Effects of single and double deletions of Trpc1 and I-mfa genes on osteoclastogenesis in vivo determined by histomorphometry. Summary data of bone volume/tissue volume (BV/TV) (A), osteoclast number/bone surface (N.Oc/BS) (B), or erosion (or osteoclast) surface/bone surface (ES/BS) (C) in tibiae of 12-week-old Trpc1^+/+;I-mfa^+/+ (WT, n = 30), Trpc1^−/−;I-mfa^+/+ (C1^−/−, n = 18), Trpc1^+/+;I-mfa^−/− (I^−/−, n = 18), and Trpc1^−/−;I-mfa^−/− (DKO, n = 17) male mice. Data were obtained by quantitative analysis of static histomorphometric indices using Goldner's Trichrome staining. Data represent mean ± S.E. ns, nonsignificant. D, representative images of Goldner's Trichrome stained sections of tibiae of four indicated strains of mice. Arrows indicate osteoclasts. Scale bar, 50 μm.

Effects of single and double deletions of Trpc1 and I-mfa genes on bone architecture determined by μCT. Summary data of bone mass as indicated by bone volume/tissue volume (BV/TV) (A), trabecular spacing (Tb Sp) (B), trabecular thickness (TbTh) (C), trabecular number (Tb N) (D), or connectivity density (Conn. Den) (E) for all four mouse strains: Trpc1^+/+;I-mfa^+/+ (WT, n = 12), Trpc1^−/−;I-mfa^+/+ (C1^−/−, n = 12), Trpc1^+/+;I-mfa^−/− (I^−/−, n = 8), and Trpc1^−/−;I-mfa^−/− (DKO, n = 17). Data represent mean ± S.E. F, representative three-dimensional images of tibiae from the indicated strain of mice obtained by μCT. Analysis of tibia trabecular architecture was initiated proximal to the growth plate. Scale bar, 100 μm.

Trpc1 and I-mfa do not genetically interact to regulate osteoblast formation in vivo. Osteoblast number/bone surface (N.Ob/BS) (A) and osteoblast surface per bone surface (ObS/BS) (B) in Trpc1^+/+;I-mfa^+/+ (WT, n = 30), Trpc1^−/−;I-mfa^+/+ (C1^−/−, n = 18), Trpc1^+/+;I-mfa^−/− (I^−/−, n = 18), and Trpc1^−/−;I-mfa^−/− (DKO, n = 17) from 12-week-old male mice were determined by bone histomorphometry. C, representative images of sections of the four indicated strains of mice. Arrows indicate osteoblasts. Scale bar, 25 μm. D, summary data and representative images of mineral apposition rate (MAR) (μm/day) determined by dynamic histomorphometry using double calcein labeling. E, bone formation rate (BFR) in μm per day. Data represent mean ± S.E.

Effects of single and double deletions of Trpc1 and I-mfa genes on osteoclastogenesis ex vivo. A, number of tartrate-resistant acid phosphatase-stained bone marrow-derived pre-osteoclasts cultured in the presence of 20 ng/ml recombinant M-CSF and 50 ng/ml RANKL for 4 days to visualize multinucleated osteoclasts. Cells with three or more nuclei were included in the analysis. Data in quadruplicates were obtained from four mice per group (n = 4). Trpc1^+/+;I-mfa^+/+ (WT), Trpc1^−/−;I-mfa^+/+ (C1^−/−), Trpc1^+/+;I-mfa^−/− (I^−/−), and Trpc1^−/−;I-mfa^−/− (DKO). B, average pit area in pixels from 100 nonoverlapping pits (n = 100) per each indicated genotype. Results from one out of two independent experiments are shown. In each experiment, 3–5 animals per genotype were used. C, representative images of resorption pits (white) left by mature and functional osteoclasts for each indicated genotype. Scale bar, 200 μm. D, representative images of phalloidin-Texas Red (1:300; Molecular Probes)-stained cells after 4 days in differentiation medium to visualize the formation of actin rings. Scale bar, 200 μm. E, representative images of tartrate-resistant acid phosphatase-stained multinucleated osteoclasts for each indicated genotype. Scale bar, 200 μm. F, size of multinucleated osteoclasts in all genotypes. Cells were classified as small (<50,000 pixels), medium (50,001–100,000 pixels), or large (>100,000 pixels) based on pixels per cell. Data represent mean ± S.E. G, in vitro derived osteoclasts effectively resorb dentin. 50,000 bone marrow-derived osteoclast precursors were plated on dentin discs in the presence of 50 ng/ml RANKL and 20 ng/ml MCSF for 10 days. Media were refreshed every 3 days. Cells were removed with a cotton swab and discs stained with Mayers hematoxylin to reveal the resorption pits (×20 magnification).

