Calreticulin regulates transforming growth factor-β-stimulated extracellular matrix production

Abstract

Endoplasmic reticulum (ER) stress is an emerging factor in fibrotic disease, although precise mechanisms are not clear. Calreticulin (CRT) is an ER chaperone and regulator of Ca(2+) signaling up-regulated by ER stress and in fibrotic tissues. Previously, we showed that ER CRT regulates type I collagen transcript, trafficking, secretion, and processing into the extracellular matrix (ECM). To determine the role of CRT in ECM regulation under fibrotic conditions, we asked whether CRT modified cellular responses to the pro-fibrotic cytokine, TGF-β. These studies show that CRT-/- mouse embryonic fibroblasts (MEFs) and rat and human idiopathic pulmonary fibrosis lung fibroblasts with siRNA CRT knockdown had impaired TGF-β stimulation of type I collagen and fibronectin. In contrast, fibroblasts with increased CRT expression had enhanced responses to TGF-β. The lack of CRT does not impact canonical TGF-β signaling as TGF-β was able to stimulate Smad reporter activity in CRT-/- MEFs. CRT regulation of TGF-β-stimulated Ca(2+) signaling is important for induction of ECM. CRT-/- MEFs failed to increase intracellular Ca(2+) levels in response to TGF-β. NFAT activity is required for ECM stimulation by TGF-β. In CRT-/- MEFs, TGF-β stimulation of NFAT nuclear translocation and reporter activity is impaired. Importantly, CRT is required for TGF-β stimulation of ECM under conditions of ER stress, as tunicamycin-induced ER stress was insufficient to induce ECM production in TGF-β stimulated CRT-/- MEFs. Together, these data identify CRT-regulated Ca(2+)-dependent pathways as a critical molecular link between ER stress and TGF-β fibrotic signaling.

Keywords: Calcium; Calreticulin; Collagen; Endoplasmic Reticulum Stress; Fibronectin; Fibrosis; NFAT Transcription Factor; Transforming Growth Factor β (TGFβ).

FIGURE 1.

TGF-β induces fibronectin and COL1A1 transcript in wild type but not CRT−/− MEFs. Wild type (gray bars, gray symbols) and CRT−/− MEFs (open bars, open symbols) were grown overnight in medium with 10% FBS, starved for 12 h in low serum (0.5% FBS) medium, and treated with increasing concentrations of TGF-β for 4 h (A and B). Cells were also treated with 100 pm TGF-β over a 24-h time period (C and D). RNA was harvested by TRIzol, and transcript levels of fibronectin (A and C) and COL1A1 (B and D) were determined by quantitative real time PCR (RQ). Values represent mean levels normalized to S9 ± S.D. of triplicate samples each performed in technical triplicates. Values for untreated wild type and CRT−/− cells were set to 1. Each assay shown is representative of three separate experiments with similar results. *, p < 0.05 versus non-treated or time zero cells.

FIGURE 2.

CRT is required for TGF-β stimulation of fibronectin and collagen I protein. A and B, wild type (gray bars) and CRT−/− MEFs (open bars) were grown overnight in medium with 10% FBS, starved for 2 h in low serum (0.5% FBS) medium, and then treated with 100 pm TGF-β with 20 μm ascorbic acid for 24 h. A, Laemmli cell lysates were immunoblotted for fibronectin and collagen. Results of a representative blot are shown. Results are the mean density of bands normalized to β-tubulin ± S.D. (n = 4 separate experiments). B, the 4% DOC soluble (cell fraction) and insoluble (extracellular matrix) fractions of cells treated as in A were separated by SDS-PAGE and immunoblotted for fibronectin. Membranes were re-probed with antibody to β-tubulin to determine loading and efficacy of fractionation of the cellular and extracellular matrix fractions. C, wild type (gray bars) and CRT−/− MEFs (open bars) were plated in medium with 10% FBS overnight, switched to low serum medium with 20 μm ascorbic acid, and treated with 10 pm TGF-β for 72 h, re-dosing TGF-β and ascorbic acid every 24 h. After 72 h, conditioned medium from triplicate samples were pooled, and levels of secreted soluble collagen were measured by Sircol assay according to the manufacturer's specifications. Results are the means ± S.D. from three separate experiments. *, p < 0.05 versus non-treated cells.

FIGURE 3.

