significantly inhibited (56-58%) CCE induced by 1a,25(OH)2D3 or thapsigargin respect to control cells microinjected with sense or scrambled ODNs (not shown). Similar results were obtained upon transfection of cells with a pool of different ODNs directed against regions of TRPC3-PCR products (Fig. 1). In agreement with these data, inhibitions of 30-50 % were observed when Mn2+ influx stimulation (measured as the quenching of fura-2 fluorescence by Mn2+) by 1a,25(OH)2D3 or thapsigargin was monitored in microinjected osteoblasts with a pool of antisense ODNs against human TRPC3 mRNA or the 201 bp TRPC3-PCR product (Fig 1). Neither basal (non-stimulated cells) rates of Mn2+ entry into cytosol nor the transient IP3-dependent phase of Ca2+ release induced as a consequence of 1a,25(OH)2D3-stimulation of the PLC pathway were significantly affected by using scrambled or anti-TRPC3 antisense ODN. The marked reduction observed upon transfection of antisense ODNs suggests the participation of TRPC3 proteins in CCE stimulated by 1a,25(OH)2D3 or thapsigargin in both cell types. Besides the fact that the efficiency of the antisense technique is far from being total, a heterotetrameric structure for the endogenous TRPC3-like channel may also account for the non-complete inhibition of CCE in anti-TRPC3 antisense-transfected cells. Alternatively, non-TRPC3-encoded channels may contribute to total CCE in these cells; in fact, pharmacological evidence indicating a heterogenous population of non-selective cation channels has been observed for CCE in osteoblasts (1). These data strongly suggest that in muscle cells and osteoblasts, the endogenous TRPC3-like protein takes part in the CCE pathway activated by 1a,25(OH)2D3.

The existence of a 1a,25(OH)2D3-mediated CCE pathway raises the possibility that the VDR could interact with TRPC proteins and takes part in the modulation of this Ca2+ entry route by the hormone. Because of this we examined the existence of protein-protein interactions between TRPC3 and VDR by coimmunoprecipitation. In muscle cells VDR and TRPC3 coprecipitated suggesting the association between both proteins (not shown). In accordance with this result, it has been recently reported that the VDR rapidly translocates from the nucleus to plasma membranes after short treatment of muscle cells with 1a,25(OH)2D3 (4).

Moreover, CCE and Mn2+ influx stimulation by 1a,25(OH)2D3 were inhibited 45% and 40%, respectively, upon transfection of cells with a pool of three anti-VDR antisense ODNs target against regions close to AUG of VDR mRNA (Fig. 1) suggesting the participation of the VDR in hormone modulation of CCE.

Different reports have shown that TRP channels can be modulated by association of macromolecules integrating signaling supramolecular complexes and the scaffold protein INAD clusters these macromolecules through its PDZ domains (6). In osteoblasts, two fragments of 150 and 550 bp were amplified by RT-PCR. These fragments showed 100% and 80% homology with human INAD-like (hINADL) and multiple-PDZ-domain protein (MPDZ), respectively. Northern blot analysis using as probe the 32P-labelled 150 bp sequence showed transcripts of 3.7 and 2.5 Kb in osteoblasts as in rat brain. The presence of two nucleotide sequences highly identical to human multiple PDZ domain sequences and two transcripts similar in size to that expressed in rat brain, is indicative of the presence of an INAD-like protein in these cells. In addition, microinjection of cells with various anti-INAD antisense ODNs decreased 1a,25(OH)2D3- and thapsigargin-dependent Sr2+ influx through CCE by 22% and 40%, respectively (Fig. 1). The latter data suggested a functional role for INAD-like protein in hormone activation of CCE in osteoblastic cells.

Figure 1. Participation of TRPC3, VDR and INAD proteins in CCE induced by 1,a25(OH)2D3 in skeletal muscle and osteoblastic cells. CCE was measured in fura-2-loaded cells microinjected with anti-TRP, anti-VDR, anti-INAD antisense, sense or scrambled (controls) oligodeoxynucleotides. Results are expressed as the percentage of SOC influx inhibition (measured at the peak of Ca2+, Sr2+ or Mn2+ influx) after the stimulation of the cells with 10-8 M 1a,25(OH)2D3 (white bars) or 1 mM thapsigargin (hatched bars) compared with controls (100%). Results are representative of three independent experiment performed in triplicate.

We speculate that a protein as INAD-like with multiple modular protein interaction domains could be well suited for recruiting different functional components of the 1a,25(OH)2D3 signal cascade.

Based in our results, we propose a model of action to explain how 1a,25(OH)2D3 operates in muscle and osteoblastic cells (Fig 2). Of relevance, this work provides for the first time molecular evidence on the existence of TRPC3 proteins in muscle and bone cells, which mediate 1a,25(OH)2D3 regulation of CCE through a complex formed at least by TRPC3 and VDR, and assembled by an INAD-like protein.

Figure 2. 1a,25(OH)2D3 non-genomic modulation of intracellular calcium concentration in skeletal muscle and osteoblastic cells. The model shows stimulation of CCE by 1a,25(OH)2D3 through activation of the PI-PLC/IP3 pathway and multiprotein complex formation integrated by VDR-TRPC3-INAD.

 

ACKNOWLEDGMENTS

This research was supported by grants from the Agencia Nacional de Promoción Científica y Tecnológica and Fundación Antorchas, Argentina.

REFERENCES

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Corresponding author: R. Boland, Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur,San Juan 670 (B8000CN), Bahía Blanca, Argentina. Phone: 0054-291-4595100, Fax: 0054-291-4595130, E-mail: rboland@criba.edu.ar

Received: December 4, 2004. Accepted: January 4, 2004.
 

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