Expression of I-mfa and TRPC1 in osteoclast precursors. A–D, expression of I-mfa or Trpc1 mRNA determined by RT-PCR (A and C) or real time quantitative PCR (n = 3) (B and D) in M-CSF-untreated (day 0) or M-CSF-treated (days 2, 4, 6, and 8) and RANKL-treated (days 4, 6, and 8) nonadherent freshly isolated bone marrow-derived cells. Asterisk indicates nonspecific band. Trpc1 mRNA was reversed-transcribed, amplified by PCR, and digested with EcoRV. EcoRV-resistant PCR fragment at day 2 was gel-purified and directly sequenced. Asterisks indicate nonspecific products. E, nucleotide and corresponding amino acid sequence of the junction between exons 4 and 5 of mouse TRPC1α and TRPC1ϵ isoforms. Deleted sequence in TRPC1ϵ isoform is boxed. Unique EcoRV site in TRPC1α is shown in red.

Inhibition of TRPC1 by I-mfa in myeloid precursors. A, time course of store-operated whole currents induced by 10 mm BAPTA in the recording pipette and inhibited by 20 μm La³⁺ applied in the extracellular solution in myeloid precursors (M-CSF-treated for 2 days) obtained from Trpc1^+/+;I-mfa^+/+ (WT, n = 8 cells), Trpc1^−/−;I-mfa^+/+ (C1^{−/ −}, n = 8 cells), Trpc1^+/+;I-mfa^−/− (I^−/−, n = 12 cells), and Trpc1^−/−;I-mfa^−/− (DKO, n = 9 cells) mice. B, current-voltage (I-V) curve (taken at 200 s) of BAPTA-induced whole cell currents in cells derived from all four mouse strains. C and D, summary data of whole cell current density (pA/picofarads (pF)) at −80 (C) or +80 mV (D) of myeloid precursor cells derived from all four strains.

Translation of TRPC1 is initiated from an upstream non-AUG site. A, nucleotide sequence upstream of first AUG of mouse TRPC1. Potential non-AUG translation start sites (Sites 1–5) are boxed. B, predicted non-AUG translation start sites (sites 1–5) of mouse TRPC1 are boxed. C, mobility of TRPC1 constructs containing various lengths of the presumed 5′-untranslated region of mouse TRCP1α mRNA. HEK293T cell lysates transfected with the α-isoform of mouse TRPC1 cDNA containing the entire 5′-untranslated region (TRPC1α, lane 1), the α-isoform of mouse TRPC1α cDNA lacking the sequence upstream of the first predicted methionine (TRPC1α-ΔNTx, lane 2), TRPC1α cDNA lacking site 1 (lane 3), TRPC1α cDNA lacking sites 1 and 2 (lane 4), TRPC1α cDNA lacking sites 1–3 (lane 5), or TRPC1α cDNA with mutated site 2 (lane 6) were separated in 6% SDS-PAGE, immunoblotted, and probed with α-TRPC1 (1F1). D, translational initiation of endogenous human TRPC1 from a non-AUG site. HEK293T cell lysates transfected with pCDNA3 (mock-transfected, lane 1), TRPC1α (lane 2), or TRPC1α-ΔNTx (lane 3) were immunoblotted with α-TRPC1 (1F1). Endogenous TRPC1 was immunoprecipitated with mouse IgG (10 μg/ml lysates, negative control, lane 4) or 1F1 (10 μg/ml lysates, lane 5) and detected using 1F1.