Impaired responsiveness to TGF-β in the CRT−/− MEFs can be rescued by transfection with CRT plasmid or CRT plasmid lacking the TSP1 binding site. A, CRT−/− MEFs stably transfected with rabbit HA-tagged CRT were grown overnight in medium with 10% FBS, starved overnight in low (0.5%) serum medium, and treated with 100 pm TGF-β for 4 h. RNA was harvested by TRIzol, and transcript levels of fibronectin (open circles) and COL1A1 (closed squares) were determined by quantitative real time PCR. B, wild type MEFS (gray bars), CRT−/− MEFs stably expressing rabbit CRT lacking the TSP1 binding site (black bars), and CRT−/− MEFs (open bars) were treated as in A, RNA was harvested, and levels of COL1A1 were determined by RTQ-PCR (RQ). Results are the means normalized to S9 levels of triplicate samples ± S.D. from one representative experiment. Experiments were repeated on three (A) or two (B) separate occasions. The means of untreated cells were set to 1. *, p < 0.05 versus untreated cells.

FIGURE 4.

Knockdown of CRT in Thy1−/− rat lung fibroblasts and human lung fibroblasts significantly inhibits TGF-β-stimulated matrix production. Thy1−/− rat lung fibroblasts were transfected with 100 nm non-targeting (NT) or CRT siRNA in medium with 10% FBS and maintained for 24 h. Cells were switched to low (0.5%) serum medium and stimulated with 100 pm TGF-β for 24 h. Laemmli cell lysates were immunoblotted for CRT (B), fibronectin (C), and collagen type I (D). A representative blot is shown in A. Results are the mean density normalized to β-actin ±S.D. from three separate experiments. E, human IPF lung fibroblasts were transfected with 200 nm non-targeting or CRT siRNA and maintained in medium with 10% FBS for 48 h. Cells were switched to low FBS medium for 6 h and then treated with 100 pm TGF-β with 2 μm ascorbic acid for 24 h. Laemmli cell lysates were separated by SDS-PAGE and immunoblotted for CRT, fibronectin, collagen type Iα2, and β-tubulin. Densitometric analysis of bands normalized to β-tubulin are indicated below each band. Results are representative of three separate experiments. *, p < 0.05 versus untreated cells.

FIGURE 5.

Overexpression of CRT increases TGF-β stimulation of ECM. Parental and CRT overexpressing L fibroblasts were grown overnight in medium with 10% FBS, starved for 2 h in medium with low (0.5%) serum, and treated with 100 pm TGF-β for 24 h for fibronectin determinations (A) or 48 h for collagen I determinations (B). Cells were harvested with Laemmli buffer, separated by SDS-PAGE, and immunoblotted for fibronectin, collagen I, and β-tubulin. C, bands were analyzed by densitometry (n = 3 separate experiments) and normalized to β-tubulin ±S.D. Untreated cells were set to 1.0. *, p < 0.05 versus parental cells. CRT overs, CRT overexpressors.

FIGURE 6.

ER stress is insufficient to drive TGF-β stimulation of ECM in CRT−/− MEFs. A, wild type (gray bars) and CRT−/− MEFs (open bars) were grown overnight in DMEM with 10% FBS and 20 μm ascorbic acid with or without 0.01 μg/ml tunicamycin. Cells were treated with 100 pm TGF-β in low (0.5%) serum medium containing 20 μm ascorbic acid for 24 h. Laemmli cell lysates were immunoblotted for collagen I, GRP78, or β-tubulin. A–C, bands were analyzed by densitometry and normalized to β-tubulin. Results are the mean densities normalized to β-tubulin ± S.D. from three separate experiments. *, p < 0.05 versus untreated cells. UT, untreated; TM, tunicamycin.

FIGURE 7.

TGF-β stimulates Smad activity in wild type and CRT−/− MEFs. A, wild type and CRT−/− MEFs were grown overnight in medium with 10% FBS, starved overnight in low (0.5%) serum medium, and treated with 100 pm TGF-β for 15, 30, or 60 min. Laemmli cell lysates containing phosphatase inhibitor were immunoblotted for phospho-Smad 3. Membranes were reprobed with antibody to Smad 2/3 or β-tubulin (data not shown) to normalize cell protein. The blot is representative of four separate experiments using 10–400 pm TGF-β. B, wild type and CRT−/− MEFs were grown overnight on glass coverslips in medium with 10% FBS, starved in low serum medium overnight, and treated with 100 pm TGF-β for 30 min. Cells were fixed, permeabilized, and incubated with antibody to phospho-Smad 3 followed by fluorescein-conjugated secondary antibody. Nuclei were stained with Hoechst. Results are representative of three separate experiments. Confocal images were obtained at an original magnification of 600×. C, wild type and CRT−/− MEFs were transfected with the Smad 2/3/4 firefly luciferase reporter construct and the control renilla luciferase construct and kept in medium with 10% FBS overnight, switched to low serum DMEM, and treated with 100 pm TGF-β for 8 h. Some cells were treated with 100 pm TGF-β in the presence of 3 μm LY364947. Cells were lysed with 1× lysis buffer (Promega), and triplicate samples were combined. Luciferase reporter activity is normalized to the renilla luciferase control. Data represent the means of samples from three separate experiments ± S.D. *, p < 0.05 versus untreated control.

