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TRPC3‐like protein is involved in the capacitative cation entry induced by 1α,25‐dihydroxy‐vitamin D 3 in ROS 17/2.8 osteoblastic cells
Author(s) -
Baldi Carolina,
Vazquez Guillermo,
Calvo Juan Carlos,
Boland Ricardo
Publication year - 2003
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.10612
Subject(s) - trpc3 , trpc , endogeny , microbiology and biotechnology , transient receptor potential channel , thapsigargin , messenger rna , western blot , biology , receptor , chemistry , biochemistry , gene , intracellular
In ROS 17/2.8 rat osteoblastic‐like cells a capacitative Ca 2+ entry (CCE) pathway operates which is activated by either 1α,25‐dihydroxy‐vitamin D 3 (1α,25(OH) 2 D 3 ) or thapsigargin (Tpg)‐induced depletion of Ca 2+ stores (Baldi et al. [2002]: J. Cell. Biochem. 86:678–687). In view of recent evidence favoring a role for transient receptor potential (TRP) proteins in mediating CCE, we investigated if channels involved in the 1α,25(OH) 2 D 3 ‐sensitive CCE in rat osteoblasts were related to an endogenous TRP‐canonical (TRPC) isoform homologue. By reverse transcription (RT)‐PCR using mRNA from ROS 17/2.8 cells and primers based on conserved regions within the mammalian TRPC3/6/7 subfamily, two fragments were amplified of 390 and 201 bp with 100 and 94% sequence identity, respectively, with human TRPC3. Northern blot analysis showed the presence of a 3.5 kb transcript and both immunobloting and immunocytochemistry using a specific anti‐TRPC3 antibody confirmed endogenous expression of a TRPC3‐like protein (∼110 kDa) with membrane localization. In ROS 17/2.8 cells intranuclearly microinjected with anti‐TRPC3 antisense oligodeoxynucleotides (ODN), both the initial rate and magnitude of CCE activated by either 1α,25(OH) 2 D 3 or Tpg were markedly reduced, whereas no changes were detected in control‐injected cells. The present findings constitute the first evidence to date suggesting that an endogenous TRPC3‐like protein is functionally involved in the CCE route activated by 1α,25(OH) 2 D 3 in a secosteroid target cell. We anticipate TRPC3 as a candidate for mediating store‐operated non‐selective cation entry into osteoblasts. J. Cell. Biochem. 90: 197–205, 2003. © 2003 Wiley‐Liss, Inc.