Generation of I_SOC by TRPC1α and amplification of I_CRAC by TRPC1ϵ. A, time course and I–V curve (taken at 200 s) of BAPTA-induced whole cell currents in cells transfected with STIM1 (1.6 μg), Orai1 (1 μg), and TRPC1α (1 μg), (n = 9). pF, picofarad. B, time course and I–V curve (taken at 200 s) of BAPTA-induced whole cell currents in cells transfected with STIM1 (1.6 μg), Orai1 (1 μg), and TRPC1α-ΔNTx (1 μg) (n = 10). C, time course and I–V curve (taken at 200 s) of BAPTA-induced whole cell currents in cells transfected with STIM1 (1.6 μg), Orai1 (1 μg), and TRPC1ϵ-ΔNTx (1 μg) (n = 14). D, time course and I–V curve (taken at 200 s) of BAPTA-induced whole cell currents in cells transfected with STIM1 (1.6 μg), Orai1 (1 μg), and TRPC1ϵ (1 μg) (OSTϵ, red, n = 11) or STIM1 (1.6 μg) and Orai1 (1 μg) (OS, blue, n = 8). E, expression levels of Orai1, STIM1, TRPC1, or CD8α in transfected HEK293T cells. Cell lysates transfected with GFP (negative control, lane 1), Orai1, STIM1, and CD8α (lane 2), Orai1, STIM1, TRPC1α-ΔNTx, and CD8α (lane 3), Orai1, STIM1, TRPC1α, and CD8α (lane 4), Orai1, STIM1, TRPC1ϵ-ΔNTx, and CD8α (lane 5), or Orai1, STIM1, TRPC1ϵ, and CD8α (lane 6) were immunoblotted with a rabbit polyclonal antibody to Orai1 (1:5000, Sigma), mouse monoclonal antibody to STIM1 (1:1000, Cell Signaling), mouse monoclonal to TRPC1 (1F1, 1:300), or rabbit polyclonal to CD8α (1:500, Santa Cruz Biotechnology). Asterisks indicate nonspecific bands. F, effects of Na⁺ to N-methyl-d-glucamine substitution on whole cell currents induced by BAPTA (10 mm in pipette) in HEK293 cells transiently transfected with Orai1 and STIM1 (OS, black, (n = 5) or Orai1, STIM1, and TRPC1ϵ (OSTϵ, red, n = 8). G, effects of Ca²⁺ to Ba²⁺ substitution on whole cell currents induced by BAPTA (10 mm in pipette) in HEK293 cells transiently transfected with Orai1 and STIM1 (OS, black, n = 7) or Orai1, STIM1, and TRPC1ϵ (OSTϵ, red, n = 8).

Cell surface expression of TRPC1α and TRPC1ϵ in transiently transfected HEK293 cells. Cells were left untrasfected (lanes 1 and 5) or transfected with STIM1 + Orai1 + TRPC1α (lanes 2, 4, 6, and 8), or STIM1 + Orai1 + TRPC1ϵ (lanes 3 and 7) in 10-cm dishes using Lipofectamine 2000. Twenty four hours following transfection, cell surface proteins were biotinylated with 0.05 mg/ml cell impermeant biotin for 30 min at room temperature in PBS (pH 8.0), washed three times with PBS plus 100 mm glycine, and captured with streptavidin beads. Biotinylated proteins were probed with rabbit α-Orai1 (1:5000 dilution, Sigma) (upper left panel), mouse monoclonal α-TRPC1 (1F1, 3.4 μg/ml) (middle left panel), or mouse monoclonal α-β-actin (1:1000 dilution, Santa Cruz Biotechnology) (lower left panel). Right panels indicate input amounts of Orai1, TRPC1, and β-actin in lysates. Cells transfected with Orai1, STIM1, and TRPC1α were stimulated by 1 μm thapsigargin (TG) in DMEM supplemented with 10%FBS for 5 min before biotinylation (lanes 4 and 8). IP, immunoprecipitation; IB, immunoblot.