FIGURE 8.

TGF-β stimulates Ca²⁺ release, and Ca²⁺-dependent fibronectin and COL1A1 transcript are impaired in the CRT−/− MEFs. Wild type (A) and CRT−/− MEFs (B) were plated overnight in DMEM with 10% FBS, washed with low (0.5%) serum medium, and loaded with 5 μm Fluo-4 AM in low serum medium with 10 mm HEPES. Cells were loaded with dye for 20 min at 37 °C. After a 5-min equilibration, cells were stimulated with 100 pm TGF-β (red squares), 1 μm ionomycin (blue squares), or low serum medium (gray circle). Cells were excited at 485 nm, and emission was read at 520 nm. Results are representative of a typical experiment repeated in quadruplicate on at least four different occasions. C and D, wild type MEFs were plated overnight in medium with 10% FBS and starved overnight in low (0.5%) serum medium. Cells were pretreated with thapsigargin (0.5 μm) for 30 min and washed with low serum medium to remove the thapsigargin. Cells were treated with or without 100 pm TGF-β for 4 h. RNA was harvested with TRIzol and transcript levels of fibronectin (C) and COL1A1 (D) were determined by RTQ-PCR. Values represent the mean expression levels normalized to S9 ± S.D. of triplicate samples from a single representative experiment. Experiments were repeated three times with similar results. E, CRT−/− MEFs were plated overnight in medium with 10% FBS, starved overnight in low serum medium, and treated with TGF-β (100 pm), ionomycin (Iono, 1 μm), or both for 4 h. RNA was harvested with TRIzol, and transcript levels of fibronectin and COL1A1 were determined by RTQ-PCR (RQ). *, p < 0.05 versus untreated control.

FIGURE 9.

CRT−/− MEFs do not stimulate NFAT activity in response to TGF-β. A, wild type and CRT−/− MEFs were grown overnight on glass coverslips in medium with 10% FBS, starved overnight in low (0. 5%) serum medium, and stimulated with 400 pm TGF-β for 5 or 15 min. After treatment, cells were fixed, permeabilized, and incubated with anti-NFATc3 antibody. Cells were washed with PBS and incubated with a fluorescein-labeled secondary antibody. Results are representative of one experiment performed on at least three separate occasions. Original magnification = 1000×. B, wild type (gray bars) and CRT−/− MEFs (open bars) were transfected with an inducible NFAT reporter firefly luciferase reporter construct and a renilla luciferase control construct overnight in medium with 10% FBS. Cells were starved for 2 h in low serum medium and then treated every 2 h with 100 pm TGF-β over an 8-h span. After 8 h, cells were lysed, and triplicate samples were combined. Luciferase reporter activity was normalized to the renilla luciferase control. Data represent the mean normalized luciferase activity ± S.D. of one representative experiment performed in triplicate. The experiment was performed on three separate occasions with similar results. *, p < 0.05 versus untreated control. C and D, wild type MEFs were grown overnight in medium with 10% FBS, starved overnight in low serum medium, and pretreated with 2 μm 11R-VIVIT for 30 min (open bars). Cells were treated with or without 100 pm TGF-β ± 11R-VIVIT peptide for 4 h, and RNA was harvested with TRIzol. Transcript levels of fibronectin (C) or COL1A1 (D) were determined by RTQ-PCR and normalized to S9 levels ±S.D. Results are from one experiment of triplicate samples. Similar results were obtained in three separate experiments. *, p < 0.05 versus untreated control.

FIGURE 10.

Model of CRT regulation of TGF-β ECM transcription. TGF-β binds to the heterotetrameric receptor complex (blue bars) to activate receptor type I kinase activity, which phosphorylates Smad2 and Smad 3. TGF-β also increases cytosolic Ca²⁺. The specific mechanism by which TGF-β regulates Ca²⁺ release and the involvement of Smad signaling in Ca²⁺ release are not yet clear (orange arrow). TGF-β stimulated Ca²⁺ release is dependent on CRT regulation of ER Ca²⁺ and possibly CRT-regulated store-operated Ca²⁺ entry (not depicted). Increased cytosolic Ca²⁺ leads to activation of calcineurin, which dephosphorylates cytoplasmic NFAT, resulting in NFAT activation and nuclear translocation. NFAT can directly stimulate transcription of ECM proteins or partner with other known matrix-inducing transcription factors such as AP-1 and SP-1. In the absence of CRT, there is a failure of TGF-β to increase cytoplasmic Ca²⁺, activate NFAT, and up-regulate ECM transcription. In contrast, under pathological ER stress, CRT expression can be up-regulated, resulting in increased TGF-β stimulated ECM.