Suppression of I_CRAC and I_SOC by I-mfa. A, time course and I–V curves (taken at 200 s) of BAPTA-induced whole cell currents in HEK293 cells transfected with STIM1 (1.6 μg), Orai1 (1 μg), and I-mfa (0.3 μg) (OSImfa, black, n = 7), STIM1 (1.6 μg), Orai1 (1 μg), TRPC1ϵ (1 μg), and I-mfa (0.3 μg) (OSTϵImfa, red, n = 10), or STIM1 (1.6 μg), Orai1 (1 μg), TRPC1ϵ (1 μg), and I-mfb (0.3 μg) (OSTϵImfb, violet, n = 8). B, time course and I–V curves (taken at 200 s) of BAPTA-induced whole cell currents in HEK293 cells transfected with STIM1 (1.6 μg), Orai1 (1 μg), TRPC1α (1 μg), and I-mfa (0.3 μg) (OSTαImfa, blue, n = 10), or STIM1 (1.6 μg), Orai1 (1 μg), TRPC1α (1 μg), and I-mfb (0.3 μg) (OSTαImfb, green, n = 8). C, expression levels of Orai1, STIM1, TRPC1ϵ, and TRPC1α in HEK293 cell lysates transfected with pCDNA3 (lane 1), Orai1, STIM1, TRPC1ϵ, and FLAG-tagged I-mfa (lane 2), Orai1, STIM1, TRPC1ϵ, HA-tagged I-mfb (lane 3), Orai1, STIM1, TRPC1α, and FLAG-tagged I-mfa (lane 4), Orai1, STIM1, TRPC1α, and HA-tagged I-mfb (lane 5), or Orai1, STIM1, and FLAG-tagged I-mfa (lane 6). Asterisk indicates a nonspecific band. D, summary data showing the effect of indicated plasmids on BAPTA-induced I_CRAC or I_SOC density at −80 mV obtained 200 s following break-in in HEK293 cells. pF, picofarad. E, I-mfa does not disrupt the association of Orai1 and TRPC1. Panel a, HEK293T cells were transfected with pCDNA3 (mock, lane 1), Orai1 (lane 2), TRPC1ϵ (lane 3), FLAG-tagged I-mfa (F-I-mfa, lane 4), Orai1 + F-I-mfa (lane 5), F-I-mfa and TRPC1ϵ (lane 6), Orai1 + F-I-mfa + TRPC1ϵ (lane 7), or Orai1 + TRPC1ϵ (lane 8). Endogenous and transfected Orai1 was immunoprecipitated (IP) with α-Orai1, and association with transfected TRPC1ϵ was determined by immunoblotting (IB) using α-TRPC1. Panel b, association of F-Imfa and TRPC1 was determined by immunoprecipitation with α-FLAG and immunoblotting using α-TRPC1. Panels c–e, input amounts of TRPC1ϵ, F-I-mfa, or Orai1.

Hypothetical model for the modulation of I_SOC and I_CRAC by TRPC1 and I-mfa in early stages of osteoclastogenesis. M-CSF primes myeloid precursors for Ca²⁺ signaling by up-regulating not only TRPC1ϵ but also its negative regulator, I-mfa. As a result, Ca²⁺ signaling is maintained at a low level in this stage. In response to RANKL, I-mfa is down-regulated, although TRPC1ϵ expression persists, setting up myeloid precursors/early pre-osteoclasts highly competent for Ca²⁺ signaling, which is crucial for the downstream activation of NFATc1 and other regulators of osteoclastogenesis.